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Neurons, especially axons, are metabolically demanding and energetically vulnerable during injury. However, the exact energy budget alterations that occur early after axon injury and the effects of these changes on neuronal survival remain unknown. Using a classic mouse model of optic nerve-crush injury, we found that traumatized optic nerves and retinas harbor the potential to mobilize two primary energetic machineries, glycolysis and oxidative phosphorylation, to satisfy the robustly increased adenosine triphosphate (ATP) demand. Further exploration of metabolic activation showed that mitochondrial oxidative phosphorylation was amplified over other pathways, which may lead to decreased retinal ganglion cell (RGC) survival despite its supplement to ATP production. Gene set enrichment analysis of a microarray (GSE32309) identified significant activation of oxidative phosphorylation in injured retinas from wild-type mice compared to those from mice with deletion of phosphatase and tensin homolog (PTEN), while PTEN-/- mice had more robust RGC survival. Therefore, we speculated that the oxidation-favoring metabolic pattern after optic nerve-crush injury could be adverse for RGC survival. After redirecting metabolic flux toward glycolysis (magnifying the Warburg effect) using the drug meclizine, we successfully increased RGC survival. Thus, we provide novel insights into a potential bioenergetics-based strategy for neuroprotection.
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AIM:To observe the mechanism of Tianma Gouteng Decoction on the protein molecular level in the optic nerve crush model rats.METHODS:Totally 36 participants 36 male Wistar rats were divided randomly into six groups(6 in every group):normal control group,negative control group,Tianma Gouteng Decoction treatment groups (con-centrations were 0.6g/mL,1.2g/mL,2.4g/mL respictively) and ginkgo biloba tablets positive control group (concentrations was 1.2mg/mL).Nothing was done in the normal control group.The optic nerve of right eye in the other groups was done with the optic nerve crush model.Normal control group and negative control group was treated only with water.The average grey scale values of the N-methyl-D-aspartic acid receptor 2B (NMDA2B) receptor protein,beta-amyloid protein (Aβ) in the average grey scale values were detected.RESULTS:The average grey scale value of Tianma Gouteng Decoction in low,medium and high dose groups about NMDA2B receptor protein was significantly less than that of the negative control group (all P＜0.001),and there was no significant difference with the positive control group (P=0.092,0.411,0.676),the difference between normal control group and negative control group was significant (P＜0.001).The high dose group of betaamyloid's average grey scale value reduced significantly than the negative control group (P=0.030,0.001).The low dose group than the negative control group was not obviously (P=0.614).The high dose group was not significantly different from the positive control group (P=0.927),the difference between normal control group and negative control group was significant (P＜0.001).CONCLUSION:Tianma Gouteng Decoction can go through the decrease of the NMDA2B receptor protein expression and the control of beta-amyloid deposition to reduce the retinal ganglion cell injury and apoptosis.
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Objectivc To investigate the protective effect of simvastatin on retinal ganglion cells (RGC) after optic nerve crush (ONC).Methods Together 50 Kunming mice were randomly divided into normal group,sham group,ONC group and simvastatin protection group.The mice in the normal group were untreated,the sham group was treated with the exposure of the optic nerve without injury,the ONC group mice underwent ONC operation on the left eyes,followed by intravitreal administration of equilibrium solvent [50 mg · mL-1 ethanol plus 1 mol · L-1 NaOH,which were activated by 1 mol · L-1 HC1 (pH 7.2)],and the simvastatin protection group was intravitreally injected different concentrations of simvastatin (0.5 g · L-1,1.0 g · L-1,1.5 g · L-1) after ONC operation.Brn3a immunofluorescence staining,HE staining and toluidine blue staining were used to detect the apoptosis of RGC and pathological changes of optic nerve.Results On day 7 after operation,in the ONC group,the apoptosis of RGC was observed obviously,with the survival rate dropping to (35.1 ± 3.9) ％,and the thickness from the retinal ganglion cell layer to outer nuclear layer was decreased from (123.13 ± 1.04) μm to (97.48 ± 2.33) μm,which was significantly thinner than that in the control group (P ＜ 0.01);moreover,the fibrous bundle of optic nerve disappeared,and the neuroglial cells were clustered into groups,as well as the axons showed swelling and serious degeneration,but after intravitreal injection of 1.0 g · L-1 simvastatin,the survival rate of retinal ganglion cells increased to (76.3 ± 3.7) ％ (P ＜ 0.05),and the aforementioned thickness was increased to (111.39 ± 4.06) μm,which was statistically significant when compared with the ONC group (P ＜ 0.01).The degeneration of optic nerve was improved,and the structure of neuroglial cell axons and the nerve fibers became normal.Conclusion Simvastatin can reduce the optic nerve degeneration and improve the survival rote of retinal ganglion cells.
