Depression marker and application thereof in preventing and treating depression

Abstract

The application discloses a marker for depression and application thereof in prevention and treatment of depression. Through the marker, whether an individual is suffering from depression can be detected and diagnosed, so that corresponding treatment strategies can be given in time, and hidden troubles brought by depression to the individual can be reduced. The application also discloses application of the marker in prevention and treatment of depression phenotype. Taking a marker with specific up-regulation as an example, through blocking the function of the marker, prevention and treatment of depression can be realized. Specifically, in addition to specific material markers, it is disclosed that abnormality of meningeal lymphatic vessels is closely related to depression phenotype, and detection of the meningeal lymphatic vessels is expected to be a new marker for early diagnosis and prediction of clinical depression treatment effectiveness. Improvement of the meningeal lymphatic vessels is expected to be applied to treatment of depression and other neurosychiatric diseases related to stress and play an effective treatment role.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of depression prevention and treatment, and more particularly, to a depression marker and application thereof in the prevention and treatment of depression. BACKGROUND

[0002] Major depressive disorder (MDD) is characterized by persistent depressed mood and loss of motivation and pleasure, and is a debilitating mental disorder affecting 4.4% of the global population, and is the leading cause of non-fatal health loss. Women are more likely to develop MDD and have more severe depressive symptoms than men. Although the gender difference in MDD is well documented in clinical reports, the underlying mechanism is still largely unknown. For the pathological development of depression, the existing technology proposes various hypotheses from different perspectives; at the circuit level, the impaired activation of ventral tegmental dopamine neurons is considered to be the key to the brain reward circuit defects and lack of motivation in patients with depression; in addition to the changes in neurons, there are also abnormalities in glial cells; for example, in the post-mortem brain of MDD patients, a decrease in the expression of astrocyte markers (such as S100β and glial fibrillary acidic protein (GFAP)) in the medial prefrontal cortex (mPFC), a brain region with stress resilience, can be observed; in rodent models, the depletion of astrocytes or the prevention of ATP release by astrocytes using drugs leads to a depressive-like phenotype (depression-like behavior). Although the existing technology proposes various hypotheses and research, the upstream factors that trigger or maintain these changes in the brain are still unclear. And the current treatment, diagnosis and other means for depression are still limited, which is difficult to meet the actual clinical needs.

[0003] On this basis, it is crucial to elucidate the difference in the manifestation of depression and its upstream mechanism in gender, because through the study of the mechanism, it is expected to provide corresponding clinically applicable markers, therapeutic drugs and treatment strategies, etc., in order to meet the actual clinical needs. SUMMARY

[0004] The present application aims to overcome at least one of the above-mentioned deficiencies of the prior art, and provides a depression marker and application thereof in the prevention and treatment of depression. Through the marker, it is beneficial to timely diagnose depression and depression-like phenotypes, and one or more embodiments of the present application disclose that by blocking one of the specifically up-regulated markers, the damage to the meningeal lymphatic vessels can be improved, and thus the depressive phenotype can be improved, i.e. the marker involved can also be applied to the prevention and treatment of meningeal lymphatic vessel damage and depression.

[0005] One object of the present application is to provide a depression marker, comprising Col6a1, Col1a2, Col3a1, Mmp23, Trpml and / or CCL6.

[0006] Further, Col6a1, Col1a2, Col3a1 and / or Mmp23 are down-regulated in the brain specifically, and Trpml and / or CCL6 are up-regulated in the brain specifically.

[0007] Another object of the present application is to provide the use of a reagent for detecting Col6a1, Col1a2, Col3a1, Mmp23, Trpml and / or CCL6 in the brain in the preparation of a drug for diagnosing depression, anxiety, depression-like behavior, anxiety-like behavior and / or meningeal lymphatic vessel injury.

[0008] Further, the depression-like behavior (depression-like phenotype / depression-like behavior) is a depression-like behavior caused by stress (stress), the anxiety-like behavior (anxiety-like phenotype / anxiety-like behavior) is an anxiety-like behavior caused by stress (stress), and the meningeal lymphatic vessel injury is a meningeal lymphatic vessel injury caused by stress (stress).

[0009] Further, the depression includes animal depression.

[0010] Another object of the present application is to provide the use of a CCL6 and / or CCL23 downstream pathway blocker in the preparation of a drug for preventing and treating depression, depression-like behavior and / or meningeal lymphatic vessel injury. Further, the CCL6 downstream pathway blocker includes a CCL6 blocker and / or a CCL6 receptor blocker. Further, the CCL6 blocker includes a CCL6 neutralizing antibody and / or a CCL6 functional antagonist; and the CCL6 receptor blocker includes a receptor CCR1 blocking antibody and / or a receptor CCR1 functional antagonist. Similarly, as a homologous gene of CCL6, CCL23 also has the same effect, and therefore, the use of a CCL23 downstream pathway blocker in the preparation of a drug for preventing and treating depression, depression-like behavior and / or meningeal lymphatic vessel injury is also achieved. And the receptor of CCL23 is also CCR1, that is, the CCL23 receptor blocker also includes a receptor CCR1 blocking antibody and / or a receptor CCR1 functional antagonist.

[0011] Further, the depression-like behavior is a depression-like behavior caused by stress (stress).

[0012] Another object of the present application is to provide the use of a meningeal lymphatic vessel imaging product in the preparation of a product for diagnosing depression. In one or more embodiments of the present application, changes and injuries of the meningeal lymphatic vessels corresponding to depression behavior are found, and therefore, by imaging the changes and injuries of the meningeal lymphatic vessels, the diagnosis of depression is facilitated.

[0013] Further, the meningeal lymphatic vessel imaging product includes a meningeal lymphatic vessel imaging agent and / or a meningeal lymphatic vessel imaging device that can be injected into the brain.

[0014] It is another object of the present application to provide use of a meningeal lymphatic vessel improving agent in the manufacture of a medicament for reducing stress sensitivity and / or improving stress adaptability in an individual. In one or more embodiments of the present application, it is found that changes in meningeal lymphatic vessels change stress sensitivity in an individual, and that when meningeal lymphatic vessels are improved, stress sensitivity (i.e., stress sensitivity) in an individual is reduced, and stress adaptability in an individual is improved.

[0015] Further, the medicament for reducing stress sensitivity and / or improving stress adaptability in an individual includes a medicament for reducing stress sensitivity and / or improving stress adaptability in a female individual.

[0016] It is another object of the present application to provide use of a meningeal lymphatic vessel improving agent in the manufacture of a medicament for preventing and / or treating mPFC astrocyte abnormalities, VTA dopaminergic neuron abnormalities, depression, anxiety, depression-like behavior, and / or anxiety-like behavior.

[0017] Further, the mPFC astrocyte abnormalities are mPFC astrocyte abnormalities caused by stress; and the VTA dopaminergic neuron abnormalities are VTA dopaminergic neuron abnormalities caused by stress.

[0018] Further, the meningeal lymphatic vessel improving agent includes VEGFC and a pharmaceutically acceptable carrier thereof.

[0019] Further, the pharmaceutically acceptable carrier includes an adenovirus vector.

[0020] It is another object of the present application to provide a medicament for preventing and / or treating meningeal lymphatic vessel damage, depression, and / or anxiety, comprising VEGFC, a CCL6 downstream pathway blocker, and / or a CCL23 downstream pathway blocker.

[0021] Further, the CCL6 downstream pathway blocker includes a CCL6 blocker and / or a CCL6 receptor blocker; and / or, the CCL23 downstream pathway blocker includes a CCL23 blocker and / or a CCL23 receptor blocker. Further, the CCL6 blocker includes a CCL6 neutralizing antibody and / or a CCL6 functional antagonist; and / or, the CCL23 blocker includes a CCL23 neutralizing antibody and / or a CCL23 functional antagonist; further, the CCL6 receptor blocker or CCL23 receptor blocker includes a receptor CCR1 blocking antibody and / or a receptor CCR1 functional antagonist.

