Use of AAV virus in preparing medicine for treating brain trauma

Abstract

The present invention relates to a new treatment method for promoting the recovery of neurological function in the subacute phase after brain injury, and specifically to the use of an AAV virus in the preparation of a drug for treating brain trauma, which can be applied to rehabilitation treatment in the subacute phase after brain trauma.

Description

Technical Field

[0001] This invention relates to a novel treatment method for promoting the recovery of neurological function in the subacute phase after brain injury. Specifically, it relates to the use of an AAV virus in the preparation of a drug for treating traumatic brain injury, which can be applied to rehabilitation treatment in the subacute phase after traumatic brain injury. Background Technology

[0002] Traumatic brain injury (TBI) is a disease with high mortality and morbidity, disrupting the homeostasis of the central nervous system (CNS) and causing primary and secondary damage, leading to severe neurological dysfunctions such as severe motor, psychological, and cognitive impairments. Therefore, most TBI survivors experience long-term disabling changes in cognition, sensorimotor function, and personality. Currently, rehabilitation training is the main treatment for the persistent damage caused by TBI; however, it is time-consuming and expensive, and its effectiveness is limited. Therefore, there is an urgent need to explore more effective treatments for TBI. Astrocytes are known to be glial cells that specifically maintain brain homeostasis. In the response to brain injury, astrocytes transition from a quiescent state to a reactive state, potentially playing a passive or active role. However, currently, there is a lack of treatments targeting astrocytes to promote neurological recovery after traumatic brain injury.

[0003] T-cell death-associated gene 8 (TDAG8, also known as GPR65) is a proton-sensitive G protein-coupled receptor widely expressed in brain tissue. Activated TDAG8 is involved in a wide range of biological processes, including neuroinflammation, cellular immunity, and apoptosis. In recent years, TDAG8 has been confirmed to be expressed in brain tissue, but reports on its function in the brain are still limited. Recent studies have indicated that TDAG8 is a potential neuroprotective target during the acute and subacute phases of cerebral ischemia. However, whether this target is an important therapeutic target for neurorepair after TBI remains unreported.

[0004] Studies have shown that in the subacute phase of cerebral frostbite (cTBI, an animal model of traumatic brain injury), transient daily CO2 inhalation treatment can enhance axonal regeneration and synaptic remodeling in the peri-infarct area, inhibit glial scar formation and microglial activation, thereby promoting the recovery of neurological function in mice. However, the therapeutic window of this CO2 inhalation treatment is 3-7 days after cTBI in mice, and there are currently no methods to further extend the therapeutic window of CO2 inhalation treatment and enhance its therapeutic effect. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide the use of AAV virus in the preparation of drugs for treating traumatic brain injury, in order to solve the problems in the prior art.

[0006] To achieve the above objectives and other related objectives,

[0007] In a first aspect, the present invention provides the use of an AAV virus in the preparation of a medicament for treating traumatic brain injury.

[0008] In one embodiment of the present invention, the traumatic brain injury is either in the subacute phase or the chronic phase of traumatic brain injury.

[0009] In one embodiment of the present invention, the AAV virus is used in the preparation of a medicament for the treatment of post-traumatic brain injury rehabilitation.

[0010] In one embodiment of the present invention, the AAV virus is AAV-GFAP-TDAG8 virus (HBAAV2 / 9-GFAP-m-GPR65-3xflag-EGFP, designed and manufactured by Shanghai Hanheng Biotechnology Co., Ltd., China).

[0011] In one embodiment of the present invention, the AAV-GFAP-TDAG8 virus is used in the preparation of a medicament to enhance the therapeutic effect of CO2 inhalation after traumatic brain injury.

[0012] In one embodiment of the present invention, the AAV-GFAP-TDAG8 virus is used in the preparation of a medicament for prolonging the time window of CO2 inhalation therapy after traumatic brain injury.

[0013] This invention utilizes TDAG8 knockout (TDAG8 - / - In mice using the classic brain injury model—controlled cortical impact (CCI)—TDAG8 was confirmed to be a key target for CO2 inhalation treatment in promoting neurological function recovery after traumatic brain injury. Furthermore, by injecting the adeno-associated virus vector AAV-GFAP-TDAG8 (HBAAV2 / 9-GFAP-m-GPR65-3xflag-EGFP, designed and manufactured by Shanghai Hanheng Biotechnology Co., Ltd., China) containing the TDAG8 gene, overexpression of TDAG8 in astrocytes surrounding infarcts further extended the CO2 treatment time window from 7 days to 14 days and significantly enhanced the therapeutic effect. This invention proposes a novel method for promoting neurological function recovery after traumatic brain injury by targeting astrocytes. This combination of endogenous protective strategies and therapeutic target overexpression holds promise for providing new treatment options for patients in the subacute to chronic phases of traumatic brain injury. Attached Figure Description

[0014] Figure 1 To verify that TDAG8 is a key target for CO2 inhalation treatment, line plots of the sliding rate of mice in each group were used. *P<0.05 vs I-WT mice CCI group; #P<0.05 vs I-TDAG8- / - Group.

