Inhibition of Reactive Gliosis Attenuates Excitotoxicity-Mediated Death of Retinal Ganglion Cells

Reactive gliosis is a hallmark of many retinal neurodegenerative conditions, including glaucoma. Although a majority of studies to date have concentrated on reactive gliosis in the optic nerve head, very few studies have been initiated to investigate the role of reactive gliosis in the retina. We have previously shown that reactive glial cells synthesize elevated levels of proteases, and these proteases, in turn, promote the death of retinal ganglion cells (RGCs). In this investigation, we have used two glial toxins to inhibit reactive gliosis and have evaluated their effect on protease-mediated death of RGCs. Kainic acid was injected into the vitreous humor of C57BL/6 mice to induce reactive gliosis and death of RGCs. C57BL/6 mice were also treated with glial toxins, alpha-aminoadipic acid (AAA) or Neurostatin, along with KA. Reactive gliosis was assessed by immunostaining of retinal cross sections and retinal flat-mounts with glial fibrillary acidic protein (GFAP) and vimentin antibodies. Apoptotic cell death was assessed by TUNEL assays. Loss of RGCs was determined by immunostaining of flat-mounted retinas with Brn3a antibodies. Proteolytic activities of matrix metalloproteinase-9 (MMP-9), tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) were assessed by zymography assays. GFAP-immunoreactivity indicated that KA induced reactive gliosis in both retinal astrocytes and in Muller cells. AAA alone or in combination with KA decreased GFAP and vimentin-immunoreactivity in Mϋller cells, but not in astrocytes. In addition AAA failed to decrease KA-mediated protease levels and apoptotic death of RGCs. In contrast, Neurostatin either alone or in combination with KA, decreased reactive gliosis in both astrocytes and Mϋller cells. Furthermore, Neurostatin decreased protease levels and prevented apoptotic death of RGCs. Our findings, for the first time, indicate that inhibition of reactive gliosis decreases protease levels in the retina, prevents apoptotic death of retinal neurons, and provides substantial neuroprotection

Recombinant Human Clusterin Seals Damage to the Ocular Surface Barrier in a Mouse Model of Ophthalmic Preservative-Induced Epitheliopathy

There is a significant unmet need for therapeutics to treat ocular surface barrier damage, also called epitheliopathy, due to dry eye and related diseases. We recently reported that the natural tear glycoprotein CLU (clusterin), a molecular chaperone and matrix metalloproteinase inhibitor, seals and heals epitheliopathy in mice subjected to desiccating stress in a model of aqueous-deficient/evaporative dry eye. Here we investigated CLU sealing using a second model with features of ophthalmic preservative-induced dry eye. The ocular surface was stressed by topical application of the ophthalmic preservative benzalkonium chloride (BAC). Then eyes were treated with CLU and sealing was evaluated immediately by quantification of clinical dye uptake. A commercial recombinant form of human CLU (rhCLU), as well as an rhCLU form produced in our laboratory, designed to be compatible with U.S. Food and Drug Administration guidelines on current Good Manufacturing Practices (cGMP), were as effective as natural plasma-derived human CLU (pCLU) in sealing the damaged ocular surface barrier. In contrast, two other proteins found in tears: TIMP1 and LCN1 (tear lipocalin), exhibited no sealing activity. The efficacy and selectivity of rhCLU for sealing of the damaged ocular surface epithelial barrier suggests that it could be of therapeutic value in treating BAC-induced epitheliopathy and related diseases

KA activates glial cells in the retina.

<p>C57BL/6 mice (n = 6) were treated by intravitreal injection of PBS or KA (10 nM). At 24, 48 and 72 h after injection, glial cell activation was assessed by immunostaining retinal flat mounts with antibodies against GFAP (top panel) or retinal cross sections (lower panel) with antibodies against GFAP (green fluorescence) and vimentin (red fluorescence); blue indicates nuclei. Immunofluorescent staining indicates that KA increases GFAP-immunoreactivity in both astrocytes and Mϋller cells. All images were acquired at 40x magnification.</p

Neurostatin alone reduces GFAP expression.

