Use of peginterferon alfa-2b in the preparation of a medicament for treating traumatic optic neuropathy

Abstract

The application belongs to the technical field of biological medicine, and discloses application of plerixafor in preparation of a medicine for treating traumatic optic neuropathy. Researches of the application show that the plerixafor has a repairing effect of promoting survival of retinal ganglion cells (RGC) and axon regeneration after optic nerve injury, and can achieve and improve the effect of treating traumatic optic nerve injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of praxaf in the preparation of drugs for treating traumatic optic neuropathy. Background Technology

[0002] Traumatic optic neuropathy (TON) is a group of diseases caused by mechanical trauma to the craniofacial region, resulting in the disruption of axonal continuity and impaired transport function of retinal ganglion cells (RGCs) through biomechanical conduction, leading to irreversible damage to the visual pathway. Although its overall incidence is low (approximately 0.7%–2.5%), about 79%–85% of patients are young and middle-aged men, placing a significant burden on the workforce and families.

[0003] Current clinical treatment strategies for tonic-clonic optic nerve (TON) mainly include optic canal decompression surgery, high-dose glucocorticoid pulse therapy, and observational supportive pharmacological therapy. However, these methods are generally ineffective in reversing the disease progression, failing to achieve substantial regeneration of the rhabdomyocartilaginous (RGC) axons and repair of dendritic structures, resulting in poor visual function prognosis for patients. In recent years, emerging strategies such as stem cell transplantation and gene therapy have focused on functional reconstruction of the optic nerve in the mid-to-late stages, but their efficacy remains severely limited due to the rapid death of the RGC after injury and its inherently limited regenerative capacity.

[0004] Therefore, more comprehensive and effective solutions need to be developed to promote the repair and treatment of optic nerve damage. Summary of the Invention

[0005] This invention aims to at least solve one of the problems existing in the prior art. To this end, this invention proposes the application of praxaf in the preparation of a medicament for treating traumatic optic neuropathy. Research in this invention shows that praxaf has a repairing effect by promoting the survival of retinal ganglion cells (RGCs) and axonal regeneration after optic nerve injury, thus achieving and improving the therapeutic effect of treating traumatic optic nerve injury.

[0006] This invention provides the use of praxavir in the preparation of medicaments for treating traumatic optic neuropathy.

[0007] Preferably, Plerixafor (AMD 3100) is a small molecule CXCR4 chemokine receptor antagonist with the molecular formula C2. 28 H 54 N8, with a molecular weight of 502.78 and CAS number 110078-46-1, has the following structural formula:

[0008]

[0009] Prexafovir, a selective CXCR4 antagonist, reversibly blocks the binding of the CXCR4 receptor to its ligand CXCL12, disrupting retention signals between stem cells and the bone marrow microenvironment, thereby efficiently mobilizing hematopoietic stem cells into the peripheral blood. Its IC50, which blocks the binding of CXCL12 to CXCR4, is significant. 50 It is 44 nM.

[0010] In the field of optic nerve injury, existing research suggests that the CXCL12 / CXCR4 signaling pathway plays a dual regulatory role: on the one hand, moderate upregulation of this pathway after injury can recruit supportive cells to participate in the local repair process, which has a positive effect on nerve repair; on the other hand, overactivation of this pathway may also form a "chemical barrier" at the injury site, which may hinder axonal regeneration and be detrimental to the recovery of visual function. Therefore, the direct role of praxavir, as a highly effective CXCR4 antagonist, in the repair of optic nerve injury is currently unclear, and it is impossible to effectively determine whether it can produce therapeutic and ameliorative effects on traumatic optic neuropathy.

[0011] This invention demonstrates through experiments that praxaf can protect retinal ganglion cells and promote optic nerve regeneration by enhancing endogenous stem cell mobilization to secrete neurotrophic factors and exert anti-inflammatory effects, thereby breaking down the optic nerve regeneration barrier and promoting retinal ganglion cell axon regeneration.

[0012] Preferably, the dosage form of the drug is a suspension.

[0013] Preferably, the route of administration of the drug is subcutaneous injection.

