Optic Nerve Crush Rat Research Articles

Type I Collagen-Adhesive and ROS-Scavenging Nanoreactors Enhanced Retinal Ganglion Cell Survival in an Experimental Optic Nerve Crush Model.

Traumatic optic neuropathy (TON) is a severe condition characterized by retinal ganglion cell (RGC) death, often leading to irreversible vision loss, and the death of RGCs is closely associated with oxidative stress. Unfortunately, effective treatment options for TON are lacking. To address this, catalase (CAT) is encapsulated in a tannic acid (TA)/poly(ethylenimine)-crosslinked hollow nanoreactor (CAT@PTP), which exhibited enhanced anchoring in the retina due to TA-collagen adhesion. The antioxidative activity of both CAT and TA synergistically eliminated reactive oxygen species (ROS) to save RGCs in the retina, thereby treating TON. In vitro experiments demonstrated that the nanoreactors preserve the enzymatic activity of CAT and exhibit high adhesion to type I collagen. The combination of CAT and TA-based nanoreactors enhanced ROS elimination while maintaining high biocompatibility. In an optic nerve crush rat model, CAT@PTP is effectively anchored to the retina via TA-collagen adhesion after a single vitreous injection, and RGCs are significantly preserved without adverse events. CAT@PTP exhibited a protective effect on retinal function. Given the abundance of collagen that exists in ocular tissues, these findings may contribute to the further application of this multifunctional nanoreactor in ocular diseases to improve therapeutic efficacy and reduce adverse effects.

Anti-inflammatory effect of glucagon-like Peptide-1 receptor agonist on the neurosensory retina in an acute optic nerve injury rat model

PurposeTo explore the possibility of using glucagon-like peptide-1 receptor agonist (GLP-1RA) as a new treatment for neuroinflammation, by analyzing retinal pathological changes in an optic nerve crush rat model. MethodsEight-week-old male Sprague-Dawley rats were divided into lixisenatide (LIX, n = 10), traumatic control (T-CON, n = 10), and normal control (n = 5) groups. The optic nerves of left eyes in the LIX and T-CON groups were crushed in a standardized manner. The LIX group was treated with subcutaneous injections of lixisenatide (200 μg/kg/day) for 5 days. One week after initiating treatment, quantitative polymerase chain reaction, Western blot, and immunohistochemistry analyses were performed on the retinal tissues of each group to identify inflammatory markers. ResultsThe LIX group showed significantly lower mRNA levels of interleukin 1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), thioredoxin interacting protein (TXNIP), and glial fibrillary acidic protein (GFAP) than the T-CON group. Also, the LIX group exhibited decreased TXNIP and GFAP expression compared with the T-CON group, and similar expression to the normal control group, according to Western blot analysis. Significantly increased immunohistochemistry staining of Brn3a and decreased TUNEL staining were seen in the LIX group compared with the T-CON group, indicating that lixisenatide contributes to retinal ganglion cell survival in cases of acute optic nerve injury. ConclusionsNeuroinflammation was significantly reduced in lixisenatide-treated retinas compared with untreated retinas in our acute optic nerve injury rat model. The neuroprotective effect of lixisenatide indicates that it can serve a new treatment option against clinically intractable traumatic optic neuropathy.

Protection of retinal ganglion cells from optic nerve injury by human umbilical cord blood stem cells transplantation

Objective To observe the protective effect of human umbilical cord blood stem cells (hUCBSC) transplantation on retinal ganglion cells (RGC) after optic nerve injury. Method 48 adult Sprague-Dawley rats were randomly divided into group A and B, therefore 24 rats in each group. Calibrated optic nerve crush injury model was induced in the left eyes, the right eyes served as a control. Medicine was injected at seventh day after optic nerve injury. PBS was injected into the eyes of Group A rats by peribulbar injection. The hUCBSCs were injected into the eyes of Group B rats by peribulbar injection. Seven days before sacrifice, 5% fluorogold was injected into superior colliculi bilaterally. At 7, 14, 21, 28 days after labeled, retinal flat mounts were observed under fluorescence microscope and optical microscope to investigate the morphological and RGC changes in density during retinal degeneration. Results The RGC number showed a tendency to decline gradually along with increases of the time in two groups, but the trend of decrease of Group B was evidently slower than that of Group A. The RGC number of the injury eye were less than the control eye in Group A and B (t=3.24, 3.15; P<0.05). At 7, 14, 21, 28 days after labeled, the RGC number (t=4.78, 4.70, 3.98, 3.27; P<0.05) and labeled RGC rate (t=4.39, 4.21, 4.36, 5.07; P<0.05) in group B were more than those in group A. After optic nerve injury, there was karyopycnosis on ganglion cell layer of retina, thinning on each layer of retina, derangement of cell and decrease in RGC. There was different degree of the above change in different time after optic nerve injury. There were the swelling, the hemorrhage, derangement of spongiocyte and the denaturation like vacuole in the spot of optic nerve injury. Moreover, they were aggravating with increases of the time after optic nerve injury. There was no pathological changes in normal eyes. Conclusion The hUCBSC can increase the survival rate of the RGC and can rescue and(or) restore the injujed RGC after transplanted into body of optic nerve crush rat model by peribulbar injection. Key words: Optic nerve injuries; Optic nerve diseases/therapy; Cord blood stem cell transplantation

