Extracranial method for promoting drainage of cerebrospinal fluid

Abstract

Described herein is a method of preventing or treating a neurodegenerative disease comprising administering to a human an agent wherein the agent causes increased cerebrospinal fluid outflow through nasopharyngeal lymphatic plexus (NPLP), or the agent causes contraction and relaxation of annular smooth muscles covering deep cervical lymphatic vessels (dcLV).

Description

Technical Field

[0001] The present application relates to a method for increasing or restoring the outflow rate or level of cerebrospinal fluid (CSF) from the central nervous system to the systemic circulation. The present application also relates to a method for diagnosing and treating neurodegenerative diseases by assessing CSF outflow levels and increasing outflow in patients identified as suffering from reduced CSF outflow levels. Background Art

[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 as incorporated by reference. The following description contains information that may be helpful 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, nor is it an admission that any publication explicitly or implicitly referenced is prior art.

[0003] The cerebrospinal fluid (CSF) volume range of adults is 140-200ml. CSF is produced from the choroid plexus with 400-600ml / day and circulates in the brain substance and spinal cord (references 1 and 2). Therefore, cerebrospinal fluid circulates 3-5 times / day. The central nervous system (CNS) includes the brain, spinal cord, optic nerve and retina, and their fine activities require high energy. CNS produces waste and antigenic macromolecules again, which include synaptic and myelin fragments and misfolded proteins, such as amyloid and hyperphosphorylated tau protein. The waste and macromolecules in these CNS sources are dissolved in the brain interstitial fluid and CSF, and they should be fully discharged into the systemic circulation. The excessive accumulation of these waste and macromolecules (including amyloid and hyperphosphorylated tau) causes neurodegenerative diseases such as Alzheimer's disease (reference 3).

[0004] Traditionally, the arachnoid villi and cribriform plate are known to be the drainage pathways of CSF (Refs. 1 and 2). However, since two research groups rediscovered the meningeal lymphatic vessels (mLVs) in the dorsal dura mater of the mouse skull (Refs. 4 and 5), the so-called “dorsal mLVs” have been Figure 1 )”, they have been considered as a new drainage route for CSF. They (4,5) showed that dorsal skull mLVs are phenotypically similar to peripheral lymphatic capillaries. They also showed that dorsal mLVs transport macromolecules and cells along the superior sagittal and transverse sinuses (4,5). However, Ma and Proulx and colleagues (ref. 6) did not find significant CSF reuptake and drainage via dorsal mLVs using controlled low-rate and low-volume stereotactic CSF tracer injections. These researchers instead proposed that CSF drains along cranial nerves as they exit the skull (5). In contrast, our group revealed that the basolateral mLVs are one of the major routes for CSF drainage ( Figure 2) and compared the morphological and functional differences of dorsal mLV and basolateral mLV in adult and aged mice ( Figure 3-5 )(6). Importantly, with aging, basolateral mLV acquire features of lymphedema and CSF drainage is delayed ( Figure 5 )(6). These findings may explain the pathological mechanism of neurodegenerative diseases such as Alzheimer's disease, whereby the drainage of CSF via the damaged basolateral mLV decreases with aging, leading to excessive accumulation of waste products and macromolecules in the brain. However, the basolateral mLV is considered to be the drainage pathway for CSF in the posterior brain region and spinal cord circulation ( Figure 6 ).

[0005] Therefore, the existence of mLVs to cover CSF drainage from the CNS and the anterior (except the lamina cribrosa) and middle cranial fossa regions of the skull is poorly understood ( Figure 6 ). If they exist, how they connect to the extracranial lymphatics is unknown. In 2009, Pan et al. (ref. 7) reported lymphatic vessels (LVs) in the human nasal cavity and nasopharynx, but they did not describe the relationship between CSF drainage and these LVs. In 2017, Ma and Proulx and colleagues (ref. 8) described that they observed bright signals in Prox1-GFP mice from the tracer P40D680 injected intracranially into the nasal cavity, as well as tracers emanating from the pharyngeal lymphatic plexus that tracked to the deep cervical lymph nodes (dcLN). Based on this finding, they proposed that CSF can be drained through the pharyngeal LVs. However, the characteristics of the pharyngeal LVs and their CSF outflow have not been fully elucidated. In 2022, Jacob and Thomas and colleagues (ref. 9) reported that OVA-A 55 After the tracer was injected into the subarachnoid space, light sheet fluorescence microscopy (LSFM) was used to image whole head specimens of mice and an anterior mLV network extending around the cavernous sinus was found, with its exit pathway passing through the foramen of the emissary vein. They also performed real-time vascular wall (VW) magnetic resonance imaging (VW-MRI) after systemic injection of gadobutrol in patients with neurological conditions. However, due to the tracking of OVA-A in fixed tissues and organs, the 555 Because VW-MRI provides low resolution for the lymphatic system, reflecting the distribution of phagocytic macrophages rather than mLVs, this study lacks detailed histological and functional information about mLVs, which are supposed to transport intracranial CSF to extracranial compartments.

[0006] U.S. Patent Application Publication No. 20190269758, "Methods and Compositions for Modulating Lymphatic Vessels in the Central Nervous System" (Reference 10), relates to manipulating mLVs to treat neurodegenerative diseases. However, this patent application does not disclose or suggest methods for increasing or restoring the rate or level of CSF outflow from the central nervous system to the systemic circulation by regulating or manipulating the nasopharyngeal lymphatic plexus (NPLP)-deep cervical lymphatic vessel (dcLV) pathway of CSF.

[0007] U.S. Patent Application Publication No. 20210311076, COMPOSITIONS AND METHODS OF DIAGNOSIS AND TREATMENT FOR NEUROLOGICAL DISEASES (Reference 11), relates to the manipulation of mLV to treat neurodegenerative diseases. However, this patent application does not disclose or suggest methods for increasing or restoring the rate or level of CSF outflow from the central nervous system to the systemic circulation by regulating or manipulating the nasopharyngeal lymphatic plexus (NPLP)-dcLV pathway of cerebrospinal fluid (CSF). Summary of the Invention

[0008] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions, and methods, which are intended to be exemplary and illustrative, not limiting in scope.

[0009] Improving cerebrospinal fluid (CSF) drainage is valuable in the prevention and treatment of neurodegenerative diseases including Alzheimer's disease. Here, the present invention provides information on extracranial approaches and methods to improve CSF drainage.

[0010] The present invention encompasses extracranial approaches to enhance CSF drainage via the nasopharyngeal lymphatic plexus (NPLP) and deep cervical lymphatic vessels (dcLV) for the prevention and treatment of neurodegenerative diseases.

[0011] 1. The applicants have newly discovered that the nasopharyngeal lymphatic plexus (NPLP) is a hub for CSF drainage through the skull base (including the cribriform plate).

[0012] 2. The applicant has newly discovered a connection pathway for CSF drainage from the intracranial cavity to the NPLP.

[0013] 3. The applicant has newly discovered a connection pathway for CSF drainage from the intracranial cavity to the submucosa of the hard palate.

[0014] 4. Applicants have newly discovered that CSF drainage via the nasopharyngeal lymphatic plexus (NPLP) / medial deep cervical lymphatic vessel (M-dcLV) route is greater in volume and faster in rate than via the basolateral mLV / lateral (L)-dcLV route, emphasizing the importance of the NPLP.

[0015] 5. Applicants have newly discovered that NPLP is impaired with aging and that exogenous agents can restore reduced lymphatic vessel area along with reduced CSF drainage to increased lymphatic vessel area along with increased CSF drainage levels.

[0016] 6. Applicants have newly discovered that the contraction and relaxation of the circular smooth muscle covering the dcLV can be regulated or modulated by stimulation and inhibition of smooth muscle cells, peripheral nerves, neurotransmitters, mechanical stimuli, and gentle massage.

[0017] 7. Applicants have newly discovered that NPLP excretion through the NPLP can be regulated by exogenous agents via extracranial routes. CS excretion can be promoted through extracranial manipulation, administration of agents, and related modulators.

[0018] In one aspect, the present invention relates to a method of increasing cerebrospinal fluid (CSF) outflow from the central nervous system, the method comprising repairing or enlarging the nasopharyngeal lymphatic plexus (NPLP), comprising: identifying a subject in need of increased CSF outflow; and administering to the subject in need thereof an effective amount of an NPLP glidant, wherein the amount of the agent repairs or enlarges the subject's NPLP, thereby increasing CSF outflow from the subject's central nervous system to the systemic circulation. Identifying a subject in need of increased CSF outflow may comprise determining that the subject has a neurodegenerative disease or condition, determining that the subject has risk factors for a neurodegenerative disease or condition, or both. The disease or condition may be cognitive decline associated with aging, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke. Specifically, 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 also be 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, neuropilin, or prostaglandin E2. The agent may be a protein, or a gene vector carrying a gene encoding an agent polypeptide. The gene may encode angiogenic factor-C, angiopoietin-1, or a Tie2 agonist.

[0019] In one aspect, the agent can be selectively administered at or near the NPLP-dcLV space. The NPLP-dcLV space can be located in the nasopharyngeal mucosa. The agent can be selectively administered at or near the hard palate submucosa-mandibular lymph node space. The hard palate submucosa-mandibular lymph node space can be located in the hard palate mucosa. The agent can be administered intrathecally to the subject's CSF space, or nasally to the subject's nasopharynx or orally to the subject's hard palate submucosa. In this regard, the subject's central nervous system can contain soluble molecules, and wherein increasing CSF outflow reduces the amount of soluble molecules in the brain. The subject's central nervous system can contain amyloid beta protein plaques, and wherein increasing CSF outflow reduces the amount of amyloid beta protein plaques in the brain.

[0020] In another aspect, the present invention relates to a method for increasing cerebrospinal fluid (CSF) outflow from the central nervous system, the method comprising increasing contraction-relaxation of the DCLV, comprising: identifying a subject in need of increased CSF outflow; and administering to the subject an effective amount of a DCLV glidant, wherein the amount increases contraction-relaxation of the subject's DCLV, thereby increasing CSF flow from the subject's central nervous system to the systemic circulation. Identifying the subject in need of increased CSF outflow may comprise determining that the subject suffers from a neurodegenerative disease or condition, determining that the subject has a risk factor for a neurodegenerative disease or condition, or both. The disease or condition may be cognitive decline associated with aging, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke. The agent may be a stimulator of the circular smooth muscle covering the DCLV. The agent may be a G protein-coupled receptor agonist such as phenylephrine or a nitric oxide donor. In one aspect, the agent may be administered transcervically, transdermally, topically to the subject's neck muscles or neck lymph nodes or neck space, or the agent may be mechanically applied to the side of the neck.

[0021] In yet another aspect, the present application relates to a method for preventing, treating, or ameliorating a neurodegenerative disease or condition in a subject, the method comprising repairing or enlarging the nasopharyngeal lymphatic plexus (NPLP), comprising: determining a subject in need of increased CSF outflow from the central nervous system; and administering an effective amount of an NPLP flow enhancer to the subject in need, wherein the amount of the agent repairs or enlarges the subject's NPLP, thereby increasing CSF outflow from the subject's central nervous system to the systemic circulation. The disease or condition may be cognitive decline associated with aging, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke. Specifically, the agent may be a VEGFR3 agonist, such as VEGF-C or VEGF-D, an analog, variant, or fragment thereof, or any combination thereof. The agent may also be fibroblast growth factor 2 (FGF-2), insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), endothelin-1 (ET-1), angiopoietin-1, a Tie2 agonist, neuropilin, or prostaglandin E2. The agent may be a protein, or a gene vector carrying a gene encoding an agent polypeptide. The gene may encode vascular growth factor-C, angiopoietin-1 or Tie2 agonist.

[0022] The agent can be selectively administered at or near the NPLP-dcLV space, including where the NPLP-dcLV space is located in the nasopharyngeal mucosa. The agent can be administered intrathecally to the CSF space of the subject or nasally to the nasopharynx of the subject. The agent can be selectively administered at or near the hard palate submucosa-mandibular lymph node space. The hard palate submucosa-mandibular lymph node space can be located in the hard palate mucosa. The agent can be administered intrathecally to the CSF space of the subject or nasally to the nasopharynx of the subject or orally to the hard palate submucosa of the subject. In this regard, the central nervous system of the subject may include soluble molecules, and wherein increasing CSF outflow reduces the amount of soluble molecules in the brain. The central nervous system of the subject may include amyloid beta protein plaques, and wherein increasing CSF outflow reduces the amount of amyloid beta protein plaques in the brain.