<p>C57BL/6 mice (n = 6) were treated by intravitreal injection of PBS, KA (10 nM), or KA plus Neurostatin (5 mM). At 24, 48 and 72 h after injection, retinal flat mounts were immunostained with antibodies against GFAP (top panel) and retinal cross sections (lower panel) with antibodies against GFAP (green) and vimentin (red fluorescence). Retinal cross sections were also counterstained with DAPI (blue). Immunostaining results indicate that Neurostatin alone reduces GFAP expression in both astrocytes and Mϋller cells. All images were acquired at 40× magnification.</p

Neurostatin attenuates KA-induced ganglion cell loss.

<p>C57BL/6 mice were treated by intravitreal injection of PBS, KA (10 nM), Neurostatin (5 mM), or KA plus Neurostatin. At 24, 48, and 72 h after the treatment, loss of RGCs was determined by immunofluorescent staining of retinal flat mounts with antibodies against Brn3a (left panel). Immunofluorescent staining and quantification of cell loss (right panel) indicate that while Brn3a-positive RGCs remained similar in both PBS and Neurostatin-treated animals, Brn3a-positive RGCs were decreased significantly in KA-treated retinas (+, p<0.05). In contrast, Brn3a-Positive RGC loss was inhibited significantly (++, P<0.05) in animals treated with KA plus Neurostatin. All images were acquired at 40× magnification.</p

Curcumin Attenuates Staurosporine-Mediated Death of Retinal Ganglion Cells

The functional effect of curcumin, a free radical scavenger and an herbal medicine from Indian yellow curry spice, Curcuma longa, on protease-mediated retinal ganglion cell death was investigated. These results show, for the first time, that curcumin indeed prevents the protease-mediated death of RGCs, both in vitro and in vivo

AAA does not inhibit KA-induced protease expression.

<p>C57BL/6 mice (n = 6) were treated by intravitreal injection of PBS, KA, AAA, or KA plus AAA. At 24, 48 and 72 h after injection, proteins were extracted from the retinas, and aliquots containing an equal amount of protein (50 ug) were subjected to zymography assays (A). The areas cleared by proteases were scanned by a densitometer and results from three independent experiments were shown as arbitrary units (B). The assays indicate that a low level of MMP-9 and tPA were expressed constitutively in the retinas treated with PBS or AAA at all time points tested. In contrast, KA increased not only MMP-9 and tPA levels, but also uPA levels, which were absent in PBS and AAA-treated retinas. Furthermore, AAA failed to decrease the KA-mediated increase in MMP-9, tPA, and uPA levels at all time points tested. *, **, *** p<0.05, compared to untreated or AAA-treated retinas.</p

Neurostatin down-regulates GFAP expression in astrocytes.

<p>C57BL/6 mice (n = 6) were treated by intravitreal injection of PBS, KA (10 nM), or KA plus Neurostatin (5 mM). At 24, 48 and 72 h after injection, glial cell activation was observed by immunostaining of retinal flat mounts with antibodies against GFAP (top panel) or retinal cross sections (lower panel) with antibodies against GFAP (green fluorescence) and vimentin (red fluorescence). Retinal cross sections were also counterstained with DAPI (blue). Immunostaining results indicate that KA induces GFAP-immunoreactivity in both astrocytes (top panel) and Mϋller cells (lower panel). Results from radial sections indicate that Neurostatin decreases KA-mediated GFAP-immunoreactivity not only in astrocytes, but also in Mϋller cells. All images were acquired at 40× magnification.</p

Neurostatin reduces KA-induced protease levels in the retina.

<p>C57BL/6 mice (n = 6) were treated by intravitreal injection of PBS, KA, Neurostatin, and KA plus Neurostatin. At 24, 48 and 72 h after injection, proteins were extracted from the retinas and aliquots containing an equal amount of protein (50 ug) were subjected to zymography assays (A). The areas cleared by proteases were scanned by a densitometer and results from three independent experiments were represented as arbitrary units (B). The assays indicate that low levels of MMP-9 and tPA were expressed constitutively in retinas treated with PBS alone. KA increased the levels not only of MMP-9 and tPA, but also of uPA levels, which were absent in PBS or Neurostatin-treated retinas at all time points tested. In contrast, when animals were treated with Neurostatin and KA, levels of all three proteases were reduced considerably. *p<0.05, compared to Neurostatin-treated and **p<0.05 compared to KA-treated retinas.</p