STAT3 combined with Y27632 to treat glaucoma by promoting axon growth of Müller differentiated retina ganglion cells

STAT3 combined with Y27632 to treat glaucoma by promoting axon growth of Müller differentiated retina ganglion cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article STAT3 combined with Y27632 to treat glaucoma by promoting axon growth of Müller differentiated retina ganglion cells Wulong Zhang, Yujue Wang, Lemeng Feng, Cheng Zhang, Weiming Zhu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3447824/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Müller differentiated RGCs have potential therapeutic value for glaucoma. However, axonal regeneration of differentiated RGCs has been a difficult problem. Retinal stem cells were differenticated from rat retinal Müller cells. The stem cells were randomly divided into five groups (control group, AAV-STAT3 group, shSTAT3 group, Y27632 group and AAV-STAT3 + Y27632 group). Stem cells in different groups were injected into rat model of glaucoma. The length of axon regeneration in STAT3 combined with Y27632 group was significantly longer than that in other experimental groups. The AAV-STAT3 transfected RGCs treated with Y27632 significantly increased the mRNA levels of Esrrb, Prdm14, Sox2, and Rex1, while decreasing the mRNA levels of Nestin, Eomes, Mixl1, and Gata4. Meanwhile, Socs3, Pten, Klf9, and Mdm4 were significantly lowered, while Dclk2, Armcx1, C-MYC, and Nrn1 were elevated. After injecting differentiated RGCs into the glaucoma model rat eyes, the axon length, RGC layer thickness and the electrophysiology were superior to the glaucoma model group. These findings suggested that STAT3 combined with Y27632 can significantly improve the axonal growth level of Müller differentiated RGCs, and reveal the potential mechanism to induce pluripotency of RGCs. Müller cells Retinal ganglion cells STAT3 Y27632 Glaucoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Glaucoma is the first irreversible blindness eye disease in the world. In 2010, there were approximately 84 million glaucoma patients. It is estimated that by 2020, the number of glaucoma patients in the world will reach 796 million( 1 , 2 ). Glaucoma is a class of neurodegenerative diseases that can be generally classified as primary glaucoma and secondary glaucoma( 3 ). The selective and progressive death of retinal ganglion cells (RGCs) is the common pathway and final outcome of optic nerve damage in glaucoma( 4 ). The goal of glaucoma treatment is to prevent the progressive loss of neurons and protect the optic nerve, thereby preserving the patient's visual function. Elevated pathological intraocular pressure is usually considered as the leading risk factor for optic nerve damage in glaucoma( 5 ). Thus, the current main treatment methods typically focus on the control of intraocular pressure. However, some patients did not receive good results through ocular hypotensive therapy, mainly due to the pathological changes in glaucoma are not only related to the changes in intraocular pressure( 6 ). Therefore, exploring the mechanisms of death of RGCs, seeking new protection to delay the death of RGCs, and repair or regenerate ganglion axons are fundamental strategies for the treatment of glaucoma. At present, the protection and treatment of the optic nerve of glaucoma mainly included: improvement of optic disc microcirculation, glutamate pathway inhibitors, neurotrophic factors, induction of heat shock protein expression, and anti-oxidation therapy( 3 , 7 – 10 ). However, these methods are still unable to prevent RGCs damage and protect the optic nerve effectively. Besides, these methods are not helpful for those patients who have lost the RGCs with advanced or absolute glaucoma. In recent years, stem cell research has brought new hope for the alternative treatment of glaucoma RGCs. Many studies found that retinal Müller cells are a kind of abundant and self-derived potential retinal stem cells( 11 , 12 ). They provide nutrient and metabolic support for the retinal neurons and neurotransmitter cycle. Therefore, they may become essential cell sources for retinal neuron regeneration. In our previous studies, we have successfully purified retinal Müller cells in vitro. The reinal stem cells also can be induced dedifferentiation( 13 ). Simultaneously, based on Müller cell-derived stem cells, it was also confirmed that the Atoh7 gene could promote a large number of RGCs directional differentiation, with a differentiation rate of 50.4%( 14 ). However, in vivo studies showed that the differentiated RGCs did not appear axon growth and just expressed the RGCs specific markers Thy1.1 and Brn-3b, which lead to function loss in conducting signals. In addition, although it was found that in vitro RGCs differentiated with axons, their length was only about 600 µm. Therefore, how to regenerate the retinal Müller cell differentiated RGCs restart the axon growth, and guide them along the correct route to the brain projection zone has become a problematic point in current research. Current research has confirmed that the reactivation of nerve growth ability is related to the transcriptional activator in nerve cells( 15 ). Among them, STAT3 plays an regulatory function in the regeneration of RGCs( 16 ). Studies have shown that the expression of the endogenous gene STAT3 is low in normal retinas. It often exists in cells in a non-phosphorylated form when cytokines or growth factors are activated by binding to receptors, activated STAT3 is transferred from the cytoplasm into the nucleus and participated in regulating the gene expression( 17 ). Research indicates that subretinal delivery of overexpressing AAV-STAT3 is protective against photoreceptor death. The results demonstrate the integration and expression of STAT3 in Müller cells and photoreceptors (PR) which is protective against neuronal insults by enhancing the STAT3 signaling( 18 ). It’s been reported that ROCK is negatively related to cell mitosis and nerve regeneration( 19 ). Rho/ROCK signaling pathway inhibitor Y27632 phosphorylates STAT3. The combination of Y27632 and STAT3 efficiently promotes neurite cell axon regeneration and significantly up-regulates its downstream genes p21, Irf1, and Sprr1a( 20 ). Therefore, this study was designed to investigate the effect of combination of Y27632 and STAT3 in regenerating ganglion cell axons derived from retinal Müller cells, and to explore its regulatory mechanisms, laying the foundation for the regeneration of glaucoma nerves. MATERRIALS AND METHODS Animals SD rats, without specific pathogen grade, were obtained from Central South University Animal Experimental Department. The Central South University Animal Care and Use Committee approved this research. Müller cells extraction The extraction and culture methods of Müller cells have been reported in our previous studies( 11 ). The eyes of P21 rats were taken and rinsed with PBS containing penicillin/streptomycin (Sigma). After removing the cornea and lens of the eyeball, the retina and retinal pigment epithelium were separated, and the complete retina of the rat was obtained. First, the rat retina was mechanically separated. Next, small tissue fragments were added to trypsinized with 0.25% trypsin-EDTA for digestion for 20 min. After digestion was terminated by adding medium, centrifugation was carried out. The obtained cells were added to DMEM containing 20% FBS for culture. The adherent growth of Müller cells was observed after 5–7 days. The adherent Müller cells were digested and re-inoculated in DMEM containing 20% fetal bovine serum for about 7 days. The cells were then inoculated in DMEM/F12 (Gibco) medium (1 × N2 supplement (Gibco), 2 × B27 supplement (Gibco), and 20 ng/ml EGF (Peprotech: Rocky Hill, NJ, USA), 10 ng/ mL bFGF (Peprotech), 2 mM glutamine (HyClone:100 µg/ mL penicillin and 100 µg/ mL streptomycin) at a density of 1 × 10 5 cells /cm 2 for 7 days. The nerve spheres were formed in about a week of culture, and the dedifferentiated medium was changed every other day. Lentivirus PGC-FU-Atoh7-GFP Construction The synthesis and construction of GC-Fu-Atoh7-IRES-GFP was performed by GeneChem (Shanghai, China). The MOI of neuroglobules transfected by lentiviral vector was 10( 14 ). Our previous studies have shown that AtoH7 induces differentiation of the neurosphere into RGC( 12 ). Recombinant AAV-STAT3 Vectors Construction AAV-STAT3 vector was prepared by plasmid co-transfection. The recombinant AAV-STAT3 was purified by gradient supercentrifugation with iodiol. The 40% iodixanol fraction was buffer-exchanged with 0.001% Tween in phosphate buffered saline (PBS) and concentrated by 100K Amicon Ultra-15 centrifugal filter units. The volume of the centrifugation filter unit is 200 ml. Then quantitative PCR was used to determine the relative standard titers of DNA-resistant viral genomes in the concentrated bacterial population. The vector concentration was calculated in terms of virus genome/mL under the condition of 2–4×10 13 vg/mL. ShRNA- STAT3 Expressing Vectors Construction The STAT3-specific shRNA carrying lentiviral vectors were bought from Santa Cruz Biotechnology, Inc (TX, USA). Add the shRNA Plasmid DNA solution (Solution A) directly to the dilute shRNA Plasmid Transfection Reagent (Solution B) using a pipette. Mix gently by pipetting the solution up and down and incubate the mixture 30 minutes at room temperature. Wash the cells twice with 2 ml of shRNA Transfection Medium. For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well. Add the 200 µl shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent Complex (Solution A + Solution B) dropwise to well, covering the entire layer. Gently mix by swirling the plate to ensure that the entire cell layer is immersed in solution. Incubate the cells 6 hours at 37° C in a CO 2 incubator or under conditions normally used to culture the cells. Following incubation, add 1 ml of normal growth medium containing 2 times the normal serum and antibiotics concentration (2x normal growth medium). Lentiviral transduction was performed by incubating RGCs with lentiviruses in growth media, and stable transfectants were selected with puromycin (2 µg/ml) for 1 week. Immunohistochemical Analysis and Axon Length Measurement Immunohistochemistry is used to analyze specific markers of cells. 4% paraformaldehyde was used to fix the cells, and the fixation time was 15 minutes. The cells were blocked with 5% goat serum, and the blocking time was 1 hour. Incubate the primary antibody overnight at 4℃. Wash away the primary antibody with PBS and wash 3 times for 10 mins each time. Incubate the cells with a fluorescent secondary antibody (KPL, Rouses Point, NY, USA) for 1 h at room temperature and protected from light. The cells were washed 3 times with PBS for 10 mins each time. DAPI was used to stain the cell nucleus. A confocal microscope (Leica TCS SP5, Leica Microsystems GmbH, Wetzlar, Germany) was used to take pictures of cell staining. Taking the center of the culture plate as the center, randomly select 5 microscope scanning field of view frames in the upper, lower, left, right, and middle five directions, collect images, and use the image analysis system (ImagePro Plus software) to measure the longest protrusion length of RGCs. Three cells were randomly selected for measurement, and the results were averaged for comparison. The experiment was repeated 5 times. Flow cytometry The purity of the extracted Müller cells was detected by flow cytometry, which has been reported in our previous studies ( 14 ). Adhesive Müller cells were digested, resuspended and gently blown into single cells. The cells were fixed with 4% PFA. Cells were sealed with a blocking solution (PBS containing 1% BSA and 0.1% TritonX-100) at 4℃ for 30 mins. After sealing, the cells were incubated with a primary antibody at 4 ℃ for 1 h. Cells were incubated with fluorescence-labeled secondary antibodies at 4℃ for 1 h. After washing the cells with PBS, they were resuspended and analyzed by flow cytometry. Edu assay The proliferation level of the stem cells was measured by the EDU assay. The EDU (RiboBio) solution was diluted with cell medium in the ratio of 1000:1 to prepare an appropriate amount of 50 µM EDU culture medium. The neurospheres were mixed with the medium containing EDU and incubated overnight at 37℃. Wash the cells with PBS twice for 5 mins each time. The neurospheres were fixed with PBS containing 4% paraformaldehyde at room temperature for 30 mins. The cells were incubated with Apollo buffer at room temperature for 30 mins in dark. Cells were incubated with PBS containing 0.5% TritonX-100 at room temperature for 10 mins. The cells were washed with PBS for 5 mins. The nuclei were stained with Hoechst 33342. Fluorescence microscopy was used to obtain the stained images. qRT-PCR Total RNA was extracted using the TRIzol reagent (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. The purity and quantity of samples were measured using a Nano spectrophotometer. qPCR was conducted to confirm the results for gene expression in samples from all the samples. The reaction conditions of PCR are carried out according to the conditions on the instructions. The RNA expression level was normalized to that of Gapdh. The corresponding quantitative RT-PCR primers were shown in Table S1. Western Blot Cells grown on plastic plates were lysed in ice-cold RIPA buffer. Lysates were placed on ice for 30 min and centrifuged at 16,000 g for 15 min at 4°C. The protein concentration was determined using the Micro BCA Protein Assay Kit. The protein is mixed with the loading buffer and heated at 95°C for 10 minutes. The sample was loaded for SDS-PAGE electrophoresis. After the electrophoresis was complete, the protein was transferred to the PVDF membrane in the transfer buffer. The membrane was sealed in PBST containing 5% milk at room temperature for 1 hour. Secondary antibodies were incubated for 1 h at room temperature. Primary antibodies, including anti-STAT3 (Cell Signaling Technology), anti-p-STAT3 (Cell Signaling Technology), Tyrosinase (Santa Cruz), mGluR6 (Abcam), Atoh7 (Abcam), CD31 (Cell Signaling Technology), Rhodopsin (Cell Signaling Technology), β-tubulin ((Cell Signaling Technology), Iba1 (Abcam), Brn3b (Abcam), Pax6 (Abcam), HPC-1 (Abcam), Sprrla1 (Santa Cruz), P21 (Santa Cruz), Irfland (Santa Cruz), Musashi1 (Abcam), Gap43 (Abcam), Ki-67 (Abcam), Nestin (Abcam), and anti-β-actin (Santa Cruz), were used in this study. The immunoreactive bands were imaged with the Bio-Rad ChemiDoc XRS system. Chronic Ocular Hypertension Glaucoma Model of Mice The establishment of a rat model of chronic ocular hypertension has been reported in the literature( 21 ). The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate. After 1% prilocaine was instilled in the right eye for topical anesthesia, the extrascleral aqueous humor drainage vein was photocoagulated by laser. The photocoagulation range is 270 degrees. The laser photocoagulation point is 20 points. The energy of the laser is 0.7W pulse lasting 0.6 seconds. Tonopen was used to detect intraocular pressure at 3, 10, 15, 20, 25, 30, 35, 40, 50, 60 days after photocoagulation. Stem Cells Transfection and Intraocular Injections Neurospheres were transfected by lentivirus PGC-FU-Atoh7-GFP for 24 h. The stem cells were divided into 4 groups: group A: PBS control group, PBS was added to medium; group B: STAT3 group, only added AAV-STAT3 to the medium; group C: Y27632 group ,only add Y27632 to the medium; group D: STAT3 + Y27632 group, added both AAV-STAT3 and Y27632 to the medium. After 24 hours of treatment, the neurospheres were isolated into individual cells by accutase. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital. Intravenous injection of 5ul differentiation medium (1 ng/mL BDNF, 30 nmol Brdurd, and 1 µM Ra) containing stem cells at a concentration of 1×10 4 cells/µL. Eyeball frozen sections were performed for Immunohistochemistry and immunofluorescence analysis after 12 days of retinal stem cell transplantation. Immunofluorescence of Eyeball Sections Immunofluorescence staining of frozen sections of eyeballs was performed as reported( 22 , 23 ). Eyeball tissue sections were sealed at room temperature for 1 h in PBS containing 3% BSA and 0.3% TritonX-100. After sealing, sections were incubated with primary antibody at 4℃ overnight. Sections were incubated with secondary antibodies at room temperature for 1 h. Eyeball tissue sections were sealed with antifade solution containing DAPI. Images were taken by fluorescence microscopy(Leica DMI4000B). Visual Evoked Potential Recording To evaluate retinal ganglion cells (RGCs) function, visual evoked potential (VEP) was employed. Briefly, Prior to VEP recordings, animals were dark -adapted for 12 hours and were prepared under dim red light. The animals were anaesthetized with ketamine and xylazine (87/13 mg/kg), and Pupils dilated by 1% tropicamide eye drops. The positive electrodes were inserted into the skin approximately 4 mm lateral to the midline over visual cortex. The reference electrodes were inserted into the skin above frontal cortex and the ground electrode was placed on the tail. To minimize the variation in the position of the needles in different sessions, their positions were marked on the skin and needles were fixed on the skin using tape. Visual stimulus was presented 60 times using a photic stimulator, with 15 dB intensity and 0.2 Hz stimulation frequency, at a distance of 15 cm. The time of measurement was 300 ms. Optical Coherence Tomography To measure thickness of RGC layer, OCT was performed. OCT was performed after anesthetization of rats followed by restraining animals in front of the OCT device with dilated pupil. All central retina OCT measurements were performed with spectral domain OCT (Heidelberg Spectralis SD-OCT; Heidelberg Engineering, Heidelberg, Germany) on each rat eyes. Spectralis software was used for retinal layer segmentation. The RGC thickness data was recorded. Statistical Analysis Data from at least three independently dissociated cultures, each measured in triplicate, were expressed as mean ± standard deviation. The data for two-group comparisons were analyzed for statistical significance using two-tailed Student’s t-test. Multiple comparisons statistical analysis was performed with one-way ANOVA using SPSS 18.0. P values are indicated with single asterisk (* <0.05), double asterisk (** <0.01) and triple asterisks (*** <0.001). RESULTS Müller Cell Culture and Identification We found that GS as Müller cell-specific marker was significantly high express in three generations cells dedifferentiated from rat retina (Fig. 1 A). Results also showed that 95.3% of the third-generation purified cells were immunoreactive for GS by Flow cytometry (Fig. 1 B). The protein expression of specific markers of other retinal cells in purified cells was accessed by WB assay. The results showed that the protein expression of Atoh7, Rhodopsin, Pax6, β-tubulin, Brn-3b, HPC1, Tyrosinase, and mGluR6 were low (Fig. 1 C-D). But the results of WB and Flow cytometry also suggest that there are a small number of other cells in Muller cells. Stem Cell Culture After cultured in DMEM/F12 dedifferentiation medium for 24 h, cells proliferate and differentiate into round shapes, and then some cells aggregate to form small cell spheres. After 48–72 h, the proliferation of cells was significantly accelerated, and the shape of single round cells was decreased compared with the former. The diameter of the cell spheres increased. After 5d, the number and the diameter of the cell sphere continued to increase, the boundary of the cell sphere was clearly defined, and the refractive index was strong. After 7 days, there was a slight increase in the volume of the cell sphere compared to before. At 10th day, the central refractivity of the visible neurosphere was reduced, showing that the cell bodies were atrophied and darkened (Fig. 2 A). The cell proliferation was observed daily under a phase-contrast microscope and the diameter of the neurosphere was measured (Fig. 2 B). We tested the mRNA level of Vsx2, Rax, Otx2 and Sox2 in Muller cells and neurosphere (Fig. S1). The results show that the mRNA level of Vsx2, Rax, Otx2 and Sox2 in neurosphere were significantly increased. When the cell diameter reached 150 µm, the cell sphere was digested with Accutase(Sigma)and passaged to obtain purified neurospheres. After three passages, the purified neurospheres were examined by immunofluorescence. Our results showed that the purified neurospheres are labeled with nestin, ki-67, pax6, and musashi1 antibodies, and the expression level of these four antibodies is approximately 90% (Fig. 3 A). WB showed that the cell spheres purified after three passages highly expressed nestin, ki-67, pax6, and musashi-1 when compared with the purified Müller cells (Fig. 3 B-D). These results indicated that the cells purified with three passages in the dedifferentiation medium are dedifferentiated into retinal stem cells. We then used Edu marker to detect the proliferation ability of these retinal stem cells. Our results showed that the proliferation ability of these purified retinal stem cells was very strong (Fig. 3 E). Effect of STAT3 and Y27632 on Axonal Regeneration of Müller Cell Differentiated RGCs After the retinal stem cells were identified, they were transfected with the lentivirus PGC-FU-Atoh7-GFP. The cells were plated onto 0.01% poly-D-lysine (Sigma)-coated 24 mm coverslips (Corning) at a concentration of 1×10 4 cells/well, and cultured in differentiation medium (1 ng/ml BDNF, 1 µM RA and 1% FBS). Three days later, stem cells were randomly divided into five groups: group A: PBS control group, PBS was added to medium; group B: AAV-STAT3 group, only added AAV-STAT3 to the medium; group C: AAV-shSTAT3 group, only added AAV-shSTAT3 to the medium; group D: Y27632 group, only add Y27632 to the medium; group E: AAV-STAT3 + Y27632 group, added both AAV-STAT3 and Y27632 to the medium (Y27632 intervened multiple times). The number of GFP labeled cells increased, the fluorescence intensity was enhanced, and the cytoplasm was uniformly distributed after 48 h intervention (Fig. 4 A). The differentiation of RGCs in each group was observed every day. With the prolongation of the differentiation time, the rounded stem cells gradually grow into a plurality of protrusions. And these cells also extended branches. The axon of the proximal cell body in these cells is thicker than that of the distal body. The axon walks more and more naturally. In the 12 days of differentiation, the length of axon in each group can be observed by IF staining (Fig. 4 B). We also detected the mRNA level of Brn3a and pax6 in RGCs and Muller cells (Fig. S2). The results show that the mRNA level of Brn3a and pax6 in RGCs were significantly increased. In the control group, the axon length of ganglion cells was 198.54 ± 9.18 µm. The axon length of the ganglion cells in AAV-STAT3 group and shSTAT3 was 265.38 ± 13.67 µm and 183.49 ± 11.64 µm respectively. In the Y27632 group, the axon length of ganglion cells was 200.35 ± 10.25 µm. In the AAV-STAT3 + Y27632 group, the axon length of ganglion cells was 437.32 ± 21.14 µm. The axon length of ganglion cells in the AAV-STAT3 group was longer than that in the control group. The results showed that the axon length of RGCs in the AAV-STAT3 + Y27632 group was significantly longer than that in the other groups (Fig. 4 C). Western blot analysis was performed on STAT3 group and STAT3 + Y27632 group on day 12. The results showed that the protein expression levels of p-STAT3, p21, GAP43, Irfland, and Sprrla1 in the STAT3 group were significantly higher than those in the control group. The protein expression levels of P-STAT3, P21, GAP43, Irfland, and Sprrla1 in the STAT3 + Y27632 group were higher than those in the STAT3 group, and the differences were statistically significant (Fig. 4 D-E). mRNA levels of axonal regeneration related proteins Socs3, Pten, Klf9, Mdm4, Dclk2, Armcx1, C-myc, and Nrn1 at D0, D3, D7, and D12 were also analyzed. The experimental results showed that the mRNA level of Socs3, Pten, Klf9 and Mdm4 in STAT3 + Y27632 group was significantly lower than that in STAT3 group, and the expression level was the lowest at D12. The mRNA levels of Dclk2, Armcx1, C-MYC and Nrn1 were significantly higher in STAT3 + Y27632 group than in STAT3 group. The mRNA level of Dclk2 was the highest at D7 and decreased at D12. The mRNA level of Nrn1 was the highest at D3, but decreased at D7 and D12. The mRNA level of Armcx1 and C-myc was highest at D12 (Fig. 4 F-M). The above results suggest that the use of STAT3 alone in vitro could promote the growth of axons. When combined with Y27632, the effect of promoting growth of axon was more substantial in RGCs. Mechanism of STAT3 Combined with Y27632 on the Regeneration of RGCs Differentiated by Müller Cell We used WB to detect the expression levels of STAT3, and p-STAT3 in each intervention groups of Müller cell differentiated RGCs (Fig. 5 . A-C). The results showed that the expression levels of STAT3 and p-STAT3 proteins in the STAT3 group, Y27632 group and STAT3 + Y27632 group were all higher than those in the control group, while the expression levels of STAT3 and p-STAT3 proteins in the shSTAT3 group were lower. There was no significant difference in STAT3 and p-STAT3 protein expression levels between the STAT3 and Y27632 group. The expression levels of STAT3 and p-STAT3 proteins in the STAT3 + Y27632 group were significantly higher than those in the other groups. mRNA levels of pluripotent related genes (Esrrb, Prdm14, Sox2, and Rex1) and differentiated related genes (Nestin, Eomes, Mixl1, and Gata4) were also detected at D12 (Fig. 5 D, E). The results showed that mRNA levels of pluripotent related genes (Esrrb, Prdm14, Sox2, and Rex1) in the STAT3, Y27632 and STAT3 + Y27632 groups were significantly increased, while mRNA levels in the shSTAT3 group were significantly decreased. mRNA levels of pluripotent related genes in the STAT3 + Y27632 group were considerably higher than that in other groups. In the STAT3, Y27632 and STAT3 + Y27632 groups, mRNA levels of differentiated related genes (Nestin, Eomes, Mixl1, and Gata4) were significantly decreased, while mRNA levels in the shSTAT3 group were significantly increased. The mRNA level of differentiated related genes in the STAT3 + Y27632 group was markedly lower than that in the other groups. We also detected the mRNA levels of axonal regeneration related proteins Socs3, Pten, Klf9, Mdm4, Dclk2, Armcx1, C-MYc, and Nrn1 at D12. The mRNA levels of Socs3, Pten, Klf9, and Mdm4 in the shSTAT3 group were increased dramatically, while those in the STAT3, Y27632, and STAT3 + Y27632 groups were significantly decreased. mRNA level of Dclk2, Armcx1, C-myc, and Nrn1 were significantly increased in the STAT3 group, Y27632 group, and STAT3 + Y27632 group, while those in the shSTAT3 group were significantly decreased. mRNA levels of pluripotent related genes (Esrrb, Prdm14, Sox2, and Rex1) in the STAT3 and STAT3 + Y27632 groups were detected at D0, D3, D7, and D12. mRNA levels of pluripotent related genes in the STAT3 group and the STAT3 + Y27632 group were higher than those in the control group at all time. The mRNA expression level of the STAT3 + Y27632 group was significantly higher than that of the STAT3 group and control group. mRNA levels of differentiated related genes (Nestin, Eomes, Mixl1, and Gata4) in the STAT3 group and the STAT3 + Y27632 group were also detected at D0, D3, D7, and D12. The results showed that the mRNA levels of differentiated genes in the STAT3 + Y27632 group and the STAT3 + Y27632 group were