System for control of autonomic dysreflexia

Abstract

The invention provides a neuromodulation / neurostimulation system (10) for stimulating sympathetic circuitry responsible for blood pressure control in a mammal with autonomic dysreflexia, said system (10) comprising: at least one control unit (12) configured and arranged to provide stimulation data, and at least one stimulation unit (14), operatively connected to the at least one control unit (12), said at least one stimulation unit (14) being configured and arranged to provide electrical stimulation to the spinal cord of said mammal, preferably at thoracic level, more preferably around spinal cord level T10-T12. The at least one stimulation unit (14) includes an implantable lead (18). The neuromodulation / neurostimulation system is configured and arranged to provide neuromodulation to said mammal to activate the sympathetic circuitry responsible for blood pressure control that mitigates autonomic dysreflexia.

Description

[0001] Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 System for control of autonomic dysreflexiaThe present invention belongs to the technical field of spinal cord stimulation for rehabilitation of an autonomic function in a mammal, in particular a human. More specifically, the present invention relates to a system for stimulation of the spinal cord, in particular for the neurorehabilitation of an autonomic function, more in particular for control of autonomic dysreflexia in a mammal.In particular, said mammal may be a human with spinal cord injury (SCI) and / or otherneurological disorders such as a stroke, multiple sclerosis, autonomic failure, autonomicneuropathy or cancer of the neurological tissue which impair operation of descending sympathetic pathways that normally facilitate control of autonomic functions. The spinal cord is an integral part of the central nervous system (CNS). SCI may result notonly in motor and sensory deficits, but also in autonomic dysfunctions. SCI may result indisconnection of some, most, or all descending sympathetic pathways that carry signalsresponsible for regulating arterial blood pressure, heart rate and / or gut and bladder function.Autonomic dysfunctions following SCI are potentially life-threatening conditions for that maylead, inter alia, to blood pressure instability and ensuing chronic dysfunction of the heart and vasculature.Most SCI patients experience multiple drastic, blood pressure fluctuations on a daily basis,and rank this as a top healthcare priority, even more than restoration of motor functions, e.g., walking. Blood pressure is the pressure of circulating blood on the walls of blood vessels. Withoutfurther specification, ‘blood pressure’ often may refer to the pressure in arteries of the systemiccirculation. Blood pressure may usually be expressed in terms of the systolic pressure, i.e. the maximum pressure during one heartbeat and / or over diastolic pressure, i.e. the minimum pressure in between two heartbeat and / or over mean arterial blood pressure, i.e. an average blood pressure in an individual during a single cardiac cycle, and may be measured in millimeters of mercury (mmHg, above the surrounding atmospheric pressure). Blood pressure monitoring may comprise monitoring a parameter value such as a diastolic blood pressure, a systolic blood pressure, a diastolic blood pressure and a systolic blood pressure, a mean arterial pressure, a blended blood pressure value or the like.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Further, perfusion pressure, i.e., spinal cord perfusion pressure, which is defined as thedifference between mean arterial blood pressure and cerebrospinal fluid pressure may be ofimportance. The latter may be monitored using intrathecal catheters placed, e.g., near the siteof injury or in the lumbar cistern of an injured mammal, in particular a human.In SCI subjects, in case of increase or decrease of blood pressure, in particular arterial bloodpressure, the spinal cord neurons responsible for blood pressure control no longer have the capacity to maintain blood pressure at a normal, physiological level. The interruption of supraspinal efferent commands to sympathetic circuits located in the spinal cord in SCI subjects prevents natural baroreflex from controlling these circuits to adjust peripheral vascular resistance, thereby generating haemodynamic instability (Furlan J. C. et al., Descending vasomotor pathways in humans: correlation between axonal preservation and cardiovascular dysfunction after spinal cord injury. J Neurotrauma, 2003 Dec; 20(12):1351-63. doi: 10.1089 / 089771503322686148).SCI subjects may suffer from extremely low blood pressure, i.e., hypotension. In particular,SCI subjects may experience very low arterial blood pressure at rest, during exercise and / orwhen assuming a seated or standing position. This may lead to dizziness, disorientation,reduction in cognitive functioning, loss of consciousness, and a predisposition to strokes andheart attacks. In particular, SCI subjects may suffer orthostatic hypotension, i.e., the inability to maintainblood pressure within a normal, physiological range when moving from lying to sitting orstanding. Gravity causes blood to pool in the lower part of the body when a subject is sitting orstanding. A reduction in arterial blood pressure or blood volume reduces baroreceptor activity. In healthy subjects, this activates the sympathetic branch of the autonomic nervous system,restoring blood pressure by increasing vascular resistance and the flow of blood back to theheart. SCI interrupts the connection between the lower brainstem, which receives the information from the baroreceptors, and the sympathetic neurons that innervate thecardiovascular system, originating from thoracic and upper lumbar spinal segments.Accordingly, baroreceptors are no longer able to work properly (P.G. Guyenet, Neuroprostheticdevice maintains blood pressure after spinal cord injury, Nature, article published online onJanuary 27, 2021). In recent times, a neuroprosthetic system and epidural electrical stimulation (EES) protocolshave been developed for minimizing orthostatic hypotension after SCI (or other neurologicaldisorders), and restore blood-pressure control.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025In case of a fall in blood pressure, said neuroprosthetic system provides an electrical stimulusthrough targeted epidural spinal stimulation (TESS) to the back side of the thoracic spinal cordto activate sympathetic neurons, so that the sympathetic neurons are enabled to send signalsto the heart and blood vessels to increase cardiac output and vascular resistance, thereby restoring normal blood pressure (Squair, J.W. et al. Neuroprosthetic baroreflex controls haemodynamics after spinal cord injury, Nature 590, 308–314 (2021). https: / / doi.org / 10.1038 / s41586-020-03180-w). On the other hand, SCI subjects may also experience dangerous elevations in blood pressure, i.e. hypertension, that may lead to heart attacks, strokes, and sub-clinical vascular consequences. A critical autonomic issue after SCI is autonomic dysreflexia, which is associated withpotentially life-threatening elevations in blood pressure due to afferent input activatingsympathetic circuitry located caudally on the spinal cord to the location of the SCI. Clinically, autonomic dysreflexia is defined as elevations in systolic blood pressure of 20 mmHg or more (WO2018148844A1).The severe, rapid and uncontrolled elevations of blood pressure that characterize autonomicdysreflexia may lead to long term damage of both the cerebral vasculature and the heart, oreven death. SCI subjects may experience autonomic dysreflexia episodes on a daily basis, up to 40 times per day. Autonomic dysreflexia is a life-threatening medical condition characterized by episodes of uncontrolled hypertension that occur in response to sensory stimuli after spinal cord injury (SCI) [1]-[7]. A detailed study of life-threatening outcomes associated with autonomic dysreflexia can be found in Wan D, Krassioukov A V., Life-threatening outcomes associated with autonomic dysreflexia: A clinical review, The journal of spinal cord medicine, 2014;37:2-10). Typically, SCI subjects are more likely to experience episodes of autonomic dysreflexia when the lesion occurs above spinal cord level T6. The higher the level of the lesion, the greater the risk with up to 90% of patients with cervical spinal or high-thoracic spinal cord injury beingsusceptible (Allen K.J. et al, Autonomic Dysreflexia, StatPearls Publishing; Jan. 2021).Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Systems and methods for control and rehabilitation of autonomic functions, e.g., bloodpressure, in a mammal, in particular a human, are known in the art. WO2018148844A1 discloses a device and algorithm for controlling an autonomic function in an individual. In particular, a controller device is disclosed that utilizes physiologicalmeasurements (such as blood pressure) to regulate spinal cord electrical stimulation tostabilize blood pressure. A control interface and algorithm for controlling an autonomic functionin a subject. In particular, an algorithm that utilizes physiological measurements (such as bloodpressure) is disclosed to regulate spinal cord electrical stimulation to stabilize blood pressure.The neuronal structures involved may be located within the T1 to S5 segments of the spinal cord, for example. Stimulation may be configured to control a particular function by selecting electrodes and / or the nature of the stimulation. US2007156200A1 discloses an apparatus and a method for controlling blood pressure by stimulating the cardiac afferent sympathetic nerves. The invention may be implemented in a medical device having a pressure sensor for sensing blood pressure, an electrode for providing electrical signals to the cardiac afferent sympathetic nerves, and a controller for providing signals to the electrode as a function of blood pressure signals received from the pressure sensor. US2011082515A1 discloses a neurostimulation device including an external neurostimulator worn by a patient using a bracing element that braces a portion of the patient's body. Theexternal neurostimulator delivers neurostimulation to modulate a cardiovascular function of thepatient. Preferably, the external stimulator delivers the neurostimulation transcutaneously to a stimulation target in the patient's body using surface stimulation electrodes placed on the body approximately over the stimulation target. US2011 / 0202107A1 relates to an electric stimulation apparatus for treating hypotension of patients suffering from SCI and a method for treating hypotension. The electric stimulation apparatus comprises: a blood pressure measuring means for continuously measuring a blood pressure of a subject; an electric current application means for intermittently applying an electric current to skin of the subject; and a control means for controlling the electric current application means so as to maintain the blood pressure at a predetermined target blood pressure value by activating the electric current application means when the subject blood pressure is equal to or less than the target blood pressure value. US2013 / 0289650A1 relates to neuromodulation for controlling hypertension and other cardio- renal disorders of a patient suffering from SCI. A neuromodulation device is delivered to aOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 patient’s body for applying electric activation to decrease renal sympathetic hyperactivity of the patient based on monitored blood pressure of the patient, substantially without thermal energization of the patient’s body by applying the electric activation. The electric activation may also depend on monitored blood volume of the patient. A feedback control module may be used to provide feedback control information for adjusting the electric activation based on the monitored blood pressure and volume of the patient. US3650277A discloses a system for reducing and controlling the blood pressure of a hypertensive patient by providing electrical pulse stimulation of the carotid-sinus nerves controlled by the arterial blood pressure of the patient in such a manner that the number of stimulation pulses within each heart cycle is determined by the arterial means blood pressure whereas the distribution of stimulation pulses over the heart cycle is a function of the arterial pulse wave shape with the pulse frequency being greater during the first portion of the heart cycle. US6058331 discloses techniques for therapeutically treating peripheral vascular disease. A sensor is implemented for sensing the extent of blood flow in a patient's limb or ischemic pain and generating a corresponding sensor signal. The signal is processed to determine the level of spinal cord stimulation or peripheral nerve stimulation to be applied. This information is provided to a signal generator which thereby provides electrical stimulation energy to one or more stimulation leads. Stimulation of the spinal cord, peripheral nerve or neural tissue ganglia thereby improves blood flow, helps restore tissue health and reduces the extent of ischemic pain in the limbs of a peripheral vascular disease patient or organs of other patients. US2007027495A1 relates to an implantable bladder sensor attachable to an exterior surface of a urinary bladder to sense bladder condition or activity for urinary incontinence, or an inability to control urinary function. The sensor includes a strain gauge that detects mechanical deformation of the bladder. Mechanical deformation may be indicative of a gradual filling of the bladder, or an instantaneous contraction indicating an imminent urine voiding event. Wireless telemetry circuitry within the sensor transmits information to implanted electrical stimulator that delivers electrical stimulation for alleviating urinary incontinence, or to an external programmer that controls the implanted stimulator. One known approach for clinical management of autonomic dysreflexia is focused on symptoms management, e.g., by using hypotension-inducing pharmacology. However, most of the antihypertensive drugs for clinical management of autonomic dysreflexiashow a delayed onset of action (about 60 minutes or more), and are thus are sub-optimalOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025especially for managing transient (i.e., within the range of seconds), but drastic, changes inblood pressure. In addition, said known antihypertensive drugs have significant side effects. Inparticular, said drugs may determine a drastic decrease in arterial blood pressure, below thedesired level, which may last even for hours and thereby require monitoring and additional clinical management (WO2018148844A1). Another known approach for clinical management of autonomic dysreflexia is focused on targeting the triggers of autonomic dysreflexia, e.g., through Botox injections in the bladder. Additional background art on clinical management of autonomic dysreflexia can be found in Krassioukov A. et al., A Systematic Review of the Management of Autonomic Dysreflexia After Spinal Cord Injury, Archives of physical medicine and rehabilitation, 2009;90:682-695. However, there is currently no therapy that is capable of leading to a sustained and robust reduction in both the number of autonomic dysreflexia episodes as well as their severity. In particular, the fragmented understanding of the mechanisms underlying autonomic dysreflexia hampered the development of therapeutic strategies to manage this condition, leaving people with SCI at daily risk of heart attack and stroke [8]-

[0018] .Thus, there is a strongly-felt need for an improved solution allowing to significantly reduceseverity and frequency of autonomic dysreflexia episodes after SCI and / or other neurologicaldisorders. It is therefore an object of the present invention to provide a neuromodulation / neurostimulation system for control of autonomic dysreflexia after SCI and / or other neurological disorders thatallows to significantly reduce severity and frequency of autonomic dysreflexia episodes.In particular, there is the need for a rationally focused system and stimulation paradigmdesigned to specifically target and activate sympathetic nervous system structures responsiblefor blood pressure control to eliminate, or at least significantly mitigate, autonomic dysreflexiaepisodes after SCI and / or other neurological disorders.This object is achieved by the provision of a neuromodulation / neurostimulation systemaccording to claim 1. The present invention provides a neuromodulation / neurostimulation system for stimulating sympathetic circuitry responsible for blood pressure control in a mammal with autonomic dysreflexia.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025In particular, said mammal may be a human affected by SCI and / or other neurologicaldisorders such as a stroke, multiple sclerosis, autonomic failure, autonomic neuropathy orcancer of neurological tissue which impair operation of descending sympathetic pathways thatnormally facilitate control of autonomic functions. The system comprises at least one control unit. The at least one control unit is configured and arranged to provide stimulation data. In one embodiment, the system may comprise a single control unit. The system further comprises at least one stimulation unit. The at least one stimulation unit is operatively connected to the at least one control unit. The at least one stimulation unit is configured and arranged to provide electrical stimulation to the spinal cord of said mammal. In one embodiment, the system may comprise a single stimulation unit.The at least one stimulation unit includes an implantable lead.Further, the neuromodulation / neurostimulation system is configured and arranged to provideneuromodulation to said mammal to activate the sympathetic circuitry responsible for blood pressure control that mitigates autonomic dysreflexia. The implantable lead is configured and arranged to provide stimulation to the spinal cord ofsaid mammal at thoracic level, especially at spinal cord level around T10-T12.The invention is based on the basic idea that a stimulation system has to be provided whichspecifically targets and modulates sympathetic circuitry responsible for blood pressure controlin a way that enables precise control over blood pressure after SCI and / or other neurologicaldisorders. In particular, a novel stimulation system and stimulation paradigm are provided fordelivering stimulation at a specific location of the spinal cord, i.e., at spinal cord level T10-T12,to activate sympathetic neuronal circuitry responsible for blood pressure control that mitigates severity and / or frequency of autonomic dysreflexia.As extensively discussed in the following, experiments carried out by the inventors exposedcomplete de novo neuronal architecture that develops after SCI and causes autonomicdysreflexia. In parallel, the inventors uncovered a competing, yet overlapping neuronalOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 architecture activated by epidural electrical stimulation of the spinal cord that safely regulates blood pressure after SCI. The discovery that these adversarial neuronal architectures converge onto a single neuronal subpopulation provided a blueprint for the design of a mechanism-based intervention that reversed autonomic dysreflexia in mice, rats, and humans with SCI. These results establish a path for the effective treatment of autonomic dysreflexia in people with SCI. The system may further include at least one implantable pulse generator (IPG). Advantageously, the at least one IPG may be programmed with a recurring EES sequence. In particular, the implantable lead may include one or more implantable electrode arrays.The one or more electrode arrays may be positioned over the dorsal side of the lower thoracicspinal cord of a subject, preferably a human, typically at spinal cord level T10-T12.The one or more electrode arrays may be connected to the at least one IPG by cables that areconfigured and arranged to carry electrical current from the at least one IPG toward theimplanted electrode arrays that deliver electrical stimulation to the spinal cord. The system may operate as an open-loop system.Alternatively, the system may operate as a closed-loop system.In this case, the system may further comprise at least one sensor unit. In one embodiment, the system may comprise a single sensor unit. Preferably, the at least one sensor unit is a real-time monitoring sensor unit. The system may further comprise a signal processing unit.The signal processing unit may be operatively connected to the at least one control unit andthe at least one sensor unit.The at least one sensor unit may be configured and arranged to measure and / or monitor ablood pressure value of said mammal, in particular a human. Additionally or alternatively, the at least one sensor unit may be configured and arranged tomeasure and / or monitor a spinal cord perfusion pressure value of said mammal, in particulara human.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025The sensor unit may be further configured and arranged to transmit the measured and / ormonitored blood pressure value and / or spinal cord perfusion pressure value to the signalprocessing unit.The signal processing unit may be configured and arranged to determine if the measuredand / or monitored blood pressure value deviates from a predetermined blood pressure targetvalue and / or blood pressure target range. Additionally or alternatively, the signal processing unit may be configured and arranged todetermine if the measured and / or monitored spinal cord perfusion pressure value deviatesfrom a predetermined spinal cord perfusion pressure target value and / or spinal cord perfusionpressure target range.In case it is determined that the measured and / or monitored blood pressure value deviatesfrom the predetermined blood pressure target value and / or blood pressure target range and / or the measured and / or monitored spinal cord perfusion pressure value deviates from the predetermined spinal cord perfusion pressure target value and / or spinal cord perfusion pressure target range, the signal processing unit transmits an output signal to the at least onecontrol unit for stimulation data adjustment.The at least one sensor unit may comprise at least one sensor.In particular, the at least one sensor may comprise at least one biocompatible implantablesensor.Alternatively or additionally, the at least one sensor may comprise at least one non-implantablesensor. The at least one control unit may be configured and arranged to control the at least one stimulation unit to provide electrical stimulation over a predefined period of time.In particular, the predefined period of time may be between 0 and 30 minutes.This allows optimizing efficiency in mitigating autonomic dysreflexia after SCI and / or otherneurological disorders. In particular, the at least one control unit may be configured and arranged to control the at least one stimulation unit to provide electrical stimulation on a daily basis, especially by stimulating the neuroprosthetic baroreflex.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 In particular, it has been observed that daily spinal cord stimulation using neuroprostheticbaroreflex significantly reduces the severity of autonomic dysreflexia episodes.The stimulation data may comprise at least frequency, amplitude and pulse width.Advantageously, the frequency may be between 10 Hz and10k Hz.Advantageously, the amplitude may be between 0 and 1A or 0 and 15 V.Advantageously, the pulse width may be between 1 and 500 ^s.The at least one control unit may comprise an oscillation control module. The oscillation control module may be configured and arranged to provide an input between0.01 Hz and 0.2 Hz low frequency oscillation in the amplitude and / or frequency.Preferably, the oscillation control module may be configured and arranged to provide an inputof 0.1 Hz low frequency oscillation in the amplitude and / or frequency.The at least one stimulation unit may be configured and arranged to provide at least one bursttrain stimulation pulse.In particular, the at least one stimulation unit may be configured and arranged to provide at least one burst of several pulses, preferably of 2 to 5 pulses. The present invention further relates to the use of a neuromodulation / neurostimulation system as described above for the treatment of a mammal, preferably a human, with autonomic dysreflexia after SCI and / or other neurological disorders. The neuromodulation / neurostimulation system of the invention may be used in a method for stimulating sympathetic circuitry responsible for blood pressure control in a mammal with autonomic dysreflexia. The method may include: -positioning the implantable lead on the body of said mammal, preferably a human, toprovide stimulation to the spinal cord of said mammal at spinal cord level T10-T12, and -controlling the at least one stimulation unit to deliver electrical stimulation to the spinalcord of said mammal, preferably a human.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 In particular, the implantable lead may include one or more implantable electrode arrays positioned over the dorsal side of the lower thoracic spinal cord of a subject, preferably ahuman, typically at spinal cord level T10-T12.In particular, the one or more electrode arrays may be connected to at least one IPG by cables that carry electrical current from the at least one IPG toward the implanted electrode arrays that deliver electrical stimulation to the spinal cord. Advantageously, the at least one IPG may be programmed with a recurring EES sequence. The method may operate the system as an open-loop system. Alternatively, the method may operate the system as a closed-loop system. In this case, the method may further include: -measuring and / or monitoring a blood pressure value and / or a spinal cord perfusionpressure value of said mammal, in particular a human, through the at least one sensor unit; -transmitting the measured and / or monitored blood pressure value and / or spinal cordperfusion pressure value to the signal processing unit;- comparing the measured and / or monitored blood pressure value to a predeterminedblood pressure target value and / or blood pressure target range, and / or- comparing the measured and / or monitored spinal cord perfusion pressure value to apredetermined spinal cord perfusion pressure target value and / or spinal cord perfusion pressure target range, and -if the comparison indicates that the measured and / or monitored blood pressure valuedeviates from the predetermined blood pressure target value and / or blood pressure target range and / or the measured and / or monitored spinal cord perfusion pressure value deviates from the predetermined spinal cord perfusion pressure target value and / or spinal cord perfusion pressure target range, providing an output signal to the control unit for stimulation data adjustment. The at least one sensor unit may comprise at least one sensor.The at least one sensor may comprise at least one biocompatible implantable sensor.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Alternatively or additionally, the at least one sensor may comprise at least one non-implantablesensor. The method may include providing electrical stimulation over a predefined period of time. In particular, the predefined period of time may range between 0 and 30 minutes. The method may include providing electrical stimulation on a daily basis, especially by stimulating the neuroprosthetic baroreflex.In particular, it has been observed that daily spinal cord stimulation using neuroprostheticbaroreflex significantly reduces the severity of autonomic dysreflexia episodes.Advantageously, the method may include delivering stimulation with frequency between 10 Hzand 10k Hz.Advantageously, the method may include delivering stimulation with amplitude between 0 and 1A or 0 and 15V. Advantageously, the method may include delivering stimulation with pulse width between 1and 500 ^s.The method may include providing, through an oscillation module, an input between 0.01 Hzand 0.2 Hz low frequency oscillation in the amplitude and / or frequency.In particular, the method may include providing an input of 0.1 Hz low frequency oscillation inthe amplitude and / or frequency.The method may include providing at least one burst train stimulation pulse.In particular, the method may include providing at least one burst of several pulses, preferably of 2 to 5 pulses. Further details and advantages of the present invention shall now be disclosed in connection with the drawings, showing: Fig. 1: Schematic overview a neuromodulation / neurostimulation system according toan embodiment of the present invention.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Fig.2: Detail of an implantable pulse generator (IPG) and stimulation electrodes of thesystem of Fig. 1, implanted over the dorsal side of the lower thoracic spinal cord of asubject, in particular a human.Fig. 3: Laboratory experiment on SCI rodents, in particular rats, by using the systemaccording to the invention over a period of six weeks where stimulation was deliveredto the T12 spinal segment for 30 minutes per day.Fig. 4: Autonomic dysreflexia triggers transcriptional activity in the lumbosacral andlower thoracic spinal cord. In detail: a) Experimental model to quantify the severity of autonomic dysreflexia in mice with complete SCI. b) Changes in blood pressure during an episode of autonomic dysreflexia elicited by a controlled colorectal distension. c) Severity of autonomic dysreflexia measured by the change in systolic blood pressure elicited by a controlled colorectal distension at different timepoints after SCI (5 days post injury-Uninjured [p-value = 0.95], 14 days post injury-Uninjured [p-value= 0.00048], 30 days post injury-Uninjured [p-value = 0.0000001], 45 days post injury- Uninjured [p-value = 0.0000001], 14 days post injury-5 days post injury [p-value = 0.0013], 30 days post injury-5 days post injury [p-value = 0.0000001], 45 days post injury-5 days post injury ([p-value = 0.0000002], 30 days post injury-14 days post injury [p-value = 0.00086], 45 days post injury-14 days post injury [p-value = 0.0024] and 45 days post injury-30 days post injury [p-value = 0.99]). d) Whole spinal cord visualizationof immunohistochemical staining for cFos

