Extracranial mechanical stimulation for facilitating cerebrospinal fluid drainage

Abstract

Described herein is a system and method of preventing or treating a neurodegenerative disease including administering pharmacological agents and / or electrical stimulations and / or mechanical stimulations to a person in which the stimulation causes modulation in outflow of cerebrospinal fluid through superficial cervical lymphatic vessels.

Description

Extracranial Mechanical Stimulation For Facilitating Cerebrospinal Fluid DrainageTECHNICAL FIELD

[0001] The present application relates to a method and device for extracranial mechanical stimulation for increasing or recovering rate of or level of outflow of cerebrospinal fluid (CSF) from the central nervous system to the systemic circulation. The present application also relates to a method of extracranial electrical stimulation or pharmacological agent for regulating rate of or level of outflow of CSF from the central nervous system to the systemic circulation. The present application also relates to a method of diagnosing neurodegenerative disease by assessing the level of CSF outflow and increasing the outflow in the patient identified as suffering from a reduced outflow level of CSF.BACKGROUND OF THE INVENTION

[0002] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] The range of CSF volume is 140-200 ml in the adult human. CSF is produced 400-600 ml / day from the choroid plexus and circulates brain parenchyma and spinal cord (Ref. 1 and 2). Thus, the fluid turns over 3-5 times / day. The central nervous system comprises the brain, spinal cord, and cranial nerves which require high energy for their delicate and fine activities. In turn, the central nervous system produces waste products and antigenic macromolecules including synaptic and myelin debris and misfolded proteins such as amyloid and hyperphosphorylated tau. These central nervous system-derived waste products and macromolecules are dissolved in brain interstitial fluids and CSF, which should be adequately drained into the systemic circulation. Excessive accumulation of these waste products and macromolecules including amyloid and hyperphosphorylated tau causes neurodegenerative diseases such as Alzheimer's disease (Ref. 3).

[0004] Traditionally, arachnoid villi and cribriform plate are known to be drainage routes of CSF (Ref. 1 and 2). However, since two research groups have rediscovered meningeal lymphatic vessels (mLVs) at the dura mater of the dorsal side skull in mice (Ref. 4 and 5), so-called "dorsal mLVs", they have been suggested as a new drainage route of CSF. However, rebuttal evidences were provided byMa and Proulx, and their colleagues (Ref. 6) These researchers instead suggested that CSF drains along cranial nerves as they exit the skull (Ref. 6). In comparison, our group uncovered the basolateral mLVs as one of the main routes of CSF drainage and compared morphologic and functional differences between the dorsal mLVs and the basolateral mLVs in adult and aged mice (Ref. 7). Importantly, the basolateral mLVs acquired lymphedematous characteristics and delayed CSF drainage with aging (Ref. 7). These findings could explain the pathologic mechanism of neurodegenerative diseases such as Alzheimer's disease, that is, overaccumulation of waste products and macromolecules in the brain due to reduced CSF drainage through the impaired basolateral mLV over aging. However, the basolateral mLVs are regarded as a drainage route of CSF that circulates the posterior area of the brain and spinal cord. Recently, our group revealed lymphatic CSF outflow route from the anterior and middle cranial fossa areas to deep cervical lymph nodes (Ref.8, Fig. 1). Briefly, we found nasopharyngeal lymphatic plexus is the main hub for CSF drainage and proved that the CSF drainage can be controlled by modulating deep cervical lymphatic vessels that connects nasopharyngeal lymphatic plexus and deep cervical lymph node. However, the CSF can be drained not only to the deep cervical lymph node, but also to superficial cervical lymph node (including lymph nodes in level I and level Ila in human). The detailed anatomy for CSF draining through superficial cervical lymphatic vessels has been elusive. Here, we provide a detailed landscape of superficial cervical lymphatic vessels which is responsible for CSF drainage. It is also disclosed herein that extracranial mechanical stimulation on superficial cervical lymphatic vessels facilitates CSF drainage (Fig. 2).

[0005] US patent application publication number 20190269758 METHODS AND COMPOSITIONS FOR MODULATING LYMPHATIC VESSELS IN THE central nervous system (Ref. 9) relates to manipulation of mLVs for treating neurodegenerative diseases. However, this patent application does not disclose or suggest a method of increasing or recovering rate or level of CSF outflow fromthe central nervous system to the systemic circulation by mechanical stimulation on extracranial lymphatics for modulating or manipulating the superficial cervical lymphatic vessels pathway of CSF.

[0006] US patent application publication number 20210311076 COMPOSITIONS AND METHODS OF DIAGNOSIS AND TREATMENT FOR NEUROLOGICAL DISEASES (Ref. 10) relates to manipulation of mLVs for treating neurodegenerative diseases. However, this patent application does not disclose or suggest a method of increasing or recovering rate or level of outflow of cerebrospinal fluid (CSF) from the central nervous system to the systemic circulation by modulating or manipulating the superficial cervical lymphatic vessels pathway of CSF with mechanical stimulation.SUMMARY OF THE INVENTION

[0007] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0008] Facilitating CSF drainage is valuable in preventing and treating neurodegenerative diseases, including Alzheimer's disease. Here, this invention provides information on how extracranial mechanical stimulation and methods can facilitate CSF drainage.

[0009] This invention encompasses extracranial devices and methods for facilitating CSF drainage through the superficial cervical lymphatic vessels and superficial cervical lymph nodes leading to preventing and treating neurodegenerative diseases.

[0010] 1. Applicant newly discovered that superficial cervical lymphatic vessels are novel and manipulatable route for CSF drainage through the skull base including the cribriform plate.

[0011] 2. Applicant newly discovered the connecting routes for CSF drainage from the intracranial cavity to the superficial cervical lymphatic system.

[0012] 3. Applicant newly discovered that CSF drainage through the superficial cervical lymphatic vessels occurs from various upstream lymphatics to the superficial cervical lymph node such as periorbital lymphatic vessels, nasal lymphatic vessels, and hard palate lymphatic vessels, and although each cervical lymphatic vessel may achieve CSF drainage at different rates, the difference between the physiological dynamics of lymphatic vessels are not significant.

[0013] 4. Applicant newly discovered that the upstream connections of superficial cervical lymphatic system such as nasal lymphatic vessels, and hard palate lymphatic vessels are impaired with aging.

[0014] 5. Applicant newly discovered that the afferent lymphatic vessels of submandibular lymph node are not significantly changed with aging.

[0015] 6. Applicant newly discovered that CSF drainage through the superficial cervical lymphatic system can be modulated by extracranial mechanical stimulation on superficial cervical lymphatic vessels.

[0016] 7. Applicant newly discovered that impaired CSF drainage with aging can be rescued by extracranial mechanical stimulation on superficial cervical lymphatic vessels.

[0017] 8. Applicant newly discovered that proper functioning of nitric oxide signaling pathway is required to achieve the full effects of mechanical stimulation on superficial cervical lymphatic vessels in order to enhance the CSF outflow.

[0018] 9. Applicant newly discovered that contraction and relaxation of the circular smooth muscles covering the superficial lymphatic vessels can be modulated or regulated by administrating pharmacological agents or electrical stimulation of innervating peripheral nerves.

[0019] In one aspect, the present invention is directed to a method and device for increasing outflow of CSF from central nervous system comprising mechanical stimulation or electrical stimulation on superficial cervical lymphatic vessels comprising: determining a subject in need of increased CSF outflow; and administering an effective amount and proper location of mechanical stimulation to the subject in need, whereby the amount of mechanical stimulation enhances lymph outflow, thereby increasing CSF outflow from the central nervous system to systemic circulation in the subject. The determining of the subject in need of increased CSF outflow may include determining the subject to have a neurodegenerative disease or condition, determining the subject to have a risk factor for the neurodegenerative disease or condition, or both. The disease or condition may be cognitive decline with aging, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke. In particular, the mechanical stimulation may include pressing or sweeping motions, and can be performed using a machine set to a specific intensity or by a trained individual.

[0020] In one aspect, the mechanical stimulation may be administered selectively at or near superficial cervical lymphatic vessels that run along the facial veins. The superficial cervical lymphatic vessels are located around facial vein beneath superficial muscular aponeurotic system (SMAS) level. The detailed pathway of the superficial cervical lymphatic vessels can be inferred by mapping the path of the facial vein. Personalized mapping of the superficial cervical lymphatic vessels can be achieved by using non-invasive methods such as doppler ultrasonography to precisely identify the path of the facial vein.

[0021] In another aspect, the present invention is directed to a method of increasing outflow of CSF from central nervous system comprising increasing contracting-relaxing of superficial cervical lymphatic vessels comprising: determining a subject in need of increased CSF outflow; and administering an effective amount of superficial cervical lymphatic vessel flow agent to the subject, whereby the amount increases lymph in superficial cervical lymphatic vessels or contracting-relaxing of superficial cervical lymphatic vessels of the subject, thereby increasing CSF flow from the central nervous system to systemic circulation in the subject. The determining the subject in need of increased CSF outflow may include determining the subject to have a neurodegenerative disease or condition, determining the subject to have a risk factor for the neurodegenerative disease or condition, or both. The disease or condition may be cognitive decline with aging, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke. The agent may be catecholamine releasing agent (such as tyramine) or nitric oxide donor or phosphodiesterase 5 (PDE5) inhibitor (such as sildenafil), G protein-coupled receptor agonist. The agent that stimulates circular smooth muscle covering the superficial cervical lymphatic vessels may increase or decrease myosin phosphorylation by activating myosin light chain kinase or activating myosin light chain phosphatase. The agent may be administered to the subject percutaneously to facial and neck muscles.

[0022] In yet another aspect, the present application is directed to a method of preventing or treating or ameliorating a neurodegenerative disease or condition in a subject comprising repairing or enlarging upstream lymphatic vessels of superficial cervical lymphatic system comprising: determining the subject in need of increased CSF outflow from the central nervous system; and administering an effective amount of upstream lymphatic reconstruction agent tothe subject in need, whereby the amount of the agent repairs or enlarges upstream lymphatic connections of the subject, thereby increasing CSF outflow from the central nervous system to systemic circulation in the subject. The disease or condition may be cognitive decline with aging, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke. In particular, the agent may be a VEGFR3 agonist, such as VEGF-C or VEGF-D, an analog, variant, or fragment thereof, or a combination of any of these. The agent may be also a fibroblast growth factor 2 (FGF-2), insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), endothelin-1 (ET- 1), angiopoietin-1, Tie2 agonist, neuropilins, or prostaglandin E2. The agent may be a protein, or a genetic vector that carries a gene encoding an agent polypeptide. The gene may encode vascular growth factor-C, angiopoietin-1, or Tie2 agonist.

[0023] The agent may be administered selectively at or near upstream lymphatic vessels of superficial cervical lymphatic system, including wherein the upstream lymphatic vessels are located in periorbital lymphatics, nasal lymphatics, and hard palate lymphatic plexus. The agent may be administered to the subject intrathecally to CSF space or trans-nasally (for nasal lymphatics) or subcutaneously (for periorbital lymphatics) or topically (for nasal and periorbital lymphatics) or submucosally (for hard palate lymphatics) or directly to hard palate submucosa. In this regard, the central nervous system of the subject may include soluble molecules, and wherein increasing the CSF outflow reduces the quantity of toxic soluble molecules in the brain. The central nervous system of the subject may include amyloid-beta plaques, and wherein increasing the CSF outflow reduces the quantity of amyloid-beta plaques in the brain.

[0024] In another aspect, the present invention is directed to a method for determining a change in CSF outflow content or rate in a subject comprising: obtaining a biological sample to obtain a specimen of nasal mucosa, periorbital mucosa or hard palate mucosa on at least two separate times, assaying for presence and amount of CSF or an assayable substance in the CSF so as to obtain a value for each time, comparing the value of amount of CSF or the assayable substance, and determining the change in the amount of the value of CSF or the assayable substance, wherein a change in value of CSF or the assayable substance over time indicates changed CSF outflow content or rate. The biological sample may be obtained by swab or biopsy. The biological sample may include the nasal cavity, hard palate, or nasopharynx. The assayingmay be carried out by imaging of nasal cavity, hard palate, or nasopharynx, or gene expression analysis or protein analysis of obtained biological samples.

[0025] According to some embodiments, a device for providing mechanical stimulation to facilitate cerebrospinal fluid drainage includes a handle member having a proximal end and a distal end, the handle member having a length; a force sensor (which may also be considered a force controller) coupled to the distal end of the handle member, the force sensor comprising a multilayer capacitive sensor having at least one silicone layer disposed between conductive fabric layers; a shaft having a first end and a second end, the shaft coupled to the force sensor at the first end and extending distally therefrom; a replaceable tip assembly removably coupled to the second end of the shaft, the tip assembly comprising a stimulation tip; and an electronic control system comprising a microcontroller electrically connected to the force sensor; a capacitance-to-digital converter interfacing between the force sensor and the microcontroller; and an amplifier unit housing the microcontroller and providing real-time force measurement data; wherein the device is configured to measure and maintain applied forces within a therapeutic range of 0.01 to 0.04 kilogram-force for facilitating cerebrospinal fluid drainage through superficial cervical lymphatic vessels.

[0026] In some examples, the force sensor includes a first conductive fabric layer; a first silicone layer having a thickness of approximately 0.5 mm; a second conductive fabric layer; a second silicone layer; and a third conductive fabric layer; wherein the layers are arranged to provide differential capacitive sensing.

[0027] In some instances, the electronic control system further includes visual indicators configured to illuminate different colors based on measured force levels. For example, a first color may indicate force within the therapeutic range of 0.01 to 0.04 kilogram-force; a second color may indicate force approaching predetermined limits; and a third color may indicate force exceeding recommended levels.

[0028] The capacitance-to-digital converter may provide at least 28-bit resolution for capacitance measurements.

[0029] In some embodiments, the microcontroller is configured to sample the force sensor at a rate of at least 100 Hz. This allows real-time force feedback to the operator.

[0030] In many cases, the tip is replaceable and is formed to have a mounting rod; and a coupling mechanism selected from a threaded engagement, bayonet coupling, or friction fit for removable attachment to the shaft. This allows the tip to be easily and quickly replaceable as desired.

[0031] The stimulation tip may be formed of a cotton material configured to provide compliance for skin interface during lymphatic stimulation. Of course, other materials are suitable for patient comfort and hygiene.

[0032] In some examples, the device includes a data logging system configured to record one or more of continuous force profiles during treatment; stroke counts; treatment duration; and timestamp data.

[0033] In some instances, the force sensor is encapsulated in a silicone material forming a flexible strap configuration while maintaining electrical connections to the microcontroller.

[0034] The device may be calibrated to provide a linear relationship between sensor readings and actual force values with a correlation coefficient greater than 0.99 across the range of 0 to 0.05 kilogram-force.

[0035] According to some embodiments, a method for facilitating cerebrospinal fluid drainage through mechanical stimulation of superficial cervical lymphatic vessels includes the steps of providing a force-controlled mechanical stimulation device comprising a handle, a force sensor, a shaft, and a replaceable tip, which may have an oval configuration; positioning the tip against intact skin overlying a superficial cervical lymphatic vessel pathway; applying mechanical stimulation strokes to three anatomical regions in sequence: Region 1: from periorbital area to mandible; Region 2: from nasal sidewall to mandible; and Region 3: along paths of superficial cervical lymphatic vessels to submandibular lymph node; maintaining applied force within a range of 0.01 to 0.04 kilogram-force as measured by the force sensor; performing the stimulation in cycles, each cycle comprising: 4 strokes to Region 1; 4 strokes to Region 2; and 2 strokes to Region 3; wherein each stroke has a duration of approximately 2 seconds.

[0036] In some instances, the mechanical stimulation is performed for a total of 20 one- minute sessions.

[0037] The method may include monitoring force feedback in real-time through an electronic control system sampling the force sensor; and adjusting applied pressure based on visual indicators that illuminate to indicate whether force is within the therapeutic range.

[0038] In some cases, two cycles of 10 strokes are performed over 40 seconds followed by a 20-second rest period.

[0039] In examples, it has been shown that the described and claimed stimulation methods enhance cerebrospinal fluid drainage by at least a 2-fold increase in lymphatic flow as measured by tracer accumulation in submandibular lymph nodes compared with no stimulation.

[0040] In some preferred instances, the mechanical stimulation is applied bilaterally to both left and right superficial cervical lymphatic vessel pathways.

[0041] The method may further include identifying the pathway of superficial cervical lymphatic vessels by mapping facial veins using doppler ultrasonography; and targeting the mechanical stimulation within 2 cm of the identified lymphatic pathway.

[0042] The method may be performed on a subject having a neurodegenerative condition selected from cognitive decline with aging, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke.

[0043] The method may include recording treatment data including one or more of: continuous force profiles throughout the treatment session; total number of strokes applied to each region; treatment duration; and protocol adherence metrics.

[0044] In some instances, the mechanical stimulation activates nitric oxide signaling pathways to enhance cerebrospinal fluid drainage through the superficial cervical lymphatic vessels.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0047] The present disclosure is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0048] Fig. 1 shows schematic diagram showing that the basolateral meningeal lymphatic vessels and nasopharyngeal lymphatic plexus are main CSF lymphatic drainage route to deep cervical lymph node. Intracranial upstream lymphatic regions #1, #2, and #3 that drain through the nasopharyngeal lymphatic plexus en route to medial deep cervical lymphatics and deep cervical lymph nodes in the neck. Upstream lymphatic region #1 includes lymphatics near the pituitary gland and cavernous sinus that drain to the nasopharyngeal lymphatic plexus. Upstream lymphatic region #2 includes lymphatics in the anterior region of basolateral dura near the middle meningeal artery and petrosquamosal sinus (PSS) that course along the pterygopalatine artery (PPA) to the nasopharyngeal lymphatic plexus. Upstream lymphatic region #3 includes lymphatics near the cribriform plate that drain to lymphatics in the olfactory mucosa en route to the posterior nasal lymphatic plexus and nasopharyngeal lymphatic plexus. In contrast, lymphatics in the posterior region of basolateral dura around the sigmoid sinus do not drain to the nasopharyngeal lymphatic plexus but instead pass through the jugular foramen to lateral deep cervical lymphatics en route to deep cervical lymph nodes. Anatomical positions are indicated in lower corner. Ant., anterior; Post., posterior; Sup., superior; Inf., inferior anatomical position.

