Contralateral Astrocyte Response to Acute Optic Nerve Damage Is Mitigated by PANX1 Channel Activity.

Glial reactivity is considered a hallmark of damage-induced innate immune responses in the central nervous system. In the visual system, unilateral optic nerve damage elicits dramatic glial reactivity in the retina directly affected by the lesion and a similar, albeit more modest, effect in the contralateral eye. Evaluation of astrocyte changes in a mouse model of optic nerve crush indicates that astrocyte reactivity, as a function of retinal coverage and cellular hypertrophy, occurs within both the experimental and contralateral retinas, although the hypertrophic response of the astrocytes in the contralateral eyes is delayed for at least 24 h. Evaluation of astrocytic reactivity as a function of Gfap expression indicates a similar, muted but significant, response in contralateral eyes. This constrained glial response is completely negated by conditional knock out of Panx1 in both astrocytes and Müller cells. Further studies are required to identify if this is an autocrine or a paracrine suppression of astroglial reactivity.

Retinal glial responses to optic nerve crush are attenuated in Bax-deficient mice and modulated by purinergic signaling pathways.

BACKGROUND: Retinal ganglion cell (RGC) soma death is a consequence of optic nerve damage, including in optic neuropathies like glaucoma. The activation of the innate immune network in the retina after nerve damage has been linked to RGC pathology. Since the eye is immune privileged, innate immune functions are the responsibility of the glia, specifically the microglia, astrocytes, and Müller cells that populate the retina. Glial activation, leading to the production of inflammatory cytokines, is a hallmark feature of retinal injury resulting from optic nerve damage and purported to elicit secondary degeneration of RGC somas. METHODS: A mouse model of optic nerve crush (ONC) was used to study retinal glial activation responses. RGC apoptosis was blocked using Bax-deficient mice. Glial activation responses were monitored by quantitative PCR and immunofluorescent labeling in retinal sections of activation markers. ATP signaling pathways were interrogated using P2X receptor agonists and antagonists and Pannexin 1 (Panx1)-deficient mice with RGC-specific deletion. RESULTS: ONC induced activation of both macroglia and microglia in the retina, and both these responses were dramatically muted if RGC death was blocked by deletion of the Bax gene. Macroglial, but not microglial, activation was modulated by purinergic receptor activation. Release of ATP after optic nerve damage was not mediated by PANX1 channels in RGCs. CONCLUSIONS: RGC death in response to ONC plays a principal stimulatory role in the retinal glial activation response.

Neuroinflammation in Glaucoma and Optic Nerve Damage.

Glaucoma is a group of optic neuropathies characterized by the degeneration of retinal ganglion cell axons and somas, ultimately preventing light signals in the retina from reaching the brain. Glaucoma is a leading cause of blindness in the world, and treatment options for patients remain limited and minimally efficacious. A number of mechanisms have been linked to glaucomatous pathophysiology. A leading role is now attributed to neuroinflammatory conditions generated by the resident innate immune cells in the optic nerve and retina. Since the eye is immune privileged, the adaptation of these innate immune cells, termed glia, is crucial following trauma. In this chapter, we discuss the mechanisms associated with normal glial function in a healthy eye, and how changes in glial activation can contribute to the process of glaucomatous neurodegeneration in both the optic nerve and retina.

Tumor necrosis factor alpha has an early protective effect on retinal ganglion cells after optic nerve crush.

BACKGROUND: Glaucoma is an optic neuropathy that is characterized by the loss of retinal ganglion cells (RGCs) initiated by damage to axons in the optic nerve. The degeneration and death of RGCs has been thought to occur in two waves. The first is axogenic, caused by direct insult to the axon. The second is somatic, and is thought to be caused by the production of inflammatory cytokines from the activated retinal innate immune cells. One of the cytokines consistently linked to glaucoma and RGC damage has been TNFα. Despite strong evidence implicating this protein in neurodegeneration, a direct injection of TNFα does not mimic the rapid loss of RGCs observed after acute optic nerve trauma or exposure to excitotoxins. This suggests that our understanding of TNFα signaling is incomplete. METHODS: RGC death was induced by optic nerve crush in mice. The role of TNFα in this process was examined by quantitative PCR of Tnfα gene expression, and quantification of cell loss in Tnfα (-/-) mice or in wild-type animals receiving an intraocular injection of exongenous TNFα either before or after crush. Signaling pathways downstream of TNFα were examined by immunolabeling for JUN protein accumulation or activation of EGFP expression in NFκB reporter mice. RESULTS: Optic nerve crush caused a modest increase in Tnfα gene expression, with kinetics similar to the activation of both macroglia and microglia. A pre-injection of TNFα attenuated ganglion cell loss after crush, while ganglion cell loss was more severe in Tnfα (-/-) mice. Conversely, over the long term, a single exposure to TNFα induced extrinsic apoptosis in RGCs. Müller cells responded to exogenous TNFα by accumulating JUN and activating NFκB. CONCLUSION: Early after optic nerve crush, TNFα appears to have a protective role for RGCs, which may be mediated through Müller cells.

Evaluation of the percentage of ganglion cells in the ganglion cell layer of the rodent retina.