[0023] In another aspect, the present invention relates to a method for preventing, treating, or ameliorating a neurodegenerative disease or condition in a subject, the method comprising repairing or enlarging the nasopharyngeal lymphatic plexus (NPLP), comprising: identifying a subject in need of increased CSF outflow from the central nervous system; and administering to the subject an effective amount of a dcLV glidant, wherein the amount increases contraction-relaxation of the subject's dcLV, thereby increasing CSF outflow from the subject's central nervous system to the systemic circulation. The disease or condition can be cognitive decline associated with aging, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke. The agent can be a stimulator of the circular smooth muscle covering the dcLV. The agent can be the G protein-coupled receptor agonist phenylephrine or a nitric oxide donor. The agent that stimulates the circular smooth muscle covering the dcLV can increase or decrease myosin phosphorylation by activating myosin light chain kinase or activating myosin light chain phosphatase. The agent can be administered transcervically, transdermally, topically to the subject's neck muscles, cervical lymph nodes, or cervical space, or mechanically applied to the side of the neck.

[0024] In another aspect, the present invention relates to a method for determining a change in CSF outflow content or rate in a subject, the method comprising: obtaining a biological sample to obtain samples of nasopharyngeal mucosa or hard palate mucosa at at least two separate times; analyzing the CSF for the presence and amount of CSF or an analyzable substance to obtain a value at each time point; comparing the values ​​of the amount of CSF or the analyzable substance; and determining a change in the value of the amount of CSF or the analyzable substance, wherein the change in the value of the CSF or the analyzable substance over time indicates a changed CSF outflow content or rate. The biological sample can be obtained by swab or biopsy. The biological sample can include the NPLP or the hard palate. The analysis can be performed by imaging the NPLP or the hard palate, or performing protein analysis on the obtained biological sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0026] The present disclosure will be better understood from the following detailed description when read in conjunction with the accompanying drawings. It should be emphasized that, as is customary, the various features in the drawings are not to scale. Rather, the dimensions of the various features have been arbitrarily enlarged or reduced for clarity. The accompanying drawings include the following figures.

[0027] Figure 1 The dorsal mLV of the mouse skull is shown. Immunofluorescence staining (IFS) images show LYVE-1+ dorsal mLV along the VE-cadherin+ superior sagittal and transverse sinuses.

[0028] Figure 2The basolateral meningeal lymphatic vessels (basolateral mLV) of the mouse skull are shown. IFS images show LYVE-1+ basolateral mLV (arrows) along the VE-cadherin+ petrosquamous and sigmoid sinuses.

[0029] Figure 3 Schematic diagrams depicting the morphological and functional differences between the dorsal mLV and basolateral mLV in adult and aged mice are shown.

[0030] Figure 4 Schematic diagram showing that the basolateral mLV is a "hot pathway" for CSF drainage (posterior brain) and degenerates in aged mice.

[0031] Figure 5 A-5B shows that CSF excretion of basolateral mLV is greatly reduced in aged mice. (A) Alexa 488 (A488)-conjugated LYVE-1 antibody was injected intracranially through the cisterna magna (CM) to visualize basolateral mLV. 12 hours later, QD705 was injected into the intracranial cavity, and 15 minutes later, the injected QD705 in the basolateral mLV was visualized by fluorescence microscopy. (B) Comparison of QD705 excretion (arrows) in A488-LYVE-1+ basolateral mLV (arrows) between 3-month-old adult mice and 27-month-old mice.

[0032] Figure 6 Shown is a schematic diagram depicting known and unknown CSF drainage pathways to the cervical lymph nodes.

[0033] Figure 7 A-7B shows the distribution of exogenously injected dextran within the intracranial cavity of adult mice. (A) Schematic depicts intracranial injection of TMR-dextran (10 kDa) via the cisterna magna of a 10-week-old Prox1-GFP mouse. 60 minutes later, the head was sampled. (B) Images show the distribution of TMR-dextran within the intracranial and extracranial cavities. Notably, strong Prox1+ expression is observed in the hippocampus, as well as in the nasopharynx, oropharynx, and the LV surrounding the hard and soft palates.

[0034] Figure 8 A-8C shows that the lymphatic plexus in the nasopharynx (but not the oropharynx) of adult Prox1-GFP mice contains CSF-derived TMR-dextran. (A) Image shows the distribution of LVs in the head and neck. (B) Image shows the lymphatic plexus in the nasopharynx and oropharynx. (C) Image shows that the lymphatic plexus in the nasopharynx (but not the oropharynx) contains CSF-derived TMR-dextran (reddish-yellow arrows). Green arrows indicate lymphatic valves.

[0035] Figure 9A-9B shows intravital imaging demonstrating the dynamic and active efflux of dextran from the intracranial cavity into the NPLP. (A) Schematic depicting intracranial injection of TMR-dextran (10 kDa) into the cisterna magna of a 10-week-old Prox1-GFP mouse. Semi-in vivo imaging was performed 30 minutes later. (B) Video showing the dynamic expulsion of CSF-derived TMR-dextran (reddish-yellow arrows) through the NPLP.

[0036] Figure 10 This image shows that in adult Prox1-GFP mice, NPLP contains abundant and selective CSF-derived fluorescent microbeads. Fluorescent microbeads were injected into the intracranial cavity via the cisterna magna, and samples were collected 6 hours later. The dotted box is the enlarged image on the right.

[0037] Figure 11 This image shows the dcLV, which has periodic lymphatic valves (green arrows) and lymphatic vessels, and contains TMR-dextran, which was injected into the intracranial cavity via the cisterna magna 30 minutes earlier. Of note, the L-dcLV is connected to the basolateral LV via the jugular foramen, while the M-dcLV is connected to the LV arising from the nasopharynx. The injected dextran was detected not only in the dcLN but also in the M-dcLV and L-dcLV.

[0038] Figure 12 A-12B shows the butterfly-shaped nasopharyngeal lymph node polyps (NPLP) located in the submucosa of the nasopharynx. (A) Images show dorsal, ventral, and lateral views of the NPLP. Green lines outline the major Prox1+ / VEGFR3+ / LYVE1+ LVs that comprise the NPLP. The flat and dense posterior nasal lymphatic plexus lies anterior to the NPLP. (B) Schematic diagram showing the structure and views of the nasopharynx.

[0039] Figure 13 Shown are coronal cross-sectional views of the NPLP (white arrows) in Prox1-GFP adult mice.

[0040] Figure 14 The schematic structure of the NPLP is shown. Dorsal, lateral, ventral, and coronal sections of the NPLP are depicted. The NPLP is divided into the head, body, and tail. The green lines outline the main LV and valves for CSF outflow.

[0041] Figure 15 A schematic diagram of the 3D structure of the NPLP is shown. The green lines and ducts represent the inflow and outflow of CSF and the main LV that constitutes the NPLP.

[0042] Figure 16A-16C shows the discovery of LVs originating from the pituitary region, which course along the cavernous sinus and connect to the NPLP in Prox1-GFP mice. (A) Schematic diagram showing the imaging area (red line). (B) Sequential images show LVs originating from the pituitary region connecting to the NPLP. (C) Magnified view of the red box in (B), showing lymphatic terminals in the Prox1+ pituitary.

[0043] Figure 17 Shows Figure 16 Shown is a photograph of the 3D structure connecting the LV to the NPLP from the pituitary region.

[0044] Figure 18 A-18B shows the finding of LV along the course of the pterygopalatine artery (PPA) connecting to the NPLP and the LV in the hard palate. (A) Schematic diagram showing the imaging area (green line). (B) Sequential images show the LV along the course of the pterygopalatine artery (PPA), connecting to the NPLP, and the LV in the submucosa of the hard palate.

[0045] Figure 19 The 3D structural photograph of the LV is shown. The LV runs along the pterygopalatine artery (PPA) and connects to the NPLP and the LV in the submucosa of the hard palate, as shown in FIG. Figure 18 shown.

[0046] Figure 20 A-20D shows the discovery of Prox1+ LVs located beneath the olfactory epithelium, connecting to the posterior nasal lymphatic plexus and the NPLP of the nasopharynx. (A) Schematic diagram showing the imaging area (green line). (B) In Prox1-GFP adult mice, LVs in the olfactory mucosa contain abundant and selective CSF-derived fluorescent microbeads. Fluorescent microbeads were injected into the intracranial cavity through the cisterna magna, and the olfactory organs were sampled 6 hours later. (C) In Prox1-GFP adult mice, LVs in the olfactory mucosa contain abundant and selective CSF-derived QD705. QD705 was injected 60 minutes before sampling. (D) Image showing two connecting LVs between the olfactory mucosa and the NPLP and the posterior nasal lymphatic plexus.

[0047] Figure 21 Schematic diagram showing the discovery of three lymphatic pathways from the intracranial cavity to the extracranial NPLP. The NPLP is the hub for CSF drainage. (1) The LV originating from the pituitary region courses along the cavernous sinus and connects to the NPLP; (2) The LV courses along the pterygopalatine artery (PPA) and connects to the NPLP; (3) The LV, located beneath the olfactory epithelium, connects to both the posterior nasal lymphatic plexus and the NPLP of the nasopharynx.

[0048] Figure 22A-22B shows that the LVs in the hard palate submucosa serve as a drainage pathway for CSF and connect to the mandibular lymph nodes (LNs). (A) The image shows that the lymphatic vessels in the hard palate submucosa connect to the mandibular LNs. Furthermore, the injected dextran is detected not only in the mandibular LNs but also in the hard palate lymphatic vessels. (B) The image shows that the lymphatic vessels in the hard palate submucosa contain CSF-derived TMR-dextran.

[0049] Figure 23 The right image shows the lymphatic plexus in the submucosa of the hard palate of a cynomolgus macaque. The right image shows the presence of CSF-derived fluorophores in the hard palate lymphatic plexus. The red arrows indicate CSF-derived fluorophores (0.1 μm).

[0050] Figure 24 A-24B shows the four lymphatic branches connecting the NPLP and dcLV in Prox1-GFP mice. (A) Image shows the four lymphatic branches connecting the NPLP and dcLV before and after soft palate removal. (B) Schematic diagram showing the four lymphatic branches connecting the NPLP and dcLV.

[0051] Figure 25 A-25B shows the presence of NPLP in the nasopharyngeal submucosa of the cynomolgus macaque. (A) Gross image of a sagittal section of the head and neck of a cynomolgus macaque. (B) Immunofluorescence images of LYVE1+NPLP and type IV collagen+BV in the nasopharyngeal submucosa. The green box in the lower image is an enlarged view. White arrows indicate lymphatic valves.

[0052] Figure 26 A-26B shows CSF drainage via the NPLP and hard palate in primates (cynomolgus macaques). (A) MRI images of monkeys depict contrast enhancement of the CSF following injection of Gadospin P via the cisterna magna. Red circles indicate regions of interest for measuring CSF tracer signal enhancement. (B) Quantification of signal enhancement from different regions over 3 hours reveals that the NPLP and hard palate are CSF drainage pathways.

[0053] Figure 27 A-27C shows that in adult Prox1-GFP mice, the medial dcLV drains more CSF than the lateral dcLV. (A) Schematic diagram depicting the junctions of L-dcLV or M-dcLV. (B) Light and fluorescence microscopy images are shown for each dcLV junction. (C) Comparison of signal intensity in dcLN. n = 9 per group, P values ​​calculated by Brown-Forsythe and Welch ANOVA tests.

[0054] Figure 28A-28D shows that in adult Prox1-GFP mice, medial dcLVs drain CSF faster than lateral dcLVs. (A) Schematic diagram depicting intracranial TMR-dextran injection and temporal analysis. (B) Image showing dextran in each dcLV. (C) Schematic diagram depicting dextran in each dcLV in (B). (D) Comparison of signal intensity in dcLVs. P values ​​were calculated using the Brown-Forsythe and Welch variance tests.

[0055] Figure 29 A-29D shows that intracisternal injection of AAV-mVEGF-C increases the area of ​​Prox1+ lymphatic vessels in the nasopharynx of Prox1-GFP mice. (A) Schematic diagram depicting intracisternal injection of AAV-mVEGF-C-mCherry and analysis time. (B) and (C) Representative images and comparison of quantitative parameters of NPLP. (D) Representative images and comparison of CSF tracer output in dcLN. P values ​​were calculated using the Mann-Whitney test.