[0015] Figure 2 To verify that TDAG8 is a key target for CO2 inhalation treatment, line plots of rotarod drop latency were used in different groups of mice. *P<0.05 vs I-WT mice CCI group; #P<0.05 vs I-TDAG8 - / - Group.

[0016] Figure 3 To verify that TDAG8 is a key target for CO2 inhalation treatment, line plots of mNSS scores were used in each group of mice. *P<0.05 vs I-WT mouse CCI group; #P<0.05 vs I-TDAG8 - / - Group.

[0017] Figure 4 Immunofluorescence typical images (scale bar: 40 μm) of the number of GFAP cells in the cerebral cortex of mice in each group in an experiment to verify that TDAG8 is a key target of CO2 inhalation treatment.

[0018] Figure 5 The graph shows the number of GFAP cells in the cerebral cortex of mice in each group during the CO2 inhalation treatment key target experiment. *P<0.05, ***P<0.001.

[0019] Figure 6 Western blot image validating AAV virus expression in an exploratory experiment to explore the beneficial role of CO2 in activating astrocyte-derived TDGA8.

[0020] Figure 7 Map of AAV virus localization in an exploratory experiment to explore the beneficial role of CO2 in activating astrocyte-derived TDGA8. (Scale bar: 40 μm)

[0021] Figure 8 Line graphs showing the sliding rate of mice in each group during an exploratory experiment to explore the beneficial effects of CO2 inhalation treatment on activated astrocyte-derived TDGA8. *P<0.05, ***P<0.001 vs positive virus CCI group; #P<0.05, ###P<0.001 vs negative virus CO2 group.

[0022] Figure 9 Line graphs of mNSS scores in mice in each group during an exploratory experiment to explore the beneficial effects of CO2 inhalation treatment on TDGA8 derived from astrocytes. ***P<0.001 vs positive virus CCI group; ###P<0.001 vs negative virus CO2 group.

[0023] Figure 10Immunofluorescence typical image of the number of GFAP cells in the cerebral cortex of mice in each group during an exploratory experiment to explore the beneficial role of CO2 in activating astrocyte-derived TDGA8. (Scale bar: 40 μm)

[0024] Figure 11 A statistical graph showing the number of GFAP cells in the cerebral cortex of mice in each group during an exploratory experiment to explore the beneficial role of CO2 in activating astrocyte-derived TDGA8. **P<0.01, ***P<0.001.

[0025] Figure 12 mRNA expression of proton-sensitive G protein-coupled receptor TDAG8. *P<0.05, **P<0.01 vs. control group.

[0026] Figure 13 Line plots showing the slippage rate for the II-CCI group, IIA-CO2 inhalation treatment group, and IIB-CO2 inhalation treatment group. *P<0.05,**P<0.01,***P<0.001 vs II-CCI group; ##P<0.01,###P<0.001 vs IIA-CO2 group.

[0027] Figure 14 Line graphs of mNSS scores for the II-CCI group, IIA-CO2 inhalation treatment group, and IIB-CO2 inhalation treatment group. **P<0.01,***P<0.001 vs II-CCI group; #P<0.05,##P<0.01 vs IIA-CO2 group.

[0028] Figure 15 Line plots showing the slip rate of the III-negative virus control group, III-positive virus control group, III-negative virus CCI group, III-positive virus CCI group, III-negative virus CO2 inhalation treatment group, and III-positive virus CO2 inhalation treatment group. **P<0.01,***P<0.001 vs III-CCI group; #P<0.05,##P<0.01 vs III-negative virus CO2 group.

[0029] Figure 16 Line graphs of mNSS neurological function scores for the III-negative virus control group, III-positive virus control group, III-negative virus CCI group, III-positive virus CCI group, III-negative virus CO2 inhalation treatment group, and III-positive virus CO2 inhalation treatment group. *P<0.05, **P<0.01, ***P<0.001 vs III-CCI group; ##P<0.01, ###P<0.001 vs III-negative virus CO2 group.