[0014] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0015] The present invention also provides the use of praxafol in the preparation of a drug that promotes the regeneration of retinal ganglion cells (RGC) axons.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] This invention experimentally demonstrates that praxavir (AMD 3100) can effectively improve the survival rate of retinal ganglion cells and promote optic nerve axon regeneration. This result provides experimental evidence and a theoretical basis for the treatment of optic nerve injury and its potential clinical applications. This technical solution has broad application prospects and significant value in the preparation of drug formulations for treating neurodegenerative diseases. Attached Figure Description

[0018] Figure 1The images show the graphic electroretinograms of the control group (PBS group) and the AMD 3100 treatment group in Example 2. In the recorded waveforms, each grid on the vertical axis represents 10 μV, and each grid on the horizontal axis represents 20 ms. a represents the preoperative ONC result of the control group (PBS group), b represents the preoperative ONC result of the AMD 3100 treatment group, c represents the result 7 days after ONC with PBS administration, d represents the result 7 days after ONC with AMD 3100 administration, e represents the result after ONC with PBS administration, and f represents the result 14 days after ONC with AMD 3100 administration.

[0019] Figure 2 The graph shows the statistical changes in retinal potentials in the control group (PBS group) and the AMD 3100 treatment group in Example 2; ns indicates not significant, and ** indicates P<0.01.

[0020] Figure 3 The changes in GAP43 staining in mice after AMD 3100 intervention for optic nerve injury in Example 3; GAP43 red fluorescent markers for regenerated axons; scale bar: 250 μm;

[0021] Figure 4 This is a statistical graph showing the change in GAP43 expression at 250 μm after AMD 3100 intervention treatment in Example 3. *** indicates P<0.001.

[0022] Figure 5 This is a statistical graph showing the change in GAP43 expression at 500 μm after AMD 3100 intervention treatment in Example 3. *** indicates P<0.001.

[0023] Figure 6 The image shows β-tubulin immunofluorescence staining of mouse retinal patches after ONC surgery and AMD 3100 intervention for 1, 7, and 14 days; scale bar: 250 μm.

[0024] Figure 7 The graph shows the RGC cell density of mouse retinal patches after ONC surgery and AMD3100 intervention for 1, 7, and 14 days. ns indicates no significance, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0025] Figure 8 The expression levels of Creb, Erk1 / 2, Stat3, Akt proteins and their phosphorylated proteins after AMD 3100 treatment of optic nerve injury in Example 5;

[0026] Figure 9This is a statistical graph showing the effects of AMD 3100 on the Creb, Erk1 / 2, Stat3, and Akt signaling pathways after treating optic nerve injury in Example 5; ns indicates no significance, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0027] Figure 10 The expression of CD90, CD34, CD29, and Sca-1 monoclonal antibodies and CD34 / Sca-1 and CD90 / CD29 dual antibodies in the AMD 3100 treatment group cells in Example 6;

[0028] Figure 11 The expression of CD90, CD34, CD29, and Sca-1 monoclonal antibodies and CD34 / Sca-1 and CD90 / CD29 double antibodies in the control group (PBS group) cells in Example 6;

[0029] Figure 12 The graph shows the percentage of CD90, CD34, CD29, and Sca-1 positive cells in the AMD 3100 treatment group and the control group (PBS group) in Example 6; ns indicates not significant, and * indicates P<0.05. Detailed Implementation

[0030] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0031] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0032] Preparatory work for the experiment

[0033] 1. Laboratory animals

[0034] Wild-type C57BL / 6 mice were purchased from Beijing Vital River Company. Animal ethics and all experimental procedures complied with the "Regulations on the Management of Laboratory Animals" issued by the State Science and Technology Commission.

