Method and device for treating motor dysfunction after spinal cord injury by targeting lateral hypothalamus

Abstract

The present invention provides a method and device for treating motor dysfunction after spinal cord injury by targeting the lateral hypothalamus (LHA). Specifically, in the present invention, by performing electrical stimulation on glutamatergic neurons in the dorsal part of the caudal LHA, the LHA-PnO-spinal cord circuit is activated to modulate limb motor function in patients with spinal cord injury. The method of the present invention has small side effects and can serve as a potential approach for restoring voluntary motor function in patients with spinal cord injury.

Description

A method and device for targeted treatment of motor dysfunction after spinal cord injury using the lateral hypothalamus. Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to a method and apparatus for treating motor dysfunction following spinal cord injury by targeting the lateral hypothalamus. Background Technology

[0002] Spinal cord injury or stroke typically results in incomplete damage to the central nervous system, characterized by incomplete damage to the site of injury while maintaining the integrity of structures outside the injury site. However, incomplete damage can lead to the disruption of upstream and downstream signals in the central nervous system, silencing of neural circuits, and ultimately, complete paralysis or disability.

[0003] After a spinal cord injury or stroke, neural remodeling occurs in the relevant brain regions, which can partially or completely restore some motor functions.

[0004] Research has found that neuromodulation techniques can activate dormant neural circuits after injury, helping patients recover motor function. Specifically, epidural stimulation (EES) can activate spinal cord sensory neurons, inducing activity of the central motor pattern generator in the spinal cord and restoring hind limb function; or deep brain stimulation (DBS) can stimulate motor-related brain regions, activating dormant neural circuits, restoring upstream and downstream neural connections at the site of injury, and rebuilding motor function.

[0005] Therapies using epidural stimulation (EES) or closed-loop epidural stimulation to restore hindlimb function in patients with motor loss have been clinically tested in multiple centers worldwide in recent years. However, cases of using deep brain stimulation to treat paralyzed or stroke patients are relatively few. This is mainly due to unclear target areas, significant side effects from parameter modulation, and the lack of systematic stimulation systems specifically designed for this indication. Following spinal cord injury or stroke, extensive remodeling occurs in the central nervous system, particularly the brain.

[0006] Although the spinal cord contains all the circuitry required for movement, patients with spinal cord injuries are unable to walk due to the lack of commands from the brain. Motor recovery can be achieved in part through rehabilitation training and neuromodulation therapy, aimed at promoting the activation of descending motor commands from the brain to the spinal cord after spinal cord injury. The lateral hypothalamus is an important subcortical motor region that has been shown to be involved in various motor behaviors in animals, and electrical stimulation of the lateral hypothalamus can manipulate the speed and pattern of movement in an intensity-dependent manner. Therefore, the lateral hypothalamus has the potential to serve as a deep brain stimulation therapeutic target for treating motor dysfunction.

[0007] Deep brain stimulation (DBS) has been widely used clinically to treat Parkinson's disease, dystonia, epilepsy, and other conditions. Recent research reports that targeting the wedge-shaped nucleus or pontine peduncle in the midbrain motor cortex may help reactivate residual neural circuits and restore motor function in patients with spinal cord injuries; however, the specific target nuclei remain controversial. Different stimulation modalities in DBS may elicit different therapeutic effects, and incorrect stimulation modalities may cause adverse reactions. For example, the subthalamic nucleus, a commonly used target for Parkinson's disease, may cause increased tremors or spasms when stimulated below 50 Hz, while above 90-170 Hz, patients may experience heaviness in the head or general discomfort. Furthermore, long-term continuous DBS stimulation may accelerate the progression of other symptoms while treating the underlying condition. For instance, some reports suggest that long-term continuous DBS may accelerate cognitive impairment while treating motor dysfunction in Parkinson's patients.

[0008] Given the currently unclear deep brain stimulation targets after spinal cord injury, the effective stimulation modes for deep brain stimulation therapy to treat motor dysfunction after spinal cord injury, and the potential adverse reactions caused by deep brain stimulation therapy, there is an urgent need in this field to explore key brain regions in the remodeling process and to use optimized deep brain stimulation systems to regulate targets, thereby bringing new therapies for the recovery of motor function in patients with spinal cord injury or stroke. Summary of the Invention

[0009] This invention discloses a deep brain stimulation method and device for treating motor dysfunction after spinal cord injury by targeting the lateral hypothalamus. Combining pathology, neuroanatomy, and neuroelectrophysiology, therapeutic targets for hindlimb function recovery after paralysis are identified through model construction, viral tracing, 3D reconstruction, and neuromodulation techniques. Then, key target EEG decoding, electromyography analysis, neural modulation, and machine learning are performed to construct optimized neural modulation parameters. Finally, a closed-loop deep brain stimulation system that decodes motor intention is used to reconstruct hindlimb motor function in paralyzed cases.

[0010] The method for promoting the recovery of motor function in patients with traumatic spinal cord injury provided by this invention can effectively reconstruct the hindlimb motor function of patients with spinal cord injury. The optimized stimulation targets and parameters will reduce the side effects of DBS therapy. The introduction of brain-computer interface into the method can prolong the treatment effect through real-time closed-loop stimulation mode.

[0011] In a first aspect of the invention, a device for treating traumatic spinal cord injury is provided, comprising:

[0012] The stimulation module is configured to electrically stimulate the lateral hypothalamus (LHA) or pontine orifice (PnO) of a subject with traumatic spinal cord injury, thereby activating the LHA-PnO-spinal cord circuit or the PnO-spinal cord circuit and promoting the recovery of the subject's limb motor function.

[0013] In another preferred embodiment, the stimulation module is further configured to activate spinal interneurons expressing Zfhx3.

[0014] In another preferred embodiment, the electrical stimulation may be replaced by light stimulation or chemical stimulation.

[0015] In another preferred embodiment, the electrical stimulation is applied via deep brain stimulation.

[0016] In another preferred embodiment, the electrical stimulation comprises applying a current with a frequency of 1-200 Hz.

[0017] In another preferred embodiment, the frequency of the current applied by the electrical stimulation is 5-150 Hz; more preferably 10-100 Hz; more preferably 10-50 Hz; and even more preferably 20-40 Hz.

[0018] In another preferred embodiment, the electrical stimulation time is 1-200 s.

[0019] In another preferred embodiment, the electrical stimulation time is 10-150 s; more preferably, the electrical stimulation time is 20-120 s; more preferably, the electrical stimulation time is 50-100 s; more preferably, the electrical stimulation time is 80-100 s; and even more preferably, the electrical stimulation time is 90-100 s.

[0020] In another preferred embodiment, the electrical stimulation is alternating between positive and negative to maintain charge balance, and the frequency is 20-40Hz.

[0021] In another preferred embodiment, the electrical stimulation is alternating between positive and negative, with a frequency of 20-40Hz and a stimulation time of 80-120s.

[0022] In another preferred embodiment, the higher the frequency and / or the longer the duration of the applied current, the greater the amplitude of the regulated movement.

[0023] In another preferred embodiment, the area where the electrical stimulation is applied is any location of the LHA; more preferably, it is the dorsal region of the LHA tail; even more preferably, it is a subregion of the dorsal region of the LHA tail rich in glutamatergic neurons.

[0024] In another preferred embodiment, the device further includes:

[0025] The data acquisition module is configured to: acquire electroencephalogram (EEG) signals from the M1 and / or LHA of the cerebral cortex; and output the voluntary movement signal when the EEG signal is identified as a voluntary movement signal.

[0026] The information processing module is configured to: denoise and decode (normalize) the autonomous motion signal, and output the stimulus value V;

[0027] An evaluation module is configured to: instruct the stimulation module to initiate electrical stimulation when the stimulation value V ≥ stimulation threshold V0; and instruct the stimulation module not to initiate electrical stimulation when the stimulation value V < stimulation threshold V0.