[0056] Figure 30 Aged mice show altered morphology and reduced lymphoid signaling in the NPLP. Images show the NPLP between 10-week-old and 80-88-week-old Prox1-GFP mice.

[0057] Figure 31 A-31C shows the transcriptomes of lymphatic endothelial cells (LECs) from young and elderly NPLP. (A) Five LEC clusters are conserved in elderly NPLP. (B-C) Lists show differentially expressed genes in elderly NPLP. These differentially expressed genes are involved in pro-apoptotic and inflammatory signaling in elderly NPLP.

[0058] Figure 32 A-32B shows that phosphorylated tau (ptau) is increased in aged NPLP and the number of apoptotic lymphatic endothelial cells is increased in aged NPLP. P values ​​were calculated using the Mann-Whitney test.

[0059] Figure 33A-33E shows the effects of AAV-mVEGF-C-mCherry infection on NPLP in aged mice. (A) Schematic diagram depicting intracisternal injection of AAV-mVEGF-C-mCherry and analysis time. (B) and (C) Representative images and comparison of the lymphatic vessel area enlarged by intracranial administration of AAV-VEGF-C-mCherry in NPLP. (D) Increased lymphatic vessels following AAV-mVEGF-C-mCherry infection serve as pathways for CSF tracers. (E) Representative images and comparison of CSF tracer drainage from dcLNs. Note that AAV-mVEGF-C-mCherry infection increases CSF drainage in aged mice after three weeks. P values ​​were calculated using a two-tailed Mann-Whitney test.

[0060] Figure 34 Figure 3: DCLVs in Prox1-GFP mice show well-developed lymphatic valves (green arrows) and lymphatic vessels, and are finely covered by circular smooth muscle cells.

[0061] Figure 35 β3-tubulin+ peripheral nerve fibers were abundantly distributed along the dcLV.

[0062] Figure 36 A-36B shows that the innervating peripheral nerve fibers are sympathetic. (A) The innervating peripheral nerves are positive for β3-tubulin and tyrosine hydroxylase (TH). TH is a marker for sympathetic nerve fibers. Vesicular acetylcholine transporter (VAChT) is negative in the innervating peripheral nerve fibers along the dcLV. (B) VAChT antibody staining was performed to confirm the presence of pulmonary bronchiolar nerves.

[0063] Figure 37 A-37B shows that the structure of lymphatic vessels downstream of the NPLP remains unchanged with aging. P values ​​were calculated using a two-tailed Mann-Whitney test. ns, p > 0.05.

[0064] Figure 38 Figure 38C shows contraction of dcLV by potassium chloride solution. (A) Schematic diagram of the experimental process. 10-week-old Prox1-GFP male mice were exposed by muscle dissection and treated with saline (0.9% NaCl solution). 5 minutes later, 0.1M or 1.0M potassium chloride (KCl) was applied, and 20 minutes later, the dcLV was washed with saline. (B) Contraction of M-dcLV in Prox1-GFP mice by KCl (white arrow). (C) Contraction of M-dcLV in Prox1-GFP mice by KCl (white arrow) after intracranial injection of QD705 2 hours before imaging.

[0065] Figure 39A-39B shows that the G protein-coupled receptor agonist phenylephrine or a nitric oxide donor (sodium nitroprusside) shrinks or dilates DCLV. (A) The image panel shows that PBS (phosphate-buffered saline) treatment does not shrink or dilate DCLV, but phenylephrine or a nitric oxide donor shrinks or dilates DCLV. (B) The graph shows the dose-dependent shrinkage of DCLV by phenylephrine or the dose-dependent dilation of DCLV by a nitric oxide donor. The amount of CSF tracer was reduced by phenylephrine, while it was increased by a nitric oxide donor. P The values ​​were calculated by two-way repeated measures analysis of variance (ANOVA) test.

[0066] Figure 40 A-40C showed that low doses of phenylephrine (10 nM) enhanced CSF drainage as measured by the outflow of CSF tracer from deep cervical lymph nodes. At higher doses (5 mM), as Figure 36 As shown, the amount of CSF tracer decreased. The nitric oxide donor sodium nitroprusside did enhance CSF outflow at 3 μM but neither enhanced nor decreased CSF outflow at 30 μM. P values ​​were calculated by two-way ANOVA followed by Dunnett's T3 multiple comparison post hoc test.

[0067] Figure 41 Extracranial regulation of CSF drainage is demonstrated.

[0068] Figure 42 The NPLP is shown to be the hub for CSF outflow. The green arrow indicates the direction of CSF outflow. Detailed Description of the Invention

[0069] All references cited herein are incorporated by reference in their entirety as if fully described in full. Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. The definition of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al., (editor), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.Meyers (editor), 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, 22nd ed., 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, 3rd ed., Revised, J. Wiley & Sons (New York, NY 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure, 7th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology, 3rd ed., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory, NY 2006). 2012), which provides those skilled in the art with a general guide to many of the terms used in this application.For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory, NY, 2013);. 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, US Patent No. 5,585,089 (December 1996); and Riechmann et al., 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); Parviz Janfaza et al., Surgical Anatomy of the Head and Neck, Harvard University Press, 2011 (ISBN 978-0674058033).

[0070] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to practice the present invention. Other features and advantages of the present 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 present invention. Indeed, the present invention is in no way limited to the methods and materials described. For convenience, certain terms used herein, in the specification, examples, and appended claims are summarized here.

[0071] Unless otherwise stated or the context indicates, the following terms and phrases include the meanings provided below. Unless otherwise expressly stated or apparent from the context, the following terms and phrases do not exclude the meanings acquired in the art to which they belong. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. It should be understood that the present invention is not limited to the particular methodology, protocols, reagents, etc. described herein and is therefore subject to change. The definitions and terms used herein are intended to help describe specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims.

[0072] As used herein, "treatment" or "treating" includes achieving a therapeutic benefit and / or a preventive benefit. A therapeutic benefit refers to eradicating or improving the underlying disorder or condition being treated. For example, in individuals suffering from general age-related cognitive impairment or Alzheimer's disease, a therapeutic benefit includes partially or completely stopping the progression of the disorder or condition, or partially or completely reversing the disorder or condition. In addition, a therapeutic benefit is achieved by eradicating or improving one or more physiological or psychological symptoms associated with the underlying condition, such that an improvement is observed in the patient, even though the patient may still be affected by the condition. The preventive benefit of treatment includes preventing the condition, delaying the progression of the condition (e.g., slowing the progression of Alzheimer's disease or slowing the decline in cognitive ability that may be due to aging), or reducing the likelihood of the condition occurring.

[0073] As used herein, the term "effective amount" can be an amount sufficient to produce a beneficial or desired outcome in the CNS after administration, such as a beneficial or desired clinical outcome, or to enhance cognition, memory, mood, or other desired CNS outcome. An effective amount is also an amount that produces a preventive effect, for example, an amount that delays, alleviates, or eliminates the occurrence of a pathological or undesirable condition. Such conditions include, but are not limited to, neurodegeneration. An effective amount can be administered in one or more administrations.

[0074] As used herein, a "subject" or "individual" is an animal, such as a mammal. In some embodiments, the "subject" or "individual" is a human. In some embodiments, the subject suffers from Alzheimer's disease or age-related cognitive impairment.

[0075] In some embodiments, the pharmaceutical composition is "peripherally administered." As used herein, these terms refer to administering an agent (e.g., a therapeutic agent) to a subject in any form that is not directly administered to the CNS, i.e., that brings the agent into contact with the non-brain side of the blood-brain barrier. As used herein, "peripheral administration" includes intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, transdermal, inhalation, buccal, intranasal, rectal, oral, parenteral, sublingual, topical, transmucosal, or nasal.

[0076] As used herein, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" 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 detailed discussion of pharmaceutically acceptable carriers / excipients can be found in Remington's Pharmaceutical Sciences, edited by Gennaro, AR, 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 organic acid salts such as acetates, propionates, malonates, benzoates, and the like. For example, the compositions of the present invention can be provided in liquid form and formulated in saline-based aqueous solutions at varying pH values ​​(5-8), with or without detergents such as 0.01-1% polysorbate-80, or carbohydrate additives such as 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. Unless any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

[0077] Exemplary pharmaceutically acceptable carriers of injectable compositions may include salts, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and organic acid salts such as acetates, propionates, malonates, benzoates, etc. Commonly used buffers include histidine, acetates, phosphates, or citrates. Under normal conditions of storage and use, these preparations may contain preservatives to prevent the growth of microorganisms. The effects of microorganisms may be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugars or sodium chloride. The absorption of the injectable composition may be prolonged by using an agent that delays absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0078] For human administration, the formulations meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA and other regulatory agencies. The active compound is typically formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, intralesional, or intraperitoneal routes. In view of the present disclosure, the preparation of aqueous compositions comprising the active ingredient or ingredients is known to those skilled in the art. Typically, such compositions can be prepared as an injection, such as a liquid solution or suspension; can also be prepared in a solid form suitable for preparing a solution or suspension after adding a liquid before injection; and the formulation can also be emulsified.

[0079] Sterile injectable solutions are prepared by combining the required amount of the active compound with the various other ingredients listed above (as needed) in an appropriate solvent, followed by filter sterilization. Typically, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle comprising a basic dispersion medium and the required other ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, preparation methods include vacuum drying and freeze drying techniques, which produce a powder of the active ingredient and any other desired ingredients from a previously filter sterilized solution.

[0080] After formulation, the solution will be administered systemically or locally in a manner compatible with the dosage form and in such an amount as to be therapeutically effective based on the criteria described herein. The formulation is readily administered in a variety of dosage forms, such as the injectable solution type described above, but drug release capsules and the like may also be employed.

[0081] "Recombinant host cell" or "host cell" refers to a cell that contains an exogenous polynucleotide, regardless of the method used for insertion, such as direct uptake, transduction, f-hybridization, or other methods known in the art for creating recombinant host cells. The exogenous polynucleotide can be maintained as a non-integrating vector (e.g., a plasmid), or alternatively, can be integrated into the host genome.

[0082] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, a description of 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, in which the amino acid residues are linked by covalent peptide bonds.

[0083] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides, and polymers thereof, in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0084] As used herein, "intracranial" refers to the material within the skull or skull, including the meninges and parenchyma, as well as other structures.

[0085] As used herein, "extracranial" refers to material not within the skull, such as the nasopharynx and the medial deep cervical lymphatics (dcLV).

[0086] As used herein, an "exogenous agent" refers to a composition or method that can be administered to a subject to increase or stabilize CSF flow. Specifically, the exogenous agent affects flow in the NPLP and / or dcLV. In this context, a "glidant" is synonymous with an exogenous agent, particularly because it increases or stabilizes CSF outflow through the NPLP and / or dcLV.

[0087] As used herein, "NPLP-dcLV" or "NPLP-dcLV pathway" refers to the extracranial region or lymphatic space or system of lymphatic structures where CSF outflow occurs from the NPLP to the deep cervical lymph nodes present in the neck along the internal jugular vein and carotid sheath.

[0088] NPLP and dcLV

[0089] As described herein, applicants have discovered a novel lymphatic system or pathway that functions to drain macromolecules and debris from the central nervous system (CNS). Specifically, reducing drainage through NPLP-dcLV vessels can reduce cerebrospinal fluid (CSF) outflow and exacerbate symptoms of neurodegenerative diseases characterized by increased concentration and / or accumulation of molecules in the CNS, such as Alzheimer's disease (AD). Modulating lymphatic vessels to increase flow can alleviate cognitive impairment conditions or AD symptoms associated with aging, including cognitive symptoms, accumulation of amyloid-beta plaques, and tau hyperphosphorylation.

[0090] Methods for treating, preventing, suppressing or ameliorating the symptoms of a neurodegenerative disease or condition associated with elevated and / or accumulated concentrations of macromolecules, cells and debris in the CNS are described. The method can increase drainage through lymphatic vessels and thus increase the flow of CSF. The methods of the present invention are advantageous because they comprise one, more or all of the following benefits: (i) increasing the outflow and drainage of CSF; (ii) reducing the accumulation of macromolecules, cells or synapses and myelin debris in the CNS (e.g., reducing the accumulation of amyloid beta protein); and (iii) maintaining or improving cognitive function (e.g., memory function) in subjects who suffer from cognitive impairment conditions due to old age or are suspected of having dementia (such as neurodegenerative diseases) and / or are at risk of dementia (such as neurodegenerative diseases).