[0049] Fig. 2 shows the complex lymphatic system for CSF drainage from the meningeal lymphatic vessels to cervical lymph nodes via different lymphatic pathways. Submandibular lymph nodes (smLNs) drain CSF via two superficial cervical lymphatic vessels (scLVs): scLV-1 and scLV-2. scLV-1 connects to periorbital lymphatic vessels, which originate from meningeal lymphatic vessels alongside pterygopalatine artery and infraorbital artery through orbital fissure. scLV-2 connects to hard palate lymphatic vessels, receiving CSF from meningeal lymphatic vessels along with pterygopalatine artery, greater palatine artery, and greater palatine nerve through greater palatine canal and from nasal lymphatic vessels through incisive foramen. Additionally, scLV-2 connects to nasal cavity lymphatics originating from meningeal lymphatic vessels near20074.002WQ0 olfactory bulb area through cribriform plate via nasal sidewall lymphatics. Accessory submandibular lymph nodes (asmLNs) drain CSF via downstream branching lymphatics of nasopharyngeal lymphatic plexus. Deep cervical lymph nodes (dcLNs) mainly drain CSF via NPLP. ptLN, parotid lymph node. dcLV, deep cervical lymphatic vessel.

[0050] Fig. 3 shows that the superficial cervical lymphatic system completely compensates for the drainage of CSF, following the complete blocking of CSF drainage to the deep cervical lymphatic system. (A) Diagram of the experimental sequence for intracisternal (i.c.) infusion of TMR-dextran through the cisterna magna at 1 ml min1for 1 min into Proxl-GFP mice at 2 wk after ligation of deep cervical lymphatic vessels (dcLVs). The fluorescence intensity in the deep cervical lymph nodes (dcLN) and superficial cervical lymph nodes (scLN) were measured at 60 min after i.c infusion. (B) Ligation of the dcLVs shifted the CSF drainage to the scLN, including both submandibular (smLN) and accessory submandibular lymph nodes. White arrowhead indicates the ligation site.

[0051] Fig. 4 shows three types of superficial cervical lymph nodes categorized by location of accessory submandibular lymph node (white dashed-line) in Proxl-GFP mice without sex difference. The superficial cervical lymph nodes are composed of submandibular lymph node (smLN), accessory submandibular lymph node (asmLN), and parotid lymph node (ptLN). Type 1, asmLN is fused with smLN; Type 2, asmLN is separated from both smLN and ptLN; Type 3, asmLN is fused with ptLN. Anatomical positions are indicated in the top left corner: A, anterior; P, posterior; L, lateral; M, medial. Pie charts depicting the distribution percentage of three types in male (n - 41), female (n - 36), and total (n - 77) mice.

[0052] Fig.5 shows anatomical variations of afferent lymphatic vessels of submandibular lymph node (smLN). Two-thirds of the smLN had three afferent superficial cervical lymphatics (Type A), scLV-1, scLV-2, and scLV-3, while one-third of the lymph node had additional superficial cervical lymphatics (Type B), scLV-4, without sex difference. Anatomical positions are indicated in the top left corner: A, anterior; P, posterior; L, lateral; M, medial. Pie charts depicting the distribution percentage of two types in female (n - 24), male (n - 16), and total (n - 40) mice.

[0053] Fig.6 shows that selective distribution of TMR-dextran in the superficial and deep cervical lymph node, and superficial cervical lymphatic vessels (scLVs) after intracisternal (i.c.)20074.002WQ0 infusion. (A) Diagram of the experimental sequence of i.c. infusion of TMR-dextran into Proxl- GFP mice through the cisterna magna at 1.0 pl min1for 1 min followed by analysis of the distribution of TMR-dextran in the cervical lymph nodes and afferent lymphatic vessels of submandibular lymph node (smLN) at 30 min and 60 min later. (B) Fluorescence image and measurement of TMR-dextran signal intensity in the superficial and deep cervical LNs at 60 min after i.c. infusion. Injected TMR-dextran (red) was drained to smLN, accessory submandibular lymph node (asmLN) and the deep cervical lymph node (dcLN) but not to parotid lymph node (ptLN). (C)Fluorescence images and comparison of TMR-dextran distribution in three different afferent lymphatic vessels (outlined by green dashed-lines) of smLN at 30 min and 60 min after i.c. infusion. Occasionally, TMR-dextran was notably detected within scLV-1 at 30 min and equally detected within scLV-1 and scLV-2 at 60 min (red arrowheads). In contrast, it was not detected within scLV-3 at 30 min and 60 min after the intracisternal infusion. Anatomical positions are indicated in the top left corner: A, anterior; P, posterior; L, lateral; M, medial.

[0054] Fig. 7 shows morphological features of afferent lymphatic vessels of submandibular lymph node (smLN). Diameters and lymphangion (lymphatic segment between the valves) lengths of scLV-1 and scLV-2 were similarly variable (62-115 pm and 58-92 pm in diameter, and 543-1,238 pm and 638-1,052 pm in length), but were larger and longer than those of scLV-3 (40-76 pm and 423-673 pm), without sex difference. These three cervical lymphatics had a dense but uneven layer of circular a-smooth muscle actin positive (aSMA+) smooth muscle cells (SMCs) along their entire length without a significant difference. The otSMA+SMC coverage was higher in the mid-lymphangion area than the perivalvular area, without sex differences in all scLVs. M; male, F; female, ML; mid-lymphangion, PV; perivalvular. Anatomical positions are indicated in the top right corner: A, anterior; P, posterior; L, lateral; M, medial.

[0055] Fig.8 shows lack of differences in physiologic properties of superficial cervical lymphatics among superficial cervical lymphatic vessels (scLVs) and sexes. Under in vivo conditions, scLVs in adult mice exhibited spontaneous contractions and relaxation under anesthesia. (A) Diagram of the experimental sequence of intravital imaging of scLVs of Proxl- GFP mice. Intravital imaging is performed for 5 min after 20 min stabilization period. (B) The mean diameter, amplitude and frequency of these spontaneous contractions and relaxations,along with the ejection fraction and functional pump flow, were similar between scLV-1 and scLV-2, with no observed sex differences. Anatomical positions are indicated in the bottom left corner: A, anterior; P, posterior.

[0056] Fig. 9 shows the submandibular lymph node received CSF drainage through scLV-1 and scLV-2 but not through scLV-3, which received lymph from the mouth floor. To find the initial lymphatics for scLV-3, we infused an Alexa-647 conjugated ovalbumin (OVA647) into several potential regions of the mouse head, with simultaneous intracisternal infusion of TMR- dextran for CSF tracing. The strong signal of OVA647 was detected in the scLV-3 and the upper portion of submandibular lymph node (smLN) when the OVA647 was infused into the mouse floor (blue arrowheads). This indicates that scLV-3 drains interstitial fluid from the mouth floor. White arrowhead indicates the exit point of scLV-3 from the mouth floor. Orange dashed-line indicates the boundary of the intraoral space visible from the neck after opening intraoral space. White dashed-line boxes mark the connecting point that is enlarged to show detail. Anatomical positions are indicated in the bottom right corner: A, anterior; P, posterior; L, lateral; M, medial.

[0057] Fig.10 shows afferent lymphatic vessels of accessory submandibular lymph node (asmLN). At 60 min after the intracisternal (i.c.) infusion of TMR-dextran, TMR-dextran was detected in one of the afferent cervical lymphatics leading to the asmLN (red arrowheads). Afferent LV of asmLN originates from below the masseter muscle. This lymphatic was traced back to a downstream branching lymphatic of the nasopharyngeal lymphatic plexus. White dashed-line box marks connecting point (white arrowhead) that is enlarged to show detail. Submandibular lymph node, smLN. Parotid lymph node, ptLN. Anatomical positions are indicated in the bottom right corner: A, anterior; P, posterior; L, lateral; M, medial.

[0058] Fig. 11 shows the CSF drainage from the periorbital area to submandibular lymph node (smLN) through periorbital lymphatics and superficial cervical lymphatic vessel-1 (scLV-1) at 60 min after the intracisternal (i.c.) infusion of TMR-dextran. (A) TMR-dextran was injected into cisterna magna and after 60 min, scLVs were dissected for observation. (B-C) TMR-dextran was distributed in lymphatic vessels in periorbital region and scLV-1 (red arrowheads). The region of the white dashed-line box is enlarged in C. (D-F) i.c. injected microbeads (FluoSpheres)were in periorbital lymphatics and lymphatic vessels running along the infraorbital artery through the orbital fissure (red arrowheads). The white dashed-lines indicate the lymphatic pathway from the orbital fissure. Orange dashed-line indicates the boundary of intracranial space. The region of the red dashed-line boxes are enlarged in E and F. (G) The schematic illustration for periorbital lymphatic vessels for the CSF drainage route. Anatomical positions are indicated in the top right corner: A, anterior; P, posterior; L, lateral; M, medial.

[0059] Fig. 12 shows that the hard palate lymphatic plexus (HPLP) drains CSF. (A) Diagram of the experimental sequence of intracisternal (i.c.) infusion of TMR-dextran or FluoSpheres into Proxl-GFP mice through the cisterna magna at 1.0 pl min1for 1 min followed by analysis of the distribution of TMR-dextran or FluoSpheres in the hard palate lymphatic plexus at 60 min later. (Band D) Injected TMR-dextran or FluoSpheres was detected in the hard palate lymphatic plexus, buccal and mandibular portion of scLV-2, and submandibular lymph node (red arrowheads), but not in the lymphatics of the soft palate (white empty arrowheads). (B) White dashed-lines indicate border of hard and soft palate. Orange arrowheads indicate the TMR- dextran signal emitted from the nasopharyngeal lymphatic plexus. (C) Hard palate lymphatic plexus has lymphatic valves (white arrowheads). Initial lymphatics (blue arrowheads) are distributed in the medial and lateral sides of hard palate. (D) TMR-dextran in the hard palate lymphatic plexus merged to the buccal portion of scLV-2 at the anterolateral portion of the hard palate and drained into the submandibular lymph node. White dashed-lines indicate border of tongue, hard palate, and pathway of scLV. Yellow arrows indicate the direction of CSF outflow. (E) The schematic illustration for hard palate lymphatic plexus for the CSF drainage route. Anatomical positions are indicated in the bottom left corner: A, anterior; P, posterior; L, lateral; M, medial.

[0060] Fig. 13 shows the morphological features of hard palate lymphatic plexus. The HPLP had semilunar or irregular, linearly shaped valves, which expressed Proxl-GFP and Iaminin-a5. Immunofluorescence images of whole mount showing absence of oc-smooth muscle actin (ocSMA) positive smooth muscle cells at mid-lymphangion and perivalvular regions of Proxl+HPLP (yellow and white arrowheads) but abundancy of (ocSMA) positive smooth muscle cells covering blood vessels (red arrowheads). White dashed-line boxed region of HPLP is enlarged inthe middle and right panels.

[0061] Fig. 14 shows the two upstream lymphatics of the HPLP for CSF drainage. (A) Diagram of the experimental sequence of intracisternal (i.c.) infusion of FluoSpheres into Proxl- GFP mice through the cisterna magna at 1.0 pl min1for 1 min followed by analysis of FluoSpheres distribution in the nasal cavity and hard palate at 60 min later. (B) Bright-field image showing the anatomical location of incisive foramen (blue dashed-elliptical circle), greater palatine foramen (red dashed-circle), and greater palatine nerve (GPN, red dashed- lines). The border of olfactory epithelium and olfactory bulb are marked by black dashed-line. White dashed-line box is enlarged and viewed under a fluorescence microscope as right two panels, showing that the nasal lymphatics connect to the HPLP through the incisive foramen. (C) Immunofluorescence image showing the FluoSpheres distributions within both nasal and the HPLP (green arrowheads). The boundary between the nasal and hard palate lymphatics is marked by orange dashed-line. (D and E) One meningeal lymphatic vessel running along the pterygopalatine artery contained FluoSpheres. The descending branch of the meningeal lymphatics runs along the greater palatine artery connected to the HPLP through the greater palatine canal. Both lymphatic branches also contained the injected FluoSpheres (green arrowheads). FluoSpheres was also detected in the lymphatics within the incisive foramen, which connects the nasal lymphatics to the HPLP (green arrowheads). The white dashed-lines indicate the lymphatic pathway from the orbital fissure and greater palatine canal. (F) The schematic illustration shows two upstream lymphatic vessels of the hard palate lymphatic plexus for the CSF drainage route. smLN, submandibular lymph node. Anatomical positions are indicated in the top right corner: A, anterior; P, posterior; S, superior; I, inferior, L, lateral; M, medial.

[0062] Figs. 15-16 shows that nasal cavity lymphatic vessels originating from meningeal lymphatic vessels near the olfactory bulb area through the cribriform plate are the route for CSF drainage. The meningeal lymphatic vessels around the olfactory bulb connected to the olfactory mucosal lymphatics through the cribriform plate (Ref. 8). As shown in Fig. 15A, these lymphatics are linked with the respiratory mucosal lymphatics, which course rostral ly, exit the nasal cavity at the bony cartilage junction, and continue to the nasal sidewall lymphatics.FluoSpheres (red) is located in the Proxl+lymphatics (green arrowheads) but not in Proxl+venous sinusoids (red arrowheads) in the nasal mucosa. White boxes mark three regions that are enlarged in the low panels to show details that the FluoSpheres are distributed inside the nasal lymphatics in various regions. White asterisk marks the connecting point of the nasal lymphatics to the hard palate lymphatic plexus at the incisive foramen.As shown in Figs. 15B, TMR-dextran (red) is strong within the nasal sidewall lymphatics (blue arrowhead), HPLP (orange arrowhead), scLV-1, and scLV-2 (green arrowheads). smLN, submandibular lymph node. As shown in Figs. 16, the nasal sidewall lymphatics then merged with scLV-2, followed the facial vein, and connected to the smLN. Infused TMR-dextran is detected in these lymphatic routes (red arrowheads). Black dashed-line box region is enlarged and viewed under a fluorescence microscope (C-E). White dashed-line box regions in (C) are enlarged (D and E). White asterisks mark the connecting point of the nasal lymphatics to nasal sidewall lymphatics. Red dashed-line marks the bony-cartilage junction of nasal bone. Black dashed-line marks the contour of nose and eye. Anatomical positions are indicated in the top right corner: A, anterior; P, posterior; S, superior; I, inferior.

[0063] Fig. 17 shows that the parotid lymph node (ptLN) is a primary node for facial lymphatic drainage. (A) Diagram of the experimental sequence of intradermal (i.d.) infusion of TMR-dextran into each facial area of Proxl-GFP mice at 0.5 pl min1for 1 min followed by analysis of the distribution of TMR-dextran in the facial skin at 15 min later. (B) The schematic illustration for the compartment of the facial area. (C) The face is compartmentalized by location, nasal and medial (med.) canthus, cheek and chin, and lateral (lat.) canthus and anterior (ant.) auricular, and their subdermal layer had a rich of lymphatic networks. Yellow dashed-lines indicate the imaginary boundary line of each area. White dashed-lines outline boarder of eye, external auditory canal (EAC), and flipped facial skin. Injected TMR-Dextran fluorescence (red arrowheads) traced with a fluorescence stereomicroscope was concentrated in each collecting lymphatics and ptLN but not in submandibular and accessory submandibular lymph nodes (smLN and asmLN). These findings indicate that the parotid lymph node is specialized in draining the fluid derived from the face. Post., posterior. Anatomical positions are indicated in the top left corner: A, anterior; P, posterior; L, lateral; M, medial.20074.002WQ0

[0064] Fig. 18 shows submandibular lymph node (smLN), parotid lymph node (ptLN) and retropharyngeal lymph node (rpLN, equivalent to deep cervical lymph node in mice) of primate (Macaca fascicularis). (A) Diagram of the experimental sequence of intracisternal (i.c.) infusion of FluoSpheres into primate, Macaca fascicularis, through the cisterna magna at 250 pl min1for 10 min followed by analysis of the distribution of FluoSpheres in the cervical lymph nodes and hard palate at 180 min later. Before infusion, 1 ml of CSF was removed through the cisterna magna at 100 pl min1for 10 min. After infusion, the primate aroused from the anesthesia. (B) Three hours after microbeads (FluoSpheres) infusion to cisterna magna, the microbeads were detected inside lymphatics of smLN and rpLN but not in ptLN (red arrowheads). These results indicate that primates have similar anatomical features regarding the CSF drainage route as rodents.

[0065] Fig. 19 shows the two upstream connections of the HPLP for CSF drainage in primates. Intracisternal infusion was conducted using the same procedure as in Fig. 18. The Incisive foramen and greater palatine canal are anatomically located similarly in primates as in rodents. Three hours after microbeads (FluoSpheres) infusion to cisterna magna, the fluorescence of FluoSpheres is strong in the incisive foramen area and greater palatine area in the hard palate. White dashed-circles indicate the anatomical location of incisive foramen and greater palatine foramen in hard palate. White dashed-line indicate the imaginary line which separates the hard palate and soft palate. Anatomical positions are indicated in the bottom left corner: A, anterior; P, posterior; L, lateral; M, medial.

[0066] Fig. 20 shows 29% delayed CSF drainage to the submandibular lymph node (smLN) in aged mice (83 weeks old) compared with adult mice (12 weeks old). TMR-dextran was intracisteranlly (i.c.) injected into C57BL / 6J mice through cisterna magna at 1.0 pl min1for 1 min followed by analysis of TMR-dextran distribution in the smLN (outlined by green dashed-lines) at 60 min later.

[0067] Fig. 21 shows aging-related alterations in the HPLP and nasal mucosal lymphatics. (A) In case of hard palate lymphatic plexus, multiple alterations are evident in the greater palatine nerve area (ROI1, white dotted-line boxes) and incisive foramen area (ROI2, white dotted-line boxes) of aged mice. Analysis of the hard palate lymphatic plexus in aged mice20074.002WQ0 revealed that VEGFR3+lymphatic area was 9-17% less, number of lymphatic valves was 43-71% less, and lymphatic diameter was 11-12% greater, but the LYVE1 staining was not different from adult mice. (B)The VEGFR3+lymphatic area in the nasal mucosa was ~80% less without the difference in the lymphatic diameter detected in aged mice than in adult mice. Anatomical positions are indicated in the top right corner: S, superior; I, inferior; A, anterior; P, posterior.

