Rat Optic Nerve Research Articles

Influence of the Neural Crest Derived Stem Cells to Optic Nerve Regeneration After Its Experimental Injury

Purpose: Orbital trauma is a challenging problem due to such severe sequel as diplopia, decrease of vision or eye motility disorder. However, the conditions of orbital soft tissue content still become underestimated. The aim of this study was to investigate structural changes in the rat optic nerve after experimental injury followed by treatment with stem cells. Materials and Ьethods: An experimental model of injury to the orbital soft tissue content in the rat was developed. Forty Wistar rats maintained under daylight were divided into two equal experimental groups. Unlike the rats of Group I, in rats of Group II, the site of injury to the orbital soft tissue mass received postnatal multipotent stem cells, epidermal neural crest stem cells (NCSCs) derived from the bulge of hair follicles. Results: Comparing the number of glial cells per certain area of the slice (NC) between group І and site without injury (control) after 3 week of observation, it was higher in group I more than 258.8% (p < 0.0001) and on 272.4% in group II (p < 0.0001). After 6 weeks NC in group I was higher than at previous terms: more then 128.9% (р < 0.0001). At the same, NC in group II was higher comparing with previous terms only on 17.1% (р = 0.0212). Between the animals of group I at terms of 12 and 24 weeks NC high and wasn’t significantly differ between this terms of observation (ANOVA p = 0.4379). In contrast, NC in group II stopped rising between 6 and 12 weeks demonstrating statistical equality (p = 0.4563). Conclusions: It can be assumed that the application of mesenchymal stem cells, derivates of the neural crest, after the experimental orbital trauma, stimulates a recovery of the optic nerve. Further studies should be performed to more deeply discover the neural crest derived stem cell populations, ivoleved into recovery of damaged optic nerves.

Optic Nerve Engraftment of Neural Stem Cells.

PurposeTo evaluate the integrative potential of neural stem cells (NSCs) with the visual system and characterize effects on the survival and axonal regeneration of axotomized retinal ganglion cells (RGCs).MethodsFor in vitro studies, primary, postnatal rat RGCs were directly cocultured with human NSCs or cultured in NSC-conditioned media before their survival and neurite outgrowth were assessed. For in vivo studies, human NSCs were transplanted into the transected rat optic nerve, and immunohistology of the retina and optic nerve was performed to evaluate RGC survival, RGC axon regeneration, and NSC integration with the injured visual system.ResultsIncreased neurite outgrowth was observed in RGCs directly cocultured with NSCs. NSC-conditioned media demonstrated a dose-dependent effect on RGC survival and neurite outgrowth in culture. NSCs grafted into the lesioned optic nerve modestly improved RGC survival following an optic nerve transection (593 ± 164 RGCs/mm2 vs. 199 ± 58 RGCs/mm2; P < 0.01). Additionally, RGC axonal regeneration following an optic nerve transection was modestly enhanced by NSCs transplanted at the lesion site (61.6 ± 8.5 axons vs. 40.3 ± 9.1 axons, P < 0.05). Transplanted NSCs also differentiated into neurons, received synaptic inputs from regenerating RGC axons, and extended axons along the transected optic nerve to incorporate with the visual system.ConclusionsHuman NSCs promote the modest survival and axonal regeneration of axotomized RGCs that is partially mediated by diffusible NSC-derived factors. Additionally, NSCs integrate with the injured optic nerve and have the potential to form neuronal relays to restore retinofugal connections.

Metformin treatment confers protection of the optic nerve following photoreceptor degeneration.

