Eye / Vision

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UNILATERAL PAPILLEDEMA: MEDICAL MANAGEMENT AND TREATMENT
(2013-04-12) Nguyen, Tran
Purpose: Introduction: Papilledema, also known as optic disc swelling, occurs in the setting of increased intracranial pressure. The etiology of papilledema may be due to several causes, and medical management varies depending on the etiology. Therefore, diagnostic testing is important in determining the source of elevated intracranial pressure. While headache is a common manifestation of papilledema, visual obscuration is most notable and frightful event for the patient. Medical management of papilledema requires serial clinical evaluation and serial visual acuity measurements, fundoscopic examination and visual field testing in order to monitor the efficacy of treatment. Methods: Single patient chart review Results: Case Description: A 30 year-old Caucasian female presents to the Emergency Department with chief complaints of acute vision loss in her right eye and periorbital headache. Physical exam was within normal limits. Brain MRI revealed right optic nerve enhancement. Patient was admitted for optic neuritis workup and received IV steroids. She was discharged after four days with a Medrol dose pack and clinic follow up. At her follow up visit at ophthalmology clinic, her vision improved slightly but she complained some of her visual field remains dark. Visual exam revealed 20/40 (OD), 20/30 (OS), 8 mm pupil OU, 2+ afferent papillary defect OD, and visual field showed inferior scotoma. Clinical differential diagnosis included idiopathic intracranial hypertension (IIH) versus optic neuritis based on neuroimaging and patient's body habitus. Patient started on corticosteroid treatment and taper, and clinician order lumbar puncture. On follow up visit 3 weeks later, patient continue to complain of visual loss OD. Visual field test revealed shifted visual defect relative to initial test. Results of lumbar puncture showed CSF opening pressure within normal limits when adjusted for patient's body habitus. Optic neuritis CSF workup revealed negative oligoclonal bands. IIH ruled out due to unilateral papilledema and normal CSF opening pressure. A working diagnosis of an independent optic neuritis was made. Patient started IV steroids, oral steroid taper, and follow up visit in 4 weeks. Conclusions: Discussion: This case is an example of optic neuritis manifestation prior to clinical and laboratory findings of Multiple Sclerosis. The case also illustrates the importance of serial clinic evaluations and neuroimaging aid in determining the etiology of papilledema.
ENDOTHELIN A RECEPTOR EXPRESSION IN RAT MODEL OF OCULAR HYPERTENSION
(2013-04-12) McGrady, Nolan
Purpose: The endothelin system of peptides and their receptors have been implicated for their neurodegenerative role in glaucoma. The purpose of this study was to determine changes in ETA receptor expression in the retina in the Morrison's elevated IOP model of glaucoma in rats. Methods: IOP was elevated in the left eye of adult male retired breeder Brown Norway rats using the Morrison's model of glaucoma (by injection of hypertonic saline through episcleral veins) while the contralateral eye served as the corresponding control. The rats were maintained for two weeks following IOP elevation and sacrificed. Retinal sections were obtained from both the control and IOP-elevated eyes and analyzed for changes in ETA receptor expression using immunohistochemistry. ETA receptor immunostaining was co-localized with β-III-Tubulin, which is selectively expressed in retinal ganglion cells. Results: Rat eyes with IOP elevation showed an increase in immunostaining ETA receptor in several retinal layers including retinal ganglion cells, the inner plexiform layer, and the outer plexiform layer. An increased co-immunostaining of ETA receptors with β-III-Tubulin was observed both in retinal ganglion cells and inner plexiform layer. Conclusions: Elevated intraocular pressure results in an increase in ETA receptor expression. Increased endothelin receptor expression is associated with neurodegeneration in glaucoma.