[0035] 2. Experimental reagents and antibodies

[0036] βIII-Tubulin antibody (Abcam); GAP43 antibody (Abcam); Plesiosafer AMD 3100 (Sigma-Aldrich); DMSO (Sigma-Aldrich); Optimal Cutting Temperature Compound (OCT) embedding buffer; 10X TBS (Solarbio); NGS (Sigma-Aldrich); BSA (Sigma-Aldrich); DAPI (Invitrogen); Goat anti-rabbit IgG secondary antibody (555); Goat anti-rabbit IgG secondary antibody (488); RIPA lysis buffer (Sigma-Aldrich); BCA protein quantification kit (Thermo Fisher Scientific); 6X SDS protein loading buffer (Zhongke Maichen); Trizol Reagent (Invitorgen); Goat anti-Rabbit secondary antibody (Invitrogen); CD29-PE antibody (Biolegend); CD34-BV421 antibody (Biolegend); CD90-APC antibody (Biolegend); Sca-1-PE antibody (Biolegend); PEHamster IgG2,λ1 Isotype antibody (Biolegend); APC Mouse IgG1,κ Isotype antibody (Biolegend); BV421 Rat IgG2a,κ Isotype antibody (Biolegend); PE-Cy™7 Rat IgG2a,κ Isotype antibody (Biolegend); Fc Block antibody (Biolegend); Red blood cell lysate (Solarbio); AKT antibody (Huaan Biotechnology); p-AKT antibody (Huaan Biotechnology); STAT3 antibody (Huaan Biotechnology); p-STAT3 antibody (Huaan Biotechnology); ERK1 / 2 antibody (Huaan Biotechnology); p-ERK1 / 2 antibody (Huaan Biotechnology); Anti-GAPDH antibody (Shanghai Kangcheng); Creb antibody (Shanghai Kangcheng); Compensation microspheres (Thermo Fisher Scientific).

[0037] 3. Buffer solution

[0038] RIPA buffer protein lysis buffer: 1 mL of 0.5 M NaCl, 0.04 mL of 2 mM ethylenediaminetetraacetic acid (EDTA), 100 μL of benzosulfonyl fluoride (PMSF), 0.5 mL of 50 mM tris(hydroxymethyl)aminomethane (Tris), 1 mL of 1% surfactant NP-40, 0.2 mL of 0.1% sodium dodecyl sulfate (SDS), and ultrapure water to a final volume of 10 mL.

[0039] 10×TAE: 11.4 mL of 17.4 M glacial acetic acid, 3.7 g of EDTA, 48.4 g of Tris, and double-distilled water to a final volume of 1.0 L.

[0040] Blocking buffer: 50 μL Triton X-100, 0.5 g fetal bovine serum albumin (BSA), and 10.0 mL phosphate buffer (PBST) or Tris-Borate-Sodium Tween-20 buffer (TBST) are added.

[0041] 2×SDS loading buffer: Tris-HCl pH 6.8, β-mercaptoethanol (200 mM), glycerol (20%), SDS (4%), bromophenol blue (0.2%).

[0042] PBS / PBST buffer: Add 16.0 g NaCl, 0.46 g NaH2PO4, 0.40 g KCl, and 0.24 g Na2HPO4 to a final volume of 2.0 L with double-distilled water. Add Tween-20 at 1‰ to prepare the buffer solution.

[0043] Electrophoresis buffer: 94 g of 1.25 M glycine, 15.1 g of 0.125 M Tris, 5 g of 0.5% (w / v) SDS, and double-distilled water to a final volume of 1 L.

[0044] Electroporation buffer: 0.125 M Tris 15.14 g, 1.25 M Glycine 72.07 g, double-distilled water 400 mL, add water to 500 mL.

[0045] 4. Experimental instruments and equipment

[0046] Graphical electroretinography (RETI scan system); Leica cryostat (CM1950, Germany); laser confocal microscope (Carl Zeiss LSM710, Germany); fluorescence microscope (A1, AxioVision, Leica DM4000, Germany); ophthalmic microsurgical instruments; 33G microinjector (Hamilton); CYTEK Aurora analytical flow cytometer (Cytek Biosciences, USA).

[0047] Leica cryostat (CM1950, Germany); laser confocal microscope (Carl Zeiss LSM710, Germany); fluorescence microscope (A1, AxioVision, Leica); RETIMAP small animal retinal imaging system (Roland Consult, Germany); ophthalmic microscopy instruments.

[0048] 5. Statistical and analytical software

[0049] Data entry and management were performed using Excel, while data analysis was conducted using SPSS 26.0 statistical software, and GraphPadPrism 10 was used for result visualization. Statistical methods were as follows: 1) Data description: Continuous data underwent a Shapiro-Wilk test for normality. Data conforming to a normal distribution were expressed as mean ± standard deviation; otherwise, the median (25th percentile, 75th percentile) was used. Categorical data were expressed as percentages. 2) Intergroup comparisons: Normally distributed continuous data were analyzed using one-way ANOVA; non-normally distributed data were analyzed using non-parametric tests; and multiple comparisons among multiple groups were performed using a post-hoc Tukey test. Significance level: A two-sided p-value < 0.05 was considered statistically significant.

