The Role of Peroxisome Proliferator-Activated Receptor and Effects of Its Agonist,Pioglitazone, on a Rat Model of Optic Nerve Crush: PPARγ in Retinal Neuroprotection

It has been shown that peroxisome proliferators-activated receptor gamma (PPARγ) is beneficial for central nervous system injury. However its role on optic nerve injury remains unknown. In the present study, we examined the change of PPARγ expression in rat retina following optic nerve injury and investigated the effect of pioglitazone (Pio), a PPARγ agonist, on retinal ganglion cells (RGCs) neuroprotection using a rat optic nerve crush (ONC) model. Our results showed that PPARγ mRNA and protein levels were increased after ONC, and most of PPARγ-immunoreactive cells colocalized with Müller cells. Pio treatment significantly enhanced the number of surviving RGCs and inhibited RGCs apoptosis induced by ONC. However, when PPARγ antagonist GW9662 was used, these neuroprotective effects were abolished. In addition, pio attenuated Müller cell activation after ONC. These results indicate that PPARγ appears to protect RGCs from ONC possibly via the reduction of Müller glial activation. It provides evidence that activation of PPARγ may be a potential alternative treatment for RGCs neuroprotection.     

Comparative Evaluation of Methods for Estimating Retinal Ganglion Cell Loss in Retinal Sections and Wholemounts

To investigate the reliability of different methods of quantifying retinal ganglion cells (RGCs) in rat retinal sections and wholemounts from eyes with either intact optic nerves or those axotomised after optic nerve crush (ONC). Adult rats received a unilateral ONC and after 21 days the numbers of Brn3a⁺, βIII-tubulin⁺ and Islet-1⁺ RGCs were quantified in either retinal radial sections or wholemounts in which FluoroGold (FG) was injected 48 h before harvesting. Phenotypic antibody markers were used to distinguish RGCs from astrocytes, macrophages/microglia and amacrine cells. In wholemounted retinae, counts of FG⁺ and Brn3a⁺ RGCs were of similar magnitude in eyes with intact optic nerves and were similarly reduced after ONC. Larger differences in RGC number were detected between intact and ONC groups when images were taken closer to the optic nerve head. In radial sections, Brn3a did not stain astrocytes, macrophages/microglia or amacrine cells, whereas βIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of βIII-tubulin⁺ RGCs was greater than Brn3a⁺ RGCs, both in retinae from eyes with intact optic nerves and eyes 21 days after ONC. Islet-1 staining also overestimated the number of RGCs compared to Brn3a, but only after ONC. Estimates of RGC loss were similar in Brn3a-stained radial retinal sections compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG, with sections having the added advantage of reducing experimental animal usage.     

Establishment of the Relationship between Tumor Size and Range of Histological Involvement to Evaluate the Rationality of Current Retinoblastoma Management

Purpose

To determine whether tumor size correlates with histopathological involvement and hence evaluate the rationality of conservative treatment for retinoblastoma.

Methods

We retrospectively studied 221 patients (221 eyes) treated for retinoblastoma with enucleation in the Zhongshan Ophthalmic Center of Sun Yat-sen University, China, from October 1995 to December 2004. Histopathological data included involvement of the anterior chamber, sclera, choroids, and optic nerve. Tumor size was measured by B-ultrasound examination.

Results

Tumor invasion of the optic nerve correlated with the Reese-Ellsworth (R-E) staging system and the International Classification for Retinoblastoma (ICRB): optic nerve involvement was significantly more frequent in R-E stage V (P = 0.009) and ICRB Group E (P = 0.002) cases. However, 19.1% of patients with R-E stage I, II and III, and 16.7% of patients with ICRB Group B and C disease showed histopathological involvement of the postlaminar optic nerve. Extraocular involvement was observed in 17.7% of tumors ≤15 mm in diameter. Tumors >15 mm in diameter showed greater extraocular involvement, including the optic nerve (P = 0.000) and sclera (P = 0.032), than tumors ≤15 mm in diameter. Postlaminar optic nerve invasion was observed in 19.6% of tumors ≤10 mm in thickness. Tumors >10 mm in thickness had sclera involvement more frequently than tumors ≤10 mm in thickness (P = 0.029). Postlaminar optic nerve invasion was noted in 17.1% of patients with tumors ≤15 mm in diameter and ≤10 mm in thickness.