[0068] Fig. 22 shows the lack of aging-related changes in morphological feature and in vivo physiological properties of superficial cervical lymphatic vessels (scLVs). (A) In comparison, there is no significant differences in the diameter, lymphangion length, and a smooth muscle actin positive (aSMA+) smooth muscle coverage on the mid-lymphangion area of scLV-1 and scLV2 between adult mice and aged mice, but the ocSMA+smooth muscle coverage on the peri- valvular area was 5.7% less in aged mice compared with that of adult mice. smLN, submandibular lymph node. L; mid-lymphangion. V; perivalvular. (B) Vital imaging analysis revealed that mean diameter (20% greater), amplitude (80% greater), and ejection fraction during spontaneous contraction and relaxation (30% larger) but no difference in frequency and fractional pump flow of scLV-1 in aged mice compared to younger adults. Nevertheless, the cross-correlations, synchronizing index for spontaneous contractions and relaxations at five locations, spaced 40 pm apart, were indifferent between adult and aged scLV-1. Thus, aged scLV-1 is relatively resilient for spontaneous contraction and relaxation. Anatomical positions are indicated in the top left corner: A, anterior; P, posterior; L, lateral; M, medial.

[0069] Fig. 23 shows that the pumping activity (contraction and relaxation) of superficial cervical lymphatic vessels (scLVs) is modulated by a pharmacological agent. scLVs are innervated by tyrosine hydroxylase positive adrenergic nerves (white arrowhead) not by vesicular acetylcholine transporter (VAChT) positive cholinergic nerves. Catecholamine releasing agent, tyramine (10 pM), modulates the pumping activity (contraction and relaxation) of scLV. The ejection fraction and fractional pump outflow were reduced, but the mean diameter was increased after tyramine treatment. Anatomical positions are indicated in the top left corner: A, anterior; P, posterior; L, lateral; M, medial.

[0070] Fig. 24 shows that pumping activity (contraction and relaxation) of superficial cervical lymphatic vessels (scLVs) is modulated by electrical stimulation. (A) To see how an20074.002WQ0 electrical stimulus affects the responses of the cervical lymphatics, we chose the relatively low current amplitude, 40 pA, after the optimization. A single pulse (40 pA for 10 sec) of the electrical current with a bipolar electrode positioned closely in the middle of scLV-1 lymphangion caused a transient, strong contraction followed by a return to the original diameter. (B) In comparison, a repeated pulse (40 pA, 0.2Hz, pulse duration of 0.5 sec) of the electrical current with a bipolar electrode closely positioned to the peri-valvular area of the lymphangion segment did not lead to significant vascular responses over 9 min.

[0071] Fig. 25 shows the specific protocol for mechanical stimulation. To determine whether mechanical stimulation enhances CSF outflow through the superficial cervical lymphatics, we developed a precision force-controlled mechano-stimulator. (A) Diagram and photograph of details for the precision force-controlled mechano-stimulator. The stimulator consisted of a replaceable tip, a handle, a force sensor, a shaft connecting the tip and the force sensor, and an amplifier. The replaceable tip was an oval-shaped cotton ball with a major axis of 1 cm and a minor axis of 0.5 cm, attached to a 1cm long rod that fits securely into the shaft. The length of the handle is 9 cm. The force sensor was made of silicone and conductive fabric and was physically connected to the replaceable tip through the shaft. The measured force was transmitted to an amplifier, and the data was relayed to a personal computer (PC). (B) Comparison showing the results of applying and measuring low-magnitude (0.01-0.02 kilogramforce, kgf) and high-magnitude (0.04-0.08 kgf) mechanical stimulation using the precision force- controlled mechano-stimulator. (C) Diagrams illustrating the procedure for mechanical stimulation on superficial cervical lymphatic vessels (scLV) over intact skin using the mechano- stimulator. Mechanical stimulation was applied to three regions, following the pathways of scLV-1 and scLV-2, as indicated by the brown dashed-arrow: Region 1 (from the periorbital area to the mandible), Region 2 (from the nasal sidewall to the mandible), and Region 3 (from rostral to caudal along the paths of scLV-1 and scLV-2 to the submandibular lymph node (smLN)). Each session consisted of two cycles of 10 strokes each of 2 seconds duration (4 strokes to region 1, 4 strokes to region 2, and 2 strokes to region 3). Two 10-stroke cycles over 40 seconds were followed by a 20-second rest period.

[0072] Fig. 26 shows that the mechanical stimulation on superficial cervical lymphaticvessels (scLVs) enhances CSF drainage to submandibular lymph node (smLN) in young adult mice (8-12 weeks old). (A) Mechanical stimulation was performed in two levels of strength: low-magnitude (0.01-0.02 kgf) and high-magnitude (0.04-0.08 kgf). To understand effect of the mechanical stimulation on scLVs, mechanical stimulation was applied to scLV-1 and scLV-2 for 5 minutes. TMR-dextran tracer was infused intracisternally 30 min before the onset of mechanical stimulation. (B) CSF drainage was assessed by measuring tracer fluorescence in scLV-1 and scLV-2 at the end of the stimulation. TMR-dextran fluorescence doubled (2.29-fold increase) in scLV-1 and scLV-2 after low-magnitude stimulation over 5 min. However, the scLV-1 and scLV-2 was constricted in places and the TMR-dextran fluorescence decreased (9.22-fold) in scLV-1 and scLV-2 after high-magnitude stimulation over 5 min. (C) To measure the effect of the mechanical stimulation on CSF drainage to the cervical lymph nodes, treatments consisted of 20 1-min sessions of mechanical stimulation was applied. TMR-dextran tracer was infused intracisternally 10 min before the onset of mechanical stimulation, and CSF drainage was assessed by measuring tracer fluorescence in submandibular lymph nodes at the end of the stimulation. TMR-dextran accumulation in the ipsilateral lymph node doubled (1.97-fold increase) after low-magnitude mechanical stimulation over 20 min, but decreased accumulation (3.11-fold)was found after high-magnitude stimulation or in the contralateral lymph node.

[0073] Fig. 27 shows that the nitric oxide signaling pathway is essential for maximum effect of the mechanical stimulation. (A) The systemic blockade of nitric oxide synthesis with N(®)- nitro L-arginine methyl ester (L-NAME, lmg / kg of body weight) decreased the mean diameter, amplitude, ejection fraction, and functional pump flow of scLV-1 during spontaneous contraction and relaxation. (B) To determine whether the mechanical stimulation-enhanced CSF drainage is involved in the nitric oxide signaling pathway, we pre-treated the mice with an intraperitoneal injection of L-NAME, 1 mg / kg of body weight) before the intracisternal infusion of TMR-dextran. Under this condition, the 20-session, low-magnitude mechanical stimulation- enhanced CSF drainage to the smLN was reduced by 55% implying that activation of the nitric oxide signaling pathway is substantially involved in the mechanical stimulation-enhanced CSF drainage. Anatomical positions are indicated in the bottom left corner: A, anterior; P, posterior.

[0074] Fig. 28 shows that the mechanical stimulation on superficial cervical lymphatic vessels (scLVs) enhances CSF drainage to submandibular lymph node (smLN) in aged mice (87- 105 weeks old). Low-magnitude mechanical stimulation was administrated using the same procedure as in Fig. 26. (A) The stimulation enhanced the TMR-dextran in the submandibular lymph node by 2.4-fold and (B) increased the TMR-dextran within the scLV by 4.7-fold in aged mice, which are similar to those in adult mice. These data imply that the low-magnitude mechanical stimulus could be a way to improve CSF drainage in the aged-related human with a declined CSF drainage function.

[0075] Fig. 29 shows that areas of the human face that should be mechanically stimulated and the direction to enhance CSF outflow. (A) The facial lymphatics and superficial cervical lymphatic vessels run alongside the facial vein. For clarity, the right side of the figure shows only the lymphatic vessels, while the left side shows only the facial vein. In reality, both are present on both sides of the body. (B) Based on mouse experiments, mechanical stimulation targets the periorbital lymphatics, which run alongside branches of the facial vein in the periorbital area, with stimulation applied from top to bottom (1&2). Next, the nasal side wall lymphatics, which run alongside the external nasal vein, are stimulated from the nose towards the cheek, following their path (3). Then, the palatal lymphatics, which run alongside the labial vein, are stimulated from the center of the lips towards the cheeks. Finally, the collecting lymphatic vessel, which drains into the submandibular lymph node, is targeted with stimulation from the cheekbone area down to the submandibular gland. For simplicity, the figure only shows arrows on one side, but stimulation is performed bilaterally.

[0076] Fig. 30 shows a schematic diagram of the electronic control system of the precision force-regulated mechano-stimulator, illustrating the force sensor, microcontroller, capacitance sensing board, and electrical connections, according to some embodiments.

[0077] Fig. 31 shows a calibration plot demonstrating the linear relationship between actual force values and sensor readings, in accordance with some embodiments.

[0078] Fig. 32 shows an exploded view of the mechanical assembly configuration of the precision force-regulated mechano-stimulator, illustrating its primary structural components, in accordance with some embodiments.

[0079] Fig. 33 shows the multilayer fabrication process for the capacitive force sensor, in accordance with some embodiments.

[0080] Fig. 34A shows a complete assembled precision force-regulated mechano-stimulator, in accordance with some embodiments.

[0081] Fig. 34B shows a schematic side view of the complete device illustrating the spatial relationship between the components, in accordance with some embodiments.

[0082] Fig. 34C shows an enlarged detail view of the replaceable tip assembly 3416, in accordance with some embodiments.

[0083] Fig. 35A shows a frontal view of a wearable mechanical stimulation device configured as a face mask with perforated framework for breathability, in accordance with some embodiments.

[0084] Fig. 35B illustrates a lateral profile view of the wearable mechanical stimulation device illustrating anatomical coverage zones, in accordance with some embodiments.

[0085] Fig. 36 shows a component diagram of a mechanical stimulation system for facilitating cerebrospinal fluid drainage, in accordance with some embodiments.

[0086] Fig. 37 shows a detailed hardware overview of a mechanical stimulation device, in accordance with some embodiments.DETAILED DESCRIPTION OF THE INVENTION

[0087] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632- 02182-9); and Robert A. Meyers (ed .), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Allen et al., Remington: The Science and Practice of Pharmacy 22nded., Pharmaceutical Press (September 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3rded., revised ed., J.Wiley & Sons (New York, NY 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7thed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3rded., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2nded., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U. S. Patent No. 5,585,089 (1996 Dec); and Riechmann etal., Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7; Paul W. Flint et al., Cummings Otolaryngology: Head and Heck Surgery, Elsevier Health Sciences, 2020 (ISBN 978-0323611794); ParvizJanfaza etal., Surgical Anatomy of the Head and Neck, Harvard University Press, 2011 (ISBN 978-0674058033).

[0088] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention. Indeed, the present invention is in no way limited to the methods and materials described. For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0089] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The definitions and terminology used herein are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims.

[0090] "Treatment" or "treating" as used herein includes achieving a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder or condition being treated. For example, in an individual with general age- related deficiency in cognition or Alzheimer's Disease, therapeutic benefit includes partial or complete halting of the progression of the disorder or condition, or partial or complete reversal of the disorder or condition. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological or psychological symptoms associated with the underlying condition such that an improvement is observed in the patient, notwithstanding the fact that the patient may still be affected by the condition. A prophylactic benefit of treatment includes prevention of a condition, retarding the progress of a condition (e.g., slowing the progression of Alzheimer's Disease or slowing the decline in cognitive abilities perhaps due to aging), or decreasing the likelihood of occurrence of a condition.

[0091] As used herein, the term "effective amount" can be an amount, which when administered, is sufficient to effect beneficial or desired results in the central nervous system, such as beneficial or desired clinical results, or enhanced cognition, memory, mood, or other desired central nervous system results. An effective amount is also an amount that produces a prophylactic effect, e.g., an amount that delays, reduces, or eliminates the appearance of a pathological or undesired condition. Such conditions include, but are not limited to, neurodegeneration. An effective amount can be administered in one or more administrations.

[0092] As used herein, the term "proper location" can be a specific area, which when administered, is sufficient to effect beneficial or desired results in the central nervous system, such as beneficial or desired clinical results, or enhanced cognition, memory, mood, or other desired central nervous system results.

[0093] A "subject" or an "individual," as used herein, is an animal, for example, a mammal. In some embodiments a "subject" or an "individual" is a human. In some embodiments, the subject suffers from Alzheimer's Disease or age-related cognitive disability.20074.002WQ0

[0094] In some embodiments, a pharmacological composition is "administered peripherally" or "peripherally administered." As used herein, these terms refer to any form of administration of an agent, e.g., a therapeutic agent, to an individual that is not direct administration to the central nervous system, i.e., that brings the agent in contact with the non-brain side of the bloodbrain barrier. "Peripheral administration," as used herein, includes intravenous, intra-arterial, subcutaneous, submucosal, subdernal, intramuscular, intraperitoneal, transdermal, by inhalation, transbuccal, intranasal, rectal, oral, trans-oral, parenteral, sublingual, topical, transmucosal or trans-nasal.

[0095] A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" herein refers to any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Such carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable carriers / excipients can be found in Remington's Pharmaceutical Sciences, Gennaro, A R, ed., 20th edition, 2000: Williams and Wilkins PA, USA. Exemplary pharmaceutically acceptable carriers can include salts, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. For example, compositions of the invention may be provided in liquid form, and formulated in saline based aqueous solution of varying pH (5-8), with or without detergents such polysorbate-80 at 0.01-1%, or carbohydrate additives, such mannitol, sorbitol, or trehalose. Commonly used buffers include histidine, acetate, phosphate, or citrate. "Pharmaceutically acceptable carrier" also includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0096] Exemplary pharmaceutically acceptable carriers for injectable compositions can include salts, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates,20074.002WQ0 benzoates, and the like. Commonly used buffers include histidine, acetate, phosphate, or citrate. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol; phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate, and gelatin.

[0097] For human administration, preparations meet sterility, pyrogenicity, general safety, and purity standards as required by FDA and other regulatory agency standards. The active compounds will generally be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, submucosal, subdermal, intranasal, transnasal, intralesional, or intraperitoneal routes. The preparation of an aqueous composition that contains an active component or ingredient will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for use in preparing solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.

[0098] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-fi Itered solution thereof.

[0099] Upon formulation, solutions will be systemically or locally administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective basedon the criteria described herein. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

[0100] A "recombinant host cell" or "host cell" refers to a cell that includes an exogenous polynucleotide, regardless of the method used for insertion, for example, direct uptake, transduction, f-mating, or other methods known in the art to create recombinant host cells. The exogenous polynucleotide may be maintained as a nonintegrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

[0101] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid, e.g., an amino acid analog. As used herein, the terms encompass amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0102] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0103] As used herein, "intracranial" refers to matter within the cranium or skull including the meninges and parenchyma as well as other structures.

[0104] As used herein, "extracranial" refers to matter that is not within the cranium, such as superficial cervical lymphatic vessels (scLV), periorbital lymphatic vessels, nasal lymphatic vessels, or hard palate lymphatic plexus.

[0105] As used herein, "exogenous agent" refers to a composition or methodology that can be applied to a subject so as to increase or stabilize flow of CSF. In particular, the exogenous agent affects flow in scLVs. In this regard, "flow agent" is synonymous with an exogenous agentin particular as it increases or stabilizes outflow of CSF through scLV to superficial cervical lymph nodes (scLN).

[0106] As used herein, "upstream lymphatics of the scLV-scLN" or "scLV-scLN path"refers to the extracranial area or space of lymph vessel or lymph structural system in which CSF outflowoccurs from the periorbital lymphatic vessels, nasal lymphatic vessels and hard palate lymphatic plexus to superficial cervical lymph node occurring in the facial along the facial veins.

[0107] scLV and scLN path

[0108] As described herein, Applicant has discovered a new lymphatic system or route that functions in draining macromolecules, and debris from the central nervous system. In particular, reducing drainage through the scLV-scLN path can reduce the outflow of CSF, and can exacerbate symptoms of neurodegenerative diseases characterized by increases in concentration and / or accumulations of molecules in theCENTRAL NERVOUS SYSTEM, for example, Alzheimer's disease (AD). Modulating lymphatic vessels to increase flow may alleviatecognitive impairment conditions due to old age, or symptoms of AD, including cognitive symptoms, accumulation of amyloid-beta plaques and hyperphosphorylated tau proteins.

[0109] Methods for treating, preventing, inhibiting, or ameliorating symptoms of neurodegenerative diseases or conditions associated with increased concentration and / or the accumulation of macromolecules, cells, and debris in the central nervous system are described. The methods may increase drainage by lymphatic vessel, and thus increase flow in CSF. The inventive methods are advantageous because they include one, several or all of the following benefits: (i) increased outflow and drainage of CSF; (ii) decreased accumulation of macromolecules, cells, or synaptic and myelin debris in the central nervous system (for example, decreased accumulation of amyloid-beta); and (iii) maintenance of or improvement in cognitive function (for example memory function) in a subject suffering from cognitive impairment condition due to old age, or is suspected of having, and / or at risk for dementia (such as in a neurodegenerative disease).

[0110] CSF Flow and Agents for Increasing or Stabilizing Flow

[0111] As used herein, "flow" refers to a rate of perfusion through an area of the central nervous system of a subject. In some embodiments of the invention, "flow" can be measured asa rate at which a label or tracer in CSF perfuses through a particular area of the central nervous system. As such, flow can be compared between two subjects or two sets of conditions by ascertaining how quickly an injected label or tracer perfuses throughout a particular area or volume of the brain and / or other portion of the central nervous system. Additionally, "outflow" refers to the drainage of CSF into systemic circulation, and in particular through the inventive the scLV-scLN pathway.

[0112] As used herein, "exogenous agent", "agent" or "flow agent" broadly refers to classes of compositions that can increase the passage of substances into and out of lymphatic vessels and thus can modulate flow in CSF. "exogenous agent", "agent" or "flow agent" means not only pharmacological agents but also devices or methods capable of providing mechanical or electrical stimulation. In particular, the invention is directed to an agent that specifically or generally increases flow of CSF in scLV ("scLV flow agent") or upstream lymphatics of the scLV-scLN ("upstream lymphatics flow agent").

[0113] Without being limited by theory, it is contemplated, according to several embodiments herein, that removal of macromolecules through the scLV-scLN path can keep their concentrations low in the CSF, allowing a gradient to clear macromolecules from the parenchyma. Furthermore, the higher the rate of fluid flow and drainage in the central nervous system, the higher the rate of clearance and / or the lower the concentration of cells, macromolecules, waste, and debris form the central nervous system.