[0030] Figure 17 Typical diagrams of neuronal tree mutations (Golgi staining) in mice of the III-negative virus control group, III-positive virus control group, III-negative virus CCI group, III-positive virus CCI group, III-negative virus CO2 inhalation treatment group and III-positive virus CO2 inhalation treatment group (scale bar: 40 μm).

[0031] Figure 18 Schematic diagram of changes in neuronal dendritic complexity in mice in the III-negative virus control group, III-positive virus control group, III-negative virus CCI group, III-positive virus CCI group, III-negative virus CO2 inhalation treatment group, and III-positive virus CO2 inhalation treatment group (scale bar: 40 μm).

[0032] Figure 19 Statistical graph of neuronal dendritic complexity in mice of the III-negative virus control group, III-positive virus control group, III-negative virus CCI group, III-positive virus CCI group, III-negative virus CO2 inhalation treatment group and III-positive virus CO2 inhalation treatment group. Detailed Implementation

[0033] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0034] An exploratory experiment on the synergistic treatment of traumatic brain injury with CO2 inhalation therapy and AAV virus therapy.

[0035] I. Methods

[0036] 1. CCI Model Establishment

[0037] This experiment used C57 / BL6 wild-type (WT) mice (purchased from the Experimental Animal Center of Shanxi Medical University) and TDAG8 mice. - / - Mice (purchased from Beijing Biocytogen Pharmaceutical Technology Co., Ltd.) were bred at the Experimental Animal Center of Shanxi Medical University. Male WT mice aged 8-10 weeks and TDAG8 mice were used... - / -Mouse brain injury was simulated using a cortical impaction (CCI) model, employing a controlled impaction device (68099Ⅱ precision impaction device, Shenzhen RWD Life Sciences, China). Mice were generally anesthetized via intraperitoneal injection of 4% sodium pentobarbital (45 mg / kg). A circular area with a diameter of 3 mm was marked with Bregma's point as the origin, centered 2 mm to the right and 0.5 mm forward, and a circular craniotomy was performed. Impact parameters were set as follows: velocity 5 m / s, residence time 500 ms, depth 1.2 mm, using a 3 mm diameter impactor for CCI modeling. The sham-operated group received the same surgical procedure but without CCI. Bone windows were sealed with bone wax (Johnson & Johnson, USA), and the wound was sutured. A heating pad was used to maintain the mice's body temperature at 36-37°C until full awakening.

[0038] 2. CO2 Inhalation Treatment Methods

[0039] The CO2 inhalation treatment method was consistent throughout this study, implemented in a closed chamber (1100 mL) using a mixed gas (10% CO2, 21% O2 supplemented with N2) at a flow rate of 1 L / min instead of air. Mice were given 10% CO2 inhalation daily starting 3 or 14 days after CCI, with specific parameters of 10 minutes of inhalation followed by 10 minutes of rest, repeated three times. Furthermore, the number of days of CO2 inhalation treatment varied depending on the experimental setup.

[0040] 3. Balance Beam Experiment

[0041] Three days before CCI modeling, mice underwent adaptive training, enabling them to independently traverse a 100cm long, 0.8cm diameter balance beam (30cm above ground) without slipping. This training was conducted three times daily. A soft pad was placed under the balance beam to prevent injury from slipping. Motor function was evaluated by performing balance beam tests on mice at 3, 7, 14, 21, and 28 days after CCI. The total number of steps and the number of slips in the left hind limb were recorded in three repeated experiments, with 5-minute intervals. The slip rate was calculated as (slips / total steps) × 100%. If some mice were unable to traverse the balance beam due to severe motor dysfunction, their slip rate was recorded as 100%. After each data collection, the balance beam was wiped with 75% alcohol, and data collection was conducted at the same time period each time.

[0042] 4. mNSS neurological function score

[0043] Mice in each group were scored using the mNSS by researchers familiar with the scoring criteria but not from the experimental group, to determine the degree of neurological impairment in rats. Motor tests included the tail-lifting test (1 point each for forelimb flexion, hindlimb flexion, and head deviation from the vertical axis >10° within 30 seconds during tail lifting) and the walking test (0 points for normal walking, 1 point for inability to walk in a straight line, 2 points for turning towards the side of mild paralysis, and 3 points for falling towards the side of mild paralysis); sensory tests included visual / tactile tests (1 point for abnormalities) and proprioceptive tests (1 point for abnormalities); the balance beam test was divided into 7 levels: stable balance posture, gripping the edge of the balance beam, clinging to the balance beam with one limb and falling from the balance beam, clinging to the balance beam with two limbs and falling from the balance beam or rotating on the balance beam (>60s), attempting to balance on the balance beam but falling (>40s), attempting to balance on the balance beam but falling (>20s), and not attempting to balance on the balance beam but falling, scored from 0 to 6 points respectively. The reflex test included 1 point for the absence of the auricular reflex, 1 point for the absence of the corneal reflex, 1 point for the absence of the startle reflex, and 1 point for the presence of epilepsy, myoclonus, or dystonia. The maximum score was 18 points, with 1-6 points indicating mild impairment, 7-12 points indicating moderate impairment, and 13-18 points indicating severe impairment. Mice were examined 3 days before CCI and at 3, 7, 14, 21, and 28 days after CCI.

