Anatomical location of injected microglia in different activation states and time course of injury determines survival of retinal ganglion cells after optic nerve crush.

Background: Activated microglia release harmful substances to retinal ganglion cells (RGCs), but may also benefit by removing cellular debris and secreting neurotrophic factors. These paradoxical roles remain controversial because the nature and time-course of the injury that defines their role is unknown. The aim of this study was to determine if pharmacological manipulation of microglia to acquire a pro-inflammatory or pro-survival phenotype will exacerbate or enhance neuronal survival after injury.Material and methods: Treated HAP I (highly aggressively proliferating immortalized) microglia were injected into the vitreous or tail vein (T V) of female Sprague-Dawley rats. Retinas were examined at 4-14 days following optic nerve crush (ONC) and the number of surviving RGCs was determined.Results: Injection of untreated HAP I cells resulted in the greater loss of RGCs early after ONC when injected into the vitreous and later after ONC when injected into the T V. LP S activated HAP I cells injected into the vitreous resulted in greater RGC loss with and without injury. When injected into the T V with ONC there was no loss of RGCs 4 days after ONC but greater loss afterwards. Minocycline treated HAP I cells injected into the vitreous resulted in greater RGC survival than untreated HAP I cells. However, when injected into the T V with ONC there was greater loss of RGCs. These results suggest that optic nerve signals attract extrinsic microglia to the retina, resulting in a proinflammatory response.Conclusion: Neuroprotection or cytotoxicity of microglia depends on the type of activation, time course of the injury, and if they act on the axon or cell body.

We show here that neuroprotection is not solely determined by the microglial activation state but factors such as the environment and time-course of the injury.Culture microglia can be treated in vitro and then injected in vivo.The cells migrate to the site of injury, cell body of retinal ganglion cells if in the vitreous or to the optic nerve if injected in the tail vein.Retinal ganglion cell death is dependent on the location the microglia act, time-course of injury, and activation state.Proinflammatory microglia can be neuroprotective early in the injury when the primary site of action is on the axons whereas hypoactivated microglia are neuroprotective early in injury when they act on the soma. Later in the injury, both become detrimental.

AP-2ß is required for formation of the murine trabecular meshwork and Schlemm's canal.

Previously, we have shown that Tfap2b, the gene encoding transcription factor AP-2ß, is needed for normal mouse eye development. Specifically, targeted loss of Tfap2b in neural crest cells (NCCs)1 and their derivatives, particularly the periocular mesenchyme (POM), resulted in anterior segment defects affecting the cornea and angle tissue. These defects were further associated with an increase in intraocular pressure (IOP). The present study investigates the underlying changes in embryonic and postnatal POM cell development and differentiation caused by loss of AP-2ß in the NCCs, particularly in the structures that control aqueous outflow, using Wnt1Cre+/-; Tfap2b-/lox; tdTomatolox/+ mice (AP-2ß neural crest cell knockout or AP-2ß NCC KO). Toluidine blue-stained sections and ultrathin sections stained with uranyl acetate and lead citrate were used to assess morphology and ultrastructure, respectively. Immunohistochemistry of KO and control eyes was performed at embryonic day (E) 15.5, E18.5, postnatal day (P) 1, P7 and P14 using phospho-histone H3 (PH3), α-smooth muscle actin (α-SMA), myocilin and endomucin antibodies, as well as a TUNEL assay. Conditional deletion of AP-2ß in the NCC-derived POM resulted in defects that appeared during both embryogenesis and postnatal stages. Fate mapping of the knockout cells in the mutants revealed that the POM migrated appropriately into the eye during embryogenesis. However, during postnatal stages a significant reduction in POM proliferation in the angle region was observed in the mutants compared to controls. This was accompanied by a lack of expression of appropriate trabecular meshwork and Schlemm's canal markers. This is the first study to show that AP-2ß is required for development and differentiation of the trabecular meshwork and Schlemm's canal. Together, these defects likely contributed to the elevated intraocular pressure (IOP) previously reported in the AP-2ß NCC KO mice.

Changes in parvalbumin immunoreactive retinal ganglion cells and amacrine cells after optic nerve injury.