Conclusions

Medium-sized retinoblastomas frequently invade outside the globe. Thus, indications for conservative treatment need improvement.     

Human induced pluripotent stem cells differentiation into oligodendrocyte progenitors and transplantation in a rat model of optic chiasm demyelination

Background

This study aims to differentiate human induced pluripotent stem cells (hiPSCs) into oligodendrocyte precursors and assess their recovery potential in a demyelinated optic chiasm model in rats.

Methodology/Principal Findings

We generated a cell population of oligodendrocyte progenitors from hiPSCs by using embryoid body formation in a defined medium supplemented with a combination of factors, positive selection and mechanical enrichment. Real-time polymerase chain reaction and immunofluorescence analyses showed that stage-specific markers, Olig2, Sox10, NG2, PDGFRα, O4, A2B5, GalC, and MBP were expressed following the differentiation procedure, and enrichment of the oligodendrocyte lineage. These results are comparable with the expression of stage-specific markers in human embryonic stem cell-derived oligodendrocyte lineage cells. Transplantation of hiPSC-derived oligodendrocyte progenitors into the lysolecithin-induced demyelinated optic chiasm of the rat model resulted in recovery from symptoms, and integration and differentiation into oligodendrocytes were detected by immunohistofluorescence staining against PLP and MBP, and measurements of the visual evoked potentials.

Conclusions/Significance

These results showed that oligodendrocyte progenitors generated efficiently from hiPSCs can be used in future biomedical studies once safety issues have been overcome.     

Intravitreal Transplantation of Human Umbilical Cord Blood Stem Cells Protects Rats from Traumatic Optic Neuropathy

Objectives

To treat traumatic optic neuropathy (TON) with transplantation of human umbilical cord blood stem cells (hUCBSC) and explore how transplanted stem cells participate in the neuron repairing process.

Methods

A total of 195 Sprague-Dawley rats were randomly assigned to three groups: sham-surgery, optic nerve injury, and stem cell transplant group. Optic nerve injury was established in rats by directly clamping the optic nerve for 30 seconds. hUCBSC was microinjected into the vitreous cavity of injured rats. Optic nerve function was evaluated by flash visual evoked potentials (F-VEP). Apoptosis in retina tissues was detected by TUNEL staining. GRP78 and CHOP gene expression was measured by RT-PCR.

Results

After injury, transplantation of hUCBSC significantly blunted a reduction in optic nerve function indicated by smaller decreases in amplitude and smaller increases in peak latency of F-VEP waveform compared to the injury alone group. Also, significant more in retinal ganglion cell (RGC) count and less in RGC apoptosis were detected after transplantation compared to injured rats. The protective effect correlated with upregulated GRP78 and downregulated CHOP mRNA expression.

Conclusion

Intravitreal transplantation of hUCBSCs significantly blunted a reduction in optic nerve function through increasing RGC survival and decreasing retinal cell apoptosis. The protective role of transplantation was associated with upregulation of GRP78 expression and downregulation of CHOP expression in retinal cells.     

Scapular Winging Secondary to Apparent Long Thoracic Nerve Palsy in a Young Female Swimmer

Background

 In neurological diseases, winging of the scapula occurs because of serratus anterior muscle dysfunction due to long thoracic nerve palsy, or trapezius muscle dysfunction due to accessory nerve palsy. Several sports can cause long thoracic nerve palsy, including archery and tennis. To our knowledge, this is the first report of long thoracic nerve palsy in an aquatic sport.

Objective

 The present study is a rare case of winging of the scapula that occurred during synchronized swimming practice.

Methods

 The patient''s history with the present illness, examination findings, rehabilitation progress, and related medical literature are presented.

Results

 A 14-year-old female synchronized swimmer had chief complaints of muscle weakness, pain, and paresthesia in the right scapula. Upon examination, marked winging of the scapula appeared during anterior arm elevation, as did floating of the superior angle. After 1 year of therapy, right shoulder girdle pain and paresthesia had disappeared; however, winging of the scapula remained.

Conclusions

 Based on this observation and the severe pain in the vicinity of the second dorsal rib, we believe the cause was damage to the nerve proximal to the branch arising from the upper nerve trunk that innervates the serratus anterior.     