CYTOSOLIC PHOSPHOLIPASE A2 ALPHA INHIBITORS ATTENUATE APOPTOSIS OF THE CORNEAL EPITHELIAL CELLS AND MITIGATE ACANTHAMOEBA KERATITIS
(2013-04-12) Tripathi, Trivendra
Purpose: The aim of this study was to explore if MIP-133 from Acanthamoeba castellanii trophozoites induces apoptosis of Chinese hamster corneal epithelial cells (HCORN) in vitro via cPLA2ɑ pathway, and to determine the efficacy of cPLA2ɑ inhibitors to alleviate AK in vivo. Methods: HCORN were incubated with or without MIP-133 at doses of 7.5, 15, and 50µg/ml for 6, 12, and 24hrs. Inhibition of cPLA2ɑ was carried out by pre-incubating HCORN for 1hr with cPLA2ɑ inhibitors (10µM MAFP and 20µM AACOCF3) with or without 15µg/ml MIP-133 for 24hrs. Chinese hamsters were injected subconjunctivally with MIP-133 at dosage 40µg/40µl and eyes were removed 3 days after infection. Chinese hamsters were infected with parasite-laden contact lens and treated with cPLA2ɑ inhibitors (AACOCF3 and CAY10650) topically 50µg/5µl three times a day for 14 days post-infection. Expression of cPLA2ɑ at mRNA and enzyme levels was examined by RT-PCR and cPLA2ɑ enzyme assay. Apoptosis was determined by DNA fragmentation assay. CXCL2 expression was examined by RT-PCR and ELISA. In vivo infections were examined by clinical severity of disease scored on a scale of 0 to 5 based on corneal infiltration, corneal neovascularization, and corneal ulceration. Clinical pathology of cornea was examined by histopathology. Results: MIP-133 induces significant increase in cPLA2ɑ and CXCL2 levels from corneal cells. Moreover, cPLA2ɑ inhibitors, MAFP and AACOCF3, significantly reduce cPLA2ɑ and CXCL2 from these cells (P<0.05). Additionally, cPLA2ɑ inhibitors significantly inhibit MIP-133-induced apoptosis in HCORN cells (P<0.05). Subconjunctival injection of purified MIP-133 produced cytopathic properties characteristic of MIP-133. Treatments of cPLA2ɑ inhibitors showed significantly less severe keratitis and decreased clinical pathology as compared with control animals. Conclusions: Results suggest that cPLA2ɑ inhibitors therapeutically attenuate apoptosis of the corneal epithelial cells and mitigate AK.
EFFECTS OF TGF-BETA2, FOLLISTATIN AND ACTIVIN A ON EXTRACELLULAR MATRIX IN NORMAL HUMAN TRABECULAR MESHWORK CELLS AND TISSUES.
(2013-04-12) Fisher, Andrew
Purpose: Primary open angle glaucoma (POAG) is characterized as a group of eye diseases resulting in optic nerve head damage and irreversible blindness. A major risk factor for developing POAG is increased intraocular pressure (IOP) leading to decreased outflow of aqueous humor (AH) through the trabecular meshwork (TM). Transforming growth factor-beta2 (TGF-β2) is increased in the AH of glaucoma patients, and causes increased extracellular matrix (ECM) protein synthesis in the TM. Bone morphogenetic protein-4 (BMP-4) has been shown to inhibit TGF-β2 actions. Follistatin (FST) is an antagonist of BMP-4 and is elevated in the glaucomatous TM. Elevated levels of FST in the TM may block BMP-4 ability to attenuate TGF-β2 induction of ECM proteins. FST may also have a direct role in regulating ECM protein expression in human TM (HTM) cells. HTM cells also express Activin A (Act A). The purpose of this study was to assess the role of FST and Act A in HTM cells as related to TGF-β2/BMP-4 signaling. Understanding these interactions may provide possible new therapeutic targets for the treatment of glaucoma. Methods: Normal HTM cell lines were cultured and treated with TGF-β2 (5ng/ml), FST-315, FST-288, or Act A (each at 50ng/ml) alone and/or simultaneously for 24 and 48 hrs. Western blot analysis was used to evaluate the effects of FST-315/288, Act A, and TGF-β2 on ECM protein synthesis including fibronectin (FN), PAI-1, and collagen1A. Results: TGF-β2 induced expression of PAI-1 and FN.. ACT-A mildly induced PAI-1 and FN proteins as compared to TGF-β2. TGF-β2 and Act A treatment appeared to have a synergistic effect on the expression of PAI-1 and FN protein as compared to individual treatment (Tx) of TGF-β2 or Act A. FST 288 does not seem to change the Act A + TGF-β2 synergism. FST 315 inhibits the synergism of Act A + TGF-β2 showing a decrease in PAI-1 and FN protein. Conclusions: FST-315 decreased the induction of ECM proteins by TGF-β2 and Act-A. FST-288 increased induction of ECM proteins in cells treated with TGF-β2 and Act A. FST-288 treatment for 24 hours induced increased ECM proteins PAI-1 and FN, but there was no induction at 48 hours. FST-315 treatment for 24 hours slightly induced ECM PAI-1 and FN, but there was no induction at 48 hours. Act A treatment for 24 and 48 hours increased induction of PAI-1 or FN. These results further our knowledge of the potential role of BMP antagonists in the human TM and their potential roles in the pathogenesis of glaucoma.