[0028] The information processing module receives signals from the data acquisition module, and simultaneously inputs the processed data into the evaluation module.

[0029] In another preferred embodiment, the data acquisition module is further configured to include: decoding discrete neuron signals with a sampling rate > 10 kHz; or continuous field potential signals with a sampling rate of 0 Hz to 1000 Hz.

[0030] In another preferred embodiment, the stimulation threshold is an empirical value.

[0031] In another preferred embodiment, the stimulation threshold is obtained through an unsupervised algorithm. When the stimulation value is greater than the average value of 0.7, the stimulation is turned on; when the stimulation value is less than the average value of 0.3, the stimulation is turned off.

[0032] In another preferred embodiment, the device further includes, prior to application:

[0033] A pre-screening module, configured to determine whether the spinal cord of a subject with traumatic spinal cord injury is completely severed:

[0034] If ≥5% of the spinal cord gray matter is preserved, preferably ≥10%, and even more preferably ≥20% (30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%), then the spinal cord is not completely severed.

[0035] If the amount of gray matter preserved in the spinal cord is <5%, preferably <4%, and even better <3% (2%, 1%, 0%), then the spinal cord is completely severed.

[0036] When the spinal cord is not completely severed, the potential for recovery of motor function is high in subjects with traumatic spinal cord injury.

[0037] When the spinal cord is completely severed, the potential for recovery of motor function in subjects with traumatic spinal cord injury is low.

[0038] In another preferred embodiment, the traumatic spinal cord injury refers to a condition in which less than 100% of the gray matter of the subject's spinal cord is preserved.

[0039] In another preferred embodiment, the traumatic spinal cord injury refers to the subject's inability to move voluntarily below the thoracic spine.

[0040] In another preferred embodiment, the traumatic spinal cord injury refers to the subject's inability to move voluntarily below the cervical spine.

[0041] In another preferred embodiment, the traumatic spinal cord injury refers to the subject's inability to move his or her lower limbs voluntarily.

[0042] In another preferred embodiment, the traumatic spinal cord injury is caused by a car accident or stroke.

[0043] In another preferred embodiment, the traumatic spinal cord injury is caused by a disease.

[0044] In another preferred embodiment, the traumatic spinal cord injury refers to the loss of partial voluntary movement ability of the subject's limbs or trunk.

[0045] In another preferred embodiment, the traumatic spinal cord injury refers to the subject's complete loss of voluntary motor function in the limbs or trunk (e.g., a vegetative state).

[0046] In another preferred embodiment, the activation of LHA glutamatergic neurons can be promoted to facilitate motor function recovery by optogenetic, chemogenetic, or pharmacological means instead of electrical stimulation.

[0047] In a second aspect of the invention, a method for assessing the recovery potential of patients with traumatic spinal cord injury is provided, comprising the following steps:

[0048] (1) Determine whether the spinal cord of a patient with traumatic spinal cord injury is completely severed;

[0049] If ≥5% of the spinal cord gray matter is preserved, preferably ≥10%, and even more preferably ≥20% (30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%), then the spinal cord is not completely severed.

[0050] If the amount of gray matter preserved in the spinal cord is <5%, preferably <4%, and even better <3% (2%, 1%, 0%), then the spinal cord is completely severed.

[0051] (2) When the spinal cord is not completely severed, the recovery potential of patients with traumatic spinal cord injury is high; when the spinal cord is completely severed, the recovery potential of patients with traumatic spinal cord injury is low.

[0052] In another preferred embodiment, the more gray matter is preserved in the spinal cord, the higher the recovery potential of patients with traumatic spinal cord injury; the less gray matter is preserved, the lower the recovery potential of patients with traumatic spinal cord injury.

[0053] In a third aspect of the invention, a method for promoting the recovery of motor function in patients with traumatic spinal cord injury is provided, comprising:

[0054] Electrical stimulation of the lateral hypothalamus (LHA) or pontine ostomy (PnO) in subjects with traumatic spinal cord injury activates the LHA-pontine ostomy-spinal cord circuit or the pontine ostomy-spinal cord circuit.

[0055] In another preferred embodiment, the area where the electrical stimulation is applied is any location of the LHA; more preferably, it is the dorsal region of the LHA tail; even more preferably, it is a subregion of the dorsal region of the LHA tail rich in glutamatergic neurons.

[0056] In another preferred embodiment, the electrical stimulation comprises applying a current with a frequency of 10-100 Hz.

[0057] In another preferred embodiment, the electrical stimulation time is 1-100 seconds.

[0058] In another preferred embodiment, the frequency of the current applied by the electrical stimulation is 10-50 Hz; more preferably 10-40 Hz; and even more preferably 15-30 Hz.

[0059] In another preferred embodiment, the electrical stimulation time is 1-50 s; more preferably, the electrical stimulation time is 1-20 s; even more preferably, the electrical stimulation time is 1-10 s.

[0060] In another preferred embodiment, the method is also used to prevent muscle atrophy.

[0061] In another preferred embodiment, the method promotes the recovery of limb motor function in the subject by activating spinal interneurons expressing Zfhx3.

[0062] In another preferred embodiment, the method includes training techniques to prevent muscle atrophy.

[0063] In another preferred embodiment, the electrical stimulation is applied at regular intervals.

[0064] In another preferred embodiment, the electrical stimulation is applied according to the needs of the subject.

[0065] In another preferred embodiment, the electrical stimulation is applied at intervals of 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, or 24h.

[0066] In another preferred embodiment, the region of electrical stimulation is a subregion of the LHA dorsal region rich in glutamatergic neurons.

[0067] In another preferred embodiment, the method further includes a brain-computer interface:

[0068] (a) Acquire EEG signals from the M1 and / or LHA of the cerebral cortex; when the EEG signals are identified as voluntary movement signals, output the voluntary movement signals;

[0069] (b) The autonomous motion signal is de-noiseed, decoded (normalized), and the stimulus value V is output;

[0070] (c) When the stimulation value V ≥ stimulation threshold V0, the instruction stimulation module starts electrical stimulation; when the stimulation value V < stimulation threshold V0, the instruction stimulation module does not start electrical stimulation.

[0071] In another preferred embodiment, the subject is a vertebrate, such as a human, mouse, rabbit, monkey, sheep, cow, or horse.

[0072] In another preferred embodiment, the subject is a patient with traumatic spinal cord injury.

[0073] In another preferred embodiment, the subject is a patient with acute or chronic traumatic spinal cord injury.

[0074] In another preferred embodiment, the subject is a paralyzed patient.

[0075] In another preferred embodiment, the subject is a person in a vegetative state.

[0076] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0077] Figure 1 shows the recovery of motor function in the T10 segment spinal cord hemisection model at week 8 post-injury. Figure 1a shows the spontaneous recovery model of motor function constructed using the T10 segment spinal cord hemisection. Figure 1b shows the plateau in motor function recovery at week 8 post-injury.

[0078] Figure 2 shows the brain regions associated with leg muscle control in the three-dimensional reconstruction of viral tracing, with a densely labeled region found in the lateral hypothalamus (LHA). Figure 2a illustrates the PRV viral tracing process. Figures 2b and 2c show the distribution of labeled neurons in the hypothalamus. Figures 2d and 2e show the distribution of labeled neurons in the lateral hypothalamus.

[0079] Figure 3 shows the leg responses induced by electrical stimulation in different brain regions during the chronic phase, with the lateral hypothalamus (LHA) exhibiting a good response index. Figure 3a shows representative electromyographic (EMG) patterns of the hindlimb induced by stimulation of different brain regions under anesthesia. Figure 3b shows the amplitude of the EMG induced by stimulation (significantly higher in LHA than in other brain regions). Figure 3c shows the delay in the EMG induced by stimulation (significantly lower in LHA than in other brain regions).