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AIM: To analyze the effect of pigment epithelium derived factor (PEDF) on nitrogen monoxide (NO) and expression of cysteine-containing, aspartate-specific proteases-3 (caspase-3) in retinal tissues of model rats with optic nerve crush injury.METHODS: A total of 60 SD rats were randomly divided into the blank control group, model group and PEDF group, with 20 rats in each group.Except the blank control group, the optic nerve crush injury rat models were established in the other groups, and left eyeballs were taken as samples.After successfully modeling, the model group were treated with intravitreal injection of 5μL of balanced salt solution while PEDF group were treated with intravitreal injection of 5μL of PEDF (0.2μg/μL).Two weeks later, the retinal tissues were collected, and changes of shape were observed under microscope after HE staining.The changes of NO level were measured by colorimetry assay, the expression of caspase-3 mRNA and caspase-3 protein was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western-blot.RESULTS: HE staining showed that retinal tissues of the blank control group arranged neatly and clearly.Retinal ganglion cells (RGCs) arranged in a monolayer, and cells were oval, uniform in size and distribution, the cell nuclei were clear, closely arranged, with clear boundaries.The retinal tissues of the model group were sparse in shape, RGCs showed vacuolar changes, the overall number of cells was reduced, and cell nuclei of residual RGCs showed pyknosis and uneven staining.RGCs in PEDF group were with slightly edema and arranged closely, and the degree of injury was significantly milder than that in the model group.Levels of Caspase-3 mRNA and protein and NO levels in the three groups showed the model group > PEDF group > blank control group (all P < 0.05).CONCLUSION: The application of PEDF can down regulate the expression of Caspase-3 and NO in rates with optic nerve injury and reduce RGCs injury.
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After a traumatic injury to the central nervous system, the distal stumps of axons undergo Wallerian degeneration (WD), an event that comprises cytoskeleton and myelin breakdown, astrocytic gliosis, and overexpression of proteins that inhibit axonal regrowth. By contrast, injured neuronal cell bodies show features characteristic of attempts to initiate the regenerative process of elongating their axons. The main molecular event that leads to WD is an increase in the intracellular calcium concentration, which activates calpains, calcium-dependent proteases that degrade cytoskeleton proteins. The aim of our study was to investigate whether preventing axonal degeneration would impact the survival of retinal ganglion cells (RGCs) after crushing the optic nerve. We observed that male Wistar rats (weighing 200-400 g; n=18) treated with an exogenous calpain inhibitor (20 mM) administered via direct application of the inhibitor embedded within the copolymer resin Evlax immediately following optic nerve crush showed a delay in the onset of WD. This delayed onset was characterized by a decrease in the number of degenerated fibers (P<0.05) and an increase in the number of preserved fibers (P<0.05) 4 days after injury. Additionally, most preserved fibers showed a normal G-ratio. These results indicated that calpain inhibition prevented the degeneration of optic nerve fibers, rescuing axons from the process of axonal degeneration. However, analysis of retinal ganglion cell survival demonstrated no difference between the calpain inhibitor- and vehicle-treated groups, suggesting that although the calpain inhibitor prevented axonal degeneration, it had no effect on RGC survival after optic nerve damage.