[0114] Exogenous agents may increase the diameter of the scLV lymphatic vessels, and / or repair the scLV-scLN lymphatic vessels, which increases or stabilizes rate or level of drainage, resulting in increased flow of the CSF. In some embodiments, exogenous agents enlarge or repair scLV or the scLV-scLN path, and / or enhance contraction and relaxation of the scLV, thus increasing net drainage, resulting in increased or stabilized flow of the CSF.

[0115] scLV Flow Agent

[0116] Examples of suitable scLV flow agents for increasing CSF flow include but are not limited to catecholamine releasing agent (Fig. 23), electrical stimulation (Fig. 24) or mechanical stimulation (Figs. 25-28) which modulate contraction and relaxation of scLV.

[0117] Contraction and relaxation of the circular smooth muscles covering the scLVs can be regulated by 1) stimulation and inhibition of the smooth muscle cells or peripheral nerves, 2) neurotransmitters, 3) mechanical stimulators, and 4) gentle massage of the facial and neck area near scLV, or 5) electrical stimulators. CSF drainage can be facilitated by extracranial manipulations, administration of agents, and regulators. Considering that there is no prominent change in aged scLVs, the reduced CSF drainage can be enhanced by extracranial manipulations of scLVs.

[0118] The administered composition may include a single unit dose of pharmacological agent effective for increasing or repairing flow, increasing clearance or reducing accumulated amyloid-beta plaques. In some embodiments, the effective amount of pharmacological agent is about 0.00015 mg / kg to about 1.5 mg / kg, including any other amount or range contemplated as a therapeutically effective amount of a compound as disclosed herein. The range may be greater or less than the range of about 0.00015 mg / kg to about 1.5 mg / kg.

[0119] The mechanical stimulation may include a single or multiple repetition for increasing or repairing flow or increasing clearance or reducing accumulated amyloid-beta plaques or phosphorylated tau in CSF. In some embodiments, the effective amount of mechanical stimulation is about under 0.04kgf, including any other range contemplated as a therapeutically effective force as disclosed herein. The range may be greater or less than about 0.04 kgf. In some embodiments, the effective acceleration of CSF through scLV is about 0.001 m / s2to about 2.5 m / s2, including any other range contemplated as a therapeutically effective acceleration as disclosed herein. The range may be greater or less than the range of about 0.001 m / s2to about 2.5 m / s2. The mechanical stimulation is not limited to mechanical pressing or sweeping motions and may include massage or any other mechanical methods to allow scLV to contract and dilate the scLV. A mechanical stimulation may be performed so as to increase the flow of CSF through the scLV. In some embodiments, the proper location of mechanical stimulation refers to the pathway of the facial and cervical lymphatic vessels, which run alongside the facial veins. The range is about within 0 cm to 2 cm in all directions from the lymphatic pathway, including any other range contemplated as a therapeutically effective area as disclosed herein.

[0120] The electrical stimulation may include a single or multiple repetition for increasing orrepairing flow or increasing clearance or reducing accumulated amyloid-beta plaques or phosphorylated tau in CSF. In some embodiments, the effective intensity of electrical stimulation is about 0.000A to 1mA (alternative current), including any other range contemplated as a therapeutically effective current as disclosed herein. An electrical stimulation may be performed so as to control the flow of CSF through the scLV.

[0121] Upstream Lymphatics Flow Agent

[0122] Examples of suitable upstream lymphatics flow agents for increasing CSF flow include, but are not limited to vascular growth factor C (VEGF-C), vascular growth factor D (VEGF-D), fibroblast growth factor 2 (FGF-2), insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), endothelin-1 (ET-1), angiopoietin-1, Tie2 agonist, neuropilins, prostaglandin E2, and further include viral vector-mediated gene transfer of VEGF-C or VEGF-D or FGF-2 or IGF-1 or HGF or ET-1 or angiopoietin-1.

[0123] VEGFR3, also known as FLT4, is a receptor tyrosine kinase, and its signaling pathway has been implicated in embryonic lymphatic development, and adult lymphangiogenesis. Upon binding of ligand, VEGFR3 dimerizes, and is activated through autophosphorylation. As such, VEGFR3 agonists are suitable for methods for treating, reducing the symptoms of, or preventing neurodegenerative diseases associated with accumulation of molecules in the brain, for example AD, in accordance with some embodiments herein. Accordingly, in some embodiments, such as methods or compositions for which increased drainage and flow are desired, a flow agent is a VEGFR3 agonist.

[0124] In particular, VEGF-C promotes the growth of lymphatic vessels (lymphangiogenesis). It acts on lymphatic endothelial cells (LECs) primarily via its receptor VEGFR-3 promoting survival, growth and migration. As such, VEGF-C is suitable as VEGFR3 agonist.

[0125] An effective amount of VEGFR3 agonist in accordance with the inventive methods can be understood in terms of its ability to enlarge or repair upstream lymphatics of superficial cervical lymphatic system including periorbital lymphatics, nasal lymphatics, and hard palate lymphatic plexus, so as to increase or stabilize flow of CSF, or to treat, ameliorate, or prevent various cognitive impairment conditions due to old age or neurodegenerative disease by increasing clearance of substances from the central nervous system. Accordingly, in the inventivemethods, an effective amount of VEGFR3 agonist enlarges upstream lymphatics of superficial cervical lymphatic system by at least about 2%, for example, at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, including ranges between any two of the listed values. In some embodiments, an effective amount of VEGFR3 agonist increases flow of the CSF by at least about 2%, for example, at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, including ranges between any two of the listed values.

[0126] In addition to VEGF-C, a VEGFR3 agonist may include VEGF-D. In one aspect, VEGF-C and VEGF-D together agonize VEGFR3, and can be provided in a single composition, or in separate compositions. In some embodiments, a VEGFR3 agonist includes an analog, variant, or functional fragment, such as a mutant, ortholog, fragment, or truncation of VEGF-C or VEGF-D.

[0127] The present application also discloses exogenous nucleotides encoding a VEGFR3 agonist, such as VEGF-C. In one aspect, a nucleotide encoding VEGF-C or VEGF-D as described herein is expressed in a subject in order to administer the VEGFR3 agonist to a subject. For example, an exogenous vector such as a retroviral, lentiviral, adeno-viral, or adeno-associated viral vector containing a nucleic acid encoding a VEGFR agonist as described herein can be inserted into a host nucleic acid of the subject. In some embodiments, the vector further comprises transcriptional machinery to facilitate the transcription of the nucleic acid encoding the VEGFR3 agonist.

[0128] In one aspect, the VEGFR3 agonist may include a modification, for example a glycosylation, PEGylation, or the like. In some embodiments, a composition for use in accordance with the methods described herein comprises the VEGFR3 agonist, and a pharmaceutically acceptable diluent or carrier.

[0129] Routes of Administration

[0130] The flow agents according to the invention may be administered to a subject using any of a number of suitable routes of administration, provided that the route of administration administers the flow agent to the scLV-scLN path of a subject.

[0131] By "administering to the scLV-scLN path of a subject," as used herein, it is not necessarily required that a flow agent be administered directly to the scLV-scLN path space, butrather, this term encompasses administering a flow agent directly and / or indirectly to the scLV- scLN path space. It is contemplated that administering the flow agent so that it is in fluid communication with the scLV-scLN path space of the subject in accordance with some embodiments herein typically by administering the flow agent optionally in the brain, the flow agent will be administered to the scLV-scLN path space. Accordingly, in one aspect, the flow agent is not administered systemically. In another aspect, the flow agent is not administered systemically, but rather is administered to a fluid, tissue, or organ in fluid communication with the scLV-scLN path space. In some embodiments, the flow agent is not administered systemically, but rather is administered to the central nervous system. In some embodiments, the flow agent is administered to the central nervous system, but is not administered to any organ or tissue outside of the central nervous system. In some embodiments, the flow agent is not administered to the blood.

[0132] In some embodiments, the flow agent is physically attached on the scLV-scLN path space to administer vibration, squeezing, massaging, or current. For example, the flow agent can be provided in a handheld force-sensing device or electrical stimulation patch.

[0133] In some embodiments, the flow agent is administered nasally or orally so as to more directly impact the scLV-scLN path. For example, the flow agent can be provided in a nasal spray, or can be contacted directly with a nasal mucous membrane.

[0134] In some embodiments, the flow agent is administered through contacting with CSF of the subject. For example, the flow agent can be directly injected into CSF of a patient (for example intratheca I ly) . Suitable apparatuses for injection can include a syringe, or a pump that is inserted or implanted in the subject and in fluid communication with CSF. In another aspect, the flow agent, may be in a slow-release gel, is implanted in a subject so that it is in fluid communication with CSF of the subject, and thus contacts the CSF. Topical administration in particular to the neck region to affect the muscle surrounding scLV is also contemplated. Nonlimiting examples for topical administration include creams, lotions, gels, salves, sprays, dispersions, suspensions, pastes and ointments.

[0135] In some aspects, the flow agent is administered transcranially. For example, the flow agent such as in a gel formulation can be placed on an outer portion of the subject's skull, andcan pass through the subject's skull. In some embodiments, the flow agent is contacted with a thinned portion of the subject's skull to facilitate transcranial delivery.

[0136] In another aspect, the flow agent is administered by expressing a nucleic acid encoding the flow agent in the subject. A vector including a nucleic acid, for example a viral vector such as a retroviral vector, lentiviral vector, or adenoviral vector, or adeno-associated viral vector (AAV) can be administered to a subject as described herein, for example via injection or inhalation. In some embodiments, expression of the nucleic acid is induced in the subject, for example via a regulator of transcription.

[0137] In some embodiments, the flow agent is administered selectively to the scLV-scLN path space of the subject. As used herein, administered "selectively" indicates that the flow agent is administered preferentially to the indicated target compared to other tissues or organs. As such, direct injection to extracranial scLV-scLN path would represent "selective" administration, whereas administration to CSF in general via a spinal injection would not. In some embodiments, the flow agent is administered selectively to the scLV-scLN path space, and not to portions of the central nervous system outside of the scLV-scLN path space, nor to any tissues or organs outside of the central nervous system. In some embodiments, the flow agent is administered selectively to the central nervous system, and not to tissue or organs outside of the central nervous system such as the peripheral nervous system, muscles, the gastrointestinal system, or musculature.

[0138] In some embodiments, the flow agent is administered to area surrounding facial and neck area of the scLV-scLN path of the subject. As used herein, surrounding facial and neck area of the scLV-scLN path indicates the tissues around lymphatic vessels from the upstream lymphatics of scLVto the superficial cervical lymph nodes.

[0139] In some embodiments, the flow agent is administered to the surrounding neck area of the mandibular lymph node. As used herein, surrounding neck area of the mandibular lymph node indicates the tissues around afferent lymphatic vessels of the mandibular lymph nodes. For example, lymphatics from the hard palate to the mandibular lymph nodes are included.

[0140] For any of the routes of administration listed herein in accordance with the method of the present invention, it is contemplated that a flow agent can be administered in a single administration, or in two or more administrations, which can be separated by a period of time.20074.002WQ0For example, in some embodiments, the flow agent as described herein can be administered via a route of administration as described herein hourly, daily, every other day, every three days, every four days, every five days, every six days, weekly, biweekly, monthly, bimonthly, and the like. In some embodiments, the flow administration is administered in a single administration, but not in any additional administrations.

[0141] Some embodiments include methods of making a composition or medicament including a flow agent as described herein suitable for administration according to a route of administration as described herein. For example, in some embodiments, catecholamine releasing agent or VEGFR3 agonist is prepared for transdermal, transcervical, transnasal, transcranial administration or administration to the CSF.

[0142] Particularly preferred methods of administration of the agents include without limitation intrathecal approach to CSF space, transdermal, transnasal or transoral approach to nasal lymphatics or hard palate lymphatics, transcervical or percutaneous or topical approach to facial, and head and neck muscles or cervical lymphatic vessels or cervical nodes (submandibular lymph node) or neck spaces.

[0143] Neurodegenerative Diseases and Conditions

[0144] The present invention is useful for treating, preventing, inhibiting, ameliorating, or reducing the symptoms of one or more neurodegenerative diseases. These diseases can occur in subjects, for example humans, as well as non-human animals, such as non-human mammals, and non-human primates in particular.

[0145] In some embodiments, neurodegenerative diseases associated with accumulation of macromolecules, cells, and debris in the central nervous system are treated, prevented, inhibited, or reduced by methods that increase flow, drainage, and / or clearance in the scLV-scLN path. In some embodiments, neurodegenerative diseases associated with accumulation of macromolecules, cells, and debris in the central nervous system are treated, prevented, inhibited, or reduced. Examples of neurodegenerative diseases include cognitive decline with aging, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke.

[0146] In some embodiments, the neurodegenerative disease can be prevented, treated, or ameliorated prophylactically. Accordingly, a subject having one or more risk factors for theneurodegenerative disease can be determined to be in need of receiving the method of treatment described herein. For example, a subject may have accumulated amyloid-beta plaques in their central nervous system, and may benefit from increased flow, increased drainage, increased clearance and / or reduction of amyloid-beta plaques, even if they do not yet have an AD diagnosis based on cognitive symptoms. A number of risk factors for AD are suitable as risk factors in accordance with methods, compositions, and uses of some embodiments herein, for example familial AD, a genetic marker for AD, or a symptom of AD such as early dementia. The foremost risk factor for sporadic AD is age. However, increased risk of this form of AD has also been attributed to diverse genetic abnormalities.

[0147] Methods for Increasing Flow

[0148] The inventive method can include determining whether the subject is in need of increased fluid flow in the central nervous system. Further, the inventive method can include determining whether the subject is in need of increased drainage of CSF. If the subject is in need of increased fluid flow or drainage, the methods can include administering an effective amount of VEGFR3 agonists, catecholamine releasing agents, nitric oxide donor, phosphodiesterase 5 (PDE5) inhibitor, electrical stimulations, or mechanical stimulations to the scLV-scLN path space of the subject. Thus, fluid flow in the central nervous system of the subject can be increased as well as level or rate of CFS outflow. In some embodiments, the VEGFR3 agonist comprises VEGF- C or VEGF-D or an analog, variant, or fragment thereof. It is also contemplated that for in some embodiments herein, FGF2 can be substituted for the indicated VEGFR3 agonist in order to increase flow or can be used in addition to a VEGFR3 agonist in order to increase flow. In some embodiments, catecholamine releasing agent comprises tyramine. In some embodiments, phosphodiesterase 5 (PDE5) inhibitor comprises sildenafil. In some embodiments, electrical stimulation can be applied to head and neck area around the scLV-scLN path. In some embodiments, mechanical stimulation can be applied to head and neck area around the scLV- scLN path.

[0149] A subject can be determined to be in need of increased fluid flow or outflow by determining whether the subject has cognitive impairment related to old age or not, a neurodegenerative disease, or is at risk of developing a neurodegenerative disease. The diseasecan be associated with the increased concentrations and / or accumulation of molecules or cells or debris in the central nervous system, for example Alzheimer's Disease (AD). In some embodiments, the subject can be determined to be at risk for the disease, for example through having familial occurrence of the disease, by having one or more genetic markers associated with the disease, through advanced age, or by exhibiting symptoms of the disease, for example early dementia in the case of AD.

[0150] As used herein, "advanced age" refers to an age characterized by a decrease in memory function, decrease in CSF production, substantial increases in neuronal senescence, and in the context of some embodiments, can include at least 65 years of age in a human, for example, at least 60, 65, 70, 75, 80, or 85, including ranges between any of these values. In some embodiments, determining whether the subject is in need of increased fluid flow or outflow comprises determining the subject to have a neurodegenerative disease such as AD. In some embodiments, determining whether the subject is in need of increased fluid flow or outflow comprises determining the subject to have a risk factor for the neurodegenerative disease associated with the increased concentration and / or accumulation of molecules or macromolecules or cells or debris in the central nervous system as described herein. In some embodiments, determining whether the subject is in need of increased fluid flow or outflow comprises determining the subject to have a risk factor, and also determining the subject to have the disease itself.

[0151] In some embodiments, the neurodegenerative disease or condition is selected from the group consisting of at least one of the following: cognitive impairment possibly due to old age, Alzheimer's disease (AD), dementia, Parkinson's disease, Huntington's disease, or stroke. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the risk factor is a risk factor for Alzheimer's disease as described herein. In some embodiments, the VEGFR3 agonist and / or FGF2 and / or catecholamine releasing agent and / or nitric oxide donor and / or phosphodiesterase 5 (PDE5) inhibitor and / or electrical stimulation and / or mechanical stimulation to the scLV-scLN path is administered to the subject after determining that the subject has a risk factor for the neurodegenerative disease (even if the subject does not necessarily have the disease itself), for example for prophylactic treatment orprevention. In some embodiments, the VEGFR3 agonist and / or FGF2 and / or catecholamine releasing agent and / or nitric oxide donor and / or phosphodiesterase 5 (PDE5) inhibitor and / or electrical stimulation and / or mechanical stimulation to the scLV-scLN path is administered to the subject after determining that the subject has the neurodegenerative disease.