[0080] Figure 4 shows the remodeling of motor-related neurons in the lateral hypothalamus (LHA) during spontaneous recovery. Figure 4a shows a schematic diagram of the electromyography (EMG) recording paradigm. Figure 4b shows the distribution (green) of labeled neurons in the LHA at 1 and 8 weeks post-injury, along with the corresponding evoked EMG intensities (as shown in the heatmap).

[0081] Figure 5 illustrates the parameter-dependent regulation of movement by activating LHA glutamatergic neurons, rather than gabatergic neurons. Figure 5a shows that photoactivation of LHA-vglut2 glutamatergic neurons can induce movement. Figure 5b shows that photoactivation of LHA-vgat neurons cannot induce movement. Figures 5c, 5d, 5e, and 5f demonstrate that photoactivation of LHAvglut2 neurons can regulate movement in a parameter-dependent manner.

[0082] Figure 6 illustrates how activation of LHA-vglut2 glutamatergic neurons promotes hindlimb function recovery after spinal cord injury. Figure 6a shows the experimental flowchart of photoactivation of vglut2 neurons promoting motor function recovery in a paralysis model. Figure 6b shows the enhanced hindlimb function of the paralysis model at 1, 4, and 8 weeks post-injury. Figure 6c shows hindlimb retraction, hindlimb extension, and a significant increase in iliac bone height.

[0083] Figure 7 illustrates motivation-related movement mediated by LHA glutamatergic neurons. Figures 7a, 7b, and 7c show multichannel electrophysiological recordings of LHA neuronal activity, where neuronal firing (green) is significantly correlated with movement speed (gray). Figures 7d and 7e show fiber optic recordings of LHA-vglut2 neuronal activity, demonstrating its correlation with movement and foraging, suggesting that this region regulates motivation-driven movement. Figure 7f shows a flowchart of ablation of LHA-vglut2 neurons and the detection of movement motivation intensity. Figure 7g shows that ablation of LHA-vglut2 neurons does not affect gait. Figures 7i, 7j, and 7h show that ablation of LHA-vglut2 neurons reduces motivation-driven movement, significantly decreasing the number of times the neuron enters the food region.

[0084] Figure 8 shows that ablation of LHA glutamatergic neurons reduces motivation-related movements in healthy and injured mice, but only affects gait in injured mice. Specifically, Figures 8a and 8b show that ablation of LHA-vglut2 neurons affects hindlimb recovery in a paralyzed model. Figures 8d and 8c show that ablation of LHA-vglut2 neurons reduces motivation-related movements in a paralyzed model.

[0085] Figure 9 shows the LHA single neuromic analysis of motor-related circuits (downstream brain regions projected by LHA neurons).

[0086] Figure 10 shows the motor response induced by photoactivation of LHA neurons with different downstream projections. Figures 10a, 10b, 10c, 10d, and 10h show the anatomical distribution of LHA-SC neurons (lateral hypothalamic-spinal projection neurons), where photoactivation does not induce motor response. Figures 10e, 10f, 10g, 10h, 10i, 10j, 10k, and 10l show that only photoactivation of LHA-PnO (lateral hypothalamic-pontine projection neurons) or LHA-MSDB (lateral hypothalamic-septal projection neurons) can induce motor response.

[0087] Figure 11 illustrates the structural connections between the PnO pontine orifice and the spinal cord, and the effect of activation on motor function. Figures 11a, 11b, 11c, and 11d show the direct anatomical connections between the PnO pontine orifice and the spinal cord, and that photoactivation of PnO-SC neurons (pontine orifice-spinal projection neurons) can induce motor function. Figures 11f and 11g show that photoactivation of LHA-PnO neurons (lateral hypothalamus-pontine projection neurons) or LHA-PnO-SC neurons (lateral hypothalamus-pontine-spinal projection neurons) can induce motor function.

[0088] Figure 12 illustrates the spontaneous recovery induced by motivation mediated by the LHA-PnO motor circuit. Figures 12a, 12b, and 12c show that ablation of LHA-PnO neurons hinders the recovery of motor function in the paralysis model; hindlimb retraction, hindlimb extension, and iliac bone height are all impaired during the recovery process. Figure 12d shows that ablation of LHA-PnO neurons reduces motivation-related movements in the paralysis model, with a significant decrease in the number of entry points into the center and the total distance traveled.

[0089] Figure 13 shows the immediate recovery of stepping function in a severe spinal cord injury model after lateral hypothalamic electrical stimulation (DBS). Figures 13a and 13b show the experimental flowchart of deep brain stimulation (DBS) promoting motor function in a T7 and T10 stage alternating hemiplegia model. Figures 13c-13e show that at weeks 4 and 10 post-injury, LHA DBS promoted hindlimb function recovery, with significant improvements in iliac bone and gait height.

[0090] Figure 14 illustrates the natural recruitment of LHA neurons by brain-computer interface-controlled DBS to support long-term rehabilitation. Figure 14a shows a schematic diagram of closed-loop DBS triggered by the brain-computer interface (brain-controlled DBS). Figure 14b shows that cortical motor intention signals triggering DBS (blue area) improves hindlimb function. Figures 14c, 14d, 14e, and 14f show that cortical motor intention signals triggering DBS do not induce anxiety-like behaviors in the paralysis model. Figures 14g, 14h, 14i, 14j, and 14k show that closed-loop DBS supported long-term motor function rehabilitation (10 weeks) in the paralysis model, with significantly higher BMS scores, iliac bone height, and gait height compared to the control group and the randomized DBS group.

[0091] Figure 15 illustrates how DBS promotes long-term rehabilitation by activating spinal cord Zfhx3 neurons. Figure 15a shows the statistical flow of the proportion of Zfhx3 neurons in spinal cord neurons (cFos, i.e., early gene expression) activated by DBS. Figure 15b shows examples of immunostaining for cFos and Zfhx3 (markers of long projection neurons). Figure 15c demonstrates how LHA-DBS significantly activates spinal cord neurons, stimulating the activated spinal cord Zfhx3 neurons in the on-group. + Positive neurons were significantly higher in the stimulation-off group than in the group with stimulation turned off.

[0092] Figure 16 illustrates the motor movements induced by different LHA electrical stimulation sites, excluding linear movements. Figure 16a shows stimulation of other lateral hypothalamic regions besides the LHA sites identified in this invention. Figures 16b and 16c show stimulation of the LHA sites identified in this invention inducing behaviors such as backward movement, circling, or combing, while other sites do not promote forward movement.

[0093] Figure 17 shows how wireless closed-loop DBS promotes the reconstruction of motor function in paralyzed mice. Detailed Implementation

[0094] Through extensive and in-depth research, the inventors have, for the first time, provided a method and device for promoting the recovery of motor function in patients with traumatic spinal cord injury (SDI). Specifically, this invention combines pathology, neuroanatomy, and neuroelectrophysiology, employing model building, viral tracing, 3D reconstruction, and neuromodulation techniques to identify therapeutic targets for hindlimb function recovery after paralysis. Then, through key target EEG decoding, electromyography analysis, neural modulation, and machine learning, optimized neural modulation parameters are constructed. Finally, a closed-loop deep brain stimulation system that decodes motor intentions is used to reconstruct hindlimb motor function in paralyzed cases. Based on this, the inventors completed this invention.

[0095] the term

[0096] The cerebral cortex (M1): A part of the cerebral cortex, specifically the primary motor cortex (M1), plays a crucial role in motor control. It is one of the most important areas of the cerebral cortex, responsible for planning, controlling, and executing voluntary movements.

[0097] M1 neurons project directly into the spinal cord, and their axons form the corticospinal tract, the main pathway by which the cerebral cortex controls skeletal muscle movement. The activity of each M1 neuron is associated with a specific movement (e.g., a specific movement of the hand), thus, the activity patterns of M1 neurons can encode complex movements.

