Unilateral Optic Nerve Transection Research Articles

Differential Responses of Retinal Neurons and Glia Revealed via Proteomic Analysis on Primary and Secondary Retinal Ganglion Cell Degeneration.

To explore the temporal profile of retinal proteomes specific to primary and secondary retinal ganglion cell (RGC) loss. Unilateral partial optic nerve transection (pONT) was performed on the temporal side of the rat optic nerve. Temporal and nasal retinal samples were collected at 1, 4 and 8 weeks after pONT (n = 4 each) for non-biased profiling with a high-resolution hybrid quadrupole time-of-flight mass spectrometry running on label-free SWATHTM acquisition (SCIEX). An information-dependent acquisition ion library was generated using ProteinPilot 5.0 and OneOmics cloud bioinformatics. Combined proteome analysis detected 2531 proteins with a false discovery rate of <1%. Compared to the nasal retina, 10, 25 and 61 significantly regulated proteins were found in the temporal retina at 1, 4, and 8 weeks, respectively (p < 0.05, FC ≥ 1.4 or ≤0.7). Eight proteins (ALDH1A1, TRY10, GFAP, HBB-B1, ALB, CDC42, SNCG, NEFL) were differentially expressed for at least two time points. The expressions of ALDH1A1 and SNCG at nerve fibers were decreased along with axonal loss. Increased ALDH1A1 localization in the inner nuclear layer suggested stress response. Increased GFAP expression demonstrated regional reactivity of astrocytes and Muller cells. Meta-analysis of gene ontology showed a pronounced difference in endopeptidase and peptidase inhibitor activity. Temporal proteomic profiling demonstrates established and novel protein targets associated with RGC damage.

Longitudinal Observation of Retinal Response to Optic Nerve Transection in Rats Using Visible Light Optical Coherence Tomography.

To characterize rat retinal responses after optic nerve transection (ONT) by visible-light optical coherence tomography (vis-OCT). Unilateral ONT was performed in Brown Norway rats (n = 8). In vivo, vis-OCT retinal imaging was performed on the experimental eyes before ONT (baseline), and two days, one week, two weeks, and four weeks (endpoint) after ONT, as well as on fellow eyes at the endpoint. The system was operated at a 70 kHz A-line sampling rate with both raster scans (512 × 2 × 512 A-lines), and circular scans (2048 × 100 A-lines) acquired around the optic disc. Retinal layers were segmented to calculate layer thicknesses and project en face images for visualization and quantifications. Vessel densities and oxygen saturation were used to evaluate the morphologic and functional impact on the retinal vasculature. After ONT, retinal nerve fiber bundles demonstrated significant degeneration, starting at two weeks, with a reduction of thicknesses quantified on the nerve fiber layer, ganglion cell complex, and total retina. Along with that, the activation of macrophage-like cells in the vitreoretinal interface was also observed. Vessel densities for all three retinal plexuses were unaffected over the period of observation. However, oxygen saturation in retinal arteries and veins was significantly reduced at four weeks after ONT. Vis-OCT can provide high-definition, in vivo characterization of retinal responses to ONT in rats. Despite a significant reduction in retinal layer thickness, this was not accompanied by alterations in vascular density. Despite this, oximetry indicates reduced retinal oxygen saturation, suggesting that altered vascular physiology is not reflected in the anatomic appearance of retinal blood vessel density alone.

Differential Retinal Protein Expression in Primary and Secondary Retinal Ganglion Cell Degeneration Identified by Integrated SWATH and Target-Based Proteomics.

