Method for verifying influence of quercetin on HIE newborn rat lymphatic system and neurological function

Abstract

The invention relates to the technical field of biology, in particular to a method for verifying influence of quercetin on HIE newborn rat lymphatic systems and neurological functions. The method comprises the following steps: a, acquiring a newborn rat for an experiment, and establishing an HIE model; b, preparing a quercetin working solution; c, after a rat in the HIE model is selected and injected with a quercetin working solution, nerve function evaluation is completed, then the brain of the rat is taken out to be sliced, TTC dyeing is completed, and the infarct area percentage is calculated; and performing brain water content detection on the rats to complete verification of influence of quercetin on lymphatic system functions in HIE. The method provided by the invention has the advantages that the steps are simple, and the influence of the quercetin on the lymphatic system and the neurological function of the HIE newborn rat can be more intuitively verified.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and is a method for verifying the effects of quercetin on the lymphatic system and nerve function of HIE neonatal rats. Background Art

[0002] Neonatal hypoxic-ischemic encephalopathy (HIE) is an acute brain injury caused by insufficient oxygen supply to the brain during the neonatal period. HIE can lead to premature death and multiple lifelong sequelae in affected infants. Currently, hypothermia therapy is the mainstay of clinical treatment for HIE. However, hypothermia does not provide complete neuroprotection, and the incidence of secondary neurological damage after treatment remains high.

[0003] Quercetin (QE) is a flavonoid compound widely found in herbaceous and woody plants. Studies have shown that quercetin acts as an antioxidant, neuroprotectant, and anti-inflammatory agent, with protective effects against inflammatory and neurological diseases. Studies have shown that in a rat model of HIE, quercetin partially reversed brain edema and morphological changes and reduced hypoxia-ischemia-induced AQP4 expression. Quercetin significantly improved cerebral blood flow in a mouse model of dementia. Cerebral arterial blood flow is the driving force for the exchange of cerebrospinal fluid and interstitial fluid in the brain, but the application of quercetin in HIE is still in the exploratory stage.

[0004] Therefore, how to verify what effect quercetin can have on the lymphatic system and neural function in the neonatal rat model of hypoxic-ischemic encephalopathy, and how it produces the effect, has become a technical problem to be solved. Summary of the Invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is how to provide a verification method and steps that can verify what effect quercetin can have on the lymphatic system and neural function in the neonatal rat model of hypoxic-ischemic encephalopathy, and how the effect is produced.

[0006] To achieve the above objectives, the present invention provides a method for verifying the effect of quercetin on the glymphatic system and neural function of neonatal rats with HIE, comprising the following steps: a. obtaining neonatal rats for experiment and establishing an HIE model; b. preparing a quercetin working solution; c. selecting rats in the HIE model and injecting the quercetin working solution, completing a neurological function assessment, then removing the rat brain for slicing and performing TTC staining, and calculating the percentage of infarct area; and performing a brain water content test on the rats to complete the verification of the effect of quercetin on the glymphatic system function in HIE; d. selecting rats in the HIE model and injecting the quercetin working solution again, collecting the rat brain tissue and preparing brain slices, observing the slices under a fluorescence microscope, and using ImageJ software to determine the colocalization coefficient of GFAP and AQP4; then injecting Evans blue into the cerebral ventricles of the neonatal rats to evaluate the diffusion of cerebrospinal fluid (CSF); and collecting the rat heart for processing and testing; and completing the verification of the effect of quercetin on the glymphatic system function and AQP4 polarity in HIE.

[0007] As an optimization, in step a, when establishing the HIE model, 10-day-old rats were anesthetized with isoflurane and placed in the supine position; the hair was shaved to expose the anterior neck area; the right common carotid artery was isolated and double-ligated, then cut between the ligature points, and finally the incision was sutured; the time from the start of anesthesia to the end of the operation was controlled within 8 minutes; 1 hour after the operation, the rats were exposed to a hypoxic environment at 37°C for 2.5 hours; at the same time, newborn rats were used to set up a sham operation group, in which only the right common carotid artery was exposed, no ligation or cutting was performed, and no hypoxic treatment was given.

[0008] Furthermore, the hypoxic environment condition is a mixed gas of 8% oxygen and 92% nitrogen.