[0091] CSF flow and agents that increase or stabilize flow

[0092] As used herein, "flow" refers to the rate of perfusion through a region of the central nervous system of a subject. In some embodiments of the present invention, "flow" can be measured as the rate at which a marker or tracer in the CSF perfuses through a specific region of the central nervous system. Thus, flow between two subjects or two groups of conditions can be compared by determining the rate at which an infused marker or tracer perfuses through a specific region or volume of the brain and / or other parts of the CNS. Additionally, "outflow" refers to the discharge of CSF into the systemic circulation, and in particular, through the NPLP-cdLV pathway of the present invention.

[0093] As used herein, "exogenous agent," "agent," or "glidant" refers broadly to any composition that increases the passage of substances into and out of lymphatic vessels and, thereby, modulates CSF flow. Specifically, the present invention relates to an agent that specifically or generally increases CSF flow in NPLP ("NPLP glidant") or dcLV ("dcLV glidant").

[0094] Without being limited by theory, according to several embodiments herein, it is contemplated that the removal of macromolecules by NPLP-dcLV lymphatics can maintain their low concentrations in the CSF, thereby allowing for a gradient of macromolecule clearance from the parenchyma. Furthermore, the higher the fluid flow and drainage rate in the CNS, the higher the clearance rate and / or lower the concentration of cells, macromolecules, waste products, and debris in the CNS.

[0095] Exogenous agents can increase the diameter of NPLP-dcLV lymphatic vessels and / or repair NPLP-dcLV lymphatic vessels, which increases or stabilizes the rate or level of drainage, resulting in increased CSF flow. In some embodiments, exogenous agents enlarge or repair NPLP and / or enhance the contraction and relaxation of dcLV, thereby increasing net drainage and resulting in increased or stabilized CSF flow.

[0096] NPLP glidant

[0097] Examples of suitable NPLP flow aids for increasing CSF flow include, but are not limited to, vascular endothelin C (VEGF-C), vascular endothelin 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 agonists, neuropilin, 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.

[0098] VEGFR3, also referred to as FLT4, is a receptor tyrosine kinase, and its signal transduction pathway is relevant with embryonic lymphatic development and adult lymphangiogenesis.After being bound to the ligand, VEGFR3 dimerizes and is activated by autophosphorylation. Therefore, according to some embodiments herein, VEGFR3 agonists are applicable to treating the neurodegenerative disease (such as AD) relevant to the accumulation of molecules in the brain, alleviating the symptoms of the neurodegenerative disease (such as AD) relevant to the accumulation of molecules in the brain, or preventing the neurodegenerative disease (such as AD) relevant to the accumulation of molecules in the brain. Therefore, in some embodiments, such as in the method or composition where discharge and flow need to be increased, flow aid is a VEGFR3 agonist.

[0099] In particular, VEGF-C promotes the growth of lymphatic vessels (lymphangiogenesis). It acts primarily on lymphatic endothelial cells (LECs) via its receptor VEGFR-3, promoting survival, growth, and migration. Therefore, VEGF-C is suitable as a VEGFR3 agonist.

[0100] According to the present method, the effective amount of a VEGFR3 agonist can be understood as its ability to expand or repair NPLP, thereby increasing or stabilizing CSF flow, or treating, improving or preventing various cognitive impairments caused by aging or neurodegenerative diseases by increasing the clearance of substances from the CNS. Therefore, in the method of the present invention, the effective amount of a VEGFR3 agonist expands NPLP 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, the effective amount of a VEGFR3 agonist increases CSF flow 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 recited values.

[0101] In addition to VEGF-C, VEGFR3 agonists may also include VEGF-D. In one aspect, VEGF-C and VEGF-D together excite VEGFR3 and can be provided as a single composition or separate compositions. In some embodiments, VEGFR3 agonists include analogs, variants, or functional fragments, such as mutants, orthologs, fragments, or truncation of VEGF-C or VEGF-D.

[0102] The application also discloses exogenous nucleotides encoding VEGFR3 agonists (such as VEGF-C). On the one hand, nucleotides encoding VEGF-C or VEGF-D as described herein are expressed in a subject so that the VEGFR3 agonist is administered to the subject. For example, an exogenous vector (such as a retrovirus, slow virus, adenovirus or adeno-associated virus vector) comprising a nucleic acid encoding a VEGFR agonist as described herein can be inserted into the host nucleic acid of the subject. In some embodiments, the vector further comprises a transcriptional mechanism to promote transcription of the nucleic acid encoding the VEGFR3 agonist.

[0103] In one aspect, the VEGFR3 agonist can include modifications, such as glycosylation, pegylation, etc. In some embodiments, a composition for use according to the methods described herein comprises a VEGFR3 agonist and a pharmaceutically acceptable diluent or carrier.

[0104] The present invention also relates to a method of applying a glidant (such as a chemical molecule or a biological molecule) and then analyzing the rate of CSF flow through an NPLP or NPLP-dcLV pathway, wherein if the flow or outflow is greater than before application of the glidant, the glidant is considered to be an effective agent in increasing CSF flow in the area.

[0105] Thus, in one aspect, the invention relates to a method of assaying a CSF agent that increases CSF efflux via the NPLP or NPLP-dcLV pathway.

[0106] dcLV glidant

[0107] Some dcLV glidants can be described as follows. Potassium chloride (KCl) at 0.1 and 1.0 M can cause strong but transient contraction of dcLV ( Figure 38 In addition, the G protein-coupled receptor agonist phenylephrine or the nitric oxide donor (sodium nitroprusside) constricts or dilates dcLV and can modulate the amount of CSF tracer ( Figure 39 Low doses of phenylephrine (10 nM) increased the amount of CSF drained, but high doses (5 mM) decreased the amount of CSF drained, as measured by CSF tracer accumulation in dcLNs ( Figure 40 Low doses of the nitric oxide donor sodium nitroprusside (3 μM) enhanced CSF drainage, but no changes were observed with 30 μM nitric oxide donor ( Figure 40 ).

[0108] Contraction and relaxation of the circular smooth muscle covering the dcLV can be modulated by: 1) stimulation and inhibition of smooth muscle cells or peripheral nerves; 2) neurotransmitters; 3) mechanical stimulants; and 4) gentle massage of the neck area adjacent to the cdLV. CSF drainage can be promoted by extracranial manipulation, administration of agents, and modulators ( Figure 41 Considering that DCLV does not change significantly in the elderly, reduced CSF drainage can be enhanced by extracranial manipulation of DCLV.

[0109] The composition to be administered may comprise a single unit dose of a glidant that is effective to increase or repair flow, increase clearance, or reduce accumulated amyloid beta plaques. In some embodiments, the effective amount of the glidant is from about 0.00015 mg / kg to about 1.5 mg / kg, including any other amount or range considered a therapeutically effective amount of a compound as disclosed herein. This range may be greater or less than the range of about 0.00015 mg / kg to about 1.5 mg / kg.

[0110] Mechanical stimulation can include single or multiple vibrations to increase or restore flow in the CSF, or to increase the clearance or reduce the accumulation of amyloid beta plaques or phosphorylated tau protein. In some embodiments, the effective acceleration of the CSF through the dcLV is about 0.001 m / s 2 to about 2.5m / s 2 , which includes any other range considered for therapeutically effective acceleration as disclosed herein. This range may be greater than or less than about 0.001 m / s 2 to about 2.5m / s 2 Mechanical stimulation is not limited to mechanical vibration and may also include massage or any other mechanical method to allow the dcLV to contract and release the dcLV. Vibration or massage may be performed to increase CSF flow through the dcLV. Vibration machines suitable for use on humans are already known in the art.

[0111] The present invention also relates to a method of applying a glidant (such as a biological or chemical molecule or physical pressure method) and then analyzing the rate of CSF flow through a dcLV, wherein if the flow or outflow of a region of the dcLV is greater than before the application of the glidant, then the glidant is considered to be an effective agent for increasing CSF flow in that region. In one aspect, the present invention relates to a method of determining CSF outflow through a dcLV by a CSF agent.

[0112] Route of administration

[0113] The glidants of the present invention can be administered to a subject using any of a variety of suitable routes of administration, provided that the route of administration administers the glidant to the NPLP-dcLV pathway of the subject.

[0114] As used herein, "administering to a subject's NPLP-dcLV access space" does not necessarily require that the glidant be administered directly to the NPLP-dcLV access space, but rather the term encompasses direct and / or indirect administration of the glidant to the NPLP-dcLV access space. According to some embodiments herein, it is contemplated that the glidant is administered so that it is in fluid communication with the subject's NPLP-dcLV access space, typically by optionally administering the glidant to the brain. Thus, in one aspect, the glidant is not administered systemically. In another aspect, the glidant is not administered systemically, but rather is administered to a fluid, tissue, or organ in fluid communication with the NPLP-dcLV access space. In some embodiments, the glidant is not administered systemically, but rather is administered to the CNS. In some embodiments, the glidant is administered to the CNS, but not to any organ or tissue outside the CNS. In some embodiments, the glidant is not administered to the blood.

[0115] In some embodiments, the glidant is administered through the nose or mouth to more directly affect the NPLP. For example, the glidant can be provided in the form of a nasal spray or can be directly contacted with the nasal mucosa.

[0116] In some embodiments, the glidant is administered by contact with the subject's CSF. For example, the glidant can be injected directly into the patient's CSF (e.g., intrathecally). A suitable injection device may include an injector or pump that is inserted or implanted into the subject and communicates with the CSF fluid. In another aspect, the glidant can be a slow-release gel that is implanted into the subject so that it communicates with the subject's CSF fluid and thus contacts the CSF. Topical administration is also contemplated, particularly to the neck region to affect the muscles surrounding the dcLV. Non-limiting examples of topical administration include creams, lotions, gels, salve, sprays, dispersions, suspensions, pastes, and ointments.

[0117] In some aspects, the glidant is administered transcranially. For example, a glidant (such as a gel formulation) can be placed outside the subject's skull and can pass through the subject's skull. In some embodiments, the glidant contacts a thinner portion of the subject's skull to facilitate transcranial delivery.

[0118] In another aspect, the glidant is administered by expressing a nucleic acid encoding the glidant in a subject. A vector comprising the nucleic acid, e.g., a viral vector, such as a retroviral vector, a lentiviral vector, or an adenoviral vector or an adeno-associated viral vector (AAV), can be administered to a subject as described herein, e.g., via injection or inhalation. In some embodiments, expression of the nucleic acid is induced in the subject, e.g., via a transcriptional regulator.

[0119] In some embodiments, the glidant is selectively administered to the NPLP-dcLV access space of a subject. As used herein, "selective" administration refers to preferential administration of the glidant to a designated target as compared to other tissues or organs. Thus, direct injection into the extracranial NPLP-dcLV access represents "selective" administration, while administration into the CSF, typically via spinal injection, does not represent "selective" administration. In some embodiments, the glidant is selectively administered to the NPLP-dcLV access space and is not administered to portions of the CNS outside the NPLP-dcLV access space, nor to any tissue or organ outside the CNS. In some embodiments, the glidant is selectively administered to the CNS and is not administered to tissues or organs outside the CNS, such as the peripheral nervous system, muscle, gastrointestinal system, or muscle tissue.

[0120] In some embodiments, the glidant is administered to the subject in the area surrounding the cervical region of the NPLP-dcLV pathway. As used herein, "the cervical region surrounding the NPLP-dcLV pathway" refers to the tissue surrounding the lymphatic vessels from the NPLP to the deep cervical lymph nodes.

[0121] In some embodiments, the glidant is administered to the cervical region surrounding the mandibular lymph nodes. As used herein, "cervical region surrounding the mandibular lymph nodes" refers to the tissue surrounding the afferent lymphatic vessels of the mandibular lymph nodes. For example, it includes lymph from the hard palate to the mandibular lymph nodes.

[0122] For any of the routes of administration listed according to the methods of the present invention, it is contemplated that the glidant can be administered in a single administration, or in two or more administrations separated by a period of time. For example, in some embodiments, the glidant described herein can be administered hourly, daily, every other day, every three days, every four days, every five days, every six days, weekly, every two weeks, monthly, every two months, etc., via the routes of administration described herein. In some embodiments, the glidant is administered in a single administration without any additional administration.

[0123] Some embodiments include methods of preparing a composition or medicament comprising a glidant as described herein, which is suitable for administration according to a route of administration as described herein. For example, in some embodiments, the VEGFR3 agonist is prepared for nasal administration, administration to CSF, or transcranial administration.