[0023] ,

[0024] in a mouse with SCI that wasexposed to repetitive episodes of autonomic dysreflexia. e) Bar plots reporting the mean number of cFos-labelled neurons for each spinal cord segment quantified in mice with SCI that were exposed to repetitive episodes of autonomic dysreflexia (n = 5; mixed effect linear model; p-value < 0.001), demonstrating a clear enrichment in the lumbosacral and lower thoracic spinal.Fig. 5: Autonomic dysreflexia triggers transcriptional activity in the lumbosacral andthoracic spinal cord. In detail: a) Experimental model of autonomic dysreflexia in mice with SCI and timeline of the experiment and final assessments. Blood pressure responses were monitored beat-by-beat using a blood pressure catheter inserted into the carotid artery. Autonomic dysreflexia was elicited using controlled colorectal distension in mice with upper-thoracic SCI. b) Baseline systolic blood pressure measured at different timepoints after SCI. Raw data and statistics provided in Table 1. c) Blood pressure recording autonomic dysreflexia elicited before, during and after a controlled colorectal distension. d) Pressor responses (Left; bold line representsOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 mean trace ± sem for each group and individual line traces are from each animal) and severity of autonomic dysreflexia (Right) measured by the change in systolic blood pressure during colorectal distension at different timepoints after SCI (n = 5 per timepoint). Raw data and statistics provided in Table 1. e) Overview of experimental protocol to identify the regions of the spinal cord activated during autonomic dysreflexia. Thirty days after receiving SCI, mice underwent repetitive episodes of autonomic dysreflexia over 90 minutes, consisting of 30s, and then deflated for 60s. Tissues were collected one hour after the exposure to autonomic dysreflexia, and then processed to visualize cFos immunoreactivity in neurons. Bottom, CLARITY-optimized light sheet microscopy of the cleared spinal cord enabled vizualisation of cFos immunoreactivityover the entire thoracolumbosacral spinal cord