[0050] Example 1

[0051] 1. Establishment and treatment of mouse optic nerve compression (ONC) model

[0052] (1) Eight-week-old wild-type C57BL / 6 mice were anesthetized by intramuscular injection of a mixture of 70 mg / mL Zoletil and 20 mg / mL Xylazine hydrochloride (dose 1.0 mL / kg);

[0053] (2) Make a horizontal incision in the conjunctiva near the fornix of the upper eyelid of the mouse's right eye. The incision is about 1 / 4 of the distance of the mouse's eyeball surface at the 12 o'clock position. The optic nerve is exposed by separating the extraocular muscles. Under the surgical microscope, the optic nerve is squeezed for 5 seconds at a distance of 1.0-1.5 mm behind the eyeball with reverse forceps.

[0054] (3) The effectiveness of optic nerve injury was confirmed by examining the clamp marks.

[0055] 2. Treatment with subcutaneous injection of praxavir (AMD 3100)

[0056] (1) Preparation of plexafor injection: Weigh plexafor powder and place it in a 15 mL centrifuge tube; add PBS to prepare a 5 mg / kg solution and mix thoroughly; prepare and use immediately, operate on ice, and store at -20℃.

[0057] (2) Experimental Groups:

[0058] The constructed mouse optic nerve compression (ONC) model was used as the experimental animal. Immediately after ONC surgery, 5 mg / kg of the corresponding drug was injected subcutaneously, and the drug was administered at a fixed time every day.

[0059] ① Control group (PBS group): 5 mg / kg once a day for 14 consecutive days;

[0060] ②Prexafos 1-day group: 5mg / kg once a day for 1 day;

[0061] ③Prexafos 7-day group: 5mg / kg once a day for 7 consecutive days;

[0062] The 14-day course of plexafort was administered once daily for 14 days at a single dose of 5 mg / kg.

[0063] (3) Administration procedure:

[0064] ① Record the mouse's weight and calculate the drug dosage;

[0065] ② Prepare two 1 mL syringes and draw plexafor and PBS respectively;

[0066] ③ Fix the mouse, inject subcutaneously through the back of the neck, and inject bolus after aspiration to ensure no blood is drawn back, to avoid spillage of the drug;

[0067] ④ Return the samples to the cages and complete the experimental record.

[0068] The experimental mice in the experimental groups of this embodiment were used to carry out the detection items of the following embodiments.

[0069] Example 2

[0070] Graphical electroretinography (PERG) was used to evaluate retinal function in mice treated with AMD 3100.

[0071] (1) Preparation: PERG was performed on C57BL / 6 mice using the RETIMAP small animal retinal imaging system (Roland Consult, Germany), and the amplitude of N1-P1 and the peak value of P1 were measured using RETI-port recording software (Roland-Consult, Germany). Mice were dark-acclimatized for 12 hours before the examination. Anesthesia was performed by intramuscular injection (a mixture of 70 mg / mL Zoletil and 20 mg / mL Xylazine, dose 1.0 mL / kg), and mydriasis was achieved with compound tropicamide eye drops.

[0072] (2) PERG detection: The entire experiment was conducted under dim red light illumination. Mice were fixed on the experimental board and electrodes were installed. A small amount of 3% hydroxypropyl methylcellulose lubricating gel was applied to the cornea to keep it moist. The recording electrode was gently placed on the cornea, the reference electrode (needle electrode) was placed subcutaneously on the ipsilateral cheek, and the ground electrode was placed subcutaneously on the tail. After dark adaptation for about 5 minutes, the relevant examinations began. Dark adaptation ERG (Scot ERG) detection and recording were performed according to the international clinical visual electrophysiology standardization protocol. The spatial frequency was maintained at 0.02 cycles / degree, the temporal frequency was maintained at 1 Hz, and the number of superpositions was maintained at 200. The PERG recording time was 200 ms, and the recording time was 20.0 cd s / m. 2 PERG waveforms of mice under varying light intensity. During recording, the contralateral eye was covered with a black cloth, and the spatial frequency, temporal frequency, and number of superpositions of the vertical grating were kept consistent. Software was used to analyze and statistically analyze the waveforms.