[0124] Particularly preferred methods of administering the agent include, but are not limited to, intrathecal routes to the CSF space, nasal or oral routes to the nasopharynx, transcervical or transdermal or topical routes to the neck muscles or neck lymph nodes or neck spaces.

[0125] Neurodegenerative diseases and conditions

[0126] The present invention can be used to treat, prevent, inhibit, improve or alleviate the symptoms of one or more neurodegenerative diseases. These diseases can occur in subjects, such as humans, and non-human animals, such as non-human mammals, and especially non-human primates.

[0127] In some embodiments, methods for increasing NPLP-dcLV lymphatic flow, drainage, and / or clearance are used to treat, prevent, inhibit, or mitigate neurodegenerative diseases associated with the accumulation of macromolecules, cells, and debris in the CNS. In some embodiments, neurodegenerative diseases associated with the accumulation of macromolecules, cells, and debris in the CNS are treated, prevented, inhibited, or mitigated. Examples of neurodegenerative diseases include cognitive decline associated with aging, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke.

[0128] In some embodiments, neurodegenerative diseases can be prevented, treated or improved prophylactically. Therefore, it can be determined that a subject with one or more disease risk factors for neurodegeneration needs to receive the treatment methods described herein. For example, a subject may have accumulated amyloid beta protein plaques in their CNS, and even if they do not yet have an AD diagnosis based on cognitive symptoms, they may benefit from increased flow, increased excretion, increased clearance and / or reduced amyloid beta protein plaques. According to the methods, compositions and uses of some embodiments herein, many AD risk factors are suitable as risk factors, such as familial AD, genetic markers for AD or symptoms of AD, such as early dementia. The primary risk factor for sporadic AD is age. However, the increased risk of this form of AD is also attributed to multiple genetic abnormalities.

[0129] Ways to increase traffic

[0130] The methods of the present invention may include determining whether the subject needs to increase fluid flow in the central nervous system. In addition, the methods of the present invention may also include determining whether the subject needs to increase the discharge of CFS. If the subject needs to increase fluid flow or discharge, the method may include administering an effective amount of a VEGFR3 agonist or applying mechanical stimulation to the subject's NPLP-dcLV pathway space. Thus, fluid flow in the subject's central nervous system and the level or rate of CFS outflow can be increased. In some embodiments, the VEGFR3 agonist comprises VEGF-C or VEGF-D or an analog, variant, or fragment thereof. It is also contemplated that in some embodiments herein, FGF2 may be used in place of the indicated VEGFR3 agonist to increase flow, or in addition to a VEGFR3 agonist to increase flow. In some embodiments, mechanical stimulation may be applied to the neck region surrounding the NPLP-dcLV pathway.

[0131] The need for increased fluid flow or outflow can be determined by determining whether the subject suffers from cognitive impairment, neurodegenerative disease, or is at risk of developing a neurodegenerative disease associated with old age. The disease can be associated with elevated concentrations and / or accumulation of molecules, cells, or debris in the CNS, such as Alzheimer's disease (AD). In some embodiments, the subject can be determined to be at risk for the disease by, for example, having a familial history of the disease, having one or more gene markers associated with the disease, advanced age, or exhibiting symptoms of the disease (e.g., early dementia in the case of AD).

[0132] As used herein, "advanced age" refers to an age characterized by a decline in memory function, a decrease in CSF production, and a significant increase in neuronal aging, and in the context of some embodiments, may include at least 65 years old for a human, for example, at least 60, 65, 70, 75, 80, or 85 years old, including the range between any of these values. In some embodiments, determining whether a subject needs to increase fluid flow or outflow includes determining that the subject suffers from a neurodegenerative disease, such as AD. In some embodiments, determining whether a subject needs to increase fluid flow or outflow includes determining that the subject has a risk factor for a neurodegenerative disease associated with an increase and / or accumulation of molecules or macromolecules or cells or debris in the CNS, as described herein. In some embodiments, determining whether a subject needs to increase fluid flow or outflow includes determining that the subject has a risk factor, and determining that the subject itself suffers from the disease.

[0133] In some embodiments, neurodegenerative disease or the patient's condition is selected from the group consisting of at least one of the following: cognitive impairment, Alzheimer's disease (AD), dementia, Parkinson's disease, Huntington's disease or stroke that may be caused by old age. In some embodiments, neurodegenerative disease is Alzheimer's disease. In some embodiments, risk factor is the risk factor for Alzheimer's disease as described herein. In some embodiments, after determining that the subject has the risk factor for neurodegenerative disease (even if the subject itself does not necessarily suffer from the disease), VEGFR3 agonists and / or FGF2 are administered to the subject, for example, for prophylactic treatment or prevention. In some embodiments, after determining that the subject suffers from neurodegenerative disease, VEGFR3 agonists and / or FGF2 are administered to the subject.

[0134] Without being limited by theory, according to several embodiments herein, it is contemplated that VEGFR3 agonists and / or FGF2 need not be administered systemically to effectively modulate NPLP-dcLV lymphatic vessel size and drainage or flow. Thus, in some embodiments, VEGFR3 agonists and / or FGF2 are selectively administered to the NPLP-dcLV pathway space of a subject. In some embodiments, VEGFR3 agonists and / or FGF2 are administered to spaces within or outside the CNS. In other embodiments, VEGFR3 agonists and / or FGF2 are administered to the NPLP-dcLV pathway space but not to the blood. In some embodiments, VEGFR3 agonist and / or FGF2 are applied to the experimenter by the approach selected from the group consisting of following at least one approach: local, intrathecal, nasal administration, oral, transcranial administration, contact with experimenter's cerebrospinal fluid (CSF), pumped into experimenter's CSF, implanted in skull or brain, the skull or skull portion of experimenter's thinning is contacted with VEGFR3 agonist and / or FGF2, or the nucleic acid encoding VEGFR3 agonist and / or FGF2 is expressed in the experimenter, or the combination of any listed approach. In some embodiments, what is administered is a VEGFR3 agonist. In some embodiments, the VEGFR3 agonist is selected from the group consisting of following at least one approach: VEGF-C, VEGF-D or its analog, variant or functional fragment.

[0135] In some embodiments, administration of a VEGFR3 agonist results in an increase in NPLP-dcLV lymphatic vessel diameter, the number of NPLP-dcLV pathway lymphatic vessels, NPLP-dcLV pathway lymphatic drainage, or an improvement in symptoms of a neurodegenerative disease or condition. For example, in some embodiments, administration of a VEGFR3 agonist increases NPLP lymphatic vessel diameter by at least about 5%, such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, including ranges between any two of the recited values. In some embodiments, the average diameter of a subject's NPLP-dcLV lymphatic vessel population is increased by a value described herein. In some embodiments, administration of a VEGFR3 agonist increases CSF flow in the central nervous system, including CSF outflow from a subject, including increasing the rate of fluid perfusion throughout a region of the subject's brain.

[0136] In some embodiments, mechanical stimulation of the cervical region surrounding the NPLP-dcLV pathway results in increased CSF flow, or improved symptoms of a neurodegenerative disease or condition. For example, in some embodiments, application of mechanical stimulation increases CSF flow (including CSF outflow) in the subject's central nervous system by at least about 5%, such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, including ranges between any two of these values. In some embodiments, the average CSF flow rate in the subject's NPLP-dcLV lymphatic vessels is increased by the values ​​described herein. Mechanical stimulation herein includes vibration, ultrasound stimulation, light stimulation, electrical stimulation, magnetic stimulation, temperature stimulation, and is not limited to the stimulations listed herein.

[0137] Improving clearance can reduce macromolecules (such as amyloid beta protein plaques) or reduce their accumulation rate. Without being limited by theory, it is envisioned that by clearing soluble amyloid beta protein from the CNS, the gradient will favor the dissolution of amyloid beta protein plaques, allowing fluid in the CNS to continue to flow and the CNS to continue to be cleared, and amyloid beta protein plaques can be reduced, or the rate of increase can be reduced. Therefore, a reduction in amyloid beta protein plaques can represent a reduction in the cause of the disease caused by amyloid beta protein plaques.

[0138] By increasing fluid flow, the amount of amyloid beta protein plaques accumulated in a subject can be reduced, or the rate of accumulation thereof can be reduced. In some embodiments, the amount of amyloid beta protein plaques accumulated or the rate of accumulation is reduced by at least 2%, such as 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 recited values.

[0139] Detecting changes in CSF flow

[0140] The NPLP is a newly discovered hub for CSF drainage through the skull base (including the cribriform plate). The submucosa of the hard palate is a newly discovered pathway for CSF drainage to the mandibular lymph nodes. Therefore, measuring changes in CSF flow rate in humans at these locations is within the scope of the present invention.

[0141] In one aspect, a swab or biopsy is performed on the nasopharyngeal mucosa or hard palate submucosal area to obtain a cell sample, which should include NPLP or hard palate submucosal lymphocytes. The CSF content in the sample is determined or measured over time, and when the determination is repeated in humans, the results are compared from time to time.

[0142] When CSF outflow is found to decrease over time, then an exogenous agent may be administered to increase CSF outflow in the subject.

[0143] The assay method may include imaging NPLP with a tracer molecule and observing it under a microscope. Other methods may include, but are not limited to, proteomic analysis of CSF-specific proteins. The level of a selected protein in the CSF may be measured from time to time to determine its amount, wherein a decrease or increase in the amount of the selected protein indicates the level and quality of CSF drainage. Examples of selected proteins to be monitored may include β2-transferrin in a CSF sample obtained. Specifically, for example, an increase in the amount of β2-transferrin in a sample over time indicates improved CSF drainage function. A decrease in the amount of β2-transferrin in a sample over time indicates impaired CSF drainage function. Other proteins may be included without limitation.

[0144] Promotes cerebrospinal fluid (CSF) drainage

[0145] Improving cerebrospinal fluid (CSF) drainage is valuable for preventing and treating neurodegenerative diseases, including Alzheimer's disease. The present invention provides information on extracranial approaches and methods for improving CSF drainage. The present invention encompasses extracranial approaches for improving CSF drainage via the nasopharyngeal lymphatic plexus (NPLP) and deep cervical lymphatic vessels (dcLV) for preventing and treating neurodegenerative diseases.

[0146] In one aspect, the present invention relates to a method for preventing or treating a central nervous system disease by improving CSF drainage in a human by repairing or sometimes expanding the NPLP. The CNS disease can be Alzheimer's disease, Parkinson's disease, or Huntington's disease. The NPLP can be restored by administering to a human in need thereof an agent such as, but not limited to, vascular endothelin-C (VEGF-C), vascular endothelin-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 agonists, neuropilin, prostaglandin E2, 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.

[0147] The contraction and relaxation of the circular smooth muscle covering the dcLV can be regulated by stimulatory and inhibitory agents of smooth muscle cells, such as, but not limited to, agents that increase or decrease myosin phosphorylation by activating myosin light chain kinase, or agents that activate myosin light chain phosphatase by activating voltage-operated Ca2+ channels (KCl) or G protein-coupled receptor agonists (phenylephrine) or stimulating cGMP-dependent protein kinases (nitric oxide or acetylcholine).

[0148] Contraction and relaxation of the circular smooth muscle covering the dcLV can be further modulated by stimulating and inhibiting peripheral nerves with agents that interfere with depolarization or exocytosis of synaptic vesicles, such as, but not limited to, botulinum toxin or tetanus toxin.

[0149] Contraction and relaxation of the circular smooth muscle covering the dcLV can also be regulated by stimulatory and inhibitory neurotransmitters, such agents including but not limited to agonists or antagonists of neurotransmitter receptors or agonists or antagonists of degradative enzymes at the circular smooth muscle covering the dcLV, such as agonists or antagonists of norepinephrine.

[0150] The contraction and relaxation of the circular smooth muscle covering the dcLV can also be modulated by mechanical stimuli such as, but not limited to, vibrators, high or low frequency electrical stimuli, optogenetic stimulation of specific wavelengths of light.

[0151] The method of delivery of the agent may be, but is not limited to, an intrathecal route to the CSF space, a nasal or oral route to the nasopharynx, a transcervical or transcutaneous or topical route to the neck muscles or neck lymph nodes or neck space.

[0152] The various methods and techniques described above provide a variety of ways to implement the present application. Of course, it will be understood that not all of the objects or advantages described can be achieved according to any particular embodiment described herein. Thus, for example, one skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one or a group of advantages described herein without necessarily achieving other objects or advantages taught or indicated herein. Various alternatives are mentioned herein. It will be understood that some preferred embodiments explicitly include one, another, or several features, while other embodiments explicitly exclude one, another, or several features, and still other embodiments mitigate particular features by including one, another, or several advantageous features.