[0022] Compared with the prior art, the application has the beneficial effects that the application discloses a marker for depression and application thereof in prevention and treatment of depression. Through the marker, it is beneficial to detect and diagnose whether an individual has depression, so that corresponding treatment strategies can be given in time, and the hidden danger brought by depression to the individual can be reduced. The application also discloses application of the marker in prevention and treatment of depression phenotype. Taking one specific up-regulated marker in the examples as an example, through blocking the function of the marker, prevention and treatment of depression can be realized. Specifically, in addition to the specific material marker, it is disclosed that the meningeal lymphatic vessel abnormality is closely related to the depression phenotype, and detection of the meningeal lymphatic vessel is expected to be a new marker for early diagnosis and prediction of clinical depression treatment effectiveness. And improvement of the meningeal lymphatic vessel is expected to be applied to treatment of depression and other neurosychiatric diseases related to stress and play an effective treatment role. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1Display: Subchronic variable stress (SCVS) damages the meningeal lymphatic vessels in female, but not in male mice. a: Timeline of the experiment with the SCVS paradigm, behavioral tests, 70 kD dextran tracer injection into the cisterna magna (i.c.m.) and tissue collection (sac). b: Schematic of i.c.m. tracer injection. c: Schematic of the dura mater with the superior sagittal sinus (SSS) and the intersection of the sinus and transverse sinuses (COS+TS) areas encircled by a red dashed line for image analysis. d-e: Representative images of LYVE1 staining (gray) and dextran tracer (red) in the SSS and COS+TS areas of the dura mater of female (d) and male (e) mice (compare non-stressed (N) and SCVS groups). Scale bar: 500 pm. f, k: Representative images of LYVE1 -labeled meningeal lymphatic vessels (mLV) at high magnification in female (f) and male (k) mice for diameter analysis. Scale bar: 200 pm. g-j, l-o: Quantification of the fluorescence intensity of LYVE1 staining (g, 1), the area covered by mLV (h, m) and the diameter of LYVE1 -labeled mLV (i, n) in the SSS and COS+TS areas of the dura mater of female (g-i: n = 9-12 / group; results from three independent experiments) and male (1-n: n = 8-10 / group; results from two independent experiments) mice. j, o: Quantification of the fluorescence intensity of dextran tracer in the SSS and COS+TS areas of the dura mater of female (j: n = 8-9 / group) and male (o: n = 7-8 / group) mice. p, r: Representative images of dextran tracer (red) and DAPI (blue) in the deep cervical lymph nodes (dCLN) of female (p) and male (r) mice. Scale bar: 500 pm. q, s: Quantification of the fluorescence intensity of dextran tracer in the dCLN of female (q: n = 8-9 / group) and male (s: n = 7-8 / group) mice. All data are presented as mean ± s.e.m. Analyses were performed by unpaired Student’s t test (g-j, m-o, q, s) or Mann-Whitney test (1).

[0024] Figure 2Intracisternal cerebello-medullary injection of AAV overexpressing VEGFC improves meningeal lymphatic vessels and stress-induced depressive-like behaviors in female mice. a: Timeline of experiments for intracisternal cerebello-medullary AAV infusion, subchronic variable stress (SCVS) paradigm, behavioral tests, dextran tracer injection into the cisterna magna (i.c.m.) and tissue collection (sac). b: Representative images of eGFP (green)-labeled AAVl-infected cells around LYVE1 (gray)-positive meningeal lymphatic vessels (mLVs) in the dura mater. Nuclei were labeled with DAPI (blue). Left: whole dura mater, scale bar: 2000 pm. Right: magnified view of the boxed area in the left image, scale bar: 200 pm. c: Representative images of LYVE1 staining (gray) and dextran tracer (red) in the SSS and COS+TS regions of the dura mater (comparing AAV-VEGFC-injected and AAV-eGFP-injected female mice); both groups received SCVS; scale bar: 500 pm. d: Representative images of LYVE1-labeled mLVs at higher magnification; scale bar: 200 pm. e-h: Quantification of LYVE1 staining fluorescence intensity (e), mLV-covered area (t), LYVE1-labeled mLV diameter (g), and dextran tracer fluorescence intensity (h) in the SSS and COS+TS regions of the dura mater. i: Representative images of dextran tracer (red) and DAPI (blue) in the deep cervical lymph nodes (dCLNs). Scale bar: 500 pm. j: Quantitative analysis of dextran tracer fluorescence intensity in the dCLNs. k: Quantitative analysis of body weight changes. I: Quantitative analysis of grooming duration in the splash test (ST). m: Quantitative analysis of immobility time in the forced swim test (FST). n: Representative traces of animal paths in the open field (OF). oq: Quantification of the percentage of the distance moved in the center zone (o), the amount of time spent in the center zone quantified as a percentage of the total time (p), and the total distance moved in the OF (q). r: Quantitative analysis of the latency to start feeding in the novel-situation feeding test (NSF) (e-h, j-m, o-r: n = 8 per group; results from two independent experiments). All data are presented as mean ± s.e.m. Analyses were performed by unpaired Student’s t test (e, g, j-m, o, p) or Mann-Whitney test (f, h, q, r).

[0025] Figure 3: Cerebellomedullary cistern injection of AAV overexpressing VEGFC in female mice effectively mitigated subchronic variable stress (SCVS)-induced impairment of astrocyte marker expression in mPFC and impairment of cFOS expression in VTA dopaminergic neurons. a: Top panel, coronal maps with brain regions highlighted in orange. Bottom panel, Representative images of OVA Alexa Fluor 647 tracer (red) distribution in brain sections after cerebellomedullary cistern injection of OVA Alexa Fluor 647 tracer at 15, 30, and 60 min, and DAPI staining (blue). Scale bar: 1000 pm. Yellow arrows indicate tracer accumulation in mPFC at 15 and 30 min post-injection, and in VTA at 30 and 60 min post-injection. b: Quantification of tracer distribution in various brain regions at different time points post-cerebellomedullary cistern injection (n = 4 per group). c: Experimental timeline for cerebellomedullary cistern AAV infusion, subchronic variable stress (SCVS) paradigm, and tissue collection (sac). d, g: Representative images of S100B (d: scale bar: 50 pm) and GFAP (g: scale bar: 200 pm) staining in mPFC. e, h: Quantification of percentage of covered area by S100B (e) and GFAP (h) staining in mPFC. f: Quantification of S100B-labeled astrocyte density in mPFC. i: Representative images of TH (red) and c-FOS (gray) staining in VTA. White arrows indicate c-FOS and TH double-labeled cells. Scale bar: 100 pm. j: Quantification of percentage of c-Fos+ neurons within TH-labeled dopaminergic neurons in VTA. (e-j: n = 5-6 / group; results from two independent experiments). All data are presented as mean ± s.e.m. and were analyzed by one-way ANOVA with Tukey’s post-hoc test.

[0026] Figure 4 : Cerebellomedullary cistern injection of AAV overexpressing VEGFR3 d1-4 in male mice impaired meningeal lymphatic vessels and promoted stress-induced depressive-like behaviors. a: Experimental timeline for AAV injection, subchronic variable stress (SCVS) paradigm, behavioral tests, dextran tracer infusion into the cerebellomedullary cistern (i.c.m.), and tissue collection (sac). b: Representative images of eGFP (green)-labeled AAV9-infected cells around intradural LYVE1 (gray)-positive meningeal lymphatic vessels (mLVs). Nuclei were stained with DAPI (blue). Left: whole dura mater, scale bar: 2000 pm. Right: magnified view of the boxed area in the left image, scale bar: 200 pm. c: Comparison of AAV-VEGFR3 d1-4 (VEGFR3 d1-4Male mice with +SCVS and those injected with AAV-VEGFR3 d4-7 (VEGFR3) d4-7 Representative images of LYVE1 staining (gray) and dextran tracer (red) in the SSS and COS+TS regions of the dura mater of male mice with SCVS. Both groups received SCVS treatment. Scale bar: 500µm. d: Representative image of LYVE1-labeled mLVs at higher magnification. Scale bar: 200µm. eh: Quantitative determination of fluorescence intensity (e), mLV coverage area (t), diameter of LYVE1-labeled mLVs (g), and fluorescence intensity (h) of dextran tracer in the SSS and COS+TS regions of the dura mater. i: Representative image of dextran tracer (red) in dCLNs. Cell nuclei were counterstained with DAPI (blue). Scale bar: 500µm. j: Quantitative analysis of fluorescence intensity of dextran tracer in dCLNs. kn: Quantitative analysis of combing duration (ST, k) in the splash test, immobility time (FST, l) in the forced swimming test, and the waiting period for initiation of feeding (NSF, m) and weight change (n) in the novelty inhibition feeding test. o: Representative trace images of animal paths in the open field. The central region is indicated by a gray box in the center. pr: Quantitative percentage of the total distance traveled in the central region (p), the percentage of the total time spent in the central region (q), and the total distance traveled in the OF (r). (e.g., kn, pr: n = 8–9 / group; h, j: n = 7–8 / group; results from two independent experiments). All data are expressed as mean ± sem and analyzed by unpaired Student's t-test (eh, jl, n, pr) or Mann-Whitney test (m).

[0027] Figure 5 Injection of VEGFR3-overexpressing cells into the cerebellomedullary cistern of male mice d1-4AAV will promote subchronic variable stress (SCVS)-induced impairments in astrocyte marker expression in the mPFC and impairments in cFOS expression in VTA dopaminergic neurons. a: Top panels are coronal maps with brain regions highlighted in orange. Bottom panels, Representative images of OVA Alexa Fluor 647 tracer (red) distribution in brain sections after cerebellomedullary cistern injection of OVA Alexa Fluor 647 tracer at 15, 30, and 60 min, and DAPI staining (blue). Scale bar: 1000 pm. Yellow arrows indicate tracer accumulation in mPFC at 15 and 30 min post-injection, and in VTA at 30 and 60 min post-injection. b: Quantification of tracer distribution in multiple brain regions at different time points after intracerebral injection (n = 4 per group). c: Experimental timeline for cerebellomedullary cistern AAV infusion, SCVS paradigm, and tissue collection (sac). d, g: Representative images depicting S100P (d: scale bar: 50 pm) and GFAP (g: scale bar: 200 pm) staining in the mPFC. e, h: Quantification of the percentage of covered area by S100P (e) and GFAP (h) staining in the mPFC. f: Quantification of S100P-labeled astrocyte density in the mPFC. i: Representative images of TH (red) and c-FOS (gray) staining in the VTA. White arrows indicate c-FOS and TH double-labeled cells. Scale bar: 100 pm. j: Quantification of the percentage of c-Fos+ neurons within TH-labeled dopaminergic neurons in the VTA. (e, f, h, j: n = 7-8 / group; results from two independent experiments). All data are presented as mean ± s.e.m. and were analyzed by one-way ANOVA and Tukey’s post-hoc test.