[0009] As an optimization, in step b, quercetin was dissolved in 1 ml of dimethyl sulfoxide (DMSO) at a concentration of 30 mg / ml to prepare a stock solution; the stock solution was then diluted with 0.9% saline; and a working solution with a final concentration of 7.5 mg / ml was prepared with saline.

[0010] As an optimization, in step c; in the neurological function assessment, 24 hours after HIE, neurological function was assessed using the righting reflex and negative geotropism tests; for the righting reflex, the rats were placed in the supine position, and the time required for them to change from supine to prone was recorded; in the negative geotropism test, the rats were placed head down on a 40-degree inclined surface, and the time required for them to turn and face upward was recorded; the maximum test time was 20 seconds; times exceeding 20 seconds were recorded as 20 seconds.

[0011] As an optimization, in step c, TTC staining was completed including: 24 hours after HIE, rats were anesthetized with isoflurane and perfused transcardially with 0.01 M phosphate buffered saline (PBS); the brain was removed and cut into 2 mm coronal sections; the sections were stained with 2% TTC; treated for 5 minutes, then washed with PBS and photographed; ImageJ software was used to analyze the non-infarct area of the ipsilateral hemisphere and the total area of the contralateral hemisphere; the percentage of infarct area of each section was calculated by the following formula: [(total area of the contralateral hemisphere)-(non-infarct area of the ipsilateral hemisphere)] / (total area of the contralateral hemisphere × 2); the average percentage of infarct area of each section of the brain of each animal was used to represent the percentage of infarct area of the animal.

[0012] As an optimization, in step c; brain water content detection, 24 hours after HIE, the rats were deeply anesthetized and brain tissue was collected; the wet weight of the tissue was measured and then dried in an oven at 95°C within 72 hours to obtain the dry weight; the brain water content was calculated using the formula [(wet weight-dry weight) / wet weight]×100%.

[0013] As an optimization, in step d; 24 hours after HIE, rats were anesthetized with isoflurane and perfused through the heart with 0.01 M PBS and 4% paraformaldehyde; brain tissues were collected, fixed in 4% paraformaldehyde overnight, and then dehydrated in 10%, 20% and 30% sucrose solutions in a gradient manner until sedimentation; brain sections (10 μm) were prepared using a freezing microtome, and then incubated with 5% bovine serum albumin at room temperature for 1 hour, followed by incubation with primary antibodies at 4°C overnight; the primary antibodies used included: mouse anti-AQP4 monoclonal antibody (1:100) and rabbit anti-GFAP monoclonal antibody (1:50); after washing with PBS, the sections were incubated with the corresponding fluorescent secondary antibodies for 1 hour; DAPI was used to stain the nucleus, the sections were observed under a fluorescence microscope, and the colocalization coefficient of GFAP and AQP4 was determined using ImageJ software.

[0014] Furthermore, in step d, Evans blue was injected into the cerebral ventricles to assess the diffusion of cerebrospinal fluid (CSF)

[11] . At 24 h after HIE, rats were deeply anesthetized with isoflurane and placed in a stereotaxic frame with 15 μl of a 0.4% Evans solution. Blue and 0.1% bovine serum albumin (in saline) were then injected into the cisterna magna at a rate of 2 μl / min for 7.5 min. After injection, the dye circulated for 1 h. 60 min after injection, rats were perfused and euthanized, and brain tissue was collected. Brain tissue was homogenized with 50% trichloroacetic acid. The samples were incubated overnight at 4°C to determine the Evans blue concentration, and the samples were centrifuged at 12,000 RPM for 30 min at 4°C. The supernatant was measured at 620 nm using a spectrophotometer and quantified using a standard curve.

[0015] Further, in step d, 24 hours after HIE, the rats were anesthetized with isoflurane, and the hearts were collected and perfused with normal saline; the brain tissue was quickly removed, homogenized with RIPA lysis buffer, and sonicated; the homogenate was centrifuged at 12000RPM for 30 minutes at 4°C and the supernatant was collected, and the collection time was measured in minutes; the protein concentration was determined using a BCA kit; equal amounts of protein samples (20 μg per well) were separated by 10% SDS-PAGE and transferred to a PVDF membrane; the membrane was lysed with a buffer (5% desorption ionization buffer) at room temperature. The membranes were incubated with 4% paraformaldehyde (0.5% glutathione) for 2 h. Primary antibodies were incubated overnight at 4°C: rabbit anti-GFAP monoclonal antibody (1:2000), mouse anti-AQP4 monoclonal antibody (1:2000), and mouse anti-α-tubulin (1:5000). After washing with PBST, the membranes were incubated on a shaker at 37°C for 60 min with HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (1:5000). The membranes were washed and then immunoreacted. Detection was performed using a chemiluminescence kit. Bands were quantified using ImageJ.