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Animals , Male , Polyvinyls/pharmacology , Retinal Ganglion Cells/drug effects , Axons/drug effects , Wallerian Degeneration/drug therapy , Glycoproteins/pharmacology , Optic Nerve Injuries/drug therapy , Axons/pathology , Immunohistochemistry , Cell Survival/drug effects , Treatment Outcome , Cell Death/drug effects , Cell Death/physiology , Rats, Wistar , Optic Nerve Injuries/pathology , Microscopy, Electron, Transmission , Nerve CrushABSTRACT
Background Toll-like receptor 4 (TLR4) is an immune related receptor.It plays an important role in inducing inflammation response.The inflammatory response secondary to optic nerve crush will results in serious retinal damage.It is worthy of studying the expression and effect of TLR4 in retina after optic nerve crush.Objective This experimental study was to explore the role of TLR4 in the loss of retinal ganglion cells(RGCs) after optic nerve crush.Methods Twenty-four SPF adult health Sprague-Dawley (SD) rats were used in the study and radomized into two groups based to the experimental time.The optical nerve crush models were established by crushing the optical nerve in the right eyes of the rats,and the left normal eyes served as controls.The rats were sacrificed by over anesthesia and retinas were isolated 3 days and 7 days after operation.Expression of TLR4 in the retinas was detected using immunofluorescence method.Reverse trancription PCR (RT-PCR) and Western blot were applied respectively for the detection of TLR4 mRNA and protein in the retinas.The apoptosis of RGCs was evaluated using TUNEL staining.The use and care of experimental animals followed theGuide for the Care and Use of Laboratory Animals of NIH.This study was approved by the Institutional Animal Care and Use Committee at the Zhongshan Ophthalmic Center.Results The expression of TLR4 in rat retinas presented with green fluorescence mainly in the inner layer of retinas.The fluorescence was enhanced in the model 3 days group and the model 7 days group compared with the corresponding control groups.The relative expressing values of TLR4 mRNA in the retinas were 2.92±0.06and 3.92±0.12 in the model 3 days and 7 days groups,respectively,which were significantly higher than 2.87±0.12and 3.44±0.17 in the control 3 days and 7 days group (t3d =-12.888,P＜0.001 ;t7d =-4.669,P=0.010).In the model 3 days group and model 7 days group,the grey values of TLR4 protein were 1.14±0.05 and 1.49±0.03,and those in the control 3 days and 7 days groups were 0.99 ± 0.09 and 1.38 ± 0.07,showing significant differences between them(t3d =-11.324,P＜0.001 ;t7d =-5.638,P=0.005).Apoptotic RGCs were obviously increased in the optic nerve damage group in comparison with the control group.Conclusions The TLR4 is over-expressed in the inner layer of retina after optic nerve crush,which suggestes that TLR4 is probably involved in the loss of RGCs after optic nerve crush.
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BackgroundOptic nerve injury lead to apoptosis of retinal ganglion cells ( RGCs ), and its mechanism of apoptosis is endoplasmic reticulum stress (ERS). So, decreasing of ERS may protect the injury of RGCs. ObjectiveThe present study was to investigate the mechanisms of endoplasmic reticulum stress(ERS) and the protective effects of human umbilical cord blood stem cells on partial optic nerve crush injury. MethodsThe optical nerves were crushed with a 40 g clip by holding for 60 seconds to establish the partial optical nerve injury model in the left eyes of 102 SPF SD rats,and 10 μl of mRNA and 10 μl of nerve growth factor were injected into the vitreous immediately after the establishment of the model. The morphological changes of retinal ganglion cells(RGCs) were examined under the light microscope after 3,7,14,21 and 28 days and the RGCs number was calculated. The apoptosis rates of RGCs were detected by the TUNEL technique after 3, 12,24,45,72 hours and 1 week. The expression levels of GRP78 mRNA and CHOP mRNA were detected by reverse transcription polymerase chain reation (RT-PCR). This procedure followed the Regulations for the Administration of Affair Concerning Experimental Animals by Hunan Provincial Science and Technology Committee.Results The number of RGCs was significantly decreased with the prolongation of time of optical nerve injury in the model injury group,whereas the number of RGCs in the human cord blood cells group was reduced at a slower rate( Ftime =20. 100,P =0. 007 ). At various time points after the injection of human cord blood cells, the survival of RGCs was evidently increased in comparison with the model group(P＜0. 01 ). The apoptosis rate of RGCs was considerably elevated with injury time prolongation both in the model group and human cord blood cells group,but no apoptosis was seen from 3-24 hours after operation,and only a small amount of apoptotic cells were found in the human cord blood cells group from 48 hours through 1 week than in the model group(P＜0. 01 ). In the human cord blood cells group,GRP78 mRNA level was significantly higher and the CHOP mRNA level was significantly lower than those in the injury group at identical time points(P＜0. 01 ). ConclusionsIn the rat optic nerve partial crush model,ERS induces the apoptosis of RGCs. Human umbilical cord blood stem cells can protect RGCs from ERS injury by inhibiting apoptosis.