[0153] In addition, those skilled in the art will recognize the applicability of various features from different embodiments. Similarly, those skilled in the art can adopt the above-mentioned various elements, features and steps in various combinations, as well as other known equivalents of each such element, feature or step, to implement the method according to the principles described herein. Among the various elements, features and steps, some will be explicitly included, and others will be explicitly excluded in different embodiments.

[0154] Although the present application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the present application extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses and modifications and equivalents thereof.

[0155] The preferred embodiments of the present application are described herein, which include the best mode of implementing the present application known to the inventor. Various modifications of those preferred embodiments will become apparent to those skilled in the art after reading the foregoing description. Those skilled in the art may adopt these modifications as appropriate, and the present application may be practiced in a manner different from that specifically described herein. Therefore, many embodiments of the present application include all modifications and equivalents of the subject matter described in the appended claims, as long as applicable law permits. In addition, unless otherwise specified herein or the context clearly contradicts, this application covers any combination of the above-mentioned elements in all possible variations thereof.

[0156] All patents, patent applications, patent application publications, and other materials, such as articles, books, specifications, publications, documents, things, and the like, cited herein are hereby incorporated by reference in their entirety for all purposes, except to the extent that any application record to which they relate is inconsistent or conflicting with this document, or that which would limit the broadest scope of the claims now or later to which they relate. For example, if there is any inconsistency or conflict between the descriptions, definitions, and / or usage of terms in any incorporated material and the descriptions, definitions, and / or usage of terms in this document, the descriptions, definitions, and / or usage of terms in this document shall control.

[0157] It should be understood that the embodiments of the present application disclosed herein are merely illustrative of the principles of the embodiments of the present application. Other modifications that may be employed are also within the scope of the present application. Therefore, by way of example, and not limitation, alternative configurations of the embodiments of the present application may be used according to the teachings herein. Therefore, the embodiments of the present application are not limited to what is shown and described in detail.

[0158] Various embodiments of the present invention have been described above in the detailed description. Although these descriptions directly describe the above-mentioned embodiments, it should be understood that those skilled in the art may conceive of modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the scope of this specification are intended to be included in this specification. Unless otherwise indicated, the inventors intend that the words and phrases in the specification and claims be given the ordinary and customary meanings by those of ordinary skill in the art.

[0159] The foregoing presents various embodiments of the present invention known to the applicant at the time of filing the application and is intended to be illustrative and descriptive. This specification is not intended to be exhaustive or to limit the invention to the precise form disclosed, and many modifications and variations are possible in light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable those skilled in the art to utilize the invention in various embodiments and modifications suitable for the specific use contemplated. Therefore, the present invention is not intended to be limited to the specific embodiments disclosed for practicing the invention. Example

[0160] The following examples are provided to better illustrate the present invention and should not be construed as limiting the scope of the invention. The specific materials mentioned herein are for illustrative purposes only and are not intended to limit the present invention. Those skilled in the art can develop equivalent methods or reactants without exercising creative ability and without departing from the scope of the present invention.

[0161] Example 1: Improving cerebrospinal fluid (CSF) drainage is valuable for preventing and treating neurodegenerative diseases, including Alzheimer's disease. Here, the present invention provides information on extracranial approaches and methods to improve CSF drainage.

[0162] The present invention encompasses extracranial approaches to promote CSF drainage via the nasopharyngeal lymphatic plexus (NPLP) and deep cervical lymphatic vessels (dcLV) for the prevention and treatment of neurodegenerative diseases.

[0163] Anesthetized mice (10-week-old male Prox-1 GFP mice) were placed prone on a stereotaxic frame and placed under a microscope equipped with a heating pad. The mouse head was adjusted to a 90° angle with the body axis, and a mouthpiece was used to facilitate access to the cerebral medullary cistern. After the skin was incised along the posterior midline of the neck, the muscle layer was carefully separated with a microreactor. A No. 33 NanoFil needle (WorldPrecision Instruments) was used to superficially puncture the atlanto-occipital membrane covering the cerebral medullary cistern, and then 3 μl of tetramethylrhodamine (TMR)-coupled 10 kDaMW dextran (Invitrogen, D1816) was infused into the subarachnoid space at 1 μl / min using a microinjector (88000, Hamilton) and a microinfusion machine (Fusion 100, Chemyx Inc). The infusion lasted for 3 min ( Figure 7A). The mouse was left in place for 5 min and the needle was slowly withdrawn to prevent CSF leakage. The muscle layer and neck skin were then sutured with 6-0 black silk (Ailee, SK617). 60 min after infusion, the head was dissected without cardiac perfusion or saline immersion. After decapitation, the head was cut in half along the sagittal plane, and then fluorescent images of the sectioned head were obtained using a fluorescent stereo zoom microscope (AxioZoom V16, Carl Zeiss) ( Figure 7 A).

[0164] The results showed that glucan accumulated highly in the olfactory bulb, cribriform plate, dorsolateral skull, upper spinal cord region, and skull base adjacent to the nasopharynx ( Figure 7 B). Notably, significant Prox1-GFP signals were detected in the nasopharynx, oropharynx, hard palate, and soft palate ( Figure 7 B), showing the abundant presence of LVs in these regions.

[0165] Example 2: Anesthetized mice (10-week-old male Prox1-GFP mice) were subjected to the same procedures as in Example 1 and sacrificed by incising the abdominal aorta 60 minutes after injection. After dissecting the surrounding muscles using an operating microscope (SZX16, Olympus), dextran in the LV, nasopharynx, and dcLN was imaged using a fluorescent stereo zoom microscope ( Figure 8 We found the presence of medial and lateral afferent LV to the dcLN and named them medial-deep cervical LV (M-dcLV) and lateral-dcLV (L-dcLV) ( Figure 8 A). L-dcLV connects to the basolateral LV via the jugular foramen, whereas M-dcLV connects to the LV arising from the nasopharynx ( Figure 8 A). Injected dextran was detected not only in dcLN but also in M- and L-dcLV ( Figure 8 A). When we examined the nasopharynx in detail, glucan was highly detected in the upper nasopharynx but not in the lower nasopharynx and oropharyngeal region ( Figure 8 Importantly, a well-organized Prox1+ lymphoid plexus was found around the nasopharynx ( Figure 8 B and 8C) and named it "nasopharyngeal lymphatic plexus (NPLP)". NPLP has valves ( Figure 8 C, green arrow), and the injected dextran was highly detected in the NPLP of the upper nasopharynx ( Figure 8 C, reddish-yellow arrow). The NPLP is connected to the oropharyngeal lymphoid plexus (OPLP), but no dextran signal was detected in the OPLP ( Figure 8 C) These findings suggest that NPLP, but not OPLP, serves as an outlet pathway for CSF connected to dcLNs via M-dcLVs.

[0166] Example 3: The same procedures as in Examples 1 and 2 were performed on anesthetized mice (10-week-old male Prox1-GFP mice), and 30 minutes after injection, time-lapse in vivo imaging of NPLP was performed using a fluorescent stereo zoom microscope ( Figure 9 A). Flow of injected dextran was detected in the NPLP of the upper nasopharynx ( Figure 9 B, reddish-yellow arrow).

[0167] Example 4: To verify the above findings, a solution containing 3 μl of fluorescent microbeads (0.5 μm diameter, F8887, Thermo-Fisher) was infused into the intracranial cavity at 1 μl / min for 3 min using a microinjector (88000, Hamilton) and an infusion machine (Fusion100, Chemyx Inc). After 6 hours, NPLP was sampled and it was found that the fluorescent microbeads were abundantly and selectively present with NPLP ( Figure 10 ), which confirms that NPLP can serve as a hub for CSF drainage.

[0168] Example 5: The procedure of Example 1 was applied to anesthetized mice (10-week-old male Prox1-GFP mice), and the mice were sacrificed by incising the abdominal aorta 60 minutes after injection. After dissecting the surrounding muscles using an operating microscope (SZX16, Olympus), the dextran in the proximal region of the LV and dcLN was imaged using a fluorescent stereo zoom microscope ( Figure 11 We found that medial and lateral afferents of LV to dcLN, namely M-dcLV and L-dcLV ( Figure 11 L-dcLV connects to the basolateral LV via the jugular foramen, whereas M-dcLV connects to the LV arising from the nasopharynx ( Figure 11 The injected dextran was detected not only in dcLN but also in M- and L-dcLV ( Figure 11 Prox1+ dense lymphatic valves are periodically distributed within the dcLV ( Figure 11 , green arrow).

[0169] Example 6: Figure 12Dorsal, ventral, and lateral views of the anterior nasopharyngeal NPLP and posterior nasal lymphatic plexus are shown. To obtain these images, immunofluorescence staining (IFS) was performed on the murine NPLP of 10-week-old male Prox1-GFP mice. After anesthesia and right atrial puncture, ice-cold phosphate-buffered saline (PBS) was perfused into the left ventricle to remove blood. After PBS perfusion, 4% paraformaldehyde solution was injected through the left ventricle to fix the tissue. With the help of an operating microscope (Stemi508, Zeiss), fine forceps and surgical microscissors were used to remove the surrounding skull, nerves, and soft tissue to separate the nasopharyngeal mucosa. The dorsal and ventral portions of the mucosa were cut in half transversely, and the lateral portion of the mucosa was cut in half longitudinally. The collected tissues were fixed with 2% paraformaldehyde solution at 4 degrees Celsius for 2 hours. After fixation, the nasopharyngeal mucosa was incubated in 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) at room temperature for 1 hour. To label lymphatic vessel markers, the nasopharyngeal mucosa was immersed in 5% normal donkey serum (containing VEGFR3 antibodies (AF743, R&D) and LYVE1 antibodies (11-034, Angiobio) at a 1:400 dilution) for 12 hours at 4°C. After washing with PBS, the tissue blocks were incubated with normal donkey serum containing Alexa-594-conjugated anti-goat IgG antibodies and Alexa-647-conjugated anti-rabbit IgG antibodies (Jackson ImmunoResearch) for 12 hours at 4°C. After washing with PBS, the tissues were covered with mounting medium (H1200, Vector). Images were acquired using a confocal microscope (LSM800, Zeiss) with a Plan-Apochromat 10x / NA 0.45 lens.

[0170] Figure 13 A cross-sectional view of the NPLP is shown (white arrow). In addition to tissue sampling and sectioning, Figure 12 method. The head was collected, the surrounding skin was peeled off after perfusion, and after fixation, it was immersed in 2% PFA solution at 4 degrees Celsius for 12 hours. For decalcification, the head was immersed in 0.5M EDTA solution at 4 degrees Celsius for 48 hours. After decalcification, the head sample was dehydrated by immersing it in 30% sucrose solution for 48 hours. The head sample was embedded and frozen in freezing section medium (Leica) and cut into 30μm slices using a Cryocut microtome (Leica). IFS was performed. The lymphatic vessels from the front to the nasopharynx constitute the posterior nasal lymphatic plexus. The NPLP has abundant lymphatic valves in the middle of the nasopharynx, and they are connected to the lymphatic vessels in the posterior nasopharynx.

[0171] based on Figure 12 and Figure 13 , the 2D and 3D schematics of NPLP are shown in Figure 14 and Figure 15 middle.

[0172] Example 7: Figure 16 As shown in Figure 2, newly discovered lymphatic vessels originating from the pituitary region and connected to the NPLP were observed. Tissue clearing was performed on 10-week-old male Prox1-GFP mice. Figure 12 The head was sampled, fixed and decalcified using the method of . After decalcification, the samples were incubated in CUBIC-L (T3740, Tokyo Chemical Industry Co. Ltd) solution at 37 degrees Celsius for one week for tissue clearing. After the tissue clearing step, the samples were washed with PBS for one day. To label the lymphatic vessels, the samples were immersed in 5% normal donkey serum (in which LYVE1 antibody (11-034, Angiobio) was dissolved at a ratio of 1:200) and gently shaken at room temperature for 7 days. After primary antibody incubation and PBS washing, the samples were incubated with normal donkey serum containing Alexa-594-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch) dissolved at 1:100 and gently shaken at room temperature for 4 days. After secondary antibody incubation and subsequent washing with PBS, the samples were immersed in D-PROTOSS solution (Ku et al., Nature methods, 2020) for 48 hours to match the refractive index. The clarified samples were imaged using a light sheet microscope (LightSheet7, Zeiss) equipped with an EC Plan Neofluar 5x / 0.16 lens. Around the pituitary region, the blunt end of the LV was observed ( Figure 16 These LVs run along the cavernous sinus and connect to the NPLP ( Figure 16 B) The 3D video of the connected LV is displayed on Figure 17 Middle (green arrow).