lower than those in the Control group at all time points, and the expression levels of mRNA in the STAT3 + Y27632 group were significantly lower than those in the STAT3 and control groups. The above results suggest that STAT3 combined with Y27632 may improve the pluripotency of Müller differentiated RGCs cells to regulate the expression level of proteins related to axon regeneration and promote its axon regeneration. Detection of Ganglion Cells in Rat Chronic Ocular Hypertension Glaucoma Model To further verified the role of STAT3 and Y27632 in promoting the growth of RGCs axon, we established rat chronic ocular hypertension glaucoma model. A digital tonometer was used to measure IOP after laser treatment at different time points. Compared with normal eyes, the mean IOP in model eyes increased significantly from 3 to 30 days after modeling. The intraocular pressure gradually increased with time, reaching its maximum value in 10–25 days. The intraocular pressure began to decrease on the 30th day and reached a normal level in about 60 days (Fig. 5 A). HE stain was used to detect the numbers of retina ganglion cell nucleus in the glaucoma rat model (Fig. 5 B). The experimental results showed that with the increase of intraocular pressure, the number of RGC cells gradually decreased. The intraocular pressure decreased to normal levels on day 60, but the number of RGCs continued to decrease (Fig. 5 C). Moreover, TUNEL staining was used to assess whether the RGCs of glaucoma rats undergo apoptosis. Experimental results show that RGCs in the retinal ganglion cell layer undergo apoptosis (Fig. 6 ). The results showed that the number of apoptotic cell nuclei gradually increased as the intraocular pressure gradually increased. At day 3, RGCs was still regularly shaped and uniformly distributed in retina ganglion cell layer. At day 10, cytoplasm and nucleus of part of RGCs began to disintegrate. At day 30, more cell debris were detected in retina ganglion cell layer. At day 60, most of RGCs disappeared with a few debris left. The proportion of TUNEL positive cells were 6.5 ± 2.1,8.7 ± 1.7,13.5 ± 3.6,57.1 ± 3.0%, respectively. In the meantime, we found an increase in apoptosis of cells in outer nuclear layer. Growth of RGC Axon in Glaucoma Rat Model Stem cells were transfected with lentivirus PGC-FU-Atoh7-GFP and cultured in differentiation medium. After 24 hours, these stem cells were randomly divided into four groups: group A: control group, PBS was added to medium; group B: AAV-STAT3 group, only added AAV-STAT3 to the medium; group C: Y27632 group, only add Y27632 to the medium; group D: AAV-STAT3 + Y27632 group, added both AAV-STAT3 and Y27632 to the medium. 24 hours later, we divided glaucoma rat models randomly into four groups as above and injected stem cells into the vitreous cavity in each group. IF staining of retinal tissue sections was performed to detect cell-specific markers GFP and ZO-1 (a kind of tight junction protein) after 12 days of retinal stem cell transplantation (Fig. 7 A). The length of axons in each group can be observed by the chemical staining of immunofluorescence cells (Fig. 7 B). In the control group, the axon length of RGCs was 360.5 µm. The axon length of RGCs in the AAV-STAT3 group was 877.3 µm. The axon length of RGCs in the Y27632 group was 377.9 µm. In the AAV-STAT3 + Y27632 group, the axon length of ganglion cells was 1986.7 µm. The axon length of RGCs in the AAV-STAT3 group was longer than that in the PBS group. The axon length of RGCs in the AAV-STAT3 + Y27632 group was significantly longer than that in the other groups. We observed that the axons of RGCs were significantly longer than those of the glaucoma model group after transplantation of Müller cell differentiated RGCs after STAT3 + Y27632 intervention by retinal lamination (Fig. 7 C). We observed that the axons of RGCs were significantly longer than those of the glaucoma model group after transplantation of Müller differentiated RGCs intervened with STAT3 + Y27632 by retinal lamination. We observed the thickness of RGCs layer in each group through OCT (Fig. 7 D). The RGCs layer of the STAT3, Y27632, and STAT3 + Y27632 groups was significantly thicker than that of the glaucoma model group (Fig. 7 F). In comparison the RGCs layer of the STAT3 + Y27632 group was significantly thicker than that of the other groups. Flash-VEP was used to detect optic nerve conduction in each group (Fig. 7 E). The optic nerve conduction function in the STAT3 and STAT3 + Y27632 groups were significantly better than that of the glaucoma model group, and the optic nerve conduction function in the STAT3 + Y27632 group was significantly better than that of the other groups (Fig. 7 G-H). STAT3 combined with Y27632 enhanced the pluripotency and inhibited the differentiation of Müller cell differentiated RGCs (Fig. 8 ). DISCUSSION Glaucoma is a main blinding disease in the world. It causes permanent damage of the optic nerve since its’ impossible for mammalian nerves regenerated. Glaucoma is characterized by progressive loss of retinal ganglion cells along with their optic nerve axons. Reduction of intraocular pressure (IOP), nutrition of optic nerve, or the antioxidation treatment are the therapies of glaucoma. These treatments postpone further death of the ganglion cell. However, these methods can not make the apoptosis RGCs regenerate. Therefore, for the patient with massive death of ganglion cells, we must find an effective therapy to activate the regeneration of ganglion cells to reestablish visual pathway and help these patients to regain their vision. Atoh7 is a member of the bHLH family, and in our previous research, it had been verified that Atoh7 regulates Müller cell-derived stem cells differentiating into RGCs both in vitro and in vivo. This makes it possible for us to replace glaucoma-induced apoptosis of RGCs cells by transforming Müller cell-derived stem cells differentiating into RGCs. However, the axonal growth of the regenerated RGCs determines the reconstruction of the visual pathway. How to promote the growth of regenerated RGCs axons becomes our research focus. Studies have shown that STAT3 plays an important role in the axon growth of retinal ganglion cells. However, the specific mechanism of STAT3 promoting RGCs axon regeneration is still unknown. STAT3 is a kind of bifunctional cytoplasmic protein coupling with tyrosine phosphorylation signal pathway in the cytoplasm ( 24 ). Normally STAT3 protein is in a non-phosphorylated form in cells, when cytokines or growth factors bind to the receptor, tyrosine kinases (JAK) that bind to receptors are activated. Activated JAK phosphorylates the 705th position of tyrosine residue (Tyr705) in the STAT3 cytoplasm. They combined to form the JAK/STAT3 homodimers. Then the homodimers entered into the nucleus, recognized specific DNA sequences and regulated the transcription of target genes. Therefore, the amount of nuclear-activated STAT3 represents the activation of the JAK/STAT3 pathway in the cell. Activated STAT3 will be dephosphorylated in the nucleus after transmitting signals and be restored to the monomeric form. And then, it will deactivate and return to the cytoplasm to participate in the next round of signal transduction. The activation degree of JAK/STAT3 in normal peripheral nerves is low. Only phosphorylated activated STAT3 may translocate into the nucleus to regulate the expression of certain genes to regenerate peripheral never. Our experimental results also confirmed that the axons of RGCs in the STAT3 overexpressed group were significantly longer than those in the STAT3 inhibited group. We hypothesized that STAT3 regulates the growth of RGCs axons through the above mechanisms. Research showed that when adeno-associated virus vector containing STAT3 gene was injected into vitreous cavity of the optic nerve contusion model rats, they found that the number of STAT3 positive cells increased significantly as well as the expression of STAT3 protein and STAT3 mRNA increased by 4 to 6 fold after 3 weeks. And the expression of GAP-43 (growth-associated protein-43), a marker of retinal ganglion cell axon regeneration, significantly increased. The optic nerve 3D image analysis which used nerve fiber anterograde tracer CTb-594 showed that the axons of ganglion cells after STAT3 gene infected increased about 200 microns than the control group. But the axon density did not increase and formed many U-turn and right-angle turn. These results indicated that STAT3 signal transduction can increase the length of retinal ganglion cell axons and promote the extension of axonal growth. Another research showed that the use of the JAK2 inhibitor AG490 in vitro in RGCs inhibited CNTF-mediated axonal growth and significantly reduced the regenerative effect of RGCs in response to inflammatory stimuli in vivo. These results confirmed that the activation of JAK and STAT3 played an important role on the axis initial stage of outgrowth ( 25 , 26 ). Studies have demonstrated that STAT3 signaling pathway plays an important role in the axon growth of RGCs( 27 , 28 ). The same results were obtained in our experiment. Deletion Pten, a negative regulator of the mammalian target of rapamycin (mTOR) pathway in adult RGCs, promotes robust axon regeneration after optic nerve injury( 29 ). A high-throughput gene profiling study revealed that the deletion of Klf9 gene substantially promotes optic nerve regeneration in adults RGCs( 30 ). Researches confirmed that a genetic deletion of PTEN, SOCS3, or PTEN/SOCS3 allows partial axon regeneration in the optic nerve after optic nerve crush( 31 , 32 ). Inhibition of Mdm4 in the eye and spinal cord promotes axonal regeneration and sprouting of the optic nerve after crush and of supraspinal tracts after spinal cord injury( 33 ). Our research showed that the mRNA level of PTEN, SOCS3, Klf9, and Mdm4 decreased significantly in AAV-STAT3 group. Overexpression of Dclk2, Armcx1, c-myc, and Nrn1 are closely related to the regeneration of RGCs axon( 34 – 37 ). The mRNA level of Dclk2, Armcx1, c-myc, and Nrn1 significantly increased in the AAV-STAT3 group. The inhibition of STAT3 expression inhibited the growth of the RGCs axon. The mRNA expression levels of RGCs axon-regenerated proteins were significantly opposite between the shSTAT3 group and the AAV-STAT3 group. All the above results confirmed that STAT3 overexpression could promote axonal regeneration of Müller differentiated RGCs. Recently years, studies have shown that Rho-associated coiled-coil-containing protein kinase (Rho-ROCK) is negatively correlated with cell mitosis and nerve regeneration. Rho is a small molecule homopolymer of the GTPases superfamily and is a mammalian gene homologue of the Ras superfamily. Its biological function is mainly through its downstream effector molecule ROCK. ROCK is a serine/threonine-protein kinase whose molecular structure includes an amino-terminal catalytic domain, an intermediate domain which combined with Rho’s α-coiled-coil, a carboxy-terminal catalytic domain, and a Cys/His region. Activated Rho-GTP activates ROCK by binding to the alpha coiled-coil domain of ROCK and exposing the catalytic center of ROCK( 38 ). Our results have shown that STAT3 could be phosphorylated by Y27632, which is an inhibitor of the Rho-ROCK signaling pathway. The STAT3 + Y27632 combination can effectively promote axon regeneration of Müller cell differentiated RGCs. The axon length of RGCs in the AAV-STAT3 + Y27632 group was the longest, and the expression of GAP-43, phosphorylated STAT3 and its downstream genes p21, Irf1 and Sprr1a was significantly increased. Inhibition of the Rho/ROCK signaling pathway not only reduced the U-turn of the axon and avoid navigation errors but also promote axon regeneration in RGCs. Our research is the first to confirmed that the combined treatment of STAT3 + Y27632 on Müller-derived RGCs can improve the differentiation rate of RGC cells and significantly increase the length of axons, which is a significant improvement for RGCs regeneration. The mRNA levels of RGCs axon-growth-related proteins Pten, Socs3, Klf9, and Mdm4 in the STAT3 + Y27632 group were significantly lower than those in the STAT3 group, while the mRNA levels of Dclk2, Armcx1, C-myc, and Nrn1 were significantly higher than those in the STAT3 intervention group. In order to analyze the mechanism underlying the effect of STAT3 + Y27632 in promoting axon growth of Müller cells differentiated RGCs, we tested the mRNA level of pluripotency genes (Esrrb, Prdm14, Sox2, and Rex1) and differentiation genes (Nestin, Eomes, Milx1, and Gata4). In the STAT3 + Y27632 group, mRNA level of pluripotency genes (Esrrb, Prdm14, Sox2, and Rex1) compared with STAT3 group increased significantly, the mRNA level of differentiation genes (Nestin, Eomes Milx1, and Gata4) decreased obviously. We concluded that the reason for overexpression of STAT3 promote axon growth could be launched Müller cells differentiated RGCs pluripotency. The combination of STAT3 and Y27632 can promote the axon growth of Müller cells differentiated RGCs better than STAT3 alone, which is the same mechanism. Furthermore, rat chronic ocular hypertension glaucoma model were made to access the mechanism in vivo. in this study. To verify whether STAT3 + Y27632 improve growth of RGCs differentiated from stem cells, the stem cells were transfected with lentivirus PGC-FU-Atoh7-GFP. The stem cells were transplanted into the vitreous cavity of the glaucoma rat model. After 14 days, retina sections of glaucoma models were used to examine immunoreactivity for RGCs-specific marker GFP and ZO-1. The result showed that length of RGCs axons was significantly longer in the STAT3 + Y27632 group, which verified that combination STAT3 with Y27632 can promote the growth of RGCs axons. OCT results showed that the RGCs cell layer in the STAT3 + Y27632 