To investigate the retinal proteins associated with primary and secondary retinal ganglion cell (RGC) degeneration and explore their molecular pathways, SWATH label-free and target-based mass spectrometry was employed to identify the proteomes in various retinal locations in response to localized optic nerve injury. Unilateral partial optic nerve transection (pONT) was performed on adult Wistar rats and their retinas were harvested 2 weeks later. To confirm the separation of primary and secondary RGC degeneration, immunohistochemistry of RNA binding protein with multiple splicing (RBPMS) and glial fibrillary acidic protein (GFAP) was performed on retinal whole-mounts. Retinal proteomes in the temporal and nasal quadrants were evaluated with high resolution hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS), and SWATH-based acquisition, and their expression was compared to the corresponding retinal quadrant in contralateral control eyes and further validated by multiple reaction monitoring mass spectrometry (MRM-MS). A total of 3641 proteins (FDR < 1%) were identified using QTOF-MS. The raw data are available via ProteomeXchange with the identifier PXD026783. Bioinformatics data analysis showed that there were 37 upregulated and 25 downregulated proteins in the temporal quadrant, whereas 20 and five proteins were upregulated and downregulated, respectively, in the nasal quadrant, respectively (n = 4, p < 0.05; fold change ≥ 1.4-fold or ≤0.7). Six proteins were regulated in both the temporal and the nasal quadrants, including CLU, GFAP, GNG5, IRF2BPL, L1CAM, and CPLX1. Linear regression analysis indicated a strong association between the data obtained by means of SWATH-MS and MRM-MS (temporal, R2 = 0.97; nasal, R2 = 0.96). Gene ontology analysis revealed statistically significant changes in the biological processes and cellular components of primary RGC degeneration. The majority of the significant changes in structural, signaling, and cell death proteins were associated with the loss of RGCs in the area of primary RGC degeneration. The combined use of SWATH-MS and MRM-MS methods detects and quantifies regional changes of retinal protein expressions after localized injury. Future investigation with this integrated approach will significantly increase the understanding of diverse processes of progressive RGC degeneration from a proteomic prospective.

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.

Changes in DTI parameters in the optic tracts of macaque monkeys with monocular blindness

For humans and non-human primates, the alteration of the visual pathway’s white matter fibers after visual deprivation has been partially explored. However, the changes in the optic tracts after the transection of the optic nerve have not been well characterized. In the current study, we attempted to investigate the differences in optic tracts between normal and unilateral optic nerve transected macaque monkeys using diffusion tensor imaging (DTI). Four healthy neonatal macaque monkeys were randomly divided into 2 groups, with 2 in each group. Group A served as a control group, and Group B underwent unilateral (right eye) optic nerve transection to produce monocular blindness. Sixteen months (Group B16M) and thirty-two months (Group B32M) after optic nerve transection, diffusion tensor imaging was performed on all monkeys. Then, we compared fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD) and radial diffusivity (RD) in bilateral optic tracts between Group A and Group B and between Group B16M and Group B32M. In both Group B16M and Group B32M, when compared with normal monkeys in Group A, FA was decreased and MD, AD and RD were increased in the bilateral optic tracts of monkeys with monocular blindness. Furthermore, compared with Group B16M, FA was reduced and MD, AD, RD were more obviously increased in the bilateral optic tracts of Group B32M, and noticeable differences in MD, AD and RD were found between the left and right optic tracts in group B32M. We believe that the results of this study would be helpful in investigation of the histological abnormalities of the integrity damage, axonal degeneration and demyelination of optic tracts in macaque monkeys with monocular blindness by DTI parameters in noninvasively and quantitatively.

Cupping in the Monkey Optic Nerve Transection Model Consists of Prelaminar Tissue Thinning in the Absence of Posterior Laminar Deformation.

PurposeTo use optical coherence tomography (OCT) to test the hypothesis that optic nerve head (ONH) “cupping” in the monkey optic nerve transection (ONT) model does not include posterior laminar deformation.MethodsFive monkeys (aged 5.5–7.8 years) underwent ONH and retinal nerve fiber layer (RNFL) OCT imaging five times at baseline and biweekly following unilateral ONT until euthanization at ∼40% RNFL loss. Retinal nerve fiber layer thickness (RNFLT) and minimum rim width (MRW) were calculated from each pre- and post-ONT imaging session. The anterior lamina cribrosa surface (ALCS) was delineated within baseline and pre-euthanasia data sets. Significant ONT versus control eye pre-euthanasia change in prelaminar tissue thickness (PLTT), MRW, RNFLT, and ALCS depth (ALCSD) was determined using a linear mixed-effects model. Eye-specific change in each parameter exceeded the 95% confidence interval constructed from baseline measurements.ResultsAnimals were euthanized 49 to 51 days post ONT. Overall ONT eye change from baseline was significant for MRW (−26.2%, P = 0.0011), RNFLT (−43.8%, P < 0.0001), PLTT (−23.8%, P = 0.0013), and ALCSD (−20.8%, P = 0.033). All five ONT eyes demonstrated significant eye-specific decreases in MRW (−23.7% to −31.8%) and RNFLT (−39.6% to −49.7%). Four ONT eyes showed significant PLTT thinning (−23.0% to −28.2%). The ALCS was anteriorly displaced in three of the ONT eyes (−25.7% to −39.2%). No ONT eye demonstrated posterior laminar displacement.ConclusionsSeven weeks following surgical ONT in the monkey eye, ONH cupping involves prelaminar and rim tissue thinning without posterior deformation of the lamina cribrosa.