[0016] Furthermore, the method further includes step e, analyzing the data using GraphPad Prism 6, wherein the quantitative data are expressed as mean ± standard deviation (SD), the comparison between two groups is performed using t-test, and the comparison between multiple groups is performed using one-way analysis of variance (ANOVA); the significance level is set at α = 0.05.

[0017] In summary, in the above method, the entire method step design is simple, and can more intuitively verify the effects of quercetin on the lymphatic system and neural function of HIE neonatal rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a two-part experimental design map.

[0019] Figure 2 The effect of quercetin on the neurological function of HIE rats; (A) Statistical analysis of negative geotaxis test time; (B) Statistical analysis of righting reflex time.

[0020] Figure 3 The effect of quercetin on infarct size and brain water content in HIE.

[0021] Figure 4 is the effect of quercetin on the distribution of Evans blue staining in HIE brain tissue.

[0022] Figure 5 The effect of quercetin on the polarized expression of AQP4 in HIE brain.

[0023] Figure 6 The effect of quercetin on the expression of AQP4 and GFAP proteins. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the accompanying drawings and examples.

[0025] A method for verifying the effect of quercetin on the glymphatic system and neural function of HIE neonatal rats; Figures 1 to 6 As shown; including the following steps;

[0026] 1. Animal handling;

[0027] All procedures in this study were approved by the Animal Ethics Committee.

[0028] A total of 162 Sprague Dawley rat pups (weighing approximately 25 ± 2 g) were purchased from the Animal Center of Chongqing Medical University. All rats were housed in a temperature- and humidity-controlled environment with a 12-h light / dark cycle and had unlimited access to food and water. Across all our experiments, the total mortality rate was 8 rats. Ten rats were excluded from this study due to unsuccessful ligation.

[0029] 2. Experimental design;

[0030] Two independent experiments were designed;

[0031] Experiment 1 investigated the effects of quercetin on neurological function, brain, ischemia-hypoxia, and brain water content after hypoxic-ischemic encephalopathy (HIE). Neurological function was assessed by neurobehavioral assessment.

[0032] Scoring, evaluation of cerebral ischemia-hypoxia status using TTC staining, measurement of brain water content, and assessment of glymphatic system function by injection of Evans blue dye into the cisterna magna. Rats were randomly divided into three groups: sham operation group, HIE+vehicle group, and HIE+QE group, with n=3 in each group.

[0033] Experiment 2 evaluated the effects of quercetin on glymphatic system function and AQP4 polarity in HIE. Glymphatic system function was assessed by injecting Evans blue dye into the cisterna magna, AQP4 polarity was measured by immunofluorescence, and GFAP and AQP4 protein expression levels were quantified using Western blot analysis. Rats were randomly divided into three groups: sham-operated group, HIE + control group, and HIE + quercetin group, with n = 3 per group.

[0034] 3. HIE model;

[0035] The HIE model was established as follows: 10-day-old rats were anesthetized with isoflurane and placed in the supine position. The hair was shaved and the anterior neck region was exposed. The right common carotid artery was isolated and double-ligated, then cut between the ligatures, and the incision was sutured. The time from the start of anesthesia to the end of the operation was controlled within 8 minutes. One hour after the operation, the rats were exposed to a hypoxic environment (8% O2 and 92% N2 mixture) at 37°C for 2.5 hours. In the sham operation group, only the right common carotid artery was exposed, without ligation or cutting, and no hypoxic treatment was given.

[0036] 4. Working solution;

[0037] A stock solution of quercetin was prepared by dissolving it in 1 ml of dimethyl sulfoxide (DMSO) at a concentration of 30 mg / ml. This stock solution was then diluted with 0.9% saline. A working solution was prepared in saline at a final concentration of 7.5 mg / ml. The HIE+vehicle group received an equal volume of DMSO and a 0.9% saline mixture. Under isoflurane anesthesia, quercetin was injected intraperitoneally at a dose of 30 mg / kg 10 minutes after HIE surgery.