Acquired or inherited or photoreceptor loss causes retinal ganglion cell loss and ultimately axonal transport alteration. Thus, therapies should be applied early during photoreceptors degeneration before the remodeling process reaches the inner retina. This study aimed to evaluate the protective effect of metformin on the rat optic nerve following photoreceptors loss induced by N-Ethyl-N-nitrosourea (ENU). Eighteen adults male Wistar rats were divided into two groups. Group I: normal vehicle control (n=6). Group II: ENU-induced photoreceptors degeneration (n=12) received a single intraperitoneal injection of ENU at a dose of 600 mg/kg. Rats in group II were equally divided into two subgroups: IIa: photoreceptor degeneration induced group and IIb: metformin treated group (200 mg/kg) for 7 days. Specimens from the optic nerve were processed for light and electron microscopy. In ENU treated group, the optic nerve revealed reduction in the diameter of the optic nerve fibers and thinning of myelin sheath with morphological changes in the glia (astrocytes, oligodendrocytes, and microglia). Caspase-3 (apoptotic marker), iNOS (oxidative stress marker) and CD68 (macrophage marker) expression increased. In metformin-treated group, the diameter of optic nerve fibers and myelin sheath thickness increased with improvement of the deterioration in the glia. Caspase-3, iNOS and CD68 expression decreased. Metformin ameliorates the histological changes of the rat optic nerve following photoreceptors loss induced by ENU.

Diterpene Ginkgolides Meglumine Injection inhibits apoptosis induced by optic nerve crush injury via modulating MAPKs signaling pathways in retinal ganglion cells

Ethnopharmacological relevanceDiterpene Ginkgolides Meglumine Injection (DGMI) is made of extracts from Ginkgo biloba L, including Ginkgolides A, B, and K and some other contents, and has been widely used as the treatment of cerebral ischemic stroke in clinic. It can be learned from the “Compendium of Materia Medica” that Ginkgo possesses the effect of “dispersing toxin”. The ancient Chinese phrase “dispersing toxin” is now explained as elimination of inflammation and oxidative state in human body. And it led to the original ideas for today's anti-oxidation studies of Ginkgo in apoptosis induced by optic nerve crush injury. Aim of the studyTo investigate the underlying molecular mechanism of the DGMI in retinal ganglion cells (RGCs) apoptosis. Materials and methodsTUNEL staining was used to observe the anti-apoptotic effects of DGMI on the adult rat optic nerve injury (ONC) model, and flow cytometry and hoechst 33,342 staining were used to observe the anti-apoptotic effects of DGMI on the oxygen glucose deprivation (OGD) induced RGC-5 cells injury model. The regulation of apoptosis and MAPKs pathways were investigated with Immunohistochemistry and Western blotting. ResultsThis study demonstrated that DGMI is able to decrease the conduction time of F-VEP and ameliorate histological features induced by optic nerve crush injury in rats. Immunohistochemistry and TUNEL staining results indicated that DGMI can also inhibit cell apoptosis via modulating MAPKs signaling pathways. In addition, treatment with DGMI markedly improved the morphological structures and decreased the apoptotic index in RGC-5 cells. Mechanistically, DGMI could significantly inhibit cell apoptosis by inhibiting p38, JNK and Erk1/2 activation. ConclusionThe study shows that DGMI and ginkgolides inhibit RGCs apoptosis by impeding the activation of MAPKs signaling pathways in vivo and in vitro. Therefore, the present study provided scientific evidence for the underlying mechanism of DGMI and ginkgolides on optic nerve crush injury.

Дослідження протекторних властивостей гідрогелю метилкремнієвої кислоти щодо нейротоксичності, індукованої Cr (VI) в зоровому нерві щурів

Дослідження протекторних властивостей гідрогелю метилкремнієвої кислоти щодо нейротоксичності, індукованої Cr (VI) в зоровому нерві щурів