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Background Researches demonstrated that epigallocatechin-3-gallate(EGCG) can protect retinal ganglion cells(RGCs) against damage induced by retinal ischemia-reperfusion and optic nerve crush(ONC),but the effect of EGCG on lateral geniculate nucleus(LGN) was under study.Objective This study was designed to detect neuroprotective effect of EGCG on LGN in the rat model with ONC.Methods Forty-eight 7-week-old female clean Wistar rats were randomly divided into normal control group,sham operation+EGCG group,ONC+normal saline(NS) group and ONC+EGCG group.ONC models were created by clamping the optical nerve for 60 seconds with the clipper with the force of 40 grams in the right eyes of 24 rats.The EGCG solution(25mg/kg) was intraperitoneally injected from 2 days before operation daily for 5 consecutively days and orally administered(2mg/kg) after that,and NS was used in the same way for ONC+NS group.Four weeks after ONC,the brain tissue of the rats was obtained,and the neurons of dorsal LGN(dLGN) were counted by Nissl staining under the light microscopy.The expression of neurofilament triplet L(NF-L) was detected by immunohistochemistry and Western blot analysis.Meanwhile,the neuronal nitric oxide synthase(nNOS) positive cells were counted.Results Compared with normal control group,no significant differences were found in neuron number both between sham operation+EGCG group or ipsilateral LGN of operative eyes in ONC+normal saline group and ONC+EGCG group(P=0.906,0.561,0.794,0.646 respectively) in 4 weeks after ONC,but loss of neurons in contralateral LGN in both ONC+normal saline group and ONC+EGCG group were observed(P=0.000,0.015 respectively).However,compared with ONC+normal saline group,the density of neurons in ONC+EGCG group was higher(P=0.007).Moreover,a higher expression level of NF-L protein was seen in ONC+EGCG group compared with ONC+normal saline group at contralateral LGN of operative eyes(P=0.002).Concerning the number of nNOS positive cells in LGN,there was no significant difference among normal control group,sham operation+EGCG group and ONC+EGCG group(P＞0.05).The number of nNOS positive cells in the contralateral LGN of operative eyes of ONC+normal saline group was higher than that of ONC+EGCG group(P=0.000).Conclusion EGCG plays the protective effect on LGN after ONC in rats through mediating the expression of nNOS.