group was significantly thicker than that in the other intervention groups. We observed a delay in peak latencies of P1 waves both in the Glaucoma group and Y27632 group. Additionally, N1-P1 amplitudes in STAT3 + Y27632 group are significantly higher compared with other groups excepted Con group. No significance was observed in the P1 latency and N1-P1 amplitude between Glaucoma group and Y27632 group. These results suggests that Müller cell differentiated RGCs transplants can integrate into preexisting retinal circui and function physiologically. In conclusion, STAT3 combined with Y27632 can significantly promote the axon growth of ganglion cells which was dedifferentiated from retina Müller cells either in vitro or in vivo. Abbreviations RGCs: retinal ganglion cells; VEP: Visual evoked potential; OCT: Optical coherence tomography; STAT3: signal transducer and activator of transcription 3; Rho-ROCK: Rho-associated coiled-coil-Containing protein kinase; MOI: multiplicity of infection; FACS: Flow cytometry; PBS: phosphate buffered saline; HE: Hematoxylin and eosin; qRT-PCR: Quantitative real time polymerase chain reaction; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IOP: intraocular pressure: BDNF: brain-derived neurotrophic factor. Declarations ACKNOWLEDGEMENTS Not applicable AUTHOR CONTRIBUTIONS Wulong Zhang:Collection and assembly of data, data analysis and interpretation, manuscript writing, Writing - Original Draft. Yujue Wang: Data Curation; Formal Analysis; Writing - Original Draft. Lemeng Feng, Cheng Zhang: Data interpretation, Visualization Weiming Zhu, Xin Li, Ye He: Collection of data, data interpretation. Weitao Song: Conception and design, Resources, Supervision, Writing - Review & Editing FUNDING This study was supported by grant from National Nature Science Fund of China（81974132），National Nature Science Fund of China（81770927），National Key R&D Program of China(2021YFA1101202)；Hunan Provincial Health Commission（20220702839）and Hunan Natural Science Foundation (No.2022JJ30076) AVAILABILITY OF DATA AND MATERIALS All the data generated or analyzed during this study are included in this published article. The use of mouse Müller cells was in accordance with the relevant guidelines and regulations, and the experimental protocols were approved by the Medical Ethics Committee of the Xiangya Hospital of Central South University. This study is in accordance with ARRIVE guidelines ETHICS APPROVAL AND CONSENT TO PARTICIPATE The use of mouse Müller cells was in accordance with the relevant guidelines and regulations, and the experimental protocols were approved by the Medical Ethics Committee of the Xiangya Hospital of Central South University. This study is in accordance with ARRIVE guidelines CONSENT FOR PUBLICATION Not applicable COMPETING INTERESTS All authors declare that they have no competing interests. AUTHOR DETAILS Hunan Key Laboratory of Ophthalmology, Eye Center of Xiangya Hospital, Central South University, Hunan, 410008, China. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. References Jonas JB, Yang D, Wang N. [Effect of intraocular pressure on glaucomatous damage to the optic nerve]. Ophthalmologe. 2014;111(2):181-8; quiz 9-90. Quigley HA, Broman AT. The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol. 2006;90(3):262-7. Tian K, Shibata-Germanos S, Pahlitzsch M, Cordeiro MF. Current perspective of neuroprotection and glaucoma. Clin Ophthalmol. 2015;9:2109-18. Medeiros FA, Lisboa R, Weinreb RN, Liebmann JM, Girkin C, Zangwill LM. Retinal ganglion cell count estimates associated with early development of visual field defects in glaucoma. Ophthalmology. 2013;120(4):736-44. Weinreb RN, Aung T, Medeiros FA. The pathophysiology and treatment of glaucoma: a review. JAMA. 2014;311(18):1901-11. Hove IV, Lefevere E, Moons L. ROCK inhibition as a novel potential strategy for axonal regeneration in optic neuropathies. Neural Regen Res. 2015;10(12):1949-50. Fang JH, Wang XH, Xu ZR, Jiang FG. Neuroprotective effects of bis(7)-tacrine against glutamate-induced retinal ganglion cells damage. BMC Neurosci. 2010;11:31. Baltmr A, Duggan J, Nizari S, Salt TE, Cordeiro MF. Neuroprotection in glaucoma - Is there a future role? 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Exogenous CNTF stimulates axon regeneration of retinal ganglion cells partially via endogenous CNTF. Mol Cell Neurosci. 2009;41(2):233-46. Muller A, Hauk TG, Fischer D. Astrocyte-derived CNTF switches mature RGCs to a regenerative state following inflammatory stimulation. Brain. 2007;130(Pt 12):3308-20. Moore DL, Goldberg JL. Multiple transcription factor families regulate axon growth and regeneration. Dev Neurobiol. 2011;71(12):1186-211. Liu K, Tedeschi A, Park KK, He Z. Neuronal intrinsic mechanisms of axon regeneration. Annu Rev Neurosci. 2011;34:131-52. Park KK, Liu K, Hu Y, Smith PD, Wang C, Cai B, et al. Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway. Science. 2008;322(5903):963-6. Moore DL, Blackmore MG, Hu Y, Kaestner KH, Bixby JL, Lemmon VP, et al. KLF family members regulate intrinsic axon regeneration ability. Science. 2009;326(5950):298-301. Mak HK, Ng SH, Ren T, Ye C, Leung CK. Impact of PTEN/SOCS3 deletion on amelioration of dendritic shrinkage of retinal ganglion cells after optic nerve injury. Exp Eye Res. 2020;192:107938. Smith PD, Sun F, Park KK, Cai B, Wang C, Kuwako K, et al. SOCS3 deletion promotes optic nerve regeneration in vivo. Neuron. 2009;64(5):617-23. Joshi Y, Soria MG, Quadrato G, Inak G, Zhou L, Hervera A, et al. The MDM4/MDM2-p53-IGF1 axis controls axonal regeneration, sprouting and functional recovery after CNS injury. Brain. 2015;138(Pt 7):1843-62. Nawabi H, Belin S, Cartoni R, Williams PR, Wang C, Latremoliere A, et al. Doublecortin-Like Kinases Promote Neuronal Survival and Induce Growth Cone Reformation via Distinct Mechanisms. Neuron. 2015;88(4):704-19. Cartoni R, Norsworthy MW, Bei F, Wang C, Li S, Zhang Y, et al. The Mammalian-Specific Protein Armcx1 Regulates Mitochondrial Transport during Axon Regeneration. Neuron. 2016;92(6):1294-307. Belin S, Nawabi H, Wang C, Tang S, Latremoliere A, Warren P, et al. Injury-induced decline of intrinsic regenerative ability revealed by quantitative proteomics. Neuron. 2015;86(4):1000-14. Zemlin WR, Daniloff RG, Shriner TH. The difficulty of listening to time-compressed speech. J Speech Hear Res. 1968;11(4):875-81. Fujita Y, Yamashita T. Axon growth inhibition by RhoA/ROCK in the central nervous system. Front Neurosci. 2014;8:338. Supplementary Files FigureS1.tif FigureS2.tif TableS1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Reject and Resubmit 07 Nov, 2023 Editor assigned by journal 18 Oct, 2023 First submitted to journal 15 Oct, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3447824","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":241093170,"identity":"4add8936-4319-4d71-a521-d6825b661979","order_by":0,"name":"Wulong Zhang","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Wulong","middleName":"","lastName":"Zhang","suffix":""},{"id":241093172,"identity":"523ad380-ac03-49de-8e53-c2683a3d3597","order_by":1,"name":"Yujue Wang","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Yujue","middleName":"","lastName":"Wang","suffix":""},{"id":241093174,"identity":"9b3b6f2a-47ca-44b0-9077-f660d7b42ced","order_by":2,"name":"Lemeng Feng","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Lemeng","middleName":"","lastName":"Feng","suffix":""},{"id":241093176,"identity":"2a30d8f7-4dfc-420c-b38e-a24d3870ee2c","order_by":3,"name":"Cheng Zhang","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Zhang","suffix":""},{"id":241093177,"identity":"bfc6677a-7db2-4ad8-bb6c-d9d57233f85e","order_by":4,"name":"Weiming Zhu","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Weiming","middleName":"","lastName":"Zhu","suffix":""},{"id":241093178,"identity":"05335a7b-869c-4693-9b17-454235286a97","order_by":5,"name":"Xin Li","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Li","suffix":""},{"id":241093179,"identity":"d9c8ff8e-f86a-40ad-b165-529809a9bde2","order_by":6,"name":"Ye He","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"He","suffix":""},{"id":241093180,"identity":"8e56ba9f-d2f0-4014-bdc2-8f6a9ab72a7d","order_by":7,"name":"weitao Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYPACCTl+ZuYDBz5UEKOYDUxaGEu2tyUenHGGeC0ViRvOnDE+zNtChA6D++0PHxf8kkicOSPnwwHeBgZ5frEDBLQc4zE2ntknYdwvkbvhgOQOBsOZsxPwazE7xsMmzdsjITtzBlCL4RmGBIPbBLWwPwNpYdxwI+fBgcQ2orQwmEnz/JBQBHqf4cBBYrTYH8sxNuZtkAAFssHBhjMShP0i2Xz84WOeP3WgqHz8+U+FjTy/NAEtYMDYBmdKEKEcDP4Qq3AUjIJRMApGJAAAsNFKFAtVjhsAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6320-3209","institution":"Xiangya Hospital Central South University","correspondingAuthor":true,"prefix":"","firstName":"weitao","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2023-10-15 08:27:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3447824/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3447824/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":45102167,"identity":"f6e72ee6-6769-4401-87f2-719a6a79fce2","added_by":"auto","created_at":"2023-10-23 17:47:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1321137,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence staining showed that the three generations cells dedifferentiated from rat retina had a positive expression of Müller cell-specific marker GS (A), FACS showed that 95.3% of the third-generation purified cells were immunoreactive for GS (B). Western blot analysis showed that the purified cells rarely express the specific markers of other cells in each layer of the retina (C, D).\u003c/p\u003e","description":"","filename":"FIGURE1.png","url":"https://assets-eu.researchsquare.com/files/rs-3447824/v1/31f1b1ddc199355a870721d3.png"},{"id":45102168,"identity":"bcf64eee-8126-4c07-8b43-0a2cd9a5372a","added_by":"auto","created_at":"2023-10-23 17:47:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1184811,"visible":true,"origin":"","legend":"\u003cp\u003eCell proliferation was observed on 3,5,7,10, and 14 days by phase contrast microscopy (A). The scale bar was 100 μm. The diameters of the neurosphere were 11.55 μm, 29.64 μm, 61.82 μm, 88.36 μm and 177.73 μm at 3,5,7,10 and 14 days, respectively (B).\u003c/p\u003e","description":"","filename":"FIGURE2.png","url":"https://assets-eu.researchsquare.com/files/rs-3447824/v1/6332e23d0bbc5c748c6c762d.png"},{"id":45102172,"identity":"738b3212-c615-4923-aedf-5ffcc5a0b1d6","added_by":"auto","created_at":"2023-10-23 17:47:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":707902,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence staining showed that the stem cells within the cell spheres had a positive expression of retinal stem cell-specific markers Nestin (90.60%), Ki67(89.20%), Pax6(91.00%), Musashi1(89.90%) (A, B). Western blot analysis was used to detect the expression of retinal stem cells makers (C, D). Immunocytochemical analysis of Edu showed that newborn cell spheres had the capacity of effective proliferation (E).\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-3447824/v1/791fb59db97d3bed8db17fb2.png"},{"id":45102173,"identity":"a8612661-d897-4afe-a731-a2011ab5b97d","added_by":"auto","created_at":"2023-10-23 17:47:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1825249,"visible":true,"origin":"","legend":"\u003cp\u003eStem cells dedifferentiated from Müller cells were transfected with AAV-STAT3 (A). Bar=100 μm. Immunocytochemical analysis showed that in the 12 days RGCs were stained with Tuj1 and Pax6 (B-C). Bar=200 μm. Western blot analysis showed that the amount of protein and mRNA of GAP-43, phosphorylated STAT3 and its downstream genes p21, Irf1 and Sprr1a increased significantly in STAT3 and STAT3+Y27632 group (D, E). Quantitative RT-PCR assay to measure the mRNA level of Socs3, Pten, Klf9, Mdm4, Dclk2, Armcx1, c-myc, and Nrn1 in control group, STAT3 group and STAT3+Y27632 group (F-M).\u003c/p\u003e","description":"","filename":"FIGURE4.png","url":"https://assets-eu.researchsquare.com/files/rs-3447824/v1/40e74f951de06081470b6520.png"},{"id":45102169,"identity":"9b592d4d-db64-4779-83c8-c85195191596","added_by":"auto","created_at":"2023-10-23 17:47:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":625449,"visible":true,"origin":"","legend":"\u003cp\u003eThe protein expression and phosphorylation levels of STAT3 in Müller differentiated RGCs in the STAT3 group, shSTAT3 group, Y27632 group and STAT3+Y27632 group (A-C). The mRNA level of axonal regeneration associated proteins Socs3, Pten, Klf9, Mdm4, Dclk2, Armcx1, C-myc, and Nrn1 in STAT3 group, shSTAT3 group, Y27632 group and STAT3+Y27632 group (D-G). Quantitative RT-PCR assay to measure the mRNA level of pluripotent associated proteins Esrrb, Prdm14, Sox2, and Rex1 in STAT3 group and STAT3+Y26632 group on D0, D3, D7, and D12 (H-K). Quantitative RT-PCR assay to measure the mRNA level of differentiation-associated proteins Nestin, Eomes, Mixl1, and Gata4 in the STAT3 group and STAT3+Y26632 group on D0, D3, D7, and D12 (L-O).\u003c/p\u003e","description":"","filename":"FIGURE5.png","url":"https://assets-eu.researchsquare.com/files/rs-3447824/v1/d7a7cbd7e5f5381fd73def91.png"},{"id":45102175,"identity":"f43d842b-7fae-4dad-8b7d-f66b1584e352","added_by":"auto","created_at":"2023-10-23 17:47:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2751355,"visible":true,"origin":"","legend":"\u003cp\u003eOcular hypertension was induced using laser photocoagulation. The mean IOP level of glaucomatous eyes were detected by a digital tonometer from day 3 to day 60 (A). HE staining of RGCs in glaucoma rat model at day 15, day 30 and day 60. The number of RGCs was also caculated (B and C). TUNEL staining of RGCs in glaucoma rat model at day 3, day 10, day 30, and day 60. Scale bar=100 μm.