[0073] Test results as follows Figure 1-2 As shown. C57BL / 6 mice in the control group (PBS group) exhibited decreased waveform amplitude and prolonged peak duration; after AMD 3100 treatment, under dark adaptation at 20.0 cd s / m 2 Improvements in the waveform amplitude and peak time of PERG were observed under all light intensities, suggesting a partial improvement in retinal nerve function.

[0074] Example 3

[0075] Immunofluorescence staining of optic nerve

[0076] (1) Immunofluorescence staining process of regenerated axons:

[0077] Buffer preparation: TBS buffer (containing 0.15 mol / L NaCl and 0.01 mol / L Tris), TBS2T buffer (containing 0.3 mol / L NaCl, 0.01 mol / L Tris and 0.1% Tween-20), blocking solution (TBS buffer containing 5% BSA), antibody buffer (TBS2T buffer containing 5% BSA);

[0078] The optic nerve slices were washed three times with TBS, each time for 5-10 minutes.

[0079] Immerse the optic nerve slices in methanol containing 0.1% H2O2 for 10 minutes. Because methanol is volatile, the solution needs to be replenished in time.

[0080] After soaking, wash the TBS three times, 5-10 minutes each time, to ensure it is clean. Wipe the surrounding area dry, add sealing solution, and seal at room temperature for 1 hour.

[0081] Discard the blocking solution, wipe the slide dry, add GAP-43 antibody diluted with antibody buffer (1:500), and incubate overnight in a humidified box at 4°C, taking care to prevent the primary antibody from evaporating;

[0082] The next day, after recovering the excess primary antibody from the slide, TBS2T was directly added to the optic nerve tissue on the slide and soaked in a humidified box at 4°C for 1 hour.

[0083] Remove TBS2T, add the primary antibody again, and incubate at room temperature for 1 hour;

[0084] After incubation, wash with TBS2T buffer at room temperature for 1 hour; then wash three times with TBS2T for 5 minutes each time.

[0085] Dilute the secondary antibody with TBS2T (1:400) and incubate at room temperature in the dark for 2 hours;

[0086] After the secondary antibody incubation is complete, wash three times with TBS2T for 5 minutes each time, then wash with TBS for 5 minutes each time, and finally mount the slide.

[0087] The results are as follows Figure 3-5 As shown, the number of regenerated axons in the control group (PBS group) was significantly lower than that in the AMD 3100 intervention group, which verifies that AMD 3100 can promote optic nerve axon regeneration after ONC.

[0088] Example 4

[0089] Retinal patch immunofluorescence staining

[0090] (1) Materials preparation: 60 mm culture dish, 1×PBS, 1 mL syringe, ophthalmic microscissors, fine ophthalmic scissors (toothed forceps and toothless forceps), 25% and 50% glycerol, 1.5 mL centrifuge tube, glass slides, coverslips. All instruments were autoclaved.

[0091] (2) Retinal separation and mounting

[0092] 1) Sample collection: After cardiac perfusion, the eyeballs of mice were removed, placed in PBS, and observed under a microscope;

[0093] 2) Separation: Under a microscope, the eyeball is fixed with toothless forceps, the cornea is circularly excised, the lens is delivered, the vitreous body is removed, and the retina is bluntly separated;

[0094] 3) Fixation: Fix in 4% paraformaldehyde for 1 hour, wash 3 times with PBS, and soak in 25% glycerol overnight at -20°C.

[0095] 4) RGC staining: ① Wash the retina 3 times with PBS; ② Block at room temperature (PBS + 0.3% Triton X-100 + 5% goat serum) for 1 hour; ③ Add antibody buffer (PBS + 0.3% Triton X-100 + 1% BSA) and incubate overnight at 4°C with β III-Tubulin primary antibody (1:500); ④ Discard the primary antibody incubation solution the next day and wash with PBS; ⑤ Incubate with secondary antibody (DAPI 1:500, goat anti-rabbit secondary antibody 1:400) at room temperature for 2 hours in the dark;

[0096] 5) Slide preparation: After staining the retina, wash it 3 times with PBS, cut the retina into a four-leaf clover shape, place it flat on the glass slide with the RGC layer facing up, and ensure that there are no curled edges or foreign objects attached.

[0097] 6) Covering: After adding 50% glycerin, place a coverslip on the slide, avoiding the formation of air bubbles or pressure on the retina during the process.