Effect of taxifolin on cisplatin-associated oxidative optic nerve damage in rats

Aim To investigate the effect of taxifolin on cisplatin-induced oxidative and proinflammatory optic nerve damage in rats. Methods A total of 18 albino Wistar male rats were assigned into 3 groups, as follows; Group 1: Control group, Group 2: Only cisplatin administered group for 14 days (Cisplatin group), and Group 3: Taxifolin + cisplatin administered group for 14 days (CIS + TAX group). Serum malondialdehyde (MDA), total Glutathione (tGSH), Nuclear Factor-Kappa B (NF-ƘB), Total Oxidative Status (TOS) and Total Antioxidant Status (TAS) levels were collected from the left eyes of rats. Rats’ right eyes were enucleated for histopathological evaluations of optic nerves. Results NF-ƘB, MDA and TOS levels were statistically significantly higher (p < 0.001) in cisplatin group when compared to other 2 groups, the tGSH and TAS levels of which were statistically significantly lower (p < 0.001). Regarding these parameters, in cisplatin group NF-ƘB, MDA and TOS levels were statistically significantly increased with cisplatin administration and giving taxifolin concomitantly with cisplatin prevented this elevation. On the other hand, tGSH and TAS levels were statistically significantly decreased with cisplatin administration and routine simultaneous application of taxifolin with cisplatin prevented this decrease. In histopathological findings, haemorrhage was observed in the perineum of the injured optic nerves in the cisplatin treated group. And also edoema and degeneration in nerve fascicles in damaged optic nerves were seen in the cisplatin group. In the taxifolin treated group histopathological examinations were close to normal appearance, except mild edoema in nerve fascicles. Conclusion Cisplatin causes oxidative stress on the rat optic nerves, and these changes lead to significant histopathological damage. Taxifolin, which we used to prevent oxidative damage to the optic nerves caused by cisplatin, has been emphasized as a powerful antioxidant agent in many previous scientific investigations. Concomitant administration of taxifolin may prevent these adverse effects of cisplatin, as well as histopathological damage. Further studies are needed to fully determine the effects of cisplatin and taxifolin on the eye.

Neuroprotective Effect of Intravitreal Single-Dose Lithium Chloride after Optic Nerve Injury in Rats

ABSTRACT Objective Lithium is an old drug to control bipolar disorder. Moreover, it presents neuroprotective effects and supports neuronal plasticity. The aim of this study was to evaluate neuroprotective effect of intravitreal lithium after optic nerve injury. Methods Three dosages of lithium chloride, including 2 pmol, 200 pmol, and 2 nmol, were injected intravitreally after rat optic nerve injury. Proteins expression were assessed by western blot. Nitric oxide (NO) metabolites were measured by Griess test. Visual evoked potential (VEP) and optical coherence tomography (OCT) measurement were performed after trauma induction, in addition to H & E and TUJ1 staining of ganglion cells. Results Western blot depicted lithium can significantly increase antiapoptotic Bcl-2 protein level and reduce p-ERK, Toll-like receptor 4 (TLR4) and proapoptotic proteins such as Bax level in retinal tissue and Griess test reflected that NO metabolites level decreased in lithium treated eyes (P < .05). While, OCT showed no significant changes (P = .36 and P = .43 comparing treated group with trauma) in retinal ganglion cell layer thickness after lithium injection, VEP P2 wave amplitude increased significantly (P < .01) in lithium-treated eyes and its latency reduced (P < .05 for N1 wave and P < .01 for P2 wave). Tuj1 antibody-labeled retinal ganglion cells analyzing showed that the number of retinal ganglion cells were significantly higher in lithium treated eyes compared to untreated eyes with optic nerve injury. Conclusion It seems intravitreally lithium has optic nerve neuroprotective effects by various mechanisms like overexpression of antiapoptotic proteins, suppressing proinflammatory molecules and proapoptotic factors, and decreasing nitric oxide.

Quantitative analysis of astrocyte and axonal density relationships: Glia to neuron ratio in the optic nerve laminar regions