Metabolic changes in the visual cortex are linked to retinal nerve fiber layer thinning in multiple sclerosis

Objective

To investigate the damage to the retinal nerve fiber layer as part of the anterior visual pathway as well as an impairment of the neuronal and axonal integrity in the visual cortex as part of the posterior visual pathway with complementary neuroimaging techniques, and to correlate our results to patients'' clinical symptoms concerning the visual pathway.

Design, Subjects and Methods

Survey of 86 patients with relapsing-remitting multiple sclerosis that were subjected to retinal nerve fiber layer thickness (RNFLT) measurement by optical coherence tomography, to a routine MRI scan including the calculation of the brain parenchymal fraction (BPF), and to magnetic resonance spectroscopy at 3 tesla, quantifying N-acetyl aspartate (NAA) concentrations in the visual cortex and normal-appearing white matter.

Results

RNFLT correlated significantly with BPF and visual cortex NAA, but not with normal-appearing white matter NAA. This was connected with the patients'' history of a previous optic neuritis. In a combined model, both BPF and visual cortex NAA were independently associated with RNFLT.

Conclusions

Our data suggest the existence of functional pathway-specific damage patterns exceeding global neurodegeneration. They suggest a strong interrelationship between damage to the anterior and the posterior visual pathway.     

Neuroprotective effect of transplanted human embryonic stem cell-derived neural precursors in an animal model of multiple sclerosis

Background

Multiple sclerosis (MS) is an immune mediated demyelinating disease of the central nervous system (CNS). A potential new therapeutic approach for MS is cell transplantation which may promote remyelination and suppress the inflammatory process.

Methods

We transplanted human embryonic stem cells (hESC)-derived early multipotent neural precursors (NPs) into the brain ventricles of mice induced with experimental autoimmune encephalomyelitis (EAE), the animal model of MS. We studied the effect of the transplanted NPs on the functional and pathological manifestations of the disease.

Results

Transplanted hESC-derived NPs significantly reduced the clinical signs of EAE. Histological examination showed migration of the transplanted NPs to the host white matter, however, differentiation to mature oligodendrocytes and remyelination were negligible. Time course analysis of the evolution and progression of CNS inflammation and tissue injury showed an attenuation of the inflammatory process in transplanted animals, which was correlated with the reduction of both axonal damage and demyelination. Co-culture experiments showed that hESC-derived NPs inhibited the activation and proliferation of lymph node–derived T cells in response to nonspecific polyclonal stimuli.

Conclusions

The therapeutic effect of transplantation was not related to graft or host remyelination but was mediated by an immunosuppressive neuroprotective mechanism. The attenuation of EAE by hESC-derived NPs, demonstrated here, may serve as the first step towards further developments of hESC for cell therapy in MS.     

Reduced Ventral Cingulum Integrity and Increased Behavioral Problems in Children with Isolated Optic Nerve Hypoplasia and Mild to Moderate or No Visual Impairment

Objectives

To assess the prevalence of behavioral problems in children with isolated optic nerve hypoplasia, mild to moderate or no visual impairment, and no developmental delay. To identify white matter abnormalities that may provide neural correlates for any behavioral abnormalities identified.

Patients and Methods

Eleven children with isolated optic nerve hypoplasia (mean age 5.9 years) underwent behavioral assessment and brain diffusion tensor imaging, Twenty four controls with isolated short stature (mean age 6.4 years) underwent MRI, 11 of whom also completed behavioral assessments. Fractional anisotropy images were processed using tract-based spatial statistics. Partial correlation between ventral cingulum, corpus callosum and optic radiation fractional anisotropy, and child behavioral checklist scores (controlled for age at scan and sex) was performed.

Results

Children with optic nerve hypoplasia had significantly higher scores on the child behavioral checklist (p<0.05) than controls (4 had scores in the clinically significant range). Ventral cingulum, corpus callosum and optic radiation fractional anisotropy were significantly reduced in children with optic nerve hypoplasia. Right ventral cingulum fractional anisotropy correlated with total and externalising child behavioral checklist scores (r = −0.52, p<0.02, r = −0.46, p<0.049 respectively). There were no significant correlations between left ventral cingulum, corpus callosum or optic radiation fractional anisotropy and behavioral scores.