Protective effect on rat retinal ganglion cells and the safety of intravitreal injected acteoside

Objective To observe the protective effect on retinal ganglion cells (RGC) and the safety of intravitreal injected acteoside in rats.Methods A total of 50 male Sprague Dawley rats with the weight of 190-210 g were used in this study.Fifteen rats were used for safety experiment of intravitreal injection of acteoside.The rats were divided into group A,B,C,control group and blank group,three rats in each group.The rats in group A,B and C were received intravitreal injection of 5 μl acteoside at the concentration of 1,2,and 5 mg/ml,respectively.Phosphate buffer solution (PBS) was injected in rats of control group.No treatment was performed for blank group.The retinal structure was examined by hematoxylin-eosin (HE) staining of retinal frozen sections at one,two and three weeks after injection.The retinal ultrastructure was examined by ultrathin section under transmission electron microscope at one and three weeks after injection.Others 35 rats were used for experiment of protective effect of acteoside on RGC.The rats were divided into operation group A and B (n=8),sham operation group C and D (n=8),and blank group (n=3).The optic nerve of rats in operation group was clamped for 10 seconds after optic nerve exposure,while the optic nerve of rats in sham operation group was exposed only.The rats in operation group A and B were received intravitreal injection with 5 μl acteoside (1 mg/ml) and 5 μl PBS respectively.The rats in sham operation group C and D were received intravitreal injection with 1 μl acteoside (1 mg/ml) and 1 μl PBS respectively.No treatment was performed for blank group.The retinal structure was examined by HE staining of retinal frozen sections at one,two and four weeks after injection.Immunohistochemistry was used to measure the expression of growth associated protein 43 (GAP-43).RGC apoptosis was assessed by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labelling (TUNEL) method.Software of SPSS 13.0 was used for the data statistical analysis in this study.Results In the safety experiment of intravitreal injected acteoside,there was no abnormity of cornea,anterior chamber,lens,vitreous cavity and retina after injection.At one,two and three weeks after injection,the retina structure was normal without significant apoptosis,necrosis and inflammatory cell infiltration.The ganglion cell layer showed slightly edema; there was no obvious change of retinal ultrastructure after injection of acteoside with 5 mg/ml and 2 mg/ml,but slight change with the format of 1 mg/ml.Transmission electron microscopy showed that intravitreal injection of 5μl acteoside at the concentration of 2 or 5 mg/ml can induce significant changes of micro-structures of retina,while injections at 1mg/ml can only induce minor changes.In the experiment of protective effect of acteoside on RGC,light microscope revealed that the cell showed typical changes of apoptosis in operation group,but not in sham operation group and blank group.At the first and second week after injection,compared with the sham operation group and blank group,the RGC number was decreased in operation group.The difference of RGC numbers between operation group A and B was statistically different (F=26.206,P＜0.05).The RGC numbers in operation group continues to decrease at the fourth week after injection,there was obvious difference compared with the first and second week after injection (F=17.364,P＜0.05),but there was no difference of RGC numbers among sham operation intra-group and between sham operation group and blank group at all the time points.Immunohistochemistry showed that at the first week after injection,the integrated absorbance (IA) value in operation group was higher than that in other groups (F=33.466,P＜0.05) ; there was no difference of IA value between operation group A and B.At the second week after injection,IA value in operation group A had slightly declined,but higher than that in operation group B (F=14.391,P＜0.05).At the fourth week after injection,IA value in operation group A declined further,but also higher than that in other groups (F=4.178,P＜0.05).TUNEL showed that on the first week after injection,RGC apoptosis rate in operation group was increased than that in other groups (F=15.365,P＜0.05).At the second week after injection,RGC apoptosis rate in operation group was decreased,and it in operation group A was lower than that in operation group B (F=15.365,P＜0.05).At the fourth week after injection,RGC apoptosis rate in operation group was decreased obviously,there was no difference compared with other groups (F =2.057,P ＞ 0.05).There was no difference of RGC apoptosis rate between sham operation group and blank group at all the time points.Conclusion Intravitreal injection of 5 μl acteoside (1mg/ml) is safe for rat retina,and can up-regulate GAP-43 expression and inhibit RGC apoptosis in optic nerve crush rats. Key words: Polyphenols/therapeutic use; Retinal ganglion cells/drug effects; Retinal ganglion cells/pathology; GAP-43 protein