[0098] Furthermore, M1 has extensive connections with other brain regions, such as the prefrontal cortex, basal ganglia, and diencephalon, which play important roles in motor planning and coordination. Therefore, M1 plays a central role in the entire motor control network.

[0099] Lateral hypothalamus (LHA): The lateral hypothalamus is a region located lateral to the fornix and is considered a major component of the diencephalon motor area. It contains various types of neurons, such as glutamatergic, GABAergic, and PV (valbumin) neurons. The lateral hypothalamus is crucial for regulating various physiological and behavioral functions, including eating, sleep-wake cycles, and motivational behavior. This functional diversity likely stems from the heterogeneous cell populations and complex cellular structures within the region.

[0100] The pontine orifice (PnO): Part of the pons (or pontine brain), it plays a crucial role in multiple functions of the nervous system. The pontine orifice processes sensory information, receiving it from the spinal cord and then transmitting it to other parts of the brain, such as the cerebral cortex. Furthermore, the pontine orifice is involved in regulating sleep and wakefulness, containing groups of neurons that play key roles in these processes. Additionally, the pontine orifice plays an important role in motor control. It receives information from the cerebral cortex and then transmits it to the spinal cord, thereby controlling voluntary movements. This includes fine motor skills, such as fine motor skills of the fingers. However, its role in hindlimb or anterior motor movements is currently lacking.

[0101] The pontoreticular tract (PnRuST) is an important component of the nervous system. It originates in a specific region of the pons and extends downwards into the spinal cord, influencing muscle movement and tone.

[0102] Spinal cord: The spinal cord is part of the central nervous system and plays a crucial role in many of the body's fundamental functions. It transmits motor commands from the brain to various parts of the body. These commands are relayed through motor neurons in the spinal cord and then through the peripheral nervous system to the muscles, causing muscle contraction and movement.

[0103] Optogenetics: Optogenetics is a technique that combines optical and genetic methods to precisely control the activity of specific neurons. This technique utilizes gene manipulation to insert exogenous light-sensitive channel proteins into viral vectors and infect cells, causing the light-sensitive channel proteins to be expressed in specific cell types. Optical fibers are implanted in the brains of experimental animals, and based on the viral expression time, the development of light-sensitive channel proteins is regulated by laser light at specific frequencies to activate or inhibit neurons.

[0104] Stimulation threshold: This is an empirical value that can be adjusted based on the subject's individual circumstances. This empirical value can typically be obtained using unsupervised algorithms. For example, by combining real-time monitored high-speed cameras with gait analysis algorithms, the effectiveness of stimulus parameters for gait reconstruction can be determined, such as stride length improvement, weight support, and gait continuity.

[0105] Spinal cord injury

[0106] Spinal cord injury (SCI) is a devastating disease that disrupts communication between the brain and spinal cord, leading to severe impairment of motor function. Despite the severity of many SCI cases, some axons often remain at the site of injury, providing a potential basis for rehabilitation. Recovery of motor function after SCI is a complex process involving the reorganization of the spinal cord and ascending circuits. Recent research has begun to elucidate how specific brain regions promote motor recovery. Among these, the midbrain motor area (MLR) has been extensively studied for its role in initiating and regulating movement. However, the contribution of another region, the hypothalamic motor area (SLR), to motor control remains less clear.

[0107] Effective treatment strategies for reconstructing or restoring motor function after spinal cord injury (SCI) remain limited. Deep brain stimulation (DBS) is a potentially effective approach to promoting SCI rehabilitation by utilizing brain neural circuits. While DBS is well-established in treating neurological diseases such as Parkinson's disease, its application in SCI research is still in its early stages. Recent studies have shown that DBS targeting specific brain regions, including MLR, can enhance motor function and promote recovery in animal models of SCI. By selectively stimulating motor-related neural circuits, DBS provides a potential strategy for reactivating potential pathways and promoting motor recovery after injury. However, several challenges remain, including optimizing stimulation parameters, understanding long-term effects, and ensuring precise targeting to avoid adverse side effects. Furthermore, combining DBS with rehabilitation strategies may further enhance functional recovery.

[0108] The lateral hypothalamus is a key component of spontaneous motor recovery (SLR), uniquely integrating motivational states (such as hunger, thirst, or reward seeking) and emotional states (such as fear, anxiety, or depression) and is closely associated with motor output. Motivation-related neurons in the LHA play a role in spontaneous motor recovery after SCI, and selective, long-term activation of these neurons—without involving neurons associated with negative emotional states—may contribute to enhanced motor function after injury.

[0109] To validate this hypothesis, this application employed a series of cutting-edge technologies, including whole-brain transsynaptic labeling, single-neuron projection analysis, and cell type-specific and projection-specific targeted modulation of the LHA. The results indicate that the LHA promotes motivated movement through an indirect pathway—specifically, the LHA-pontine-spinal circuit—rather than through direct projection to the spinal cord or the medial septal nucleus-Bloka's oblique band (MSDB). This indirect modulation of the spinal cord circuitry provides a novel target for therapeutic intervention. Furthermore, this application explores the therapeutic potential of targeting the LHA with deep brain stimulation (DBS) to promote motor rehabilitation, with the ultimate goal of developing a transferable strategy to restore motor function in patients with severe spinal cord injury (SCI).

[0110] In one embodiment, the method of the present invention for promoting the recovery of motor function in patients with traumatic spinal cord injury is for patients with spinal cord injury who have not completely severed the spinal cord and whose motor function is impaired, regardless of the cause.

[0111] In another embodiment, the method of the present invention is applicable to patients with traumatic spinal cord injury in which the gray matter of the spinal cord is preserved by more than 1%; preferably, patients with traumatic spinal cord injury in which the gray matter of the spinal cord is preserved by more than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% is preserved.

[0112] In another embodiment, the method of the present invention is applicable to subjects with impaired motor function.

[0113] In another embodiment, the method of the present invention is applicable to patients with paralysis caused by stroke.

[0114] In another embodiment, the method of the present invention is applicable to patients with postoperative paralysis.

[0115] In another embodiment, the method of the present invention is applicable to subjects without voluntary motor function (such as vegetative state patients).

[0116] Closed-loop deep brain stimulation (DBS) system

[0117] Deep brain stimulation (DBS) is a neurosurgical procedure that involves implanting electrodes to stimulate specific brain regions in order to improve or control symptoms of neurological disorders. Doctors implant one or two thin electrodes into the patient's brain. These electrodes are connected via wires to a pulse generator that produces electrical impulses. These impulses are delivered through the electrodes to specific areas of the brain to alter neural activity there. Currently, DBS is primarily used to treat Parkinson's disease, including tremors, bradykinesia, and difficulty walking. It mainly stimulates the striatum or globus pallidus in the brain to improve these symptoms. It is also used for other conditions such as dystonia, epilepsy, and some mental illnesses.

[0118] Specifically, the electrical stimulation method used in this invention includes:

[0119] The location of the stimulating electrode implantation was determined by electromyography under anesthesia; the activity of neurons in the hind limb region of the mouse motor cortex was acquired in real time using a multi-channel brain-computer interface and the motor intention was decoded online; the closed loop of the decoded stimulation command was opened to regulate deep brain stimulation at specific frequencies and intensities (specific stimulation patterns) to promote the recovery of motor function in animals with spinal cord injury.

[0120] This invention identifies deep brain stimulation targets in the lateral hypothalamus for treating motor dysfunction by measuring hindlimb electromyography intensity; it activates silent residual neural circuits in the central nervous system after spinal cord injury through specific stimulation patterns, promoting the reconstruction of motor function in animals with spinal cord injury; and it uses a brain-computer interface to decode the activity of motor cortex neurons, realizing a neuromodulation device for voluntary motor intention control after spinal cord injury, restoring voluntary motor function after spinal cord injury, and alleviating possible adverse symptoms during treatment.