[0028] Figure 6Surgical ligation of the afferent lymphatic vessels of the deep cervical lymph nodes (dCLN) in male mice promotes depression-like behavior induced by subchronic variable stress (SCVS) and reduces expression of S100B in the mPFC and c-FOS in dopaminergic neurons of the VTA. a: Timeline of experiments for surgical ligation of the afferent lymphatic vessels of the dCLN, subchronic variable stress (SCVS) paradigm, behavioral tests, injection of dextran tracer into the cisterna magna (i.c.m.) and tissue collection (sac). b: Representative images depicting dextran tracer (red) and DAPI staining (blue) in the dCLN. Scale bar: 500 µm. c: Quantitative analysis of dextran tracer fluorescence intensity in the dCLN. d: Quantitative analysis of grooming duration in the splash test (ST). e: Quantitative analysis of immobility time in the forced swim test (FST). f: Quantitative analysis of latency to start feeding in the novelty suppressed feeding test (NSF). g: Quantification of SCVS body weight change. h: Representative traces of animal paths in the open field (OF). i-k: Quantification of the percentage of the distance moved in the center zone over the total distance moved (i), the percentage of time spent in the center zone over the total time (G), and the total distance moved in the OF (k). l,o: Representative images of S100B (I: scale bar: 50 µm) and GFAP (o: scale bar: 200 µm) staining in the mPFC. m,p: Quantification of the percentage of area covered in the mPFC by S100B (m) and GFAP (p) staining. n: Quantitative analysis of S100B-labeled astrocyte density in the mPFC. q: Representative images of TH (red) and c-FOS (gray) staining in the VTA. White arrows indicate c-FOS and TH double-labeled cells. Scale bar: 100 µm. r: Quantification of the percentage of c-FOS+ neurons in TH-labeled dopaminergic neurons in the VTA. (c-g, i-k, p, r: n = 8 / group; m, n: n = 7-8 / group; results from two independent experiments). All data are presented as mean ± s.e.m. and were analyzed by unpaired Student’s t test.

[0029] Figure 7 Subchronic variable stress (SCVS) induces transcriptional changes in meningeal lymphatic endothelial cells in female mice. a: Schematic of the isolation of meningeal lymphatic endothelial cells (LECs) from the dura mater, fluorescence-activated cell sorting (FACS), and RNA sequencing. b: Representative dot plots and heatmap showing the gating strategy for isolation of meningeal LECs (CD45 - PDPN + CD31 +). c-f: Quantification of the percentage of LECs (c), vascular endothelial cells (BECs, channel CD45-PDPN-CD31+) (d), CD45+ leukocytes (e), and other stromal cells (CD45-CD31-) (f) in live cells, comparing female SCVS mice with non-stressed control mice (N) (5 biological replicates per group. Each replicate is from 3-4 animals). Data are presented as mean ± s.e.m. and analyzed by unpaired Student’s t-test. g: Heatmap showing the relative expression levels of differentially expressed genes (DEGs, by Deseq2 adjusted p<0.1) in meningeal LECs of control (N), SCVS groups. Color scale values represent standard rlog-transformed values across samples. h: Volcano plot showing the difference in gene expression between control (N) and SCVS groups. Blue and red dots represent significantly down- and up-regulated genes, respectively. i: Gene Set Enrichment Analysis (GSEA) showing the most enriched (normalized enrichment score, NES>0; false discovery rate, FDR<0.25) and depleted (NES<0, FDR<0.25) gene sets in SCVS samples. GO-BP: Gene Ontology - Biological Process, GO-CC: Gene Ontology - Cellular Component, GO-MF: Gene Ontology - Molecular Function, KEGG: Kyoto Encyclopedia of Genes and Genomes pathway database. j: Heatmap of DEGs enriched in the serotonin uptake pathway. k: Heatmap of DEGs co-regulated by stress and aging. 1: Heatmap of DEGs common to LECs of female mice after SCVS and depressed human brain regions. AI: anterior insular lobe; NAc: nucleus accumbens; vSUB: ventral subiculum; dlPFC: dorsal lateral PFC; vmPFC: ventromedial PFC; OFC: orbitofrontal cortex.

[0030] Figure 8Cerebellomedullary cistern injection of CCL6 neutralizing antibody (CCL6 mAb) effectively alleviated subchronic variable stress (SCVS)-induced depressive-like behaviors and meningeal lymphatic vessel (mLV) impairment in female mice. a: Experimental timeline for cerebellomedullary cistern injection of CCL6 mAb or control immunoglobulin (IgG), SCVS paradigm, behavioral tests, and tissue collection (sac). b: Representative heat map of animal traces in the social interaction (SI) test. c-d: Quantification of total exploration time and difference index (DI) in SI comparing female mice cerebellomedullary cistern injection of CCL6 mAb or control IgG. Both groups of mice received SCVS treatment. e: Quantification of immobility time in the forced swim test (FST). (c-e: n=7-9 per group; results from two independent experiments). f: Representative images of LYVE1 staining (gray) in the dural SSS and COS+TS regions. Scale bar: 500 µm. g: Representative images of LYVE1-labeled mLV at higher magnification. Scale bar: 200 µm. h-j: Quantification of LYVE1 staining fluorescence intensity (h), area covered by mLV (i), and diameter of LYVE1-labeled mLV in the dural SSS and COS+TS regions (j). (h-j: n=4-5 per group). All data are presented as mean ± s.e.m. and analyzed by unpaired Student’s t test.

[0031] Figure 9 Subchronic variable stress (SCVS) for six days induced depressive and anxiety-like behaviors in female mice. a: Experimental timeline for SCVS paradigm and behavioral tests. b: Quantification of body weight change compared to non-stressed control mice (N). c: Quantification of grooming duration in the splash test (ST). d: Quantification of immobility time in the forced swim test (FST). e: Quantification of latency to start feeding in the novel suppressed feeding test (NSF). f: Representative traces of animal paths in the open field. The central region is indicated by the gray box in the center. g-i: Quantification of the percentage of central zone movement distance over total movement distance (g), percentage of time spent in the central zone over total time (h), and total movement distance in the OF (i) (b-e, g-i: n=10-11 per group; results from two independent experiments). All data are presented as mean ± s.e.m. and analyzed by unpaired Student’s t test (b, c, g-i) or Mann Whitney test (d, e).

[0032] Figure 10: Six days of subchronic variable stress (SCVS) did not induce depressive- or anxiety-like behaviors in male mice. a: Experimental timeline of the SCVS paradigm and behavioral tests. b: Quantification of body weight change in SCVS mice compared to non-stressed control mice (N). c: Quantification of grooming duration in the splash test (ST). d: Quantification of immobility time in the forced swim test (FST). e: Quantification of latency to start feeding in the novelty suppressed feeding test (NSF). f: Representative traces of animal paths in the open field. The central area is indicated by the gray box in the center. g-i: Quantification of the percentage of the total distance moved spent in the center area (g), the percentage of the total time spent in the center area (h), and the total distance moved in the OF (b-e: n = 8-9 / group; g-i: n = 7-8 / group; results from two independent experiments). All data are presented as mean ± s.e.m. and analyzed by unpaired Student’s t test.

[0033] Figure 11 : Subchronic variable stress (SCVS) induced sex differences in changes of meningeal lymphatic vessel marker LYVE1 mRNA expression (compare meningeal lymphatic vessels (mLV) in control female (N) and male mice). a: Experimental timeline of the SCVS paradigm and tissue collection (sac). b-c: Quantification of fold change in Lyve1 mRNA levels in the dura mater of female (b: n = 13 / group; results from three independent experiments) and male (c: n = 7-8 / group; results from two independent experiments) mice (compare SCVS and N mice). d-f: Quantification of the fluorescence intensity of LYVE1 staining (d), the area covered by mLV (e), and the diameter of LYVE1 -labeled mLV in the superior sagittal sinus (SSS) in N female and male mice (f) at the junction of the cerebral and transverse sinuses (COS+TS). g: Quantitative analysis of the fluorescence intensity of dextran tracer injected in the dura mater SSS and COS+TS regions. h: Quantitative analysis of the fluorescence intensity of dextran tracer in the dCLN (d-h: n = 6-7 / group; results from two independent experiments). All data are presented as mean ± s.e.m. and analyzed by unpaired Student’s t test.