[0044] 5. Fatigue Rotation Test

[0045] The rotating bar test is used to assess balance and motor coordination. Mice underwent four consecutive accelerated tests (from 10 to 30 rpm) three days prior to CCI to record baseline latency, followed by a slow test (20 rpm) to familiarize them with the test. Data were collected at 3, 7, and 14 days after CCI. During the test, mice were placed on a rotating bar, which was accelerated from 4 revolutions to 40 revolutions within 5 minutes, and the time it took for the mouse to fall was recorded. The test was repeated three times, with 10-minute intervals between each test. Each test lasted a maximum of five minutes, or until the mouse fell off the bar.

[0046] 6. AAV virus injection

[0047] The AAV viruses injected in this experiment included AAV-GFAP-control negative virus (HBAAV-2 / 9-GFAP-EGFP) and AAV-GFAP-TDAG8 positive virus (HBAAV2 / 9-GFAP-m-GPR65-3xflag-EGFP). After anesthesia with sodium pentobarbital (45 mg / kg, ip), WT and TDAG8 were... - / -Male C57BL / 6 mice were general anesthetized. The mice were fixed in a stereotaxic apparatus, and the scalp was cut approximately 2 cm along the midline of the brain to fully expose the skull. The skull surface was wiped dry with sterile cotton swabs. Referring to the mouse brain stereotaxic atlas, three injection sites for the AAV carrier in the right brain parenchyma were precisely marked, using the Bregma (anterior fontanelle) as the origin: (0.5 mm right anterior fontanelle, 1 mm anterior, 1.5 mm depth), (0.5 mm right anterior fontanelle, 0.5 mm posterior, 1.25 mm depth), and (3.3 mm right anterior fontanelle, 0.3 mm posterior, 1.8 mm depth). A cranial drill was then used to drill holes at these sites. The AAV carrier was injected into the brain parenchyma using a microinjection pump at a rate of 0.1 μl / min, with an injection volume of 1 μl per site. After 10 minutes of injection at each site, the needle was allowed to remain in place for 10 minutes before being slowly withdrawn. Finally, suture the scalp and disinfect it. Remove the mouse from the adapter and use a heating pad to maintain the mouse's body temperature at 36-37°C until it is fully awake.

[0048] 7. Tissue immunofluorescence

[0049] Three weeks after injection of AAV viral vector, a CCI model was constructed in mice. Two weeks after CCI, brain tissue was perfused and collected, then fixed in 4% paraformaldehyde and dehydrated in a gradient of 15% and 30% sucrose solutions until the brain tissue was completely settled. The brain tissue was cut into a series of 20 μm thick coronal sections using a cryostat. The brain sections were repaired with sodium citrate buffer (0.01 M, pH 6.0) at 80 °C for 30 min, permeated with 10% Triton X-100 for 30 min, blocked with 10% donkey serum at room temperature for 2 h, and incubated overnight at 4 °C with primary antibodies rabbit anti-NeuN (1:200, Sigma, Abn78), rabbit anti-GFAP (1:2000, Abcam, Ab278054), and goat anti-Iba-1 (1:400, Novus Biologicals, NB100-1028). Subsequently, the slides were incubated with the corresponding secondary antibodies, donkey anti-rabbit IgG Alexa Fluor 594 (1:400, Yeasen, 34212ES60) and donkey anti-sheep IgG Alexa Fluor 594 (1:400, Yeasen, 34312ES60), at room temperature for 2 hours. Finally, they were stained with 4′,6-diamino-2-phenylindole (DAPI) solution and incubated at room temperature for 10 minutes, then mounted with anti-fluorescence attenuation mounting medium. Fluorescence signals in the peri-infarct region were captured using a Leica fluorescence microscope to observe the colocalization of the AAV virus's EGFP green fluorescence signal with the red fluorescence signals of NeuN, GFAP, and Iba-1, as well as changes in the number of astrocytes in the peri-infarct region.