[0121] In one embodiment of the present invention, a method for implanting deep brain stimulation electrodes in the lateral hypothalamus for the treatment of motor function after spinal cord injury is provided, the implantation method comprising:

[0122] Deep brain stimulation electrodes step stimulation is used to output electrical stimulation pulses to activate the hypothalamic motor area;

[0123] Hippolimb electromyography (EMG) recording is used to acquire the intensity of EMG activity induced by stimulating electrodes.

[0124] Real-time analysis of electromyographic intensity is used to analyze electrophysiological signals to identify effective implantation targets.

[0125] In another embodiment of the present invention, a method for measuring the stimulation pattern evoked by lateral hypothalamic motor function is provided:

[0126] Multi-channel adjustable stimulation electrodes are used to generate specific stimulation pulses at different depths and levels to activate the hypothalamus and regulate different motor behaviors.

[0127] In another embodiment of the present invention, a closed-loop deep brain stimulation system is provided for real-time detection of neural activity and decoding of motor intentions via a brain-computer interface, thereby enabling the recovery of motor function under conscious control after spinal cord injury.

[0128] By using multi-channel electrodes implanted in the hind limb representative area of ​​the gross motor area, neural signals are acquired and decoded in real time. The decoded motor intentions can activate brain stimulation devices to promote the recovery of motor function.

[0129] The brain-computer interface samples the motor area neurons at a frequency of 25kHz, and then triggers brain-controlled DBS stimulation with a decoding speed of less than 50ms using an unsupervised algorithm.

[0130] In this study, long-term brain-controlled DBS stimulation triggered by a brain-computer interface was used to assist in motor rehabilitation training, ultimately restoring motor function in an animal model of spinal cord injury and, to some extent, reshaping the connection between the higher central nervous system and spinal cord nerves outside the injury site.

[0131] Compared with the prior art, the main advantages of the present invention include:

[0132] (1) This invention provides an effective deep brain stimulation area that targets the hypothalamus for motor regulation, so as to effectively activate the residual descending circuits in the brain after spinal cord injury, thereby treating motor dysfunction; the confirmation of specific stimulation patterns can effectively improve the therapeutic effect of brain stimulation after spinal cord injury; the closed-loop regulation of the deep brain stimulation system that detects neural activity and decodes motor intentions in real time through brain-computer interface can realize voluntary motor control after spinal cord injury and alleviate possible adverse symptoms during treatment.

[0133] (2) The closed-loop deep brain stimulation system provided by the present invention can effectively reconstruct the hindlimb motor function of patients with spinal cord injury; the optimized stimulation targets and parameters reduce the side effects of DBS therapy, and the real-time closed-loop stimulation mode prolongs the treatment effect.

[0134] (3) The closed-loop deep brain stimulation system of the present invention can also restore motor function in stroke patients.

[0135] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0136] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0137] Example 1: Spontaneous recovery of motor function in a T10 segment spinal cord hemisection model

[0138] Hemilateral spinal cord injury (Brown-Séquard syndrome) deprives the ipsilateral spinal cord of input, leading to ipsilateral limb motor dysfunction. However, both humans and animals can spontaneously recover motor function after hemilateral spinal cord injury. Extensive brain remodeling occurs during this process, but the underlying key brain regions remain unclear. To explore the key brain regions involved in remodeling, this application describes a spontaneous recovery model by hemisection of the T10 segment of the mouse spinal cord. Behavioral tests were used to identify a plateau in the spontaneous recovery of motor function after injury.

[0139] This study investigated the basis of spontaneous recovery following spinal cord injury (SCI) in mice through a comprehensive analysis of behavior, kinesiology, and anatomy. Specifically, a hemisection at T10 in the thoracic region resulted in damage to the descending tract on the affected side (Fig. 1a). In the first week post-injury, mice exhibited severe motor dysfunction in the ipsilateral hindlimb, with complete loss of walking ability in the extensor (gastrocnemius) and flexor (tibialis anterior) muscles, while contralateral muscle function remained intact. Following the acute phase, various motor functions showed varying degrees of recovery. As shown in Fig. 1b, significant recovery was observed in the chronic phase (8 weeks post-injury), characterized by weight-bearing stepping ability, particularly in the coordinated standing and swinging phases of the ipsilateral hindlimb. By week 12, interlimb coordination had essentially fully recovered, showing no significant difference compared to pre-injury levels (NS).

[0140] Example 2: Brain regions undergoing remodeling during spontaneous recovery

[0141] To explore neuroanatomical plasticity of the brain after injury and identify which descending pathways contribute to spontaneous functional recovery, this invention employs retrograde and transparency tracking techniques. Pseudorabies virus (PRV) encoding EGFP is a retrograde tracking virus that can label brain regions controlling hindlimb movement. In this invention, PRV encoding EGFP was injected into the ipsilateral hindlimb muscles of mice to retrogradely label upstream neurons, utilizing whole-brain transparency, imaging, and 3D reconstruction techniques. The ClearMap pipeline enabled automated registration of complete mouse light slices, providing sufficient detail and contrast for comprehensive anatomical evaluation (Figure 2a).

[0142] When spontaneous recovery of motor function reaches a plateau (8 weeks post-injury), neural remodeling in the brain is essentially complete. At different stages of the spontaneous recovery process, viral tracing was used to track brain regions undergoing neural remodeling, and 3D reconstruction was used to quantify the results. In healthy mice (control group), mice in the acute phase of injury (1 WPI) (acute group), and mice in the chronic phase of injury (8 WPI) (chronic group), pseudorabies virus (PRV) encoding EGFP was injected into multiple muscles of the hind limbs.

[0143] The results showed that PRV-labeled neurons were widely distributed throughout the brain of healthy mice, with significant concentrations in the cortex (41.77% of all labeled neurons), hypothalamus (2.90%), midbrain (12.88%), cerebellum (2.18%), anterior pons (16.01%), and posterior pons and medulla oblongata (24.26%).

[0144] Mice injected with PRV immediately after injury (acute group) showed a significant reduction in neuronal labelling throughout the brain compared to uninjured healthy mice (control group), indicating impaired descending pathways connecting to impaired motor circuits. Conversely, as shown in Figure 2, mice injected with tracers 8 weeks after injury (chronic group) exhibited a significant increase in the number of labeled neurons, highlighting a major reorganization of the brain during functional recovery, particularly in the lateral hypothalamus (LHA). This reorganization was also evident in the main motor cortex (M1), subthalamic nucleus (STN), midbrain reticular nucleus (MRN), and midbrain motor area (MLR) (Figures 2b and 2c). The LHA had significantly more labeled neurons than other brain regions, suggesting it is a key area for neural remodeling after paralysis, and the predominantly posterolateral location of the labeled neurons in the LHA suggests this region as a potential site for intervention.

[0145] Meanwhile, nuclear density estimation (KDE) was used to quantify the spatial center distribution of neurons, showing that the peak was located (relative to AP+1.5mm, ML±1.0mm, DV-4.9mm), indicating that the highest density of neurons was located in the posterior dorsal region of LHA, suggesting that neurons in this LHA subregion are significantly correlated with hindlimb motor function (Fig. 2d, and 2e).

[0146] Example 3: Brain regions mediating hindlimb function during spontaneous recovery

[0147] The relationship between these brain regions and hindlimb muscle movement was explored by electrically stimulating the lateral hypothalamus (LHA), main motor cortex (M1), subthalamic nucleus (STN), midbrain reticular nucleus (MRN), and midbrain motor area (MLR) of anesthetized mice, while simultaneously recording the electromyographic activity of the hindlimb GS induced by the electrical stimulation.

[0148] As shown in Figure 3a, the experimental results showed that stimulation of M1, STN, ZI, LHA and MLR induced significant electromyographic responses in the hind limb muscles of anesthetized mice. Among them, as shown in Figures 3b and 3c, stimulation of LHA produced the largest amplitude and shorter latency, suggesting that activation of LHA is a key target for reconstructing motor function after spinal cord injury.