Conclusions

Our findings suggest that children with optic nerve hypoplasia and mild to moderate or no visual impairment require behavioral assessment to determine the presence of clinically significant behavioral problems. Reduced structural integrity of the ventral cingulum correlated with behavioral scores, suggesting that these white matter abnormalities may be clinically significant. The presence of reduced fractional anisotropy in the optic radiations of children with mild to moderate or no visual impairment raises questions as to the pathogenesis of these changes which will need to be addressed by future studies.     

Baculovirus as an Ideal Radionuclide Reporter Gene Vector: A New Strategy for Monitoring the Fate of Human Stem Cells In Vivo

Purpose

Radionuclide reporter gene imaging holds promise for non-invasive monitoring of transplanted stem cells. Thus, the feasibility of utilizing recombinant baculoviruses carrying the sodium iodide symporter (NIS) reporter gene in monitoring stem cell therapy by radionuclide imaging was explored in this study.

Methods

Recombinant baculoviruses carrying NIS and green fluorescent protein (GFP) reporter genes (Bac-NIS and Bac-GFP) were constructed and used to infect human induced pluripotent stem cells (hiPSCs), human embryonic stem cells (hESCs) and human umbilical cord blood mesenchymal stem cells (hUCB-MSCs). Infection efficiency, total fluorescence intensity and duration of transgene expression were determined by flow cytometry. Cytotoxicity/proliferative effects of baculovirus on hUCB-MSCs were assessed using CCK-8 assays. ¹²⁵I uptake and perchlorate inhibition assays were performed on Bac-NIS-infected hUCB-MSCs. Radionuclide imaging of mice transplanted with Bac-NIS-infected hUCB-MSCs was performed by NanoSPECT/CT imaging.

Results

Infection efficiencies of recombinant baculovirus in hESCs, hiPSCs and hUCB-MSCs increased with increasing MOIs (27.3%, 35.8% and 95.6%, respectively, at MOI = 800). Almost no cytotoxicity and only slight effects on hUCB-MSCs proliferation were observed. Obvious GFP expression (40.6%) remained at 8 days post-infection. The radioiodide was functionally accumulated by NIS gene products and specifically inhibited by perchlorate (ClO₄ ^-). Radioiodide uptake, peaking at 30 min and gradually decreasing over time, significantly correlated with hUCB-MSCs cell number (R² = 0.994). Finally, radionuclide imaging showed Bac-NIS-infected hUCB-MSCs effectively accumulated radioiodide in vivo, which gradually weakened over time.

Conclusion

Baculovirus as transgenic vector of radionuclide reporter gene imaging technology is a promising strategy for monitoring stem cell transplantation therapy.     

A novel animal model of partial optic nerve transection established using an optic nerve quantitative amputator

Background

Research into retinal ganglion cell (RGC) degeneration and neuroprotection after optic nerve injury has received considerable attention and the establishment of simple and effective animal models is of critical importance for future progress.

Methodology/Principal Findings

In the present study, the optic nerves of Wistar rats were semi-transected selectively with a novel optic nerve quantitative amputator. The variation in RGC density was observed with retro-labeled fluorogold at different time points after nerve injury. The densities of surviving RGCs in the experimental eyes at different time points were 1113.69±188.83 RGC/mm² (the survival rate was 63.81% compared with the contralateral eye of the same animal) 1 week post surgery; 748.22±134.75 /mm² (46.16% survival rate) 2 weeks post surgery; 505.03±118.67 /mm² (30.52% survival rate) 4 weeks post surgery; 436.86±76.36 /mm² (24.01% survival rate) 8 weeks post surgery; and 378.20±66.74 /mm² (20.30% survival rate) 12 weeks post surgery. Simultaneously, we also measured the axonal distribution of optic nerve fibers; the latency and amplitude of pattern visual evoke potentials (P-VEP); and the variation in pupil diameter response to pupillary light reflex. All of these observations and profiles were consistent with post injury variation characteristics of the optic nerve. These results indicate that we effectively simulated the pathological process of primary and secondary injury after optic nerve injury.

Conclusions/Significance

The present quantitative transection optic nerve injury model has increased reproducibility, effectiveness and uniformity. This model is an ideal animal model to provide a foundation for researching new treatments for nerve repair after optic nerve and/or central nerve injury.     