PURPOSE: Retinal ganglion cells comprise a percentage of the neurons actually residing in the ganglion cell layer (GCL) of the rodent retina. This estimate is useful to extrapolate ganglion cell loss in models of optic nerve disease, but the values reported in the literature are highly variable depending on the methods used to obtain them. METHODS: We tested three retrograde labeling methods and two immunostaining methods to calculate ganglion cell number in the mouse retina (C57BL/6). Additionally, a double-stain retrograde staining method was used to label rats (Long-Evans). The number of total neurons was estimated using a nuclear stain and selecting for nuclei that met specific criteria. Cholinergic amacrine cells were identified using transgenic mice expressing Tomato fluorescent protein. Total neurons and total ganglion cell numbers were measured in microscopic fields of 10(4) µm(2) to determine the percentage of neurons comprising ganglion cells in each field. RESULTS: Historical estimates of the percentage of ganglion cells in the mouse GCL range from 36.1% to 67.5% depending on the method used. Experimentally, retrograde labeling methods yielded a combined estimate of 50.3% in mice. A retrograde method also yielded a value of 50.21% for rat retinas. Immunolabeling estimates were higher at 64.8%. Immunolabeling may introduce overestimates, however, with non-specific labeling effects, or ectopic expression of antigens in neurons other than ganglion cells. CONCLUSIONS: Since immunolabeling methods may overestimate ganglion cell numbers, we conclude that 50%, which is consistently derived from retrograde labeling methods, is a reliable estimate of the ganglion cells in the neuronal population of the GCL.

Adoptive transfer of lymphocytes isolated from simian immunodeficiency virus SIVmac239Δnef-vaccinated macaques does not affect acute-phase viral loads but may reduce chronic-phase viral loads in major histocompatibility complex-matched recipients.

The live attenuated simian immunodeficiency virus (SIV) SIVmac239Δnef is the most effective SIV/human immunodeficiency virus (HIV) vaccine in preclinical testing. An understanding of the mechanisms responsible for protection may provide important insights for the development of HIV vaccines. Leveraging the uniquely restricted genetic diversity of Mauritian cynomolgus macaques, we performed adoptive transfers between major histocompatibility complex (MHC)-matched animals to assess the role of cellular immunity in SIVmac239Δnef protection. We vaccinated and mock vaccinated donor macaques and then harvested between 1.25 × 10(9) and 3.0 × 10(9) mononuclear cells from multiple tissues for transfer into 12 naive recipients, followed by challenge with pathogenic SIVmac239. Fluorescently labeled donor cells were detectable for at least 7 days posttransfer and trafficked to multiple tissues, including lung, lymph nodes, and other mucosal tissues. There was no difference between recipient macaques' peak or postpeak plasma viral loads. A very modest difference in viral loads during the chronic phase between vaccinated animal cell recipients and mock-vaccinated animal cell recipients did not reach significance (P = 0.12). Interestingly, the SIVmac239 challenge virus accumulated escape mutations more rapidly in animals that received cells from vaccinated donors. These results may suggest that adoptive transfers influenced the course of infection despite the lack of significant differences in the viral loads among animals that received cells from vaccinated and mock-vaccinated donor animals.

Nuclear atrophy of retinal ganglion cells precedes the bax-dependent stage of apoptosis.

PURPOSE: Retinal ganglion cells atrophy during the execution of the intrinsic apoptotic program. This process, which has been termed the apoptotic volume decrease (AVD) in other cell types, has not been well-characterized in ganglion cells. METHODS: Acute optic nerve crush was used to examine neuronal atrophy in the ganglion cell layer in wild-type and Bax-deficient mice. Nuclear size was measured from retinal wholemounts. Heterochromatin formation was assessed using transmission electron microscopy, whereas histone H4 acetylation was monitored using immunofluoresence. Ganglion cell and retinal transcript abundance was measured using quantitative PCR. RESULTS: Nuclear and soma sizes linearly correlated in both control and damaged retinas. Cells in wild-type mice exhibited nuclear atrophy within 1 day after optic nerve damage. Three days after crush, nuclear atrophy was restricted to ganglion cells identified by retrograde labeling, while amacrine cells also exhibited some atrophy by 5 days. Similar kinetics of nuclear atrophy were observed in cells deficient for the essential proapoptotic gene Bax. Bax-deficient cells also exhibited other nuclear changes common in wild-type cells, including the deacetylation of histones, formation of heterochromatin, and the silencing of ganglion cell-specific gene expression. CONCLUSIONS: Retinal ganglion cell somas and nuclei undergo the AVD in response to optic nerve damage. Atrophy is rapid and precedes the Bax-dependent committed step of the intrinsic apoptotic pathway.

High viremia is associated with high levels of in vivo major histocompatibility complex class I Downregulation in rhesus macaques infected with simian immunodeficiency virus SIVmac239.

Human and simian immunodeficiency viruses (HIV and SIV) downregulate major histocompatibility complex class I (MHC-I) molecules from the surface of infected cells. Although this activity is conserved across viral isolates, its importance in AIDS pathogenesis is not clear. We therefore developed an assay to detect the level of MHC-I expression of SIV-infected cells directly ex vivo. Here we show that the extent of MHC-I downregulation is greatest in SIVmac239-infected macaques that never effectively control virus replication. Our results suggest that a high level of MHC-I downregulation is a hallmark of fast disease progression in SIV infection.