[0023] ,

[0024] in mice with SCI and micewith SCI that underwent repetitive episodes of autonomic dysreflexia. f) Quantification of cFos immunoreactivity in mice with SCI only and mice with SCI that underwent repetitive episodes of autonomic dysreflexia (mixed effect linear model; t = 6.60; p- value = 0.000213). g) Quantifications of cFos expression over the whole spinal cord were confirmed with immunohistochemistry and labelling for cFos on longitudinal sections of spinal cord, as illustrated in the representative photomicrographs of spinal cord sections from mice with SCI and mice with SCI that underwent repetitive episodes of autonomic dysreflexia (mixed effect linear model; t = 6.11; p-value = 0.000287). h) Schematic overview of experiment to assess blood pressure responses to colorectal distension in mice with SCI after bilateral dorsal rhizotomies at T11-T13. i) Severity of autonomic dysreflexia measured by the change in systolic blood pressure during colorectal distension in injured mice with and without bilateral dorsal rhizotomies at T11-T13 (n = 4; independent samples t-test; t = 2.1255 ; p-value = 0.079). j) Quantificationsof cFos expression over the whole spinal cord were confirmed with immunohistochemistry and labelling for cFos on longitudinal sections of spinal cord, as illustrated in the representative photomicrographs of spinal cord sections from mice with SCI and mice with SCI that underwent repetitive episodes of autonomic dysreflexia (mixed effect linear model; t = 14.082; p-value < 0.0001). Fig. 6: The neurons activated by autonomic dysreflexia. In detail: a) Schematic overview of experiments to reveal the phenotype of the neurons that are activated during autonomic dysreflexia. Vglut2Cre::Ai9(RCL-tdT), VgatCre::Ai9(RCL-tdT)andChatCre::Ai9(RCL-tdT) were subjected to repetitive episodes of autonomic dysreflexiaat 30 days post-injury. Longitudinal sections of the spinal cord from T9 to L3 and L4 to S4 were immunohistochemically stained for cFos. Right, Quantification ofOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 colocalisation of cFos-labelled neurons and endogenous fluorescence-tagged neurons (Vglut2ON, VgatONand ChatON) was performed with automated spot detection (Imaris,Bitplane v.9.8.2). b) Next experiments have been performed to determine the role ofthese neuronal subpopulations in triggering autonomic dysreflexia. To manipulate the activity of Vglut2ONand VgatONneurons, AAV5-hSyn-DIO-hm4D(Gi)-mCherry was infused in either the lower thoracic spinal cord (T11-T13) or lumbosacral spinal cord (L5-S1) of either Vglut2Creor VgatCremice prior to performing the SCI to express DREADDs in glutamatergic or gabaergic neurons. Photomicrographs of coronal sections from either the lower thoracic or lumbosacral spinal cord in Vglut2Creand VgatCremice reveal the robust expression of mCherry and thus DREADDs in thetargeted neurons. c) Chemogenetic inactivation restricted to Vglut2ON neurons locatedin the lumbosacral (n = 5; paired samples t-test; t = -10.4; p = 0.00048) or lower thoracic spinal cord (n = 5; paired samples t-test; t = -17.5; p = 0.00001) blunted autonomic dysreflexia. In contrast, chemogenetic silencing of VgatONneurons in the lumbosacral (n = 3; paired samples t-test; t = -1.53; p = 0.267) or lower thoracic spinal cord (n = 4; paired samples t-test; t = 0.269; p = 0.805) failed to modulate the severity of autonomic dysreflexia. Pressor responses (Left; bold line represents mean trace ± sem for each group and individual line traces are from each animal) and severity of autonomic dysreflexia (bar graph, Right) measured by the change in systolic blood pressure duringcolorectal distension. d) Next, the Inventors aimed to expose the projections fromVglut2ONneurons located in the lumbosacral spinal cord to the Vglut2ONneuronslocated in the lower thoracic spinal cord. For this, the Inventors labeled the axons andsynapses of Vglut2ONneurons with infusions of AAV-DJ-hSyn-flex-mGFP-2A- synaptophysinmRuby into the lumbosacral spinal cord of Vglut2Cre. The transfected cell bodies in the lumbosacral spinal cord in both intact and injured mice were quantified (n = 4; independent samples t-test; t = 0.473; p-value = 0.654). Photomicrographs of the lower thoracic spinal cord from representative mice demonstrate increases in the density of axons (Left; n = 5; independent samples t-test; t = 5.92; p-value = 0.0047) and synaptic puncta (Right; n = 5; independent samples t-test; t = 3.47; p-value = 0.027) emanating from Vglut2ONneurons located in the lumbosacral spinal cord after SCI. Fig.7: Comparative single-nucleus RNA sequencing atlas of perturbation-responsive neuronal subpopulations during autonomic dysreflexia. In detail: a) Scheme illustrating the experimental protocol followed by single-nucleus RNA sequencing. Mice receivedupper-thoracic SCI. After 30 days, half of the mice underwent repetitive episodes ofautonomic dysreflexia during 90 minutes. The lumbosacral spinal cord and the lowerOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025thoracic were dissected from the mice according to standard procedures. b) High-quality transcriptomes from 64,739 nuclei were obtained, that were evenly representedacross experimental conditions and spatial locations. c) Number of unique molecularidentifiers (UMIs) per nucleus. Inset text shows the median number of UMIs. d) Numberof genes detected per nucleus. Inset text shows the median number of genes detected. e) Proportion of mitochondrial counts per nucleus. Inset text shows the medianproportion of mitochondrial counts. f) Number of UMIs quantified per nucleus in eachmajor cell type of the mouse spinal cord. g) Number of genes detected per nucleus ineach major cell type of the mouse spinal cord. h) Proportion of mitochondrial countsper nucleus in each major cell type of the mouse spinal cord. i) Uniform manifoldapproximation and projection (UMAP) visualization of 64,739 nuclei colored by majorcell type, segregated by the location of spinal cord tissues (L6, T12) and experimentalcondition (SCI only, exposure to repeated episode of autonomic dysreflexia, AD). jProportions of nuclei from each major cell type depending on the location of spinal cordtissues and experimental conditions. k) UMAP visualization showing expression of keymarker genes for the major cell types of the mouse spinal cord. l) UMAP visualizationof 29,144 neuronal nuclei colored by neuronal subpopulations, split by experimentalcondition. m) UMAP visualization showing expression of key marker genes for themajor neuronal subpopulation classifications of the mouse spinal cord. n) UMAPvisualization and dendrograms showing cell type prioritizations assigned by Augur across the neuronal taxonomy of the lower thoracic (Top) and lumbosacral (Bottom) spinal cord. Raw AUC values and confidence values are provided in Table 2. o) Lollipop plot illustrating the statistical significance of upregulated Gene Ontology (GO) modules associated with circuit reorganization and increased neuronal excitability in Vsx2ONneurons. p) Photomicrographs of the lower thoracic and lumbosacral spinal cord afterrepetitive episodes of autonomic dysreflexia. Vsx2ONneurons were labelled with immunohistochemistry. Long-distance projecting (Zfhx3, lumbosacral spinal cord) andlocally-projecting (Nfib, lower thoracic spinal cord) were additionally colocalized withim- munohistochemistry labelling of cFos.Fig. 8: The neuronal architecture of autonomic dysreflexia. In detail: a) Schematic overview of the single-nucleus sequencing experiment. UMAP visualization of 64,739 neuronal nuclei, colored by neuronal subpopulation identity. Middle, UMAP visualizations of neuronal subpopulations in the lower thoracic (top) and lumbosacral (bottom) spinal cord. Right, Ranking neuronal subpopulations most responsive to autonomic dysreflexia with Augur. b) Schematic overview of the neuronal architectureOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 of autonomic dysreflexia, including the nodes (numbers) that are dissected anatomically and functionally in the subsequent panels. c) Whole spinal cordvisualization of projections from SCLUMBAR::Vsx2 neurons located in the lumbosacralspinal cord that project to SCTHORACIC::Vsx2neurons located in the lower thoracic spinal cord. Insets illustrate the synaptic-like appositions of CalcaONprojections labeled with immunohistochemistry onto SCLUMBAR::Vsx2neurons in the lumbosacral spinal cord. d) CalcaONprojections labeled with immunohistochemistry onto SCLUMBAR::Vsx2neurons in the lumbosacral spinal cord, including insets showing synaptic-like appositions. e) Barplot reporting the severity of autonomic dysreflexia, quantified as the mean change insystolic blood pressure in response to colorectal distension before and after the ablation of CalcaONneurons located in the dorsal root ganglia inCalcaCre::AdvilFlpO::iDTR mice (n = 5; independent samples t-test; t = -6.0; p-value =0.0006). f) Severity of autonomic dysreflexia before and after chemogenetic silencing of Vsx2ONneurons located in the lumbosacral spinal cord in Vsx2Cre(n = 5; paired samples t-test; t = -9.47; p-value = 0.00069). g) Projections from SCLUMBAR::Vsx2in lower thoracic spinal cord colabeled with SCTHORACIC::Vsx2neurons and their local projections as well as immunohistochemical labelling of ChatON. Insets show synaptic- like appositions from SCLUMBAR::Vsx2neurons onto SCTHORACIC::Vsx2, and synaptic-like appositions of projections from SCTHORACIC::Vsx2to ChatONsympathetic preganglionic neurons located in the intermediolateral column. h) Severity of autonomic dysreflexia before and after chemogenetic silencing of Vsx2ONneurons located in the lower thoracic spinal cord in Vsx2Cre(n = 5; paired samples t-test; t = -9.39; p-value = 0.00072). i) Severity of autonomic dysreflexia before and after chemogenetic silencing of ChatONneurons located in the lower thoracic spinal cord in ChatCremice (n = 5; paired samples t-test; t = -8.03; p-value = 0.00048). Fig. 9: The first and second nodes of the neuronal architecture of autonomic dysreflexia. In detail: a) Schematic overview of the neuronal architecture of autonomic dysreflexia. b) Zoom on the first node of the neuronal architecture of autonomic dysreflexia that involves the growth of projections from CalcaONneurons onto Vsx2ONneurons with long-distance projections, named SCLUMBAR::Vsx2neurons. This growth was assessed on tissues collected 30 days after SCI in wild-type mice. c) Photomicrograph taken at L6 spinal segment from a mouse with an intact spinal cord and a mouse with a chronic SCI in which CalcaONaxons were labelled with immunohistochemistry. d) Bar plots reporting the density of CalcaONaxonal projections into the intermediate laminae of the spinal cord in uninjured mice and mice with chronic SCI (n = 4; independentOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 samples t-test; t = 9.38; p-value = 0.000086). e) Overview of the experimental protocol to test the severity of autonomic dysreflexia after the ablation of CalcaONand PVONneurons. To achieve the ablation of these neurons exclusively in the dorsal root ganglia,we used a Cre- and Flp-dependent strategy in CalcaCre::AvilFlpO::iDTR andPVCre::AvilFlpO::iDTR mice that allowed the expression of diphtheria toxin receptors(DTR) in CalcaON and PVON neurons located in the dorsal root ganglia, respectively. f)Pressor responses (Left; bold line represents mean trace ±standard error of mean (sem) for each group and individual line traces are from each mouse) and severity of autonomic dysreflexia (Right) measured by the change in systolic blood pressure during colorectal distension in mice without diphtheria toxin-induced ablation of either CalcaONneurons or PVONneurons, mice with diphtheria toxin-induced ablation of CalcaONneurons and mice with diphtheria toxin-induced ablation of PVONneurons (n =5; independent samples t-test; t= -5.9998; p-value = 0.00064, independent samples t-test; t= -9.3261; p-value = 0.00014). g) Zoom on the second node of the neuronalarchitecture of autonomic dysreflexia that involves SCLUMBAR::Vsx2neurons projecting to the low thoracic spinal cord. An intersectional viral labelling strategy was used to label the axons of SCLUMBAR::Vsx2neurons located in the lumbosacral spinal cord and that establish projections in the lower thoracic spinal cord. Vsx2Cremice received SCI and were injected with Retro-AAV-DIO-FlpO into the lower thoracic spinal cord and AAV8-Con / Fon-EYFP into the lumbosacral spinal cord. h) Photomicrograph of the L6 spinalsegment from a Vsx2Cremouse with an intact spinal cord and a Vsx2Cremouse with a chronic SCI that received intersectional viral tracing to label SCLUMBAR::Vsx2neurons. Axons from CalcaONwere also labelled with immunohistochemistry. Insets showsynaptic-like appositions from CalcaON axons onto SCLUMBAR::Vsx2 neurons. i) Thenecessary role of SCLUMBAR::Vsx2neurons in autonomic dysreflexia was evaluated usingCre-dependent expression of Gi DREADDs in SCLUMBAR::Vsx2 neurons. j)Photomicrograph showing the expression of DREADD (Gi) receptors in SCLUMBAR::Vsx2neurons. k) Left, changes in systolic blood pressure in response to colorectal distension(shared area). Bold line represents mean trace ±sem for each group and individual line traces are from each mouse) and severity of autonomic dysreflexia. Right, Severity of autonomic dysreflexia in Vsx2Cre mice before and after chemogenetic silencing of Vsx2ONneurons located in the lumbosacral spinal cord (n = 5; paired samples t-test; t= -9.47; p-value = 0.00069). l) The sufficient role of SCLUMBAR::Vsx2 neurons in triggeringautonomic dysreflexia was evaluated using optogenetic activation of SCLUMBAR::Vsx2neurons in Vsx2Cremice injected with AAV-Syn-flex-ChrimsonR-tdTomato into at the lumbosacral spinal cord.30 days after SCI, blood pressure responses were monitoredOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 beat-by-beat using a blood pressure catheter inserted into the carotid artery. Red- shifted light was shined over the lumbosacral spinal cord for 60 seconds during eachtrial. m) Photomicrograph showing the expression of ChrimsonR in SCLUMBAR::Vsx2neurons. n) Left, Changes in systolic blood pressure in response to thephotostimulation of SCLUMBAR::Vsx2neurons in mice with intact spinal cord and with chronic SCI. Bold line represents mean trace ±sem for each group and individual linetraces are from each mouse) and blood pressure responses due to optogeneticactivation of SCLUMBAR::Vsx2neurons. Right, Bar plots reporting mean changes in blood pressure in Vsx2Cremice with intact spinal cord and with SCI during optogenetic activation of SCLUMBAR::Vsx2neurons (n = 5; independent samples t-test; t = 5.14; p-value = 0.00496).Fig. 10: The third and fourth nodes of the neuronal architecture of autonomicdysreflexia. In detail: a) Zoom on the third node of the neuronal architecture of autonomic dysreflexia that involves SCTHORACIC::Vsx2neurons located in the lower thoracic spinal cord. b) Overview of intersectional viral tracing strategy to label projections from SCLUMBAR::Vsx2into the lower thoracic spinal cord concomitantly to the labelling of SCLUMBAR::Vsx2 1, AAV5-CAG-flex-tdTomato was infused into the lower thoracic spinal cord of Vsx2-Cre mice to label SCTHORACIC::Vsx2. Step 2, Retro-AAV-DIO- FlpO was infused into the lower thoracic spinal cord and AAV8-Con / Fon-EYP into thelumbosacral spinal cord to label the projections from SCLUMBAR::Vsx2 neurons located inthe lumbosacral spinal cord and that project in the lower thoracic spinal cord. c)Photomicrographs of the lower thoracic spinal cord with intersectional viral tracing labelling projections from SCLUMBAR::Vsx2, SCTHORACIC::Vsx2neurons and their projectionsfrom a representative mouse with an intact spinal cord and mouse with SCI. d) Barplots reporting the mean density of projections from SCLUMBAR::Vsx2neurons in the grey matter of the lower thoracic spinal cord in mice with an intact spinal cord and withchronic SCI (n = 5; independent samples t-test; t = -3.09; p-value = 0.0162). e) Wholespinal cord visualization of projections from SCLUMBAR::Vsx2neurons located in the lumbosacral spinal cord (red) and visualization of SCTHORACIC::Vsx2neurons (blue) locatedin the lower thoracic spinal cord in mice with chronic SCI. f) Schematic overview of theexperimental protocol to monosynaptically label SCLUMBAR::Vsx2after lower thoracic spinal cord infusions targeting the virus to SCTHORACIC::Vsx2neurons. Step 1, AAV8-hSyn- dlox-TVA950-2A-EGFP was infused into the lower thoracic spinal cord of Vsx2Cre mice to express TVA950 (Avian Tumor Virus A Receptor variant), EGFP (enhanced green fluorescent protein), and oGrev (optimized rabies virus glycoprotein) simultaneously.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 Step 2, EnvA-∆G-Rabies-mCherry was infused two weeks after to monosynaptically restrict EnvA pseudotyped and G-protein deleted rabies expressing mCherry and label direct connections from SCLUMBAR::Vsx2neurons onto SCTHORACIC::Vsx2neurons. Representative photomicrographs of two neurons infected with the EnvA-∆G-Rabies-mCherry and positive to Vsx2 immunohistochemistry. g) The necessary role ofSCTHORACIC::Vsx2neurons in autonomic dysreflexia was evaluated using Cre-dependentexpression of Gi DREADDs in SCTHORACIC::Vsx2 neurons. h, Photomicrograph showingthe expression of Gi DREADD receptors in SCTHORACIC::Vsx2 neurons. changes insystolic blood pressure in response to colorectal distension. Bold line represents mean trace ±sem for each group and individual line traces are from each mouse) and severity of autonomic dysreflexia. Right, Severity of autonomic dysreflexia in Vsx2Cre mice before and after chemogenetic silencing of Vsx2ON neurons located in the lowerthoracic spinal cord (n = 5; paired samples t-test; t = -9.39; p-value = 0.00072). j) Thesufficient role of SCTHORACIC::Vsx2neurons in autonomic dysreflexia was evaluated using optogenetic activation of SCTHORACIC::Vsx2neurons in Vsx2Cre mice injected with AAV- Syn-flex-ChrimsonR-tdTomato into the lower thoracic spinal cord.30 days after SCI, blood pressure responses were monitored beat-by-beat using a blood pressure catheter inserted into the carotid artery. Red-shifted light was shine over thelumbosacral spinal cord for 60 seconds during each trial. k) Photomicrograph showingthe expression of ChrimsonR in SCTHORACIC::Vsx2 neurons. l) changes in systolicblood pressure in response to colorectal distension. Bold line represents mean trace ±sem for each group and individual line traces are from each mouse) and blood pressure responses due to optogenetic activation of SCTHORACIC::Vsx2neurons. Right, Blood pressure responses in Vsx2Cre mice with intact spinal cord and with chronic SCI during optogenetic activation of SCTHORACIC::Vsx2neurons (n = 5; independent samples t-test; t = 15.4; p-value = 0.0000148). m) Zoom on the fourth node of the neuronalarchitecture of autonomic dysreflexia that involves ChatONsympathetic preganglionicneurons. n) Overview of experimental protocol to label projections from SCTHORACIC::Vsx2neurons located in the lower thoracic spinal cord in Vsx2Cre mice with SCI. Thirty daysafter SCI and viral tracing, the spinal cord tissues were collected and processed. o)Photomicrograph of the lower thoracic spinal cord from a mouse with an intact spinal cord and a mouse with chronic SCI in which the projections of SCTHORACIC::Vsx2werelabelled concomitantly to the immunohistochemical labelling of ChatON neurons. p) Thenecessary role of ChatONneurons in autonomic dysreflexia was evaluated using Cre-dependent expression of Gi DREADDs in ChatON neurons. q) Photomicrographillustrating the expression of Gi DREADD receptors in SCTHORACIC::Vsx2 neurons. r) As inOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 h), for ChatONneurons located in the lower thoracic spinal cord (n = 5; paired samples t-test; t = -8.03; p-value = 0.00048).Fig.11: Comparative single-nucleus RNA sequencing atlas of perturbation-responsiveneuronal subpopulations during epidural electrical stimulation. In detail: a) Schemeillustrating the experimental protocol followed by single-nucleus RNA sequencing. Daysafter SCI for half of the mice, EES was applied continuously over the lower thoracicspinal cord during 45 minutes. The lower thoracic spinal cord was harvested from themice according to standard procedures. b) High-quality transcriptomes were obtainedfrom 21,098 nuclei that were evenly represented across experimental conditions andspatial locations. c) Number of unique molecular identifiers (UMIs) per nucleus. Insettext shows the median number of UMIs. d) Number of genes detected per nucleus.Inset text shows the median number of genes detected. e) Proportion of mitochondrialcounts per nucleus. Inset text shows the median proportion of mitochondrial counts. f)Number of UMIs quantified per nucleus in each major cell type of the mouse spinalcord. g) Number of genes detected per nucleus in each major cell type of the mousespinal cord. h) Proportion of mitochondrial counts per nucleus in each major cell typeof the mouse spinal cord. i) UMAP visualization of 21,098 nuclei colored by major celltype, split by experimental condition. j) Proportions of nuclei from each major cell typeacross all experimental conditions. k) UMAP visualization showing expression of keymarker genes for the major cell types of the mouse spinal cord. l) UMAP visualizationof 8,471 neuronal nuclei colored by neuronal subpopulations, split by experimentalcondition. m) UMAP visualization showing expression of key marker genes for themajor neuronal subpopulation classifications of the mouse spinal cord. n) UMAPvisualization and dendrograms showing cell type prioritizations assigned by Augur across the neuronal taxonomy of the lower thoracic spinal cord. Raw AUC values and confidence values are provided in Table 2.Fig. 12: The neuronal architecture of epidural electrical stimulation (EES)-inducedpressor responses. In detail: a) Schematic overview of experiments to trigger pressorresponses with EES in mice with SCI. b) Pressor response induced by continuous (40 Hz) EES in a mouse with SCI. c) Uniform manifold approximation and projection (UMAP) visualization of 21,098 neuronal nuclei, colored by neuronal subpopulation identity. Right, Identification of perturbation-responsive neuronal subpopulations with Augur. d-f) Schematic overview of the successive nodes constituting the neuronal architecture though which EES applied over the low thoracic spinal cord induces pressor responses. d) EES-induced pressor responses before and after the ablation ofOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025PVON neurons located in the dorsal root ganglia in PVCre::AdvilFlpO::iDTR mice (n = 5;independent samples t-test; t = -5.41; p-value = 0.0043). e) EES-induced pressor responses before and after chemogenetic silencing of Vsx2ONlocated in the lower thoracic spinal cord in Vsx2Cremice (n = 5; paired samples t-test; t = -4.21; p-value = 0.014). f) EES-induced pressor responses before and after chemogenetic silencing of ChatONneurons located in the lower thoracic spinal cord in ChatCremice (n = 5; paired samples t-test; t = -7.07; p-value = 0.0021). g) Photomicrograph of the lower thoracic spinal cord demonstrating vGlut1ONsynaptic puncta and synaptic-like appositions from large-diameter afferent neurons onto SCTHORACIC::Vsx2neurons labelled with in situhybridization (Left) or viral tract tracing (Right) in the lower thoracic spinal cord ofPVCre::AdvilFlpO::tdTomato mice.Fig. 13: The neuronal architecture activated by EES to induce pressor response. Indetail: a) Schematic overview of the neuronal architecture through which EES inducespressor responses. b) Zoom on the first node of the neuronal architecture of EES-induced pressor responses that involves PVON. c) Overview of the experimentalprotocol to test the involvement of afferent fibers from PVONand CalcaONneurons in EES-induced pressor responses. To achieve the ablation of these neurons exclusivelyin the dorsal root ganglia, we used a Cre- and Flp-dependent strategy inCalcaCre::AvilFlpO::iDTR and PVCre::AvilFlpO::iDTR mice that allowed the expression ofdiphtheria toxin receptors (DTR) in these specific neurons. d) EES-induced pressorresponses (Left; bold line represents mean trace ±sem for each group and individual line traces are from each mouse) (Right) measured by the change in systolic bloodpressure during EES in mice without any ablation, mice with diphtheria toxin-inducedablation of PVON neurons and mice with diphtheria toxin-induced ablation of CalcaONneurons (n = 5; independent samples t-test; t= -5.4141; p-value = 0.0043, independentsamples t-test; t= 6.3166; p-value = 0.0020). e) Overview of the experiment strategy tovisualize large-diameter PVONfibers in PVCre::AvilFlpO::Ai9(RCL-tdT)mice and confirmed that they established vGlut1ONsynaptic-appositions onto SCTHORACIC::Vsx2neurons. Thirtydays after SCI, spinal cord tissues were collected and processed. f) Photomicrographof the lower thoracic spinal cord showing vGlut1 synaptic puncta and synaptic-like appositions from large-diameter afferent neurons (PVCre::AdvilFlpO::Ai9(RCL-tdT)mice) ontoSCTHORACIC::Vsx2 neurons labelled with in situ hybridization (Left) or viral tract tracing(Right). g) Photomicrograph of the SCTHORACIC::Vsx2 neurons labelled with viral tracttracing and vGlut1ONsynapses labelled with immunohistochemistry. Quantification of vGlut1ONsynaptic-appositions onto SCTHORACIC::Vsx2neurons and ChatONneurons inOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025(PVCre::AdvilFlpO::Ai9(RCL-tdT) mice with an intact spinal cord and with a chronic SCI. h)Schematic overview of the experimental protocol to monosynaptically label PVONneurons in the dorsal root ganglia after lower thoracic spinal cord infusions targeting the virus to SCTHORACIC::Vsx2neurons. Step 1, AAV8-hSyn-dlox-TVA950-2A-EGFP was infused into the lower thoracic spinal cord of Vsx2Cremice to express TVA950 (Avian Tumor Virus A Receptor variant), EGFP (enhanced green fluorescent protein), and oGrev (optimized rabies virus glycoprotein) simultaneously. Step 2, EnvA-∆G-Rabies- mCherry was infused two weeks after to monosynaptically restrict EnvA pseudotyped and G-protein deleted rabies expressing mCherry and label direct connections from PVONneurons onto SCTHORACIC::Vsx2neurons. Representative photomicrograph showing neurons in the lower thoracic dorsal root ganglion infected with the EnvA-∆G-Rabies-mCherry and labelled with fluorescence in situ hybridization. i) Zoom on the secondnode of the neuronal architecture of EES-induced pressor responses that involves SCTHORACIC::Vsx2. The necessary role of SCTHORACIC::Vsx2neurons in EES-induced pressor response was evaluated using Cre-dependent expression of Gi DREADDs inSCTHORACIC::Vsx2 neurons. j) EES-induced pressor responses (Left; bold line representsmean trace ±sem for each group and individual line traces are from each mouse) (Right) measured by the change in systolic blood pressure during EES in the same mice before and after chemogenetic silencing of Vsx2ONneurons located in the lowerthoracic spinal cord (n = 5; paired samples t-test; t = -4.21; p-value = 0.014). k) Zoomon the third node of the neuronal architecture of EES-induced pressor responses that involves ChatONsympathetic preganglionic neurons. As in h, for ChatONneuronslocated in the lower thoracic spinal cord. l), As in j), for ChatON neurons in the lowerthoracic spinal cord (n = 5; paired samples t-test; t = -7.07; p-value = 0.0021). m)Photomicrograph showing the expression of ChrimsonR in Vsx2ONneurons and thetract resulting from the insertion of one electrode shank. n, Schematic overview ofexperiments to record the activity of SCTHORACIC::Vsx2during the application of EES andduring episodes of autonomic dysreflexia. o) Top, the waveforms display spikes andfiring rate evoked by optogenetic stimulation of Vsx2ONneurons by the application of continuous EES over the lower thoracic spinal cord, and by colorectal distention. Heatmap of neuronal clusters activated by EES, activated by EES and colorectal distension, activated by EES and tagged as Vsx2ONneurons by optogenetic stimulationand EES, and activated by EES and colorectal distension and tagged as Vsx2ONneurons activated by optogenetic stimulation.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Fig. 14: Competitive neuronal architectures converge on SCTHORACIC::Vsx2 neurons. Indetail: a) Schematic overview of autonomic neurorehabilitation and paradigm toquantify the severity of autonomic dysreflexia. b, Pressor responses (Left; individualmice and mean trace) and severity of autonomic dysreflexia (Right) in 5 with chronic SCI and 5 mice that underwent autonomic neurorehabilitation for 4 weeks, starting 1 week after SCI (independent samples t-test; t = -7.45; p-value = 0.00056). c) Schematic overview illustrating the competitive (overlapping) neuronal architectures of autonomic dysreflexia and EES-induced pressor responses, and their rearrangement after autonomic neurorehabilitation. d, vGlut1ONsynaptic puncta and synaptic-like appositions from SCLUMBAR::Vsx2neurons onto SCTHORACIC::Vsx2neurons in in mice with SCI and mice with SCI that underwent autonomic neurorehabilitation. (top) Bar plots reporting the mean density of axonal projections from SCLUMBAR::Vsx2neurons in the thoracic spinal cord in mice with SCI and mice with SCI that underwent autonomic neurorehabilitation (n = 5; independent samples t-test; t = 2.51; p-value = 0.0369).(bottom) (bottom) Bar plots reporting the mean number of vGlut1ON synaptic punctaapposing SCTHORACIC::Vsx2neurons (n = 5; independent samples t-test; t = 4.44; p-value = 0.0055). e) Schematic overview of experiments in which EES was applied daily over the lumbosacral spinal cord of mice with SCI, and paradigm to quantify the severity ofautonomic dysreflexia. f) As in b) for mice with SCi that were subjected to the dailyapplication of EES over the lumbosacral spinal cord (n = 5; independent samples t-test; t = 5.82; p-value = 0.00070).Fig. 15: Autonomic neurorehabilitation reversed autonomic dysreflexia in mice withSCI. In detail: a) Overview of the experimental protocol to deliver autonomicneurorehabilitation in mice with SCI. Step 1. Mice received a complete transection ofthe spinal cord at the level of the T4 segment. Step 2. Intersectional viral tracing byinfusing Retro-AAV-DIO-FlpO into the lower thoracic spinal cord and AAV8-Con / Fon- EYP into the lumbosacral spinal cord to label SCLUMBAR::Vsx2neurons located in the lumbosacral spinal cord that project onto SCTHORACIC::Vsx2neurons located in the lower thoracic spinal cord. Step 3. One week after SCI, electrodes were implanted over theT12 spinal segment to deliver EES. Step 4. EES was applied for 30 minutes every dayfor 4 weeks. Step 5. Severity of autonomic dysreflexia was assessed during terminal experiments conducted in mice with chronic SCI and mice with chronic SCI that underwent autonomic neurorehabilitation. Step 6. Spinal cord tissues were collectedand processed. b) Changes in systolic blood pressure (Left; bold line represents meantrace ± sem for each group and individual line traces are from each animal) and severityOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 of autonomic dysreflexia (Right) measured by the change in systolic blood pressure during colorectal distension in mice with and without autonomic neurorehabilitation (n= 5; independent samples t-test; t = -7.45; p-value = 0.00056). c) Left,Photomicrographs of the lower thoracic spinal cord in mice with chronic SCI and mice with chronic that underwent autonomic neurorehabilitation in which SCLUMBAR::Vsx2neurons located in the lumbosacral spinal cord were labelled with an intersection virus strategy concomitantly to the labelling of SCTHORACIC::Vsx2neurons. (Right) Photomicrographs of the lower thoracic spinal cord with intersectional viral labelling combined with immunohistochemical labelling of vGlut1ONsynapses in mice with chronic SCI and mice with chronic SCI that underwent autonomic neurorehabilitation. vGlut1ONsynaptic puncta and synaptic-like appositions from SCLUMBAR::Vsx2neurons onto SCTHORACIC::Vsx2neurons in mice with chronic SCI and mice with chronic SCI thatunderwent autonomic neurorehabilitation. d) Left, Bar plots reporting the mean numberof vGlut1ON synaptic puncta apposing SCTHORACIC::Vsx2 neurons (n = 5; independent samples t-test; t = 4.44; p-value = 0.0055), right, and the mean density of axonal projections from SCLUMBAR::Vsx2 neurons in the grey matter of the lower thoracic spinal cord in mice with chronic SCI and mice with chronic SCI that underwent autonomic neurorehabilitation (n = 5; independent samples t-test; t = 2.51; p-value =0.0369). e) As in a), for mice subjected to daily application of EES over the lumbosacralspinal cord. f) As in b), for mice subjected to daily application of EES over thelumbosacral spinal cord (n = 5; independent samples t-test; t = 5.82; p-value = 0.00070). g, As in c, for mice subjected to daily application of EES over the lumbosacralspinal cord. h) As in d), for mice subjected to daily application of EES over thelumbosacral spinal cord.Fig. 16: Longitudinal monitoring of autonomic neurorehabilitation in rats. In detail: a)Schematic overview of autonomic neurorehabilitation in rats. A wireless telemetry system was implanted chronically to acquire longitudinal recordings of hemodynamic parameters. An electronic dura mater (e-dura) designed to target the dorsal roots projecting to the T11, T12, and T13 spinal segments was then implanted over the hemodynamic hotspot to regulate blood pressure. b) Augmentation of systolic blood pressure within a target range using a proportional integral (PI) controller that adjusts the amplitude of EES in closed loop. c) Line graph reporting the severity of autonomic dysreflexia that was assessed weekly using colorectal distension in rats with SCI and rats with SCI that were undergoing autonomic neurorehabilitation. Raw data and statistics provided in Table 1. d) Whole spinal cord visualization of projections fromOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 neurons located in the lumbosacral spinal cord that establish neurons into the lower thoracic spinal cord. e) Axonal projections and synaptic puncta from neurons located in the lumbosacral spinal cord and projecting to the lower thoracic spinal cord, shown in rats with SCI and rats with SCI that underwent autonomic neurorehabilitation. f) Density of axonal projections from lumbosacral neurons in the thoracic spinal cord before and after autonomic neurorehabilitation (n = 5; independent samples t-test; t = -4.03; p-value = 0.0081). g) Synaptic-like appositions from neurons located in thelumbosacral spinal cord onto SCTHORACIC::Vsx2neurons combined with the labelling of vGlut1ONsynaptic puncta form large-diameter afferent fibers in a rat with chronic SCIand a rat with SCI that underwent autonomic neurorehabilitation. h) Bar plots reportingthe mean density of vGlut1ONsynaptic puncta onto SCTHORACIC::Vsx2neurons in rats with SCI and rats with SCI that underwent autonomic neurorehabilitation (n = 5; independent samples t-test; t = 12.71; p-value = 2.78e-06).Fig. 17: Autonomic neurorehabilitation reversed autonomic dyresflexia in rats withcontusion SCI. In detail: a) Overview of the experimental protocol to deliver autonomic neurorehabilitation in rats with SCI. Step 1. Rats received a severe contusion (380 Kdyn) of the spinal cord at the level of T3 segment. Step 2. AAV-DJ-hSyn-flex-mGFP- 2A-Synaptophysin-mRuby and an AAV-Cre were co-infused into the L6 segment of the spinal cord to label the projections from neurons located in the lumbosacral spinal cord. Step 3. A wireless telemeter recording system, including a blood pressure cannula inserted into the abdominal aorta and microelectrodes sutured over the sympathetic renal nerve, was implanted chronically to monitor hemodynamics and sympathetic nerve activity, respectively. Step 4. Seven days after SCI, an electronic dura mater (e- dura) designed to target the dorsal roots projecting to the T11, T12, and T13 spinal segments was implanted over the hemodynamic hotspot to regulate blood pressure. Step 5. EES was applied for 30 minutes every day during 6 weeks using a proportional- integral (PI) controller that adjusted the amplitude of EES in closed-loop to augment the systolic blood pressure to a target range. Step 6. The severity of autonomic dysreflexia, induced by colorectal distension, was assessed every week for 6 weeks. Step 7. After 6 weeks of autonomic neurorehabilitation, a final assessment was performed to test the severity of autonomic dysreflexia in all groups, which included rats with intact spinal cord, rats with chronic SCI and rats with chronic SCI that underwent autonomic neurorehabilitation. Step 8. Spinal cords were collected and processed. b) Changes in systolic blood pressure in response to colorectal distension(Left; bold line represents mean trace ± sem for each group and individual line tracesOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 are from each rat) and bar plots reporting the severity of autonomic dysreflexia (Right) measured by the change in systolic blood pressure during colorectal distension overthe course of 6 weeks in rats with intact spinal cord, rats with chronic SCI and rats withchronic SCI that underwent autonomic neurorehabilitation. Raw data and statistics provided in Table 1. c) Whole spinal cord visualization of projections from neuronslocated in the lumbosacral spinal cord. d) Plots reporting density of axonal projections(top) and synaptic punta (bottom) from neurons located in the lumbosacral spinal cord into the grey matter of the lower thoracic spinal cord in rats with intact spinal cord, rats with chronic SCI and rats with chronic SCI that underwent autonomic neurorehabilitation. e) Micrographs of the lower thoracic spinal cord in which the axonal projections and synaptic puncta from neurons located in the lumbosacral spinal cord are labelled for the three groups of rats. f) Bar plots reporting the mean density of axonal projections and synaptic puncta from neurons located in lumbosacral spinal cord into the grey matter of the lower thoracic spinal cord for the three groups of rats. Raw data and statistics are provided in Table 1. g) Micrographs of the lower thoracic spinal cord in which axonal projections and synaptic puncta from neurons located in the lumbosacral spinal cord are labelled concomitantly to vGlut1ONsynapses from large- diameter afferents and Vsx2ONneurons. The density of vGlut1ONsynapses onto Vsx2ONneurons is reconstructed for a rat with chronic SCI and a rat with chronic SCI that underwent autonomic neurorehabilitation. h) Bar plots reporting the density of synaptic- like appositions from neurons located in the lumbosacral spinal cord onto Vsx2ONneurons in rats with intact spinal cord, rats with chronic SCI, and rats with chronic SCI that underwent autonomic neurorehabilitation. Raw data and statistics are provided in Table 1. i), As in i), for vGlut2ONsynaptic puncta onto SCHoxa7::Nfib::Vsx2neurons. Rawdata and statistic provided are in Table 1. j) Quantification of vGlut1ON synaptic punctafrom large-diameter afferents Vsx2ONin rats with chronic SCI and rats with chronic SCI that underwent autonomic neurorehabilitation (n = 5; independent samples t-test; t = 12.71; p-value = 2.78e-06).Fig.18: Reduced severity of autonomic dysreflexia in people with chronic SCI followingautonomic neurorehabilitation. In detail: a) Prevalence of autonomic dysreflexia and management efficacy quantified in 1,479 individuals with SCI from the Rick Hansen Spinal Cord Injury Registry