MOLECULAR MECHANISMS THAT CONTRIBUTE TO THE VISION LOSS IN T17M RHO MICE.
(2013-04-12) Nashine, Sonali
Purpose: The T17M RHO transgenic mice carry a mutated human rhodopsin transgene, expression of which leads to protein misfolding, activation of the unfolded protein response (UPR) and progressive autosomal dominant retinitis pigmentosa (ADRP). Our previous study confirmed that the CHOP protein, a pro-apoptotic UPR marker, cannot be considered as a therapeutic target for ADRP photoreceptors expressing misfolded RHO. The purpose of this study is to determine the mechanism of accelerated retinal degeneration in T17M CHOP knock-out mice. Methods: T17M RHO CHOP+/+ and T17M RHO CHOP-/- mice were recruited in this study. Protein extracts were isolated from retinas of one-month-old mice to perform western blotting and detect the level of p-eIF2ɑ, XBP1, Rhodopsin, Histone deacetylase1 and P300 proteins. Results: An 8-fold increase of p-eIF2ɑ (PERK pathway) in T17M RHO CHOP-/- retina compared to T17M RHO retina (0.5 ± 0.1 arbitrary units (a.u.) vs. 0.04 ± 0.1 a.u., P=0.01) was observed. The level of spliced Xbp1 (IRE1 pathway) in the T17M RHO retina was significantly reduced by 30% (0.6 ± 0.03 a.uvs 0.4 ± 0.03 a.u, P=0.004). The T17M RHO CHOP-/- retina was characterized by 78% reduction in the level of P300 protein compared to T17M RHO (0.3 ± 0.1 a.u vs. 0.1 ± 0.02 a.u.in, P=0.02). In contrast, the Hdac1 protein was significantly increased by 245% in T17M RHO CHOP-/- retinas compared to that in T17M RHO retina. The level of Hdac1 was 0.2 ± 0.05 a.u. in T17M RHO and 0.5 ± 0.1 a.u., in T17M RHO CHOP-/- (P=0.03). Conclusions: The one -month old T17M CHOP-/- mouse retina mostly likely experiences a global transcriptional inhibition that leads to a reduction in the expression of photoreceptor-specific transcription factors (ARVO 2012). Increase in histone deacetylation and decrease in P300 protein suggests that the deacetylation process prevails in these mice.
LENS SPECIFIC RLIP76 TRANSGENIC MICE DEVELOP MICROPHTHALMIA: A NOVEL ANIMAL MODEL TO STUDY LENS DEVELOPMENT
(2013-04-12) Sahu, Mukesh
Purpose: The direct role of RLIP76 in eye development has not been investigated yet. In order to elucidate the role of RLIP76 in lens development, we have engineered lens specific RLIP76 Transgenic (RLIP76 Tg) mice and investigated the effect of RLIP76 overexpression on lens development. Methods: Lens specific RLIP76 Tg mice were generated in collaboration with Dr.Wakamia Maki. Mouse RLIP76 cDNA, 1885 base pairs long, was cloned in δenɑA promoter containing vector. Five founders were identified by southern blot analysis, and bred with C57BL/6J mice. Genotyping was performed to confirm the identity of offspring's by polymerase chain reaction using genomic DNA purified from mouse tail. Lens tissue organization of RLIP76 Tg and wild type mice was compared by Hematoxylin & eosin (H&E) staining. DNA microarray was used to compare the gene expression profiles of lens in RLIP76 Tg and wild type mice. The expression levels of various transcription factors such as Pax6 HSF1 and HSF4b was also determined using western blot and immunohistochemistry. Results: The most striking result from these studies was that the eyes of RLIP76 Tg mice were smaller in size as compared to those of wild type. In RLIP76 Tg mice the weight of the eyes was smaller and the lens development was impaired. RLIP76 Tg mice exhibited disruption in lens tissues organization and the expression of a multitude of genes was affected in lens of these mice. The expression of transcription factors Pax6, HSF1 and HSF4b, involved in eye development was suppressed in RLIP76 Tg mice. Actin cytoskeleton organization in RLIP76 Tg lens was severely disrupted. Cdc42, a regulator of actin cytoskeleton organization, activation level was reduced in RLIP76 Tg lens compared to wild type lens. Conclusions: The eyes of RLIP76 Tg exhibit features of microphthalmia and show poor lens development. These mice provide a good animal model for elucidating the mechanisms involved in lens development. Our results suggest that the lens developmental abnormalities in RLIP76 Tg mice may be due to the suppression of Pax6, HSF1, HSF4b and HSP's expression resulting in actin cytoskeleton disorganization.