Astrocytes are critical for the maintenance of retinal ganglion cell (RGC) axonal function and viability, and form a key component of the functional neurovascular unit. Recently, we described the quantitative properties of astrocytes in relation to the capillary distributions in optic nerve laminar regions. Here, we provide a quantitative analysis of astrocytes and RGC axons in longitudinal sections of optic nerve tissue. Histological and immunocytochemical techniques are used to demonstrate the density of astrocytes, RGC axons and glia-neuron ratios across the pre laminar, lamina cribrosa and post laminar compartments of the optic nerve head (ONH). A study of human, pig, horse and rat optic nerves was performed and comparisons are made between species. This study demonstrates that the distribution of astrocytes correlates closely with the density of axonal processes, in accordance with the functional requirement of different regions of the ganglion cell axon. There was a consistency of glia-neuron ratios in the majority of laminar compartments, except for the human and rat prelaminar regions, which demonstrated lower ratios of astrocyte to axonal processes. The distribution of astrocytes may reflect a functional susceptibility to development of disease in the prelaminar region of the optic nerve. Interspecies comparison at the lamina cribrosa showed strikingly consistent glia-neuron ratios. Collectively, our findings suggest there may be a critical ratio of glia to neuron needed to maintain healthy cellular physiology across different laminar compartments of the optic nerve, with particular importance for the health of the lamina cribrosa region. It is possible that, in disease processes, the glia-neuron relationships across the different laminar compartments may be perturbed and this may be relevant for the development of glaucoma. Emerging technologies may further aid our understanding in how the physiology of optic nerve tissue cellular structure may be affected by changes to ONH characteristics and elevated intraocular pressure induced damage. Such findings may also permit the early identification of RGC axonal injury by identifying quantifiable changes in structural tissue architecture when pathophysiological pathways predominate.

Philanthotoxin-343 attenuates retinal and optic nerve injury, and protects visual function in rats with N-methyl-D-aspartate-induced excitotoxicity.

Retinal ganglion cell (RGC) loss and optic neuropathy, both hallmarks of glaucoma, have been shown to involve N-methyl-D-aspartate receptor (NMDAR)-mediated excitotoxicity. This study investigated the neuroprotective effects of Philanthotoxin (PhTX)-343 in NMDA-induced retinal injury to alleviate ensuing visual impairments. Sprague-Dawley rats were divided into three; Group I was intravitreally injected with phosphate buffer saline as the control, Group II was injected with NMDA (160 nM) to induce retinal excitotoxic injury, while Group III was injected with PhTX-343 (160 nM) 24 h prior to excitotoxicity induction with NMDA. Rats were subjected to visual behaviour tests seven days post-treatment and subsequently euthanized. Rat retinas and optic nerves were subjected to H&E and toluidine blue staining, respectively. Histological assessments showed that NMDA exposure resulted in significant loss of retinal cell nuclei and thinning of ganglion cell layer (GCL). PhTX-343 pre-treatment prevented NMDA-induced changes where the RGC layer morphology is similar to the control. The numbers of nuclei in the NMDA group were markedly lower compared to the control (p<0.05). PhTX-343 group had significantly higher numbers of nuclei within 100 μm length and 100 μm2 area of GCL (2.9- and 1.7-fold, respectively) compared to NMDA group (p<0.05). PhTX-343 group also displayed lesser optic nerve fibres degeneration compared to NMDA group which showed vacuolation in all sections. In the visual behaviour test, the NMDA group recorded higher total distance travelled, and lower total immobile time and episodes compared to the control and PhTX-343 groups (p<0.05). Object recognition tests showed that the rats in PhTX-343 group could recognize objects better, whereas the same objects were identified as novel by NMDA rats despite multiple exposures (p<0.05). Visual performances in the PhTX-343 group were all comparable with the control (p>0.05). These findings suggested that PhTX-343 inhibit retinal cell loss, optic nerve damage, and visual impairments in NMDA-induced rats.

AxoNet: A deep learning-based tool to count retinal ganglion cell axons

In this work, we develop a robust, extensible tool to automatically and accurately count retinal ganglion cell axons in optic nerve (ON) tissue images from various animal models of glaucoma. We adapted deep learning to regress pixelwise axon count density estimates, which were then integrated over the image area to determine axon counts. The tool, termed AxoNet, was trained and evaluated using a dataset containing images of ON regions randomly selected from whole cross sections of both control and damaged rat ONs and manually annotated for axon count and location. This rat-trained network was then applied to a separate dataset of non-human primate (NHP) ON images. AxoNet was compared to two existing automated axon counting tools, AxonMaster and AxonJ, using both datasets. AxoNet outperformed the existing tools on both the rat and NHP ON datasets as judged by mean absolute error, R2 values when regressing automated vs. manual counts, and Bland-Altman analysis. AxoNet does not rely on hand-crafted image features for axon recognition and is robust to variations in the extent of ON tissue damage, image quality, and species of mammal. Therefore, AxoNet is not species-specific and can be extended to quantify additional ON characteristics in glaucoma and potentially other neurodegenerative diseases.

Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo, enhances translation, and modulates splicing

Axonal degeneration is a key and early pathological feature in traumatic and neurodegenerative disorders of the CNS. Following a focal lesion to axons, extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, the accumulation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we study the effect of ULK1 inhibition in different models of lesion-induced axonal degeneration in vitro and in vivo. Overexpression of a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons attenuates axotomy-induced AAD in vitro. Both ULK1.DN and the ULK1 inhibitor SBI-0206965 protect against AAD after rat optic nerve crush in vivo. ULK1.DN additionally attenuates long-term axonal degeneration after rat spinal cord injury in vivo. Mechanistically, ULK1.DN decreases autophagy and leads to an mTOR-mediated increase in translational proteins. Consistently, treatment with SBI-0206965 results in enhanced mTOR activation. ULK1.DN additionally modulates the differential splicing of the degeneration-associated genes Kif1b and Ddit3. These findings uncover ULK1 as an important mediator of axonal degeneration in vitro and in vivo, and elucidate its function in splicing, defining it as a putative therapeutic target.

Factors affecting optic nerve head biomechanics in a rat model of glaucoma.

Glaucoma is the leading cause of irreversible blindness and is characterized by the death of retinal ganglion cells, which carry vision information from the retina to the brain. Although it is well accepted that biomechanics is an important part of the glaucomatous disease process, the mechanisms by which biomechanical insult, usually due to elevated intraocular pressure (IOP), leads to retinal ganglion cell death are not understood. Rat models of glaucoma afford an opportunity for learning more about these mechanisms, but the biomechanics of the rat optic nerve head (ONH), a primary region of damage in glaucoma, are only just beginning to be characterized. In a previous study, we built finite-element models with individual-specific rat ONH geometries. Here, we developed a parametrized model of the rat ONH and used it to perform a sensitivity study to determine the influence that six geometric parameters and 13 tissue material properties have on rat optic nerve biomechanical strains due to IOP elevation. Strain magnitudes and patterns in the parametrized model generally matched those from individual-specific models, suggesting that the parametrized model sufficiently approximated rat ONH anatomy. Similar to previous studies in human eyes, we found that scleral properties were highly influential: the six parameters with highest influence on optic nerve strains were optic nerve stiffness, IOP, scleral thickness, the degree of alignment of scleral collagen fibres, scleral ground substance stiffness and the scleral collagen fibre uncrimping coefficient. We conclude that a parametrized modelling strategy is an efficient approach that allows insight into rat ONH biomechanics. Further, scleral properties are important influences on rat ONH biomechanics, and additional efforts should be made to better characterize rat scleral collagen fibre organization.

QuPath Automated Analysis of Optic Nerve Degeneration in Brown Norway Rats.