[0050] 9. Western blot

[0051] Three weeks after AAV vector injection in mice, tissue samples were collected. Mice were euthanized by cervical dislocation, and the brains were quickly decapitated. Cortical tissue from the AAV-injected region was separated. The tissue was lysed in RIPA lysis buffer containing protease and phosphatase inhibitors. Equal volumes of proteins were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated with 5% skim milk at room temperature for 2 hours, then placed on primary antibodies (mouse anti-flag, 1:500, Cell Signaling, 8146) and rabbit anti-GAPDH, 1:6000, Bioworld, Ap0063, and incubated overnight at 4°C. Subsequently, it was incubated with goat anti-mouse (1:5000, Boster, Ba1050) and goat anti-rabbit (1:5000, Boster, Ba1054) horseradish peroxidase (HRP) secondary antibodies at room temperature for 2 hours. Visualize TDAG8-flag or GAPDH immunoblot images using the ECL hypersensitive chemiluminescence kit on the Bio-Rad imaging system.

[0052] 10. Golgi staining

[0053] Golgi staining was performed using the FD Rapid Golgi Astain Kit (PK401). Mice euthanized 28 days after CCI by cervical dislocation, and brain tissue was rinsed and fixed in a solution of equal proportions of Solution A and Solution B at room temperature in the dark for 2 weeks. The tissue was then transferred to Solution C and fixed for 3 days at room temperature in the dark. After rapid freezing with dry ice and isopentane, 100 μm thick sections were cut and fixed for 3 days at room temperature in the dark. Sections were rinsed with Milli-Q water and placed in a mixture of Solution D, Solution E, and Milli-Q (mixed in a 1:1:2 ratio). Sections were rinsed again with Milli-Q water and dehydrated using graded ethanol (50%-100%). The sections were cleared with xylene and mounted with neutral resin. Sections were photographed using a Leica fluorescence microscope, and neuronal branch complexity was calculated using ImageJ.

[0054] II. Experimental Design

[0055] 1. Experiments to investigate the beneficial effects of CO2 inhalation treatment after CCI and to verify that TDAG8 is a key target for CO2 inhalation treatment.

[0056] WT mice and TDAG8 - / - After CCI modeling, mice were divided into I-WT mouse CCI group and I-TDAG8 group. - / -Mouse CCI group, I-WT mouse CO2 inhalation treatment group and I-TDAG8 - / - Mouse CO2 inhalation treatment groups, including I-WT mice CO2 inhalation treatment group and I-TDAG8 mice. - / - Mice in the CO2 inhalation treatment group were treated with 10% CO2 inhalation daily for 3-14 days post-injury (10% CO2, 10′ / 10′*3), three times a day, for 10 minutes each time, followed by a 10-minute rest. Balance beam tests, mNSS neurological scores, and fatigue rotation tests were performed on days 3, 7, and 14 post-injury to assess the recovery of motor function. Tissue samples were collected from all animals 14 days after CCI. GFAP immunofluorescence staining was used to observe changes in the number of astrocytes in the peri-infarct area. The astrocyte density in the proximal and distal infarct regions of the peri-infarct area was statistically analyzed to clarify the role of CO2 inhalation treatment in promoting motor function recovery after CCI, and to identify TDAG8 as a key target for the beneficial effects of CO2 inhalation treatment.

[0057] 2. An exploratory experiment on the beneficial effects of CO2 inhalation treatment on astrocyte-derived TDGA8.

[0058] Three weeks after injection of either the negative virus (AAV-GFAP-Control) or the positive virus (AAV-GFAP-TDAG8), brain tissue was harvested, and Western blot analysis was used to verify whether TDAG8 was successfully overexpressed. - / - Mouse cerebral cortex tissue. Simultaneously, to verify whether TDAG8 is specifically overexpressed in astrocytes, a CCI model was constructed 3 weeks after AAV viral vector injection. Brain tissue was harvested 14 days post-injury, and frozen sections were then subjected to immunofluorescence staining to observe the co-localization of cells successfully expressing the AAV viral vector (i.e., EGFP-positive cells) with neuronal marker NeuN, microglia marker Iba-1, and astrocyte marker GFAP.