Parvalbumin (PARV) is a Ca(2+)-binding protein that may offer resistance to cell death as it primarily functions to maintain Ca(2+) homeostasis. The purpose of this study was to investigate whether PARV expressing retinal ganglion cells (RGCs) would be more resistant to cell death than RGCs that do not express PARV. RGCs in Sprague-Dawley rats were retrogradely labeled with Fluorogold (FG). After 2-28 days following an optic nerve crush (ONC) injury immunohistochemistry was performed on the sections using antibodies against PARV and markers of RGCs. The proportion of retinal ganglion cell layer cells labeled with PARV colocalized with FG or Brn3a and labeled only with PARV (displaced amacrine cells; dACs) were analyzed. PARV staining intensity was measured in ACs, dACs, and RGCs. Double labeling studies revealed that 49% of RGCs and 22% of dACs expressed PARV. There was an immediate reduction in RGC PARV staining after ONC but the overall rate of cell death after 28 days was similar in PARV and non-PARV expressing RGCs. There was no change in PARV AC or dAC number or staining intensity. Although this study suggests that there is no selective survival of the subpopulation of RGCs that contain PARV, there is down-regulation of PARV expression by these RGCs. This suggests that down-regulation of PARV may contribute to RGC death due to a compromised Ca(2+) buffering capacity. Maintaining PARV expression after injury could be an important neuroprotective strategy to improve RGC survival after injury.

Progressive Loss of Retinal Ganglion Cells in Activating Protein-2ß Neural Crest Cell Knockout Mice.

Purpose: Our lab has shown that conditionally disrupting the transcription factor activating protein 2ß (Tfap2b) gene, responsible for the activating protein-2ß (AP-2ß) transcription factor, exclusively in cranial neural crest cells (AP-2ß NCC KO), leads to anterior segment dysgenesis and a closed angle phenotype. The purpose of the current study is to determine if there is a progressive loss of retinal ganglion cells (RGCs) in the mutant over time and whether this loss was associated with macroglial activity changes and elevated intraocular pressure (IOP).Methods: Using the Cre-loxP system, we generated a conditional knockout of Tfap2b exclusively in cranial NCC (AP-2ß NCC KO). Immunohistochemistry was performed using anti-Brn3a, anti-GFAP and anti-Vimentin antibodies. IOP was measured using a tonometer and the data was analyzed using GraphPad Prism software. Brn3a and DAPI positive cells were counted using Image-J and statistical analysis was performed with GraphPad Prism software.Results: Our findings revealed that while no statistical difference in Brn3a expression was observed between wild-type and mutant mice at postnatal day (P) 4 or P10, at P40 (p < .01) and P42 (p < .0001) Brn3a expression was significantly reduced in the mutant retina at the region of the ONH. There was also increased expression of glial fibrillary acidic protein (GFAP) by Müller cells in the AP-2ß NCC KO mice at P35 and P40, indicating the presence of neuroinflammation. Moreover, increased IOP was observed starting at P35 and continuing at P40 and P42 (p < .0001 for all three ages examined).Conclusions: Together, these findings suggest that the retinal damage observed in the KO mouse becomes apparent by P40 after increased IOP was observed at P35 and progressed over time. The AP-2ß NCC KO mouse may therefore be a novel experimental model for glaucoma.

Proliferating brain cells are a target of neurotoxic CSF in systemic autoimmune disease.

Brain atrophy, neurologic and psychiatric (NP) manifestations are common complications in the systemic autoimmune disease, lupus erythematosus (SLE). Here we show that the cerebrospinal fluid (CSF) from autoimmune MRL-lpr mice and a deceased NP-SLE patient reduce the viability of brain cells which proliferate in vitro. This detrimental effect was accompanied by periventricular neurodegeneration in the brains of autoimmune mice and profound in vivo neurotoxicity when their CSF was administered to the CNS of a rat. Multiple ionic responses with microfluorometry and protein peaks on electropherograms suggest more than one mechanism of cellular demise. Similar to the CSF from diseased MRL-lpr mice, the CSF from a deceased SLE patient with a history of psychosis, memory impairment, and seizures, reduced viability of the C17.2 neural stem cell line. Proposed mechanisms of cytotoxicity involve binding of intrathecally synthesized IgG autoantibodies to target(s) common to different mammalian species and neuronal populations. More importantly, these results indicate that the viability of proliferative neural cells can be compromised in systemic autoimmune disease. Antibody-mediated lesions of germinal layers may impair the regenerative capacity of the brain in NP-SLE and possibly, brain development and function in some forms of CNS disorders in which autoimmune phenomena have been documented.

Delivery of ciliary neurotrophic factor via lentiviral-mediated transfer protects axotomized retinal ganglion cells for an extended period of time.