Total Retinal Blood Flow in a Nonhuman Primate Optic Nerve Transection Model Using Dual-Beam Bidirectional Doppler FD-OCT and Microsphere Method.

We validated noninvasive Doppler-optical coherence tomography (OCT) blood flow measurements against the terminal microsphere method in a surgical induced optic nerve transection nonhuman primate model. In 6 nonhuman primates, total retinal blood flow (TRBF) was measured with a custom-built dual-beam bidirectional Doppler Fourier Domain (FD)-OCT. Peripapillary retinal nerve fiber layer thickness (RNFLT) was measured by Spectralis spectral-domain (SD)-OCT. Measurements were performed every 10 to 15 days before and after unilateral optic nerve transection (ONT) until RNFLT was reduced by more than 40% from baseline. Before the animals were killed, TRBF was measured using the microsphere technique. A significant correlation between all arterial and venous Doppler OCT TRBF measurements was found in ONT and contralateral control eyes (both P < 0.01, n = 6). The Bland-Altman analysis showed a bias of 0.57 in the ONT group and 0.02 in the contralateral control group. Also, excellent agreement was observed between Doppler OCT and microsphere measurements (P < 0.01, r = 0.976, bias = 0.54). After ONT, TRBF and RNFLT decreased by -51% ± 42% and -44% ± 2% (n = 5), respectively. In the contralateral control eyes, TRBF and RNFLT were unchanged. Very good accordance was found between TRBF measurements, obtained with dual-beam bidirectional Doppler FD-OCT and the microsphere method. It also was possible to monitor changes over time in TRBF after ONT with Doppler OCT. These findings highlight the accuracy and potential of noninvasive Doppler OCT to provide valuable information for detecting early changes in ocular disease in future.

Compromised Optic Nerve Blood Flow and Autoregulation Secondary to Neural Degeneration.

To test the hypothesis that optic nerve head (ONH) blood flow (BF) and autoregulation compromise are consequences of optic nerve degeneration induced by surgical optic nerve transection (ONT). In both eyes of five nonhuman primates, peripapillary retinal nerve fiber layer thickness (RNFLT) was measured by spectral-domain optical coherence tomography. Optic nerve head BF and dynamic autoregulation responses to a rapid manometric IOP increase (from 10-40 mm Hg) were measured by Laser Speckle Flowgraphy. The measurements were conducted every 10 to 15 days before and after unilateral ONT. Post-ONT measurements were repeated until RNFLT in the ONT eye was reduced by more than 40% of baseline value. After ONT, RNFLT, and ONH BF progressively declined over time (P < 0.0001 and P = 0.02, respectively). Longitudinal changes between the two were highly correlated (P < 0.0001). When data was grouped by test session, the first significant decreases for RNFLT and BF were found 13 ± 0.8 and 24 ± 3.2 days post ONT, respectively (P < 0.05, both). At the final time point (55 ± 0.5 days post ONT), RNFLT, and BF were reduced by 44% ± 2.0% and 38 ± 5.0% from baseline, respectively. Dynamic autoregulation analysis showed marginal increased response time in post-ONT eyes (P = 0.05). Control eyes showed no longitudinal changes for any parameter. The close association between RNFLT loss and ONH BF decrease following optic nerve degeneration demonstrated a clear cause and effect relationship. Increased BF response time appears to be a sign of dynamic autoregulation dysfunction in this ONT model.

Bilateral retinal microglial response to unilateral optic nerve transection in rats