\u003c/p\u003e","description":"","filename":"FIGURE6.png","url":"https://assets-eu.researchsquare.com/files/rs-3447824/v1/94e82b1a86904fed16b343db.png"},{"id":45102174,"identity":"570daddd-94d7-4138-8df8-4d09d1a6aa14","added_by":"auto","created_at":"2023-10-23 17:47:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3010548,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentiation and transplantation of retinal stem cells in the glaucoma rat model. Immunofluorescence staining of retinal tissue sections showed the GFP positive cells in four groups. Group A: control group; group B: AAV-STAT3 group; group C: Y27532 group; group D: AAV-STAT3+ Y27532 group (A-B). IF staining of TUJ-1 protein in the retina of control group, Glaucoma group, and STAT3+Y27632 group mouse models (C). Fundus photography and OCT examination in the control group, glaucoma group, AAV-STAT3 group, Y27632 group, and AAV-STAT3+ Y27632 group rat models. RGC layer thickness was measured (D and F). Flash-VEP examination in control group, glaucoma group, AAV-STAT3 group, Y27632 group, and AAV-STAT3+Y27632 group rat models (E). P1 latency in the control group, glaucoma group, AAV-STAT3 group, Y27632 group, and AAV-STAT3+Y27632 group (G). N1-P1 amplitude in the control group, glaucoma group, AAV-STAT3 group, Y27632 group, and AAV-STAT3+Y27632 group. Scale bar=100 μm.\u003c/p\u003e","description":"","filename":"FIGURE7.png","url":"https://assets-eu.researchsquare.com/files/rs-3447824/v1/9b743f211a4554d78aab4a74.png"},{"id":45102170,"identity":"6a47ee88-fd9a-424d-81c7-63b4d5518008","added_by":"auto","created_at":"2023-10-23 17:47:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":405525,"visible":true,"origin":"","legend":"\u003cp\u003eSTAT3 combined with Y27632 enhanced the pluripotency and inhibited the differentiation of Müller cell differentiated RGCs. By this mechanism, mRNA levels of axon growth-related proteins are regulated, and axon regeneration is promoted.\u003c/p\u003e","description":"","filename":"FIGURE8.png","url":"https://assets-eu.researchsquare.com/files/rs-3447824/v1/9e62a93cf868c07c14f90d29.png"},{"id":45102741,"identity":"b8385baf-b887-47bd-907d-c0ebd9432ec5","added_by":"auto","created_at":"2023-10-23 18:03:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4361542,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3447824/v1/e7b291df-a48f-49fc-bcc0-65c8c4c332ff.pdf"},{"id":45102165,"identity":"b77bddd1-a380-4d58-b2e1-39d5a529651b","added_by":"auto","created_at":"2023-10-23 17:47:17","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":126668,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3447824/v1/270e7bc916d69b68127a3fba.tif"},{"id":45102399,"identity":"00ec9b6e-9382-4f41-9897-d6f38ad5051c","added_by":"auto","created_at":"2023-10-23 17:55:17","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":84350,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3447824/v1/c2ba8bd440aca26d19fefdd9.tif"},{"id":45102164,"identity":"fd5cb42b-9192-4a85-b9d3-4c7d11d74ce9","added_by":"auto","created_at":"2023-10-23 17:47:17","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15391,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3447824/v1/6e668abeece990865b061007.docx"}],"financialInterests":"","formattedTitle":"STAT3 combined with Y27632 to treat glaucoma by promoting axon growth of Müller differentiated retina ganglion cells","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eGlaucoma is the first irreversible blindness eye disease in the world. In 2010, there were approximately 84\u0026nbsp;million glaucoma patients. It is estimated that by 2020, the number of glaucoma patients in the world will reach 796\u0026nbsp;million(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Glaucoma is a class of neurodegenerative diseases that can be generally classified as primary glaucoma and secondary glaucoma(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The selective and progressive death of retinal ganglion cells (RGCs) is the common pathway and final outcome of optic nerve damage in glaucoma(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The goal of glaucoma treatment is to prevent the progressive loss of neurons and protect the optic nerve, thereby preserving the patient's visual function. Elevated pathological intraocular pressure is usually considered as the leading risk factor for optic nerve damage in glaucoma(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Thus, the current main treatment methods typically focus on the control of intraocular pressure. However, some patients did not receive good results through ocular hypotensive therapy, mainly due to the pathological changes in glaucoma are not only related to the changes in intraocular pressure(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Therefore, exploring the mechanisms of death of RGCs, seeking new protection to delay the death of RGCs, and repair or regenerate ganglion axons are fundamental strategies for the treatment of glaucoma. At present, the protection and treatment of the optic nerve of glaucoma mainly included: improvement of optic disc microcirculation, glutamate pathway inhibitors, neurotrophic factors, induction of heat shock protein expression, and anti-oxidation therapy(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). However, these methods are still unable to prevent RGCs damage and protect the optic nerve effectively. Besides, these methods are not helpful for those patients who have lost the RGCs with advanced or absolute glaucoma.\u003c/p\u003e \u003cp\u003eIn recent years, stem cell research has brought new hope for the alternative treatment of glaucoma RGCs. Many studies found that retinal M\u0026uuml;ller cells are a kind of abundant and self-derived potential retinal stem cells(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). They provide nutrient and metabolic support for the retinal neurons and neurotransmitter cycle. Therefore, they may become essential cell sources for retinal neuron regeneration. In our previous studies, we have successfully purified retinal M\u0026uuml;ller cells in vitro. The reinal stem cells also can be induced dedifferentiation(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Simultaneously, based on M\u0026uuml;ller cell-derived stem cells, it was also confirmed that the Atoh7 gene could promote a large number of RGCs directional differentiation, with a differentiation rate of 50.4%(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). However, in vivo studies showed that the differentiated RGCs did not appear axon growth and just expressed the RGCs specific markers Thy1.1 and Brn-3b, which lead to function loss in conducting signals. In addition, although it was found that in vitro RGCs differentiated with axons, their length was only about 600 \u0026micro;m. Therefore, how to regenerate the retinal M\u0026uuml;ller cell differentiated RGCs restart the axon growth, and guide them along the correct route to the brain projection zone has become a problematic point in current research.\u003c/p\u003e \u003cp\u003eCurrent research has confirmed that the reactivation of nerve growth ability is related to the transcriptional activator in nerve cells(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Among them, STAT3 plays an regulatory function in the regeneration of RGCs(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Studies have shown that the expression of the endogenous gene STAT3 is low in normal retinas. It often exists in cells in a non-phosphorylated form when cytokines or growth factors are activated by binding to receptors, activated STAT3 is transferred from the cytoplasm into the nucleus and participated in regulating the gene expression(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Research indicates that subretinal delivery of overexpressing AAV-STAT3 is protective against photoreceptor death. The results demonstrate the integration and expression of STAT3 in M\u0026uuml;ller cells and photoreceptors (PR) which is protective against neuronal insults by enhancing the STAT3 signaling(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). It\u0026rsquo;s been reported that ROCK is negatively related to cell mitosis and nerve regeneration(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Rho/ROCK signaling pathway inhibitor Y27632 phosphorylates STAT3. The combination of Y27632 and STAT3 efficiently promotes neurite cell axon regeneration and significantly up-regulates its downstream genes p21, Irf1, and Sprr1a(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Therefore, this study was designed to investigate the effect of combination of Y27632 and STAT3 in regenerating ganglion cell axons derived from retinal M\u0026uuml;ller cells, and to explore its regulatory mechanisms, laying the foundation for the regeneration of glaucoma nerves.\u003c/p\u003e"},{"header":"MATERRIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eSD rats, without specific pathogen grade, were obtained from Central South University Animal Experimental Department. The Central South University Animal Care and Use Committee approved this research.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eM\u0026uuml;ller cells extraction\u003c/h2\u003e \u003cp\u003eThe extraction and culture methods of M\u0026uuml;ller cells have been reported in our previous studies(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The eyes of P21 rats were taken and rinsed with PBS containing penicillin/streptomycin (Sigma). After removing the cornea and lens of the eyeball, the retina and retinal pigment epithelium were separated, and the complete retina of the rat was obtained. First, the rat retina was mechanically separated. Next, small tissue fragments were added to trypsinized with 0.25% trypsin-EDTA for digestion for 20 min. After digestion was terminated by adding medium, centrifugation was carried out. The obtained cells were added to DMEM containing 20% FBS for culture. The adherent growth of M\u0026uuml;ller cells was observed after 5\u0026ndash;7 days. The adherent M\u0026uuml;ller cells were digested and re-inoculated in DMEM containing 20% fetal bovine serum for about 7 days. The cells were then inoculated in DMEM/F12 (Gibco) medium (1 \u0026times; N2 supplement (Gibco), 2 \u0026times; B27 supplement (Gibco), and 20 ng/ml EGF (Peprotech: Rocky Hill, NJ, USA), 10 ng/ mL bFGF (Peprotech), 2 mM glutamine (HyClone:100 \u0026micro;g/ mL penicillin and 100 \u0026micro;g/ mL streptomycin) at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells /cm\u003csup\u003e2\u003c/sup\u003e for 7 days. The nerve spheres were formed in about a week of culture, and the dedifferentiated medium was changed every other day.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLentivirus PGC-FU-Atoh7-GFP Construction\u003c/h2\u003e \u003cp\u003eThe synthesis and construction of GC-Fu-Atoh7-IRES-GFP was performed by GeneChem (Shanghai, China). The MOI of neuroglobules transfected by lentiviral vector was 10(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Our previous studies have shown that AtoH7 induces differentiation of the neurosphere into RGC(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRecombinant AAV-STAT3 Vectors Construction\u003c/h2\u003e \u003cp\u003eAAV-STAT3 vector was prepared by plasmid co-transfection. The recombinant AAV-STAT3 was purified by gradient supercentrifugation with iodiol. The 40% iodixanol fraction was buffer-exchanged with 0.001% Tween in phosphate buffered saline (PBS) and concentrated by 100K Amicon Ultra-15 centrifugal filter units. The volume of the centrifugation filter unit is 200 ml. Then quantitative PCR was used to determine the relative standard titers of DNA-resistant viral genomes in the concentrated bacterial population. The vector concentration was calculated in terms of virus genome/mL under the condition of 2\u0026ndash;4\u0026times;10\u003csup\u003e13\u003c/sup\u003e vg/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eShRNA- STAT3 Expressing Vectors Construction\u003c/h2\u003e \u003cp\u003eThe STAT3-specific shRNA carrying lentiviral vectors were bought from Santa Cruz Biotechnology, Inc (TX, USA). Add the shRNA Plasmid DNA solution (Solution A) directly to the dilute shRNA Plasmid Transfection Reagent (Solution B) using a pipette. Mix gently by pipetting the solution up and down and incubate the mixture 30 minutes at room temperature. Wash the cells twice with 2 ml of shRNA Transfection Medium. For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well. Add the 200 \u0026micro;l shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent Complex (Solution A\u0026thinsp;+\u0026thinsp;Solution B) dropwise to well, covering the entire layer. Gently mix by swirling the plate to ensure that the entire cell layer is immersed in solution. Incubate the cells 6 hours at 37\u0026deg; C in a CO\u003csub\u003e2\u003c/sub\u003e incubator or under conditions normally used to culture the cells. Following incubation, add 1 ml of normal growth medium containing 2 times the normal serum and antibiotics concentration (2x normal growth medium). Lentiviral transduction was performed by incubating RGCs with lentiviruses in growth media, and stable transfectants were selected with puromycin (2 \u0026micro;g/ml) for 1 week.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical Analysis and Axon Length Measurement\u003c/h2\u003e \u003cp\u003eImmunohistochemistry is used to analyze specific markers of cells. 