[0149] Example 4: The dorsal and lateral sides of the tail in LHA were identified as the regions most strongly correlated with motor function.

[0150] As shown in Figure 4, the lateral hypothalamus was stimulated in a paralysis model where motor function had recovered to a plateau stage under anesthesia, while electromyography (EMG) of the tibialis anterior muscle was recorded. The resulting thermograms demonstrate the intensity of stepping induced by electrical stimulation at various locations in the lateral hypothalamus. Notably, a stimulation focus appeared in the posterolateral direction during the recovery phase. Comparative analysis of the thermograms from the acute and chronic phases revealed a key subregion in the posterolateral region of the lateral hypothalamus that promotes hindlimb function recovery after spinal cord injury.

[0151] Example 5: LHA glutamatergic neurons can regulate movement in a parameter-dependent manner.

[0152] Cre-induced virus (AAV-DIO-ChR2) expressing ChR2-YFP was injected into the ligands in vglut2-cre (glutamatergic neuron) and vgat-cre (Gabatergic neuron) mice, thereby specifically expressing the photosensitive protein ChR2 in vglut2 or vgat neurons within the LHA. Two weeks after viral expression, optical activation assays were performed on cells transfected with the photosensitive protein in both open-field and linear raceway modes. Two weeks later, optical fibers were implanted in the LHA and the cells were stimulated with 473 nm blue light.

[0153] As shown in Figure 5a, the results showed that stimulation of glutamatergic neurons at a frequency of 20 Hz for 3 seconds triggered forward movement and gait synchronization in both settings. The frequency and intensity of stimulation modulated the velocity parameters, which in turn affected the movement delay. Conversely, as shown in Figure 5b, stimulation of GABAergic neurons during the resting phase did not induce movement. In freely moving intact mice, activation of lateral hypothalamic vGLUT2 neurons induced movement in a frequency- and light intensity-dependent manner.

[0154] When the mouse is at rest, as shown in Figures 5c-5f, specific light stimulation of LHA excitatory neurons can induce the mouse to move, and the movement stops when the stimulation stops. At the same time, as the stimulation frequency and intensity increase, the maximum speed and average speed of the mouse movement increase, and the latency from rest to movement also shortens.

[0155] The above results indicate that glutamatergic neurons in the LHA are crucial for spontaneous recovery following incomplete spinal cord injury.

[0156] Example 6: LHA glutamatergic neurons can promote motor recovery

[0157] ChR2 was targeted to glutamatergic neurons by injecting AAV carrying Cre-dependent ChR2-mCherry into vglut2-IRES-Cre mice. These mice underwent optogenetic stimulation following incomplete spinal cord injury and were tested at weeks 1, 2, 4, 6, and 8.

[0158] As shown in Figures 6a-6c, at all test time points, mice with lateral hypothalamic glutamatergic neurons expressing ChR2 exhibited significantly improved hindlimb movement during light stimulation. Detailed motor analysis revealed enhanced body weight support and restored hindlimb stepping ability, indicated by increased maximum iliac crest height and toe elevation. These results suggest that activation of lateral hypothalamic glutamatergic neurons can promote the recovery of motor function.

[0159] Example 7: The relationship between lateral hypothalamic glutamatergic neurons and motivation-related motor behavior

[0160] As shown in Figures 7a-7c, a group of neurons in the lateral hypothalamus regulate motor speed under physiological conditions, and their firing frequency is significantly correlated with motor speed. To investigate the involvement of LHA neurons in motivation-related behaviors, AAV-DIO-GCaMP6s were injected into the LHA of vglut2-Cre mice, thereby enabling GCaMP6s to be specifically expressed in LHA glutamatergic neurons and facilitating fiber optic photometric imaging.

[0161] As shown in Figures 7d-7e, experiments observed that glutamatergic neurons are activated at the onset of food-seeking behavior. These findings suggest that glutamatergic neurons in the LHA play a crucial role in regulating motivation-related movement.

[0162] Example 8: Verifying the necessity of glutamatergic neurons in LHA for functional recovery and motivational movement.

[0163] This embodiment measured motor function and motivational activity in healthy mice and mice that spontaneously recovered after hemisection of SCI before and after fasting-triggered food-finding behavior, prior to and after conditional genetic ablation of LHA glutamatergic neurons.

[0164] Adeno-associated virus (AAV) was injected into the LHA of healthy vglut2-Cre mice and vglut2-Cre mice that spontaneously recovered after hemisection of SCI, respectively. This resulted in the expression of diphtheria toxin A (DTA) in the LHA of the mice, leading to the ablation of LHA vglut2 neurons.

[0165] As shown in Figure 8, mice with ablated LHA vglut2 neurons exhibited reduced motivation for food, manifested as decreased total movement distance and less time spent in the open field center, despite the presence of food, the total food intake remained essentially unchanged. Chemogenetic repression also showed a similar trend. Notably, based on hindlimb kinematic analysis, genetic ablation of LHA glutamatergic neurons did not significantly affect motor function.

[0166] In mice with spinal cord injury, ablation of LHA glutamatergic neurons also reduced motivation for food, manifested in less central movement and reduced distance traveled. However, it is noteworthy that gait analysis revealed impaired gait in mice in the chronic post-injury phase, characterized by delayed motor function, marked paw dragging, and impaired hindlimb extension due to loss of innervation, although overall weight support remained intact. This impaired recovery may be related to a long-term decline in motivated movement.

[0167] Example 9: Single neuron projection group (fmost) analysis to resolve motor-related hypothalamic circuits

[0168] This embodiment analyzes the single-neuron project database of LHA and studies the main brain regions receiving LHA neuronal input based on sparse labeling data from 916 neurons. Similarity scores were calculated based on the shortest distance between neuron pairs, and these neurons were classified into four categories and 24 subtypes according to their axonal projection patterns.

[0169] As shown in Figure 9, among these 916 LHA neurons, subtypes 1-4 (148 neurons) belong to class 1, projecting to the anterior part of the brain, including specific projections to the thalamus and subcortical nuclei. Subtypes 5-7 (115 neurons) constitute class 2, projecting mainly within the hypothalamus with shorter axons. Class 3 neurons (420 neurons), including 8-15 subtypes, project to the caudal region and represent the largest neuronal group with extensive connections to the midbrain and pons. Class 4 neurons (233 neurons) have a wider projection distance, with 16-19 subtypes specifically projecting to the cortex and medulla oblongata, while 20-24 subtypes are more predominantly medulla oblongata.

[0170] Example 10: Activation of LHA neurons projecting from SC does not induce motor movement, while activation of LHA neurons projecting from PnO or MSDB does induce motor movement.

[0171] Furthermore, this invention investigated the projection relationships between glutamatergic neurons in the LHA and neurons in the spinal cord. Since lumbar spinal cord circuits are known to generate rhythmic movement patterns, the coordinates of each spinal cord neuron were analyzed.

[0172] As shown in Figure 9, the results indicate that almost half of the fourth-order neurons (111 out of 233) project directly to the spinal cord, suggesting the existence of neurons in the LHA that project to the spinal cord. This finding was confirmed by tracing the axons projecting from mCherry-labeled LHA glutamatergic neurons to the dorsal and lumbar spinal cord.

[0173] Example 11: LHA-PnO circuit modulates movement without inducing anxiety-like behavior

[0174] The communication between PnO and MSDB (medial septal-Broca's band (MSDB) and the hippocampus has been extensively studied in learning and memory, and recent studies have also demonstrated the role of this brain region in motor control.

[0175] Activation of neurons in these different regions during light stimulation had varying effects on movement distance and maximum velocity. Notably, as shown in Figure 10h, activation of only LHA neurons projecting to PnO or MSDB could initiate movement. However, activation of MSDB resulted in high-speed escape-like behavior (Figures 10h-10k), contrasting with the milder response elicited by PnO activation (Figures 10h-10k).