[0034] Figure 12The dural lymphatic vessels of female mice were damaged 24 hours after subchronic variable stress (SCVS). a: Timeline of the experiment with the SCVS paradigm and tissue collection (sac). b: Representative images of LYVE1 staining (gray) in the superior sagittal sinus (SSS) and at the junction of the dural sinuses and transverse sinus (COS+TS) of female mice (compare non-stressed control (N) and SCVS groups). Scale bar: 500 pm. c: Representative images of LYVE1 -labelled dural lymphatic vessels (mLV) of female mice at high magnification. Scale bar: 200 pm. d-f: Quantification of the fluorescence intensity (d), the area covered by mLV (e), and the diameter of LYVE1 -labelled mLV (f) in the SSS and COS+TS regions of the dura mater of female mice (d-f: n = 6 / group; results from two independent experiments). All data are presented as mean ± s.e.m. and were analyzed by unpaired Student’s t test.

[0035] Figure 13Cerebellomedullary cistern injection of VEGFC-overexpressing AAVs improved meningeal lymphatic vessels but did not affect depressive-like behaviors and astrocytic protein expression in the mPFC, c-FOS expression in VTA dopaminergic neurons in non-stressed female mice. a: Timeline of cerebellomedullary AAV infusion, behavioral tests, and tissue collection (sac). b: Representative images of LYVE1 staining (gray) at the superior sagittal sinus (SSS) on the dura and at the confluence of sinuses plus transverse sinus (COS+TS) (compare female mice injected with AAV-VEGFC (VEGFC+control) with female mice injected with AAV-eGFP (eGFP+control)). Scale bar: 500 pm. c: High magnification images of LYVE1 -labeled mLVs. Scale bar: 200 pm. d-f: Quantitative analysis of LYVE1 staining fluorescence intensity (d), mLVs covered area (e), and LYVE1 -labeled mLVs diameter (t). g-i: Quantification of the duration of grooming (ST, g), the immobility time in the forced swim test (FST, h), and the latency to start feeding in the novel suppressed feeding test (NSF, i). j: Representative traces of animal paths in the open field. k-m: Quantification of the percentage of the distance moved in the center zone over the total distance moved (k), the percentage of time spent in the center zone over the total time (I), and the total distance moved in the OF (m). n, q: Representative images of S100P (n: scale bar: 50 pm) and GFAP (q: scale bar: 200 pm) staining in the mPFC. o, r: Quantification of the percentage of covered area in the mPFC by S100P (o) and GFAP (r) staining. p: Quantification of S100P-labeled astrocyte density in the mPFC. s: Representative images of TH (red) and c-FOS (gray) staining in the VTA. White arrows indicate c-FOS and TH double-labeled cells. Scale bar: 100 pm. t: Quantification of the percentage of c-Fos+ neurons within TH-labeled dopaminergic neurons in the VTA (d-f: n = 6 per group; g-i, k-m, o-p, r, t: n = 7 per group; results from two independent experiments). All data are presented as mean ± s.e.m. and were analyzed by unpaired Student’s t test (d-i, k-m, o-p) or Mann Whitney test (r, t).

[0036] Figure 14: Modulation of meningeal lymphatic vessels can modulate subchronic variable stress (SCVS)-induced social impairment and sucrose preference. a, f: Experimental timelines of AAV injection in the cerebellomedullary cistern, subchronic variable stress (SCVS) paradigm, social behavior (SI), and sucrose preference (SP) behavioral tests in female (a) and male (f) mice. b, g: Representative heatmaps of traces from female (b) and male (g) mice in SI. cd: Quantification of total exploration time and difference index (DI) in SI (comparing female mice injected with AAV-eGFP (eGFP+N) in the cerebellomedullary cistern, female mice injected with AAV-eGFP+ and experienced SCVS (eGFP+SCVS), and female mice injected with AAV-VEGFR3+ and experienced SCVS (VEGFC+SCVS). e: Quantification of sucrose preference among the three groups of female mice (ce: n=5-6 per group; results from two independent experiments). hi: Quantification of total exploration time and DI in SI (comparing AAV-VEGFR3 injection). d4-7 Control male mice (VEGFR3) d4-7 +N), injection of AAV-VEGFR3 d4-7 And male mice that have undergone SCVS (VEGFR3) d4-7 +SCVS) and injection of AAV-VEGFR3 d1-4 And male mice that have undergone SCVS (VEGFR3) d1-4 +SCVS). j: Quantification of sucrose preference among the three groups of male mice (hj: n=7-8 per group; results of two independent experiments). All data are expressed as mean ± sem and analyzed by one-way ANOVA using Tukey's post-hoc test.

[0037] Figure 15Distribution of tracer injected into the cisterna magna or intracerebrally in female mice after subchronic variable stress (SCVS). a, d, f, I: Experimental timeline for cisterna magna AAV infusion, subchronic variable stress (SCVS) paradigm, intracerebral (i.c.m.) ovalbumin (OVA) or Evans Blue (EB) injection and tissue collection (sac). b: Representative images of the distribution (red) of OVA-Alexa Fluor 647 tracer in brain sections after cisterna magna injection (compare control mice injected with AAV-eGFP (eGFP+N), mice injected with AAV-eGFP + subjected to SCVS (eGFP+SCVS) and mice injected with AAV-VEGFC + subjected to SCVS (VEGFC+SCVS). Nuclei were counterstained with DAPI (blue). Scale bar: 1000 pm. c: Quantification of the distribution of OVA tracer injected intracerebral ly into the cisterna magna in the brain parenchyma of three groups of mice (n=8-10 per group; results from three independent experiments). e: Quantification of the distribution of EB tracer injected into the cisterna magna in brain homogenates (containing EB diffused in the brain parenchyma and in the ventricles) of control female mice (N) and mice subjected to SCVS (N=8-9 per group; results from two independent experiments). g: Dural schematic with the region of the torcular Herophili (TS) marked with a red dashed line for image analysis. h: Representative images of LYVE1 (gray), OVA tracer (red) and eGFP-labeled macrophages (green) immunofluorescence staining in the dural TS region. Scale bar: 100 pm. i-k: Quantification of the percentage of area covered by total OVA tracer (i), OVA tracer in eGFP-labeled macrophages (j) and percentage of OVA tracer in eGFP-labeled macrophages over total OVA tracer (k) in the dural TS region (n=7-8 per group; results from two independent experiments). I: Experimental timeline for SCVS paradigm, infusion of 40 kDa and 70 kDa dextran tracers into the mPFC and VTA, tissue collection (sac). m: Representative images of tracer distribution in the mPFC and VTA (compare N and SCVS groups). Scale bar: 500 pm. n-q: Quantification of 40 kDa (n, p) or 70 kDa (o, q) dextran tracers remaining in the mPFC (n, o) or VTA (p, q) at 2.5 hours post-injection (n=9-11 per group; results from three independent experiments). All data are presented as mean ± s.e.m. and were analyzed by one-way ANOVA with Tukey’s post-hoc test or unpaired Student’s t-test.

[0038] Figure 16: Subchronic variable stress (SCVS) did not significantly change GFAP expression in the VTA or c-FOS expression in the mPFC of female mice. a: Coronal map highlighting the VTA in red. b-c: Representative images (b: scale bar = 50 pm) and quantification (c) of GFAP (green) immunofluorescence staining in the VTA (compare SCVS-experienced female mice to non-stressed control mice (N)). Dopaminergic neurons were co-stained with TH (gray). d: Coronal map highlighting the mPFC in red. e-f: Representative images (e: 200 pm.) and quantification (t) of c-FOS immunofluorescence staining in the mPFC (compare SCVS female mice and non-stressed control mice (N)) (c, f: n = 7-8 mice per group; results from two independent experiments). All data are represented as mean ± s.e.m and analyzed by unpaired Student’s t test.

[0039] Figure 17 : Subchronic variable stress (SCVS) and cerebellomedullary cistern injection of AAV-VEGFC altered the lipidomic profile of the mPFC, VTA and dura mater of female mice a: Experimental timeline of AAV infusion, SCVS paradigm and tissue sac. b: Score plot of partial least squares discriminant analysis (PLS-DA) of the lipidomic profile in the mPFC, VTA and dura mater (compare control mice injected with AAV-eGFP (eGFP+N), mice injected with AAV-eGFP + experienced SCVS (eGFP+SCVS) and mice injected with AAV-VEGFC + experienced SCVS (VEGFC+SCVS)). c-e: Heatmap showing the significantly changed lipid species and their relative expression levels (VIP > 1 and p < 0.05) in the three groups of samples. Color scale represents the standard rlog-transformed values across samples (3 biological replicates per group, each replicate from samples collected from 4 animals) f: Representative images of S100P staining in the mPFC. Scale bar: 100 pm. g: Quantification of S100P-labeled astrocyte density in the mPFC. h: Representative images of TH (red) and c-FOS (gray) staining in the VTA. White arrows indicate c-FOS and TH double-labeled cells. Scale bar: 100 pm. i: Quantification of the percentage of c-Fos+ neurons within TH-labeled dopaminergic neurons in the VTA (g, i: n = 5 / group; results from two independent experiments). All data are represented as mean ± s.e.m and analyzed by unpaired Student’s t test (g) or Mann-Whitney test (i).