[0038] 5. Neurological function assessment;

[0039] Neurological function was assessed 24 hours after HIE using the righting reflex and negative geotropism tests. For the righting reflex, the rat was placed in the supine position, and the time required to turn from supine to prone was recorded. For the negative geotropism test, the rat was placed headfirst on a 40-degree inclined surface, and the time required to turn and face upward was recorded. The maximum test time was 20 seconds; any time exceeding 20 seconds was recorded as 20 seconds.

[0040] 6. TTC staining;

[0041] Twenty-four hours after HIE, rats were anesthetized with isoflurane and perfused transcardially with 0.01 M phosphate-buffered saline (PBS). The brains were removed and cut into 2-mm coronal sections. The sections were stained with 2% TTC.

[0042] The cells were treated for 5 minutes, then washed with PBS and photographed. ImageJ software was used to analyze the non-infarcted area of the ipsilateral hemisphere and the total area of the contralateral hemisphere. The percentage of infarct area in each section was calculated using the following formula: [(total area of the contralateral hemisphere) - (non-infarcted area of the ipsilateral hemisphere)] / (total area of the contralateral hemisphere × 2). The average percentage of infarct area across each animal's brain section was used to represent the percentage of infarct area for that animal.

[0043] 7. Brain water content;

[0044] Twenty-four hours after HIE, rats were deeply anesthetized and brain tissue was collected. The wet weight of the tissue was measured and then dried in a 95°C oven; dry weight was obtained within 72 hours. Brain water content was calculated using the formula [(wet weight - dry weight) / wet weight] × 100%.

[0045] 8. Double immunofluorescence;

[0046] Twenty-four hours after HIE, rats were anesthetized with isoflurane and perfused transcardially with 0.01 M PBS and 4% paraformaldehyde. Brain tissue was collected and fixed overnight in 4% paraformaldehyde, followed by dehydration in a gradient of 10%, 20%, and 30% sucrose solutions until sedimentation. Brain sections (10 μm) were prepared using a freezing microtome and incubated with 5% bovine serum albumin for 1 hour at room temperature, followed by incubation with primary antibodies overnight at 4°C. The primary antibodies used included mouse anti-AQP4 monoclonal antibody (1:100) and rabbit anti-GFAP monoclonal antibody (1:50). After washing with PBS, sections were incubated with corresponding fluorescent secondary antibodies for 1 hour. DAPI was used to stain nuclei, and sections were observed under a fluorescence microscope. The colocalization coefficient of GFAP and AQP4 was determined using ImageJ software.

[0047] 9. Evans Blue Cisterna Magna Injection;

[0048] Evans blue was injected into the cerebral ventricles to assess the diffusion of cerebrospinal fluid (CSF)

[11] . 24 hours after HIE, the rats were deeply anesthetized.

[0049] The subjects were anesthetized with isoflurane and placed in a stereotaxic frame. A 15 μl solution of the dye (0.4% Evans blue) and 0.1% bovine serum albumin (dissolved in saline) was injected into the cisterna magna at a rate of 2 μl / min for 7.5 minutes. The dye was allowed to circulate for 1 hour after injection.

[0050] 60 minutes after injection, rats were perfused and euthanized, and brain tissue was collected and homogenized with 50% trichloroacetic acid.

[0051] Incubate overnight at 4°C to determine the Evans blue concentration.

[0052] The samples were centrifuged at 12,000 RPM for 30 minutes at 4° C. The supernatant was measured using a spectrophotometer at 620 nm and quantified using a standard curve.

[0053] 10. Western Blot;

[0054] 24 hours after HIE, rats were anesthetized with isoflurane and the heart was harvested and perfused with normal saline. Brain tissue was quickly removed, homogenized with RIPA lysis buffer, and sonicated. The homogenate was centrifuged at 12,000 RPM for 30 minutes, and the supernatant was collected at 4°C. The collection time was measured in minutes. Protein concentration was determined using a BCA kit. Equal amounts of protein samples (20 μg per well) were separated by 10% elution.