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Background Our previous study demonstrated that epigallocateehin-gallate(EGCG),an active ingredient of green tea,has protective effect on optical nerve after optic nerve crush.Astrocyte was proved to play key role in the repair of nerve tissue,but the influence of EGCG on astrocyte is unclear.Glial flbrillary acidic protein (GFAP) is a special marker for astrocyte. Objective The aim of this study was to investigate the effect of EGCG on the expression of GFAP in optic nerve tissue after optic nerve crush. Methods Seventy-two clean Wistar rats were randomly divided into normal control group,sham+EGCG group,optic nerve crush+normal saline group(vehicle group),optic nerve crush+EGCG group.Optic nerve crush models were established by clamping optical nerve for 60 seconds by minitype optic nerve clipper with the force of 40 gram.Only ocular tissue was cut in the rats in sham group.Normal saline solution or EGCG(25 mg/kg)was intraperitoneally injected daily for 5 days consecutively and orally administered(2 mg/kg)daily afterwards.The expression of GFAP in optic nerve was detected by immunohistochemistry and quantified by Western blotting analysis on day 7,14 and 28 after modeling. Results lmmunochemistry showed that GFAP were weakly expressed in the rats of both normal group and sham+EGCG group with the sliSht brown staining in optic nerve tissue.The deeply brown staining for GFAP was seen in vehicle group,and the staining intensity weakened in optic nerve crush+EGCG group compared to vehicle group on days 7,14 and 28 after modeling.Western blotting analysis revealed that the expression level of GFAP in rat optic nerve tissue of vehicle group was significantly enhanced in comparison with normal control group(P＜0.01).On day 7 and 14 after optic nerve modeling,the expression levels of GFAP were evidently decreased in optic nerve crush+EGCG group in comparison with vehicle group(P＜0.05).However,on day 28 after modeling,no significant difference wag found in the expression levels of GFAP between vehicle group and optic nerve erush+EGCG group(P＞0.05). Conclusion EGCG down-regulates optic nerve crush-induced of GFAP in the optic nerve and therefore attenuates the activity of astrocytes,suggesting that EGCG might reduce the formation of glial scar.
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Objective Present study aimed to research the mechanism of the use of 0.2% brimonidine tartrate eye drops in the treatment of optic nerve crush injury of rat.MethodsAnimal models of optic nerve crush injury were created in 60 SD female rats by clipping the exposed optic nerve at 2 mm in retrobulbar for 6 seconds with 78 grams of reverse forceps.The successful model was identified as Marcus-gun pupil without bleeding of fundus after operation.The animals were randomly assigned to model group and brimonidine treating group,and another 30 normal SD rats were used as the normal control group.The 0.2% brimonidine tartrate eye drops was topically administered at 2 hours before operation and after operation twice per day in the brimonidine treating group.The retinas from 18 rats were isolated after 3,7,21 days for RGCs counting by H&E staining,and the retinal ultrastructure was examined under the transmission electron microscope.The retinas from the other 72 SD rats (including normal,model and brimonidine groups) were prepared for the detection of bcl-2 and bax using immunohistochemistry l,3,5,7,14,21 days after the operation.ResultsNormal and almost normal retina structure was exhibited in rats of the normal group and brimonidine treating group,but disorder of cellular arragement and decrease of retinal thickness were found in the model rats under the optical microscope.The RGCs counting was significantly different among the three groups from 3 days through 21 days after operation with the considerably declination in the model group and brimonidine treating group compared with the normal control group (P＜0.05-0.01).However,that of the brimonidine treating group was obviously increased in comparison with the model group (P＜0.01).The expression of bax in rat retina was obviously reduced (P＜0.01),but the expression of bcl-2 was increased in brimonidine group compared with the model group from 5 through 7 days after operation (P＜0.01).ConclusionThe 0.2% brimonidine tartrate eye drops has a preventive effect on optic nerve crush injury of rat,and its inhibition on apoptosis is one of the mechanisms.
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In order to evaluate the efficacy of traditional paeonia extract paeoniflorin against optic nerve crush, 16 Brown Norway rats were divided into two groups at random, with 8 rats in each group. In paeoniaflorin-treated group, 2 mg paeoniaflorin (total volum: 1 mL) was injected into rat's peritoneum one time a day for a period of 8 days. Rats in untreated group were given a single dose of vehicle. The optic nerve was crushed by a special forceps for 30 s in the left eye and a sham proce-dure was performed in the right eye on the 2nd day after the first injection. The retrograde fluorogold labeling of ganglion cells was conducted 5 days after optic nerve crush. The whole retina was flat-mounted thereafter. The ganglion cells that survived the crush were counted under fluorescent microscope by using an automatic counting software. As compared with the contralateral eye, the survival rate of ganglion cells in the left eye increased from 40.22% to 64.53% with a significant dif- ference found between them (t=2.55, P=0.023). The results suggested that the paeonia extract paeoniflorin possessed a protective effect against optic nerve crush.