[0176] To further verify the involvement of the LHA-PnO pathway, retrograde transporter AAV carrying Cre was injected into PnO and MSDB, respectively, while Cre-dependent ChR2 was expressed in the LHA of wild-type mice. Stimulation of LHA neuronal cell bodies projected to PnO or MSDB successfully elicited immediate movement in freely moving mice.

[0177] The results indicate that the glutamate pathway from LHA to PnO plays a crucial role in regulating positive motivation-related movement.

[0178] Example 12: Activation of the LHA vglut2-PnO-spinal pathway elicits motor responses in normal and paralyzed mice.

[0179] This embodiment explores the relationship between PnO and hindlimb movement. Experiments showed that PnO has dense axonal ends in the lumbar spinal cord, which are key execution centers for initiating movement.

[0180] To confirm that LHA neurons indeed contain brainstem neurons projecting from the lumbar spinal cord, this invention conducted a three-step monosynaptic retrograde tracing experiment based on rabies virus. First, retroAAV-hSyn-Cre was injected into the lumbar spinal cord. Two weeks later, AAV expressing Cre-dependent specific avian retroviral receptor (TVA) and rabies virus glycoprotein (RVG) was injected into PnO. Subsequently, RV-EnvA-dsRed was injected into PnO, resulting in the simultaneous expression of EGFP and dsRed in PnO, identifying these neurons as "promoter cells" in the monosynaptic rabies tracing method. As shown in Figures 11a-11e, the abundance of transsynaptic labeled neurons in the LHA confirmed the existence of the LHA-PnO-spinal cord pathway.

[0181] Next, an AAV carrying the Cre gene, which is an ascending transsynaptic AAV (AAV1 type), was expressed in the LHA, while a Cre-dependent ChR2-encoding AAV was expressed in the PnO, to stimulate PnO neurons receiving LHA input. Both optogenetic stimulation of LHA neurons projecting from the PnO and PnO neurons receiving LHA input produced motor effects similar to those observed when stimulating LHA glutamatergic terminals in the PnO, thus confirming that the LHA vglut2-PnO pathway is crucial for movement (Figure 11f).

[0182] To investigate whether the PnO-to-spinal cord pathway directly controls movement, retroAAV-DIO-ChR2 was injected into the spinal cord of vglut2-Cre mice. Following retrograde labeling of ChR2 expression in cells with PnO, optogenetic stimulation (10-millisecond pulses of 20 Hz light) was applied for 5 seconds using blue light (470 nm). This stimulation reliably transitioned the mice from a resting state to a running state. The effect of the stimulation was quantified by analyzing the maximum speed and distance traveled during running compared to baseline activity levels. These results indicate that the LHA vglut2-PnO-spinal cord pathway significantly promotes movement induction in intact mice (Figure 11g).

[0183] In this study, stimulation of PnO projections to the terminals of LHA neurons did not elicit a significant stress response. As shown in Figure 10i, in contrast, optical stimulation of LHA terminals located in the septum and diagonal band nucleus (MSDB) resulted in high-speed escape-like movements. While DBS technology itself can produce side effects, its effect on the LHA-Pno circuitry does not.

[0184] The distribution of neurons corresponding to two projections within the LHA was analyzed using single-neuron projection group data. As shown in Figure 9, the results indicate that these two projections exhibit a clear spatial front-to-back arrangement. This spatial organization suggests that these two types of neurons may play different functional roles, which helps explain the observed differences in escape-like behavior.

[0185] In summary, through single-neuron projection group data analysis and optogenetic activation experiments, this invention confirmed the corresponding neuronal distribution between LHA, PnO, and MSDB, and showed that targeted stimulation of LHA promotes motor function rehabilitation without producing side effects, which is the LHA-PnO circuit.

[0186] Example 13: Spontaneous recovery induced by LHA-PnO motor loop regulation

[0187] Having confirmed that LHA-to-PnO neurons can induce movement, the goal of this invention is to investigate their effects on motivation-regulated natural repair. As shown in Figure 12a, bilateral injection of the rAAV2-retro-Cre vector into PnO and the rAAV-DIO-DTR vector into LHA ​​neurons induced projection-specific lesions in LHA neurons targeting PnO, thereby precisely assessing their roles in motor function and motivation. Gene ablation of PnO projections into LHA ​​neurons significantly impaired functional recovery of ground-based movement.

[0188] The applicant quantified hindlimb contraction, hindlimb extension, and iliac process height. Ablation of neurons specifically targeting PnO resulted in a significant reduction in food motivation, manifested as a decrease in total walking distance and a reduction in the number of times entering the center of open space, as shown in Figures 12b and 12c.

[0189] The above findings indicate that PnO projections onto LHA ​​neurons encode motivation-related movements during spontaneous recovery of motor function. Statistical analysis using t-tests revealed significant deficits in the reduced group, highlighting the importance of PnO projections onto LHA ​​neurons in motor control.

[0190] Example 14: Immediate recovery of stepping function by lateral hypothalamic electrical stimulation (DBS) in a severe spinal cord injury model with alternating hemisection

[0191] This invention investigates whether deep brain stimulation (DBS) improves motor function in mice with severe spinal cord injury (SCI). Previous studies have shown that stimulation of the LHA-MSDB pathway can induce movement, but this is accompanied by significant anxiety behaviors that are detrimental to rehabilitation. Therefore, this invention aims to selectively stimulate the LHA-PnO pathway by modulating the stimulation sites within the LHA.

[0192] Based on the somatic cell distribution of LHA neurons projecting to PnO and MSDB, this invention identified neurons projecting to PnO, while those projecting to MSDB are primarily located in the anterior ventral region. AAVs carrying GCamp6s were injected into PnO and MSDB, and activation of these downstream brain regions was recorded during stimulation at different locations along the anterior and posterior axes of the LHA. Results showed that stimulation of the anterior LHA (1.0 mm from bregma) significantly activated the MSDB, while stimulation of the posterior LHA resulted in greater activation of PnO. These fiber photometric data suggest that application of LHA DBS at specific locations can promote rehabilitation following severe spinal cord injury.

[0193] Dislocation injuries were created at the T7 and T10 levels, comprising two lateral hemisections. The T7 injury extended slightly beyond the midline, while the T10 injury terminated at the midline. This bilateral hemisection paradigm deprived the lumbar spinal cord below the injury site of all direct brain innervation while preserving potential neural circuits. A timeline experiment was conducted to assess the effects of LHA-DBS on motor function, evaluating animals treated with and without LHA-DBS at weeks 1, 4, and 8 post-injury, and analyzing the direct effects on hindlimb gait responses.

[0194] As shown in Figure 13, initially, no hind limb response was observed with LHA-DBS during the first 7 days post-injury. However, at 4 weeks post-injury, animals exhibited significantly improved walking ability during LHA-DBS. Sustained DBS (20 Hz, 500 μs, 30–50 μA) promoted marked motor improvement, manifested as a significant increase in iliac crest height and toe elevation. At 10 weeks post-injury, motor ability continued to improve under LHA DBS. However, the therapeutic effect did not persist once stimulation was discontinued, prompting further investigation into the long-term potential of LHA-DBS in promoting gait training and motor function recovery.

[0195] Example 15: Natural recruitment of LHA neurons via brain-computer interface-controlled electrical stimulation

[0196] Sustained DBS stimulation promoted hind limb function recovery at four weeks post-injury. However, this randomized DBS resulted in higher stress levels in mice, as confirmed by quantitative assessment of stress responses. Sustained electrical stimulation of non-selectively activated LHA neurons reduced center time in the open field test and open arm time in the elevated cross maze test, indicating increased anxiety.

[0197] To reduce side effects, LHA-DBS is activated based on the captured voluntary motor intentions by monitoring the cerebral cortex.