[0040] Figure 18 : Cerebellomedullary cistern injection of AAV-VEGFR3 in control (naive) male mice d1-4or in male mice subjected to subchronic variable stress (SCVS) injected in the cerebellomedullary cistern with AAV-VEGFC did not change depressive and anxious-like behaviors. a: Experimental timeline of cerebellomedullary cistern injection with AAV and behavioral tests. b-d: Quantification of grooming duration in the splash test (ST, b), immobility time in the forced swim test (FST, c) and start feeding latency in the novelty suppressed feeding test (NSF, d) (control male mice injected in the brain with AAV-VEGFR3 d4-7 (VEGFR3d 4-7 + naive) as control injected with AAV-VEGFR3 d1-4 (VEGFR3 d1-4 + naive). e: Representative traces of animal paths in the open field (OF). f-h: Quantification of the percentage of the distance moved in the center zone over the total distance moved (t), the percentage of time spent in the center zone over the total time (g) and the total distance moved in the OF (h) (b-d, f-h: n = 7-9 / group; results from two independent experiments). i: Experimental timeline of cerebellomedullary cistern injection with AAV, SCVS and behavioral tests. j-l: Quantification of grooming duration in the ST (j), immobility time in the FST (k) and start feeding latency in the NSF (I) (male mice injected in the cerebellomedullary cistern with AAV-eGFP (eGFP + SCVS) as control of AAV-VEGFC (VEGFC + SCVS), both groups of male mice were subjected to SCVS). m: Quantification of SCVS body weight changes. n: Representative traces of animal paths in the open field. o-q: Quantification of the percentage of the distance moved in the center zone over the total distance moved (o), the percentage of time spent in the center zone over the total time (p) and the total distance moved in the OF (q) (jm, o-q: n = 6-9 / group; results from two independent experiments). All data are represented as mean ± s.e.m. and were analyzed by unpaired Student’s t test.

[0041] Figure 19Corticosterone (CORT) does not participate in the sex-differential impairment of meningeal lymphatic vessels induced by subchronic variable stress (SCVS). a: Experimental timeline of the SCVS paradigm and blood collection. b-c: Quantitative analysis of serum corticosterone concentration in female (b) and male (c) mice before and after SCVS treatment at 8:00 AM, 8:00 PM (n = 10 per group; results from two independent experiments). d: Experimental timeline of intraperitoneal injection of vehicle (Veh) or CORT in male mice, behavioral tests, and tissue collection (sac). e: Quantitative analysis of grooming duration in the splash test (ST). f: Quantitative analysis of immobility time in the forced swimming test (FST). g: Representative images of LYVE1 staining (gray) in the dural SSS and COS+TS regions (compare vehicle- and CORT-injected male mice). Scale bar: 500 pm. h: Representative images depicting LYVE1 -labeled mLVs at higher magnification. Scale bar: 200 pm. i-k: Quantitative analysis of the fluorescence intensity of LYVE1 staining (i), the area covered by mLVs (j), and the diameter of LYVE1 -labeled mLVs (k) (e-f, i-k: n = 7-8 per group; results from two independent experiments). All data are represented as mean ± s.e.m. and were analyzed by two-way ANOVA followed by Tukey’s post-hoc test (b-c) or unpaired Student’s t-test (e-f, i-k). DETAILED DESCRIPTION

[0042] It should be noted that the following detailed description is merely illustrative in nature and is not intended to limit the examples of the application, as described herein. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0043] It should be noted that the terms used herein are merely for the purpose of describing specific embodiments and are not intended to limit the examples of the application. As used herein, the singular form is intended to include the plural form, unless the context clearly indicates otherwise, and it should be further understood that the terms "comprise" and / or "include" when used herein, specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0044] The application will be further described with reference to the following examples, which are intended to be purely exemplary of the application, and not intended to limit the same. The test samples and test procedures used in the following examples include the following (if the specific experimental conditions are not specified in the examples, the general conditions are usually used, or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified).

[0045] Animals used in this example include three-month-old female and male C57BL / 6J mice, and female CX3CR1-GFP transgenic mice (Cat #005582; Jackson Laboratory) for experiments. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University. After intra-cisterna magna injection of tracers, penetration of tracers was imaged by Nikon Eclipse Ni-U fluorescence microscope. Lipidomic analysis was performed by common methods including LC-MS / MS. Serum corticosterone levels were detected by ELISA kit (Abeam, Ab108821).

[0046] In the RNA extraction and quantitative real-time polymerase chain reaction (qPCR) analysis involved in this example, total RNA was extracted using Trizol (Thermo Scientific, Waltham, MA, US) and cDNA was synthesized using NovoScript Plus All-in-on Strand cDNA Synthesis Supermix (Novoprotein, Suzhou, China) according to the manufacturer's instructions. For qPCR analysis, the following primers were used: Lyve1 primers (forward: 5'CAGCACACTAGCCTGGTGTTA-3 ', reverse: 5 '-CGCCCATGATTCTGCATGTAGA-3 '), Vegfc primers (forward: 5 '-GTGAGGTGTGTATAGATGTGGGG-3 ', reverse: 5 '-ACGTCTTGCTGAGGTAACCTG-3 '), Vegfr3 primers (forward: 5 '-CTGGCAAATGGTTACTCCATGA-3 ', reverse: 5 '-ACAACCCGTGTGTCTTCACTG-3 '), Gapdh primers (forward: 5 '-GAACATCATCCCTGCATCCA-3 ', reverse: 5 'CCAGTGAGCTTCCCGTTCA-3 '). cDNA amplification was performed using NovoStart SYBR qPCR SuperMix Plus (Novoprotein, Suzhou, China).

[0047] For infusion experiments of CCL6 neutralizing antibody, rat anti-CCL6 IgG (MAB487, R&D system, USA) and rat isotype control IgG2b (MAB0061, R&D system, USA) were used.

[0048] AAV viral vector construction: For the AAV viral vectors used in this study, eGFP (AAV-CMV-eGFP-3FLAG, AAV-CAG-eGFP), VEGFC (AAV-CMV-VEGFC-P2A-eGFP-3FLAG) and two VEGFR3 mutants (AAV-CAG-VEGFR3d d1-4 -IgG Fc and AAV-CAG-VEGFR3d d4-7 -IgG Fc) were custom-made by OBiO (Shanghai, China). Briefly, a PCR amplified fragment of mouse Vegfc coding sequence (NM_009506.2) was cloned into a pAAV-CMV-eGFP-3FLAG backbone vector (CMV: cytomegalovirus promoter; eGFP: eGFP, enhanced green fluorescent protein; 3FLAG: 3 copies of FLAG epitope tag) with a self-cleaving 2A peptide (P2A) between the sequences encoding VEGFC and eGFP (AAV-CMV-VEGFC-P2A-eGFP-3FLAG). A PCR amplified fragment of mouse VEGFR3 coding sequence (NM_008029.3) (encoding amino acid sequence 45-415 of mouse VEGFR3 domain 1-4 (VEGFR3d 1-4 ) and amino acid sequence 331-764 of VEGFR3 domain 4-7 (VEGFR3d 4-7 )) was cloned into a pAAV-CAG-IgG Fc-HA backbone vector and fused in-frame with mouse IgG Fc domain (CAG: CMV early enhancer / chicken beta-actin promoter; IgG: mouse IgG Fc domain; HA: hemagglutinin epitope tag) to construct pAAV-CAG-VEGFR3d 1-4 -IgG Fc and pAAV-CAG-VEGFR3d 4-7 -IgG Fc plasmids. Plasmids of AAV-CMV-eGFP-3FLAG and AAV-CMV-VEGFC-P2A-eGFP-3FLAG were packaged into AAV serotype 1 (AAV1). Plasmids of AAV-CAG-VEGFR3d 1-4 -IgG Fc, AAV-CAG-VEGFR3d 4-7 -IgG Fc and AAV-CAG-eGFP were packaged into AAV serotype 9 (AAV9).

[0049] Example 1

[0050] I. Subchronic variable stress (SCVS) damages meningeal lymphatic vessels in female mice, but not male mice.

[0051] To investigate the role of meningeal lymphatic vessels in pressure sensitivity between sexes, we employed a 6-day SCVS paradigm (Fig. 9a, 2a). Consistent with previous reports, both sexes experienced significant weight loss after SCVS. Figure 9 (b, 2b). However, females exhibited depressive-like behaviors, including reduced grooming behavior in the Splash test, increased immobility in the Forced swim test, and an increased waiting period to begin feeding in the Novelty-Suppressed Feeding test, which were not observed in male mice. Figure 9 ce, 2c-e). Furthermore, stressed female mice were less likely to venture into the central area of ​​open fields (ce, 2c-e). Figure 9 fi (open field test). Therefore, SCVS induced depressive and anxiety-like behaviors (i.e., depression-like and anxiety-like behaviors) in female mice. Conversely, stressed male mice showed a significant increase in walking distance in the central area of ​​the open field, indicating that male mice possess an anti-anxiety phenotype. Figure 10 fi).

[0052] To investigate whether behavioral phenotypes in different sexes are associated with changes in meningeal lymphatic vessels, we measured LYVE1 mRNA levels in mice after SCVS or in non-stressed control mice. LYVE1 is a marker of dural lymphatic endothelial cells (LECs). qPCR analysis showed that SCVS significantly reduced LYVE1 mRNA levels in the dura mater of female mice, while this was not observed in male mice (Figures 11a-c).

