Browsing by Subject "Complement"
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Item C1Q EXPRESSION AND GLIAL ACTIVITY IN THE MOUSE RETINA FOLLOWING ISCHEMIA/REPERFUSION INJURY(2014-03) Silverman, Sean; Kim, Byung-Jin; Wordinger, Robert J.; Clark, Abbot F.We are using a mouse model whereby blood flow to the eye is blocked by raising the pressure in the eye in order to mimic damage caused by glaucoma. Our interest is to see how levels of C1q, a protein typically associated with the immune system as well as injury responsive cells of the eye are changed. Purpose (a): The complement cascade has become of increasing interest in several neurodegenerative diseases, including glaucoma, a leading cause of blindness. C1q has been observed as one of the earliest upregulated genes in the optic nerve head, the initial site of glaucoma injury preceding pathological changes. Here we use a glaucoma-like model of retinal ischemia/reperfusion (I/R) to mimic clinical changes in visual function and cellular loss. Methods (b): Deeply anesthetized C57BL/6J received a cannula to the anterior chamber of their left eye, through which their intraocular pressure (IOP) was raised to 120mmHg for 60 minutes leading to complete retinal ischemia. The cannula was then removed and blood flow was naturally reperfused. The right eye was uninjured as a contralateral control. Mice were sacrificed and enucleated at 3, 7, 14, 21, and 28 days. Eyes were fixed in 4% PFA and frozen for immunofluorescence or in situ hybridization studies. Microglia and astrocytes were identified using Iba1 and GFAP, respectively. Quantifications were performed using ImageJ Analysis software(NIH). Results (c): Initial changes in C1q expression were observed as early as 72 hours following injury, with a nearly two-fold increase compared to uninjured controls. Upregulated C1q was observed only in the ganglion cell (GCL) and inner plexiform (IPL) layers. Maximum intensity of C1q expression was observed 14 days post injury. Fluorescent in situ hybridization (FISH) studies reveal primarily microglia, not astrocytes, colocalized with expression of C1q in the retina. Conclusions (d): Following retinal I/R injury, C1q expression is actively upregulated, which appears to spatio-temporally correlate with changes in microglial, astrocyte, and Mueller cell homeostasis. Our FISH studies identify microglial cells as the primary producers of C1q following I/R injury. This suggests the elevated levels of C1q may stimulate astrocyte activation. There appears to be an interplay between microglia and astrocytes, both of which have been directly implicated in neurodegenerative diseases, including loss of RGCs in glaucoma. We propose C1q is an integral part of this mechanism, and by removing C1q we hope to preserve visual function and prevent degeneration in the visual system following injury.Item COMPLEMENT AND GLIAL ACTIVITY IN THE RETINOCOLLICULAR PATHWAY OF MICE USING A NOVEL MODEL OF GLAUCOMA(2013-04-12) Silverman, SeanPurpose: Glaucoma is a leading cause of irreversible visual impairment and blindness throughout the world. C1q is responsible for axonal pruning in early ocular development and is upregulated in glaucomatous eyes of mice, non-human primates, and humans. We used an inducible mouse model of human primary open angle glaucoma with elevated intraocular pressure (IOP) to examine expression levels of C1q in the retina and superior colliculus (SC), as well as identify changes in cellular homeostasis. Methods: Anesthetized A/J mice were given a single intravitreal injection of Ad5.MYOC.Y437H (5x107 pfu), a mutant glaucoma gene, or Ad5.null control virus. Following injections, conscious IOPs were measured weekly, using a TonoLab tonometer (iCare). Mice were sacrificed at time points between 3 days and 8 weeks. Brains and retinas were harvested for immunofluorescence or immunoblotting studies. Microglia and astrocytes cells were identified using Iba1 and GFAP, respectively. All quantifications were performed using ImageJ Analysis software(NIH). Results: IOPs were significantly increased in the Ad5.MYOC.Y437H eyes (p<0.01) compared to the contralateral un-injected eye and eyes receiving Ad5.null. Clq expression was significantly upregulated in retinas receiving Ad5.MYOC.Y437H (2.69-fold±0.38, p<0.0001) compared to contralateral control retinas (0.7-fold±0.29). Clq upregulation was additionally observed in SC hemispheres receiving neural connections from injected eyes. Mice given Ad5.null vector displayed no elevation of Clq in the visual axis. Additionally, colocalization studies demonstrated significant increases of inner retinal microglia density beginning 2 weeks post injection (0.61%±0.07, p<0.001) and continuing at 4 weeks (0.87%±0.09, p<0.0001) compared to untreated retinas (0.4l%±0.03 and 0.44%±0.03, respectively). No signs of astrogliosis were detected. Conclusions: C1q is actively upregulated in the retina and SC, following mutant myocilin induced ocular hypertention, whereas adenovirus alone had no effect. An increased microglial population in the retina accompanied these changes. This suggests that microglia may sense the increased IOP and play a role in upregulating endogenous C1q. Early glaucoma pathogenesis may result from the reactivation of the ocular developmental roles of C1q and microglia, suggesting new therapeutic targets for future neuroprotective studies.