Oral Microbiome Link to Neurodegeneration in Glaucoma

Background

Glaucoma is a progressive optic nerve degenerative disease that often leads to blindness. Local inflammatory responses are implicated in the pathology of glaucoma. Although inflammatory episodes outside the CNS, such as those due to acute systemic infections, have been linked to central neurodegeneration, they do not appear to be relevant to glaucoma. Based on clinical observations, we hypothesized that chronic subclinical peripheral inflammation contributes to neurodegeneration in glaucoma.

Methods

Mouthwash specimens from patients with glaucoma and control subjects were analyzed for the amount of bacteria. To determine a possible pathogenic mechanism, low-dose subcutaneous lipopolysaccharide (LPS) was administered in two separate animal models of glaucoma. Glaucomatous neurodegeneration was assessed in the retina and optic nerve two months later. Changes in gene expression of toll-like receptor 4 (TLR4) signaling pathway and complement as well as changes in microglial numbers and morphology were analyzed in the retina and optic nerve. The effect of pharmacologic blockade of TLR4 with naloxone was determined.

Findings

Patients with glaucoma had higher bacterial oral counts compared to control subjects (p<0.017). Low-dose LPS administration in glaucoma animal models resulted in enhancement of axonal degeneration and neuronal loss. Microglial activation in the optic nerve and retina as well as upregulation of TLR4 signaling and complement system were observed. Pharmacologic blockade of TLR4 partially ameliorated the enhanced damage.

Conclusions

The above findings suggest that the oral microbiome contributes to glaucoma pathophysiology. A plausible mechanism by which increased bacterial loads can lead to neurodegeneration is provided by experiments in animal models of the disease and involves activation of microglia in the retina and optic nerve, mediated through TLR4 signaling and complement upregulation. The finding that commensal bacteria may play a role in the development and/or progression of glaucomatous pathology may also be relevant to other chronic neurodegenerative disorders.     

A Laser-Induced Mouse Model with Long-Term Intraocular Pressure Elevation

Purpose

To develop and characterize a mouse model with intraocular pressure (IOP) elevation after laser photocoagulation on the trabecular meshwork (TM), which may serve as a model to investigate the potential of stem cell-based therapies for glaucoma.

Methods

IOP was measured in 281 adult C57BL/6 mice to determine normal IOP range. IOP elevation was induced unilaterally in 50 adult mice, by targeting the TM through the limbus with a 532-nm diode laser. IOP was measured up to 24 weeks post-treatment. The optic nerve damage was detected by electroretinography and assessed by semiautomatic counting of optic nerve axons. Effects of laser treatment on the TM were evaluated by histology, immunofluorescence staining, optical coherence tomography (OCT) and transmission electron microscopy (TEM).

Results

The average IOP of C57BL/6 mice was 14.5±2.6 mmHg (Mean ±SD). After laser treatment, IOP averaged above 20 mmHg throughout the follow-up period of 24 weeks. At 24 weeks, 57% of treated eyes had elevated IOP with the mean IOP of 22.5±2.5 mmHg (Mean ±SED). The difference of average axon count (59.0%) between laser treated and untreated eyes was statistically significant. Photopic negative response (PhNR) by electroretinography was significantly decreased. CD45+ inflammatory cells invaded the TM within 1 week. The expression of SPARC was increased in the TM from 1 to 12 weeks. Histology showed the anterior chamber angle open after laser treatment. OCT indicated that most of the eyes with laser treatment had no synechia in the anterior chamber angles. TEM demonstrated disorganized and compacted extracellular matrix in the TM.

Conclusions

An experimental murine ocular hypertension model with an open angle and optic nerve axon loss was produced with laser photocoagulation, which could be used to investigate stem cell-based therapies for restoration of the outflow pathway integrity for ocular hypertension or glaucoma.     

Ultrastructural Features of Ischemic Tissue following Application of a Bio-Membrane Based Progenitor Cardiomyocyte Patch for Myocardial Infarction Repair

Background and Objective

Implantation of cell-sheets into damaged regions of the heart after myocardial infarction (MI) has been shown to improve heart function. However, the tissue morphology following application of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) has not been studied in detail at the level afforded by electron microscopy. We hypothesized that increasing the number of CM derived from iPSC would increase the effectiveness of cell-sheets used to treat ischemic cardiomyopathy. We report here on the ultrastructural features after application of a bio-membrane ‘cell patch’.