[0055] SDS-PAGE was performed and transferred to a PVDF membrane. The membrane was incubated with buffer (5% skim milk) for 2 hours at room temperature. Primary antibodies were incubated overnight at 4°C: rabbit anti-GFAP monoclonal antibody (1:2000), mouse anti-AQP4 monoclonal antibody (1:2000), and mouse anti-α-tubulin (1:5000). After washing with PBST, the membrane was incubated on a shaker at 37°C for 60 minutes with HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (1:5000). The membrane was washed and then immunoreactions were performed.

[0056] 11. Statistical analysis;

[0057] Data were analyzed using GraphPad Prism 6. Quantitative data are expressed as mean ± standard deviation (SD). Comparisons between two groups were performed using the t-test, while comparisons between multiple groups were performed using one-way analysis of variance (ANOVA). The significance level was set at α = 0.05.

[0058] 12. Analysis of neurological function after quercetin improved HIE;

[0059] Compared with the sham-operated group, the negative chemotaxis test time of HIE rats was significantly increased (P<0.05, Figure 2 A). Compared with the HIE group, the negative chemotaxis test time in the quercetin group was significantly reduced (P<0.05, Figure 2 A). In the righting reflex test, HIE rats required longer time to right themselves compared with the sham-operated group (P<0.05, Figure 2 B). However, the righting time in the quercetin group was significantly shorter than that in the HIE group (P<0.05, Figure 2 B). Data are expressed as mean ± standard error, n = 3 per group. *P < 0.05 control group, #P < 0.05 HIE+ control group.

[0060] 13. Quercetin can reduce infarct size and cerebral edema after HIE;

[0061] TTC staining showed that the infarct area in the HIE group was significantly increased by 30.25% compared with the sham group (P < 0.05, Figure 3In contrast, the infarct size in the quercetin group was significantly reduced to 12.32% compared with the HIE group (P < 0.05, Figure 3 In addition, the brain water content in the HIE group was significantly increased compared with the sham group (P<0.05), whereas the brain water content in the quercetin group was significantly decreased compared with the HIE group (P<0.05, Figure 3 C).

[0062] Chemiluminescence detection was performed using a chemiluminescence kit, and bands were quantified using ImageJ. (A) Representative TTC-stained images from the sham, HIE+Vehicle, and HIE+QE groups. (B) Statistical analysis of infarct size in the sham, HIE+Vehicle, and HIE+QE groups. (C) Statistical analysis of brain water content in the sham, HIE+Vehicle, and HIE+QE groups. Data are presented as mean ± standard error, n = 3 per group. *P < 0.05 vs. sham group, #P < 0.05 vs. HIE+Vehicle group.

[0063] 14. Quercetin improves lymphatic system dysfunction after HIE;

[0064] The results of intraperitoneal injection of Evans blue dye in rats showed that the transport efficiency of dye in brain tissue of sham operation group was higher ( Figure 4 A, 4B, indicating an intact lymphatic system. In contrast, the transport efficiency of the HIE group was significantly reduced (P < 0.05, Figure 4 A, 4B), indicating glymphatic system damage. Compared with the HIE group, the quercetin group showed significantly improved glymphatic system dysfunction in HIE rats (P < 0.05, Figure 4 A, Representative images show the extent of Evans blue dye diffusion in brain tissue from the sham, HIE+Vehicle, and HIE+QE groups. (B) Statistical analysis of Evans blue dye diffusion in brain tissue from the sham, HIE+Vehicle, and HIE+QE groups. Data are presented as mean ± standard error, n = 3 per group. *P < 0.05 vs. sham group, #P < 0.05 vs. HIE+Vehicle group.

[0065] 15. Quercetin enhances the polarized expression of AQP4 after HIE;

[0066] Immunofluorescence double-labeling method was used to evaluate the expression of GFAP and AQP4 in the brain. In the sham operation group, AQP4 was mainly located at the foot of astrocytes. Compared with the sham operation group, at 24 hours after HIE, AQP4 was displaced from the end of astrocytes to the cell body, and the polarized expression around the cortex was significantly reduced, with an increase in the R score. On the contrary, compared with the HIE group, quercetin after HIE led to the localization of AQP4 at the end of astrocytes, enhanced the polarization of cortical AQP4, and reduced the R score.