[0084] ,

[0085] . b) Percentage of individuals with tetraplegia experiencing each symptom of autonomic dysreflexia scored in the Autonomic Dysfunction following SCI (ADFSCI) across various daily activities (n = 107). c) Implantable system to regulate blood pressure with EES, including a paddle lead withOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 optimized electrode configurations to target the dorsal roots projecting to the hemodynamic hotspot, an implantable pulse generator, communication hub, and external smartwatch to operate the various programs of the therapy. d) Post-operative reconstruction of the final position of the electrodes following the implantation of the paddle lead. e) Changes in blood pressure from a representative participant during an orthostatic challenge without EES and with continuous EES applied over the hemodynamic hotspot. The bar plots report the average drop in systolic blood pressure during orthostatic challenge (n = 10, paired samples two-tailed t-test; t = 4.7774 ; p = 0.00101) and average tilt duration without EES and with EES applied over the hemodynamic hotspot (n = 10, paired samples two-tailed t-test; t = 14.33100 ; p < 0.001). Kaplan-Meier plot of exposure status to time, segregated by the presence or absence of EES. f) ADFSCI autonomic dysreflexia score before implantation and after at least 6 months but up to 2 years after implantation of the system and daily use to regulate blood pressure (n = 11, paired samples one-tailed t-test; t = 2.33 ; p =0.021). g) Percentage of individuals (n = 11) experiencing each symptom described in the ADFSCI autonomic dysreflexia section before implantation (before) and at the latesttimepoint of ARCIM Therapy (after).Fig. 19: Population-level data of self-reported experiences of autonomic dysreflexiasymptoms and clinical validation of autonomic neurorehabilitation. In detail: a) The prevalence of autonomic dysreflexia and management efficacy in people with SCI (n= 1479) acquired with the Spinal Cord Injury Community Survey (SCICS). b) Percentage of individuals with SCI experiencing autonomic dysreflexia scored in the ADFSCI (n=107). c) Percentage of autonomic dysreflexia in individuals with spinal cord injuries split between tetraplegia (top) (n = 52) and paraplegia (bottom) (n = 34) acquired with the SCICS. d) Percentage of individuals experiencing each symptom described in the ADFSCI autonomic dysreflexia section split between all participants (top) and tetraplegic individuals with complete SCI (bottom). e) Timeline of a clinical trial conducted in Lausanne, Switzerland [NCT05111093, CHUV, Lausanne, Switzerland],denoted with the name HemON. f) Bar plots reporting the average daily usage of thesystem per participant (left), and the usage of the system throughout the hours of theday (right) for the 7 participants. g) Bar plots reporting the ADFSCI autonomicdysreflexia score for each symptom before implantation and at the latest timepoint ofhome use of the system to regulate blood pressure (n = 11, paired samples one tailedt-test; p-value = 0.0885, p-value = 0.01833, p-value = 0.08896, p-value = 0.05529, p-value = 0.08963).Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 Fig. 20: Overview of the experimental setup used for the HemON clinical trial,schematically illustrated in e) of Fig.19.Fig. 1 shows a schematic overview of a neuromodulation / neurostimulation system 10according to an embodiment of the present invention.In particular, the present invention relates to a neuromodulation / neurostimulation system 10for stimulating sympathetic circuitry responsible for blood pressure control in a mammal.In particular, said mammal may be a human affected by SCI and / or other neurologicaldisorders such as a stroke, multiple sclerosis, autonomic failure, autonomic neuropathy or cancer of the neurological tissue which impair operation of descending sympathetic pathways that normally facilitate control of autonomic functions. The system 10 includes at least one control unit 12. In the shown embodiment, the system 10 includes a single control unit 12. The control unit 12 is configured and arranged to provide stimulation data. The system 10 further includes at least one stimulation unit 14. In the shown embodiment, the system 10 includes a single stimulation unit 14. The stimulation unit 14 is operatively connected to the control unit 12.Connection between the stimulation unit 14 and the control unit 12 may be established by awireless link WL. Alternatively, also a cable bound and / or unidirectional and / or indirect connection between thestimulation unit 14 and the control unit 12 could be generally possible.The stimulation unit 14 is configured and arranged to provide electrical stimulation to the spinalcord of said mammal, in particular a human. To this end, the stimulation unit 14 includes an implantable lead 18. In particular, the implantable lead 18 is configured an arranged to provide stimulation to the spinal cord of said mammal at spinal cord level T10-T12.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 The system 10 is specifically designed based on anatomical and physiological features to allow activating sympathetic circuitry that is responsible for blood pressure control that mitigatesautonomic dysreflexia after SCI and / or other neurological disorders.Accordingly, severity and frequency of autonomic dysreflexia episodes after SCI and / or other neurological disorders can be significantly reduced. The system 10 may include at least one implantable pulse generator (IPG) 15. In the shown embodiment, the system 10 includes one IPG 15 (Fig.2).Advantageously, the IPG 15 may be programmed with a recurring EES sequence.In the present embodiment, the implantable lead 18 includes one or more implantableelectrode arrays 18.1 (Fig.2).As shown in Fig.2, the one or more electrode arrays 18.1 are positioned over the dorsal sideof the lower thoracic spinal cord of a subject, preferably a human, typically at spinal cord levelT10-T12.In the present embodiment, the one or more electrode arrays 18.1 are connected to the IPG15 by cables that are configured and arranged to carry electrical current from the IPG 15 towardthe implanted electrode arrays 18.1 that deliver electrical stimulation to the spinal cord. Notshown is that the system 10 may operate as an open-loop system. In the shown embodiment, the system 10 operates as a closed-loop system.The system 10 comprises at least one sensor unit 16.In the shown embodiment, the system 10 further comprises a single sensor unit 16.Preferably, the sensor unit 16 is a real-time monitoring sensor unit 16. In the shown embodiment, the system 10 further comprises a signal processing unit 20. The signal processing unit 20 is operatively connected to the control unit 12 and the sensor unit 16. Connection between the signal processing unit 20 and the sensor unit 16 can be a direct and bidirectional connection.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 In particular, the connection between the signal processing unit 20 and the sensor unit 16 canbe established by a wireless link WL.Alternatively, a cable bound and / or unidirectional and / or indirect connection between the signalprocessing unit 20 and the sensor unit 16 unit could be generally possible.In the present embodiment, the sensor unit 16 is configured and arranged to: -measure and / or monitor a blood pressure value of said mammal, and- transmit the measured and / or monitored blood pressure value to the signal processingunit 20. The sensor unit 16 may transmit the measured and / or monitored blood pressure value to the signal processing unit 20 in real time. Alternatively, the sensor unit 16 may transmit the measured and / or monitored blood pressurevalue to the signal processing unit 20 close to real-time or with time delay.The signal processing unit 20 is configured and arranged to determine if the measured and / or monitored blood pressure value deviates from a predetermined blood pressure target value and / or blood pressure target range.In case it is determined that the measured and / or monitored blood pressure value deviatesfrom the predetermined blood pressure target value and / or blood pressure target range, thesignal processing unit 20 transmits an output signal to the control unit 12 for stimulation dataadjustment. In principle, the blood pressure target value and / or blood pressure target range may be predetermined by a patient and / or a medical professional (e.g. a therapist, a nurse, a physiotherapist, a physician, a pharmacist, a physician-aid or any other trained operator). Further, the blood pressure target value and / or blood pressure target range may be changed and / or reset at any time point, depending on a current condition of the subject. Not shown is that the sensor unit 16 comprises at least one sensor.Not shown is that the at least one sensor may comprise at least one biocompatible implantablesensor.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Not shown is that, additionally or alternatively, the at least one sensor may comprise at leastone non-implantable sensor.As an example, the sensor unit 16 may include an upper arm blood pressure monitor system or a wrist blood pressure monitor system or a finger blood pressure monitor system.The one or more sensors may be configured and arranged to measure and / or monitor a bloodpressure signal indicative for a blood pressure measurement.In general, the at least one sensor may provide continuous monitoring of blood pressure and / orsporadic monitoring of blood pressure and / or measuring or monitoring blood pressure at presettime intervals.Not shown is that the sensor unit 16 may include an invasive arterial line. In particular, theinvasive arterial line may monitor blood pressure directly and in real-time. The sensor unit 16 may be configured and arranged to measure and / or monitor systolic and / or diastolic and / or mean arterial pressure.The sensor unit 16 may also be configured and arranged to report pulse rate.Additionally or alternatively, the sensor unit 16 may be configured and arranged to measure and / or monitor perfusion pressure, in particular spinal cord perfusion pressure.The sensor unit 16 may be configured and arranged to transmit a measured and / or monitoredspinal cord perfusion pressure value to the signal processing unit 20. The sensor unit 16 may transmit the measured and / or monitored spinal cord perfusionpressure value to the signal processing unit 20 in real time.Alternatively, the sensor unit 16 may transmit the measured and / or measured and / or monitoredspinal cord perfusion pressure value to the signal processing unit 20 close to real-time or withtime delay.The signal processing unit 20 may be configured and arranged to determine if the measuredspinal cord perfusion pressure value deviates from a predetermined spinal cord perfusionpressure target value and / or spinal cord perfusion pressure target range.In case it is determined that the measured and / or monitored spinal cord perfusion pressurevalue deviates from the predetermined spinal cord perfusion pressure target value and / orOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 spinal cord perfusion pressure target range, the signal processing unit 20 transmits an output signal to the control unit 12 for stimulation data adjustment. Advantageously, the control unit 12 may be configured and arranged to control the stimulation unit 14 to provide electrical stimulation over a predefined period of time. Preferably, the predefined period of time ranges between 0 and 30 minutes. The control unit 12 may be configured and arranged to control the stimulation unit to provide stimulation on a daily basis, especially by stimulating the neuroprosthetic baroreflex. In particular, it has been observed that daily spinal cord stimulation using neuroprostheticbaroreflex significantly reduces the severity of autonomic dysreflexia episodes.The stimulation data may comprise at least frequency, amplitude and pulse width.Advantageously, stimulation may be delivered with a frequency between 10 Hz and10k Hz.Advantageously, stimulation may be delivered with amplitude between 0 and 1 A or 0 and 15V.Advantageously, stimulation may be delivered with the pulse width between 1 and 500 ^s.Not shown is that the control unit 12 may comprise an oscillation control module. In particular, the oscillation control module may be configured and arranged to provide an inputbetween 0.01 Hz and 0.2 Hz low frequency oscillation in the amplitude and / or frequency.Preferably the oscillation control module may be configured and arranged to provide an inputof 0.1 Hz low frequency oscillation in the amplitude and / or frequency.Not shown is that the stimulation unit 14 may be configured and arranged to provide at least one burst train stimulation pulse.In particular, the stimulation unit 14 may be configured and arranged to provide at least oneburst of several pulses, preferably of 2 to 5 pulses.More in particular, the burst train stimulation may comprise a series of 3 to 5 (or even more)pulses delivered at, e.g., 200 Hz to 700 Hz, repeated at a frequency of e.g. 10-120 Hz.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 A method for stimulating neuronal circuitry responsible for blood pressure control in a mammalwith autonomic dysreflexia by using the neuromodulation / neurostimulation system 10 of theinvention will be described in the following.In particular, said mammal may be a human affected by SCI and / or other neurologicaldisorders such as a stroke, multiple sclerosis, autonomic failure, autonomic neuropathy or cancer of neurological tissue which impair operation of descending sympathetic pathways that normally facilitate control of autonomic functions. The method includes: -positioning the implantable lead 20 on the body of said mammal, preferably a human,to provide stimulation to the spinal cord of said mammal at spinal cord level T10-T12, and -controlling the at least one stimulation unit 14 to deliver electrical stimulation to thespinal cord of said mammal, preferably a human.In particular, the implantable lead 18 may include one or more implantable electrode arrays18.1, positioned over the dorsal side of the lower thoracic spinal cord of a subject, preferably a human, typically at spinal cord level T10-T12.In particular, the one or more electrode arrays 18.1 may be connected to at least one IPG 15by cables that carry electrical current from the at least one IPG 15 toward the implantedelectrode arrays 18.1 that deliver electrical stimulation to the spinal cord. Advantageously, the at least one IPG 15 may be programmed with a recurring EES sequence. The method may operate the system 10 as an open-loop system. Alternatively, the method may operate the system 10 as a closed-loop system. In this case, the method further includes: -measuring and / or monitoring a blood pressure value and / or a spinal cord perfusionpressure value of said mammal, in particular a human, through the at least one sensor unit; -transmitting the measured and / or monitored blood pressure value and / or spinal cordperfusion pressure value to the signal processing unit;Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 -comparing the measured and / or monitored blood pressure value to a predeterminedblood pressure target value and / or blood pressure target range, and / or -comparing the measured and / or monitored spinal cord perfusion pressure value to apredetermined spinal cord perfusion pressure target value and / or spinal cord perfusion pressure target range, and -if the comparison indicates that the measured and / or monitored blood pressure valuedeviates from the predetermined blood pressure target value and / or blood pressure target range and / or the measured and / or monitored spinal cord perfusion pressure value deviates from the predetermined spinal cord perfusion pressure target value and / or spinal cord perfusion pressure target range, providing an output signal to the control unit for stimulation data adjustment. The at least one sensor unit may comprise at least one sensor. The at least one sensor may comprise at least one biocompatible implantable sensor. Alternatively or additionally, the at least one sensor may comprise at least one non-implantable sensor. The method may include providing electrical stimulation over a predefined period of time. In particular, the predefined period of time may range between 0 and 30 minutes. The method may include providing electrical stimulation on a daily basis, especially by stimulating the neuroprosthetic baroreflex. In particular, it has been observed that daily spinal cord stimulation using neuroprostheticbaroreflex significantly reduces the severity of autonomic dysreflexia episodes.Advantageously, the method may include delivering stimulation with frequency between 10 Hzand 10k Hz.Advantageously, the method may include delivering stimulation with amplitude between 0 and1A or 0 and 15 V.Advantageously, the method may include delivering stimulation with pulse width between 1and 500 ^s.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Advantageously, the method may include providing, through an oscillation module, an inputbetween 0.01 Hz and 0.2 Hz low frequency oscillation in the amplitude and / or frequency.In particular, the method may include providing an input of 0.1 Hz low frequency oscillation inthe amplitude and / or frequency.The method may include providing at least one burst train stimulation pulse.In particular, the method may include providing at least one burst of several pulses, preferablyof 2 to 5 pulses. Experimental model in rats Experiments on rodents, specifically rats, have been carried out by the inventors to define an optimal stimulation site on the spinal cord for activating sympathetic neuronal circuitry responsible for blood pressure control that mitigates autonomic dysreflexia.Fig.3 is a diagram showing a laboratory experiment on SCI rodents, in particular rats, by usingthe system according to the invention over a period of six weeks. Rats received a robotically-controlled, severe clinically-relevant contusion onto upper thoracic (T3) segments.Within two weeks after the induced injury (prior to onset of treatment), rats developed theclassic phenotype of autonomic dysreflexia. To characterize the natural history of autonomic dysreflexia after SCI, the inventors implanted rats with a wireless system that enabled 24 / 7 monitoring of arterial blood pressure using tireless telemetry (Fig.3).As shown in Fig.3 rats were outfitted with spinal epidural electrical spinal stimulation (ESS) atT12. Following SCI, autonomic dysreflexia has been weekly induced and monitored by using colorectal distension.From two weeks post-SCI, autonomic neurorehabilitation EES has been carried out on a dailybasis for 30 minutes per day. The SCI rats have been monitored over a time period of 6 weeks (Fig.3).Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Based on experimental evidence, the inventors have found that, by delivering stimulation atspinal cord level T12, the severity of autonomic dysreflexia episodes at the end of theexperiment (week 6) was significantly mitigated (Fig.2).EXPERIMENTAL EVIDENCE Introduction Autonomic dysreflexia is a life-threatening medical condition characterized by episodes of uncontrolled hypertension that occur in response to sensory stimuli after spinal cord injury(SCI) [1]-[7].The fragmented understanding of the mechanisms underlying autonomic dysreflexia hampers the development of therapeutic strategies to manage this condition, leaving people with SCI atdaily risk of heart attack and stroke [8]-

[0018] .In the following, the complete de novo neuronal architecture

[0019] that develops after SCI andcauses autonomic dysreflexia will be exposed.In parallel, a competing, yet overlapping neuronal architecture activated by epidural electrical stimulation of the spinal cord will be uncovered, that safely regulates blood pressure after SCI. The discovery that these adversarial neuronal architectures converge onto a single neuronal subpopulation provided a blueprint for the design of a mechanism-based intervention that reversed autonomic dysreflexia in mice, rats, and humans with SCI. These results establish a path for the effective treatment of autonomic dysreflexia in people with SCI. Spinal cord injury (SCI) disrupts the communication between the brainstem vasomotor centers and the regions of the spinal cord that regulate hemodynamics

[0020] . The resulting isolation of neurons in the spinal cord triggers a progressive maladaptive reorganization of neuronal projections throughout the spinal cord below the injury that permits the insidious emergence of uncontrolled hypertensive episodes, known as autonomic dysreflexia [1]-[7].Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025The consequence of these hypertensive episodes is a daily risk of life-threateningcardiovascular events [8]-

[0018] .The inventors reasoned that disentangling the specific neuronal sub- populations involved inthe emergence of autonomic dysreflexia, and how these neurons and their projection patterns reorganize after SCI, would uncover key principles to target these neurons therapeutically and thus eliminate autonomic dysreflexia due to SCI. Spatial organization of neurons involved in autonomic dysreflexia In humans with SCI, episodes of autonomic dysreflexia are most commonly triggered by bladder or bowel distension, lower urinary tract infections, and pressure sores [2]. The inventors reasoned that identifying the neurons triggering autonomic dysreflexia would require a preclinical model that provokes reliable, repeatable, and predictable episodes of autonomic dysreflexia. To establish this model, autonomic dysreflexia was elicited using colorectal distension in mice

[0021] -

[0022] with complete upper-thoracic SCI and pressor responses were monitored with beat-by-beat blood pressure monitoring (Fig.4a-b, Fig.5a-d). In addition to the expected reduction in resting blood pressure following SCI, the inventors detected the emergence of autonomic dysreflexia, starting approximately two weeks after SCI (Fig.4c, Fig.5d). The amplitude of these pressor responses increased gradually during the following weeks until reaching a plateau by one month after SCI, which persisted when tested at 6 weeks after SCI (Fig.4c, Fig.5d). To identify the regions of the spinal cord activated during autonomic dysreflexia, the inventors optimized immunolabeling-enabled three dimensional imaging of solvent-cleared organs(iDISCO+)

[0023] -

[0024] to achieve whole-spinal cord labeling of Fos-a marker of neuronal activity-induced transcription

[0025] -

[0026] , and combined this whole spinal cord visualization with high-resolution CLARITY-optimized light-sheet microscopy

[0027] (Fig. 4d and Fig. 5e), andautomated 3D nuclear spot detection and quantification

[0028] (Fig.5f).The inventors quantified activity-induced transcription in the spinal cords of mice with SCI that underwent repetitive autonomic dysreflexia over 90 minutes (Fig.4e, Fig.5f) [3].Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 The inventors found that autonomic dysreflexia triggered massive transcriptional activation throughout the spinal cord (Fig. 5f). However, disproportionate enrichments of neuronalactivity were detected in two well-defined regions. The first enrichment occurred in thelumbosacral segments that receive the sensory afferents conveying information from the colorectal stimulus (Fig.4d). The second enrichment emerged within the lower thoracic spinal cord, which is referred to as the hemodynamic hotspot

[0020] . This region hosts a dense concentration of sympathetic preganglionic neurons that access the splanchnic vasculature through ganglionic neurons to elicit powerful pressor responses

[0020] . The inventors confirmed these segment-specific enrichments of neuronal activity with classical immunofluorescence of cFos on sectioned tissue (Fig.5g).The presence of discrete yet distant enrichments in neuronal activity demonstrated thatautonomic dysreflexia due to bowel distension activates neuronal subpopulations in thelumbosacral spinal cord, and suggested that these neurons may establish axonal projectionsonto neurons located within the lower thoracic spinal cord, coinciding with the hemodynamic hotspot that regulates blood pressure. However, the bowel is innervated by sensory afferents that project to both the lumbosacral and lower thoracic spinal cord