[0098] 7) Record: Label sample information, store in the dark, and take immunofluorescence images as soon as possible to prevent fluorescence quenching.

[0099] Experimental results are as follows Figure 6-7 As shown, the number of surviving RGCs showed a cumulative dose-dependent effect with the extension of AMD 3100 administration time; both the 7-day and 14-day administration groups significantly improved the number of surviving RGCs, with the 14-day administration group showing the most significant increase.

[0100] Example 5

[0101] Western blot detection of related proteins

[0102] Protein extraction: 1) After euthanizing the mice by dislocation, the eyeballs were quickly removed, the retina was separated on ice and transferred to EP tubes; 2) 200 μL of lysis buffer was added, and the liquid was thoroughly ground with an electric grinder until it became a fine white liquid. The grinding was repeated twice to avoid splashing; 3) The liquid was allowed to stand on ice for 20 minutes; then centrifuged at 12,000 rpm for 15 minutes at 4°C; 4) 4 μL of the supernatant was diluted in 396 μL of double-distilled water, and the sample was stored at -80°C.

[0103] BCA method for protein concentration determination: 1) Prepare BSA standard gradient and BCA chromogenic agent; 2) Add the standard and the sample to be tested to a 96-well plate, add an equal volume of chromogenic agent to each well, and incubate at 37℃ for 1 hour; 3) Return to room temperature and measure at 562nm using an ELISA reader; plot the standard curve and calculate the sample concentration; 4) Add 1× SDS loading buffer and boil the protein for 5 minutes before loading.

[0104] Mixing the adhesive: Clean the mold and check for leaks; add the separating adhesive to 2 / 3 of the height and the concentrating adhesive to the top 1 / 3; select a suitable comb and insert it into the adhesive; let it stand at room temperature for about 1 hour to solidify; remove the comb vertically; and correct the comb holes if necessary.

[0105] Sample loading and electrophoresis: Install the gel, add 1× electrophoresis buffer, check for leaks before loading the sample; mark the order on both sides of the sample with markers; connect the electrodes correctly, set the voltage to 200-250V, and electrophoresis until the bromophenol blue reaches near the bottom of the gel.

[0106] Transfer: Prepare 1× rapid transfer buffer and soak the nitrocellulose membrane for 10 minutes; assemble the transfer sandwich in sequence and then transfer the membrane immediately, using a current of 300 mA and a time depending on the molecular weight of the target protein.

[0107] Blocking and antibody incubation: 1) After transfer, the membrane was slashed and labeled; 2) Washed three times with TBST, then blocked at room temperature for 1 hour with 5% skim milk (prepared with 1×TBST); 3) Rinsed twice quickly with TBST, then incubated overnight at 4°C with primary antibody, followed by incubation at room temperature for 1 hour the next day with secondary antibody; 4) Developed with developer, and images were captured using the ChemiDoc™ XRS+ imaging system. The images were analyzed in ImageJ software.

[0108] Precautions: ① Ensure complete removal of pigments, vitreous humor, and other substances during retinal separation; use a different container for each retina. ② Prepare 2-3 BCA standard protein curves to avoid excessive errors between standard proteins. ③ Aliquot proteins as early as possible and store at -80℃, avoiding repeated freeze-thaw cycles.

[0109] Experimental results are as follows Figure 8-9 As shown in the figure. The results suggest that AMD 3100 promotes optic nerve injury repair by specifically activating the Creb / Creb and p-Erk1 / Erk1 signaling pathways.

[0110] Example 6

[0111] Peripheral blood collection from mouse periorbital vein

[0112] Materials required: Promecaine eye drops, sterile cotton swabs, capillary glass tubes (75-100 µL), grinding wheel, 1.5ml heparinized EP tube, 1ml pipette, 75% alcohol, EDTA anticoagulant tube, double-distilled water, red blood cell lysis buffer, 4% paraformaldehyde, 15ml centrifuge tube.