[0053] To further verify the damage of SCVS to the meningeal lymphatic vessels in female but not male mice, we injected 70kD dextran-Texas red tracer into the cerebellomedullary cistern 72 hours after the last behavioral test. Mice were sacrificed 1 hour later. Figure 1a-c). We found that SCVS significantly reduced the intensity of LYVE1 immunofluorescent staining and the diameter of LYVE1 -labeled meningeal lymphatic vessels in the superior sagittal sinus (SSS), transverse sinus (TS), and confluens sinuum (COS) in female mice. There was also a trend of reduced coverage area of meningeal lymphatic vessels (Fig. 1 d-i). Consistent with the structural defects of meningeal lymphatic vessels, significantly less intracavital tracer was detected in the SSS, TS, and COS regions of the dura mater where meningeal lymphatic vessels reside after SCVS (Fig. lj). The amount of tracer drained to the dCLNs was also significantly reduced (Fig. lp-q). Notably, the morphological impairment of meningeal lymphatic vessels in female mice was observed as early as 24 hours after the last SCVS (Fig. 12).

[0054] In contrast, after SCVS in male mice, the intensity of LYVE1 immunofluorescent staining, the coverage area and diameter of meningeal lymphatic vessels in the SSS, TS, and COS regions of the dura mater remained unchanged (Fig. le, k-n). SCVS also did not change the amount of 70kD dextran-Texas Red tracer in the SSS, TS, and COS regions of the dura mater and in the dCLNs in male mice (Fig. lo, r-s). Although meningeal lymphatic vessels in female and male mice exhibited different sensitivities to stress, we found no significant difference in the overall morphology of meningeal lymphatic vessels and the drainage of intracavital tracer into the dCLNs between unstressed adult female and male mice (Fig. 1 m, n). Figure 11 d-h). Consistent with previous reports, these data suggest that the basic properties of meningeal lymphatic vessels are similar in adult female and male mice.

[0055] Overall, these data reveal that SCVS causes impairment of meningeal lymphatic vessels in female but not male mice.

[0056] II. Improving meningeal lymphatic vessels can reduce the susceptibility of female mice to stress

[0057] Vascular endothelial growth factor-C (VEGFC) is a secreted protein that binds to vascular endothelial growth factor receptor 3 (VEGFR3), which is mainly expressed on LECs, to promote the growth of lymphatic vessels. Although there are reports in the prior art of the link between VEGF and depression, the specific VEGF is usually VEGFA, and the prior art does not report the link between different types of VEGFC and depression. And because it is different from the traditional VEGF (i.e. VEGFA), the link between it and depression is still unpredictable. There is also no literature to disclose that VEGFC can be used on animal models, and the prior art cannot predict whether it has the function of preventing, treating depression and the like. The present application creatively uses VEGFC for related experiments to explain its possible mechanism and influence. In the present embodiment, the adeno-associated virus (AAV) overexpressing VEGFC is injected into the cerebellomedullary cistern, which can promote the growth of meningeal lymphatic vessels and drainage to dCLNs without affecting the meningeal blood vessels. It is worth noting that although VEGFC can signal to other cells expressing VEGFR2 or VEGFR3 in the brain, the cells infected by AAV injection into the cerebellomedullary cistern are mainly cells around the meningeal lymphatic vessels in the dura mater, which may be due to the direction of cerebrospinal fluid (CSF) flow; and the coverage of meningeal vessels or the proliferation of neural stem cells in the hippocampus is not affected, which may be due to the mediation of VEGFC signaling. Therefore, this method provides a way to deliver secreted VEGFC to meningeal lymphatic vessels with relatively spatial specificity. Therefore, we injected AAV overexpressing VEGFC or enhanced green fluorescent protein (eGFP) labeled AAV as a control into the cerebellomedullary cistern of female mice. After one month, both groups of mice received a 6-day SCVS (Figure 2a). Consistent with previous reports, eGFP-labeled AAV-infected cells were mainly detected in TS, COS, and part of SSS covering the olfactory bulb, and were surrounded by LYVE1-labeled meningeal lymphatic vessels (b). We did not observe infected cells in the brain parenchyma, except for a small number of cells observed occasionally in the cerebellum near the needle insertion site. Overexpression of VEGFC significantly enhanced the intensity of LYVE1 immunofluorescence staining in the dura mater SSS and TS / COS, as well as the diameter and coverage area of meningeal lymphatic vessels (c-g). In addition, these mice showed an increase in the drainage of the tracer injected into the cerebellomedullary cistern to the dura mater and dCLNs (Figure 2c, h, i-j). Figure 2 b}). We did not observe infected cells in the brain parenchyma, except for a small number of cells observed occasionally in the cerebellum near the needle insertion site. Overexpression of VEGFC significantly enhanced the intensity of LYVE1 immunofluorescence staining in the dura mater SSS and TS / COS, as well as the diameter and coverage area of meningeal lymphatic vessels (c-g). In addition, these mice showed an increase in the drainage of the tracer injected into the cerebellomedullary cistern to the dura mater and dCLNs (Figure 2c, h, i-j). Figure 2 b}). We did not observe infected cells in the brain parenchyma, except for a small number of cells observed occasionally in the cerebellum near the needle insertion site. Overexpression of VEGFC significantly enhanced the intensity of LYVE1 immunofluorescence staining in the dura mater SSS and TS / COS, as well as the diameter and coverage area of meningeal lymphatic vessels (c-g). In addition, these mice showed an increase in the drainage of the tracer injected into the cerebellomedullary cistern to the dura mater and dCLNs (Figure 2c, h, i-j).

[0058] Importantly, female mice treated with VEGFC showed significantly alleviated body weight loss after SCVS (Fig. 2k). They also showed significantly enhanced grooming duration in the splash test, reduced immobility in the forced swim test, and increased exploration of the central area in the open field test (Figs. 21-p). Notably, neither the novelty suppressed feeding test nor the latency to start feeding in general locomotion (as measured by the total distance traveled in the open field) were affected (Figs. 2q-r). In non-stressed female mice, cerebello-medullary injection of AAV-VEGFC promoted meningeal lymphatic growth without affecting the above behaviors (Figs. 13a-m), suggesting that the enhancement of meningeal lymphatics alone was not enough to change depression or anxiety, but rather improved their resilience to stress.

[0059] Social avoidance and anhedonia are common symptoms of depression. Female mice that experienced SCVS showed significantly reduced preference for social interaction and sucrose water compared to non-stressed groups, and both cases could be prevented by intracerebello-medullary injection of AAV-VEGFC (Figs. 14a-e).

[0060] In summary, these results suggest that AAV overexpressing VEGFC via intracerebello-medullary injection can improve meningeal lymphatics to prevent stress-induced depressive-like and anxiety-like behaviors in female mice.

[0061] III. Improved meningeal lymphatics can alleviate stress-induced mPFC, VTA changes in female mice

[0062] Previous studies have shown that meningeal lymphatics are connected to the lymphatic system, which is a perivascular space in the brain parenchyma arranged at the end of astrocytes to facilitate the clearance of molecules from the brain parenchyma to meningeal lymphatics and further to peripheral lymph nodes. Changes in meningeal lymphatics can change the rate of cerebrospinal fluid (CSF) influx and interstitial fluid (ISF) diffusion in the brain parenchyma. To examine whether CSF influx and diffusion in the brain parenchyma would be affected by SCVS and VEGFC treatment, we injected ovalbumin (OVA)-Alexa Fluor 647 tracer into the cerebello-medullary pool of female mice and perfused the mice after 1 hour. The results showed that the tracer found in the brain parenchyma was significantly reduced after SCVS, and this condition could be prevented by improving meningeal lymphatics via intracerebello-medullary injection of AAV-VEGFC (Figs. 15a-c).

[0063] To assess the accumulation of tracer content in the brain parenchyma and ventricles, we collected whole brains (excluding the skull) from another cohort of female mice and measured tracer content in tissue homogenates from control and SCVS-treated mice. There was no difference between the two groups ( Figure 15 d-e). Since SCVS reduced the amount of tracer detected in the brain parenchyma, near the meningeal lymphatic vessels, and in dCLNs (Fig. lj and p-q, Fig. 15a-c), these data suggest that SCVS increased the accumulation of tracer in the ventricles and subarachnoid space of female mice.

[0064] It has been reported that a large amount of tracer reaching the dura mater can be phagocytosed by macrophages. Can SCVS change the phagocytic activity of macrophages near the meningeal lymphatic vessels? Using CX3CR1-eGFP labeled mice macrophages, we found that less tracer was detected in the dura mater TS, and less tracer was detected in the nearby macrophages. However, the percentage of tracer in macrophages out of total tracer did not significantly change after SCVS (Fig. 15f-k), suggesting that changes in macrophage phagocytic activity are unlikely to be the cause of the reduction in tracer detected near the meningeal lymphatic vessels.

[0065] Impaired CSF perfusion is often associated with insufficient efflux of ISF macromolecules from the brain parenchyma. To examine whether SCVS also affects the efflux of ISF molecules, we injected 70kD and 40kD dextran tracers into the mPFC and VTA. For both regions, SCVS significantly reduced the clearance of these tracers (Fig. 151-q). Overall, these data suggest that SCVS causes reduced CSF perfusion in female mice, likely due to impaired meningeal lymphatic vessels. Reduced CSF perfusion is accompanied by reduced efflux of ISF macromolecules and reduced CSF reaching the vicinity of meningeal lymphatic vessels and dCLNs.