[0173] Example 8: Newly discovered connection between the LV around the pterygopalatine artery (PPA) and the posterior nasal lymphatic plexus connected to the NPLP ( Figure 18 The steps of Example 7 were applied to 10-week-old male Prox1-GFP mice. Here, the Prox1+ / LYVE1+LV is located along the pterygopalatine artery (PPA), has a blunt end at the basilar meninges, and is connected to the posterior nasal lymphatic plexus. Lymphatic valve (white arrow, high Prox1 + Signal) at the connection point ( Figure 18 In addition, some of these LVs pass through the greater palatine foramen and connect with the lymphatic plexus in the submucosa of the hard palate ( Figure 18 and Figure 19 ).

[0174] Example 9: Prox1+LV located below the olfactory epithelium is connected to the posterior nasal lymphatic plexus, which is connected to the NPLP ( Figure 20, reddish-yellow and white arrows). These findings suggest that CSF may be drained through LVs in the olfactory mucosa and posterior nasal lymphatic plexus. Figure 21 A schematic diagram shows three new lymphatic pathways from the intracranial cavity to the extracranial NPLP. These three pathways are: (1) the LV originating from the pituitary region connects to the NPLP; (2) the LV running along the pterygopalatine artery (PPA) connects to the NPLP; and (3) the LV at the anterior skull base runs along the lamina cribrosa foramen and connects to lymphatic vessels within the olfactory mucosa, which in turn connects to the posterior nasal lymphatic plexus and the NPLP. Thus, the NPLP serves as a hub for CSF drainage.

[0175] Example 10: The same procedure as in Example 1 was applied to anesthetized 10-week-old male Prox-1GFP mice, and the mice were then killed by incising the abdominal aorta 60 min after injection. LV and dextran in the hard palate submucosa and mandibular LN were imaged using a fluorescent stereo zoom microscope ( Figure 22 ). We found that the LV in the submucosa of the hard palate was connected to the mandibular LN. The injected dextran was detected not only in the mandibular LN but also in the lymphatic vessels in the submucosa of the hard palate ( Figure 22 A and Figure 22 B).

[0176] Under isoflurane anesthesia, fluorescent microspheres (ThermoFisher, F8801) were infused into primates (cynomolgus monkeys) via the cisterna magna (2 mL, 25 μL / min). Six hours after infusion, blood was perfused with PBS and 2% PFA, and the hard palate was collected. We found that CSF-derived fluorescent microspheres were located in the lymphatic vessels of the hard palate ( Figure 23 These findings suggest that LVs in the hard palate serve as CSF drainage pathways, connecting to mandibular LNs. Furthermore, these LVs could be regulated to promote CSF drainage.

[0177] Example 10: IFS analysis revealed four lymphatic branches connected to NPLP and dcLV in Prox1-GFP mice ( Figure 24 ). Removing the soft palate clearly reveals the four lymphatic branches connected to the NPLP and dcLV ( Figure 24 ), which suggests that CSF flows out to the dcLV via the NPLP.

[0178] Example 11: In collaboration with the National Primate Center of the Korea Institute of Bioscience and Biotechnology, nine cynomolgus macaque heads were obtained in 2022. The samples were perfused with ice-cold saline. The decapitated head samples were soaked in 4% paraformaldehyde (PFA) at 4 degrees Celsius for 2 hours. Then, the samples were soaked in 2% PFA at 4 degrees Celsius for 12 hours. Then, the retropharyngeal LN (equivalent to dcLN in mice) was harvested. After removing the lymph nodes, the heads were soaked in 0.5M EDTA, pH 8.0 (Welgene) at 4 degrees Celsius for 3 weeks. The EDTA solution was replaced with fresh EDTA every 4 days. After decalcification, the heads were trimmed. The anterior boundary was the choanae, and the posterior boundary was the occipital bone. The dorsal boundary was the optic nerve, and the ventral boundary was the uvula. The trimmed heads were cut in half along the sagittal plane. The brain was removed from the skull. The samples were soaked in 30% sucrose solution for 72 hours.

[0179] The nasopharynx is located just below the clivus, which is one of the components of the skull base ( Figure 25 The ventral part of the nasopharyngeal mucosa is clearly distinguished from the soft palate ( Figure 25 The retropharyngeal lymph nodes are presumably located in Figure 25 The white dotted circle in .

[0180] To obtain IFS images, fixed and dehydrated nasopharyngeal sections were cut coronally using a blade, and the samples were embedded and frozen in freezing section medium (Leica) and cut into 30 μm sections using a cryocut microtome (Leica). To obtain full-field tissue images, the nasopharyngeal mucosa was carefully separated from the skull base and soft palate. After washing with PBS, the samples were incubated in 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) for 1 hour at room temperature. To label lymphatic vessel markers, the nasopharyngeal mucosa was immersed in 5% normal donkey serum (in which LYVE-1 antibody (DP3500, OriGene) and type IV collagen antibody (AB769, Sigma) were dissolved at 1:400) for 12 hours at 4 degrees Celsius. After washing with PBS, the tissue blocks were incubated with normal donkey serum containing Alexa-594-conjugated anti-goat IgG antibodies and Alexa-647-conjugated anti-rabbit IgG antibodies (Jackson ImmunoResearch) at 4 degrees Celsius for 12 hours. After washing with PBS, the tissue blocks were incubated with mounting medium (H1200, Vector). Images were acquired using a confocal microscope (LSM 800, Zeiss) with a Plan-Apochromat 10x / NA 0.45 lens.

[0181] Similar to mice, NPLP is present in the nasopharyngeal submucosa of cynomolgus monkeys ( Figure 25 ).

[0182] In addition, contrast-enhanced MR images were obtained using cynomolgus monkeys after infusion of the MR contrast enhancer Gadospin P into the cisterna magna. To infuse Gadospin P, computed tomography (CT) was used to locate the infusion needle. After confirming the needle position, Gadospin P (10% dissolved in saline, 25 μL / min) was infused. Images were obtained using FLAIRT2 MRI (3T, Philips Medical Systems) 3 hours and 30 minutes before and after the infusion of Gadospin P. Primates were anesthetized with isoflurane and were physiologically monitored by a veterinarian. We found that the signal in the nasopharynx of primates was enhanced ( Figure 26 ).

[0183] Example 12: Functional studies: To assess which side of the dcLV drains more CSF, unilateral dcLV ligation was performed in 10-week-old Prox1-GFP mice of both sexes. After careful dissection of the sternocleidomastoid muscles on both sides of the dcLV, one side of the dcLV was ligated with 10-0 polypropylene sutures (W2794, Ethicon), while the other side of the dcLV was sham-ligated. One day after ligation, TMR-dextran was injected into the cisterna magna according to the method of Example 1. Here, M-dcLV ligation significantly reduced the dextran signal in the dcLN, while L-dcLV ligation slightly reduced the dextran signal in the dcLN compared to the dextran signal in the sham operation ( Figure 27 These results indicate that CSF drainage via the NPLP / M-dcLV pathway is greater than via the basolateral mLV / L-dcLV pathway, highlighting the importance of the NPLP. P values ​​are calculated using one-way ANOVA followed by Turkey's post hoc test.

[0184] Ten-week-old Prox1-GFP mice of both sexes were instilled intracranially to measure the rate of CSF drainage into the dcLV. Figure 27 The dextran signal in dcLV was measured. Compared with the dextran signal in L-dcLV, the dextran signal was 7.9-fold, 5.2-fold, and 2.1-fold higher at 30, 60, and 120 min after injection (n=10-12 per group) ( Figure 28 These results indicate that CSF drainage via the NPLP / M-dcLV pathway is faster than via the basolateral mLV / L-dcLV pathway, emphasizing the importance of the NPLP. P values ​​were calculated by one-way analysis of variance followed by Turkey's post hoc test.

[0185] Example 13: To test whether NPLP can be improved by exogenous agents, 1.1x10 13GC / mL of AAV-mVEGF-C-mCherry (1.0 μl / min, 3 min) was injected into the intracranial cavity of 10-week-old Prox1-GFP female mice via the cisterna magna. As a control, the same amount of AAV empty vector was injected. Three weeks after injection, the NPLP of the two groups was compared. Compared with the AAV empty group, the AAV-mVEGF-C group showed an increased Prox1+ area ( Figure 29 These findings suggest that NPLP can be improved by exogenous agents. The amount of CSF discharged from dcLN in the AAV-VEGF-C group was enhanced ( Figure 29 ). This finding suggests that improvements in CSF lymphatic drainage pathways, including NPLP, may enhance CSF drainage.

[0186] Example 14: To examine whether aging alters the NPLP, we performed IFS on the NPLP of adult and aged mice. Compared to adult mice (10-week-old males), aged mice (80-88 weeks old) showed: 1) decreased lymphatic valves in the NPLP, 2) decreased Prox1 + Lymphatic vessel regression, 3) increased LYVE1, 4) fragmentation of the dorsal NPLP at the skull base ( Figure 30 ).

[0187] In addition, we analyzed the transcriptome of lymphatic endothelial cells (LECs) in young and elderly NPLP. After anesthesia, mice were perfused with ice-cold PBS, and the nasopharyngeal submucosa was isolated and collected in DMEM / F12 medium (Gibco). The nasopharyngeal submucosa was cut into small pieces and incubated at 37°C for 30 minutes in dissociation buffer containing 1 mg / ml collagenase IV (Roche), 1 mg / ml dispase (Gibco), and 0.1 mg / ml DNase I (Gibco), gently inverting every 10 minutes. The digested sample was filtered through a 70 μm filter and 2% FBS was added to stop the digestion. The cells were centrifuged at 500 x g for 8 minutes and then resuspended in PBS for washing. To exclude dead cells, 1:1000 Ghost dye (TONBO bioscience) was added to the resuspended cells at 4°C for 15 minutes. Then, PBS was added for washing and then stained with phycoerythrin / Cy7 anti-mouse CD326 (Ep-CAM, 118216, Biolegend) antibody, APC anti-mouse podoplanin antibody (127410, Biolegend) and phycoerythrin-labeled anti-mouse CD31 antibody (102508, Biolegned). CD31+PDPN+ cells were considered LECs and sorted by FACS AriaFusion (Beckton Dickinson). The sorted LECs were directly inoculated into each well of a 96-well plate containing lysis buffer. The culture plates were quickly frozen with liquid nitrogen and stored at -80°C. Following the Smart-Seq3 protocol, a single-cell library based on the culture plate was generated. Soon, the mRNA from the lysed cells was reverse transcribed. cDNA was amplified and purified using Ampure XP beads (BeckmanCoulter). The purified cDNA was diluted (100 pg / μl) and enzymatically fragmented using Tn5 tagmentation mix (Illumina). The enzymatically fragmented products were amplified using custom index primers and then merged into a single tube. After final purification with Ampure XP beads, the libraries were analyzed by tapestation for quality control. Libraries that passed quality control were sequenced using the Illumina High-X platform. The sequencing libraries were demultiplexed and aligned to the mouse reference genome (mm10) using STAR (version 2.7.9.a). The featureCount (version v2.0.1) function of the Subread package was then used to merge the aligned files and construct the raw read count matrix. To control cell quality, cells with fewer than 2,000 genes detected and more than 10% of the total reads mapped to mitochondrial genes were considered low quality / dead cells and discarded.At the gene level, genes expressed in fewer than three cells were removed from the expression matrix. For clustering and visualization of individual cells, the R package Seurat (version 4.1.0) was used. Briefly, each count for a gene in a cell was divided by the total number in a given cell and log2 normalized, multiplied by 1 x 10. 4 A pseudocount of 1 was added. The resulting expression matrix was thus transformed to have values ​​similar to log-transformed counts per million. The top 2,000 genes with the highest variability in each dataset were then selected using the FindVariableFeatures function with the option selection.method="vst". These highly variable genes were scaled and centered, and confounding variables such as the total number of counts and the percentage of reads mapping to mitochondrial genes were regressed out. Furthermore, module scores for dissociation-induced genes and ribosomal genes were calculated using the AddModuleScore function and subjected to regression analysis.