When retinal ganglion cells undergo apoptosis after optic nerve (ON) injury, microglial cells proliferate and promptly clear the degenerated debris in the ipsilateral retina. However, microglial changes in the contralateral retina have not been fully elucidated. This study characterized the long-term bilateral retinal microglial responses after unilateral ON transection. We analyzed the time course of proliferation and morphology changes of microglial cells, between 3days and 12weeks post ON transection, of undisturbed and reactive microglia in bilateral retinas of adult Fischer rats with unilateral ON transection. Microglia in retinas without ON transection were distributed homogeneously and possessed a highly ramified morphology, as judged by immunohistochemistry for ionized calcium-binding adapter molecule 1 (Iba1). After ON transection, microglia density in the ipsilateral retina increased gradually from 3days to 2weeks, and decreased from 3weeks to 12weeks, along with dramatic inverted alteration of process branch points of microglia in the ganglion cell layer (GCL). Transformation of ramified microglia into ameboid-like macrophages with few branching processes was observed in the ipsilateral retina from 1week to 3weeks. Though an increase in microglial density was weak in the contralateral retina and could only be statistically detected in the central retina, the morphological alteration over time was obvious and similar to that of the ipsilateral retina. In the inner plexiform layer (IPL), cell density and morphological changes of microglia in both the ipsilateral and contralateral retina were not prominent. These findings indicates that, though proliferation of microglial cells is weak in the contralateral retina after unilateral ON transection, conspicuous alterations in microglial morphology occur bilaterally. These suggest that using the contralateral retina as a control in studies of retinal degeneration should be considered with caution.

Changes in mRNA Expression of Class 3 Semaphorins and Their Receptors in the Adult Rat Retino-Collicular System after Unilateral Optic Nerve Injury

Increasing interest in the role of Class 3 Semaphorins (Sema3s) in plasticity and repair in the injured mammalian central nervous system prompted us to characterize changes in Sema3 expression after optic nerve (ON) injury. We used unilateral ON transection (ONT) and ON crush (ONC) models in conjunction with quantitative polymerase chain reaction (qPCR), and in situ hybridization (ISH) to characterize postinjury changes in the expression of the Sema3s and their receptors in the rat retina, optic nerve, and superior colliculus (SC). We observed no changes in mRNA expression in axotomized retinas at 1 or 14 days after ONT, but there was a transient increase for Sema3b, Sema3f, L1cam, and Plxna3 at 3 days postinjury. There was no change in transcript expression in the deafferented contralateral SC 1 day following ONT, but there was a transient increase in Plxna2 at 3 days, and a decrease in Sema3e, L1cam, and Plxna4a mRNA levels by 14 days. There were also several changes in transcript expression in the unlesioned contralateral retina and ipsilateral SC that differed from those seen in axotomized retina and contralateral SC. At the injury site after ONC, there was a reduction in Sema3b and Sema3f mRNA at 6 hours, returning to control levels by 1 day, and a transient increase in SEMA3A immunoreactivity at 6 hours. These new data on Class 3 Semaphorins and their receptors provide more information about the complex reactive events that occur bilaterally in the retino-collicular system following unilateral adult ON injury.

Dose-dependent and combined effects of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine on the survival of retinal ganglion cells in adult hamsters.

This study investigated the effects of daily intraperitoneal injections of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine (L-NA) on the survival of retinal ganglion cells (RGCs) at 1 and 2 weeks after unilateral optic nerve transection in adult hamsters. The left optic nerves of all animals were transected intraorbitally 1 mm from the optic disc and RGCs were retrogradely labeled with Fluorogold before they received different daily dosages of single MK-801 or L-NA as well as daily combinational treatments of these two chemicals. All experimental and control animals survived for 1 or 2 weeks after optic nerve transection. Our results revealed that the mean numbers of surviving RGCs increased and then decreased when the dosage of MK-801 (1.0, 3.0 and 4.5 mg/kg) and L-NA (1.5, 3.0, 4.5 and 6.0 mg/kg) increased at both 1 and 2 weeks survival time points. Daily combinational use of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA lead to a highest RGC number that was even higher than the sum of the RGC numbers in 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA subgroups at 2 weeks. These findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these chemicals possesses a potentiative and protective effect.

Retinal proteomic changes following unilateral optic nerve transection and early experimental glaucoma in non-human primate eyes