[0029] -

[0030] . Consequently, we could not rule out the possibility that autonomic dysreflexia emerged from the sensory volleys travelling to the lower thoracic spinal cord when eliciting colorectal distension. To address this possibility, we tested the blood pressure responses to colorectal distension in mice with SCI that also underwent bilateral dorsal rhizotomies at T11-T13, thus eliminating all afferent projections to these segments. In every tested mouse, quantifications revealed that the severity of autonomic dysreflexia was comparable to mice without rhizotomies (Fig.5h-j). Moreover, the pattern of transcriptional activation to colorectal distension was indistinguishable to that of mice without rhizotomies (Fig.5j). These experiments indicate that the emergence of autonomic dysreflexia following colorectal distension in mice with SCI is not dependent on afferent input from the bowel to the lower thoracic spinal cord. Instead, we interpreted these results as evidence that neuronal subpopulations in the lumbosacral spinal cord must access neurons in the lower thoracic spinalcord via projections travelling within the spinal cord in order to trigger autonomic dysreflexia.The neurons activated by autonomic dysreflexiaOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 It was anticipated that understanding the emergence of maladaptive communication betweenthe lumbosacral and lower thoracic spinal cord would be contingent on identifying the neuronalsubpopulations in each region that are activated during autonomic dysreflexia. The neuronal subpopulations embedded within the spinal cord are parcellated into a hierarchical organization that arises from their neurotransmitter expression, developmental transcription factors, and projection patterns

[0031] -

[0033] . This hierarchical organization dictates that identifying the neurons involved in specific neurological functions must follow the logical progression along the cardinal classes. To follow this progression, the inventors first asked whether the neurons embedded in the lumbosacral and lower thoracic spinal cords, and activated during autonomic dysreflexia, exhibited an excitatory or inhibitory phenotype. To answer this question, the inventors exposed Vglut2Cre::Ai9(RCL-tdT)and VgatCre::Ai9(RCL-tdT)mice to repetitive autonomic dysreflexia. The inventors quantified the proportion of activated neurons that colocalized with each neurotransmitter phenotype in the lumbosacral and lower thoracic spinal cords (Fig.6a-b). In particular, the inventors found that autonomic dysreflexia triggered a disproportionate activation of Vglut2ONneurons in the lumbosacral and lower thoracic spinal cords compared to VgatON(Fig.6b) [3], [7]. Next, the inventors sought to determine the causal role of these neuronal subpopulations in triggering autonomic dysreflexia. To manipulate each neuronal subpopulation in each location, the inventors expressed inhibitory Designer Receptors Exclusively Activated by DesignerDrugs (DREADDs)

[0034] in Vglut2ON, VgatON or ChatON neurons located either in thelumbosacral or lower thoracic spinal cord using targeted infusions of AAV5-hSyn-DIO-hM4Di-mCherry in Cre-driver mouse lines (Fig. 6c)

[0035] .Chemogenetic inactivation restricted to Vglut2ON, neurons located in the lumbosacral or lower thoracic spinal cord blunted autonomic dysreflexia (Fig. 6c). In contrast, chemogenetic silencing of VgatON, neurons had no effect (Fig.6c). These experiments suggested that Vglut2ONneurons located in the lumbosacral spinal cord must establish axonal projections within the lower thoracic spinal cord.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025To expose these putative projections, the axons and synapses of Vglut2ON neurons werelabeled with infusions of AAV-DJ-hSyn-flex-mGFP-2A-synaptophysin-mRuby

[0036] into thelumbosacral spinal cord of Vglut2Cre mice (Fig. 6d). The inventors detected long-distanceprojecting axons that established synapses throughout the thoracolumbar spinal cord. SCI altered this projection pattern, whereby the density of synaptic terminals increased significantly within the lower thoracic spinal cord segments (Fig.6d).The results thus far revealed that Vglut2ON neurons located in the lumbosacral spinal cordpropel axonal projections to the lower thoracic spinal cord where Vglut2ONneurons are activated to trigger autonomic dysreflexia. However, Vglut2ONneurons comprise diverse neuronal subpopulations that collectivelyencompass more than 50% of neurons in the spinal cord

[0031] -

[0033] . Consequently, theinventors felt compelled to descend the hierarchical organization of neurons in the spinal cordin order to identify the precise neuronal subpopulations that govern the emergence ofautonomic dysreflexia. However, this descent is contingent on an atlas that catalogs themolecular perturbation elicited by autonomic dysreflexia across the compendium of neuronalsub- populations in the spinal cord.To establish this comparative atlas, the inventors profiled the lumbosacral and lower thoracicspinal cord of mice exposed to repetitive episodes of autonomic dysreflexia using single-nucleus RNA sequencing (snRNA-seq)

[0037] -

[0041] . High-quality transcriptomes from 64,739nuclei were obtained, that were evenly represented across experimental conditions and spatiallocations (Fig. 7a-b). All of the major cell types of the mouse spinal cord were identified (Fig.7c-k). Then, the inventors integrated this dataset within previous atlases of the mouse spinalcord

[0028] ,

[0032] -

[0033] ,

[0042] -

[0043] , which enabled to annotate highly specific neuronalsubpopulations that parcellated into dorsal versus ventral, excitatory versus inhibitory, and local (Nfib) versus long-projecting (Zfhx3) populations (Fig.8a, Fig.7l-m). To identify the neuronal subpopulations perturbed by autonomic dysreflexia, the inventorsapplied cell type prioritization

[0042] ,

[0044] . The principle of cell type prioritization was captured ina machine-learning method called Augur, which identifies cell types undergoing transcriptional responses to a perturbation by ranking cell types that are increasingly more separable within the highly multidimensional space of gene expression. This prioritization exposed Vsx2ONexcitatory neurons as the most transcriptionally responsive neuronal sub-population during autonomic dysreflexia (Fig.8a, Fig.7n-o). Although Vsx2ONneuronal subpopulations were prioritized both in the lumbosacral (neurons defined by the expression of Hoxa10) and lowerOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 thoracic (Hoxa7) spinal cords, the prioritized neurons in the lumbosacral region were consistent with long-projecting Vsx2ONneurons, since they expressed the marker Zfhx3 (SCHoxa10::Zfhx3::Vsx2), while the most perturbed neuronal subpopulation in the lower thoracic spinal cord instead expressed markers of locally-projecting neurons Nfib (SCHoxa7::Nfib::Vsx2) (Fig.8a, Fig. 7n)

[0031] .The transcriptional prioritization of (SCHoxa7::Nfib::Vsx2) and (SCHoxa10::Zfhx3::Vsx2) neurons coincidedwith a pronounced upregulation of synaptic plasticity pathways, neuron projection guidanceprograms, and dendritic arborization—an ensemble of gene programs associated with circuitreorganization and increased neuronal excitability (Fig. to). Visualization of the activity- dependent marker cFos confirmed the activation of (SCHoxa10::Zfhx3::Vsx2) and (SCHoxa7::Nfib::Vsx2) neurons in the lumbosacral and lower thoracic spinal cord, respectively, in response to autonomic dysreflexia (Fig.7p). Although developmentally defined V2a neurons that express Vsx2 (previously known asChx10)

[0045] -

[0050] have been implicated in the production of reaching

[0051] -

[0053] and walking

[0050] -

[0051] ,

[0054] -

[0063] , Vsx2ON neuronal subpopulations have not been demonstrated toparticipate in the regulation of blood pressure. Nonetheless, the results of comparative snRNA- seq experiments dictate that the activation of SCHoxa7::Nfib::Vsx2 neurons and SCHoxa10::Zfhx3::Vsx2 neurons triggers autonomic dysreflexia. The neuronal architecture of autonomic dysreflexia Since autonomic dysreflexia only emerges after SCI, the inventors reasoned that the injury must induce the formation of a maladaptive neuronal architecture that incorporates SCTHORACIC::Vsx2neurons and SCLUMBAR::Vsx2neurons, and possesses the anatomical and functional features compatible with the requirements to trigger autonomic dysreflexia. To expose this architecture, sequential anatomical and functional experiments were conducted, that aimed to reconstruct the successive nodes composing the blueprint of the neuronal architecture responsible for autonomic dysreflexia (Fig.8b-c, Fig.9a). Small diameter nociceptive afferents act as the primary source of sensory input responsiblefor triggering autonomic dysreflexia [5],

[0016] ,

[0064] -

[0065] . Consequently, nociceptive neurons arepositioned as the first node within the neuronal architecture of autonomic dysreflexia, implying that SCLUMBAR::Vsx2neurons are likely to receive synaptic projections from these afferents. To expose close appositions from these afferents onto SCLUMBAR::Vsx2neurons, the inventorsOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 labeled synapses from CalcaONaxonal projections. While SCLUMBAR::Vsx2neurons in the lumbosacral spinal cord did not receive any CalcaONaxonal projections in uninjured mice, it was found that SCI triggered the invasion of CalcaONaxons into intermediate laminae, wherethey established de novo synaptic-like appositions onto SCLUMBAR::Vsx2neurons (Fig. 8c, Fig.9b-d). This anatomical reorganization suggested that CalcaONneurons trigger autonomic dysreflexia through the activation of SCLUMBAR::Vsx2neurons. To expose this causality, CalcaONneurons in the dorsal root ganglia were ableted with diphtheria toxin in CalcaCre::AdvilFlpO::iDTRmice

[0066] . The ablation of this afferent subpopulation abolished autonomic dysreflexia. In contrast, the ablation of parvalbumin (PVON) neurons in the dorsal root ganglia, which convey proprioceptive information along large diameter afferents that project into the spinal cord, failed to influence autonomic dysreflexia (Fig.8d, Fig.9e-f).Next, the inventors posited that SCLUMBAR::Vsx2 act as the second node in the neuronalarchitecture of autonomic dysreflexia (Fig. 8b-c, Fig. 9g-h). To expose the necessity of SCLUMBAR::Vsx2 neurons in triggering autonomic dysreflexia, AAV5-hSyn-DIO-hM4D(Gi)-mCherry

[0067] was infused into the lumbosacral spinal cord of Vsx2Cre mice. Inactivation ofSCLUMBAR::Vsx2neurons blunted the severity of autonomic dysreflexia (Fig.8f, Fig.9i-k). Then, the inventors sought to establish the sufficiency SCLUMBAR::Vsx2neurons to triggerautonomic dysreflexia. To expose this sufficiency, we infused AAV5-hSyn-flex- Chrimson-tdTomato

[0068] into the lumbar spinal cord of Vsx2Cre mice to express excitatory opsins inSCLUMBAR::Vsx2neurons. Optogenetic stimulation of SCLUMBAR::Vsx2neurons immediately triggered autonomic dysreflexia. This increase in blood pressure contrasted with the absence of pressor responses when the same optogenetic manipulation of SCLUMBAR::Vsx2neurons was performed in uninjured mice (Fig.9l-n). The necessity and sufficiency of SCLUMBAR::Vsx2neurons to trigger autonomic dysreflexia implied that SCI must also provoke SCLUMBAR::Vsx2neurons to propel axonal projections to the lowerthoracic spinal cord. To uncover these putative projections, rAAV2- flex-hSyn-FlpO wasinfused into the lower thoracic spinal cord followed by infusions of AAV5-hSyn-Con / Fon-eYFP(enhanced yellow fluorescent protein)

[0069] into the lumbosacral spinal cord of Vsx2Cre mice.This neuroanatomical tracing strategy exposed de novo long-distance projections from SCLUMBAR::Vsx2neurons that established synaptic-like appositions ontoSCTHORACIC::Vsx2neurons in the lower thoracic spinal cord (Fig.8c, Fig.10a-e).Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 Monosynaptically-restricted transsynaptic tracing with avian enveloped G-deleted Rabies confirmed that SCLUMBAR::Vsx2neurons established direct synaptic projections onto SCTHORACIC::Vsx2neurons (Fig.10f). Since SCTHORACIC::Vsx2neurons in the lower thoracic spinal cord were transcriptionally perturbed by autonomic dysreflexia and received direct synaptic projections from SCLUMBAR::Vsx2neurons,it was hypothesized that SCTHORACIC::Vsx2 neurons must act as the third node in the neuronalarchitecture of autonomic dysreflexia (Fig. c, Fig. 10a-f). To establish the necessity andsufficiency of this node, the activity of SCTHORACIC::Vsx2 neurons was manipulated. Chemogeneticinactivation of these neurons blunted the severity of autonomic dysreflexia (Fig.8g-h, Fig. 10fg-i). In turn, optogenetic activation of SCTHORACIC::Vsx2neurons immediately triggered pressor responses (Fig.10ij-l).Next, the inventors asked whether the projection pattern of SCTHORACIC::Vsx2 neurons in the lowerthoracic spinal cord would be compatible with an involvement in autonomic dysreflexia. Toanswer this question, AAV5- hSyn-flex-tdTomato was infused into Vsx2Cre mice and the densityof projections that formed synaptic-like appositions was quantified with ChatONneurons in the intermediolateral column of lower thoracic spinal segments, where ChatONsympathetic preganglionic neurons reside (Fig.10m-o). While SCTHORACIC::Vsx2neurons primarily projected ventrally, the inventors also detected projections that expanded laterally where they established synaptic-like appositions with the majority of ChatONneurons located in the intermediolateral column of the lower thoracic spinal cord. ChatONsympathetic preganglionic neurons are embedded in the intermediolateral column, andit was found that they receive projections from SCTHORACIC::Vsx2 neurons. Consequently, theinventors surmised that ChatON neurons act as the fourth and final node in the neuronalarchitecture of autonomic dysreflexia. Indeed, chemogenetic inactivation of ChatONneurons located in the lower thoracic spinal cord abolished autonomic dysreflexia elicited by bowel distension (Fig.8h, Fig.10p-r). These sequential anatomical and functional experiments exposed the complete neuronal architecture that causes autonomic dysreflexia. The building blocks of this architecture are precipitated by SCI, wherein specific neuronal nodes form de novo maladaptive connections that permit and exacerbate the emergence of autonomic dysreflexia. This architecture has its foundation in CalcaONneurons located in the dorsal root ganglia, which establish maladaptive projections to SCLUMBAR::Vsx2neurons in the lumbosacral spinal cord. In turn, an SCI provokes these neurons to propel axons to the hemodynamic hotspot located within the lower thoracicOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 spinal cord, where they form de novo synaptic connections with SCTHORACIC::Vsx2neurons. Theselocally- projecting neurons establish connections onto ChatON sympathetic preganglionicneurons, which permit massive blood pressure elevations through the recruitment of neurons in splanchnic ganglia and subsequent alpha1receptor activation throughout the dense splanchnic vasculature. The consequence of this aberrant neuronal architecture is the emergence of life-threatening autonomic dysreflexia. Competitive neuronal architectures converge on SCTHORACIC::Vsx2neuronsObtained results demonstrated that SCI precipitates the formation of an aberrant neuronalarchitecture that exploits the natural connections from SCTHORACIC::Vsx2neurons onto sympathetic preganglionic neurons located in the hemodynamic hotspot to trigger autonomicdysreflexia. The inventors reasoned that competitive engagement of the same neuronal sub-populations with an intervention that mediates beneficial as opposed to maladaptive reorganization of synaptic projections onto SCTHORACIC::Vsx2neurons could prevent theemergence of autonomic dysreflexia. Using molecular cartography, it was recentlydemonstrated that epidural electrical stimulation (EES) of the lumbar spinal cord restores walking through the activation of locally-projecting Vsx2ONneurons in the lumbar spinal cord

[0028] ,

[0042] . The inventors surmised that the same principle must exist in the lower thoracicspinal cord, since it has been previously shown that EES applied over this hemodynamichotspot triggers robust pressor responses after SCI

[0020] . Thus, it was hypothesized that EESapplied over the lower thoracic spinal cord activates SCTHORACIC::Vsx2neurons, and that EEScould thus compete with SCLUMBAR::Vsx2 neurons to modulate SCTHORACIC::Vsx2 neurons.To identify the neuronal subpopulations engaged by EES applied to the lower thoracic spinalcord, an additional comparative snRNA-seq experiment was performed (Fig. 11a-k).Concretely, neuronal nuclei was profiled from mice with SCI that had received EES over thelower thoracic spinal cord during 30 minutes. As previously described in rats, non-humanprimates, and humans with SCI

[0020] , all the mice exhibited robust pressor responses whendelivering EES over the lower thoracic spinal cord, referred to as the hemodynamic hotspot(Fig.12a-b). High- quality transcriptional profiles were obtained from 21,098 nuclei (Fig.12c).Integration of this dataset

[0070] with previous atlases

[0028] ,

[0032] -

[0033] ,

[0042] -

[0043] and data fromthe experiments conducted on mice that were exposed to repetitive autonomic dysreflexia enabled to identify and evaluate the same neuronal subpopulations. Cell type prioritization

[0042] ,

[0044] revealed that locally-projecting SCHoxa7::Nfib::Vsx2 neurons exhibited the mostOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 pronounced transcriptional response across the compendium of neuronal subpopulations embedded in the lower thoracic spinal cord of mice that had received EES targeting the hemodynamic hotspot (Fig.12c, Fig.11l-n).Since it was previously found that Vsx2ON neurons were recruited in response to EES in thelumbar spinal cord

[0028] ,

[0042] , these comparative snRNA-seq experiments indicate that theprinciple through which EES recruits specific neuronal subpopulations is conserved across the thoracolumbar spinal cord. Moreover, this observation nominates SCHoxa7::Nfib::Vsx2neurons asa convergence node that is not only recruited during autonomic dysreflexia, but can also beengaged by the neuronal architecture that prevents orthostatic hypotension when the delivery of EES targets the hemodynamic hotspot.The discovery of this intersection compelled the inventors to expose the entire neuronalarchitecture activated by EES. Therefore, sequential anatomical and functional experiments were conducted that aimed to reconstruct the successive nodes involved in the pressor responses during the delivery of EES targeting the hemodynamic hotspot.It has been previously shown that EES applied over the lower thoracic spinal cord activatesafferent fibers in the posterior roots to trigger pressor responses

[0020] , and mounting evidencesuggests that EES restores walking through the recruitment of large-diameter fibers wherethey bend to enter the spinal cord through the dorsal root entry zones