[0188] For visualization in two-dimensional space, principal component analysis was performed and the first 15 principal components were used as input to Uniform Manifold Approximation and Projection (UMAP). For neighborhood identification and cluster assignment, a shared nearest neighbor graph was constructed using the first 15 principal components and the Louvain algorithm was applied. To identify differentially expressed genes between cells, we used the FindMarkers function in Seurat with the following options: test.use = "MAST", logfc.threshold = 0.3, min.pct = 0.3. When performing differential expression tests, we excluded dissociation-induced genes, mitochondrial and ribosomal genes. Batch correction methods were not used when merging adult and aged mouse datasets because no obvious batch effects were observed for clustering. We found that the five LEC clusters were retained, but the expression of genes involved in apoptosis and inflammation was different, which can be explained by the fact that aged NPLP is more pro-apoptotic and pro-inflammatory ( Figure 31 ).

[0189] We also performed IFS in aged NPLP to measure phosphorylated tau (ptau) and apoptotic lymphatic endothelial cells using the same method as described in Example 6. Ptau was detected using anti-phospho-tau (mouse monoclonal (AT8) antibody, MN1020, Thermo) and was increased in aged NPLP ( Figure 32 The number of apoptotic lymphatic endothelial cells was detected using a TUNEL kit (12156792910, Merck) and increased with aging ( Figure 32These results suggest that increased apoptosis and its accompanying changes (upregulated type I interferon signaling) can be caused by increased CSF ptau.

[0190] Example 15: To test whether NPLP in elderly patients can be improved by exogenous agents, 1.1x10 13 GC / mL of AAV-VEGF-C-mCherry (1.0 μl / min for 3 minutes) was injected into the intracranial cavity of 75-78 week old Prox1-GFP female mice via the cisterna magna. As a control, the same amount of AAV empty vector was injected. Three weeks after injection, the NPLP of the two groups was compared. Compared with the AAV empty vector group, the AAV-VEGF-C-mCherry group showed: 1) Prox1 + Increased area and 2) enhanced CSF drainage in dcLNs ( Figure 33 Furthermore, CSF tracers were observed in lymphatic vessels following AAV-mVEGF-C infection. These findings suggest that NPLP, which is impaired with aging, can be improved by exogenous agents.

[0191] Example 16: IFS analysis revealed that dcLV had well-developed semi-cavitary lymphatic valves and lymphatic vessels, and was densely covered by annular smooth muscle cells ( Figure 34 Peripheral nerve fibers are distributed along the dcLV ( Figure 35 The peripheral nerve fibers that innervate the brain are sympathetic, not parasympathetic. Figure 36 There were no significant changes in the structure of peripheral smooth muscle cells and the number of lymphatic valves in aged Prox1-GFP mice ( Figure 37 ).

[0192] 0.1 and 1.0 M potassium chloride (KCl) can cause strong but transient contraction of dcLV ( Figure 38 In addition, the G protein-coupled receptor agonist phenylephrine or the nitric oxide donor (sodium nitroprusside) constricts or dilates dcLV and can modulate the amount of CSF tracer ( Figure 39 Furthermore, we found that low doses of phenylephrine (10 nM) increased the amount of drained CSF, but high doses (5 mM) decreased the amount of drained CSF, as measured by accumulation of CSF tracers in dcLNs ( Figure 40 Low doses of the nitric oxide donor sodium nitroprusside (3 μM) enhanced CSF drainage, but no changes were observed with 30 μM nitric oxide donor ( Figure 40 ).

[0193] These findings suggest that contraction and relaxation of the circular smooth muscle covering the dcLV can be modulated by: 1) stimulation and inhibition of smooth muscle cells or peripheral nerves; 2) neurotransmitters; 3) mechanical stimulants; and 4) gentle massage. Essentially, CSF drainage can be promoted by extracranial manipulation, administration of agents, and modulators ( Figure 41 Considering that DCLV does not change significantly in the elderly, reduced CSF drainage can be enhanced by extracranial manipulation of DCLV.

[0194] In summary, these examples provide information about where CSF drainage occurs and how to promote CSF drainage through extracranial pathways and methods. Since the present invention discloses that 1) NPLP is a newly discovered hub for CSF drainage through the skull base and cribriform plate; 2) the submucosa of the hard palate is a newly discovered pathway for CSF drainage to the mandibular LN; 3) CSF drainage pathways can be restored by exogenous agents (such as VEGF-C), thereby enhancing CSF drainage that is impaired with aging; 4) contraction and relaxation of the circular smooth muscle covering the dcLV can be regulated by stimulating and inhibiting intrathecal smooth muscle cells, peripheral nerves, neurotransmitters and mechanical stimulants, because CSF drainage can be promoted by extracranial manipulation, administration of agents and related regulators, it is used to prevent and treat neurodegenerative diseases including Alzheimer's disease ( Figure 42 ).

[0195] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art, based on the teachings herein, that changes and modifications may be made without departing from this invention and its broader aspects, and it is therefore intended that the appended claims cover within their scope all such changes and modifications as fall within the true spirit and scope of this invention. Cited references 1. Ransohoff, RM, & 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 / nri3265 2.Proulx,S.T.(2021).Cerebrospinal fluid outflow:a review of thehistorical 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 Alzheimerdisease-a research prospectus.Nat Rev Neurol,17(11),689-701.https: / / doi.org / 10.1038 / s41582-021-00549-x 4.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 drainsbrain interstitial fluid and macromolecules.J Exp Med,212(7),991-999. https: / / doi.org / 10.1084 / jem.20142290 5.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 lymphaticvessels.Nature,523(7560),337-341. https: / / doi.org / 10.1038 / nature14432 6.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).Meningeallymphatic vessels at the skull base drain cerebrospinal fluid.Nature,572(7767),62-66.https: / / doi.org / 10.1038 / s41586-019-1419-5 7.Pan,W.R.,Suami,H.,Corlett,R.J.,&Ashton,M.W.(2009).Lymphaticdrainage of the nasal fossae and nasopharynx:preliminary anatomical andradiological study with clinical implications.Head Neck,31(1),52-57.https: / / doi.org / 10.1002 / hed.20926 8.Ma,Q.,Ineichen,B.V.,Detmar,M.,&Proulx,S.T.(2017).Outflow ofcerebrospinal fluid is predominantly through lymphatic vessels and is reducedin aged mice.Nat Commun,8(1),1434.https: / / doi.org / 10.1038 / s41467-017-01484-69.Jacob,L.,de Brito Neto,J.,Lenck,S.,Corcy,C.,Benbelkacem,F.,Geraldo,L.H.,Xu,Y.,Thomas,J.M.,El Kamouh,M.R.,Spajer,M.,Potier,M.C.,Haik,S.,Kalamarides,M.,Stankoff,B.,Lehericy,S.,Eichmann,A.,&Thomas,J.L.(2022).Conserved meningeallymphatic drainage circuits in mice and humans.J Exp Med,219(8).https: / / doi.org / 10.1084 / jem.20220035 10.Kipnis,J.,Louveau,A.,Da Mesquita,S.(2019).US 2019-0269758 A1,University of Virginia Patent Foundation(UN162191):U.S.Patent and TrademarkOffice 11.Kipnis,J.Da Mesquita,S.(2021),US 2021-0311076 A1,University ofVirginia Patent Foundation(UN162191):U.S.Patent and Trademark Office

Claims

1. A method of increasing cerebrospinal fluid (CSF) outflow from the central nervous system, the method comprising repairing or enlarging the nasopharyngeal lymphatic plexus (NPLP), comprising: Identify subjects who need increased CSF outflow; as well as An effective amount of a NPLP glidant is administered to a subject in need thereof, wherein the amount of glidant repairs or enlarges the subject's NPLP, thereby increasing CSF outflow from the subject's central nervous system to the systemic circulation.

2. A method of increasing cerebrospinal fluid (CSF) outflow from the central nervous system, the method comprising increasing systole-relaxation of the dcLV, comprising: Identify subjects who need increased CSF outflow; as well as An effective amount of a dcLV glidant is administered to the subject, wherein the amount increases contraction-relaxation of the subject's dcLV, thereby increasing CSF outflow from the subject's central nervous system to the systemic circulation.

3. A method for preventing, treating, or ameliorating a neurodegenerative disease or condition in a subject, the method comprising repairing or enlarging the nasopharyngeal lymphatic plexus (NPLP), comprising: Identify subjects who need to increase CSF outflow from the central nervous system; as well as An effective amount of an NPLP glidant is administered to a subject in need thereof, wherein the amount of the agent repairs or enlarges the subject's NPLP, thereby increasing CSF outflow from the subject's central nervous system to the systemic circulation.

4. A method for preventing, treating, or ameliorating a neurodegenerative disease or condition in a subject, the method comprising repairing or enlarging the nasopharyngeal lymphatic plexus (NPLP), comprising: Identify subjects who need to increase CSF outflow from the central nervous system; as well as An effective amount of a dcLV glidant is administered to the subject, wherein the amount increases contraction-relaxation of the subject's dcLV, thereby increasing CSF outflow from the subject's central nervous system to the systemic circulation.

5. The method of claim 1 or 2, wherein determining the subject to be in need of increased CSF outflow comprises determining the subject has a neurodegenerative disease or condition, determining the subject has a risk factor for a neurodegenerative disease or condition, or both.

6. The method of any one of claims 1-4, wherein the disease or condition is cognitive decline associated with aging, Alzheimer's disease, Parkinson's disease, Huntington's disease, or stroke.

7. The method of claim 1 or 3, wherein the agent is a VEGFR3 agonist.

8. The method of claim 7, wherein the agent is VEGF-C or VEGF-D, an analog, variant or fragment thereof, or a combination of any of these.

9. The method of claim 1 or 3, wherein the agent is 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, neuropilin, or prostaglandin E2.

10. The method according to claim 1 or 3, wherein the agent is a protein or a gene vector carrying a gene encoding an agent polypeptide.

11. The method of claim 10, wherein the gene encodes angiogenic factor-C, angiopoietin-1, or Tie2 agonist.

12. The method of claim 1 or 3, wherein the agent is selectively administered at or near the NPLP-dcLV space.

13. The method of claim 1 or 3, wherein the NPLP-dcLV space is located in the nasopharyngeal mucosa.

14. The method of claim 1 or 3, wherein the agent is selectively administered at or near the hard palate submucosa-mandibular lymph node space.

15. The method of claim 14, wherein the hard palate submucosa-mandibular lymph node space is located in the hard palate mucosa.

16. The method of claim 1 or 3, wherein the agent is administered intrathecally to the CSF space of the subject, nasally to the nasopharynx of the subject, or orally to the submucosa of the hard palate of the subject.

17. The method of claim 1 or 3, wherein the subject's central nervous system comprises soluble molecules, and wherein increasing CSF outflow reduces the amount of soluble molecules in the brain.

18. The method of claim 1 or 3, wherein the subject's central nervous system comprises amyloid beta plaques, and wherein increasing CSF outflow reduces the amount of amyloid beta plaques in the brain.

19. The method of claim 2 or 4, wherein the agent is the G protein-coupled receptor agonist phenylephrine or a nitric oxide donor.

20. The method of claim 2 or 4, wherein the agent is administered transcervically, transdermally, topically to the subject's neck muscles, neck lymph nodes, or neck space, or wherein the agent is mechanically applied to the side of the neck.

21. The method of claim 2 or 4, wherein the agent is a stimulator of the circular smooth muscle covering the dcLV.

22. The method of claim 21, wherein the agent that stimulates the circular smooth muscle covering the dcLV increases or decreases myosin phosphorylation by activating myosin light chain kinase or activating myosin light chain phosphatase.

23. A method for determining a change in the amount or rate of CSF outflow in a subject, the method comprising: Obtaining biological samples to obtain samples of nasopharyngeal mucosa or hard palate mucosa on at least two separate occasions; analyzing the CSF or the presence and amount of an analyzable substance in the CSF to obtain a value at each time; Comparison of values ​​of CSF or amount of analyzable material; as well as A change in the value of the amount of CSF or analyte is determined, wherein a change in the value of the CSF or analyte over time is indicative of a changing CSF outflow level or rate.

24. The method of claim 23, wherein the biological sample is obtained by swab or biopsy.

25. The method of claim 23, wherein the biological sample comprises NPLP or hard palate.

26. The method of claim 23, wherein the analysis is performed by imaging the NPLP or hard palate, or performing protein analysis on the obtained biological sample.

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