[0067] Colocalization of AQP4 and GFAP in the sham operation group, HIE+Vehicle group and HIE+QE group. Scale bar = 100 μm. The rightmost column shows the Pearson correlation coefficient (denoted as R) between AQP4 and GFAP (0 < R < 1, the higher the R value, the stronger the colocalization). The number of samples in each group n = 3.

[0068] 16. Quercetin reduces the expression levels of AQP4 and GFAP proteins in HIE;

[0069] Compared with the sham operation group, the expression levels of AQP4 and GFAP proteins were significantly increased at 24 hours after HIE (P < 0.05, Figure 6 A, 6B, 6C). However, in the quercetin treatment group, the expression level of GFAP protein was significantly compared with the HIE group, and the expression level of AQP4 protein was significantly reduced (P < 0.05, Figure 6 A, 6B), and was also significantly reduced (P < 0.05, Figure 6 A, 6C). After HIE. (A) Representative Western blot bands of AQP4 and GFAP in the sham operation, HIE+Vehicle and HIE+QE groups. (B) Sham operation, HIE+vehicle, and HIE+QE groups. (C) Statistical analysis of AQP4 in the sham operation group, HIE+vehicle and HIE+QE groups. Data are presented as mean ± SEM, n = 3 for each group. *P < 0.05 compared with the sham operation group, #P < 0.05 compared with the HIE+Vehicle group.

[0070] 17. Discussion;

[0071] Studies have found that the glymphatic system plays a key role in post-stroke pathological responses, particularly in reducing edema, inflammation, and cell death. After stroke, the efficiency of the glymphatic system in clearing metabolic waste from the brain parenchyma is significantly reduced, leading to the accumulation of toxic metabolites and proteins, exacerbating neuronal damage and neuroinflammation. Therefore, understanding how to restore the glymphatic system is crucial for treating HIE. This study established a rat HIE model and evaluated the effects of quercetin using neurological function scores, TTC staining, brain water content measurements, and Evans blue staining. Cisternaria injection, double immunofluorescence, and Western blot analysis were also performed. Results showed that intraperitoneal administration of quercetin after HIE improved neurological deficits, reduced infarct size, decreased brain water content, alleviated post-HIE brain edema, reduced the expression of AQP4 and GFAP, and improved glymphatic system dysfunction and AQP4 polarity expression. This suggests that quercetin has a neuroprotective effect in this HIE model, consistent with Cardozo's findings. This study investigated the effects of quercetin on the polarity distribution of AQP4 and the cerebrospinal fluid (CSF) system. Results showed that quercetin improved the ischemic-hypoxic state of rats, enhancing the polarity expression of AQP4 in the brain and improving the transport function of the CSF system. AQP4 plays a key role in the brain, particularly in regulating the function of the CSF system. Water molecules move freely between cells through AQP4, maintaining a balance of water in brain tissue. After stroke, this balance is disrupted, leading to increased edema and accumulation of metabolic waste, further damaging brain tissue. Studies have found that the expression of AQP4 and GFAP is negatively correlated with cerebrospinal fluid permeability. By regulating the changes in AQP4 polarity or expression after cerebral ischemia, quercetin can promote cerebrospinal fluid flow, help clear metabolic waste, reduce brain edema, and contribute to the recovery of damaged glymphatic system function.

[0072] In conclusion, this study demonstrated that quercetin can reduce glymphatic system and neural damage and alleviate brain edema after HIE, and its mechanism of action may be related to quercetin enhancing the polarity expression of AQP4.

[0073] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A method for verifying the effect of quercetin on the lymphatic system and neural function of HIE neonatal rats, comprising the following steps: a. obtaining neonatal rats for experiment and establishing an HIE model; b. preparing a quercetin working solution; characterized in that ； c. After injecting quercetin working solution into rats in the HIE model, neurological function was assessed, and the rat brains were removed, sliced, and stained with TTC, and the percentage of infarct area was calculated. Brain water content was also tested to verify the effect of quercetin on glymphatic system function in HIE. d. After injecting quercetin working solution into rats in the HIE model, brain tissue was collected and brain slices were prepared. The slices were observed under a fluorescence microscope, and the colocalization coefficient of GFAP and AQP4 was determined using ImageJ software. Evans blue was then injected into the cerebral ventricles of newborn rats to assess the diffusion of cerebrospinal fluid (CSF). The rat hearts were collected for processing and testing; and the effects of quercetin on the lymphatic system function and AQP4 polarity in HIE were verified.