[0152] Without being limited by theory, it is contemplated, according to several embodiments herein, that systemic administration is not required for the VEGFR3 agonist and / or FGF2 and / or catecholamine releasing agent and / or nitric oxide donor and / or phosphodiesterase 5 (PDE5) inhibitor to effectively modulate lymphatic vessel size of the scLV-scLN path, or flow in the scLV-scLN path. Accordingly, in some embodiments, the VEGFR3 agonist and / or FGF2and / or catecholamine releasing agent and / or nitric oxide donor and / or phosphodiesterase 5 (PDE5) inhibitor and / or electrical stimulation and / or mechanical stimulation is administered selectively to the scLV-scLN path space of the subject, but is not administered to blood. In some embodiments, VEGFR3 agonist and / or FGF2 and / or catecholamine releasing agent and / or nitric oxide donor and / or phosphodiesterase 5 (PDE5) inhibitor is administered to the space, inside or outside the central nervous system. In some embodiments, the VEGFR3 agonist and / or FGF2 and / or catecholamine releasing agent and / or nitric oxide donor and / or phosphodiesterase 5 (PDE5) inhibitor is administered to the subject by a route selected from the group consisting of at least one of the following: topical, intrathecal, intranasal administration, transdermal, transcervical, transnasal, transoral, transcranial administration, contact with CSF of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the VEGFR3 agonist and / or FGF2 and / or catecholamine releasing agent and / or, nitric oxide donor and / or phosphodiesterase 5 (PDE5) inhibitor, or expression in the subject of a nucleic acid encoding the VEGFR3 agonist and / or FGF2 and / or catecholamine releasing agent and / or nitric oxide donor and / or phosphodiesterase 5 (PDE5) inhibitor, or a combination of any of the listed routes. In some embodiments, it is the VEGFR3 agonist that is administered. In some embodiments, the VEGFR3 agonist is selected from the group consisting of at least one of the following: VEGF-C, VEGF-D, or an analog, variant, or functional fragment thereof. In some embodiments, it is the catecholamine releasing agent that is administered.20074.002WQ0

[0153] In some embodiments, the administration of the VEGFR3 agonist results in an increase in lymphatic vessel diameter of the scLV-scLN path, lymphatic vessel number of the scLV-scLN path, the scLV-scLN path lymphatic vessel drainage, or amelioration of symptoms of a neurodegenerative disease or condition. For example, in some embodiments, the administration of the VEGFR3 agonist increases diameter of the lymphatic vessel in the scLV-scLN path is increased by at least about 5%, for example at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%, including ranges between any two of the listed values. In some embodiments, an average diameter of a population of lymphatic vessels of the scLV-scLN path of the subject is increased by a value noted herein. In some embodiments, the administration of the VEGFR3 agonist increases CSF flow in the central nervous system, including CSF outflow in the subject, comprising increasing a rate of perfusion of fluid throughout an area of the subject's brain.

[0154] In some embodiments, the administration of the catecholamine releasing agent and / or nitric oxide donor and / or phosphodiesterase 5 (PDE5) inhibitor results in an increase in lymphatic vessel diameter of the scLV-scLN path, lymphatic vessel number of the scLV-scLN path, the scLV-scLN path lymphatic vessel drainage, or amelioration of symptoms of a neurodegenerative disease or condition. For example, in some embodiments, the administration of the catecholamine releasing agent and / or nitric oxide donor and / or phosphodiesterase 5 (PDE5) inhibitor increases diameter of the lymphatic vessel in the scLV-scLN path is increased by at least about 5%, for example at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%, including ranges between any two of the listed values. In some embodiments, an average diameter of a population of lymphatic vessels of the scLV-scLN path of the subject is increased by a value noted herein. In some embodiments, the administration of the catecholamine releasing agent and / or nitric oxide donor and / or phosphodiesterase 5 (PDE5) inhibitor increases CSF flow in the central nervous system, including CSF outflow in the subject, comprising increasing a rate of perfusion of fluid throughout an area of the subject's brain.

[0155] In some embodiments, the mechanical stimulations to surrounding facial, and head and neck area of the scLV-scLN path results in an increase of CSF flow, or amelioration of symptoms of a neurodegenerative disease or condition. For example, in some embodiments, the administration of the mechanical stimuli increases CSF flow in the central nervous system,including CSF outflow in the subject, by at least about 5%, for example at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%, including ranges between any two of the listed values. In some embodiments, an average CSF flow rate of lymphatic vessels of the scLV-scLN path of the subject is increased by a value noted herein. Here, mechanical stimulations include squeezing, pressing, or sweeping motions, vibration, ultrasound stimulation, light stimulation, magnetic stimulation, temperature stimulation and are not limited to the stimulation listed herein. In some embodiments, the administration of mechanical stimulations increases CSF flow in the central nervous system, including CSF outflow in the subject, comprising increasing a rate of perfusion of fluid throughout an area of the subject's brain.

[0156] In some embodiments, the electrical stimulations to surrounding facial, and head and neck area of the scLV-scLN path results in an increase of CSF flow, or amelioration of symptoms of a neurodegenerative disease or condition. For example, in some embodiments, the administration of the electrical stimuli increases CSF flow in the central nervous system, including CSF outflow in the subject, by at least about 5%, for example at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%, including ranges between any two of the listed values. In some embodiments, an average CSF flow rate of lymphatic vessels of the scLV-scLN path of the subject is increased by a value noted herein. Here, electrical stimulations include single, multiple, or specific programmed stimulation and are not limited to the stimulation listed herein. In some embodiments, the administration of electrical stimulations increases CSF flow in the central nervous system, including CSF outflow in the subject, comprising increasing a rate of perfusion of fluid throughout an area of the subject's brain.

[0157] Increasing clearance can reduce macromolecules such as amyloid beta plaques, or decrease the rate of their accumulation. Without being limited by theory, it is contemplated that by clearing soluble amyloid beta from the central nervous system, a gradient will favor solubilization of amyloid beta plaques, so that fluids in the central nervous system continue to flow and the central nervous system continues to be cleared, amyloid beta plaques can diminish, or the rate of increase can be reduced. Thus, decreases of amyloid-beta plaques can represent a decrease in an etiology of a disease caused by amyloid-beta plaques.

[0158] Through increased fluid flow, the quantity of accumulated amyloid-beta plaques in the subject can be reduced, or the rate of accumulation can be reduced. In some embodiments, the quantity of accumulated amyloid- beta plaques, or the rate of accumulation, is reduced by at least 2%, for example, at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% including ranges between any two of the listed values.

[0159] Detecting Alteration in CSF Flow

[0160] The scLV-scLN path is a newly discovered routes for CSF drainage through the skull base including cribriform plate. The periorbital lymphatics, nasal lymphatics and hard palate submucosa is a newly discovered route for CSF drainage to the submandibular lymph node. Therefore, it is within purview of the present invention to assay for a change in CSF flow rate in a person at these loci.

[0161] In one aspect, a swab, biopsy, or optical method is used to obtain a specimen from the periorbital tissue, nasal mucosa or hard palate submucosa area. This specimen should include periorbital lymphatic cells, nasal lymphatic cells, or hard palate submucosa lymphatic cells. The optical method is employed to measure the emission wavelengths of biomarkers present in these lymphatic cells. The CSF content of the sample is assayed or measured separately in time, and compared from time to time as the assay is repeated in a person and the results are compared.

[0162] As it is discovered that CSF outflow has lessened over a time, then an exogenous agent may be administered to increase CSF outflow in the subject.

[0163] The assay method may include imaging the periorbital tissue, nasal mucosa or hard palate submucosa with a tracer molecule and viewing through a microscope. Other methods may include without limitation, proteomic analysis of CSF specific proteins. Levels of selected proteins in the CSF can be measured from time to time to determine their amounts, wherein as the amounts of selected proteins are decreased or increased, indicates the status of level and quality of drainage of CSF. An example of a selected protein to monitor may include [32 transferrin in the obtained CSF sample. In particular, and for example, increased amount of [32 transferrin present in the sample over time indicates improved CSF drainage function.Decreased amount of ( 2 transferrin present in the sample over time indicates impaired CSF drainage function. Other proteins can be included without limitation.

[0164] Facilitating Cerebrospinal Fluid (CSF) Drainage

[0165] Facilitating CSF drainage is valuable in preventing and treating neurodegenerative diseases, including Alzheimer' disease. Here, this invention provides information on how extracranial approaches and methods can facilitate CSF drainage. This invention encompasses the extracranial methods facilitating CSF drainage through the superficial cervical lymphatic vessels (scLVs) to superficial cervical lymph nodes (scLNs) for preventing and treating neurodegenerative diseases.

[0166] In one aspect, the invention is directed to a method of preventing or treating a central nervous system disease by improving CSF drainage in a person by repairing or at times enlarging the scLV-scLN path. The central nervous system disease may be Alzheimer's disease, Parkinson's disease, or Huntington's disease. The scLV-scLN path may be recovered by administering to a person in need thereof an agent such as without limitation vascular growth factor C (VEGF-C), vascular growth factor D (VEGF-D), fibroblast growth factor 2 (FGF-2), insulinlike growth factor 1 (IGF-1), hepatocyte growth factor (HGF), endothelin-1 (ET-1), angiopoietin- 1, Tie2 agonist, neuropilins, prostaglandin E2, tyramine, sildenafil, viral vector-mediated gene transfer of VEGF-C or VEGF-D or FGF-2 or IGF-1 or HGF or ET-1 or angiopoietin-1 or tyramine or sildenafil.

[0167] The contraction and relaxation of the circular smooth muscles covering the scLVs may be regulated by stimulation and inhibition of smooth muscle cells, such agent being without limitation an agent increasing or decreasing myosin phosphorylation by activating myosin light chain kinase or activating myosin light chain phosphatase by activating catecholamines

[0168] The contraction and relaxation of the circular smooth muscles covering the scLVs may be regulated further by stimulation and inhibition of peripheral nerves, such agent being able to interfere with depolarization or exocytosis of synaptic vesicles such as without limitation tyramine, botulinum toxin or tetanus toxin.20074.002WQ0

[0169] The contraction and relaxation of the circular smooth muscles covering the scLVs may be regulated also by stimulation and inhibition of neurotransmitter, such agent being without limitation agonist, antagonist of neurotransmitter receptor or agonist, antagonist of degradation enzymes at the circular smooth muscle covering the scLV such as agonist or antagonist of norepinephrine.

[0170] The contraction and relaxation of the circular smooth muscles covering the scLVs may be regulated also by regulation of nitric oxide signaling pathway, such agent being without limitation nitric oxide donor or phosphodiesterase 5 (PDE5) inhibitor such as sildenafil.

[0171] The contraction and relaxation of the circular smooth muscles covering the scLVs may be regulated also by mechanical stimulators such as without limitation vibrators, massage device, squeezing device, or optogenetic stimulations by specific wavelength lights.

[0172] As shown in Fig. 29, which shows areas of the human face that should be mechanically stimulated and the direction to enhance CSF outflow. The facial lymphatics and superficial cervical lymphatic vessels run alongside the facial vein. Based on mouse experiments, mechanical stimulation targets the periorbital lymphatics, which run alongside branches of the facial vein in the periorbital area, with stimulation applied from top to bottom (1&2). The nasal side wall lymphatics, which run alongside the external nasal vein, are stimulated from the nose towards the cheek, following their path (3). The palatal lymphatics, which run alongside the labial vein, are stimulated from the center of the lips towards the cheeks. Finally, the collecting lymphatic vessel, which drains into the submandibular lymph node, is targeted with stimulation from the cheekbone area down to the submandibular gland. For simplicity, the figure only shows arrows on one side, but stimulation is performed bilaterally. It is to be understood that the stimulation may occur in any order and any manner for however long it is required so long as mechanical stimulation occurs to direct and enhance CSF outflow in a person.

[0173] The contraction and relaxation of the circular smooth muscles covering the scLVs may be regulated also by high- or low-frequency electrical stimulators.

[0174] Delivery method of the agents may be without limitation, intrathecal approach to CSF space, transnasal or transoral approach to the scLV-scLN path, transdermal, transcervical orpercutaneous or topical approach to facial, and head and neck muscles or cervical lymphatic vessels or cervical lymph nodes or neck spaces.

[0175] The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.

[0176] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

[0177] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses and modifications and equivalents thereof.

[0178] Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

[0179] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and / or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and / or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and / or the use of the term in the present document shall prevail.

[0180] It is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

[0181] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0182] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes20074.002WQ0 of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0183] EXAMPLES

[0184] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0185] Example 1: Facilitating cerebrospinal fluid (CSF) drainage is valuable in preventing and treating neurodegenerative diseases, including Alzheimer' disease. Here, this invention provides information on how extracranial approaches and methods can facilitate CSF drainage.

[0186] This invention encompasses extracranial methods that facilitate CSF drainage through the superficial cervical lymphatic vessels (scLVs) and the superficial cervical lymph nodes (scLNs) to prevent and treat neurodegenerative diseases.

[0187] An anesthetized mouse (10 weeks-old male or femaleProx-1 GFP mouse) was laid prone on a stereotaxic frame under a microscope equipped with a heating pad. The head was adjusted to a 90° angle to the body axis with the help of a mouthpiece to facilitate access to the cisterna magna. After a skin incision along the midline of the posterior neck, muscle layers were carefully separated with microreactors. The atlanto-occipital membrane overlying the cisterna magna was superficially penetrated using a 33-gauge NanoFil needle (World Precision Instruments). Then lp.1 of tetramethlyrhodamine (TMR)-conjugated 10 kDa MW dextran (Invitrogen, D1816) was injected into the subarachnoid space at 1 p.l / min for 1 min using a microsyringe (88000, Hamilton) and a micro-infusion machine (Fusion 100, Chemyx Inc). The needle was slowly removed after the mouse was left in position for five minutes to prevent CSF leakage.20074.002WQ0The muscle layers and neck skin were sutured with 6-0 black silk (Ailee, SK617). At 60 min after the infusion, the head was dissected without cardiac perfusion or soaking in saline. The fluorescence image of the dissected head was acquired using a fluorescence stereo zoom microscope (AxioZoom V16, Carl Zeiss).

[0188] Superficial cervical lymph nodes are composed of submandibular lymph node, accessory submandibular lymph node and parotid lymph node, with variations in the location of accessory submandibular lymph node (Fig. 4). Two-thirds of the submandibular lymph node had three afferent superficial cervical lymphatics, scLV-1, scLV-2, and scLV-3, while one-third had additional superficial cervical lymphatics, scLV-4, without sex differences (Fig. 5)

[0189] Example 2: The same procedure as in Example 1 was applied to an anesthetized mouse (10 weeks-old male Proxl-GFP mouse). The mouse was sacrificed by cutting the abdominal aorta at 30 or 60 min after the injection (Fig. 6A). After dissection of surrounding muscles with a surgical microscope (SZX16, Olympus), highly accumulated TMR-dextran in the submandibular lymph node, accessory submandibular lymph node, and afferent lymphatic vessels (scLVs) was imaged using a fluorescence stereo zoom microscope (Fig. 6B). TMR-dextran was detected in the submandibular lymph node and accessory submandibular lymph node but not in the parotid lymph node (Fig.6B).TMR-dextran was notably detected within scLV-1 at 30 min and equally within scLV-1 and scLV-2 at 60 min. In contrast, it was not detected within scLV- 3 at either time point after the intracisternal infusion (Fig. 6C). These data revealed that about 50% of total CSF outflow into the cervical lymph nodes is drained into the superficial cervical lymph nodes.

[0190] Example 3: Anesthetized mouse (10 weeks old male Proxl-GFP mouse) was subjected to scLV and smLN tissue collection for diameter and lymphangion length measurement and immunofluorescence staining (IFS). For IFS, mice were anesthetized and perfused with ice-cold phosphate buffer saline (PBS) through the left ventricle after puncturing the right atrium. After PBS perfusion, 2% paraformaldehyde solution was injected through the left ventricle to fix the tissue. The scLVs were dissected and collected. The collected tissues were fixed with 2% paraformaldehyde solution for 2 hours at 4 Celsius degree. After the fixation, the scLVs were incubated in 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) for 1 hour atroom temperature. To label asmooth muscle actin (otSMA) positive smooth muscle cell, the scLV was immersed in 5% normal donkey serum containing otSMA-Cy3 antibody (C6198, Sigma) dissolved at 1:2000 at 4 Celsius degree for 12 hours. After PBS washing, the tissues were covered with a mounting medium (H1200, Vector). The image was acquired by using a fluorescence stereo zoom microscope (AxioZoom V16, Carl Zeiss) or confocal microscope (LSM 880, Zeiss) with Plan- Apochromat lOx / NA 0.45 lens. Diameter, lymphangion length, and smooth muscle coverage were quantified. As shown images and comparisons in Fig. 7, the mean diameters, lymphangion length, a-smooth muscle actin positive smooth muscle coverage at the mid-lymphangion and peri-valvular areas of scLV-1 and scLV-2 were similar. However, scLV-3 showed less, shorter, and similar measurements, without sex differences. These results support that the CSF can be drained through different upstream routes in similar quantity.

[0191] Example 4: To quantify dynamics of scLVs, intravital imaging of scLVs of Proxl-GFP mice was performed. An anesthetized mouse (10 weeks-old male or female Prox-1 GFP mouse) was laid in a head-back supine position. The anesthetic drug was a mixture of urethane (1.5mg / kg) and ketamine / xylazine (10 mg / kg, 1 mg / kg). Under a fluorescence surgical microscope (SZX16, Olympus), the skin was removed and underlying scLV-1 and scLV-2 were exposed. Sterile saline was supplied to prevent drying. After 20 min for stabilization of scLVs, intravital imaging was acquired using a high-speed confocal microscope (IVIM-CM3, IVIM Technology). Images were acquired at 20 frames per second for five minutes with a 10x / 0.45 lens. Images for one minute were randomly selected from the five minutes. Mean diameter, frequency of contraction and relaxation, amplitude of contraction and relaxation, ejection fraction, and fractional pump outflow were measured with custom MATLAB code. As shown in Fig. 8, spontaneous contraction and relaxation were imaged, and mean diameter, frequency of contraction and relaxation, amplitude of contraction and relaxation, ejection fraction, and fractional pump outflow were not different between scLV-1 and scLV-2, without sex difference. Example 5: The same procedures in Examples 1 and 2 were applied to an anesthetized mouse (10 weeks-old male or female Proxl-GFP mouse). The scLVs were dissected and imaged using a fluorescence stereo zoom microscope (Fig. 10). One of the afferent cervical lymphatics to the accessory submandibular lymph node was found to contain TMR-dextran 60 min afterintracisterna I infusion, which was derived from a downstream branching lymphatic of nasopharyngeal lymphatic plexus (Ref. 8) (Fig. 10). These findings demonstrate that the superficial and deep cervical lymphatic systems for CSF drainage are connected through this connecting lymphatic.

[0192] Example 6: To verify the afferent lymphatic connections to the submandibular lymph node, the same procedure as in Example 1 was applied to an anesthetized mouse (10 weeks-old male or female Proxl-GFP mouse). The mouse was sacrificed by cutting the abdominal aorta 60 min after the injection. The scLV-1 was connected to lymphatic vessels in the periorbital region, and injected TMR-dextran was detected inside the periorbital lymphatics and scLV-1 (Figs. 11B- C). To verify the finding, 1 pl of the fluorescent microbeads (0.5 pm in diameter, F8887, ThermoFisher) was injected into the intracranial cavity at 1 pl / min for 1 min using a micro-syringe (88000, Hamilton) and infusion machine (Fusion 100, Chemyx Inc). One hour later, the periorbital lymphatics were sampled and imaged with confocal microscope (LSM 880, Zeiss). We found that the fluorescent microbeads were abundantly and selectively present in the periorbital lymphatic vessels and the lymphatic vessels running along the infraorbital artery through the orbital fissure (Figs. 11D-F), confirming that the periorbital lymphatics are connecting lymphatics to scLV-1 to submandibular lymph node for CSF drainage (Fig. 11G).