Ciliary neurotrophic factor (CNTF) has recently been demonstrated to be one of the most promising neurotrophic factors to improve both the survival and regeneration of injured retinal ganglion cells (RGCs). In the present study, we used optic nerve transection as an in vivo model to evaluate the effectiveness of a self-inactivating, replication-deficient lentiviral-mediated transfer of human ciliary neurotrophic factor (SIN-PGK-CNTF) on the survival of axotomized adult rat RGCs. Counts of dextran-fluorescein isothiocyanate conjugated (D-FITC)-retrogradely labeled RGCs revealed that the percentage of RGCs was drastically reduced (<90% cell death) 21 days after optic nerve transection. Retinal sections stained with X-gal revealed that intravitreal injection of the control LacZ-expressing lentiviral vector (LV-LacZ) resulted in the transduction of RGCs and retinal pigment epithelium (RPE) cells. A single intravitreal injection of LV-CNTF at the time of axotomy significantly enhanced RGC survival at 14 and 21 days postaxotomy compared to controls. These results demonstrate for the first time that rapid and prolonged delivery of CNTF using lentiviral-mediated gene transfer to the retina is an effective treatment for rescuing axotomized RGCs for an extended period of time. These results suggest that early and continuous administration of CNTF could serve as a potential treatment for retinal disorders involving optic neuropathy and RGC injury such as in glaucoma.

Characterization of the optic nerve and retinal ganglion cell layer in the dysmyelinated adult Long Evans Shaker rat: evidence for axonal sprouting.

Myelin in the central nervous system (CNS) is hypothesized to help guide the growth of developing axons by inhibiting sprouting of aberrant neurites. Previous studies using animal models lacking CNS myelin have reported that increasing capacity for sprouting axons is negatively correlated with the degree of myelination. In the present study, we investigated the optic nerves of the recently identified Long Evans Shaker (LES) rat with prolonged dysmyelination of adult axons to determine whether the lack of myelin basic protein (MBP) in adult LES rats could manifest as increases in the population of CNS axons. We observed numerous small, unmyelinated axon profiles (<0.3 microm in diameter) clustered in bundles alongside normal caliber axons in dysmyelinated LES rats but not in normal myelinated Long Evans (LE) rats. These putative axon profiles resembled sprouting axons previously described in the CNS. Moreover, the high number of small putative axon profiles could not be accounted for by any significant increases in the number of ganglion cells and displaced amacrine cells in the ganglion cell layer when compared with normal rats as evaluated by using a variety of techniques. This finding suggests that the observed clusters of putative axon profiles were not due to developmental abnormalities in the retina but to the lack of myelin in the optic nerves of LES rats. The adult LES rat, therefore, may serve as a useful model to study the role of myelin in regulating axon development or axon regeneration after CNS injury in the adult mammalian system.

Neurturin enhances the survival of axotomized retinal ganglion cells in vivo: combined effects with glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor.

In the present study we localized glial cell line-derived neurotrophic factor (GDNF), and the high affinity receptor for GDNF (GFRalpha-1) in the rat retina. We also examined the effects of neurturin on the survival of axotomized retinal ganglion cells (RGCs) and compared neurturin-mediated RGC rescue to GDNF and brain-derived neurotrophic factor (BDNF) neuroprotection. We administered combined injections of neurturin with BDNF or GDNF in order to determine if these factors rescue RGCs by different mechanisms. GDNF immunoreactivity was localized to RGCs, photoreceptors, and retinal pigment epithelial cells. GFRalpha-1 immunoreactivity was localized to RGCs, Müller cells, and photoreceptors. RGC densities in control retinas decreased from the original value of 2481+/-121 (RGCs/mm(2)+/-S.D.) to 347+/-100 at 14 days post-axotomy. Neurturin treatment significantly increased RGC survival after axotomy (745+/-94) similar to GDNF (868+/-110). BDNF treatment resulted in higher RGC survival (1109+/-156) than either neurturin or GDNF. Combined administration of neurturin with BDNF had additive effects on the survival of axotomized RGCs (1962+/-282), similar to combined administration of GDNF and BDNF (1825+/-269). Combined administration of neurturin and GDNF (1265+/-178) had an enhanced effect on RGC survival. These results suggest that neurturin, GDNF, and BDNF act independently to rescue injured RGCs. Our results also suggest that RGCs and retinal Müller cells may be responsive to GDNF because they both express GFRalpha-1. The present findings have implications for the rescue of injured retinal ganglion cells, as well as other CNS neurons that are responsive to neurturin, GDNF, and BDNF, including midbrain dopaminergic neurons and motor neurons.

Model of retinal surface area and neuron distribution in the avian eye.