[0071] -

[0072] . Thus, theinventors asked whether large-diameter afferent neurons act as the first node of the neuronalarchitecture that enables EES targeting the hemodynamic hotspot to trigger pressor responses (Fig 13a-b). To answer this question, we ablated proprioceptive or nociceptive neurons with the administration of diphtheria toxin to PVCre::AdvilFlpO::iDTR mice and CalcaCre::AdvilFlpO::iDTR mice, and applied EES targeting the hemodynamic hotspot. The ablation of PVONneurons in the dorsal root ganglia abolished pressor responses to EES, whereas the ablation of CalcaONneurons had no effect (Fig. 12d, Fig. 13c-d). We then verified that large-diameter afferentsestablish synaptic projections onto SCTHORACIC::Vsx2 neurons in the lower thoracic spinal cord.To expose this connectome, we visualized large-diameter afferent fibers in PVCre::AdvilFlpO::Ai9(RCL-tdT)mice and confirmed that these fibers established vGlut1ON synapticappositions onto SCTHORACIC::Vsx2neurons (Fig. 13e-g). The dense projections from PVONneurons onto SCTHORACIC::Vsx2neurons contrasted with the absence of projections from PVONneurons onto ChatONneurons in the intermediolateral column of the lower thoracic spinal cord, both before or after SCI (Fig.13e-g).Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 Monosynaptically-restricted transsynaptic tracing with avian enveloped G-deleted Rabies confirmed that PVONneurons in the dorsal root ganglia established direct synaptic projections onto SCTHORACIC::Vsx2neurons (Fig.13h). Since SCHoxa7::Nfib::Vsx2neurons underwent the greatest transcriptional perturbation following EES targeting the hemodynamic hotspot and receive direct projections from large-diameterafferent fibers that are recruited by EES, the inventors anticipated that SCTHORACIC::Vsx2 neuronsact as the second node in the neuronal architecture that enables EES to trigger pressorresponses. To expose the necessary role of SCTHORACIC::Vsx2 neurons, AAV5-hSyn- DIO-hM4Di-mCherry was infused into the lower thoracic spinal cord of Vsx2Cre mice. Inactivation ofSCTHORACIC::Vsx2neurons blunted the pressor responses triggered by EES (Fig.12e, Fig.13i- j).The inventors surmised that ChatON sympathetic preganglionic neurons act as the third andfinal node in the neuronal architecture that enables EES targeting the hemodynamic hotspot to trigger pressor responses. Indeed, chemogenetic inactivation of ChatONneurons located in the lower thoracic spinal cord abolished pressor responses triggered by EES (Fig.12f, Fig. 13k-l). Together these results uncovered the neuronal architecture that enables EES targeting thehemodynamic hotspot of the lower thoracic spinal cord to trigger pressor responses. EESdirectly recruits large diameter afferents to activate SCTHORACIC::Vsx2neurons, which project to ChatONsympathetic preganglionic neurons to elicit pressor responses through the activation of ganglionic neurons, and subsequent alpha1 receptor activation to induce vasoconstriction.Finally, the inventors surmised that for the neuronal architecture enabling EES to elicit pressorresponses to compete with the neuronal architecture that triggers autonomic dysreflexia, these two architectures must intersect on the same SCTHORACIC::Vsx2neurons in the lower thoracic spinal cord. To expose this anatomical and functional intersection, first AAV5-hSyn-eGFP (enhancedgreen fluorescent protein) was infused into the lumbosacral spinal cord ofPVCre::AdvilFlpO::Ai9(RCL-tdT)mice with SCI coupled to labeling of SCTHORACIC::Vsx2neurons. Theinventors found that the same SCTHORACIC::Vsx2 neurons received direct projections from large-diameter afferents emanating from PVONneurons located in dorsal root ganglia, and from axons projecting from the lumbosacral spinal cord after SCI (Fig.12g). Second, the inventors aimed to confirm that these two distinct axonal projections were able to regulate the activity of the same SCTHORACIC::Vsx2neurons. Single-unit recordings of optogenetically-identifiedOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 SCTHORACIC::Vsx2neurons was conducted in the lower thoracic spinal cord in response toautonomic dysreflexia and EES. It was found that both paradigms elicited short-latencyresponses in the recorded SCTHORACIC::Vsx2neurons that were compatible with a direct activationof the same SCTHORACIC::Vsx2 neurons (Fig. 13o).Neuronal architecture competition to reverse autonomic dysreflexia The intersection between the neuronal architecture that enables EES targeting the hemodynamic hotspot to elicit pressor responses and the neuronal architecture that triggers autonomic dysreflexia opened the intriguing possibility that the sustained modulation of SCTHORACIC::Vsx2neurons with EES could compete with the detrimental activity emanating from aberrant axonal projections of SCLUMBAR::Vsx2neurons, ultimately reversing autonomic dysreflexia. To test this possibility, mice were subjected to autonomic neurorehabilitation, which consisted of daily sessions during which EES targeting the hemodynamic hotspot was delivered over the lower thoracic spinal cord during the course of one month (Fig.14a, Fig.15a). Autonomic neurorehabilitation abolished autonomic dysreflexia in all tested mice (Fig.14b, Fig.15b). It was suspected that the suppression of autonomic dysreflexia would emerge from the competitive advantage of large-diameter fiber projections over aberrant axonal projections from SCLUMBAR::Vsx2neurons to form synaptic connections with SCTHORACIC::Vsx2neurons (Fig. 14c). To expose this adversarial anatomy, SCTHORACIC::Vsx2and SCLUMBAR::Vsx2neurons werelabeled with intersectial viral tracing strategies. Then, the relative density of synapticappositions onto SCTHORACIC::Vsx2 neurons was quantified, both with both vGlut1 synapsesemanating from PVONlarge-diameter afferent fibers and axonal projections from SCLUMBAR::Vsx2. Autonomic neurorehabilitation reduced the number of aberrant projections from SCLUMBAR::Vsx2neurons onto SCTHORACIC::Vsx2neurons, while concomitantly augmenting the density of vGlut1ONsynaptic appositions onto SCTHORACIC::Vsx2neurons embedded in the hemodynamic hotspot (Fig.14d, Fig.15c-d). Mistargeted stimulation exacerbates autonomic dysreflexiaOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 Recent case studies reported transient increases in blood pressure in response to EES appliedover the lumbosacral spinal cord of people with SCI

[0077] -

[0082] , and that the long-term deliveryof EES over this location led to improvements in resting hypotension and orthostatic hypotension in these individuals. Since a paucity of neurons involved in the control of bloodpressure reside in the lumbosacral spinal cord, the activation and reinforcement of theneuronal architecture that causes autonomic dysreflexia is the most likely mechanism toaccount for these observations. Therefore, it has been hypothesized that the daily activationof SCLUMBAR::Vsx2neurons in response to EES applied over the lumbosacral spinal would provide a competitive advantage to vGlut2ONsynapses emanating from these neurons and projecting onto SCLUMBAR::Vsx2neurons compared to vGlut1ONsynapses received from local PVONlarge- diameter afferent fibers, and that ultimately, this shift in the synaptic innervations of SCLUMBAR::Vsx2neurons would exacerbate autonomic dysreflexia.To test this hypothesis, a new cohort of mice was subjected to daily exposure to EES, butinstead of delivering EES over the lower thoracic spinal cord to target the hemodynamic hotspot, EES was applied over the lumbosacral spinal cord for 30 minutes during the course of one month (Fig. 14e, Fig. 15e). This mistargeted stimulation doubled the severity of autonomic dysreflexia in all tested mice (Fig. 14f, Fig. 15f). As anticipated, a concomitant increase in the density of axonal projections from SCLUMBAR::Vsx2neurons onto SCTHORACIC::Vsx2neurons was observed, combined with a decrease in the density of vGlut1ON synapses fromPVONlarge-diameter afferent fibers (Fig.15g-h). The dramatic exacerbation of autonomic dysreflexia triggered by the mistargeted delivery of EES demonstrates that attempts to restore hemodynamic stability with EES applied over the lumbosacral spinal cord are not only less efficacious than EES targeting the hemodynamic hotspot located in the lower thoracic spinal cord, but also proved unsafe, since the mechanism by which hemodynamics can be modulated when stimulating this region is by triggering and reinforcing the severity of life-threatening autonomic dysreflexia. Longitudinal monitoring of autonomic neurorehabilitation While autonomic neurorehabilitation suppressed autonomic dysreflexia in mice, it has been recognized that the longitudinal monitoring of the safety and efficacy of this treatment over a long period of time would be important to inform the design of a therapy for people living with SCI.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Therefore, the inventors leveraged the chronic rat model of high-thoracic contusion SCI thatenables 24 / 7 monitoring of hemodynamic parameters without the constraints of tetheredelectronics

[0020] ,

[0083] (Fig. 16a, Fig. 17a). An electronic dura mater (e-dura) implant with anoptimized configuration of electrodes was inserted to target the dorsal root entry zones projecting to the last three thoracic segments of the spinal cord, which coincide with the hemodynamic hotspot. Closed-loop adjustment of EES amplitudes with a proportional-integralcontroller enabled the maintenance of blood pressure to a predefined target

[0020] (Fig. 16b).The efficacy of this technology was previously established for the treatment of orthostatichypotension, named the neuro- prosthetic baroreflex

[0020] .Rats with SCI were exposed to daily sessions of autonomic neurorehabilitation. During thesesessions, the closed-loop controller was configured to augment blood pressure to a predefined target. Careful monitoring of hemodynamics during autonomic neurorehabilitation revealed that blood pressure was maintained within and did not exceed the defined target range, indicating that EES targeting the hemodynamic hotspot promoted controlled elevations ofblood pressure that were not reminiscent of autonomic dysreflexia (Fig.16b). Moreover, bloodpressure returned to baseline levels as soon as EES was switched off.Formal weekly hemodynamic assessments were conducted to quantify the severity ofautonomic dysreflexia (Fig. 16c, Fig. 17b). These assessments revealed that autonomic dysreflexia vanished in all tested rats. Rats with chronic SCI exhibited an increase in the number of axonal projections emanating from neurons located in lumbosacral segments that formed synaptic appositions with Vsx2ONneurons in the lower thoracic spinal cord, comparable to observations in mice (Fig.16d, Fig. 17c-f). Autonomic neurorehabilitation reversed this aberrant connectome, and in turn augmented the density of vGlut1ONsynaptic-appositions from PVONneurons onto Vsx2ONneu-rons located in the lower thoracic spinal cord (Fig. 16g-h, Fig. 17g-j).These results reinforced the hypothesis that autonomic neurorehabilitation is safe and enables large-diameter afferents to compete for the innervation of Vsx2ONneurons located in the hemodynamic hotspot, and ultimately, to overcome the aberrant neuronal architecture responsible for the emergence of autonomic dysreflexia. The consequence is the reversal of autonomic dysreflexia. Clinical necessity of therapies for autonomic neurorehabilitationOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 While mice and rat experiments documented the safety and efficacy of EES targeting the hemodynamic hotspot to reduce the severity of autonomic dysreflexia, the neurosurgical intervention necessary to deploy this therapy must be weighed against the risks and benefits of the procedure. Establishing this balance requires an understanding of the prevalence, symptomatology, and effectiveness of current management strategies.Consequently, the inventors leveraged the Spinal Cord Injury Community Survey (SCICS),which includes self-reported information on the presence of symptoms of autonomicdysreflexia, and demographic information about the level of SCI (n = 1,479)

[0084] -

[0085] . Thesedata revealed that 82% of individuals with tetraplegia had been told by a medical practitionerthat they present autonomic dysreflexia. The inventors found that only 30% of the individualswere being treated for autonomic dysreflexia, yet 98% of these treated individuals still experienced symptoms (Fig.18a, Fig.19a). To further characterize the pattern of hypertensive-related symptoms in individuals with SCI,the inventors next surveyed a diverse sample of 254 individuals with SCI from 50 differentcountries, and included responses to the Autonomic Dysfunction Following Spinal Cord Injury(ADFSCI) rating scale. When considering individuals with tetraplegia, the inventors found that79% of these individuals experienced symptoms of autonomic dysreflexia (Fig.18b, Fig.19b- d). These symptoms were primarily characterized by vasogenic manifestations including headaches and heart palpitations, sudomotor manifestations including goose bump and sweating, as well as anxiety (Fig.18b). This symptomatology combined with the risk of life-threatening episodes of autonomic dysreflexia and the absence of adequate therapeutic management justified evaluating the impact of autonomic neurorehabilitation on the severity of autonomic dysreflexia in humans living with chronic SCI. Preliminary clinical validation of autonomic neurorehabilitationNext, the inventors aimed to conduct a preliminary clinical evaluation to assess whether thelong-term application of EES targeting the hemodynamic hotspot to reduce hypotensivesymptomatology also reduces the severity of autonomic dysreflexia in humans with chronic SCI.To carry out this assessment, the inventors leveraged ongoing clinical studies (namelyHemON [NCT05111093, CHUV, Lausanne, Switzerland] (see Fig 19e) and HemON-NLOur file: 37465.EPF.P110PCOfficial file: tba March 10, 202505941819, Sint Maartenskliniek, Ubber- gen, Netherlands]) focused on the development ofpurpose-built technologies to restore hemodynamic stability based on EES targeting thehemodynamic hotspot (

[0086] .The experimental setup used in the HemON clinical trial (Fig.19e) is schematically illustrated in Fig.20. These studies enrolled patients with cervical SCI who presented with medically-refractory orthostatic hypotension. When exposed to orthostatic challenges during a tilt-table test, all the participants exhibited a rapid decline in blood pressure that required the early termination of the tilt-test. Following this verification of their eligibility, the participants underwent aneurosurgical intervention to implant an electrode array targeting the dorsal root entry zonesinnervating the last three thoracic segments and a neurostimulator to deliver EES. Post- operative evaluations confirmed that EES targeting the hemodynamic hotspot elicited robust pressor responses that reduced the severity of orthostatic hypotension. The participants then learned how to operate the therapy in order to regulate their blood pressure throughout the day for up to two years. While these studies focused on the long-term reduction of hypotensive symptomatology, theseverity of autonomic symptoms was assessed concurrently with the ADFSCI rating scale

[0087] .Consequently, the context of these studies allowed the inventors to assess how thesymptomatology related to autonomic dysreflexia evolved in eleven participants.Quantification of the autonomic dysreflexia subscore within the ADFSCI revealed a decrease in symptomatology related to autonomic dysreflexia after long-term use of EES targeting thehemodynamic hotspot (Figs. 18f-g, Fig. 19f-g). Secondary analyses of vasogenic andsudomotor manifestations revealed that the long-term use of EES targeting the hemodynamichotspot led to a reduction in headaches and heart palpitations, which are the main vasogenic symptoms of autonomic dysreflexia. In stark contrast, sudomotor manifestations and anxiety remained unchanged from base-line. These clinical outcomes provide preliminary evidence that the daily application of EEStargeting the hemodynamic hotspot reduces the severity of autonomic dysreflexia in humanswith chronic SCI, as quantified functionally and anatomically in mice and rats. DiscussionOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 Here, the complete, aberrant neuronal architecture that develops after SCI and causes autonomic dysreflexia has been exposed. While all the neurons constituting this neuronal architecture exists in healthy mice, the SCI precipitates the development of de novo connections between nociceptive neurons and Vsx2-expressing neurons, as well as between Vsx2-expressing neurons located in the lumbosacral and lower thoracic spinal cord that provoke the emergence of autonomic dysreflexia. This architecture incorporates locally-projecting, Vsx2-expressing neurons located in the lower thoracic spinal cord that are also embedded in the neuronal architecture that enables EEStargeting the hemodynamic hotspot to achieve safe and well-controlled increases of bloodpressure. Since these adversarial architectures converged onto a single neuronalsubpopulation, their relative activation determined the prevailing architecture in thiscompetitive interaction. Exposing these adversarial architectures allowed to design a safe intervention that reversed autonomic dysreflexia in mice, rats, and humans with SCI by applying EES targeting the hemodynamic hotspot located in the lower thoracic spinal cord. Conversely, the mistargeted application of EES over the lumbosacral spinal cord reinforced the anatomical and functional connectivity of the neuronal architecture responsible for autonomic dysreflexia, which augmented the severity of these symptoms. These mechanisms establish that the long-term delivery of EES over the lumbosacral spinal cord to stabilize hemodynamic stability iscontraindicated for people who suffer from autonomic dysreflexia after SCI

[0077] -

[0082] .Indeed, a previously unrecognized increase in the incidence of autonomic dysreflexia episodeswas recently detected in people with cervical SCI who were exposed to EES over thelumbosacral spinal cord to restore lower limb movements

[0088] . The involvement of Vsx2-expressing neurons in the emergence of autonomic dysreflexia exposed the unique properties of these neurons in the regulation of neurological functions andcircuit reorganization mechanisms after neurological disorders and neurotraumas. Indeed, theinventors recently demonstrated that Vsx2-expressing neurons located in the spinal cord areinherently primed to overexpress circuit reorganization gene program - thus becomingrecovery-organizing neurons after SCI that guide the spontaneous and therapeutically-enhanced recovery of walking after both incomplete and complete SCI

[0028] ,

[0032] -

[0033] . Here,the inventors extend the role of these cells by demonstrating that since Vsx2-expressingneurons are primed to reorganize in response to injury, but are lacking molecular cues to directtheir reorganization after severe SCI

[0089] , they can be naturally coerced towards undirectedOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 and aberrant neuronal architectures that drive the emergence of maladaptive neurological functions. Future work must understand whether Vsx2-expressing neurons drive the reorganization of the neuronal architecture responsible for the development of autonomic dysreflexia, and whether these same neurons participate in multiple beneficial or maladaptive neurologicalfunctions after SCI. Moreover, it cannot be excluded that other neuronal subpopulations andpathways in the spinal cord also contribute to the emergence of autonomic dysreflexia. The activation of alternative spinal cord segments via other noxious stimuli such as pressure ulcerscould also alter the neuronal architecture, although the inventors anticipate the four nodesidentified in their work will remain involved in autonomic dysreflexia triggered by these stimuli.Autonomic dysreflexia is a life-threatening medical condition that can lead to stroke, heartattack, and death [1]-[7]. Consequently, people living with SCI and medical practitioners aretaught to identify warning signs such as headaches, sweating, and goosebumps, since these signs inform on the presence of a noxious stimulus that is triggering autonomic dysreflexia, and must therefore be localized to resolve the ongoing hypertensive episode as quickly aspossible

[0090] . Analysis of self-reported symptoms in patients from four clinical studies showedthat EES targeting the hemodynamic hotspot reduced headaches and heart palpitations, which are both directly related to dangerous increases in blood pressure. Instead, this therapy had no detectable impact on the other warning signs such as goosebumps and sweating that couldstill inform on the presence of noxious stimulus. The most likely explanation for this dichotomyis that sympathetic preganglionic neurons responsible for hemodynamic regulation areconcentrated within the penultimate segment of the thoracic spinal cord

[0020] ,

[0091] , which isdirectly targeted by EES. Instead, the sympathetic preganglionic neurons triggering symptoms such as sweating and goosebumps are distributed uniformly throughout the thoracic spinal cord and are consequently poorly targeted by EES restricted to the lower thoracic spinal cord.The inventors took advantage of ongoing clinical trials to collect preliminary evidence on thepotential for EES targeting the hemodynamic hotspot to reduce autonomic dysreflexia in humans with SCI. However, these trials were not designed to demonstrate the safety and efficacy of this therapy, and consequently, the next steps must include the assessment of the safety and efficacy of EES targeting the hemodynamic hotspot to reduce autonomic dysreflexia in a pivotal clinical trial. Materials and MethodsOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 Mouse and rat models. Adult male or female C57BL / 6 mice (15-25 g body weight, 8-15 weeks of age) or transgenic mice were used for all experiments. Vglut2Cre(Jackson Laboratory 016963), Vsx2Cre(MMMRRC 36672, also called Chx10Cre), ChatCre, VgatCre, Ai65(RCFL-tdT)(Jackson Laboratory 021875), Par- valbumin (PV)Cre (Jackson Laboratory 017320), AdvillinFlpO(a gift from V. Abraira), iDTR, CalcaCretransgenic mouse strains were bred and maintained on a mixed genetic background (C57BL / 6). Adult female Lewis rats (180–220 g body weight, 14– 30 weeks of age) were used for the rat experiments. Housing, surgery, behavioral experiments and euthanasia were all performed in compliance with the Swiss Veterinary Law guidelines.Manual bladder voiding and all other animal care was performed twice daily throughout theentire experiment. All procedures and surgeries were approved by the Veterinary Office of the Canton of Geneva (Switzerland; authorization GE67). Viral vectors and vector production. Viruses used in this study were either acquired commercially or produced at the EPFL core facility. The following AAV plasmids were used and detailed sequence information is available as detailed or upon request: AAVDJ-hSyn-flex-mGFP-2A-synaptophysin- mRuby

[0036] (Stanford Vector Core Facility, reference AAV DJGVVC- AAV-100), AAV9-CMV-Cre (Vector Biolabs 7014), AAV5-hSyn- eGFP (Addgene50465-AAV5), AAV5-Syn-flex-ChrimsonR-tdT (Addgene 62723), AAV5-hSyn-DIO-hm4D

[0034] (Gi)-mCherry (Addgene 44362, 7x1012 vg / mL), AAV5-CAG-flex-tdTomato (a gift from S.Arber), AAV5-hSyn-Con / Fon-eYFP (Addgene 55650) and rAAV2-EF1a- DIO-Flpo (Addgene87306), EnvA-∆GRabies-mCherry (a gift from S. Arber) and AAV8-hSyn-dlox TVA950-2A- EGFP-2A-oGrev-dlox-WRPE-bGHp (a gift from S. Arber).. Spinal cord injury (SCI) models. For mouse spinal cord injuries, a laminectomy was performed on the T4 vertebra to expose the T4 spinal segment. Complete transections were performed using angled microscissors. Rat spinal cord injuries were performed according to apreviously published work

[0020] . In brief, a laminectomy was performed on the T3 vertebra toexpose the T3 spinal segment. Following this, the rat was transferred to the Infinite-Horizons(IH-0400 Impactor, Precision Systems and Instrumentation LLC) impactor

[0020] stage, wherethe T2 and T4 spinous processes were securely clamped using modified Allis forceps

[0020] .The rat was stabilized on the platform and the impactor tip (2.5 mm) was properly aligned using a three-dimensional coordinate system moving platform. The IH system was set to deliver animpact force of 400 kdyn, with a 5 s dwell time

[0020] . Analgesia (buprenorphine, Essex ChemieAG, 0.01–0.05 mg per kg, subcutaneously) and antibiotics (amoxicillin 200 mg per 4 ml, Sandoz, 200 mg l-1ad libitum) were provided for 3 and 5 days after surgery, respectively.Bladders were manually expressed twice a day until the end of the experiment

[0092] .Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Rodent anesthesia use. All non-terminal experiments were conducted by anaesthetizinganimals with isoflurane (initial induction 5% and maintained on a Bain’s system at 2%). Terminal hemodynamic assessments were carried out as previously described four weeks after SCI. In brief, animals were anaesthetized with urethane (1.5 g kg-1; intraperitoneally)

[0020] . Depth of anesthesia was continually monitored by assessing withdrawal reflexes andrespiratory rate. Hemodynamic monitoring. In mice, carotid artery catheterization was performed. After induction of anesthesia, the hair on the neck was shaved, and the surgical site cleaned with alcohol and betadine. The right common carotid artery was exposed and isolated from the internal jugular vein using blunt dissection. The rostral portion of the carotid artery immediately below bifurcation was permanently occluded, while the caudal portion of the vessel wastemporarily occluded using 5-0 silk sutures. A bent tip 30- gauge needle was used to create asmall hole on top of the carotid artery. The blood pressure sensor was inserted into the carotid artery and advanced 0.5 cm caudally. The catheter was then secured with 25-0 silk sutures. In rats, the procedure for the telemeter implantation was performed according to our previously published work