4% paraformaldehyde was used to fix the cells, and the fixation time was 15 minutes. The cells were blocked with 5% goat serum, and the blocking time was 1 hour. Incubate the primary antibody overnight at 4℃. Wash away the primary antibody with PBS and wash 3 times for 10 mins each time. Incubate the cells with a fluorescent secondary antibody (KPL, Rouses Point, NY, USA) for 1 h at room temperature and protected from light. The cells were washed 3 times with PBS for 10 mins each time. DAPI was used to stain the cell nucleus. A confocal microscope (Leica TCS SP5, Leica Microsystems GmbH, Wetzlar, Germany) was used to take pictures of cell staining. Taking the center of the culture plate as the center, randomly select 5 microscope scanning field of view frames in the upper, lower, left, right, and middle five directions, collect images, and use the image analysis system (ImagePro Plus software) to measure the longest protrusion length of RGCs. Three cells were randomly selected for measurement, and the results were averaged for comparison. The experiment was repeated 5 times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eThe purity of the extracted M\u0026uuml;ller cells was detected by flow cytometry, which has been reported in our previous studies (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Adhesive M\u0026uuml;ller cells were digested, resuspended and gently blown into single cells. The cells were fixed with 4% PFA. Cells were sealed with a blocking solution (PBS containing 1% BSA and 0.1% TritonX-100) at 4℃ for 30 mins. After sealing, the cells were incubated with a primary antibody at 4 ℃ for 1 h. Cells were incubated with fluorescence-labeled secondary antibodies at 4℃ for 1 h. After washing the cells with PBS, they were resuspended and analyzed by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEdu assay\u003c/h2\u003e \u003cp\u003eThe proliferation level of the stem cells was measured by the EDU assay. The EDU (RiboBio) solution was diluted with cell medium in the ratio of 1000:1 to prepare an appropriate amount of 50 \u0026micro;M EDU culture medium. The neurospheres were mixed with the medium containing EDU and incubated overnight at 37℃. Wash the cells with PBS twice for 5 mins each time. The neurospheres were fixed with PBS containing 4% paraformaldehyde at room temperature for 30 mins. The cells were incubated with Apollo buffer at room temperature for 30 mins in dark. Cells were incubated with PBS containing 0.5% TritonX-100 at room temperature for 10 mins. The cells were washed with PBS for 5 mins. The nuclei were stained with Hoechst 33342. Fluorescence microscopy was used to obtain the stained images.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using the TRIzol reagent (Sigma, St. Louis, MO, USA) according to the manufacturer\u0026rsquo;s instructions. The purity and quantity of samples were measured using a Nano spectrophotometer. qPCR was conducted to confirm the results for gene expression in samples from all the samples. The reaction conditions of PCR are carried out according to the conditions on the instructions. The RNA expression level was normalized to that of Gapdh. The corresponding quantitative RT-PCR primers were shown in Table S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot\u003c/h2\u003e \u003cp\u003eCells grown on plastic plates were lysed in ice-cold RIPA buffer. Lysates were placed on ice for 30 min and centrifuged at 16,000 g for 15 min at 4\u0026deg;C. The protein concentration was determined using the Micro BCA Protein Assay Kit. The protein is mixed with the loading buffer and heated at 95\u0026deg;C for 10 minutes. The sample was loaded for SDS-PAGE electrophoresis. After the electrophoresis was complete, the protein was transferred to the PVDF membrane in the transfer buffer. The membrane was sealed in PBST containing 5% milk at room temperature for 1 hour. Secondary antibodies were incubated for 1 h at room temperature. Primary antibodies, including anti-STAT3 (Cell Signaling Technology), anti-p-STAT3 (Cell Signaling Technology), Tyrosinase (Santa Cruz), mGluR6 (Abcam), Atoh7 (Abcam), CD31 (Cell Signaling Technology), Rhodopsin (Cell Signaling Technology), β-tubulin ((Cell Signaling Technology), Iba1 (Abcam), Brn3b (Abcam), Pax6 (Abcam), HPC-1 (Abcam), Sprrla1 (Santa Cruz), P21 (Santa Cruz), Irfland (Santa Cruz), Musashi1 (Abcam), Gap43 (Abcam), Ki-67 (Abcam), Nestin (Abcam), and anti-β-actin (Santa Cruz), were used in this study. The immunoreactive bands were imaged with the Bio-Rad ChemiDoc XRS system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eChronic Ocular Hypertension Glaucoma Model of Mice\u003c/h2\u003e \u003cp\u003eThe establishment of a rat model of chronic ocular hypertension has been reported in the literature(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate. After 1% prilocaine was instilled in the right eye for topical anesthesia, the extrascleral aqueous humor drainage vein was photocoagulated by laser. The photocoagulation range is 270 degrees. The laser photocoagulation point is 20 points. The energy of the laser is 0.7W pulse lasting 0.6 seconds. Tonopen was used to detect intraocular pressure at 3, 10, 15, 20, 25, 30, 35, 40, 50, 60 days after photocoagulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStem Cells Transfection and Intraocular Injections\u003c/h2\u003e \u003cp\u003eNeurospheres were transfected by lentivirus PGC-FU-Atoh7-GFP for 24 h. The stem cells were divided into 4 groups: group A: PBS control group, PBS was added to medium; group B: STAT3 group, only added AAV-STAT3 to the medium; group C: Y27632 group ,only add Y27632 to the medium; group D: STAT3\u0026thinsp;+\u0026thinsp;Y27632 group, added both AAV-STAT3 and Y27632 to the medium. After 24 hours of treatment, the neurospheres were isolated into individual cells by accutase. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital. Intravenous injection of 5ul differentiation medium (1 ng/mL BDNF, 30 nmol Brdurd, and 1 \u0026micro;M Ra) containing stem cells at a concentration of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/\u0026micro;L. Eyeball frozen sections were performed for Immunohistochemistry and immunofluorescence analysis after 12 days of retinal stem cell transplantation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence of Eyeball Sections\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining of frozen sections of eyeballs was performed as reported(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Eyeball tissue sections were sealed at room temperature for 1 h in PBS containing 3% BSA and 0.3% TritonX-100. After sealing, sections were incubated with primary antibody at 4℃ overnight. Sections were incubated with secondary antibodies at room temperature for 1 h. Eyeball tissue sections were sealed with antifade solution containing DAPI. Images were taken by fluorescence microscopy(Leica DMI4000B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eVisual Evoked Potential Recording\u003c/h2\u003e \u003cp\u003eTo evaluate retinal ganglion cells (RGCs) function, visual evoked potential (VEP) was employed. Briefly, Prior to VEP recordings, animals were dark -adapted for 12 hours and were prepared under dim red light. The animals were anaesthetized with ketamine and xylazine (87/13 mg/kg), and Pupils dilated by 1% tropicamide eye drops. The positive electrodes were inserted into the skin approximately 4 mm lateral to the midline over visual cortex. The reference electrodes were inserted into the skin above frontal cortex and the ground electrode was placed on the tail. To minimize the variation in the position of the needles in different sessions, their positions were marked on the skin and needles were fixed on the skin using tape. Visual stimulus was presented 60 times using a photic stimulator, with 15 dB intensity and 0.2 Hz stimulation frequency, at a distance of 15 cm. The time of measurement was 300 ms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eOptical Coherence Tomography\u003c/h2\u003e \u003cp\u003eTo measure thickness of RGC layer, OCT was performed. OCT was performed after anesthetization of rats followed by restraining animals in front of the OCT device with dilated pupil. All central retina OCT measurements were performed with spectral domain OCT (Heidelberg Spectralis SD-OCT; Heidelberg Engineering, Heidelberg, Germany) on each rat eyes. Spectralis software was used for retinal layer segmentation. The RGC thickness data was recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData from at least three independently dissociated cultures, each measured in triplicate, were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The data for two-group comparisons were analyzed for statistical significance using two-tailed Student\u0026rsquo;s t-test. Multiple comparisons statistical analysis was performed with one-way ANOVA using SPSS 18.0. P values are indicated with single asterisk (* \u0026lt;0.05), double asterisk (** \u0026lt;0.01) and triple asterisks (*** \u0026lt;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eM\u0026uuml;ller Cell Culture and Identification\u003c/h2\u003e \u003cp\u003eWe found that GS as M\u0026uuml;ller cell-specific marker was significantly high express in three generations cells dedifferentiated from rat retina (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Results also showed that 95.3% of the third-generation purified cells were immunoreactive for GS by Flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The protein expression of specific markers of other retinal cells in purified cells was accessed by WB assay. The results showed that the protein expression of Atoh7, Rhodopsin, Pax6, β-tubulin, Brn-3b, HPC1, Tyrosinase, and mGluR6 were low (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). But the results of WB and Flow cytometry also suggest that there are a small number of other cells in Muller cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStem Cell Culture\u003c/h2\u003e \u003cp\u003eAfter cultured in DMEM/F12 dedifferentiation medium for 24 h, cells proliferate and differentiate into round shapes, and then some cells aggregate to form small cell spheres. After 48\u0026ndash;72 h, the proliferation of cells was significantly accelerated, and the shape of single round cells was decreased compared with the former. The diameter of the cell spheres increased. After 5d, the number and the diameter of the cell sphere continued to increase, the boundary of the cell sphere was clearly defined, and the refractive index was strong. After 7 days, there was a slight increase in the volume of the cell sphere compared to before. At 10th day, the central refractivity of the visible neurosphere was reduced, showing that the cell bodies were atrophied and darkened (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The cell proliferation was observed daily under a phase-contrast microscope and the diameter of the neurosphere was measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We tested the mRNA level of Vsx2, Rax, Otx2 and Sox2 in Muller cells and neurosphere (Fig. S1). The results show that the mRNA level of Vsx2, Rax, Otx2 and Sox2 in neurosphere were significantly increased. When the cell diameter reached 150 \u0026micro;m, the cell sphere was digested with Accutase(Sigma)and passaged to obtain purified neurospheres. After three passages, the purified neurospheres were examined by immunofluorescence. Our results showed that the purified neurospheres are labeled with nestin, ki-67, pax6, and musashi1 antibodies, and the expression level of these four antibodies is approximately 90% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). WB showed that the cell spheres purified after three passages highly expressed nestin, ki-67, pax6, and musashi-1 when compared with the purified M\u0026uuml;ller cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D). These results indicated that the cells purified with three passages in the dedifferentiation medium are dedifferentiated into retinal stem cells. We then used Edu marker to detect the proliferation ability of these retinal stem cells. Our results showed that the proliferation ability of these purified retinal stem cells was very strong (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEffect of STAT3 and Y27632 on Axonal Regeneration of M\u0026uuml;ller Cell Differentiated RGCs\u003c/h2\u003e \u003cp\u003eAfter the retinal stem cells were identified, they were transfected with the lentivirus PGC-FU-Atoh7-GFP. The cells were plated onto 0.01% poly-D-lysine (Sigma)-coated 24 mm coverslips (Corning) at a concentration of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well, and cultured in differentiation medium (1 ng/ml BDNF, 1 \u0026micro;M RA and 1% FBS). Three days later, stem cells were randomly divided into five groups: group A: PBS control group, PBS was added to medium; group B: AAV-STAT3 group, only added AAV-STAT3 to the medium; group C: AAV-shSTAT3 group, only added AAV-shSTAT3 to the medium; group D: Y27632 group, only add Y27632 to the medium; group E: AAV-STAT3\u0026thinsp;+\u0026thinsp;Y27632 group, added both AAV-STAT3 and Y27632 to the medium (Y27632 intervened multiple times). The number of GFP labeled cells increased, the fluorescence intensity was enhanced, and the cytoplasm was uniformly distributed after 48 h intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The differentiation of RGCs in each group was observed every day.\u003c/p\u003e \u003cp\u003eWith the prolongation of the differentiation time, the rounded stem cells gradually grow into a plurality of protrusions. And these cells also extended branches. The axon of the proximal cell body in these cells is thicker than that of the distal body. The axon walks more and more naturally. In the 12 days of differentiation, the length of axon in each group can be observed by IF staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). We also detected the mRNA level of Brn3a and pax6 in RGCs and Muller cells (Fig. S2). The results show that the mRNA level of Brn3a and pax6 in RGCs were significantly increased.