In this work we compared proteomic changes in non-human primate (NHP) retinas at the onset of early experimental glaucoma (EEG) and 3 weeks after optic nerve transection (ONT), as a means to identify regulators in the retina’s response to EEG and ONT. Both eyes of 7 NHPs with either unilateral EEG ( n = 4) or ONT ( n = 3) were enucleated. Proteins were analyzed by a label-free quantitative mass spectrometry system and the abundance of identified retinal proteins was compared between the treated eye and its contralateral control for each NHP. Cellular processes associated with regulated proteins were identified using the MetaCore program. As a result, a total of 209 and 200 proteins were identified in EEG and ONT retinas, respectively. The EEG eyes exhibited two distinguishable levels of maximum IOP: the highest IOP <27 mmHg ( n = 2) and >45 mmHg ( n = 2), termed mild IOP EEG and high IOP EEG eyes, respectively. A limited overlap of proteins regulated in the same direction was seen between the high IOP EEG and either the mild IOP EEG eyes or ONT eyes. Most of the proteins that were up regulated in the high IOP EEG eyes were down regulated in the mild IOP EEG eyes; the latter showed an overall down regulation that was not seen in the other two conditions. An association with cytoskeleton regulation was recognized for up-regulated proteins in the high IOP EEG eyes. We conclude that mild IOP EEG, high IOP EEG and ONT retinas exhibited condition-specific proteomic changes with little overlap between conditions. Cytoarchitecture regulation appears to be a component of the early retinal response to chronic experimental IOP elevation.

Ontogeny of the conus papillaris of the lizard Gallotia galloti and cellular response following transection of the optic nerve: an immunohistochemical and ultrastructural study

Spontaneous regrowth of the axons of retinal ganglion cells (RGC) occurs after unilateral optic nerve transection (ONT) in the lizard Gallotia galloti. We have performed an immunohistochemical and ultrastructural study of the conus papillaris (CP) of this lizard during ontogeny and after ONT in order to characterize its cell subpopulations, innervation and putative blood-brain barrier (BBB) and to evaluate changes occurring throughout regeneration. Proliferating PCNA(+) cells were abundant between embryonic stage 33 (E33) and hatching. From E33, we observed Pax2(+)/GS(+) glial cells in the primitive CP, which became increasingly pigmented and vascularised from E35. Conal astrocytes coexpressing Pax2 with vimentin and/or GFAP were identified from E37-E38. GluT-1(+)/LEA(+)/Pax2(-) endothelial cells (ECs) formed a continuous endothelium with tight junctions and luminal and abluminal microfolds. In adults, the peripheral blood vessels showed a thinner calibre, stronger GluT-1 staining and more abundant microfolds than those of the central CP indicating the higher specialization involved during transport within the former. Occasional pericytes, abundant Pax2(+) pigment cells, LEA(+) microglia/macrophages, unmyelinated Tuj1(+) nerve fibres and SV2(+) synaptic vesicles were also observed in the perivascular zone. After ONT, the expression of GluT-1 and p75(NTR) persisted in ECs, suggesting the preservation/early recovery of the BBB. Relevant ultrastructural alterations were observed at 0.5months postlesion, although, by 3months, the CP had recovered the ultrastructure of controls indicating tissue recovery. Abnormal newly formed blood vessels had developed in the CP-optic nerve junction. Thus, the CP is a central nervous system structure whose regenerating capacity might be key for the nutritional support of regenerating RGCs in G. galloti.

Expression of immediate-early genes in primary visual cortex of rat early after acute optic nerve injury

To investigate the differential expression of immediate-early genes c-jun and c-fos in primary visual cortex of rat early after acute optic nerve injury and the relationship between immediate-early gene expression and injury degree of optic nerve. This was an factorial design of two factors. 55 male Sprague-Dawley rats were divided into groups randomized. Unilateral optic nerve crush injury or transection was performed in rats to obtain acute partial or complete monocular optic nerve injury models. Frozen sections through visual cortex were cut in normal rats and model rats respectively at 2 h, 1 d, 3 d, 1 week and 1 month after operation. Expression of c-Jun and c-Fos was detected in primary visual cortex by means of immunohistochemistry. Statistical comparisons were made using variance analysis of factorial design. Statistically significant different of c-Jun expression existed between optc nerve crash injury and transection models (F = 50.344, P = 0.000). Increased expression of c-Jun in primary visual cortex could be observed at 2 h postoperation, and reached peak value at 1d postoperation. The extent of increased c-Jun expression was much higher in optic nerve transection models than crush injury models. Statistically significant different of c-Fos expression existed between optc nerve crash injury and transection models (F = 62.232, P = 0.000) Decreased expression of c-Fos in primary visual cortex could be observed at 2 h postoperation in optic nerve crush injury models, and reached bottom at 3 d postoperation. The extent of decrease of c-Fos expression was lower in optic nerve transection models than crush injury models and transient increased expression could be observed at 2 h postoperation. The expression of immediate-early genes c-Jun and c-Fos changed shortly after optic nerve injury. They may act in opposite direction in the primary visual cortex.