[0198] Specifically, the steps include the following:

[0199] As shown in Figure 14, a signal was obtained in healthy mice to capture the intention to move voluntarily. The signal was then processed, decoded, and quantified into an index, and LHA-DBS was activated.

[0200] We hypothesized that the initiation of locomotion could be predicted via multi-unit activity (MUA) in the leg region of the motor cortex. A 32-channel microarray electrode was implanted in the M1 region of healthy mice to record MUA and decode motor intention. Analysis showed that MUA exhibited an increasing emissivity (157 ± 68 ms, n = 7) prior to hindlimb initiation. This suggests that decoding real-time motor intention from motor cortex activity to initiate LHA-DBS may promote voluntary movement and reduce stress.

[0201] Advantages: Under brain-controlled electrical stimulation, LHA-DBS did not elicit a stress response in either healthy or injured mice. These results suggest that brain-controlled DBS can recruit LHA neurons more naturally and promote long-term recovery.

[0202] Example 16: LHA-DBS triggered by brain-computer interface to support long-term rehabilitation training and enhance motor function

[0203] As shown in Figure 14a, to assess the therapeutic potential of long-term training facilitated by brain-controlled LHA-DBS, animals with dislocation injuries were assigned to three groups: (i) daily brain-controlled training with stimulation on, (ii) daily brain-controlled training with stimulation off, or (iii) observation of spontaneous recovery without training. Results showed that brain-controlled DBS further reduced anxiety-like behavior in the paralysis model when stimulation was on (Figures 14c, 14d, and 14e).

[0204] Kinetic analysis of gait performance at key time points showed a significant improvement in gait ability after long-term training compared to the control group (Figs. 14h, 14i, 14j, and 14k). Furthermore, mice exhibited improved motor performance when stimulation was applied during long-term training of LHA-DBS triggered by the brain-computer interface. As shown in Figs. 15a and 15b, c-fos and immunostaining were performed on perfused spinal cord tissue. The results, as shown in Fig. 15c, indicate that DBS-mediated gait reconstruction is mediated by activation of zfhx3 neurons (long projection neurons) in the spinal cord.

[0205] These findings indicate that targeted LHA-DBS can effectively promote motor rehabilitation in dislocation injury models, while reducing anxiety-related side effects through brain-controlled stimulation strategies. Further stimulation of the lateral hypothalamus, in addition to the stimulation sites targeted in this invention, induced other behaviors that do not promote positive movement, such as backward movement, combing, and exploratory behaviors (Figures 16a-16c). Furthermore, this invention replicated the above treatment process using an optimized, miniaturized DBS device and could promote motor function in both acute and chronic paralysis models (Figure 17). In summary, these methods hold promise for developing transformable therapeutic interventions that can promote the recovery of motor function after spinal cord injury.

[0206] discuss

[0207] The precise roles of different brain regions in motor function recovery after spinal cord injury (SCI) remain unclear. Using whole-brain circuit tracing, we identified the lateral hypothalamus (LHA) as a key region regulating spontaneous motor recovery after SCI in mice. Notably, activation of glutamate neurons in the LHA enhanced motivation-driven (food-finding) movement. Through single-neuron projection omics analysis, transsynaptic tracing, and optogenetic manipulation, we found that the LHA promotes motivated movement via the pontoretinospinal tract (PnRuST) rather than direct spinal cord (SC) projection or pressure-mediated septal broca's band (MSDB). Importantly, LHA activation of PnO (pontine orifice), followed by PnO projection onto spinal cord axons, activates Zfhx3-expressing SC interneurons to initiate movement and promote functional recovery. Finally, LHA-controlled closed-loop deep brain stimulation (DBS) controlled by motor cortex signals significantly promoted long-term recovery of hindlimb motor function after severe SCI. This approach enables targeted training and promotes long-term recovery of lower limb motor function after severe SCI. In summary, the findings of this application provide a promising and potentially translatable strategy for restoring motor function following severe spinal cord injury.

[0208] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A device for treating traumatic spinal cord injury, characterized in that, include: The stimulation module is configured to electrically stimulate the lateral hypothalamus (LHA) or pontine orifice (PnO) of a subject with traumatic spinal cord injury, thereby activating the LHA-PnO-spinal cord circuit or the PnO-spinal cord circuit and promoting the recovery of the subject's limb motor function.

2. The device as described in claim 1, characterized in that, The electrical stimulation involves applying a current with a frequency of 1-200 Hz.

3. The device as described in claim 1, characterized in that, The duration of electrical stimulation is 1-200 seconds.

4. The device as described in claim 1, characterized in that, The area where the electrical stimulation is applied is any location of the LHA; preferably, it is the dorsal region of the LHA tail; more preferably, it is a sub-region of the dorsal region of the LHA tail rich in glutamatergic neurons.

5. The device as described in claim 1, characterized in that, The device also includes: The data acquisition module is configured to: acquire electroencephalogram (EEG) signals from the M1 and / or LHA of the cerebral cortex; and output the voluntary movement signal when the EEG signal is identified as a voluntary movement signal. The information processing module is configured to: denoise and decode (normalize) the autonomous motion signal, and output the stimulus value V; An evaluation module is configured to: instruct the stimulation module to initiate electrical stimulation when the stimulation value V ≥ stimulation threshold V0; and instruct the stimulation module not to initiate electrical stimulation when the stimulation value V < stimulation threshold V0. The information processing module receives signals from the data acquisition module, and simultaneously inputs the processed data into the evaluation module.

6. The device as described in claim 1 or 5, characterized in that, The device, before application, also includes: A pre-screening module, configured to determine whether the spinal cord of a subject with traumatic spinal cord injury is completely severed: If ≥5% of the spinal cord gray matter is preserved, preferably ≥10%, and even more preferably ≥20% (30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%), then the spinal cord is not completely severed. If the amount of gray matter preserved in the spinal cord is <5%, preferably <4%, and even better <3% (2%, 1%, 0%), then the spinal cord is completely severed. When the spinal cord is not completely severed, the potential for recovery of motor function is high in subjects with traumatic spinal cord injury. When the spinal cord is completely severed, the potential for recovery of motor function in subjects with traumatic spinal cord injury is low.

7. A method for assessing the recovery potential of patients with traumatic spinal cord injury, characterized in that, Includes the following steps: (1) Determine whether the spinal cord of a patient with traumatic spinal cord injury is completely severed; If ≥5% of the spinal cord gray matter is preserved, preferably ≥10%, and even more preferably ≥20% (30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%), then the spinal cord is not completely severed. If the amount of gray matter preserved in the spinal cord is <5%, preferably <4%, and even better <3% (2%, 1%, 0%), then the spinal cord is completely severed. (2) When the spinal cord is not completely severed, the recovery potential of patients with traumatic spinal cord injury is high; when the spinal cord is completely severed, the recovery potential of patients with traumatic spinal cord injury is low.

8. A method for promoting the recovery of motor function in patients with traumatic spinal cord injury, characterized in that, include: Electrical stimulation of the lateral hypothalamus (LHA) or pontine ostomy (PnO) in subjects with traumatic spinal cord injury activates the LHA-pontine ostomy-spinal cord circuit or the pontine ostomy-spinal cord circuit.

9. The method as described in claim 8, characterized in that, The method described is also used to prevent muscle atrophy.

10. The method as described in claim 8, characterized in that, The method also includes brain-computer interfaces: (a) Acquire EEG signals from the M1 and / or LHA of the cerebral cortex; when the EEG signals are identified as voluntary movement signals, output the voluntary movement signals; (b) The autonomous motion signal is de-noiseed, decoded (normalized), and the stimulus value V is output; (c) When the stimulation value V ≥ stimulation threshold V0, the instruction stimulation module starts electrical stimulation; when the stimulation value V < stimulation threshold V0, the instruction stimulation module does not start electrical stimulation.

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