Methods

iPSC-derived progenitor cells were transduced using lentivirus vectors with or without NCX1 promoter. iPSC-CM sheets were transplanted over the transmural MI region in a mouse model of regional ischemic cardiomyopathy. Mice were divided into four groups, 1) Sham; 2) MI; 3) MI + iPSC without NCX1 treated cells (MI + iPSC^Null) and 4) MI + iPSC receiving NCX1 promoter treated cells (MI + iPSC^NCX1). Echocardiography was performed 4 weeks after cell patch application, followed by histological and transmission electron microscopy (TEM) analysis.

Results

Large numbers of transplanted CM were observed with significant improvements in left ventricular performance and remodeling in group 4 as compared with group 3. No teratoma formation was detected in any of the treatment groups.

Conclusion

Manipulation of iPSC yields large numbers of iPSC-CM and favorable morphological and ultrastructural tissue changes. These changes have the potential to enhance current methods used for restoration of cardiac function after MI.     

Estimated Rates of Retinal Ganglion Cell Loss in Glaucomatous Eyes with and without Optic Disc Hemorrhages

Purpose

To evaluate whether optic disc hemorrhages are associated with faster rates of estimated retinal ganglion cell (RGC) loss in glaucoma.

Methods

A longitudinal observational cohort study of 222 eyes of 122 patients with glaucoma recruited from the Diagnostic Innovations Glaucoma Study (DIGS) followed for an average of 3.74±0.85 years. All subjects had optical coherence tomography and standard automated perimetry during follow up. Optic disc hemorrhages were detected by masked evaluation of stereophotographs. Rates of change in estimated numbers of RGCs were determined using a previously described method. A random coefficients model was used to investigate the relationship between disc hemorrhages and rates of change in estimated RGC counts over time.

Results

19 eyes of 18 subjects had at least one disc hemorrhage during follow up. At baseline, average estimated RGC counts in eyes with and without disc hemorrhages were 677,994 cells and 682,021 cells, respectively (P = 0.929). Eyes with optic disc hemorrhages during follow-up had significantly faster rates of estimated RGC loss than eyes without disc hemorrhages (22,233 cells/year versus 10,704 cells/year, P = 0.020). The effect of disc hemorrhages on the rates of estimated RGC loss remained significant after adjusting for confounding variables.

Conclusion

Eyes with disc hemorrhages showed faster rates of RGC loss compared to eyes without disc hemorrhages. These results provide further evidence that disc hemorrhages should be considered as an indicator of increased risk for faster neural loss in glaucoma.     

Comparison of Longitudinal In Vivo Measurements of Retinal Nerve Fiber Layer Thickness and Retinal Ganglion Cell Density after Optic Nerve Transection in Rat

Purpose

To determine the relationship between longitudinal in vivo measurements of retinal nerve fiber layer thickness (RNFLT) and retinal ganglion cell (RGC) density after unilateral optic nerve transection (ONT).

Methods

Nineteen adult Brown-Norway rats were studied; N = 10 ONT plus RGC label, N = 3 ONT plus vehicle only (sans label), N = 6 sham ONT plus RGC label. RNFLT was measured by spectral domain optical coherence tomography (SD-OCT) at baseline then weekly for 1 month. RGCs were labeled by retrograde transport of fluorescently conjugated cholera toxin B (CTB) from the superior colliculus 48 hours prior to ONT or sham surgery. RGC density measurements were obtained by confocal scanning laser ophthalmoscopy (CSLO) at baseline and weekly for 1 month. RGC density and reactivity of microglia (anti-Iba1) and astrocytes (anti-GFAP) were determined from post mortem fluorescence microscopy of whole-mount retinae.

Results

RNFLT decreased after ONT by 17% (p<0.05), 30% (p<0.0001) and 36% (p<0.0001) at weeks 2, 3 and 4. RGC density decreased after ONT by 18%, 69%, 85% and 92% at weeks 1, 2, 3 and 4 (p<0.0001 each). RGC density measured in vivo at week 4 and post mortem by microscopy were strongly correlated (R = 0.91, p<0.0001). In vivo measures of RNFLT and RGC density were strongly correlated (R = 0.81, p<0.0001). In ONT- CTB labeled fellow eyes, RNFLT increased by 18%, 52% and 36% at weeks 2, 3 and 4 (p<0.0001), but did not change in fellow ONT-eyes sans CTB. Microgliosis was evident in the RNFL of the ONT-CTB fellow eyes, exceeding that observed in other fellow eyes.