Serial multifocal electroretinograms during long-term elevation and reduction of intraocular pressure in non-human primates

The purpose of this study was to evaluate the relationship between elevations of intraocular pressure (IOP) and the multifocal electroretinogram (mfERG) in non-human primates. Experimental glaucoma was induced in 4 rhesus and 4 cynomolgus monkeys by laser trabecular meshwork destruction (LTD) in one eye. To evaluate the contribution of ganglion cells to mfERG changes, one monkey of each species had previously underwent unilateral optic nerve transection (ONT). After >or=44 weeks of elevation, the IOP was reduced by trabeculectomy in 2 non-transected animals. In the intact (non-transected) animals, there was an increase in the amplitude of the early mfERG waveforms (N1 and P1) of the first-order kernel (K1) throughout the period of IOP elevation in all of the rhesus, but not all of the cynomolgus monkeys. A species difference was also present as a decrease of the second-order kernel, first slice (K2.1) in all of the cynomolgus monkeys but only in 1 of the rhesus monkeys (the 1 with the ONT). Similar IOP effects on the mfERG were seen in the ONT animals. Surgical lowering of IOP resulted in a return of the elevated K1 amplitudes to baseline levels. However, the depressed K2.1 RMS in the cynomolgus monkeys did not recover. These results demonstrate species-specific changes in cone-driven retinal function during periods of elevated IOP. These IOP-related effects can occur in the absence of retinal ganglion cells and may be reversible.

Relative Course of Retinal Nerve Fiber Layer Birefringence and Thickness and Retinal Function Changes after Optic Nerve Transection

To test the hypothesis that alterations of RNFL birefringence precede changes in RNFL thickness in an experimental model of RGC injury and, secondarily, to determine the time course of RGC functional abnormalities relative to RNFL birefringence and thickness changes. RNFL birefringence was measured by scanning laser polarimetry (GDx VCC; Carl Zeiss Meditec, Inc., Dublin, CA). RNFL thickness was measured by spectral domain optical coherence tomography (SD-OCT, Spectralis HRA+OCT; Heidelberg Engineering, GmbH, Heidelberg, Germany). Retinal function was assessed by three forms of electroretinography (ERG): slow-sequence multifocal (mf)ERG (VERIS; EDI, San Mateo, CA); pattern-reversal (P)ERG (Utas-E3000; LKC Technologies, Inc. Gaithersburg, MD); and photopic full-field flash (ff)ERG (Utas-E3000; LKC Technologies). All measurements were obtained in both eyes of four adult rhesus macaque monkeys (Macaca mulatta) during two baseline sessions, and again 1 week and 2 weeks after unilateral optic nerve transection (ONT). ONT was successfully completed in three subjects. RNFL birefringence declined by 15% 1 week after ONT (P = 0.043), whereas there was no significant change in RNFL thickness (+1%, P = 0.42). Two weeks after ONT, RNFL retardance had declined by 39% (P = 0.018), whereas RNFL thickness had declined by only 15% (P = 0.025). RGC functional abnormalities were present 1 week after ONT, including decreased amplitudes relative to baseline of the mfERG high-frequency components (-65%, P = 0.018), the PERG N95 component (-70%, P = 0.007), and the photopic negative response of the ffERG (-44%, P = 0.005). RNFL birefringence declined before and faster than RNFL thickness after ONT. RGC functional abnormalities were present 1 week after ONT, when RNFL thickness had not yet begun to change. RNFL birefringence changes after acute RGC injury are associated with RGC dysfunction. Together, they reflect RGC abnormalities that precede axonal caliber changes and loss.

Enhanced survival of melanopsin-expressing retinal ganglion cells after injury is associated with the PI3 K/Akt pathway.

In the present study, we studied the factors that contribute to the injury-resistant property of melanopsin-expressing retinal ganglion cells (mRGCs). Since phosphatidylinositol-3 kinase (PI3 K)/Akt signaling pathway is one of the well-known pathways for neuronal cell survival, we investigated the survival of mRGCs by applying the PI3 K/Akt specific inhibitors after injury. Two injury models, unilateral optic nerve transection and ocular hypertension, were adopted using Sprague-Dawley rats. Inhibitors of PI3 K/Akt were injected intravitreally following injuries to inhibit the PI3 K/Akt signaling pathway. Retinas were dissected after designated survival time, immunohistochemistry was carried out to visualize the mRGCs using melanopsin antibody and the number of mRGCs was counted. Co-expression of melanopsin and phospho-Akt (pAkt) was also examined. Compared to the survival of non-melanopsin-expressing RGCs, mRGCs showed a marked resistance to injury and co-expressed pAkt. Application of PI3 K/Akt inhibitors decreased the survival of mRGCs after injury. Our previous study has shown that mRGC are less susceptible to injury following the induction of ocular hypertension. In this study, we report that mRGCs were injury-resistant to a more severe type of injury, the optic nerve transection. More importantly, the PI3 K/Akt pathway was found to play a role in maintaining the survival of mRGCs after injury.