[0059] To observe the recovery of TDAG8 using the positive virus (AAV-GFAP-TDAG8) - / - Whether the expression of TDAG8 in astrocytes in the peri-infarct region of mouse cerebral infarction can restore the therapeutic effect of CO2 inhalation treatment was investigated. - / -Mice were randomly divided into six groups: a negative virus control group (AAV-GFAP-Control), a negative virus CCI group (AAV-GFAP-Control CCI), and a negative virus CO2 group (AAV-GFAP-Control CO2); a positive virus control group (AAV-GFAP-TDAG8), a positive virus CCI group (AAV-GFAP-TDAG8CCI), and a positive virus CO2 group (AAV-GFAP-TDAG8 CO2). The negative and positive virus CO2 groups received daily CO2 inhalation treatment starting 3 days after CCI and continuing until day 14. The daily treatment parameters were: 10% CO2 inhalation for 10 minutes, followed by a 10-minute rest period, repeated three times. Balance beam and mNSS neurological function scores were assessed at days 3, 7, and 14 after CCI to evaluate the recovery of motor function in each group. Fourteen days after CCI, tissue samples were collected from each group of animals, and GFAP immunofluorescence staining was used to observe the changes in the number of astrocytes in the peri-infarction area. The changes in the number of GFAPs near and far from the injury area were counted, and the effect of CO2 inhalation treatment on TDAG8 activation of astrocytes was evaluated.

[0060] 3. Exploratory experiment on the therapeutic time window of CO2 inhalation treatment

[0061] In WT mice, TDAG8 mRNA expression in the peri-infarct area was detected at 1, 3, 7, 14, and 28 days after CCI. Based on the time-series changes in TDAG8 expression, the following experimental protocol was developed: WT mice were randomly divided into three groups: II-CCI group, IIA-CO2 inhalation treatment group, and IIB-CO2 inhalation treatment group. The treatment duration for the IIA-CO2 inhalation treatment group was 3-28 days after CCI. The treatment duration for the IIB-CO2 inhalation treatment group was 14-28 days after CCI. Mice in all groups underwent the balance beam test and mNSS neurological function score assessment at 3, 7, 14, 21, and 28 days after CCI to evaluate the recovery of motor function and determine the treatment time window.

[0062] Furthermore, WT mice were randomly divided into 6 groups: III-negative virus control group, III-negative virus CCI group, III-negative virus CO2 group; III-positive virus control group, III-positive virus CCI group, and III-positive virus CO2 group. One week before CCI, WT mice were injected with AAV virus into the brain parenchyma. Starting 14 days after CCI, daily CO2 inhalation treatment was administered until 28 days. The balance beam test and mNSS neurological function score were performed at 3, 7, 14, 21, and 28 days after CCI to assess the recovery of motor function, in order to determine whether overexpression of TDAG8 on astrocytes could prolong the therapeutic window of CO2 inhalation treatment.

[0063] All animals were sampled 28 days after CCI, and the number of astrocytes in the peri-infarct area was observed using GFAP immunofluorescence staining. Golgi staining was also used to observe the neuronal branching complexity in the peri-infarct area.

[0064] result

[0065] 1. The beneficial effects of CO2 inhalation after CCI, and validation that TDAG8 is a key target for CO2 inhalation treatment.

[0066] We use TDAG8 - / - We used mice to investigate whether TDAG8 is associated with the therapeutic effect of CO2 inhalation treatment. First, we assessed how CCI mice regained their motor function, specifically as follows: Figures 1-3 , Figure 1 To verify that TDAG8 is a key target for CO2 inhalation treatment, line graphs of the sliding rate of mice in each group were used in the experiment. Figure 2 To verify that TDAG8 is a key target for CO2 inhalation treatment, line graphs showing the rotarod drop latency of mice in each group were used in the experiment. Figure 3 To verify that TDAG8 is a key target for CO2 inhalation treatment, line graphs of mNSS scores were used to analyze the mice in each group.

[0067] We found that, compared with the CCI group in WT mice, CO2 inhalation treatment in WT mice significantly reduced the rate of slippage in the left hind limb after injury. Figure 1 This increases the latency period for the spinner to fall. Figure 2 ), improved mNSS neurological function score ( Figure 3 This indicates that CO2 inhalation treatment can alleviate motor dysfunction after CCI in WT mice. However, this therapeutic effect was not observed in TDAG8 mice. - / - No results were observed in mice. Therefore, these results demonstrate that TDAG8 is a key target for the therapeutic effect of CO2 inhalation treatment after CCI.

[0068] To determine how CO2 inhalation exerts its beneficial effects by activating TDAG8, we collected brain tissue samples 14 days after CCI and performed immunofluorescence staining with an antibody against the astrocyte marker GFAP to identify reactive astrocytes. Figure 4 To verify that TDAG8 is a key target for CO2 inhalation treatment, we observed immunofluorescence typical images of the number of GFAP cells in the cerebral cortex of mice in each group during the experiment. We found that 14 days after CCI, the GFAP-positive area in the peri-infarct region was larger than the corresponding area in the control group. Figure 5 This is a statistical graph showing the number of GFAP cells in the cerebral cortex of mice in each group. In WT mice, CO2 inhalation significantly reduced the number of GFAP-positive astrocytes 14 days after injury, but in TDAG8 mice...- / - In mice, this effect was significantly inhibited. Therefore, CO2 inhalation treatment may regulate synaptic plasticity and neural network remodeling in the peri-infarct region by activating TDAG8 in astrocytes, which is beneficial for motor recovery and neural repair after CCI.