PurposeA novel application of QuPath open-source digital analysis software is used to provide in-depth morphological analysis of progressive optic nerve (ON) degeneration in rats.MethodsQuPath software was adapted to assess axon and gliotic morphology in toluidine blue-stained, Brown Norway rat ON light micrographs. QuPath axon numbers, density, size distributions, and gliotic areas were obtained from test images and ON cross-sections separated by damage grade. QuPath results were compared with manual counting, AxonJ, and electron microscopy axon estimates.ResultsQuPath-derived axon number, density, and diameter decreased with increasing ON damage. Axon density negatively correlated with gliotic areas in test images (R2 = 0.759; P < 0.0001; N = 40) and in ON cross-sections (R2 = 0.803; P < 0.0004; N = 10). Although axon losses occurred across most axon diameters, large axons were more susceptible to degeneration. The exception was swollen axons > 2 µm, which increased in moderately but not severely damaged images. QuPath axon counts correlated strongly with manual counts of test images (R2 = 0.956; P < 0.0001). QuPath outperformed AxonJ on test images and total ON axon counts. Compared to electron microscopy analysis, QuPath undercounted ON axons; however, correlation between the methods was robust (R2 = 0.797; P < 0.001; N = 10).ConclusionsQuPath analysis reliably identified axon loss, axon morphology changes, and gliotic expansion that occurred in degenerating ONs.Translational RelevanceQuPath is a valuable tool for rapid, automated, analysis of healthy and degenerating ONs. Reproducible preclinical studies for new glaucoma treatments depend on unbiased in-depth analysis of ON pathology. This was provided by the QuPath approach.

Neuroprotective effects of brain-derived neurotrophic factor against amyloid beta 1-40-induced retinal and optic nerve damage.

Brain-derived neurotrophic factor (BDNF) could be considered a potential neuroprotective therapy in amyloid beta (Aβ)-associated retinal and optic nerve degeneration. Hence, in this study we investigated the neuroprotective effect of BDNF against Aβ1-40-induced retinal and optic nerve injury. In this study, exposure to Aβ1-40 was associated with retinal and optic nerve injury. TUNEL staining showed significant reduction in the apoptotic cell count in the BDNF-treated group compared with Aβ1-40 group. H&E-stained retinal sections also showed a striking reduction in neuronal cells in the ganglion cell layer (GCL) of retinas fourteen days after Aβ1-40 exposure. By contrast, number of retinal cells was preserved in the retinas of BDNF-treated animals. After Aβ1-40 exposure, visible axonal swelling was observed in optic nerve sections. However, the BDNF-treated group showed fewer changes in optic nerve; axonal swelling was less frequent and less marked. In the present study, exposure to Aβ was associated with oxidative stress, whereas levels of retinal glutathione (GSH), superoxide dismutase (SOD) and catalase were significantly increased in BDNF-treated than in Aβ1-40-treated rats. Both visual object recognition tests using an open-field arena and a Morris water maze showed that BDNF improved rats' ability to recognise visual cues (objects with different shapes) after Aβ1-40 exposure, thus demonstrating that the visual performance of rats was relatively preserved following BDNF treatment. In conclusion, intravitreal treatment with BDNF prevents Aβ1-40-induced retinal cell apoptosis and axon loss in the optic nerve of rats by reducing retinal oxidative stress and restoring retinal BDNF levels.

Intravitreal application of AAV-BDNF or mutant AAV-CRMP2 protects retinal ganglion cells and stabilizes axons and myelin after partial optic nerve injury

Secondary degeneration following an initial injury to the central nervous system (CNS) results in increased tissue loss and is associated with increasing functional impairment. Unilateral partial dorsal transection of the adult rat optic nerve (ON) has proved to be a useful experimental model in which to study factors that contribute to secondary degenerative events. Using this injury model, we here quantified the protective effects of intravitreally administered bi-cistronic adeno-associated viral (AAV2) vectors encoding either brain derived neurotrophic factor (BDNF) or a mutant, phospho-resistant, version of collapsin response mediator protein 2 (CRMP2T555A) on retinal ganglion cells (RGCs), their axons, and associated myelin. To test for potential synergistic interactions, some animals received combined injections of both vectors. Three months post-injury, all treatments maintained RGC numbers in central retina, but only AAV2-BDNF significantly protected ventrally located RGCs exclusively vulnerable to secondary degeneration. Behaviourally, treatments that involved AAV2-BDNF significantly restored the number of smooth-pursuit phases of optokinetic nystagmus. While all therapeutic regimens preserved axonal density and proportions of typical complexes, including heminodes and single nodes, BDNF treatments were generally more effective in maintaining the length of the node of Ranvier in myelin surrounding ventral ON axons after injury. Both AAV2-BDNF and AAV2-CRMP2T555A prevented injury-induced changes in G-ratio and overall myelin thickness, but only AAV2-BDNF administration protected against large-scale myelin decompaction in ventral ON. In summary, in a model of secondary CNS degeneration, both BDNF and CRMP2T555A vectors were neuroprotective, however different efficacies were observed for these overexpressed proteins in the retina and ON, suggesting disparate cellular and molecular targets driving responses for neural repair. The potential use of these vectors to treat other CNS injuries and pathologies is discussed.