[0020] . In brief, blood pressure was recorded using wireless telemeters (TRM56SP SNA and Pressure Telemeter, Kaha Sciences). A midline abdominal incision was made to expose the peritoneal cavity, followed by a blunt dissection to reach the descending aorta. The aorta was temporary occluded using a 4-0 silk, one to two millimeters rostral to the iliac bifurcation. The pressure sensor was inserted in the aorta so that the tip was just caudal to the renal artery, and fixed with a surgical mesh and biocompatible surgical glue. Colorectal distension to induce autonomic dysreflexia Foley Catheter Cysto-Care® 1.5 ml and Foley Catheter CystoCare® 3 ml were used for mice and rats, respectively, to perform colorectal distension to induce autonomic dysreflexia

[0093] . The catheter was inserted into the rectum and colon until the balloon was no longer exposed. During colorectal distension assessments, the balloon was inflated (up to 0.7 ml for mice and up to 2.5 ml in rats) for 60 s. Subsequent trials were only initiated after blood pressure returned to the baseline value. For repetitive autonomic dysreflexia experiments, the balloon was inflated for 30 s and deflated for 60 s and this protocol cycled for 90 minutes [3]. Epidural electrical stimulation implants. All the procedures have been detailed previously

[0020] ,

[0028] ,

[0094] -

[0098] . To position electrodes to deliver epidural electrical stimulation (EES) in mice, laminotomies (removal of only the connective tissue in between the bones, but not bones) were performed at T9-T10 and T12-T13 to expose the spinal cord. Teflon-coatedOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025stainless-steel wires connected to a percutaneous connector (Omnetics ConnectorCorporation) were inserted rostrally and passed between the spinal cord and the vertebral bones to the other opening. A small part of insulation was removed and the exposed stimulation sites were positioned over T12-T13. A common ground was inserted subcutaneously. The percutaneous connector was cemented to the skull. This stimulation protocol was subsequently used for all acute and chronic experiments. In rats, the implantation of the e-dura was performed according to a previously published work from the inventors

[0020] . To insert and stabilize e-dura implants into the epidural space two partial laminectomies were performed at vertebrae levels L1–L2 and T8–T9 to create entry and exit points for the implant. The implant was gently pulled above the dura mater using a surgical suture. Electrophysiological testing was performed intra-operatively to fine-tune positioning of electrodes. The connector of the implant was secured into a protective cage plastered using freshly mixed dental cement on top of the L2–L3 vertebra. Stimulation was then delivered as described previously

[0020] . The headstage was plastered using freshly mixed dental cement on the dorsal side of the skull where three stainless steel screws were placed. Autonomic neurorehabilitation. In mice, EES was delivered at conventional stimulationprotocols

[0020] consisted of continuous EES delivered at 50 Hz with 5 ms pulses at 100–150µA (2100 Isolated Pulse Stimulator, A-M Systems). Mice underwent autonomic neurorehabilitation consisting of EES applied for 30 minutes each day for 4 weeks, starting 1 week after SCI.. In rats, a closed-loop controlled stimulation was applied using a proportional integral (PI) controller that adjusts the amplitude of traveling EES waves over the threehemodynamic hotspots. A +10 mmHg systolic blood pressure target was applied from thebaseline acquired at the beginning of each autonomic neurorehabilitation session

[0020] . Ratsreceived 30 minutes of closed-loop controlled stimulation for 6 weeks, starting one week after SCI. Neuron-specific ablation, chemogenetics and optogenetics. For ablation experiments withthe diphtheria toxin, the inventors used PVCre::AdvilinFLPo::iDTR and CalcaCre::AdvillinFLPo::iDTRmice. Four weeks after the spinal cord injuries (T4 spinal level complete transection), mice received intraperitoneal injections of diphtheria toxin (Sigma, D0564) diluted in saline (100 µgkg1) to target respectively PVONor CalcaONneurons. Mice were tested two weeks post- injection. To manipulate the activity of Vglut2ON, VgatONneurons, AAV5-hSyn-DIO-hm4D was infused in either the lower thoracic spinal cord (T11-T13) or lumbosacral spinal cord (L5-S1) of either Vglut2Creor VgatCremice prior to performing the spinal cord injury. To manipulateChatON neuronal activity, AAV5-hSyn- DIO-hm4D was infused (0.15 µl per injection) at twodepths (0.8 mm and 0.4 mm below the dorsal surface) and separated by 1 mm in the lowerOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 thoracic spinal cord (T11-T13) or lumbosacral spinal cord (L5-S1) of either Vglut2Creor VgatCremice prior to performing the spinal cord injury. To manipulate ChatONneuronal activity, AAV5- hSyn-DIO-hm4D was infused (0.15 µl per injection) at two depths (0.8 mm and 0.4 mm below the dorsal surface) and separated by 1 mm in the lower thoracic spinal cord (T11-T13) of ChatCremice prior to performing the spinal cord injury. To manipulate SCTHORACIC::Vsx2or SCLUMBAR::Vsx2neurons, AAV5-hSyn-DIO-hm4D was infused in either the lower thoracic spinal cord (T11-T13) or lumbosacral spinal cord (L5-S1) (injection depths were 0.8 mm and 0.4 mm below the dorsal surface; separated by 1 mm; 0.15 µl per injection), respectively, in Vsx2Cre mice prior to performing the spinal cord injury. After four weeks, autonomic dysreflexia or EES- induced pressor response were assessed before and between 30–45 min after intraperitoneal injections of 5 mgkg1clozapine N-oxide (CNO) (Carbosynth, CAS: 34233-69-7, suspended in 2% DMSO in saline). Optogenetic manipulations. To optogenetically manipulate Vsx2ONneurons, AAV5-Syn-flex-Chrimson (Addgene 62723- AAV5, 5x1012 vg / mL) was infused in either the lower thoracicspinal cord (T11-T13) and the lumbosacral spinal cord (L5-S1), in Vsx2Cremice prior to performing the spinal cord injury. After six weeks, laminectomies were made over T11 / T12 / T13 and L5 / L6 / S1 spinal segments. 5-ms pulses were delivered at 50 Hz from a 635 nm laser (LaserGlow Technologies LRD-0635-PFR-00100-03). Laser light was delivered to the surfaceof the spinal cord through a fiber optic cable attached to 400 µm, 0.39 NA cannula with a 5 mmtip (Thorlabs). Optical power was set to 2.35 mW at the tip. Spinal injections for exclusive labeling of Vsx2ONneurons. To exclusively label Vsx2ONneurons in the lumbosacral spinal cord with long-distance projections to the lower-thoracicregion SCLUMBAR::Vsx2), the inventors leveraged Boolean logic viral strategies

[0069] . Partiallaminectomies were made over the T11 / T12 / T13 and L5 / L6 / S1 spinal segments of Vsx2Cremice. Two sets of bilateral injections of AAV5-hSyn-Con / Fon-eYFP (Addgene 55650)

[0069] were made over the L5 / L6 / S1 spinal segments (0.25 µl per injection) at a depth of 0.6 mm below the dorsal surface and separated by 1 mm. Two sets of bilateral injections of rAAV2- EF1a-DIO-Flpo (Addgene 87306) were made over the T11 / T12 / T13 spinal segments (0.15 µl per injection) at two depths (0.8 mm and 0.4 mm below the dorsal surface) and separated by 1 mm. Animals were perfused four weeks later. To label lower-thoracic Vsx2ONneurons(SCTHORACIC::Vsx2), two sets of bilateral injections of AAV- flex-tdTomato were made overT11 / T12 / T13 spinal levels (0.15 µl per injection) at two depths (0.8 mm and 0.4 mm below the dorsal surface) and separated by 1 mm. Spinal injections for exclusive labeling of Vglut2ONneurons.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 To exclusively label Vglut2ONneurons in the lumbosacral spinal cord with long-distance projections to the lower-thoracic region, the inventors leveraged Boolean logic viral strategies

[0069] . Partial laminectomies were made over the L5 / L6 / S1 spinal segments of Vglut2Cre mice. Two sets of bilateral injections of AAVDJ-hSyn-flex-mGFP-2Asynaptophysin-mRuby 33 (Stanford Vector Core Facility, reference AAV DJ GVVC-AAV-100) were made over the L5 / L6 / S1 spinal segments (0.15 µl per injection) at a depth of 0.6 mm below the dorsal surface and separated by 1 mm. Animals were perfused four weeks later. Injection site quantification. To determine the number of transfected neurons within the injection site of the spinal cord, the inventors implemented the spot detection function in Imaris. Following the semi-automatic detection of transfected neurons within representative sections per animal, the inventors quantified the neurons by compiling the exported text file from Imaris. Perfusions. Animals were perfused at the end of the experiments. Animals were deeply anesthetized by an intraperitoneal injection of 0.2 m sodium pentobarbital (50 mg / ml). Animals were transcardially perfused with Phosphate buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Tissues were removed and post-fixed overnight in 4% paraformaldehyde before being transferred to PBS or cryoprotected in 30% sucrose in PBS.Immunohistochemistry. Immunohistochemistry was performed as described previously

[0097] ,

[0099] ,

[0100] . Perfused post-mortem tissue was cryoprotected in 30% sucrose in PBS for 48 hours before being embedded in cryomatrix (Tissue Tek O.C.T, Sakura Finetek Europe B.V.) and freezing. The inventors used two procedures to identify the segment of the spinal cord. First, the inventors identify the dorsal roots in the unsectioned spinal cord. Based on thelocation of the dorsal root entry zones, the inventors prepared well-defined blocks of spinalcord. Then, the Inventors confirm that the grey matter of the segments possesses the expectedlaminar organization and morphology. 30 µm thick transverse or horizontal sections of the spinal cord were cut on a cryostat (Leica), immediately mounted on glass slides and dried or in free floating wells containing PBS + 0.03% sodium azide. The sections were incubated with following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-cFos (1:500), chicken anti-vGlut1 (1:500), goat anti-Chat (1:100), rabbit anti-Chx10 (now known as Vsx2) (1:500, Synaptic Systems Gmbh). Fluorescent secondary antibodies were conjugated to Alexa 488, or Alexa 405 or Alexa 555, or Alexa 647 (ThermoFisher Scientific, USA). Nuclear stain: 4’,6’-diamidino-2-phenylindole dihydrochloride (DAPI; 2ng / ml; Molecular Probes). Sections were imaged digitally using a slide scanner (Olympus VS-120 Slide scanner) or confocal microscope (Zeiss LSM880 + Airy fast module with ZEN 2 Black software (Zeiss,Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Oberkochen, Germany). Images were digitally processed using ImageJ (ImageJ NIH) softwareor Imaris (Bitplane, v.9.8.2).Fluorescence in situ hybridization (FISH). The inventors performed in situ hybridization ofcell type markers and using RNAscope (Advanced Cell Diagnostics). Lists of putative marker genes were obtained from snRNA-seq data, as described below, and cross-referenced against a list of validated probes designed and provided by Advanced Cell Diagnostics, Inc. Probes were obtained for the following genes: Chat, catalog no. 408731; and Vsx2, catalog no.438341, Slc17a6, catalog no. 319171; Slc6a5, catalog no.409741. Then 12 µm cryosectionswere generated from fixed-frozen spinal cords as previously described

[0091] and performedFISH for each probe according to the manufacturer’s instructions, using the RNAscope HiPlex kit (cat no.324106). iDISCO+. Mice underwent a 90-min colorectal distension protocol (30 s inflate then 60 s deflaterepeatedly) [3] and were perfused

[0023] -

[0024] 30 min later with 0.1 M PBS followed by 4% PFA(in 0.1 M PBS). Spinal cords were dissected and post-fixed in 4% PFA (in 0.1 M PBS) at 4 ◦C overnight and placed in 0.1 M PBS containing 0.03% sodium azide. Immunolabelling of the samples was performed by first pretreating with methanol in 5 ml Eppendorf tubes by dehydrating with a methanol / H2O series at 1 h each at room temperature with shaking at 60 RPM: 20%, 40%, 60%, 80% and 100%. This procedure was followed by 1 h washing with 100% methanol before chilling the samples at 4°C. Samples were then incubated overnight with shaking in 66% dicholoromethane / 33% methanol at room temperature. The samples were washed twice in 100% methanol with shaking at room temperature and then bleached in chilledfresh 5% H2O2 in methanol overnight at 4°C. Samples were rehydrated with a methanol / H2Oseries: 80%, 60%, 40%, 20% and 0.1M PBS, each for 1h at room temperature under shaking. Samples were washed for 1 h × 2 at room temperature in PTx.2 buffer (0.1 M PBS with 0.2% Triton X-100) under shaking. This was followed by an incubation in 5 ml of permeabilization solution (400 ml PTx.2, 11.5 g glycine, 100 ml DMSO for a total stock volume of 500 ml) for 2 days at 37 C with shaking at 60 RPM. Samples were incubated in 5 ml of blocking solution (42 ml PTx.2, 3 ml of normal donkey serum, 5 ml of DMSO for a total stock volume of 50 ml) for 2 days at 37°C with shaking. The samples were incubated for 7 days at 37°C with shaking inprimary antibody solution consisting of PTwH (0.1 M PBS, 2 ml Tween-20, 10 mgl-1 heparin,5% dimethyl sulfoxide, 3% normal donkey serum), and cFos antibody (1:2000, Synaptic Sys- tems, 226003) for a total volume of 5 ml per sample. Samples were washed in PTwH for 24 hwith shaking and incubated for 7 days at 37°C with shaking in secondary antibody solutionconsisting of PTwH, 3% normal donkey serum and donkey anti-rabbit Alexa Fluor 647 (1:400, ThermoFisher Scientific) in a total volume of 5 ml per sample. Samples were washed in PTwHOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 for 24 h with shaking at room temperature. Clearing of the samples was performed by first dehydrating the samples in a methanol / H2O series as follows: 20%, 40%, 60%, 80% and 100% twice each for 1 h with shaking at room temperature followed by a 3 h incubation with shaking in 66% dichloromethane / 33% methanol at room temperature. Samples were incubated in 100% dichloromethane 15 min twice with shaking to wash residual methanol. Finally, samples were incubated in 100% dibenzyl ether without shaking for refractive index matching of the solution for at least 24 h prior to imaging.Tissue clearing (CLARITY)

[0027] ,

[0097] ,

[0101] . Samples were incubated in X-CLARITYhydrogel solution (Logos Biosystems Inc., South Korea) for 24 h at 4°C with gentle shaking. Samples were degassed and polymerized using the X-CLARITY Polymerisation System (Logos Biosystems Inc., South Korea), followed by washes in 0.001 M PBS for 5 minutes at room temperature. Samples were next placed in the X-CLARITY Tissue Clearing System(Logos Biosys- tems Inc., South Korea), set to 1.5 A, 100 RPM, 37°C, for 29 h. Clearingsolution was made in-house with 4% sodium dodecyl sulfate (SDS), 200mM boric acid with dH2O, pH adjusted to 8.5. Following this, samples were washed for at least 24 h at room temperature with gentle shaking in 0.1 M PBS solution containing 0.1% Triton X-100 to remove excess SDS. Finally, samples were incubated in 40 g of Histodenz dissolved in 30 ml of 0.02M PB, pH 7.5, 0.01% sodium azide (refractive index 1.465) for at least 24 h at room temperature with gentle shaking prior to imaging.3D imaging. Imaging of cleared tissue was performed using either a customized mesoSPIM

[0027] ,

[0102] and CLARITY-optimized lightsheet microscope (COLM)

[0027] . A custom-builtsample holder was used to secure the central nervous system in a chamber filled with RIMS.Samples were imaged using either a 1.25x or 2.5x objective at the mesoSPIM

[0027] ,

[0102] anda 4x or 10x objective at the COLM

[0027] with one or two light sheets illuminating the samplefrom both the left and right sides. The voxel resolution in the x-, y- and z directions was 5.3 µmx 5.3 µm x 5 µm for the 1.25x acquisition and 2.6 µm x 2.6 µm x 3 µm for the 2.5x acquisition. The voxel resolution of the COLM was 1.4 µm x 1.4 µm x 5 µm for the 4x and 0.59 µm x 0.59 µm x 3 µm for the 10x acquisition. Images were generated as 16-bit TIFF files and then stitched using Arivis Vision4D (Arivis AG, Munich, Germany).3D reconstructions and optical sections of raw images were generated using Imaris (bitplane, v.9.8.2) software.cFos quantifications

[0028] . For the cleared spinal cords, cFos positive neurons of clearedsamples were quantified using Arivis Vi- sion4D (Arivis). After defining a region of interestaround the grey matter, each sample was subjected to a custom-made pipeline. Morphology, denoising, and normalization filters were applied to enhance the signal of bright objects andOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 homogenized the background. Threshold-based segmentation of the cFos signal was applied within predefined 3D regions to quantify the total number of cFos positive cells. Image analysis parameters were kept constant among all samples. The number of cFos positive cells and their coordinates enabled to quantify the neuronal activity segment by segment. For the classic immunohistochemistry, the quantification was done on Imaris (bitplane, v.9.8.2) using the spot detection function. 3D reconstruction and quantification. The inventors used the “Add New Surfaces” tool in Imaris to select the channel of interest (i.e., neurons of interest), setting the “Surface detail” to 0.5 microns for a more detailed surface. The “Threshold (Absolute intensity)” was adjusted to capture the full shape of the neuron accurately. The inventors applied the “Number of Voxels Img=1” filter, selecting the appropriate threshold values to include the reconstructed neuron. After rendering the surface, we made aesthetic adjustments, choosing the “Transparent 3 - Glass” material and setting the color to RGB values of (1, 1, 1) to maintain transparency and highlight viral expression. For reconstructing synaptic-like appositions, the inventors created “Spots” based on the channel of interest using Imaris’s spot detection algorithm. The “Estimate XY diameter” was set between 1.5 and 2 micrometers, with the “Quality” filter applied to capture all synaptic-like appositions in the image. The “Points Style / Quality” was set to “Sphere” with a “Radius Scale” of 0.5 micrometers, using "Phong_basic" as the material for the synapses. To filter synapticlike appositions to neurons of interest, we used the “Find spots close tosurface” function with a 1 micrometer threshold and selected only the “Spots close to theSurfaces” to display the synaptic-like appositions on neurons of interest.Axon and synapse quantification

[0032] . To determine spatial enrichment of axon andsynapse density within the grey horn of the spinal cord the inventors implemented a customimage analysis pipeline that includes preprocessing, registration and combination ofhistological images from different animals. In brief, all preprocessing was implemented in Fiji,and all registration procedures in R, using the image analysis package ‘imageR’, and medical image registration package ‘RNiftyReg’. After dynamic registration, all data were summarized and final quantifications were completed using custom R scripts.Opto-tagging-based neuron-specific recordings and analysis

[0028] . Spinal cord injury atT4 and infusion of AAV5-Syn-flex-Chrimson was made in the lower thoracic spinal cord of Vsx2Cremice at least four weeks prior to terminal experiments. Mice were anesthetized with urethane and isoflurane. Two-shank, multi-site electrode arrays (NeuroNexus A2x32-6mm-35- 200-177) were lowered into the spinal cord to a depth of 1000 µm, with shanks arrangedlongitudinally at 350 µm from midline. Signals were recorded with a NeuroNexus Smart- boxOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 Pro using a common average reference and while applying 50 Hz notch and 450–5,000 Hz bandpass filters. Stimulation was controlled with a Multi-Channel Systems STG 4004 and MC Stimulus II software. ChrimsonR-expressing neurons were identified using optogenetic stimulation. Twenty pulse trains of 10-ms pulses were delivered at 10 Hz from a 635 nm laser (LaserGlow Technologies LRD-0635-PFR-00100-03). Laser light was delivered to the surfaceof the spinal cord through a fiber optic cable attached to 400 µm, 0.39 NA cannula with a 5 mmtip (Thorlabs). Optical power was set to 2.35 mW at the tip. Electrical stimulation (EES) consisted of 5 ms pulses delivered every at 1 Hz. EES was delivered with a micro fork probe (Inomed, 45 mm straight, item no. 522610) positioned along the midline just caudal to therecording array. Spike sorting was per- formed with SpyKING CIRCUS v.1.0.773. The median-based aver- age electrical stimulation artifacts for each channel were subtracted from therecordings prior to sorting. Due to the size and variabil- ity of the artifacts, periods containingresidual stimulation artifacts were not sorted (–0.5 to +1.5 ms and -0.5 to + 1 ms around stimulus onset for EES and laser stimulation onset, respectively). Sorting results weremanually curated using Phy (https: / / github.com / cortex- lab / phy). Single unit clusters wereselected for analysis based on their biphasic waveforms and template amplitudes above 50 µV, as well as strong refractory period dips in their spike autocorrelograms. Similar clusterswere merged according to the Phy manual clustering guide. ChrimsonR-expressing putativeSCTHORACIC::Vsx2neurons were identified based on their low-latency and low-jitter responses tolight pulses. Neurons responding to EES or tail pinch were identified by a one-sided Wilcoxonsigned-rank test to compare the instantaneous firing rate of units 100 ms before and 100 ms after (EES) or 2 s before and 2 s after (pinch) stimulus onset. For EES, a post-stimulus onset firing rate increase of P value less than 0.001 was used, while for pinch a P value of 0.05 was used due to the necessarily lower number of trials and larger calculation window (minimum 6 trials for pinch, 60 for EES). Statistics, power calculations, group sizes and reproducibility. All data are reported as mean values and individual data points. No statistical methods were used to predeterminesample sizes, but the used sample sizes are similar to those reported in previous publications