[0069] 2. CO2 inhalation exerts its therapeutic effect by activating TDAG8 on astrocytes.

[0070] Figure 6 Western blot images were used to verify the AAV virus overexpression in an experiment exploring the beneficial role of CO2 in activating astrocyte-derived TDGA8. Figure 7 AAV virus localization map in an exploratory experiment to explore the beneficial role of CO2 in activating astrocyte-derived TDGA8. Figure 8 Line graphs showing the glide rate of mice in each group during an exploratory experiment to explore the beneficial effects of CO2 inhalation treatment on TDGA8, which is derived from astrocytes. Figure 9 Line graphs of mNSS scores in mice from different groups in an exploratory experiment to explore the beneficial effects of CO2 inhalation treatment on TDGA8, which is derived from astrocytes.

[0071] pass Figure 6 We found that TDAG8-flag was successfully overexpressed in the AAV-GFAP-TDAG8 group, while no positive expression of this protein was observed in the AAV-GFAP-Control group. Figure 7 It is evident that the green fluorescent protein EGFP expressed by the AAV virus completely co-localizes with the astrocyte marker GFAP, but does not overlap with the microglia marker Iba-1 or the neuronal marker NeuN. Therefore, this AAV viral vector can selectively express TDAG8 on astrocytes. Figure 8 and Figure 9 Behavioral assessment revealed that the TDAG8 group of the positive viral CO2 group (AAV-GFAP-TDAG8 CO2) had TDAG8. - / - In mice, daily CO2 inhalation treatment starting 3 days after CCI significantly reduced their slippage rate and mNSS score at days 7 and 14 post-CCI. However, CO2 inhalation treatment had a negative effect on TDAG8 mice treated with negative viruses. - / - The study showed no significant improvement in the motor function of mice.

[0072] Furthermore, we examined the density of astrocytes in the proximal and distal segments of the infarct periphery. Figure 10 Immunofluorescence typical image of the number of GFAP cells in the cerebral cortex of mice in each group during an exploratory experiment to explore the beneficial role of CO2 in activating astrocyte-derived TDGA8. (Scale bar: 40 μm) Figure 11 A statistical graph showing the number of GFAP cells in the cerebral cortex of mice in each group during an exploratory experiment to explore the beneficial role of CO2 in activating astrocyte-derived TDGA8.

[0073] like Figure 10 and Figure 11 As shown, CO2 inhalation treatment significantly reduced TDAG8 levels after treatment with positive viruses. - / - Astrocyte density in mice 14 days after CCI, but for TDAG8 mice treated with negative virus. - / - No significant effect was observed in mice. These results further indicate that CO2 inhalation treatment promotes the recovery of neurological function after CCI in mice by activating TDAG8 in astrocytes and inhibiting its overactivation.

[0074] 3. Activation of TDAG8 on astrocytes can prolong the therapeutic window of CO2 inhalation.

[0075] To further test whether the treatment time window for CO2 inhalation could be further extended, we compared the effects of starting CO2 inhalation treatment 3 days and 14 days after CCI in WT mice. Figure 12 This shows the mRNA expression status of TDAG8. Figure 13 Line graphs showing the slip rate for the II-CCI group, IIA-CO2 inhalation treatment group, and IIB-CO2 inhalation treatment group. Figure 14 Line graphs of mNSS scores for the II-CCI group, IIA-CO2 inhalation treatment group, and IIB-CO2 inhalation treatment group.

[0076] By detecting the mRNA expression of TDAG8 after CCI ( Figure 12 We found that TDAG8 expression began to upregulate 1 day after CCI, peaked at 3-7 days, and then gradually decreased. While expression showed a trend of being higher than the control group at 14 days, the difference was not significant. Behavioral results showed that the group receiving CO2 inhalation treatment starting at 14 days had significantly lower neurological function recovery than the group receiving CO2 inhalation treatment starting at 3 days. Figure 13 and 14 Therefore, the underexpression of TDAG8 14 days after CCI may be the main reason for the poor efficacy of CO2 inhalation treatment starting 14 days after CCI.