[0113] Operating Procedures: ① Anticoagulant Tube Preparation: Rinse EDTA anticoagulant tubes with double-distilled water, dispense 200µL into 1.5ml EP tubes, store at -20℃, and thaw on ice before use. ② Pre-collection Blood Examination: Ensure the mouse's eyes are free of atrophy, ulcers, or other abnormalities. ③ Mouse Fixation: Grasp the neck skin to make the eyeballs protrude, fix the tail, and apply promethacin to the operated eye for surface anesthesia. ④ Blood Collection: Insert a capillary glass tube into the periorbital venous plexus at a 30-45° angle, adjusting until blood flows into the capillary; collect blood into a heparinized EP tube and shake well. ⑤ Hemostasis and Observation: Apply pressure with cotton swabs to stop bleeding, observe the mouse's vital signs, and then return it to its cage. ⑥ Blood Sample Processing: Transfer the blood sample to a 15ml centrifuge tube and add 3 times the volume of erythrocyte lysis buffer. Centrifuge at 450×g and 4℃ for 5 minutes, repeat 3 times, discard the clear supernatant, and collect the precipitate; perform flow cytometry analysis as soon as possible to avoid prolonged sample storage.

[0114] Peripheral blood flow cytometry analysis

[0115] Peripheral blood was collected from each group of mice, approximately 2 mL per mouse. The recorder did not have information about the grouping of the experimental animals, and the experiment was conducted in a blinded manner.

[0116] Materials required: 1.5 mL ep cytometer tubes, trypan blue staining solution, Neubauer counting chamber, 38-color CYTEK Aurora analytical flow cytometer, 12×75 mm flow cytometer tubes, cell staining buffer, centrifuge, pipette, clean bench, cell filter paper, flow cytometry antibodies (CD29-PE, CD34-BV421, CD90-APC, Sca-1-PE), Fc Block antibody, and compensating microspheres.

[0117] Staining and Instrumentation: 1) Mix 10 μL of cell pellet with 10 μL of trypan blue and count viable cells. 2) Resuspend cells in 1 mL of pre-chilled Cell Staining Buffer (CSB), centrifuge at 1200 rpm for 5 minutes at 4°C, discard the supernatant, and repeat the washing process 3 times. 3) Add 100 μL of CSB, gently pipette, add 2 μL of Fc Block antibody, and incubate on ice for 15 minutes. 4) Resuspend cells in CSB, centrifuge, and aliquot into flow cytometry tubes or EP tubes according to their groups. 5) Add anti-mouse antibody and incubate on ice in the dark for 30 minutes. 6) Add 1 mL of CSB, centrifuge, discard the supernatant, and resuspend. Repeat 3 times, with the last resuspending using 300 μL of CSB. 7) Filter the cells through a cell filter into flow cytometry tubes for analysis. The antibody incubation and washing method for compensating microspheres is the same as above.

[0118] Grouping:

[0119] ① Blank tube: without antibody;

[0120] ② Single positive tube: Add only one fluorescent channel antibody (CD29-PE, CD34-BV421, CD90-APC, Sca-1-PE);

[0121] ③ Compensation microspheres: Add CD29-PE, CD34-BV421, CD90-APC, and Sca-1-PE antibodies respectively;

[0122] ④ Fully stained tubes: Add fluorescent antibodies to all channels in both groups.

[0123] Precautions: ① If there is not enough time to load the stained cells, fix them with 4% paraformaldehyde for 20-30 minutes, centrifuge, wash, discard the supernatant, resuspend in 500 μL CSB, and store at 4℃ in the dark for 48 hours. ② The compensating microspheres have extremely high viscosity; shake thoroughly before loading to facilitate spectral analysis. ③ Load the microsphere blank tubes first, adjusting the FSC / SSC voltage, followed by the antibody-stained microsphere tubes.

[0124] Experimental results are as follows Figure 10-12 As shown. The results indicate that the AMD 3100 may primarily utilize CD29⁺ and Sca-1. + Cells, but changes in CD90 and CD34 cells were not statistically significant.

[0125] The embodiments of this application have been described in detail above with reference to the accompanying drawings. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this application.

Claims

1. Application of praxavir in the preparation of drugs for treating traumatic optic nerve injury.

2. The application according to claim 1, characterized in that, The molecular formula of the praxaviva is C. 28 H 54 N8, CAS number 110078-46-1.

3. The application according to claim 1, characterized in that, The drug is in the form of a suspension.

4. The application according to claim 1, characterized in that, The drug is administered via subcutaneous injection.

5. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients.

6. Application of plexafor in the preparation of drugs that promote the regeneration of retinal ganglion cell axons.

Images