Electrical synapses interconnecting axons in the optic nerve head–a novel model of optic nerve function

AbstractPurposeAxons in the optic nerve are arranged in bundles and conducting action potential with resistance related to their membrane. Here, we aim to characterize the function of newly identified gap junctions coupling optic nerve axons with each other’s.MethodsProximal and distal rat optic nerve stumps were processed for immunostainings, electron microscopy (EM), RT‐PCR and western blots (WB). Additional 20 animals were deeply anesthetized, and stainless‐steel screw electrodes were drilled in skulls in standard locations to record fVEPs. Recordings were performed in untouched animals, after retrobulbar injection of saline (negative control) or meclofenamic acid solution (gap junctions’ blocker). Human paraffin cross‐sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research, University of Toronto, Canada.ResultsImmunostaining of rat and human samples revealed presence of Cx45 and 36 in axonal membranes, colocalizing with β3‐tubulin, but not with GFAP. In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 ± 0.005 ‐ proximal stump; 0.44 ± 0.02 ‐ distal stump), Cx45 showed higher levels (0.68 ± 0.0003 ‐ proximal stump; 0.9 ± 0.07 ‐ distal stump). Additionally, we detected transcript of Cx45 and Cx36 in RT‐PCR analysis of optic nerve homogenates. In immunogold‐EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) that are directly coupling neighboring axons. In fVEPs, blocking of gap junctions’ with meclofenamic acid resulted in prolongation of the latency of P1 wave up to 140% after 5 min from retrobulbar injection of this blocker (p &lt; 0.001) and 170% after 30 min (p &lt; 0.001).ConclusionsOptic nerve axons do not form absolutely independent conductive channels. They are directly coupled by gap junctions formed in majority by neuronal Cx45. Coupling of axons allows to reduce axonal membrane resistance and accelerates transduction of visual signal.

Electrical synapses interconnecting axons revealed in the optic nerve head - a novel model of gap junctions' involvement in optic nerve function.

PurposeTo characterize newly discovered electrical synapses, formed by connexin (Cx) 36 and 45, between neighbouring axons within the optic nerve head.MethodsTwenty‐five Wistar rats were killed by CO 2 inhalation. Proximal and distal optic nerve (ON) stumps were collected and processed for immunostainings, electron microscopy (EM) with immunogold labelling, PCR and Western blots (WB). Additional 15 animals were deeply anaesthetized, and flash visual evoked potentials (fVEP) after retrobulbar injection of saline (negative control) or 100 μ m meclofenamic acid solution (gap junctions’ blocker) were recorded. Human paraffin cross‐sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research.ResultsImmunostainings of both rat and human ON revealed the presence of Cx45 and 36 colocalizing with β3‐tubulin, but not with glial fibrillary acidic protein (GFAP). In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 ± 0.005 in proximal stump and 0.44 ± 0.02 in distal stump), Cx45 showed higher levels (0.68 ± 0.01 in proximal stump and 0.9 ± 0.07 in distal stump). In immunogold‐EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) directly coupling neighbouring axons. In fVEP, blocking of gap junctions with meclofenamic acid resulted in significant prolongation of the latency of P1 wave up to 160% after 30 min (p < 0.001).ConclusionsOptic nerve (ON) axons are equipped with electrical synapses composed of neuronal connexins, especially Cx45, creating direct morphological and functional connections between each other. This finding could have substantial implications for understanding of the pathogenesis of various optic neuropathies and identifies a new potential target for a therapeutic approach.