[0097] . Hemodynamic assays were replicated three to five times, depending on the experiment, and averaged per animal. Statistics were then performed over the mean of animals. All statistical analysis was performed in R using the base package ‘stats’, with primaryimplementation through the ‘tidyverse’ and ‘broom’ packages. Tests used included one or two-tailed paired or independent samples Student’s t-tests, one-way ANOVA forneuromorphological evaluations with more than two groups, and one- or two-way repeated-measures ANOVA for hemodynamic assessments, when data were distributed normally,Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 tested using a Shapiro–Wilk test. Post hoc Tukey tests were applied when appropriate. For regressions, mixed model linear regression was used in cases of multiple observations, or elsestandard linear modelling. In cases where group size was equal to or less than three nullhypothesis testing was not completed. The significance level was set as P < 0.05. Exclusions of data are noted in the relevant methods sections. Unless stated otherwise, experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment. Single-nucleus RNA sequencing. Single-nucleus dissociation of the mouse lower thoracicand lumbosacral spinal segments was performed according to established procedures

[0042] ,

[0044] . Following euthanasia by isoflurane inhalation and cervical dislocation, the lumbar spinal cord site was immediately dissected and frozen on dry ice. Spinal cords were doused in 500 µl sucrose buffer (0.32 M sucrose, 10 mM HEPES [pH 8.0], 5 mM CaCl2, 3 mM Mg acetate, 0.1 mM EDTA, 1 mM DTT) and 0.1% Triton X-100 with the Kontes Dounce Tissue Grinder.2ml of sucrose buffer was then added and filtered through a 40-µm cell strainer. The lysate wascentrifuged at 3200 g for 10 min at 4°C. The supernatant was then decanted, and 3 ml ofsucrose buffer was added to the pellet for 1 min. The pellet was homogenized using an Ultra-Turrax and 12.5 ml of density buffer (1 M sucrose, 10 mM HEPES [pH 8.0], 3 mM Mg acetate, 1 mM DTT) was added below the nuclei layer. The tube was centrifuged at 3200 g at 4°C and supernatant poured off. Nuclei on the bottom half of the tube wall were collected with 100 µlPBS with 0.04% BSA and 0.2 U / µl RNase inhibitor. Finally, nuclei were resuspended througha 30 µm strainer, and adjusted to 1000 nuclei / µl. Library preparation. snRNA-seq library preparation was carried out using the 10x GenomicsChromium Single Cell Kit Version 3.1. The nuclei suspension was added to the Chromium RTmix to achieve loading numbers of 2000-5000. For downstream cDNA synthesis (13 PCR cycles), library preparation and sequencing, the manufacturer’s instructions were followed.Read alignment. Reads were aligned to the most recent Ensembl release (GRCm38.93) usingCell Ranger, and obtained a matrix of unique molecular identifier (UMI) counts. Seurat

[0070] was used to calculate quality control metrics for each cell barcode, including the number of genes detected, number of UMIs, and proportion of reads aligned to mitochondrial genes. Low- quality cells were filtered by removing cells expressing less than 200 genes or with more than 5% mitochondrial reads. Genes expressed in less than three cells were likewise removed.Clustering and integration. Prior to clustering analysis, the inventors first performed batcheffect correction and data integration across the two different experimental conditions asOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025previously described

[0070] . Gene expression data was normalized using regularized negativebinomial models

[0103] , then integrated across batches using the data integration workflowwithin Seurat. The normalized and integrated gene expression matrices were then subjected to clustering to identify cell types in the integrated dataset, again using the default Seurat workflow. Cell types were manually annotated on the basis of marker gene expression, guidedby previous studies of the mouse spinal cord

[0042] ,

[0104] -

[0106] . Local and projecting neuronalsubpopulations were annotated on the basis of Nfib and Zfhx3 expression, respectively

[0031] .Following the inventor’s projection-specific snRNA-seq experiment in uninjured mice, eachsubsequent experiment was reintegrated with this dataset prior to subpopulation annotation.This enabled the identification of the same

[0031] neuronal subpopulations across the threedistinct experiments

[0070] .Cell type prioritization with Augur. To identify neuronal sub-populations perturbed duringnatural repair, machine-learning method Augur

[0042] -

[0043] was implemented. Augur was runwith default parameters for all comparisons. To evaluate the robustness of cell type prioritizations to the resolution at which neuronal subtypes were defined in the snRNA-seqdata, Augur was applied at various clustering resolutions, and the resulting cell typeprioritizations were visualized both on a hierarchical clustering tree

[0107] of neuron subtypesand as a progression of UMAPs. The key assumption underlying Augur is that cell types undergoing a profound response to a perturbation should become more separable, within thehighly multi-dimensional space of gene expression, than less affected cell types. Briefly, Augurwithholds a proportion of sample labels, then trains a random forest classifier to predict the condition from which each cell was obtained. The accuracy with which this prediction can be made from single-cell gene expression measurements is then evaluated in cross-validation,and quantified using the area under the receiver operating characteristic curve (AUC) (Table2). Clinical studies design and objectives. All experiments were carried out as part of a clinicalsafety (primary objective) and preliminary efficacy (secondary objectives) trial, in particular the:HemON clinical trial (NCT05111093, CHUV, Lausanne, Switzerland) mentioned above. Thetrial received approval by the local ethical committees and national competent authorities. All participants signed a written informed consent before their participation. All participants had the option to indicate consent for the publication of identifiable images or videos. All surgical and experimental procedures were performed at the Neurosurgery Department of the Lausanne University Hospital (CHUV). The study involved eligibility and baseline assessmentsbefore surgery, the surgical implantation of the respective investigational devices, a post-operative period during which EES protocols were configured, and long-term follow-up periods.Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 Study participants. The studies involved females and males who had suffered a traumatic spinal cord injury. Demographic data and neurological status, evaluated according to theInternational Standards for Neurological Classification of Spinal Cord Injury

[0108] , can befound Table 3. Neurosurgical intervention. The participant is put under general anesthesia and is placed in a prone position. Preoperative surgical planning informs the neurosurgeon about the vertebral entry level and predicted optimal position. Based on this knowledge, lateral and anteroposterior fluoroscopy x-rays are performed intraoperatively to guide the location of the laminotomies. A midline skin incision of approximately 5 cm on the back is performed, the fascia opened and the muscles retracted bilaterally. Excision of the midline ligamentous structures and alaminotomy at the desired entry level enables the insertion of a paddle array at the spinalthoracic level. The paddle lead(s) (e.g. the ARCIM Thoracic Lead, ONWARD Medical N.V,Eindhoven, Netherlands, Fig.20) are inserted and placed over the midline of the exposed dura-mater and advanced rostrally to the target position guided by repeated fluoroscopies. Electrophysiological recordings are conducted using standard neuromonitoring systems (IOMAX, Cadwell Industries, Kennewick, USA or ISIS Xpress, Inomed Medizintechnik, Emmendingen, Germany). Single-pulses of EES (0.5 Hz) are delivered at increasing amplitude to elicit muscle responses that are recorded from subdermal (Neuroline Twisted Pair Subdermal, 12 x 0.4 mm, Ambu A / S, Ballerup, Denmark) or intramuscular needle electrodes (Inomed SDN electrodes, 40 x 0.45 mm, Inomed Medizintechnik, Emmendingen, Germany) to correct for lateral and rostrocaudal positioning. When the paddles are deviating from a straight midline position, small additional laminotomies are made to remove bony protrusions and guide the paddle to a midline placement. Once the final position is achieved, the leads are anchored to the muscular fascia. In HemON and HemON-NL trials, the back opening is temporarily closed and the participant is put in lateral decubitus. Subsequently, the back incision is reopened and an abdominal incision of about 5 cm is made per IPG and asubcutaneous pocket is created. The IPGs (e.g., the ARCIM IPG, ONWARD Medical N.V.,Eindhoven, Netherlands, Fig.20) are implanted in the sub-cutaneous pockets and all incisionsare finally closed. Stimulation optimization. Spatial mapping was guided by the pre-clinical mechanismsdescribed previously

[0020] , and from prior clinical mappings

[0086] , and was conducted in threesteps: 1) intra-operative mapping to identify which rows of electrodes target the hemodynamichotspots

[0020] and elicit the largest pressor response in the thoracic spinal cord (T10, T11,T12), 2) post-operative imaging and spinal reconstructions were used to estimate the electrodes that maximize recruitment of the hemodynamic hotspots, 3) a single 2-hour, post-Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 operative mapping session was done to test each row of electrodes on the lead, and pick the three configurations with the largest pressor responses. These configurations were tested in both multipolar and monopolar settings, and were validated by personalized simulations toensure that the hemodynamic hotspots were optimally targeted. Stimulation frequency wasdefined empirically at 120 Hz for the spatial mapping

[0020] ,

[0086] . The pulse-width was 300 µs.The amplitude was set by incrementally increasing the current per configuration until thesystolic pressure increased by 20 mmHg, the diastolic pressure increased by 10 mmHg, or thepatient did not report any discomfort such as muscle contractions, or sensation such astingling. These mappings were done in a seated position to mimic relevant, daily life orthostaticchallenges.Hemodynamic monitoring. Beat- to-beat blood pressure and heart rate were obtained viafinger plethysmography (Finometer, Finapres Medical Systems; Amsterdam, Netherlands).Beat-by-beat blood pressure was calibrated to brachial artery blood pressure collected usingan arm cuff embedded and synchronized with the Finometer

[0109] -

[0112] . Brachial arterialpressure was sampled at 200 Hz, while the systolic, diastolic and mean arterial pressure were extracted from the calibrated arterial pressure at 1 Hz. The heart rate was also sampled at 1 Hz. Raw data and automatically extracted hemodynamic parameters were saved and exported from the Finometer. Orthostatic challenge with tilt table test. Participants were transferred to a supine position on a table capable of head-up tilt. Restraint straps were applied to secure the patient below the knees, across the thighs, and the trunk, with the feet stabilized. Resting supine blood pressure was recorded continuously for approximately 5-10 minutes to establish baseline values. Then, the patient was tilted upright up to a maximum of 70 degrees while recording hemodynamic values and symptoms of orthostatic tolerance. The time to reach the desired tilt angle from supine was achieved in less than 45 seconds. Participants were tilted until reaching their tolerance threshold or for a maximum duration of 10 minutes. They were asked not to talk during the test except to inform and grade symptoms. The participant was asked to report any symptoms every 1-3 minutes. The participant was asked to rank their symptoms between 1- 10, 1 being no symptoms at all, and 10 being feelings of dizziness, lightheadedness

[0080] , or nausea

[0080] ,

[0113] . The patient was instructed to notify the research team if they needed to be returned to the supine position. Postoperative blood pressure data. During a tilt test (See Orthostatic challenge with tilt table test), changes in blood pressure were recorded without stimulation or in response to different types of stimulation (continuous or closed-loop stimulation) using the Finometer (seeOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 Post-operative hemodynamic monitoring). Change in blood pressure or heart rate was defined as the difference in the average of a 60-second window before the start of the tilt and a 20-second window at 3 minutes of the challenge. If the participant could not tolerate at least 3 minutes of the test due to low blood pressure or other symptoms, an average of a 20-second window before the end of the tilt was used. All measurements in seated position were measured with stimulation on for 3-5 minutes. Change in blood pressure, or heart rate, was defined as the difference in the average of a 20-second window before the start of EES and the average of a 20-second window at 3 minutes, prior to stopping stimulation. All signals were smoothed over a 10-second window for illustration. The same processing was used for post- operative, day 1 quantification. In the present study, the inventors report on blood pressure data on the 2 study participants implanted with the full ARCIMimplantable system. Purpose-built IPG and communication ecosystem for restoring hemodynamic stability. The purpose-built ARCIMIPG developed by ONWARD Medical (Fig.20) is a novel 16-channel IPG developed to deliver targeted epidural electrical stimulation. It controls and delivers current-controlled stimulation pulses according to predefined stimulation programs or through commands received in real-time to monopolar or multipolar electrode configurations on 16 channels. The IPG consists of a hermetically sealed, biocompatible can that surrounds the electrical components and a rechargeable battery that enables its function. The IPG is composed of two main components: the header containing the connector block that enables connection with 28-contact lead connectors as well as 2 coils for charging and communication, and the can with a rechargeable battery and electronics circuits. The IPG was developed according to all applicable standards for medical device development. Conventionalbiomedical technologies were used to fabricate the IPG and extensive bench and in vivotesting was performed to verify its performance. The IPG is implanted subcutaneously at the abdominal level and communicates wirelessly with the ARCIMHub with Near Field Magnetic Induction (NMFI). This wearable device is worn on a belt over or in proximity to the IPG location and is responsible for wirelessly charging the IPG’s battery and for programming the IPG with stimulation settings received from several user interfaces. The communication between the hub and IPG provides real-time control of stimulation parameters (as fast as 25ms between command and stimulation execution), allowing integration with a fast closed-loop neuromodulation system. The ARCIMHub contains a Bluetooth Low Energy (BLE) chip to enable fast, reliable wireless communication with external programmers such as the ARCIMClinician Programmer (Fig. 20), an Android app designed for clinicians to configure and testthe implanted system and personalized stimulation programs. When a stimulation program isOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 deemed safe for personal use, the Clinician Programmer can be used to make this stimulation program available to the patient. The patients, or their caregivers, can control the systemthrough the ARCIM Personal Programmer (Fig. 20). This Android Watch application allowsusers to select, start, and stop stimulation programs, as well as modulate stimulation amplitudes within predefined safety limits ad-hoc. Device errors, paddle lead impedances, and daily stimulation utilization were extracted from usage logs across all devices. Furthermore, the Clinician Programmer includes an application programming interface (the ARCIMAPI) thatenables other programming software to control the stimulation, e.g. for closed-loop control ofthe stimulation. All devices and software are adherent to the applicable standards and theirperformance was extensively tested. The entire system, including the IPG, received the equivalent of an investigational device exemption from the competent, Swiss authorities. Purpose-built paddle lead. The ARCIMThoracic Lead (Fig. 20) developed by ONWARDMedical is a new 16-electrode paddle lead that is designed for selective recruitment of thedorsal root entry zones of the low-thoracic spinal cord with optimal coverage of the T10-T12 spinal levels. More detailed information about this paddle lead can be found in other publications

[0086] . Spinal Cord Injury Community Survey (SCICS). Individuals with SCI (n = 1,479) across Canada were recruited using a national consumer awareness campaign and provided writteninformed consent

[0076] -

[0077] . The survey consisted of a series of variables identified byhealthcare and service providers, researchers, as well as individuals with SCI, including demographics, secondary health complications and comorbidities, SCI-related needs, healthcare utilization, community participation, quality of life, as well as overall health ratings

[0084] -

[0085] . Participants were asked how often they had experienced symptoms related to autonomic dysreflexia in the past 12 months and responses were ranked on a 6-point ordinal severity scale ranging from zero (i.e., "Never") to 5 (i.e., "Every day"). Participants were alsoasked if they received or sought out treatment in relation to these symptoms on a two- pointscale (i.e., “Yes” or “No”), along with the degree to which it limited activities from zero (i.e., "Never") to 5 (i.e., "Every day"). Participants were also asked if they had experienced specific problems, such as heart disease, in the past 12 months. Participants’ American Spinal Injury Association Impairment Scale (AIS) were estimated using responses to questions about lesionlevel and sensorimotor / mobility capabilities

[0084] . A binary approach was used for theevaluation of outcome variables including the level of injury (i.e., cervical SCI vs. non-cervical SCI), the severity of injury (i.e., complete vs. incomplete), presence of autonomic dysreflexia (i.e., yes vs. no), and autonomic dysreflexia symptoms (i.e., yes vs. no). For variables rankedOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 on a 6-point ordinal scale, lower scores (i.e., 0-3) were categorized as “No” and higher scores(i.e., 4-5) were categorized as “Yes”

[0114] .Autonomic Dysfunction following SCI questionnaire (ADF-SCI). The ADFSCI is a 24-item questionnaire divided into four sections: demographics, medication, autonomic dysreflexia, and orthostatic hypotension. The autonomic dysreflexia section consists of 7 items. Each item employs a 5-point scale to measure the frequency and severity of symptoms related to autonomic dysreflexia, including headaches, goosebumps, heart palpitations, sweating and anxiety, across different situational contexts. Participants were categorized as experiencing symptoms if the item score was higher than 2 or not experiencing symptoms otherwise.

[0002] Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Table 1 – Supplementary Data 1Referring to Figs.4-5 Referring to Figs.16-17 Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 Referring to Figs.16 and 17h-i

[0003] Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Table 2 – Supplementary Data 2 Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025Table 3 – Supplementary Data 3

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[0005] Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 -85 -References 10 Neuromodulation / neurostimulation system12 Control unit14 Stimulation unit16 Sensor unit18 Lead20 Signal processing unitT12 Thoracic spinal segmental level 12

Claims

Our file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 Claims1. A neuromodulation / neurostimulation system (10) for stimulating sympathetic circuitryresponsible for blood pressure control in a mammal with autonomic dysreflexia, said system (10) comprising: -at least one control unit (12) configured and arranged to provide stimulation data, and- at least one stimulation unit (14), operatively connected to the at least one control unit(12), said at least one stimulation unit (14) being configured and arranged to provide electrical stimulation to the spinal cord of said mammal, wherein the at least one stimulation unit (14) includes an implantable lead (18), and wherein neuromodulation / neurostimulation system (10) is configured and arranged to provide neuromodulation to said mammal to activate the sympathetic circuitry responsible for blood pressure control that mitigates autonomic dysreflexia.

2. The neuromodulation / neurostimulation system (10) according to claim 1,characterized in that the implantable lead (18) is configured and arranged to providestimulation to the spinal cord of said mammal at thoracic level, especially around spinal cord level T10-T12.

3. The neuromodulation / neurostimulation system (10) according to claim 1 or claim 2,characterized in that it further comprises at least one implantable pulse generator (IPG) (15).

4. The neuromodulation / neurostimulation system (10) according to any one of thepreceding claims, characterized in that it further comprises:- at least one sensor unit (16), preferably a real-time monitoring sensor unit (16), and- a signal processing unit (20), operatively connected to the at least one control unit(12) and the at least one sensor unit (16),wherein the at least one sensor unit (16) is configured and arranged to: -measure and / or monitor a blood pressure value and / or a spinal cord perfusionpressure value of said mammal, andOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 -transmit the measured and / or monitored blood pressure value and / or spinalcord perfusion pressure value to the signal processing unit (20), and wherein the signal processing unit (20) is configured and arranged to: -determine if the measured and / or monitored blood pressure value deviatesfrom a predetermined blood pressure target value and / or blood pressure target range, and / or -determine if the measured and / or monitored spinal cord perfusion pressurevalue deviates from a predetermined spinal cord perfusion pressure target value and / or spinal cord perfusion pressure target range, and in case it is determined that the measured and / or monitored blood pressure value deviates from the predetermined blood pressure target value and / or blood pressure target range and / or the measured and / or monitored spinal cord perfusionpressure value deviates from the predetermined spinal cord perfusion pressure target value and / or spinal cord perfusion pressure target range, transmit an output signal to the at least one control unit (12) for stimulation data adjustment.

5. The neuromodulation / neurostimulation system (10) according to claim 4,characterized in that the at least one sensor unit (16) includes at least one biocompatibleimplantable sensor.

6. The neuromodulation / neurostimulation system (10) according to claim 4 or 5,characterized in that the at least one sensor unit (16) includes at least one non-implantablesensor.

7. The neuromodulation / neurostimulation system (10) according to any one of thepreceding claims, characterized in that the at least one control unit (12) is configured andarranged to control the at least one stimulation unit (14) to provide electrical stimulation over a predefined period of time.

8. The neuromodulation / neurostimulation system (10) according to claim 7,characterized in that the predefined period of time is between 0 and 30 minutes.

9. The neuromodulation / neurostimulation system (10) according to any one of thepreceding claims, characterized in that the at least one control unit (12) is configured andOur file: 37465.EPF.P110PCOfficial file: tba March 10, 2025 arranged to control the at least one stimulation unit (14) to provide electrical stimulation on a daily basis, especially by stimulating the neuroprosthetic baroreflex.

10. The neuromodulation / neurostimulation system (10) according to any one of thepreceding claims characterized in that the implantable lead (18) includes one or moreimplantable electrode arrays (18.1).

11. The neuromodulation / neurostimulation system (10) according to any one of thepreceding claims characterized in that the stimulation data comprise at least frequency,amplitude and pulse width, wherein the frequency is between 10Hz and10k Hz, the amplitude is between 0 and 1A or 0 and 15 V and the pulse width is between 1 and 500^s.

12. The neuromodulation / neurostimulation system (10) according to any one of thepreceding claims characterized in that the at least one control unit (12) comprises anoscillation control module, wherein the oscillation control module is configured and arrangedto provide an input between 0.01Hz and 0.2Hz, preferably of 0.1Hz, low frequency oscillationin the amplitude and / or frequency.

13. The neuromodulation / neurostimulation system (10) according to any one of thepreceding claims characterized in that the at least one stimulation unit (14) is configured andarranged to provide at least one burst train stimulation pulse.

14. The neuromodulation / neurostimulation system (10) according to claim 13characterized in that the at least one stimulation unit (14) is configured and arranged toprovide at least one burst of several pulses, preferably of 2 to 5 pulses.

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