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PURPOSE: To make an optic nerve crush injury model and to investigate the neuroprotective effect of intravitreally injected ciliary neurotrophic factor (CNTF) on rat retinal ganglion cells (RGCs) in the model. METHODS: The optic nerves of 12 Sprague-Dawley rats were crushed at 3 mm posterior to the eyeball for 1 minute using aneurysm clip (110 g). Two micrograms of CNTF in 2 micro liter of vehicle was injected intravitreally in one group (n=6) and 2 micro liter of PBS was injected in the control group (n=6) at 4, 7, and 10 days after the optic nerve injury. After 2 weeks, the retrograde labeling of the RGCs was done by the dextran tetramethylrhodamine. Twenty-four hours after the labeling, the retina was wholly mounted and the labeled RGCs were counted under the fluorescence microscope. RESULTS: The death of RGCs in this model began at 1 week and continued for 3 weeks. The number of labeled RGCs in CNTF-injected group (510+/-139/mm2) were significantly higher than that in control group (345+/-87/mm2)(p<0.05). CONCLUSIONS: The optic nerve crush injury model was established by use of aneurysm clip. In this model, the intravitreally injected CNTF had a neuroprotective effect on the rat RGCs.
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Animals , Rats , Aneurysm , Ciliary Neurotrophic Factor , Dextrans , Fluorescence , Neuroprotective Agents , Optic Nerve Injuries , Optic Nerve , Rats, Sprague-Dawley , Retina , Retinal Ganglion Cells , RetinaldehydeABSTRACT
PURPOSE: To investigate the neuroprotective effect of Ginexin (Ginkgo biloba extract) on rat retinal ganglion cell in the optic nerve crush injury model. METHODS: From 1 week before the optic nerve injury, Ginexin 100 mg/kg/day (100 mg group), 200 mg/kg/day (200 mg group) or carboxymethylcellulose as a control group (CMC group) was administrated orally 4 weeks in Spraque-Dawley rats. At 3 weeks after the optic nerve injury, the RGCs were counted and the concentration of intravitreal glutamate was measured. RESULTS: The RGC density of CMC group (103+/-22 cells/mm2) was significantly lower than that of 100 mg group (182+/-39 cells/mm2) and 200 mg group (201+/-63 cells/mm2). There was no difference between the 100 mg group and 200 mg group. The concentration of invtravitreal glutamate did not significantly differ among normal, CMC group, 100 mg group and 200 mg group. CONCLUSIONS: Ginexin increased the survival of the retinal ganglion cell in rat optic nerve crush injury model, but, did not influence on the concentration of intravitreal glutamate.
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Animals , Rats , Carboxymethylcellulose Sodium , Glutamic Acid , Neuroprotective Agents , Optic Nerve Injuries , Optic Nerve , Retinal Ganglion Cells , RetinaldehydeABSTRACT
Objective To observe the morphological change of retina and its affection on visual function after optic nerve crush in adult rat. Methods The models of optic nerve crush in rats were made. The change of retinas in different time (1,3 ,5 ,7,9,14,28,56,84d respectively) after injury were observed by light microscope, and the flash visual evoked potential (F-VEP) was recorded in normal and injured rats. Result The number of retinal ganglion cells (RGCs) was significantly reduced in the partial lesion of optic nerve crush compared with normal optic nerves without injury. The initial loss of RGCs was accelerated during 3-7days after nerve crush and then declined gradually by 14 days, and no change was observed after 28 days. The wave of F-VEP became lower and wider in one day after the crush, and the latency and amplitude of F-VEP declined gradually from one day to 14 days, and recovery response was observed by the time of 4 weeks after injury. Conclusion The secondary degeneration of RGCs following optic nerve crush is the important morphological foundation of fall of visual function, which have relativity with the regular decline of visual function.