[0066] While SCVS and VEGFC treatment affected CSF perfusion throughout the brain, we noticed that, in control female mice, cerebello-medullary cisterna injection of tracer was not uniformly distributed in the brain parenchyma, being enriched in the mPFC 15-30 min after injection, and in the hypothalamic and midbrain regions, including the VTA, 30-60 min after injection ( Figure 3 a-b). Importantly, both the mPFC and VTA are closely associated with emotional regulation.

[0067] The mPFC is thought to be critical in providing resilience against stress. Moreover, previous studies have found that astrocyte function is reduced in the mPFC of post-mortem brains of MDD patients. Since the glymphatic system is formed by the arrangement of astrocytic end-feet, we hypothesized that impairment of meningeal lymphatic drainage by stress could lead to accumulation of macromolecules in the mPFC glymphatic system, which in turn could cause damage to mPFC astrocytes. Consistent with clinical observations, we found that SCVS significantly reduced the expression of astrocyte markers S100β and GFAP in the female mPFC, as well as the density of S100β -labeled astrocytes. Improvement of meningeal lymphatic vessels by injection of AAV overexpressing VEGFC in the cistema magna could prevent stress-induced reduction of S100β and GFAP levels and astrocyte density in the mPFC Figure 3 d-h).

[0068] Moreover, reduction of VTA dopaminergic neuron activity has been consistently linked to the development of depression. We also found that SCVS reduced the expression of c-FOS, a marker of neuronal activity, in tyrosine hydroxylase positive (TH + ) dopaminergic neurons in the female VTA. AAV-mediated VEGFC overexpression in the dura mater significantly enhanced c-FOS expression in TH + neurons, indicating improved VTA dopaminergic neuron activity (Fig. 3i-j). Notably, SCVS did not significantly change c-FOS expression in the mPFC or GFAP expression in the VTA (Fig. 16). Moreover, VEGFC treatment alone did not change astrocyte protein expression in the mPFC or c-FOS expression in VTA dopaminergic neurons in non-stressed female mice (n-t), suggesting that enhancement of meningeal lymphatic vessels alone was not enough to change mPFC astrocyte and VTA dopaminergic neuron activity. Figure 13 n-t), suggesting that enhancement of meningeal lymphatic vessels alone was not enough to change mPFC astrocyte and VTA dopaminergic neuron activity.

[0069] One of the main physiological functions of lymphatic vessels is to absorb and transport lipids. On the other hand, lipids are enriched in the brain and participate in a wide range of physiological functions. Previous studies reported that chronic unpredictable stress changes the lipidomic profile of the prefrontal cortex and that the activity of dopaminergic neurons in the VTA can be modulated by triacylglycerols (TAGs). Since ISF macromolecule outflow is reduced after SCVS (Fig. 151-q), we hypothesized that the damage to the meningeal lymphatic vessels after SCVS could lead to the accumulation of lipids in the brain parenchyma, resulting in abnormalities in the mPFC and VTA, and that VEGFC treatment could prevent these changes. To this end, we performed lipidomic analysis of the mPFC, VTA and dura mater of three groups of female mice: (1) control mice injected with AAV-eGFP, (2) mice injected with AAV-eGFP and subjected to SCVS, and (3) mice injected with AAV-VEGFC and subjected to SCVS Figure 17 a). Partial least squares discriminant analysis (PLS-DA) score plot showing clear separation between the three groups Figure 17 b). After SCVS, there was a significant increase in a variety of lipids in the mPFC, including long-chain TAGs, and a small increase in a subset of TAGs in the VTA after SCVS, and this change was partially prevented by VEGFC treatment (Fig. 17c-d). Importantly, VEGFC treatment significantly increased a group of lipid molecules in the dura mater, including long-chain TAGs (Fig. 17e). These findings are consistent with the idea that VEGFC treatment increases meningeal lymphatic drainage, thereby promoting the outflow of lipids and reducing the accumulation of lipid molecules in the mPFC and VTA of stressed female mice.

[0070] To investigate the functional links between the identified lipids that were significantly altered in the mPFC and VTA of female mice by SCV, we focused on long-chain TAGs, which here include: long-chain TAGs that accumulated in both brain regions after SCVS, and that were partially prevented from accumulating by VEGFC treatment. The results showed that direct injection of this class of TAGs into the mPFC led to a significant loss of astrocytes, while in the VTA, injection reduced TH + Expression of c-FOS in dopaminergic neurons Figure 17 f-i).

[0071] In summary, the above findings indicate that enhancing meningeal lymphatic vessels by VEGFC prevents SCVS-induced abnormalities in mPFC astrocytes and VTA dopaminergic neurons in female mice, and that this process can be achieved by promoting the outflow of long-chain TAGs from both brain regions.

[0072] Four, damage to the meningeal lymphatic vessels makes male mice sensitive to stress

[0073] Does damage to the cerebral meningeal lymphatic vessels in male mice promote SCVS-induced depressive-like phenotypes? VEGFC binds to VEGFR3 to regulate lymphangiogenesis. Intracisternal cerebellomedullary injection of VEGFR3 d1-4 AAV expressing VEGFR3 d1-4 -IgG Fc contains VEGFR3 ectodomains 1-4 that bind to VEGFC, but the intracellular domain is replaced by the immunoglobulin fragment crystallizable domain (Fc). Thus, VEGFR3 d1-4 -IgG Fc competes with endogenous VEGFR3 for VEGFC binding but cannot initiate signaling. Control virus expresses the IgG Fc fusion ectodomain of VEGFR3 4-7, which does not bind to VEGFC. We will express VEGFR3 d1-4 -IgG Fc or as a control VEGFR3 d4-7 -IgG Fc into the cerebellomedullary cistern of male mice (Fig. 4a). AAV-infected cells were mainly located in the TS / COS, parts of the SSS that cover the olfactory bulb dura, and in the vicinity of LYVE1 -labeled meningeal lymphatic vessels (Fig. 4b). Figure 4 b). VEGFR3 d1-4 -IgG Fc overexpression isolates VEGFC in the microenvironment and prevents VEGFC binding to VEGFR3 on meningeal LECs. Both groups of mice were then subjected to SCVS for 6 days (Fig. 4c). Figure 4 a). The results show that VEGFR3 d1-4 -IgG Fc overexpression significantly reduced LYVE1 expression in the dura SSS and TS / COS, as well as the diameter and area covered by LYVE1 -labeled meningeal lymphatic vessels (Fig. 4c-g). Figure 4 c-g). Meanwhile, the amount of cerebellomedullary cisternal injection tracer detected in the dura and dCLN was also significantly reduced (Fig. 4c, h-j).

[0074] Importantly, VEGFR3 d1-4 -IgG Fc overexpression in male mice exhibited significantly reduced grooming time in the splash test, increased immobility time in the forced swim test, and impaired social interaction after SCVS (Fig. 4k-l, Figure 14 f-i). VEGFR3 d1-4 , VEGFR3 d4-7There were no significant differences in the latency to start feeding in the novelty suppressed feeding test, body weight change, overall locomotion and center zone exploration in the open field test, and sucrose preference test among the overexpressing male groups (Fig. 4m-r, Fig. 14f,j).

[0075] Notably, VEGFR3d1-4 treatment alone did not affect the performance of non-stressed male mice in the depression-like behavior tests in the absence of SCVS (Fig. 18a-d), suggesting that damage to the meningeal lymphatic vessels alone is not sufficient to induce depression-like behavior in male mice, but increases their susceptibility to stress. For the VEGFR3d1-4 treated group, there was a trend of decreased time spent in the center zone of the open field (p = 0.055), suggesting a potential anxiety phenotype Figure 18 e-h). Moreover, cerebromedullary cistern injection of AAV overexpressing VEGFC did not affect the performance of male mice in the depression and anxiety-like behavior tests and body weight change (Fig. 18i-q), suggesting a “ceiling effect” in the role of meningeal lymphatic vessels in modulating depression and anxiety-like behavior.

[0076] Similar to female mice (Fig. 3a-b), male mice showed tracer enrichment in the mPFC 15-30 minutes after injection, and in the hypothalamus and VTA 30-60 minutes after injection (Fig. 5a-b). SCVS did not significantly change the expression of astrocytic proteins in the mPFC, nor did it change TH + c-FOS expression in dopaminergic neurons. However, VEGFR3 d1-4 Treated male mice showed significantly reduced S100β and GFAP expression after SCVS, and s100β + Decreased density of astrocytes, reduced TH + c-FOS expression in dopaminergic neurons (Fig. 5c-j).

[0077] As concerns about the impact of manipulating VEGFC-VEGFR3 signaling on stress susceptibility also include lymphatic-independent effects, we also investigated whether ligation of the lymphatic vessels connecting the dCLN to the meninges (preventing the drainage pathway of the brain parenchyma through the meningeal lymphatic vessels) would make male mice susceptible to SCVS. Compared to sham-operated mice, male mice that received ligation surgery showed a significant reduction in the drainage of an intracerebral injection of tracer to the dCLN (Fig. 6a-c). Moreover, these mice showed a decrease in grooming time in the splash test and an increase in immobility in the forced swim test after SCVS, while there were no significant changes in behavior and body weight changes in the novel suppressed feeding test and open field test (Fig. 6d-k). These mice also showed a decrease in S100β and GFAP expression after SCVS, a decrease in astrocyte density in the mPFC and a decrease in TH + dopaminergic neurons c-FOS expression in the VTA + Figure 6 l-r).