Global changes in optic nerve head gene expression after exposure to elevated intraocular pressure in a rat glaucoma model.

In glaucoma, the optic nerve head (ONH) is the likely site of initial injury and elevated intraocular pressure (IOP) is the best-known risk factor. This study determines global gene expression changes in the pressure-injured ONH. Unilateral sustained IOP elevation (glaucoma, n = 46) or optic nerve transection (n = 10) was produced in rats. ONHs were removed, and the retrobulbar optic nerves were graded for degeneration. Gene expression in the glaucomatous ONH with extensive injury was compared with that in the fellow ONH (n = 6/group), by using cDNA microarrays. Data from 12 arrays were normalized, significant differences in gene expression determined, and significantly affected gene classes identified. For the remaining ONH, grouped by experimental condition and degree of injury, quantitative reverse transcriptase-PCR (qPCR) and ANOVA were used to compare selected message levels. Microarray analysis identified more than 2000 significantly regulated genes. For 225 of these genes, the changes were greater than twofold. The most significantly affected gene classes were cell proliferation, immune response, lysosome, cytoskeleton, extracellular matrix, and ribosome. A 2.7-fold increase in ONH cellularity confirmed glaucoma model cell proliferation. By qPCR, increases in levels of periostin, collagen VI, and transforming growth factor beta1 were linearly correlated to the degree of IOP-induced injury. For cyclinD1, fibulin 2, tenascin C, TIMP1, and aquaporin-4, correlations were significantly nonlinear, displaying maximum change with focal injury. In the ONH, pressure-induced injury results in cell proliferation and dramatically altered gene expression. For specific genes, expression levels were most altered by focal injury, suggesting that further array studies may identify initial, and potentially injurious, altered processes.

Pax7 expression in the adult rat superior colliculus following optic nerve injury

The transcription factor Pax7 has been implicated in the normal development of the superior colliculus and continues to be expressed in the adult superior colliculus, where it is concentrated in the retino-recipient laminae. Here we assessed, immunohistochemically, Pax7 expression in the adult rat superior colliculus after unilateral intraorbital optic nerve transection. We show that after optic nerve transection, the number of Pax7-expressing cells increased to re-establish the developmental rostral-caudal gradient and that these Pax7-expressing superior colliculus cells were neurons. These findings may have implications for the design of therapeutic interventions to restore functional connections in the adult visual pathway.

Unilateral optic nerve transection up-regulate Hsp70 protein expression in lateral geniculate nucleus of rats

Studies have demonstrated that optic nerve transection results in apoptotic cell death of retinal ganglion cells (RGCs) and neurons within lateral geniculate nucleus (LGN). Heat shock protein (Hsp) 70 was reported to be involved in protecting cells from injury under various pathological conditions in vivo and in vitro. To determine the involvement of Hsp70 in protecting neurons within LGN against damage or loss induced by optic nerve injuries, we observed the changes in protein expression and distribution of Hsp70 in LGN at days 1, 3, 7, 14 and 28 after unilateral optic nerve transection in the left eye of Sprague–Dawley rats by using Western blot analysis and immunohistochemical staining. We found that the levels of Hsp70 protein expression increased significantly ( p < 0.05, n = 6 for each group) in both right and left LGN of rats following left optic nerve transection 1–7 days. The maximum of Hsp70 expression reached at day 3. However, Hsp70 protein expression levels in both right and left LGN returned to control levels at 14 and 28 days after left optic nerve lesion. In addition, the increased Hsp70 expression, which mainly localized in the intergeniculate leaflet of LGN, was also observed by immunostaining in right LGN at the end of day 3 after the lesion. These results suggest that increased expression of Hsp70 may be involved in protecting neurons within LGN against damage or loss induced by left optic nerve transection at early stage.