\u003c/p\u003e \u003cp\u003eIn the control group, the axon length of ganglion cells was 198.54\u0026thinsp;\u0026plusmn;\u0026thinsp;9.18 \u0026micro;m. The axon length of the ganglion cells in AAV-STAT3 group and shSTAT3 was 265.38\u0026thinsp;\u0026plusmn;\u0026thinsp;13.67 \u0026micro;m and 183.49\u0026thinsp;\u0026plusmn;\u0026thinsp;11.64 \u0026micro;m respectively. In the Y27632 group, the axon length of ganglion cells was 200.35\u0026thinsp;\u0026plusmn;\u0026thinsp;10.25 \u0026micro;m. In the AAV-STAT3\u0026thinsp;+\u0026thinsp;Y27632 group, the axon length of ganglion cells was 437.32\u0026thinsp;\u0026plusmn;\u0026thinsp;21.14 \u0026micro;m. The axon length of ganglion cells in the AAV-STAT3 group was longer than that in the control group. The results showed that the axon length of RGCs in the AAV-STAT3\u0026thinsp;+\u0026thinsp;Y27632 group was significantly longer than that in the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eWestern blot analysis was performed on STAT3 group and STAT3\u0026thinsp;+\u0026thinsp;Y27632 group on day 12. The results showed that the protein expression levels of p-STAT3, p21, GAP43, Irfland, and Sprrla1 in the STAT3 group were significantly higher than those in the control group. The protein expression levels of P-STAT3, P21, GAP43, Irfland, and Sprrla1 in the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group were higher than those in the STAT3 group, and the differences were statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E). mRNA levels of axonal regeneration related proteins Socs3, Pten, Klf9, Mdm4, Dclk2, Armcx1, C-myc, and Nrn1 at D0, D3, D7, and D12 were also analyzed. The experimental results showed that the mRNA level of Socs3, Pten, Klf9 and Mdm4 in STAT3\u0026thinsp;+\u0026thinsp;Y27632 group was significantly lower than that in STAT3 group, and the expression level was the lowest at D12. The mRNA levels of Dclk2, Armcx1, C-MYC and Nrn1 were significantly higher in STAT3\u0026thinsp;+\u0026thinsp;Y27632 group than in STAT3 group. The mRNA level of Dclk2 was the highest at D7 and decreased at D12. The mRNA level of Nrn1 was the highest at D3, but decreased at D7 and D12. The mRNA level of Armcx1 and C-myc was highest at D12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-M).\u003c/p\u003e \u003cp\u003eThe above results suggest that the use of STAT3 alone in vitro could promote the growth of axons. When combined with Y27632, the effect of promoting growth of axon was more substantial in RGCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMechanism of STAT3 Combined with Y27632 on the Regeneration of RGCs Differentiated by M\u0026uuml;ller Cell\u003c/h2\u003e \u003cp\u003eWe used WB to detect the expression levels of STAT3, and p-STAT3 in each intervention groups of M\u0026uuml;ller cell differentiated RGCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. A-C). The results showed that the expression levels of STAT3 and p-STAT3 proteins in the STAT3 group, Y27632 group and STAT3\u0026thinsp;+\u0026thinsp;Y27632 group were all higher than those in the control group, while the expression levels of STAT3 and p-STAT3 proteins in the shSTAT3 group were lower. There was no significant difference in STAT3 and p-STAT3 protein expression levels between the STAT3 and Y27632 group. The expression levels of STAT3 and p-STAT3 proteins in the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group were significantly higher than those in the other groups. mRNA levels of pluripotent related genes (Esrrb, Prdm14, Sox2, and Rex1) and differentiated related genes (Nestin, Eomes, Mixl1, and Gata4) were also detected at D12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). The results showed that mRNA levels of pluripotent related genes (Esrrb, Prdm14, Sox2, and Rex1) in the STAT3, Y27632 and STAT3\u0026thinsp;+\u0026thinsp;Y27632 groups were significantly increased, while mRNA levels in the shSTAT3 group were significantly decreased. mRNA levels of pluripotent related genes in the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group were considerably higher than that in other groups. In the STAT3, Y27632 and STAT3\u0026thinsp;+\u0026thinsp;Y27632 groups, mRNA levels of differentiated related genes (Nestin, Eomes, Mixl1, and Gata4) were significantly decreased, while mRNA levels in the shSTAT3 group were significantly increased. The mRNA level of differentiated related genes in the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group was markedly lower than that in the other groups. We also detected the mRNA levels of axonal regeneration related proteins Socs3, Pten, Klf9, Mdm4, Dclk2, Armcx1, C-MYc, and Nrn1 at D12. The mRNA levels of Socs3, Pten, Klf9, and Mdm4 in the shSTAT3 group were increased dramatically, while those in the STAT3, Y27632, and STAT3\u0026thinsp;+\u0026thinsp;Y27632 groups were significantly decreased. mRNA level of Dclk2, Armcx1, C-myc, and Nrn1 were significantly increased in the STAT3 group, Y27632 group, and STAT3\u0026thinsp;+\u0026thinsp;Y27632 group, while those in the shSTAT3 group were significantly decreased. mRNA levels of pluripotent related genes (Esrrb, Prdm14, Sox2, and Rex1) in the STAT3 and STAT3\u0026thinsp;+\u0026thinsp;Y27632 groups were detected at D0, D3, D7, and D12. mRNA levels of pluripotent related genes in the STAT3 group and the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group were higher than those in the control group at all time. The mRNA expression level of the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group was significantly higher than that of the STAT3 group and control group. mRNA levels of differentiated related genes (Nestin, Eomes, Mixl1, and Gata4) in the STAT3 group and the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group were also detected at D0, D3, D7, and D12. The results showed that the mRNA levels of differentiated genes in the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group and the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group were lower than those in the Control group at all time points, and the expression levels of mRNA in the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group were significantly lower than those in the STAT3 and control groups. The above results suggest that STAT3 combined with Y27632 may improve the pluripotency of M\u0026uuml;ller differentiated RGCs cells to regulate the expression level of proteins related to axon regeneration and promote its axon regeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eDetection of Ganglion Cells in Rat Chronic Ocular Hypertension Glaucoma Model\u003c/h2\u003e \u003cp\u003eTo further verified the role of STAT3 and Y27632 in promoting the growth of RGCs axon, we established rat chronic ocular hypertension glaucoma model. A digital tonometer was used to measure IOP after laser treatment at different time points. Compared with normal eyes, the mean IOP in model eyes increased significantly from 3 to 30 days after modeling. The intraocular pressure gradually increased with time, reaching its maximum value in 10\u0026ndash;25 days. The intraocular pressure began to decrease on the 30th day and reached a normal level in about 60 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eHE stain was used to detect the numbers of retina ganglion cell nucleus in the glaucoma rat model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The experimental results showed that with the increase of intraocular pressure, the number of RGC cells gradually decreased. The intraocular pressure decreased to normal levels on day 60, but the number of RGCs continued to decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Moreover, TUNEL staining was used to assess whether the RGCs of glaucoma rats undergo apoptosis. Experimental results show that RGCs in the retinal ganglion cell layer undergo apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results showed that the number of apoptotic cell nuclei gradually increased as the intraocular pressure gradually increased. At day 3, RGCs was still regularly shaped and uniformly distributed in retina ganglion cell layer. At day 10, cytoplasm and nucleus of part of RGCs began to disintegrate. At day 30, more cell debris were detected in retina ganglion cell layer. At day 60, most of RGCs disappeared with a few debris left. The proportion of TUNEL positive cells were 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1,8.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7,13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6,57.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0%, respectively. In the meantime, we found an increase in apoptosis of cells in outer nuclear layer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eGrowth of RGC Axon in Glaucoma Rat Model\u003c/h2\u003e \u003cp\u003eStem cells were transfected with lentivirus PGC-FU-Atoh7-GFP and cultured in differentiation medium. After 24 hours, these stem cells were randomly divided into four groups: group A: control group, PBS was added to medium; group B: AAV-STAT3 group, only added AAV-STAT3 to the medium; group C: Y27632 group, only add Y27632 to the medium; group D: AAV-STAT3\u0026thinsp;+\u0026thinsp;Y27632 group, added both AAV-STAT3 and Y27632 to the medium. 24 hours later, we divided glaucoma rat models randomly into four groups as above and injected stem cells into the vitreous cavity in each group. IF staining of retinal tissue sections was performed to detect cell-specific markers GFP and ZO-1 (a kind of tight junction protein) after 12 days of retinal stem cell transplantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The length of axons in each group can be observed by the chemical staining of immunofluorescence cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). In the control group, the axon length of RGCs was 360.5 \u0026micro;m. The axon length of RGCs in the AAV-STAT3 group was 877.3 \u0026micro;m. The axon length of RGCs in the Y27632 group was 377.9 \u0026micro;m. In the AAV-STAT3\u0026thinsp;+\u0026thinsp;Y27632 group, the axon length of ganglion cells was 1986.7 \u0026micro;m. The axon length of RGCs in the AAV-STAT3 group was longer than that in the PBS group. The axon length of RGCs in the AAV-STAT3\u0026thinsp;+\u0026thinsp;Y27632 group was significantly longer than that in the other groups. We observed that the axons of RGCs were significantly longer than those of the glaucoma model group after transplantation of M\u0026uuml;ller cell differentiated RGCs after STAT3\u0026thinsp;+\u0026thinsp;Y27632 intervention by retinal lamination (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). We observed that the axons of RGCs were significantly longer than those of the glaucoma model group after transplantation of M\u0026uuml;ller differentiated RGCs intervened with STAT3\u0026thinsp;+\u0026thinsp;Y27632 by retinal lamination. We observed the thickness of RGCs layer in each group through OCT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The RGCs layer of the STAT3, Y27632, and STAT3\u0026thinsp;+\u0026thinsp;Y27632 groups was significantly thicker than that of the glaucoma model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). In comparison the RGCs layer of the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group was significantly thicker than that of the other groups. Flash-VEP was used to detect optic nerve conduction in each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). The optic nerve conduction function in the STAT3 and STAT3\u0026thinsp;+\u0026thinsp;Y27632 groups were significantly better than that of the glaucoma model group, and the optic nerve conduction function in the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group was significantly better than that of the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG-H). STAT3 combined with Y27632 enhanced the pluripotency and inhibited the differentiation of M\u0026uuml;ller cell differentiated RGCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eGlaucoma is a main blinding disease in the world. It causes permanent damage of the optic nerve since its\u0026rsquo; impossible for mammalian nerves regenerated. Glaucoma is characterized by progressive loss of retinal ganglion cells along with their optic nerve axons. Reduction of intraocular pressure (IOP), nutrition of optic nerve, or the antioxidation treatment are the therapies of glaucoma. These treatments postpone further death of the ganglion cell. However, these methods can not make the apoptosis RGCs regenerate. Therefore, for the patient with massive death of ganglion cells, we must find an effective therapy to activate the regeneration of ganglion cells to reestablish visual pathway and help these patients to regain their vision.\u003c/p\u003e \u003cp\u003eAtoh7 is a member of the bHLH family, and in our previous research, it had been verified that Atoh7 regulates M\u0026uuml;ller cell-derived stem cells differentiating into RGCs both in vitro and in vivo. This makes it possible for us to replace glaucoma-induced apoptosis of RGCs cells by transforming M\u0026uuml;ller cell-derived stem cells differentiating into RGCs. However, the axonal growth of the regenerated RGCs determines the reconstruction of the visual pathway. How to promote the growth of regenerated RGCs axons becomes our research focus. Studies have shown that STAT3 plays an important role in the axon growth of retinal ganglion cells. However, the specific mechanism of STAT3 promoting RGCs axon regeneration is still unknown.