Effects of Erythropoietin-Dextran Microparticle-Based PLGA/PLA Microspheres on RGCs

We explored the neuroprotective effects of erythropoietin (EPO)-loaded dextran microparticle-based Poly(DL-lactide-co-glycolide)/Poly(DL-lactide) (PLGA/PLA) microspheres (EPO-dextran PLGA/PLA microspheres) on retinal ganglion cells (RGCs) in optic nerve crush rats for a prolonged period of time. EPO-dextran PLGA/PLA microspheres were prepared first by a novel solid-in-oil-in-water (S/O/W) technique. Then, the in vitro EPO release profile was assessed. Afterward, the bioactive effect of EPO released from EPO-dextran PLGA/PLA microspheres was explored in vitro on the retinal explants. Lastly, the neuroprotective effects of EPO-dextran PLGA/PLA microspheres on RGCs were evaluated in optic nerve crush rats with TUNEL staining for apoptotic RGCs. The level of glial fibrillary acidic protein (GFAP) expressed in retina was explored by immunohistochemistry staining. Survival RGCs were observed by DiI retrograde labeling using a DiI fluorescent tracer (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). The results demonstrated that a sustained release of EPO from PLGA/PLA microspheres could last for at least 60 days. EPO released from the microspheres showed as efficaciously neuroregenerative as EPO protein solution on retinal explants (P = 0.2554 for neurite density, P = 0.1004 for neurite length). TUNEL staining revealed that EPO-dextran PLGA/PLA microspheres remarkably reduced RGCs death when compared to the control (untreated) group (P < 0.01 at five days and one week post-crush, P < 0.05 at two weeks post-crush). Increased GFAP expression in retina was reduced greatly in EPO-dextran PLGA/PLA microspheres-administrated rats two weeks post optic nerve crush. DiI retrograde labeling revealed that a single injection of EPO-dextran PLGA/PLA microspheres significantly promoted RGCs survival (P < 0.01 at four and eight weeks post-crush). A single intravitreal injection of EPO-dextran PLGA/PLA microspheres appeared to have a prolonged protective effect on RGCs in optic nerve crush rats. The PLGA/PLA microspheres may be a feasible protein delivery system, such as EPO, to intravitreal injection for retinal degeneration diseases.

Neuroprotective mechanisms of human umbilical cord blood stem cells to retinal ganglion cells in a partial optic nerve crush rat model

Background Optic 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. Objective The 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. Methods The 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 ). Conclusions In 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. Key words: Human umbilical cord blood stem cell; Endoplasmic reticulum stress; GRP78; CHOP; Optic nerve/crush

Neuroprotective effect of epigallocatechin-3-gallate on retinal ganglion cells in optic nerve crush rat model

Objective To investigate the neuroprotective effect of epigallocatechin-gallate (EGCG),the major catechin found in green tea, in an optic nerve crush (ONC) rat model. Methods Seventy-two Wistar rats were divided randomly into four groups: normal control, sham-operation+ EGCG, ONC+vehicle, and ONC+ EGCG groups. Animals were treated intraperitoneally and orally with either vehicle (normal saline) or EGCG (25 mg/kg, injected daily for up to five days, and orally 2 mg/kg daily afterwards). Two days after the first injection, an ONC injury using a micro optic nerve clipper was performed at approximately 2 mm from the optic nerve head for 60 seconds. Fluorogold was injected into bilateral superior colliculi five days before sacrifice and fluorescent gold labeled retinal ganglion cells (RGC)were counted under fluorescence microscopy on days seven, 14 and 28 after optic nerve crush. Meanwhile,the expression of neurofilament triplet L (NF-L) was measured by immunohistochemistry and Western blotting. Results In the ONC + vehicle group, a progressive loss of RGC was observed;however,compared with the ONC+vehicle group, RGC density was significantly higher on day seven (t=3. 216, P=0. 009), day 14 (t=2. 609, P=0. 026) and day 28 (t=2. 792, P=0. 019) , and a significant increase of theNF-L protein expression has been observed on days seven, 14 and 28 after optic nerve crush in the ONC+EGCG group. Conclusions EGCG has a protective effect to the bodies and axons of the RGC after optic nerve crush. Key words: Optic nerve injuries; Callic acid /therapeutic use; Retinal ganglion cells; Animal experimentation