[0077] To compensate for insufficient TDAG8 expression by overexpressing TDAG8 in astrocytes in the peri-infarct region 14 days after CCI, we injected WT mice with negative virus (AAV-GFAP-Control) and positive virus (AAV-GFAP-TDAG8) one week before CCI. We observed changes in motor function using the balance beam test and mNSS neurological function score to determine whether overexpression of TDAG8 in astrocytes could enhance the therapeutic effect of CO2 inhalation treatment and prolong its therapeutic window.

[0078] Figure 15 Line graphs showing the slip rate for the III-negative virus control group, III-positive virus control group, III-negative virus CCI group, III-positive virus CCI group, III-negative virus CO2 inhalation treatment group, and III-positive virus CO2 inhalation treatment group. Figure 16 Line graphs of mNSS neurological function scores for the III-negative virus control group, III-positive virus control group, III-negative virus CCI group, III-positive virus CCI group, III-negative virus CO2 inhalation treatment group, and III-positive virus CO2 inhalation treatment group.

[0079] The results showed that, compared with the III-negative virus CCI group, the III-negative virus CO2 inhalation treatment group showed no significant behavioral changes at 21 days post-CCI, and only slight improvement at 28 days. Meanwhile, compared with the III-positive virus CCI group, CO2 inhalation treatment starting 14 days post-CCI combined with AAV-GFAP-TDAG8 injection significantly reduced the slip rate and mNSS neurological function score on the balance beam test at 21 and 28 days post-CCI in mice, and this treatment effect was significantly stronger than that in the III-negative virus CO2 inhalation treatment group. Figure 15 and 16 Therefore, overexpression of TDAG8 on astrocytes can enhance the therapeutic effect of CO2 inhalation, promote motor function recovery after CCI, and extend its treatment window from 3-7 days to at least 14 days. Thus, injection of AAV-GFAP-TDAG8 virus into patients in the chronic phase of traumatic brain injury can improve the efficacy of CO2 inhalation treatment.

[0080] Figure 17 Typical neuronal tree mutations (Golgi staining) in mice of the III-negative virus control group, III-positive virus control group, III-negative virus CCI group, III-positive virus CCI group, III-negative virus CO2 inhalation treatment group, and III-positive virus CO2 inhalation treatment group. Figure 18This diagram illustrates the neuronal dendritic complexity of mice in the III-negative virus control group, III-positive virus control group, III-negative virus CCI group, III-positive virus CCI group, III-negative virus CO2 inhalation treatment group, and III-positive virus CO2 inhalation treatment group. Figure 19 Statistical graph of neuronal dendritic complexity in mice of the III-negative virus control group, III-positive virus control group, III-negative virus CCI group, III-positive virus CCI group, III-negative virus CO2 inhalation treatment group and III-positive virus CO2 inhalation treatment group.

[0081] After Golgi staining, we found that in the AAV-GFAP-TDAG8 group and the AAV-GFAP-Control group treated with CO2 inhalation, the AAV-GFAP-TDAG8 group showed better overall neuronal morphological recovery. Figure 17 ), with higher complexity ( Figure 18-19 Therefore, it can be concluded that overexpressing TDAG8 on astrocytes using AAV virus combined with CO2 inhalation treatment can better promote the recovery of neurological function and nerve repair in the subacute and chronic phases after CCI.

[0082] This study is the first to demonstrate that delayed CO2 inhalation treatment can promote neurological repair and improve neurological dysfunction after traumatic brain injury (CCI) by activating TDAG8 in astrocytes. Furthermore, overexpression of TDAG8 on astrocytes enhances the therapeutic effect of CO2 inhalation, promotes motor function recovery after CCI, and extends the treatment window from 3-7 days to at least 14 days. Therefore, AAV-GFAP-TDAG8 combined with CO2 inhalation therapy has a synergistic effect, extending the treatment window of CO2 inhalation treatment while also enhancing its therapeutic efficacy in the chronic phase of CCI.

[0083] In summary, AAV virus combined with CO2 inhalation therapy has a synergistic effect, and its combined use can further extend the therapeutic window of CO2 inhalation therapy. Therefore, this invention effectively overcomes the various shortcomings of the prior art and has high industrial application value.

[0084] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. Use of AAV-GFAP-TDAG8 virus in the preparation of a drug for enhancing the therapeutic effect of CO2 inhalation after brain trauma, wherein the AAV-GFAP-TDAG8 virus is an AAV-GFAP-TDAG8 virus that overexpresses TDAG8.

2. The use according to claim 1, characterized in that The AAV-GFAP-TDAG8 virus is used to prepare a drug for prolonging the CO2 inhalation treatment time window after brain trauma.

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