[0078] Overall, the above data suggest that impairment of the meningeal lymphatic vessels or reduction in meningeal lymphatic drainage increases the susceptibility of male mice to stress and promotes stress-induced abnormalities in the mPFC and VTA.

[0079] V. Subchronic variable stress alters the transcriptional profile of meningeal lymphatic endothelial cells in female mice

[0080] Corticosterone is considered an important stress hormone for depression and anxiety, and to explore the mechanisms by which stress regulates the meningeal lymphatic vessels, we wondered whether the differential impairment of the meningeal lymphatic vessels in female mice, but not in male mice, after SCVS was related to the gender differences in corticosterone levels. To this end, we measured serum corticosterone levels in female and male mice before and after completion of SCVS Figure 19 a). In agreement with previous reports about the circadian oscillation of corticosterone levels, serum corticosterone levels were higher at 8:00 PM compared to 8:00 AM in control female and male mice, and no gender differences were detected. Notably, when comparing serum corticosterone levels at 8:00 AM, corticosterone levels were significantly increased in female and male mice after SCVS compared to control (naive) mice. However, when checking serum corticosterone levels at 8:00 PM, we found that corticosterone levels were significantly decreased in female and male mice after SCVS Figure 19 ​bc). Therefore, the diurnal oscillation of serum corticosterone levels was weakened after SCVS, with similar amplitudes in female and male mice. To directly examine the effects of corticosterone on meningeal lymphatic vessels, male mice were injected with corticosterone for 28 consecutive days, producing the previously reported depressive-like phenotype (Fig. 19d-f). However, by detecting LYVE1 expression, the coverage of LYVE1-labeled meningeal lymphatic vessels, and the diameter of meningeal lymphatic vessels, it was found that chronic corticosterone administration did not cause detectable changes in meningeal lymphatic vessels ( Figure 19 Therefore, our findings suggest that corticosterone is unlikely to cause sex-dependent meningeal lymphatic vessel damage induced by SCVS.

[0081] To further explore the molecular mechanisms by which stress affects meningeal lymphatic vessels, we analyzed meningeal lymphatic vessels (LECs) from female mice receiving SCVS and non-stressed mice. - CD31 + PDPN + RNA-seq analysis was performed (Figures 7a-b). We found that the percentage of total viable cells (LECs) in the dura mater of stressed female mice was significantly reduced compared to non-stressed mice, further supporting our observation that SCVS damages meningeal lymphatic vessels. Figure 7 c). No significant changes were detected in other meningeal cell types, including blood endothelial cells (CD45-CD31+PDPN+) and leukocytes (CD45-CD31+PDPN+). + ) or other stromal cells (CD45) - CD31 - (Mainly composed of fibroblast-like cells) Figure 7 df).

[0082] Deseq2 analysis with a cutoff value of p-adjusted < 0.1 identified 236 differentially expressed genes (DEGs) in meningeal LECs, of which 156 were upregulated and 80 were downregulated by SCVS. Figure 7 Notably, epidermal growth factor receptor (Egfr) expression is one of the most downregulated genes under stress, indicating insufficient signaling in support of lymphangiogenesis. Figure 7 h). We further performed gene set enrichment analysis (GSEA), which showed that gene sets involved in regulating DNA replication, sphingolipid biosynthesis, and serotonin uptake were enriched in the meningeal lymphatic vessels of stressed female mice, while gene sets involved in proteasome assembly, ribosome breakdown, and platelet-derived growth factor binding were de-enriched, indicating that stress causes profound functional changes in the meningeal lymphatic vessels. Figure 7i). Notably, a serotonin uptake gene set was enriched (normalized enrichment score = 2.14, FDR = 0.059) in stressed female mouse meningeal LECs, suggesting that serotonin, a neurotransmitter heavily implicated in depression, can be involved in mediating stress-induced meningeal lymphatic vessel damage (Figures 7i-j)

[0083] Previous studies have reported that aging impairs meningeal lymphatic vessels. In addition, stress accelerates brain aging and progression of aging-related neurodegenerative diseases. Therefore, we wondered whether stress and aging have any common mechanisms in the regulation of meningeal LECs. To this end, we compared the DEGs identified in the current study with those identified in a previous study that examined how aging affects the transcriptome of meningeal LECs. Interestingly, we found that several genes encoding collagens, including Col6a1, Col1a2, and Col3a1, and the gene Mmp23, which encodes a matrix metalloproteinase that processes collagens, were significantly downregulated during both stress and aging, suggesting that dissociation from the extracellular matrix can be a key event in mediating meningeal lymphatic vessel damage (Figures 7k-l). Figure 7 k). To further explore whether the gene expression changes found in stressed female mice in the current study have any similarities with those associated with human depression, we compared the DEGs found in the current study with those found in a previous study by Labonte et al. that explored transcriptional profiling changes in 6 brain regions in post-mortem tissues of female MDD patients. This analysis identified 12 upregulated and 9 downregulated DEGs in common, and female mouse LECs that received SCVS also harbored these DEGs obtained by Labonte et al. in at least one brain region examined. Among these DEGs, the most striking was Trpml, which encodes a transient receptor potential cation channel, and in our study, it was upregulated in LECs of female mice that received SCVS, as well as in the ventral subiculum, dorsolateral prefrontal cortex, nucleus accumbens, and prefrontal cortex of female MDD patients, suggesting that it is a candidate gene worth further exploring for the development of depression. We also noted that Col1a2, a downregulated gene in common with aging and stress in LECs, was also downregulated in the nucleus accumbens of female MDD patients (Figure 7l).

[0084] From the RNA-seq data, we found that SCVS stress can strongly induce an increase in the expression of chemokine (C-C motif) ligand 6 (CCL6) in female mouse meningeal lymphatics (Figure 7h).

[0085] To examine the potential regulatory role of CCL6 in meningeal lymphatic impairment, we injected CCL6 neutralizing antibodies into the cistema magna to block the associated signaling that disrupts CCL6 activation of the receptor CCR1. The results showed that functional blockade of CCL6-CCR1 prevented the depressive-like phenotype and meningeal lymphatic impairment (Fig. 8). These findings suggest that CCL6 upregulation and CCL6-mediated activation of CCR1 function are biomarkers and upstream pathogenesis of stress-induced reduction of meningeal lymphatic coverage and increased susceptibility to initiate depressive-like phenotypes.

[0086] In summary, the present application discloses that SCVS disrupts the meningeal lymphatic structure and brain drainage function in female mice (but not in male mice) with profound transcriptome changes in meningeal LECs. Cistema magna injection of AAV overexpressing VEGFC improves meningeal lymphatics and alleviates stress-induced depressive and anxiety-like behaviors and changes in the mPFC and VTA of female mice; whereas intracerebral injection of AAV overexpressing VEGFR3 d1-4 impairs meningeal lymphatics or reduces meningeal lymphatic drainage by ligating the dCLN-connected lymphatic vessels, increases the susceptibility of male mice to SCVS. These findings reveal the functional role of meningeal lymphatics in the sex difference in stress susceptibility. Based on the present study, because the drainage function of meningeal lymphatics can be assessed by clinically available imaging techniques, such as magnetic resonance imaging (MRI) and computed tomography (CT), meningeal lymphatic abnormalities are expected to be a new biomarker for early diagnosis and prediction of the effectiveness of clinical depression treatment. Moreover, improvement of meningeal lymphatics can have therapeutic potential for the treatment of depression and other neuropsychiatric diseases associated with stress.

[0087] Obviously, the above-mentioned embodiments of the present application are only examples for clearly illustrating the technical solutions of the present application, and are not intended to limit the specific implementation manners of the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Use of a reagent for detecting expression levels of Col6a1, Col1a2, Col3a1, Mmp23, Trpml and CCL6 in the brain in the preparation of a drug for diagnosing depressive-like behavior, anxiety-like behavior and / or meningeal lymphatic vessel damage. The depressive-like behavior is stress-induced depressive-like behavior, the anxiety-like behavior is stress-induced anxiety-like behavior, and the meningeal lymphatic vessel damage is stress-induced meningeal lymphatic vessel damage.

2. Use of a meningeal lymphatic vessel improving drug in the preparation of a drug for treating depressive-like behavior and / or anxiety-like behavior, the depressive-like behavior being stress-induced depressive-like behavior; the meningeal lymphatic vessel improving drug being VEGFC and a pharmaceutically acceptable overexpression vector thereof.

3. Use according to claim 2, characterized in that, The pharmaceutically acceptable overexpression vector includes an adenovirus vector.

4. Use of a CCL6 downstream pathway blocker in the preparation of a drug for treating meningeal lymphatic vessel damage, depression and / or anxiety; The CCL6 downstream pathway blocker is a CCL6 neutralizing antibody.

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