[0193] Example 7: To verify the scLV-2 connections to submandibular lymph node, tissue collection and IFS were performed using the same procedures as in Example 3 on an anesthetized mouse (10 weeks-old male or female Proxl-GFP mouse). The scLV-2 was connected to lymphatic vessels in the nasal cavity and hard palate, and injected TMR-dextran was detected inside the nasal cavity lymphatics, hard palate lymphatic plexus, and scLV-2 (Fig. 12, Figs. 14-16). Injected microbeads were observed in the lymphatic vessels running along the great palatine artery through the great palatine canal, in the lymphatic vessels within the incisive foramen, which serves as a passage to the nasal cavity, in the hard palate lymphatic plexus connecting from these two pathways, and in scLV-2 connecting from the anterolateral portion of the hard palate lymphatic plexus (Fig. 14). Additionally, injected microbeads were observed in the olfactory mucosal lymphatic vessels, respiratory mucosal lymphatic vessels, and nasal sidewall lymphatic vessels, all connected from the meningeal lymphatic vessels surrounding the olfactory bulb toscLV-2 (Figs. 15-16). To characterize the hard palate lymphatic plexus, the hard palate tissue was immersed in 5% normal donkey serum containing LYVE1 antibody (11-034, Angiobio), VEGFR3 antibody (AF743, R&D), CD31 antibody (AF806, R&D), or Laminin-a5 antibody (EWL004, kerafast) dissolved at 1:400 at 4 Celsius degree for 12 hours. To label a smooth muscle actin (otSMA) positive smooth muscle cell, the scLV was immersed in 5% normal donkey serum containing otSMA-Cy3 antibody (C6198, Sigma) dissolved at 1:2000 at 4 Celsius degree for 12 hours. After PBS washing, the tissue was incubated in normal donkey serum containing Alexa-594 or Alexa- 647 conjugated anti-rabbit or anti-goat or anti-hamster IgG antibody (Jackson ImmunoResearch) for 12 hours at 4 Celsius degrees. The hard palate lymphatic plexus had semilunar or irregular, linearly shaped valves, which stained for Proxl-GFP and Iaminin-a5, but no smooth muscle coverage was evident after a smooth muscle actin staining. The segments between valves (lymphangions) were unusually short (Fig. 13).

[0194] Example 8: An anesthetized mouse (10 weeks-old male or female Proxl-GFP mouse) was laid laterally under a microscope equipped with a heating pad. Then, 0.5 pl of TMR- conjugated 10 kDa MW dextran (Invitrogen, D1816) was injected into the subdermal layer of various skin region (nasal and medial canthus region, lateral canthus and anterior auricular region, and cheek and cheen region) with 1 pl / min. After 15 min, the skin was dissected and underlying lymphatic vessels were imaged with a fluorescence stereo zoom microscope (AxioZoom V16, Carl Zeiss). As a result, TMR-dextran was detected only in parotid lymph node, not in submandibular lymph node and accessory submandibular lymph node (Fig. 17). These results indicate that the parotid lymph node is a primary node for facial lymphatic drainage.

[0195] Example 9: To determine whether the findings observed in rodents also apply to primates, Macaca fascicularis was subjected to CSF injection experiments. An anesthetized primate (6-8 years-old male or female) was positioned in the sphinx posture using a custom-built stereotaxic frame for an image-guided stereotactic system under isoflurane anesthesia (1.5% in 2 L / min oxygen). The correct placement of the needle tip within the cisterna magna was confirmed using XperCT (Phillips) imaging, followed by CSF release to verify accurate positioning. Oxygen saturation (SpO2), heart rate, respiration rate, and blood pressure were continuously monitored and maintained within normal ranges. Before injection of fluorescent microbeads (0.520074.002WQ0 pm in diameter, F8887, Thermo-Fisher), CSF was released in a volume of 1 ml using a 23-gauge needle connected to a 10 ml Hamilton syringe via a Dual Removable Needle Coupler (Hamilton, Franklin, MA, USA). After CSF release, fluorescent microbeads were injected into the subarachnoid space at 250 pl min1for 10 min using a micro-infusion machine (World Precision Instruments, Sarasota, FL, USA). The needle was left in the position for 20 min and slowly removed from the monkey to prevent a CSF leakage. After infusion, the monkey was weaned off anesthesia and maintained in an awake state under vital monitoring. At 3 hours after the injection, the carotid artery was cannulated for ice-cold PBS perfusion. After PBS perfusion, 4% paraformaldehyde solution was perfused. The submandibular lymph nodes, parotid lymph nodes and retropharyngeal lymph node were collected (Fig. 18). After fixation, the lymph nodes were incubated in 2% paraformaldehyde solution for 24 hours at 4 Celsius degree, washed with PBS, and dehydrated with 30% sucrose solution for at least three days at 4 Celsius degree. The dehydrated lymph nodes were embedded with optimal cutting temperature compound solution (Leica) and frozen for cryosection. The lymph nodes were sectioned to 20 pm thickness and immersed in 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) for 1 hour at room temperature. To label lymphatic vessel markers, the tissue was immersed in 5% normal donkey serum containing LYVE1 antibody (DP3500, Origene) dissolved at 1:400 at 4 Celsius degree for 12 hours. After PBS washing, the tissue was incubated in normal donkey serum containing Alexa-488 conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch) for 12 hours at 4 Celsius degree. After PBS washing, the tissues were covered with a DAPI containing mounting medium (H1200, Vector). Image was acquired by a confocal microscope (LSM 880, Zeiss) with a Plan-Apochromat lOx / NA 0.45 lens. As shown in Fig. 18, the intracisternal injected microbeads were detected within the submandibular lymph nodes and retropharyngeal lymph nodes but not in the parotid lymph nodes.

[0196] Example 10: To verify the role of hard palate lymphatic plexus as a CSF drainage route in primates, we collected hard palate mucosa and submucosa of Macaca fascicularis. With the same procedure as in Example 9, the hard palate was perfused with PBS and 4% PFA. The hard palate was dissected from the hard palate bone (Fig. 19). The isolated hard palate mucosa and submucosa were imaged with a fluorescence stereo zoom microscope (AxioZoom V16, Carl20074.002WQ0Zeiss). As a result, red microbeads were presented in the hard palate submucosa (Fig. 19). These results confirm that the CSF is drained to the submandibular lymph node through the same routes observed in rodents.

[0197] Based on Figs. 3-19, schematic diagrams of the scLV-scLN path are shown in Fig. 1 and 2.

[0198] Example 11: To verify decreased CSF drainage to the submandibular lymph node in aged mice, TMR-dextran was injected into the cisterna magna using the same procedure as in Example 1. After one hour, TMR-dextran signal intensity was measured in the submandibular lymph node of both in adult (8-12 weeks-old) and aged (82-105 weeks-old) C57BL / 6J mice. As shown in Fig. 20, the signal intensity of TMR-dextran was decreased in aged submandibular lymph node.

[0199] Example 12: To verify the effect of aging on upstream CSF draining lymphatic routes, we collected periorbital tissue (containing lymphatics), nasal mucosa and hard palate mucosa from aged mice (82-105 weeks-old male or female Proxl-GFP mice). Tissue collection and IFS were performed using the same procedure as in Example 3 and 7. As shown in Fig. 21A, analysis of the hard palate lymphatic plexus in aged mice revealed that the VEGFR3+lymphatic area was 9-17% less, the number of lymphatic valves was 43-71% less, and the lymphatic diameter was 11- 12% greater, but the LYVE1 staining was not different from that in adult mice (Fig. 21A). Nasal lymphatics, upstream connection of the scLV, were also compared by age and lymphatic area was ~80% less (Fig. 21B).

[0200] Example 13: To determine the effect of aging on scLVs, upstream lymphatic vessels of aged mice were collected and compared. Young (8-12 weeks-old male or female Proxl-GFP mice) and aged (82-105 weeks-old Proxl-GFP mice) mice were sacrificed. After the same procedure as in Example 7, the scLVs and the submandibular lymph node were collected. The collected tissues were fixed by 2% paraformaldehyde solution for 2 hours ay 4 Celsius degree. After the fixation, the collected tissue was incubated in 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) for 1 hour at room temperature. To label a smooth muscle cells, the collected tissue was immersed in 5% normal donkey serum containing otSMA-Cy3 antibody (C6198, Sigma)dissolved at 1:2000 at 4 Celsius degree for 12 hours. After PBS washing, the tissues were20074.002WQ0 covered with a mounting medium (H1200, Vector). The image was acquired by a confocal microscope (LSM 880, Zeiss) with a Plan-Apochromat lOx / NA 0.45 lens. As shown in Fig. 22A, lymphangion length, diameter and smooth muscle coverage at mid-lymphangion were not different in aged scLVs.

[0201] Example 14: To determine ageing effect on scLVs, intravital imaging was performed using the same procedure as in Example 4 on aged mice (82-105 weeks-old male or female Proxl- GFP mice). As shown in Fig.22B, differences were found in the mean diameter (20% greater), amplitude (80% greater), and ejection fraction during spontaneous contraction and relaxation (30% larger) but no difference in frequency and fractional pump flow of scLV-1 in aged mice compared to younger adults. To measure synchronous contraction and relaxation along the length of scLVs, cross correlation of diameter changes at five location, spaced 40 pm apart, was measured. The cross correlation was not changed in aged scLVs.

[0202] Based on Figs. 20-22, ageing decreases CSF drainage through the superficial cervical lymphatic vessels to superficial cervical lymph nodes (scLV-scLN path). The upstream connections of the scLV-scLN path are damaged and atrophied, but scLVs were not. Therefore, restoring upstream lymphatic connections or manipulating scLVs may increase reduced CSF drainage through the scLV-scLN path.

[0203] Example 15: Pharmacological stimulation (example ofscLVflow agent): To determine the effect of catecholamine releasing agents on scLV for modulating CSF drainage to submandibular lymph node, adult mice (8-10 weeks-old male or female Proxl-GFP mice) were used. To confirm which type of nerve fiber, adrenergic or cholinergic, is innervating the scLVs, we performed IFS using the same procedure as in Example 7. The scLVs and submandibular lymph nodes were collected and the tissues were fixed by 2% paraformaldehyde solution for 2 hours at 4 Celsius degree. After the fixation, the collected tissue was incubated in 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) for 1 hour at room temperature. To label sympathetic nerves, the collected tissue was immersed in 5% normal donkey serum containing tyrosine hydroxylase antibody (AB152, Merck) dissolved at 1:400 at 4 Celsius degree for 12 hours. To label parasympathetic nerves, the collected tissue was immersed in 5% normal donkey serum containing vesicular acetylcholine transporter (VAChT) antibody (ABN100, Merck) dissolved at20074.002WQ01:400 at 4 Celsius degree for 12 hours. After PBS washing, the tissue was incubated in normal donkey serum containing Alexa-647 conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch) or anti-goat IgG antibody (Jackson ImmunoResearch) for 12 hours at 4 Celsius degrees. After PBS washing, the tissues were covered with a mounting medium (H1200, Vector). The image was acquired by a confocal microscope (LSM 880, Zeiss) with a Plan-Apochromat lOx / NA 0.45 lens. As shown in Fig. 23, the scLVs were innervated by tyrosine hydroxylase positive sympathetic nerves, but were not innervated by VAChT positive parasympathetic nerves. To determine whether catecholamine releasing agent, tyramine (10 pM, T90344, Sigma) affect spontaneous contraction and relaxation of scLV, tyramine was topically applied to scLVs under a high-speed confocal microscope (IVIM-CM3, IVIM Technology). As shown in Fig. 23, topical tyramine generated variable changes in the mean diameter, amplitude of contraction and relaxation, ejection fraction, and fractional pump flow on scLV-1, indicating that control of contraction or relaxation of scLV is feasible.

[0204] Example 16: Electrical stimulation (example of scLV flow agent): To determine the effect of electrical stimulation on scLV for modulating CSF drainage to submandibular lymph node, adult mice (8-10 weeks-old male or female Proxl-GFP mice) were used. A electrical current pulse was delivered via a bipolar electrode connected to an isolated pulse stimulator (Model 2100, A-M systems). Under a high-speed confocal microscope (IVIM-CM3, IVIM Technology), the bipolar electrode was located. The concentric bipolar electrode (CBBPE75, FHC Inc.) was placed at the middle of the scLV and single electric current stimulation was applied with the following settings: 10 seconds of stimulation duration, 40 pA, pulse duration of 10 seconds. Applying a single electrical stimulation to scLV-1 caused a transient, strong contraction followed by a return to the original diameter (Fig. 24A). For repeated electrical stimulation, the parallel bipolar electrode (30210, FHC Inc.) was located along the entire length of one lymphangion of scLV-1 and the electric current stimulation was applied with the following settings: 9 minutes of stimulation duration, 40 pA, 0.2 Hz, pulse duration of 0.5 seconds. The mean diameter increased, but amplitude, frequency, ejection fraction and fractional pump flow showed variable changes with this electrical stimulation (Fig. 24B). These results may indicate that electrical stimulation affects physiological properties of scLV.20074.002WQ0

[0205] Example 17: Mechanical stimulation (example of scLV flow agent): To determine the effect of mechanical stimulation on scLV for increasing CSF drainage to submandibular lymph node, adult mice (8-10 weeks-old male or female Proxl-GFP mice) were used.

[0206] For precise administration of mechanical stimulation and quantification of its force, we developed a custom force measurement device as shown in Fig. 25A. The device measures the force of mechanical stimulation in real time. Mechanical stimulation was performed in two intensities: low-magnitude (under 0.04 kgf) and high-magnitude (over 0.04 kgf) (Fig. 25B). Taking advantage of what we learned about the distribution of head and neck lymphatics that carry CSF to superficial cervical lymph nodes in mice, we compared the effects of direct, downward sweepingstrokes by the mechano-stimulator on three regions of intact skin: (1) from the periorbital area to the mandible; (2) from the nasal sidewall to the mandible; and (3) from rostral to caudal along the path of scLV-1 and scLV-2 to the submandibular lymph node (Fig. 25C). Each session consisted of two cycles of 10 strokes each of 2 sec duration (4 strokes to region 1, 4 strokes to region 2, and 2 strokes to region 3). Two 10-stroke cycles over 40 sec were followed by a 20-sec rest period.

[0207] As shown in Fig. 26A, to determine the effect of mechanical stimulation on scLV for increasing CSF drainage within scLV, the TMR-dextran was injected using the same procedure as in Example 1. The stimulation was applied either low-magnitude (under 0.02kgf) or high-magnitude (over 0.04kgf). At 30 min after cisterna magna injection of TMR-dextran, mechanical stimulation was applied for five minutes. The skin was then removed to visualize scLVs, and the TMR-dextran signal in scLVs was imaged and measured with a fluorescence stereo zoom microscope (AxioZoom V16, Carl Zeiss). TMR-dextran fluorescence doubled (2.29-fold increase) in scLV-1 and scLV-2 after low-magnitude stimulation over 5 min. However, the scLV-1 and scLV-2 was constricted in places and the TMR-dextran fluorescence decreased (9.22-fold) in scLV-1 and scLV- 2 after high-magnitude stimulation over 5 min (Fig. 26B). TMR-dextran accumulation in the ipsilateral lymph node doubled (1.97-fold increase) after low-magnitude mechanical stimulation over 20 min, but decreased accumulation (3.11-fold) was found after high-magnitude stimulation or in the contralateral lymph node (Fig. 26C).

[0208] Example 18: To determine the underlying mechanism of mechanical stimulation, we blocked the nitric oxide signaling pathway with N-Nitro L-arginine methyl ester (L-NAME). L-20074.002WQ0NAME was injected intraperitoneally (1 mg / kg). Intravital imaging was performed using the same procedure in Example 4. Intravital imaging analysis showed that in L-NAME treated mice compared to saline treated mice, the mean diameter, amplitude, ejection fraction, and fractional pump flow was decreased, while the frequency remained unchanged in scLV-1 (Fig. 27A). The L- NAME was injected intraperitoneally (1 mg / kg) at 90 min before TMR-dextran injection into cisterna magna using the same procedure as in Example 1. After injection, the low-magnitude (under 0.02 kgf) mechanical stimulation was administrated to the mice using the same procedure as in Example 17. L-NAME reduced by 55% the accumulation of TMR-dextran in the submandibular lymph node after 20 1-min sessions of low-magnitude stimulation. These data indicate that mechanical stimulation enhances CSF drainage and that nitric oxide signaling is essential for the maximum effect of the stimulation.

[0209] Example 19: To determine whether the low-magnitude (under 0.02 kgf) mechanical stimulus enhances CSF outflow to the submandibular lymph node in aged mice, the low- magnitude (under 0.02 kgf) mechanical stimulation was administrated to aged mice (82-105 weeks- old Proxl-GFP mice) using the same procedure as in Example 17. The low-magnitude (under 0.02 kgf) mechanical stimulus enhanced the TMR-dextran signal of the submandibular lymph node by 2.4-fold in aged mice (Fig. 28A). The low-magnitude (under 0.02 kgf) stimulation also increased the TMR-dextran intensity in scLVby 4.7-fold in aged mice (Fig. 28B).

[0210] Based on Examples 15-19, these findings imply that the contraction and relaxation of the circular smooth muscles covering the scLVs can be regulated by 1) pharmacologically (catecholamine releasing agent or nitric oxide signaling pathway related agent or smooth muscle contraction / relaxation related agent such as PDE5 inhibitor), 2) electrically, 3) mechanically. Basically, CSF drainage can be facilitated by extracranial manipulations, administration of agents, and regulators. Considering that there is no prominent change in aged scLVs, reduced CSF drainage can be enhanced by extracranial manipulations of scLVs.