Conclusions

In vivo measurements of RNFLT and RGC density are strongly correlated and can be used to monitor longitudinal changes after optic nerve injury. The strong fellow eye effect observed in eyes contralateral to ONT, only in the presence of CTB label, consisted of a dramatic increase in RNFLT associated with retinal microgliosis.     

Anterograde Degeneration along the Visual Pathway after Optic Nerve Injury

Purpose

To investigate anterograde degenerative changes along the visual pathway in a rat model of optic nerve axotomy.

Methods

Optic nerve transection was performed in adult Sprague-Dawley rats. Animals were sacrificed at regular time intervals and tissues harvested. Immunoblotting followed by densitometric analysis was used to determine the phosphorylation profile of Akt in the dorsal lateral geniculate nucleus (dLGN) and the primary visual cortex (V1). The neuronal cell size and cell density were measured in the dLGN and the V1 using Nissl staining. The prevalence of apoptosis was characterized by terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end labelling (TUNEL) histochemistry. Caspase-3 antibodies were also used to identify apoptotic cells. Neurons and astrocytes were detected using NeuN and glial fibrillary acidic protein (GFAP), respectively.

Results

An early and sustained loss of Akt phosphorylation was observed after optic nerve transection in both dLGN and V1. At week one, a decrease in the neuronal cell size (50.5±4.9 vs 60.3±5.0 µm², P = 0.042) and an increase of TUNEL positive cells (7.9±0.6 vs 1.4±0.5 ×10² cells/mm², P<0.001) were evident in the dLGN but not in V1. A significant decline in neuronal cell number (14.5±0.1 vs 17.4±1.3 ×10² cells/mm², P = 0.048), cell size (42.5±4.3 vs 62.1±4.7 µm², P = 0.001) and an increase in apoptotic cells (5.6±0.5 vs 2.0±0.4 ×10² cells/mm², P<0.001) appeared in V1 initially at one month post-transection. The changes in the visual pathway continued through two months. Both neuronal cells and GFAP-positive glial cells were affected in this anterograde degeneration along the visual pathway.

Conclusions

Anterograde degeneration along the visual pathway takes place in target relay (LGN) and visual cortex following the optic nerve injury. Apoptosis was observed in both neural and adjacent glial cells. Reduction of Akt phosphorylation preceded cellular and apoptotic changes.     

A novel high-content flow cytometric method for assessing the viability and damage of rat retinal ganglion cells

Purpose

The aim of the study was to develop a high-content flow cytometric method for assessing the viability and damage of small, medium, and large retinal ganglion cells (RGCs) in N-methyl-D-aspartic acid (NMDA)-injury model.

Methods/Results

Retinal toxicity was induced in rats by intravitreal injection of NMDA and RGCs were retrogradely labeled with Fluoro-Gold (FG). Seven days post-NMDA injection, flatmount and flow cytometric methods were used to evaluate RGCs. In addition, the RGC area diameter (D_(a)) obtained from retinal flatmount imaging were plotted versus apparent volume diameter (D_(v)) obtained from flow cytometry for the same cumulative cell number (sequentially from small to large RGCs) percentile (Q) to establish their relationship for accurately determining RGC sizes. Good correlation (r = 0.9718) was found between D_(a) and apparent D_(v). Both flatmount and flow cytometric analyses of RGCs showed that 40 mM NMDA significantly reduced the numbers of small and medium RGCs but not large RGCs. Additionally, flow cytometry showed that the geometric means of FG and thy-1 intensities in three types of RGCs decreased to 90.96±2.24% (P<0.05) and 91.78±1.89% (P>0.05) for small, 69.62±2.11% (P<0.01) and 69.07±2.98% (P<0.01) for medium, and 69.68±6.48% (P<0.05) and 69.91±6.23% (P<0.05) for large as compared with the normal RGCs.

Conclusion

The established flow cytometric method provides high-content analysis for differential evaluation of RGC number and status and should be useful for the evaluation of various models of optic nerve injury and the effects of potential neuroprotective agents.