Neuroprotective effects of tempol on retinal ganglion cells in a partial optic nerve crush rat model with and without iron load

Iron overload can contribute to oxidative stress in many tissues. We studied the effects of pretreatment with iron dextran on RGC loss in a calibrated partial optic nerve crush (PONC) model in rats, along with the protection offered by tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl, a membrane-permeable superoxide dismutase mimetic and free-radical scavenger), in the same experimental paradigm. A total of 40 rats in 6 groups of 5–8 animals each underwent PONC in one eye and sham crush in the other. Animals were pretreated with a single iron dextran load 24 h prior to PONC, and treated with tempol 6 h before and then once daily after PONC. Control animals were treated with PBS. RGC were retrogradely labeled with a fluorescent marker; all data are expressed in percent of the RGC count in the respective sham-treated eye. Immunohistochemistry was performed to visualize 3-nitrotyrosine, a marker of nitroxidative stress. PONC without iron pretreatment resulted in the survival of only 31.4% of labeled RGC after 7 days. Even fewer RGC (12.7%) survived after PONC with iron pretreatment. However, tempol in doses of 20 mg/kg of body weight (BW) significantly attenuated this effect when given as described above; in the group without iron pretreatment the number of surviving RGC doubled from 31.4% to 62.1%. In the group with iron pretreatment the survival rate of RGC increased even more pronouncedly, from 12.7% without tempol to 46.2% with tempol. Tempol in doses of 1 mg/kg BW and 5 mg/kg BW showed no significant rescue of RGC. Immunostaining showed nitrotyrosine-positive RGCs in PONC but not in sham-treated eyes and an increase in positive cells after iron load. Tempol treatment reduced nitrotyrosine staining in both the iron and non-iron groups. Our results demonstrate that PONC results in significantly greater RGC damage when iron pretreatment is performed, and that the compound tempol may provide additional protection for RGC in cases of neuronal damage both with and without prior iron treatment.

Neuroprotective effect of transpupillary thermotherapy in the optic nerve crush model of the rat

Transpupillary thermotherapy (TTT) has been shown to induce heat shock protein (Hsp) 72 in optic nerve head tissue. The neuroprotective effect of TTT was investigated in an optic nerve crush rat model. TTT was performed onto the optic nerve head in the right eye of subject rats. After 24 h, an optic nerve crush injury using an aneurysm clip was performed at 2 mm from the optic nerve head for 60 s. At 7 and 14 days later, retrograde labelling of retinal ganglion cells (RGCs) with DTMR crystal was carried out and the density of the surviving RGCs was evaluated. Immunohistochemical staining was performed to confirm the expression of Hsp72. At 7 days after optic nerve crush injury, the mean density of surviving RGCs was higher in TTT group (372.7+/-149.8 per mm(2)) than in optic nerve crush group (252.9+/-96.7 per mm(2)) with borderline significance. In the retinal areas at 1 mm from the optic nerve head, a significant increase in surviving RGCs from TTT treated eyes was observed at both 7 and 14 days after optic nerve crush injury. However, no significant differences in surviving RGCs were demonstrated 2 and 3 mm from the optic nerve head. These results demonstrate that TTT aimed onto the optic nerve head showed a neuroprotective effect.

Rat retinal morphological and visual function changes after optic nerve crush

目的 观察视神经夹挫伤后视网膜形态学和视功能动态变化,为视功能评价和视神经保护研究提供依据.方法大鼠视神经夹挫伤后1d、3d、5d、7d、9d、2周、4周、8周、12周,光镜观察视网膜神经节细胞(RGC)改变,闪光视觉诱发电位(FVEP)检测视功能状况.结果视神经部分损伤后3d到1周内视网膜神经节细胞快速减少,2周以后缓慢减少,4周几乎无明显变化;视神经损伤1d,F-VEP波形变得低而宽,前2周呈进行性下降期,4周后变化平稳,并显示恢复迹象.结论神经节细胞继发性损伤是视功能进行性下降的重要原因,一定数量存活的视网膜节细胞是视功能恢复的基础;神经损伤变化和视功能变化与时间具有一定的相关性,这些对于正确评价视功能状况和预后有极重要的意义。