Compressive mechanical properties of rat and pig optic nerve head

Glaucoma is the leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP), the primary risk factor for glaucoma, is thought to induce abnormally high strains in optic nerve head (ONH) tissues, which ultimately result in retinal ganglion cell damage and vision loss. The mechanisms by which excessive deformations result in vision loss remain incompletely understood. The ability of computational and in vitro models of the ONH to provide insight into these mechanisms, in many cases, depends on our ability to replicate the physiological environment, which in turn requires knowledge of tissue biomechanical properties. The majority of mechanical data published to date regarding the ONH has been obtained from tensile testing, yet compression has been shown to be the main mode of deformation in the ONH under elevated IOP. We have thus tested pig and rat ONH tissue using unconfined cyclic compression. The material constants C1, obtained from fitting the stress vs. strain data with a neo-Hookean material model, were 428 [367, 488] Pa and 64 [53, 76] Pa (mean [95% Confidence Interval]) for pig and rat optic nerve head, respectively. Additionally, we investigated the effects of strain rate and tissue storage on C1 values. These data will inform future efforts to understand and replicate the in vivo biomechanical environment of the ONH.

The effects of dexamethasone on 17β-HSD1 levels at the rat optic nerve.

Dexamethasone (DEX) is associated with many inflammation and metabolic diseases. We analyzed the effects of DEX on the expression of estrogen metabolism enzyme 17-HSD1 at the optic nerve. Rats were treated with different concentrations of intraperitoneal DEX. Western Blot analysis showed that 17-HSD protein was expressed in the optic nerve tissue. The enzyme was detected by immunohistochemistry on the terminal foot of Muller cells from the ganglion cell layer of rat retina. ELISA analysis showed that the 17-HSD1 protein expression of DEX-treated group is 2.4 fold comparing to the control group. The results indicated that DMXS sodium phosphate might modulate the expression of 17-HSD1 protein in optic tissue. This study sheds light on understanding of the relationship among DEX, 17-HSD presence and distribution of visual neural systems. At the same time, DEX treatment affects the athletic ability and memory of the animals. Compared with the control group, the experimental group showed slow response to stimulation, inertia, depression, cowardice and lack of appetite. The results of ethology experiments showed that all the parameters decreased by 15–30%.

Critical Role of Monocyte Recruitment in Optic Nerve Damage Induced by Experimental Optic Neuritis.

Neuroinflammatory diseases are characterized by blood-brain barrier disruption (BBB) and leukocyte infiltration. We investigated the involvement of monocyte recruitment in visual pathway damage provoked by primary optic neuritis (ON) induced by a microinjection of bacterial lipopolysaccharide (LPS) into the optic nerve from male Wistar rats. Increased Evans blue extravasation and cellularity were observed at 6h post-LPS injection. In WT-GFPþ/WT chimeric rat optic nerves, the presence of GFP(+) neutrophils and GFP(+) monocytes, and in wild-type rat optic nerves, an increase in CD11b+CD45low and CD11b+CD45high cell number, were observed at 24h post-LPS. Gamma-irradiation did not affect the increase in BBB permeability, but significantly lessened the decrease in pupil light reflex (PLR), and retinal ganglion cell (RGC) number induced by LPS. At 6h post-LPS, an increase in chemokine (C-C motif) ligand 2 (CCL2) immunoreactivity co-localized with neutrophils (but not microglia/macrophages or astrocytes) was observed, while at 24h post-injection, an increase in Iba-1-immunoreactivity and its co-localization with CCL2 became evident. The co-injection of LPS with bindarit (a CCL2 synthesis inhibitor) lessened the effect of LPS on PLR, and RGC loss. The treatment with etoposide or gadolinium chloride that significantly decreased peripheral monocyte (but not neutrophil or lymphocyte) percentage decreased the effect of LPS on PLR, and RGC number. Moreover, a negative correlation between PRL and monocyte (but not lymphocyte or neutrophil) percentage was observed at 7days post-LPS. Taken together, these results support that monocytes are key players in the initial events that take place during primary ON.