2. The method for verifying the effect of quercetin on the lymphatic system and neural function of HIE neonatal rats according to claim 1, characterized in that: In step a, when establishing the HIE model, 10-day-old rats were anesthetized with isoflurane and placed in the supine position; the hair was shaved to expose the anterior neck area; the right common carotid artery was isolated and double-ligated, then cut between the ligature points, and finally the incision was sutured; the time from the start of anesthesia to the end of the operation was controlled within 8 minutes; 1 hour after the operation, the rats were exposed to a hypoxic environment at 37°C for 2.5 hours; at the same time, newborn rats were used to set up a sham operation group, in which only the right common carotid artery was exposed, no ligation or cutting was performed, and no hypoxic treatment was given.

3. The method for verifying the effect of quercetin on the lymphatic system and neural function of HIE neonatal rats according to claim 1, characterized in that: In step b, quercetin was dissolved in 1 ml of dimethyl sulfoxide (DMSO) at a concentration of 30 mg / ml to prepare a stock solution; the stock solution was then diluted with 0.9% saline; and a working solution was prepared with saline to obtain a final concentration of 7.5 mg / ml.

4. The method for verifying the effect of quercetin on the lymphatic system and neural function of HIE neonatal rats according to claim 1, characterized in that: In step c, neurological function was assessed 24 hours after HIE using righting reflex and negative geotropism tests. For righting reflex, the rats were placed in the supine position, and the time required to change from supine to prone position was recorded. In the negative geotropism test, the rat is placed head-down on a 40-degree inclined surface, and the time required for it to turn and face upward is recorded; the maximum test time is 20 seconds. Times exceeding 20 seconds were recorded as 20 seconds.

5. The method for verifying the effect of quercetin on the lymphatic system and neural function of HIE neonatal rats according to claim 1, characterized in that: In step c, TTC staining was performed, including: 24 hours after HIE, anesthetizing the rats with isoflurane and perfusing them transcardially with 0.01 M phosphate buffered saline (PBS); removing the brains and cutting them into 2 mm coronal sections; staining the sections with 2% TTC; treating for 5 minutes, then washing with PBS and photographing; ImageJ software was used to analyze the non-infarct area of the ipsilateral hemisphere and the total area of the contralateral hemisphere. The percentage of infarct area in each section was calculated using the following formula: [(total area of the contralateral hemisphere)-(non-infarct area of the ipsilateral hemisphere)] / (total area of the contralateral hemisphere × 2). The average percentage of infarct area in each brain section of each animal was used to represent the percentage of infarct area in that animal.

6. The method for verifying the effect of quercetin on the lymphatic system and neural function of HIE neonatal rats according to claim 1, characterized in that: In step c, brain water content was detected. 24 hours after HIE, rats were deeply anesthetized and brain tissue was collected. The wet weight of the tissue was measured and then dried in an oven at 95°C for 72 hours to obtain the dry weight. The brain water content was calculated using the formula [(wet weight - dry weight) / wet weight] × 100%.

7. The method for verifying the effect of quercetin on the lymphatic system and neural function of HIE neonatal rats according to claim 1, characterized in that: In step d, 24 hours after HIE, rats were anesthetized with isoflurane and perfused through the heart with 0.01 M PBS and 4% paraformaldehyde. Brain tissue was collected and fixed in 4% paraformaldehyde overnight, followed by gradient dehydration in 10%, 20%, and 30% sucrose solutions until sedimentation. Brain sections (10 μm) were prepared using a freezing microtome and then incubated with 5% bovine serum albumin at room temperature for 1 hour, followed by incubation with primary antibodies at 4°C overnight. The primary antibodies used included mouse anti-AQP4 monoclonal antibody (1:100) and rabbit anti-GFAP monoclonal antibody (1:50). After washing with PBS, the sections were incubated with the corresponding fluorescent secondary antibodies for 1 hour. DAPI was used to stain the nuclei, and the sections were observed under a fluorescence microscope. The colocalization coefficient of GFAP and AQP4 was determined using ImageJ software.

Images