[0211] Together, these examples provide information on where CSF drainage occurs and how extracranial approaches and methods can facilitate it. This invention discloses that 1) the scLV- scLN path is a newly discovered route for CSF drainage through the skull base and cribriform plate, 2) the periorbital lymphatics, nasal lymphatics and hard palate lymphatic plexus are newlydiscovered upstream lymphatics of the scLV-scLN path for CSF drainage, and 3) scLVs can be regulated by stimulation and inhibition of the ensheathing smooth muscle cells, peripheral nerves, neurotransmitters, and electrical or mechanical stimulation as CSF drainage can be facilitated by extracranial manipulations, administration of agents, and related regulators for preventing and treating neurodegenerative diseases including Alzheimer's disease.

[0212] Precision Force-Regulated Mechano-Stimulator

[0213] In some embodiments, a precision force-regulated mechano-stimulator for applying controlled mechanical stimulation can be used by applying the device to superficial cervical lymphatic vessels to facilitate cerebrospinal fluid drainage. The device enables precise, reproducible mechanical stimulation with real-time force monitoring and feedback control, as demonstrated in the experimental results shown in Figures 25-28.

[0214] Referring to Figure 30, the electronic control system of the precision force-regulated mechano-stimulator comprises a force sensor 3002, a microcontroller 3004, which in some cases may be an Arduino Nano board, and a capacitance sensing board, such as, for example, an FDC2214EVM board sold by Texas Instruments. The force sensor 3002 may be configured as a capacitive sensor that detects mechanical deformation caused by applied force during stimulation. The microcontroller 3004 may be configured to provide real-time data acquisition and processing capabilities with sufficient computational power for sensor signal processing and communication tasks.

[0215] In some instances, the capacitance sensing board (e.g., FDC2214EVM board) 3006 functions as a capacitance-to-digital converter that interfaces with the force sensor 3002 to measure changes in capacitance resulting from applied force. The capacitance sensing board 3006 provides high-resolution capacitance measurements, and in some cases, provides 28-bit resolution and low noise characteristics, enabling precise force detection in the therapeutic range.

[0216] According to some embodiments, the system architecture establishes electrical connections 3008 between components as follows: the force sensor 3002 connects to input terminals 3010 of the capacitive sensing board 3006 through shielded cables to minimize electromagnetic interference. The capacitive sensing board 3006 communicates with the20074.002WQ0 microcontroller 3004 via l2C communication lines 3012, specifically through SDA (serial data) and SCL (serial clock) connections, enabling high-speed bidirectional data transfer at rates up to 400 kHz. A power source, such as an AC to DC 12V adapter provides appropriate voltage levels to each component, with the microcontroller 3004 supplying regulated 5V and / or 3.3V power rails as required. The microcontroller 3004 may connect to an external computer or display device, such as through a USB interface, for data logging, real-time visualization, and user interface functionality.

[0217] Calibration and Performance Characteristics

[0218] Referring to Figure 31, the calibration plot 3100 demonstrates the relationship between sensor readings and actual applied force values. The horizontal axis 3102 represents the actual force values in kilogram-force (kgf), while the vertical axis 3104 represents the corresponding sensor readings in kgf. The calibration data points 3106 show measurements taken at multiple force levels within the operational range of 0 to 0.05 kgf.

[0219] The calibration curve 3108 demonstrates excellent linearity between sensor readings and actual force values, with the sensor readings adjusted to be directly proportional to the actual values. This linear relationship indicates a correlation coefficient greater than 0.99, enabling accurate and reliable force measurements throughout the therapeutic range. The calibration encompasses both the low-magnitude stimulation range 3110 (0.01-0.04 kgf) and extends to cover the high-magnitude stimulation range 3112 (0.04-0.05 kgf), ensuring accurate force monitoring across all therapeutic applications.

[0220] In some cases, the calibration process involves applying known reference forces using calibrated weights or a precision force gauge, recording the corresponding sensor output values, and generating a calibration function that converts raw sensor readings to calibrated force values. The calibration parameters, which may include offset and gain values, are stored in the microcontroller's non-volatile memory and applied in real-time during device operation.

[0221] Mechanical Assembly Configuration

[0222] Referring to Figure 32, the diagram illustrates an exploded view showing the assembly configuration of the precision force-regulated mechano-stimulator's mechanical components 3200. The device comprises four primary structural parts 3202, 3204, 3206, and 3208, each ofwhich may be manufactured through any suitable manufacturing technique, including additive manufacturing processes (e.g., 3D printing), material removal processes (e.g., machining), molding, casting, or other manufacturing techniques.

[0223] The first component 3202 serves as the main housing, containing the electronic control system. The second component 3204 functions as the sensor mounting bracket, securing the force sensor in proper alignment with the mechanical load path. The third component 3206 operates as the shaft, providing a rigid mechanical link between the sensor assembly and the tip holder while maintaining precise axial alignment and enabling quick attachment and removal of various stimulation tip configurations. The fourth component 3208 acts as the handle providing an ergonomic grip surface.

[0224] In some embodiments, the assembly 3200 utilizes interlocking features between adjacent components, which may aid in proper alignment and secure mechanical coupling. In some cases, the modular design incorporates alignment guides that facilitate proper component orientation during assembly. Cable routing channels may be integrated into the structural components to protect sensor wiring from mechanical damage and electromagnetic interference.

[0225] The components, which may be 3D printed, are preferably fabricated using biocompatible materials such as medical-grade ABS or polycarbonate, with layer resolution on the order of 0.1-0.2 mm to ensure smooth surface finish and dimensional accuracy. The design files may enable customization of component dimensions to accommodate different hand sizes or specific clinical requirements.

[0226] Force Sensor Fabrication Process

[0227] Referring to Figure 33, the sensor fabrication process creates a multilayer capacitive force sensor with high sensitivity and repeatability. The process begins with preparing a silicone base layer 3302, which may have a thickness of on the order of about 0.5 mm using a film applicator 3304. In some examples, the silicone material comprises Ecoflex™ 00-10 (Smooth-On Inc.), a two-part platinum-catalyzed silicone rubber that provides appropriate flexibility and mechanical properties. The base layer 3302 may be cured in an oven at 70°C for 10 minutes.20074.002WQ0

[0228] A thin adhesion layer 3306, which may be of the same silicone material, may have a thickness of about 0.15 mm, and may be applied to the cured base layer 3302. A first conductive fabric layer 3308, which may comprise a stretchable conductive fabric, which in some cases may be 4800 (Holland Shielding Systems) is positioned on the adhesion layer 3306. A roller 3310 may be used to apply uniform pressure to ensure complete bonding and eliminate air bubbles between layers.

[0229] The assembly may be flipped and the process continues with application of a second silicone layer 3312 and a second conductive fabric layer 3314 using the same techniques. The assembled layers may be optionally inverted, and a third adhesive layer 3315 and a third conductive fabric layer 3316 is attached to the opposite surface, creating a five-layer structure with the configuration: fabric / silicone / fabric / silicone / fabric. This symmetric arrangement provides differential capacitive sensing while minimizing environmental effects.

[0230] The multilayer structure may be subject to cutting, such as by laser cutting 3318 to achieve desired dimensions. In some embodiments, the structure is cut to a size of 70 mm x 5 mm, although it should be appreciated that any suitable size can be fabricated. Electrical connections may be established through any suitable technique, which may include using conductive clamps 3320 attached to each conductive fabric layer. Insulated wires 3322 connect to the clamps 3320, and may use heat shrink tubing to provide strain relief and electrical insulation.

[0231] In some examples, the sensor assembly is placed in a strap mold 3326 for final encapsulation. In some embodiments, an encapsulating material, such as, for example, Dragon Skin™ 10 silicone (Smooth-On Inc.) is poured into the mold 3326, surrounding the sensor while maintaining electrical connections. The encapsulation may cure at room temperature, forming a durable, flexible strap configuration that protects the sensor elements while allowing mechanical deformation during force measurement.

[0232] Complete Device Assembly

[0233] Referring to Figures 34A, 34B, and 34C, the complete precision force-regulated mechano-stimulator 3400 is shown. Specifically, the assembled device is shown in Figure 34A,while a schematic view of the components is shown in Figure 34B and a closeup of the replaceable tip is shown in Figure 34C.

[0234] The device comprises an amplifier unit 3402 that houses the electronic control system including the microcontroller. In some examples, the amplifier unit 3402 features a compact enclosure with dimensions optimized and may include indicator lights 3404 for power status and / or operational feedback.

[0235] The force sensor 3406 is preferably positioned between the amplifier unit 3402 and the handle 3408, within the mechanical load path to accurately measure applied forces. The force sensor 3406, which may be fabricated according to the process shown in Figure 33, comprises the multilayer silicone and conductive fabric structure that provides capacitive force sensing. The sensor 3406 is mechanically coupled to both the handle 3408 and the shaft 3410 to ensure all applied forces are transmitted linearly through the sensing element.

[0236] The handle 3408 may extend any suitable length to provide for comfortable usage, and in some cases, is approximately 9 cm in length, providing an ergonomic gripping surface for the operator. In some cases, the handle 3408 incorporates textured surfaces or rubber overmolding to enhance grip security during treatment procedures. The internal cavity 3414 of the handle 3408 accommodates routing of sensor cables and may house additional components such as wireless communication modules or battery power supplies in portable configurations.

[0237] The shaft 3410 connects the force sensor 3406 to the tip assembly 3416, transmitting mechanical forces while maintaining precise positional control. In some cases, the shaft 3410 comprises a rigid member, potentially hollow to accommodate cable routing, with sufficient strength to prevent deflection under the maximum applied forces. The length and diameter of the shaft 3410 may be selected to provide adequate reach while maintaining operator control and comfort.

[0238] In embodiments, the replaceable tip assembly 3416 includes a mounting rod 3418 of approximately 1 cm length that securely connects to the distal end of the shaft 3410. The connection mechanism may comprise a threaded engagement, bayonet coupling, friction fit, or some other removable coupling, thus enabling quick tip changes for different treatment applications or hygiene requirements.

[0239] In some embodiments, the stimulation tip 3422 comprises an oval-shaped cotton ball with a major axis 3424 of approximately 1 cm and a minor axis 3426 of approximately 0.5 cm. This elliptical configuration provides a desirable contact area for targeted stimulation of superficial cervical lymphatic vessels while maintaining patient comfort. The cotton material offers appropriate compliance and texture for skin interface, though alternative materials such as medical-grade silicone, memory foam, hydrogel, or other materials may be substituted based on specific therapeutic requirements.

[0240] Communication cables may be used to connect the amplifier unit 3402 to a personal computer or data acquisition system, enabling real-time force monitoring, data logging, and treatment parameter adjustment.

[0241] Operational Characteristics and Clinical Application

[0242] The precision force-regulated mechano-stimulator 3400 may be configured to operate within specific force ranges optimized for lymphatic stimulation. For example, the low- magnitude stimulation mode may utilize forces between 0.01-0.04 kgf, which experimental data has shown to produce optimal enhancement of CSF drainage with a 2.29-fold increase in lymphatic flow. The high-magnitude stimulation mode may employ forces between about 0.04- 0.08 kgf, though experimental results indicate this range may be less desirable due to potential vessel constriction which may impede lymphatic drainage enhancement.

[0243] During operation, the microcontroller samples the force sensor at a desirable sample rate, which may be up to 100 Hz, or more, thus providing real-time force feedback to guide the operator in maintaining therapeutic force levels. The system may incorporate visual indicators 3404 on the amplifier unit 3402, such as LEDs that illuminate green when force is within the therapeutic range, yellow when approaching limits, and red when exceeding recommended levels, in some examples.

[0244] Auditory feedback may supplement visual indicators, with distinct tones indicating proper force application versus excessive or insufficient force. This multimodal feedback may be used to promote consistent treatment delivery even when the operator's visual attention is focused on tip positioning rather than the display.20074.002WQ0

[0245] The device implements the validated stimulation protocol targeting three anatomical regions: the periorbital area to mandible pathway, the nasal sidewall to mandible pathway, and the pathway along superficial cervical lymphatic vessels to the submandibular lymph node. According to some embodiments, the treatment sessions consists of systematic strokes applied with the calibrated force, following a predetermined pattern, which in some cases is 4 strokes to the periorbital region, 4 strokes to the nasal sidewall region, and 2 strokes along the cervical lymphatic vessels, with this cycle repeated twice followed by a rest period.

[0246] It should be apparent to those of skill in the art that the described and shown device 3400 is merely shown as an example, and the actual device may be formed with substantially different shapes, sizes, and configurations without departing from the spirit of the disclosure and the device's ability to deliver targeted force within a narrow low-pressure range to produce therapeutic force levels to encourage drainage.

[0247] Data logging capabilities may record comprehensive treatment information including timestamp data, continuous force profiles, stroke counts, treatment duration, and protocol adherence metrics. This information supports clinical documentation, enables outcome analysis, and facilitates treatment optimization through evidence-based protocol refinement.

[0248] The modular design philosophy extends throughout the device architecture, enabling component upgrades, repairs, replacements, and customization without complete device replacement. Firmware updates can be deployed to the microcontroller, such a through a USB connection, allowing implementation of improved algorithms, additional safety features, or modified treatment protocols based on emerging clinical evidence.

[0249] Figures 35A and 35B illustrate alternative embodiments of a wearable mechanical stimulation device 3500 for facilitating cerebrospinal fluid drainage through superficial cervical lymphatic vessels. The device comprises a face mask configuration with integrated stimulation elements 3504 positioned to target specific anatomical regions associated with the superficial cervical lymphatic system.

[0250] Fig. 35A shows a frontal view of the device 3500 worn on a subject's face. The mask structure may include a perforated framework 3502 that allows for breathability while maintaining proper positioning of the stimulation elements 3504. The perforations are arrangedin a pattern that provides structural support while minimizing material coverage over non-target areas. The mask extends from the forehead region above the wearer's nose down to the submandibular area, with lateral extensions covering the periorbital and nasal sidewall regions. One or more registration device 3506 may be provided, such as a temple marker, or other marker to assist the clinician in positioning the device properly and repeatedly on the patient. This aids in placing the stimulation elements in the proper anatomical locations on the patient.

[0251] Fig. 35B illustrates a lateral profile view demonstrating the anatomical coverage and positioning of the device 3500. The device is positioned and configured to apply stimulation to the primary stimulation zones where mechanical pressure or vibration is applied. The upper region corresponds to the nasal sidewall lymphatic pathway, extending from the nasal bridge area laterally toward the cheek. The lower region targets the submandibular lymphatic drainage area, positioned over the superficial cervical lymphatic vessels (scLV-1 and scLV-2) that drain into the submandibular lymph node.

[0252] The device may include inflatable bladders that inflate to apply pressure to the target zone. In some cases, a series of inflatable bladders may be inflated sequentially to encourage fluid flow in a desired direction.

[0253] The device 3500 may be designed to provide hands-free operation, allowing for extended treatment sessions without requiring manual manipulation. The stimulation elements 3504 embedded within the mask structure can be configured to deliver various forms of mechanical stimulation, including static pressure, rhythmic compression, vibration, or pulsatile forces within the therapeutic range of 0.01-0.04 kilogram-force previously established as optimal for enhancing CSF drainage.

[0254] The anatomical targeting of this wearable embodiment aligns with the validated stimulation protocol, simultaneously addressing the periorbital lymphatic vessels that connect to scLV-1, the nasal sidewall lymphatics that merge with scLV-2, and the collecting lymphatic vessels that drain into the submandibular lymph node. This multi-point stimulation approach enables comprehensive activation of the superficial cervical lymphatic drainage pathway in a single treatment session.

[0255] The mask design allows for customization based on individual facial anatomy, with adjustable positioning of stimulation elements 3504 to accommodate variations in lymphatic vessel locations. The device can incorporate sensors similar to those described in the handheld embodiment (reference numerals 3002-3006) to monitor and regulate applied forces, ensuring treatment remains within therapeutic parameters throughout the session.

[0256] In some embodiments, the stimulation elements 3504 comprise inflatable air bladders positioned at strategic locations within the mask structure. Each bladder may consist of a biocompatible thermoplastic polyurethane (TPU) membrane, forming chambers that can be individually inflated and deflated. The bladders may be connected to a miniaturized pneumatic control system comprising micropumps and solenoid valves that regulate air pressure within each chamber.

[0257] The pneumatic system operates by cyclically inflating the bladders to apply pressure within the therapeutic range of 0.01-0.04 kgf, followed by controlled deflation to create a rhythmic compression-relaxation cycle. The inflation sequence can be programmed to create a "milking" action that follows the anatomical pathways of the lymphatic vessels - first inflating bladders in the periorbital region, then progressing to the nasal sidewall, and finally to the submandibular area. This sequential compression promotes directional lymphatic flow toward the submandibular lymph nodes. Each bladder incorporates a pressure sensor that provides feedback to maintain force within therapeutic limits.

[0258] Other embodiments may utilize piezoelectric actuators embedded within the mask structure to generate controlled mechanical vibrations. The actuators may comprise lead zirconate titanate (PZT) ceramic discs. When an alternating voltage is applied, the piezoelectric material undergoes mechanical deformation, creating vibrations that are transmitted to the skin surface.

[0259] The piezoelectric elements may operate at frequencies between 20-100 Hz, with amplitude modulation to maintain applied forces to the patient within the 0.01-0.04 kgf range. The vibration pattern can be customized to include continuous, pulsed, or swe pt-frequency modes. Multiple actuators may be arranged in arrays corresponding to the lymphatic drainage pathways, with phase control enabling traveling wave patterns that encourage directional fluidmovement. The low power consumption of piezoelectric actuators (typically less than 100 mW per element) allows for battery-powered operation that can provide treatment for several hours in between charges.

[0260] Other embodiments may incorporate shape memory alloy (SMA) wires, specifically nickel-titanium (NiTi) alloy, as active stimulation elements. The SMA wires may be arranged in a serpentine pattern within flexible polymer carriers positioned at the stimulation zones. When electrical current passes through the wires, resistive heating causes them to contract, applying mechanical pressure to the underlying tissue.

[0261] The SMA actuators provide silent operation with precise force control through modulation of the applied current. The wires, typically 0.1-0.3 mm in diameter, can generate forces up to 0.05 kgf, which is regulated through pulse-width modulation to maintain therapeutic levels. The contraction-relaxation cycle can be controlled by alternating heating and cooling phases, with typical cycle times of 2-5 seconds matching the optimal stroke duration identified in manual stimulation protocols. Arrays of SMA wires can be activated in sequence to create progressive compression patterns along the lymphatic pathways.