Changes in retinal neuron distribution may reflect normal or pathological changes in retinal function. The quantitative study of retinal neurons provides a better understanding of anomalous mechanisms, such as those controlling ocular growth and refractive error development in experimental animals. We developed a method to facilitate the quantitative analysis of amacrine neuron populations in wholemount chick retinae, since the domestic chicken is used extensively as an animal model in myopia studies. This method involved automated cell counting from confocal microscopic images and mathematical estimation of total cell numbers based on image cell density and retinal surface areas. Cell densities and cell counts were obtained from immunohistochemically-labeled amacrine neuron populations, using derived formulae to calculate retinal surface area based on vitreous chamber depth, equatorial width, ora serrata diameter and scleral thickness. Normalized total cell counts in each eye were compared, rather than cell densities, since changes in eye growth can affect cell densities. We also compared neuron distribution in central versus peripheral portions of the retina. This is an alternative technique for retinal analysis that supplements traditional anatomical cell counting methods, allowing higher numbers of specimens to be rapidly analyzed.

Downregulation of BM88 after optic nerve injury.

PURPOSE: BM88 is a cell-cycle exit and neuronal differentiation protein that has been used as a marker of surviving retinal ganglion cells (RGCs) after optic nerve injury. Thy1.1 has also been used as a marker for RGC loss, but after optic nerve crush (ONC) a decrease in Thy1.1 expression precedes the loss of RGCs. The purpose of this study was to determine if BM88 expression was correlated with RGC loss after ONC and optic nerve transection (ONT) injuries. METHODS: Rats were injected with Fluorogold (FG) into the superior colliculus to label RGCs and received ONC or ONT 7 days later. Eyes were collected 2 to 28 days after injury. Retinas were labeled with BM88 and intensity of the BM88 cell labeling was measured. RESULTS: In control retinas, 98.9% of RGCs were immunoreactive (-IR) for BM88. There was a significant downregulation of BM88 by 52% to 80% of RGCs 7 days after ONC or ONT. The staining intensity of the remaining labeled cells was reduced to 41% to 51% of the control after 28 days of optic nerve injury. However, early in the injury there was a significant increase in the staining intensity of BM88. CONCLUSIONS: Nearly all BM88-IR RGCs colocalized with FG-labeled RGCs in control retinas. However, both the number of BM88-IR RGCs and their intensity decreased gradually between 4 and 28 days, preceding the loss of FG-labeled cells. These findings indicate that BM88 is not a good marker of surviving RGCs but may indicate abnormal RGC functioning, which precedes cell death.

Effects of acute delivery of endothelin-1 on retinal ganglion cell loss in the rat.

The vasoconstrictive peptide, Endothelin-1 (ET-1) has been found at elevated levels in glaucomatous eyes. In this study, a single 5mul intraocular injection of ET-1 was injected into the rat eye in order to characterize an in vivo retinal ganglion cell (RGC)-specific cell death model. The most effective concentration of ET-1 at inducing RGC loss at 2 weeks post-injection was determined using 5, 50 and 500mum concentrations of ET-1. The density of surviving RGCs was determined by counting Fluorogold labelled RGCs. A significant loss (25%) of RGCs was observed using only the 500mum concentration when compared to PBS-injected controls. GFAP immunohistochemistry revealed an increase in GFAP expression in Müller cell end-feet, as well as a total increase in GFAP expression (80%), following ET-1 treatment. These changes in GFAP expression are indicative of glial hyperactivity in response to stress. The specificity of ET-1 mediated cell death for RGCs was determined by measuring the changes in retinal thickness and TUNEL labeling. Retinal thickness was quantified using confocal and light microscopy. In confocal measurements, Yo Pro-1 was used to stain nuclear layers and the thickness of retinal layers determined from reconstructions. No significant loss in thickness was observed in any retinal layers. The same observations were seen in semi-thin sections when viewed by conventional transmitted light microscopy. The lack of significant thickness changes in the outer nuclear, outer plexiform or inner nuclear layer suggests that there was no significant cell loss in the retina other than in the RGC layer. Exclusive co-localization of TUNEL-labelled nuclei with Fluorogold-labelled cytoplasm provided additional evidence for RGC-specific death that most likely occurs via an apoptotic mechanism. A cell death time course was performed to determine RGC loss over time. RGC losses of 25, 25, 36 and 44% were observed at 1, 2, 3 and 4 weeks post-ET-1 injection, compared to PBS-injected controls. The total number of remaining RGC axons was determined by multiplying the number of optic nerve (ON) axons per unit area, by the cross-sectional area. There was a 31% loss in total ON axons in ET-1 treated eyes at 3 weeks post injection. Functional integrity of the visual system was determined by observing changes in the pupillary light reflex. ET-1 treatment resulted in a slowing of the pupil velocity by 31% and an average increase in the duration of contraction of 1.85sec (32% increase). These experiments provide evidence that acute ET-1 injections can produce RGC-specific cell death and many cellular changes that are similar to glaucoma. This potential glaucoma model leaves the optic nerve intact and may be used in subsequent experiments, which are involved in increasing RGC survival and functional recovery.