\u003c/p\u003e \u003cp\u003eSTAT3 is a kind of bifunctional cytoplasmic protein coupling with tyrosine phosphorylation signal pathway in the cytoplasm (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Normally STAT3 protein is in a non-phosphorylated form in cells, when cytokines or growth factors bind to the receptor, tyrosine kinases (JAK) that bind to receptors are activated. Activated JAK phosphorylates the 705th position of tyrosine residue (Tyr705) in the STAT3 cytoplasm. They combined to form the JAK/STAT3 homodimers. Then the homodimers entered into the nucleus, recognized specific DNA sequences and regulated the transcription of target genes. Therefore, the amount of nuclear-activated STAT3 represents the activation of the JAK/STAT3 pathway in the cell. Activated STAT3 will be dephosphorylated in the nucleus after transmitting signals and be restored to the monomeric form. And then, it will deactivate and return to the cytoplasm to participate in the next round of signal transduction. The activation degree of JAK/STAT3 in normal peripheral nerves is low. Only phosphorylated activated STAT3 may translocate into the nucleus to regulate the expression of certain genes to regenerate peripheral never. Our experimental results also confirmed that the axons of RGCs in the STAT3 overexpressed group were significantly longer than those in the STAT3 inhibited group. We hypothesized that STAT3 regulates the growth of RGCs axons through the above mechanisms.\u003c/p\u003e \u003cp\u003eResearch showed that when adeno-associated virus vector containing STAT3 gene was injected into vitreous cavity of the optic nerve contusion model rats, they found that the number of STAT3 positive cells increased significantly as well as the expression of STAT3 protein and STAT3 mRNA increased by 4 to 6 fold after 3 weeks. And the expression of GAP-43 (growth-associated protein-43), a marker of retinal ganglion cell axon regeneration, significantly increased. The optic nerve 3D image analysis which used nerve fiber anterograde tracer CTb-594 showed that the axons of ganglion cells after STAT3 gene infected increased about 200 microns than the control group. But the axon density did not increase and formed many U-turn and right-angle turn. These results indicated that STAT3 signal transduction can increase the length of retinal ganglion cell axons and promote the extension of axonal growth. Another research showed that the use of the JAK2 inhibitor AG490 in vitro in RGCs inhibited CNTF-mediated axonal growth and significantly reduced the regenerative effect of RGCs in response to inflammatory stimuli in vivo. These results confirmed that the activation of JAK and STAT3 played an important role on the axis initial stage of outgrowth (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Studies have demonstrated that STAT3 signaling pathway plays an important role in the axon growth of RGCs(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). The same results were obtained in our experiment. Deletion Pten, a negative regulator of the mammalian target of rapamycin (mTOR) pathway in adult RGCs, promotes robust axon regeneration after optic nerve injury(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). A high-throughput gene profiling study revealed that the deletion of Klf9 gene substantially promotes optic nerve regeneration in adults RGCs(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Researches confirmed that a genetic deletion of PTEN, SOCS3, or PTEN/SOCS3 allows partial axon regeneration in the optic nerve after optic nerve crush(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Inhibition of Mdm4 in the eye and spinal cord promotes axonal regeneration and sprouting of the optic nerve after crush and of supraspinal tracts after spinal cord injury(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Our research showed that the mRNA level of PTEN, SOCS3, Klf9, and Mdm4 decreased significantly in AAV-STAT3 group. Overexpression of Dclk2, Armcx1, c-myc, and Nrn1 are closely related to the regeneration of RGCs axon(\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). The mRNA level of Dclk2, Armcx1, c-myc, and Nrn1 significantly increased in the AAV-STAT3 group. The inhibition of STAT3 expression inhibited the growth of the RGCs axon. The mRNA expression levels of RGCs axon-regenerated proteins were significantly opposite between the shSTAT3 group and the AAV-STAT3 group. All the above results confirmed that STAT3 overexpression could promote axonal regeneration of M\u0026uuml;ller differentiated RGCs.\u003c/p\u003e \u003cp\u003eRecently years, studies have shown that Rho-associated coiled-coil-containing protein kinase (Rho-ROCK) is negatively correlated with cell mitosis and nerve regeneration. Rho is a small molecule homopolymer of the GTPases superfamily and is a mammalian gene homologue of the Ras superfamily. Its biological function is mainly through its downstream effector molecule ROCK. ROCK is a serine/threonine-protein kinase whose molecular structure includes an amino-terminal catalytic domain, an intermediate domain which combined with Rho\u0026rsquo;s α-coiled-coil, a carboxy-terminal catalytic domain, and a Cys/His region. Activated Rho-GTP activates ROCK by binding to the alpha coiled-coil domain of ROCK and exposing the catalytic center of ROCK(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Our results have shown that STAT3 could be phosphorylated by Y27632, which is an inhibitor of the Rho-ROCK signaling pathway. The STAT3\u0026thinsp;+\u0026thinsp;Y27632 combination can effectively promote axon regeneration of M\u0026uuml;ller cell differentiated RGCs. The axon length of RGCs in the AAV-STAT3\u0026thinsp;+\u0026thinsp;Y27632 group was the longest, and the expression of GAP-43, phosphorylated STAT3 and its downstream genes p21, Irf1 and Sprr1a was significantly increased. Inhibition of the Rho/ROCK signaling pathway not only reduced the U-turn of the axon and avoid navigation errors but also promote axon regeneration in RGCs. Our research is the first to confirmed that the combined treatment of STAT3\u0026thinsp;+\u0026thinsp;Y27632 on M\u0026uuml;ller-derived RGCs can improve the differentiation rate of RGC cells and significantly increase the length of axons, which is a significant improvement for RGCs regeneration. The mRNA levels of RGCs axon-growth-related proteins Pten, Socs3, Klf9, and Mdm4 in the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group were significantly lower than those in the STAT3 group, while the mRNA levels of Dclk2, Armcx1, C-myc, and Nrn1 were significantly higher than those in the STAT3 intervention group. In order to analyze the mechanism underlying the effect of STAT3\u0026thinsp;+\u0026thinsp;Y27632 in promoting axon growth of M\u0026uuml;ller cells differentiated RGCs, we tested the mRNA level of pluripotency genes (Esrrb, Prdm14, Sox2, and Rex1) and differentiation genes (Nestin, Eomes, Milx1, and Gata4). In the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group, mRNA level of pluripotency genes (Esrrb, Prdm14, Sox2, and Rex1) compared with STAT3 group increased significantly, the mRNA level of differentiation genes (Nestin, Eomes Milx1, and Gata4) decreased obviously. We concluded that the reason for overexpression of STAT3 promote axon growth could be launched M\u0026uuml;ller cells differentiated RGCs pluripotency. The combination of STAT3 and Y27632 can promote the axon growth of M\u0026uuml;ller cells differentiated RGCs better than STAT3 alone, which is the same mechanism.\u003c/p\u003e \u003cp\u003eFurthermore, rat chronic ocular hypertension glaucoma model were made to access the mechanism in vivo. in this study. To verify whether STAT3\u0026thinsp;+\u0026thinsp;Y27632 improve growth of RGCs differentiated from stem cells, the stem cells were transfected with lentivirus PGC-FU-Atoh7-GFP. The stem cells were transplanted into the vitreous cavity of the glaucoma rat model. After 14 days, retina sections of glaucoma models were used to examine immunoreactivity for RGCs-specific marker GFP and ZO-1. The result showed that length of RGCs axons was significantly longer in the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group, which verified that combination STAT3 with Y27632 can promote the growth of RGCs axons. OCT results showed that the RGCs cell layer in the STAT3\u0026thinsp;+\u0026thinsp;Y27632 group was significantly thicker than that in the other intervention groups. We observed a delay in peak latencies of P1 waves both in the Glaucoma group and Y27632 group. Additionally, N1-P1 amplitudes in STAT3\u0026thinsp;+\u0026thinsp;Y27632 group are significantly higher compared with other groups excepted Con group. No significance was observed in the P1 latency and N1-P1 amplitude between Glaucoma group and Y27632 group. These results suggests that M\u0026uuml;ller cell differentiated RGCs transplants can integrate into preexisting retinal circui and function physiologically.\u003c/p\u003e \u003cp\u003eIn conclusion, STAT3 combined with Y27632 can significantly promote the axon growth of ganglion cells which was dedifferentiated from retina M\u0026uuml;ller cells either in vitro or in vivo.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRGCs: retinal ganglion cells; VEP: Visual evoked potential; OCT: Optical coherence tomography; STAT3: signal transducer and activator of transcription 3; Rho-ROCK: Rho-associated coiled-coil-Containing protein kinase; MOI: multiplicity of infection; FACS: Flow cytometry; PBS: phosphate buffered saline; HE: Hematoxylin and eosin; qRT-PCR: Quantitative real time polymerase chain reaction; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IOP: intraocular pressure: BDNF: brain-derived neurotrophic factor.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWulong Zhang:Collection and assembly of data, data analysis and interpretation, manuscript writing, Writing - Original Draft.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYujue Wang: Data Curation; Formal Analysis; Writing - Original Draft.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLemeng Feng, Cheng Zhang: Data interpretation, Visualization\u003c/p\u003e\n\u003cp\u003eWeiming Zhu, Xin Li, Ye He: Collection of data, data interpretation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWeitao Song: Conception and design, Resources, Supervision, Writing - Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grant from National Nature Science Fund of China（81974132），National Nature Science Fund of China（81770927），National Key R\u0026amp;D Program of China(2021YFA1101202)；Hunan Provincial Health Commission（20220702839）and Hunan Natural Science Foundation (No.2022JJ30076)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAVAILABILITY OF DATA AND MATERIALS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data generated or analyzed during this study are included in this published article. The use of mouse Müller cells was in accordance with the relevant guidelines and regulations, and the experimental protocols were approved by the Medical Ethics Committee of the Xiangya Hospital of Central South University. This study is in accordance with ARRIVE guidelines\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe use of mouse Müller cells was in accordance with the relevant guidelines and regulations, and the experimental protocols were approved by the Medical Ethics Committee of the Xiangya Hospital of Central South University. This study is in accordance with ARRIVE guidelines\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR DETAILS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHunan Key Laboratory of Ophthalmology, Eye Center of Xiangya Hospital, Central South University, Hunan, 410008, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJonas JB, Yang D, Wang N. 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Front Neurosci. 2014;8:338.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Müller cells, Retinal ganglion cells, STAT3, Y27632, Glaucoma","lastPublishedDoi":"10.21203/rs.3.rs-3447824/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3447824/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eM\u0026uuml;ller differentiated RGCs have potential therapeutic value for glaucoma. However, axonal regeneration of differentiated RGCs has been a difficult problem. Retinal stem cells were differenticated from rat retinal M\u0026uuml;ller cells. The stem cells were randomly divided into five groups (control group, AAV-STAT3 group, shSTAT3 group, Y27632 group and AAV-STAT3\u0026thinsp;+\u0026thinsp;Y27632 group). Stem cells in different groups were injected into rat model of glaucoma. The length of axon regeneration in STAT3 combined with Y27632 group was significantly longer than that in other experimental groups. The AAV-STAT3 transfected RGCs treated with Y27632 significantly increased the mRNA levels of Esrrb, Prdm14, Sox2, and Rex1, while decreasing the mRNA levels of Nestin, Eomes, Mixl1, and Gata4. Meanwhile, Socs3, Pten, Klf9, and Mdm4 were significantly lowered, while Dclk2, Armcx1, C-MYC, and Nrn1 were elevated. After injecting differentiated RGCs into the glaucoma model rat eyes, the axon length, RGC layer thickness and the electrophysiology were superior to the glaucoma model group. These findings suggested that STAT3 combined with Y27632 can significantly improve the axonal growth level of M\u0026uuml;ller differentiated RGCs, and reveal the potential mechanism to induce pluripotency of RGCs.\u003c/p\u003e","manuscriptTitle":"STAT3 combined with Y27632 to treat glaucoma by promoting axon growth of Müller differentiated retina ganglion cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-23 17:47:12","doi":"10.21203/rs.3.rs-3447824/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Reject and Resubmit","date":"2023-11-07T21:06:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-10-18T04:53:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2023-10-15T04:26:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2b5c9593-2dcb-4a67-a772-3e83c5e4ffb3","owner":[],"postedDate":"October 23rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2023-10-23T17:47:13+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-23 17:47:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3447824","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3447824","identity":"rs-3447824","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}