[0262] Other embodiments may incorporate dielectric elastomer actuators (DEAs) as stimulation elements. These may consist of a soft elastomer membrane (such as silicone or acrylic) sandwiched between compliant electrodes made from carbon grease or conductive rubber. When high voltage (typically 1-5 kV at microampere currents) is applied across the electrodes, electrostatic forces cause the elastomer to compress in thickness and expand in area.

[0263] The DEAs may be configured as multilayer stacks to amplify the mechanical output, with each layer contributing to the total displacement. The actuators can achieve strains exceeding 100%, enabling significant mechanical deformation from thin, lightweight structures. The applied force is controlled by modulating the voltage amplitude, with integrated force sensors providing feedback to maintain therapeutic pressure levels. The fast response time (milliseconds) of DEAs enables high-frequency stimulation patterns and rapid transitions between compression and relaxation phases.

[0264] Of course, embodiments may incorporate a multi-modal mechanical output and may combine multiple stimulation technologies to provide optimal treatment flexibility. For example,pneumatic bladders provide baseline compression while piezoelectric elements add high- frequency microvibrations that may enhance lymphatic vessel contractility. This hybrid approach allows for customization based on individual patient response and specific therapeutic goals.

[0265] The control system for hybrid configurations employs a hierarchical architecture where a master controller coordinates the operation of different stimulation modalities. Realtime feedback from embedded force sensors, accelerometers, and potentially bioimpedance sensors that monitor lymphatic flow, enables adaptive treatment protocols that adjust stimulation parameters based on physiological response.

[0266] In many cases, the stimulation elements 3504 are embedded within a biocompatible substrate material that provides comfort during extended wear while maintaining proper positioning. The substrate may include moisture-wicking fabrics, breathable membranes, or medical-grade silicone, selected based on the specific actuator requirements and intended use duration.

[0267] Figure 36 illustrates the system architecture of an automated mechanical stimulation device 3600 that provides precise force control for facilitating cerebrospinal fluid drainage through superficial cervical lymphatic vessels. The system represents an advancement over manual stimulation techniques by incorporating real-time force feedback and / or automated position adjustment, in some examples, to maintain therapeutic force levels consistently throughout treatment sessions.

[0268] The device hardware 3600 forms the physical interface between the therapeutic system and the patient's skin surface overlying the superficial cervical lymphatic vessels. The hardware components are designed to accommodate both handheld operation, where a user manipulates the device along predetermined lymphatic pathways, and stationary configurations where the patient's skin is brought into contact with a fixed device that provides automated stimulation patterns.

[0269] The force sensor 3606 constitutes a sensing element that continuously measures the contact force between the device and the skin surface. In some embodiments, the force sensor 3606 may comprise a strain gauge-based load cell sensor, which provides high sensitivity in the therapeutic force range of 0.01-0.04 kilogram-force. The load cell operates on the principle ofresistance change in response to mechanical deformation, with a Wheatstone bridge configuration providing temperature compensation and signal amplification. The sensor 3606 is mechanically coupled between the contact unit and the linear actuator to ensure all applied forces pass through the sensing element.

[0270] The linear actuator 3608 provides automated position control of the contact unit based on force feedback from the sensor 3606. In some implementations, the actuator comprises a miniature electric linear actuator, many of which are sold commercially, and may offer up to 50 mm stroke length or more, providing precise positional control with resolution better than 0.5 mm. The actuator 3608 may incorporate an integrated position sensor that reports absolute position to the control system, enabling accurate tracking of contact unit displacement during treatment.

[0271] The force control algorithm 3610, in some cases, implements a closed-loop control system that maintains contact force within the therapeutic range despite variations in skin compliance, user movement, or tissue properties. The algorithm may operate at a sampling rate of at least 100 Hz or more to provide rapid response to force variations. When the measured force exceeds the upper threshold (for example, 0.04 kgf), the algorithm commands the actuator 3608 to retract, reducing contact pressure. Conversely, when the contact force falls below the lower threshold (for example, 0.01 kgf), the actuator extends to increase contact pressure. This bidirectional adjustment ensures consistent therapeutic force application regardless of manual handling variations or patient movement.

[0272] The force control algorithm 3610 may implement proportional-integral-derivative (PID) control to optimize system response while preventing oscillation or overshoot. The proportional gain may determine the response magnitude to force errors, the integral term eliminates steady-state errors, and the derivative term provides damping to prevent oscillations. These control parameters can be tuned based on the mechanical properties of lymphatic tissue and typical usage conditions to achieve stable force regulation with settling times under 100 milliseconds, in some cases.

[0273] In some examples, the monitoring system 3612 provides real-time visualization and data logging of treatment parameters. The system can display current contact force as bothnumerical values and graphical trends, with color-coded indicators showing whether force is within the therapeutic range (green), approaching limits (yellow), or outside acceptable bounds (red). Actuator displacement may be similarly displayed, providing feedback on the depth of tissue compression and the dynamic response of the control system.

[0274] The monitoring system 3612 may incorporate data storage capabilities that record comprehensive treatment information including timestamp data, continuous force profiles with millisecond resolution, actuator position trajectories, cumulative dose metrics (force x time), and protocol compliance indicators. This data can be used for post-treatment analysis to assess treatment quality, identify areas for technique improvement, and correlate treatment parameters with clinical outcomes. In particular, this data can be utilized for evaluating whether lymphatic flow velocity has been increased or for assessing cerebrospinal fluid drainage function based on the increased flow velocity

[0275] Figure 37 provides a detailed view of the mechanical stimulation device hardware, showing the integration of components that enable controlled therapeutic stimulation of superficial cervical lymphatic vessels.

[0276] The housing 3700 serves as the structural framework that maintains proper alignment of internal components while providing an ergonomic interface for the user. The housing may be manufactured from biocompatible materials such as medical-grade ABS or polycarbonate, with surface textures to provide grip security during extended treatment sessions. The housing geometry is designed to accommodate the mechanical envelope of the linear actuator while minimizing overall device dimensions for maneuverability around facial contours.

[0277] The handle structure 3702 extends from the main housing 3700 to provide a comfortable gripping surface for handheld operation. The handle incorporates ergonomic features such as finger grooves, thumb rests, and balanced weight distribution to reduce user fatigue during the typical 20-minute treatment sessions. The handle may include integrated controls such as power switches, force range selectors, and / or emergency stop buttons that allow immediate user intervention if needed.

[0278] The contact unit 3704 forms the therapeutic interface with the patient's skin, transmitting controlled mechanical forces to the underlying lymphatic vessels. The contact unit3704 may be manufactured from various materials depending on specific therapeutic requirements. Ceramic materials offer excellent biocompatibility, smooth surface finish, and thermal stability. Alternative materials include medical-grade stainless steel for durability, silicone elastomers for compliance matching with soft tissue, or specialized polymers with integrated heating or cooling capabilities for combined thermal-mechanical therapy.

[0279] The geometry of the contact unit 3704 may be configured for different anatomical regions and treatment objectives. For targeting the narrow pathways of facial lymphatics, a small spherical tip, such as with 5-10 mm diameter, provides precise localization. For broader coverage of the submandibular region, an elongated oval configuration, such as with 10 mm x 20 mm contact area, enables efficient treatment of larger vessel networks. The contact surface may incorporate textures such as gentle ridges or dimples that enhance grip on the skin while promoting directional lymphatic drainage when moved along vessel pathways.

[0280] The force sensor 3706 is positioned in the mechanical load path between the contact unit 3704 and the linear actuator 3708, ensuring accurate measurement of forces transmitted to the tissue. The sensor mounting may incorporate compliance elements such as elastomeric bushings that protect the sensor from lateral forces or moments that could affect measurement accuracy. Signal conditioning electronics, including instrumentation amplifiers and analog-to- digital converters, may be positioned close to the sensor to minimize noise and maintain signal integrity.

[0281] The linear actuator 3708 is rigidly mounted within the housing 3700, such as through mounting brackets that maintain alignment while allowing for thermal expansion. In some examples, the actuator's lead screw mechanism converts rotational motor motion to linear displacement of the contact unit 3704, with anti-backlash nuts ensuring precise bidirectional positioning. The actuator may incorporate limit switches that prevent over-travel in both extension and retraction directions, protecting both the mechanism and the patient from excessive forces.

[0282] In some cases, the device includes power management systems that may comprise rechargeable lithium-ion batteries for portable operation. A charging port, which may be USB-C or other type connector, may be provided to enable convenient recharging between sessions.Power conditioning circuits may be implemented to provide stable voltage supplies to the actuator motor, sensor electronics, and control systems despite battery voltage variations during discharge.

[0283] Communication interfaces enable data transfer between the device hardware and external monitoring systems. Wired connections such as USB provide reliable, high-bandwidth communication for real-time force and position data streaming. Wireless options including Bluetooth Low Energy or WiFi enable untethered operation, particularly beneficial when treating multiple anatomical sites or when patient movement is required during treatment.

[0284] The precision force-regulated mechano-stimulator embodiments described herein represent a significant technological advancement in the non-invasive treatment of neurodegenerative conditions, providing quantifiable, reproducible mechanical stimulation to enhance cerebrospinal fluid drainage through the superficial cervical lymphatic system. The device's combination of precise force control, real-time feedback, and validated treatment protocols enables effective therapeutic intervention for conditions associated with impaired CSF clearance.

[0285] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects. Therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention.Cited References1. Ransohoff, R. M., & Engelhardt, B. (2012). The anatomical and cellular basis of immune surveillance in the central nervous system. Nat Rev Immunol, 12(9), 623-635. https: / / doi.org / 10.1038 / nri32652. Proulx, S. T. (2021). Cerebrospinal fluid outflow: a review of the historical and contemporary evidence for arachnoid villi, perineural routes, and dural lymphatics. Cell Mol Life Sci, 78(6), 2429-2457. https: / / doi.org / 10.1007 / s00018-020-03706-53. Bettcher, B. M., Tansey, M. G., Dorothee, G., &Heneka, M. T. (2021). Peripheral and central immune system crosstalk in Alzheimer disease - a research prospectus. Nat Rev Neurol, 17(11), 689-701. https: / / doi.org / 10.1038 / s41582-021-00549-x4. Aspelund, A., Antila, S., Proulx, S. T., Karlsen, T. V., Karaman, S., Detmar, M., Wiig, H., &Alitalo, K. (2015). A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J Exp Med, 212(7), 991-999. https: / / doi.org / 10.1084 / iem.201422905. Louveau, A., Smirnov, I., Keyes, T. J., Eccles, J. D., Rouhani, S. J., Peske, J. D., Derecki, N. C., Castle, D., Mandell, J. W., Lee, K. S., Harris, T. H., & Kipnis, J. (2015). Structural and functional features of central nervous system lymphatic vessels. Nature, 523(7560), 337-341. https: / / doi.org / 10.1038 / naturel44326. Ma, Q., Ineichen, B. V., Detmar, M., & Proulx, S. T. (2017). Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice. Nat Commun, 8(1), 1434. https: / / doi.org / 10.1038 / s41467-017-01484-67. Ahn, J. H., Cho, H., Kim, J. H., Kim, S. H., Ham, J. S., Park, I., Suh, S. H., Hong, S. P., Song, J. H., Hong, Y. K., Jeong, Y., Park, S. H., & Koh, G. Y. (2019). Meningeal lymphatic vessels at the skull base drain cerebrospinal fluid. Nature, 572(7767), 62-66. https: / / doi.org / 10.1038 / s41586-019- 1419-58. Yoon, J. H., Jin, H., Kim, H.J., Hong, S.P., Yang, M.J., Ahn, J.H., Kim, Y.C., Seo, J., Lee, Y., McDonald, D.M., Davis, M.J., & Koh, G.Y. (2024). Nasopharyngeal lymphatic plexus is a hub for cerebrospinal fluid drainage. Nature 625(7996): 768-777. https: / / doi.org / 10.1038 / s41586-023- 06899-49. Kipnis, J., Louveau, A., Da Mesquita, S. (2019). US 2019-0269758 Al, University of Virginia Patent Foundation (UN162191): U.S. Patent and Trademark Office10. Kipnis, J. Da Mesquita, S. (2021), US 2021-0311076 Al, University of Virginia Patent Foundation (UN162191): U.S. Patent and Trademark Office

Claims

1. Claims1. A device for providing mechanical stimulation to facilitate cerebrospinal fluid drainage, the device comprising: a handle member having a proximal end and a distal end, the handle member having a length; a force sensor (3406) coupled to the distal end of the handle member, the force sensor comprising a multilayer capacitive sensor having at least one silicone layer disposed between conductive fabric layers; a shaft (3410) having a first end and a second end, the shaft coupled to the force sensor at the first end and extending distally therefrom; a replaceable tip assembly (3416) removably coupled to the second end of the shaft, the tip assembly comprising a stimulation tip (3422); and an electronic control system (3000) comprising: a microcontroller (3004) electrically connected to the force sensor; a capacitance-to-digital converter (3006) interfacing between the force sensor and the microcontroller; and an amplifier unit (3402) housing the microcontroller and providing realtime force measurement data; and wherein the device is configured to measure and maintain applied forces within a therapeutic range of 0.01 to 0.04 kilogram-force for facilitating cerebrospinal fluid drainage through superficial cervical lymphatic vessels.

2. The device of claim 1, wherein the force sensor comprises: a first conductive fabric layer (3308); a first silicone layer (3302) having a thickness of approximately 0.5 mm; a second conductive fabric layer (3314); a second silicone layer (3312); and a third conductive fabric layer (3316); wherein the layers are arranged to provide differential capacitive sensing.

3. A device for providing mechanical stimulation to facilitate cerebrospinal fluid drainage, the device comprising: a handle member having a proximal end and a distal end, the handle member having a length; a force sensor (3606 or 3706) coupled to the distal end of the handle member, the force sensor comprising a load cell sensor; a shaft having a first end and a second end, the shaft coupled to the force sensor at the first end and extending distally therefrom; a stimulation tip (3704) coupled to the second end of the shaft; and an electronic control system (3610 and 3612) comprising: a force controller (3610) electrically connected to a linear actuator (3608); and a monitoring system (3612) for monitoring applied force and displacement of the linear actuator; and wherein the device is configured to measure and maintain applied forces on the stimulation tip within a therapeutic range of 0.01 to 0.04 kilogram-force for facilitating cerebrospinal fluid drainage through superficial cervical lymphatic vessels.

4. The device of claim 1,2 or 3, wherein the electronic control system further comprises visual indicators (3404 or 3612) configured to illuminate different colors based on measured force levels, wherein: a first color indicates force within the therapeutic range of 0.01 to 0.04 kilogramforce; a second color indicates force approaching predetermined limits; and a third color indicates force exceeding recommended levels.

5. The device of any one of claims 1 to 4, wherein the capacitance-to-digital converter provides at least 28-bit resolution for capacitance measurements.

6. The device of any one of claims 1 to 5, wherein the microcontroller is configured to sample the force sensor at a rate of at least 100 Hz.

7. The device of any one of claims 1 to 6, wherein the replaceable tip assembly comprises: a mounting rod (3418); and a stimulation tip (3704); and a coupling mechanism selected from a threaded engagement, bayonet coupling, or friction fit for removable attachment to the shaft.

8. The device of any one of claims 1 to 7, wherein the stimulation tip comprises a material configured to provide compliance for skin interface during lymphatic stimulation.

9. The device of any one of claims 1 to 8, further comprising a data logging system configured to record one or more of: continuous force profiles during treatment; stroke counts; treatment duration; and timestamp data.

10. The device of any one of claims 1 to 9, wherein the force sensor is encapsulated in a silicone material forming a flexible strap configuration while maintaining electrical connections to the microcontroller.

11. The device of any one of claims 1 to 10, wherein the device is calibrated to provide a linear relationship between sensor readings and actual force values with a correlation coefficient greater than 0.99 across the range of 0 to 0.05 kilogram-force.

12. A method for facilitating cerebrospinal fluid drainage through mechanical stimulation of superficial cervical lymphatic vessels, the method comprising: providing a force-controlled mechanical stimulation device comprising a handle, a force sensor (3406 or 3706), a shaft (3410), a linear actuator (3708) and a replaceable tip (3422 or 3704) having an oval configuration; positioning the tip against intact skin overlying a superficial cervical lymphatic vessel pathway; applying mechanical stimulation strokes to three anatomical regions in sequence:Region 1 : from periorbital area to mandible;Region 2: from nasal sidewall to mandible; andRegion 3: along paths of superficial cervical lymphatic vessels to submandibular lymph node; maintaining applied force within a range of 0.01 to 0.04 kilogram-force as measured by the force sensor; performing the stimulation in cycles, each cycle comprising:4 strokes to Region 1 ;4 strokes to Region 2; and2 strokes to Region 3; wherein each stroke has a duration of approximately 2 seconds.

13. The method of claim 12, wherein the mechanical stimulation is performed for a total of 20 one-minute sessions.

14. The method of claim 12 or 13, further comprising: monitoring force feedback in real-time through an electronic control system (3000 and 3610) sampling the force sensor; and adjusting applied pressure based on visual indicators (3404 and 3612) that illuminate to indicate whether force is within a therapeutic range.

15. The method of any one of claims 11 to 14, wherein two cycles of 10 strokes are performed over 40 seconds followed by a 20-second rest period.

16. The method of any one of claims 11 to 15, wherein the stimulation enhances cerebrospinal fluid drainage by at least a 2-fold increase in lymphatic flow as measured by tracer accumulation in submandibular lymph nodes compared with no stimulation.

17. The method of any one of claims 11 to 16, wherein the mechanical stimulation is applied bilaterally to both left and right superficial cervical lymphatic vessel pathways.

18. The method of any one of claims 11 to 17, further comprising: identifying the pathway of superficial cervical lymphatic vessels by mapping facial veins using doppler ultrasonography; andtargeting the mechanical stimulation within 2 cm of the identified lymphatic pathway.

19. The method of any one of claims 11 to 18, wherein the method is performed on a subject having a neurodegenerative condition selected from cognitive decline with aging, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke.

20. The method of any one of claims 11 to 19, further comprising recording treatment data including one or more of: continuous force profiles throughout a treatment session; total number of strokes applied to each region; treatment duration; and protocol adherence metrics.

21. The method of any one of claims 11 to 20, wherein the mechanical stimulation activates nitric oxide signaling pathways to enhance cerebrospinal fluid drainage through the superficial cervical lymphatic vessels.

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