DETERMINATION OF PROTEINS INVOLVED IN THE FORMATION OF CROSS-LINKED ACTIN NETWORKS IN THE TRABECULAR MESHWORK
(2013-04-12) Bermudez, Jaclyn Y.
Purpose: Glaucoma is a leading cause of blindness worldwide and the primary risk factor of glaucoma is increased intraocular pressure (IOP). IOP is determined by the equilibrium of aqueous humor (a fluid that fills the anterior segment of the eye) production and outflow. In glaucoma patients, the outflow resistance through the trabecular meshwork (TM), a special tissue located at the angle between the cornea and iris, is abnormally elevated. Inside TM cells, actin proteins form cross-linked actin networks (CLANs). Excessive formation of these unique structures can be found in the glaucomatous TM. They can also be induced by dexamethasone (DEX) and TGFβ2. It is suggested that CLANs increase cell rigidity and therefore elevate aqueous humor outflow resistance. However, the proteins that are involved in CLANs formation are not fully identified. We hypothesize that by comparing CLANs enriched and un-enriched TM cells, we will be able to identify the proteins that are involved in CLANs formation. Methods: We treated cultured mouse TM cells with DEX (100 nM), TGFβ2, (5 ng/mL), or ethanol (as vehicle control), evaluated CLANs formation, and separated the cell proteins by 2D gel electrophoresis. Differentially expressed proteins were analyzed by the Redfin software, and gel spots were picked and assessed by spectrometry analysis. Western blotting was used to confirm 2D gel results. Results: We found a subset of proteins that are differentially expressed in CLANs-enriched TM cells, compared to non-enriched samples. Among these proteins, ferritin heavy chain, glial fibrillary acidic protein (GFAP), and integrin alpha V, were confirmed by mass spectrometry and Western immunoblot. Conclusions: We found a subset of proteins that are differentially expressed in CLANs-enriched mouse TM cells. Their contributions and involvements in CLANs formation and regulation of TM cell morphology and functions are being investigated. They may provide new insight of the pathogenesis of glaucoma.
EFFECTS OF TGF-BETA2 ON THE ELASTIC MODULUS OF TRABECULAR MESHWORK TISSUE
(2013-04-12) Baradia, Hussein
Purpose: The primary risk factor for developing glaucoma is elevated intraocular pressure (IOP.) The key outflow passage for aqueous humor (AH) in the human eye involves the trabecular meshwork (TM) and the Schlemm's canal (SC) with the main regulator of IOP being at the junction of the two. Elevated IOP has been attributed to increased resistance to flow at the TM/SC junction. Aqueous humor levels of transforming growth factor beta-2 (TGF-b2) are elevated in glaucoma patients, suggesting its contribution to the progression of the disease. Studies using cultured TM cells as well as ex vivo tissue have shown that TGF-b2 induces extracellular matrix (ECM) remodeling. A separate study used atomic force microscopy (AFM) to measure the elastic modulus (e.g. stiffness) of TM tissue obtained from glaucoma patients compared to age-matched controls. A marked increase in stiffness in the glaucoma tissue was observed compared to non-glaucomatous controls. Taken together, these findings imply that an elevated level of TGF-b2 may lead to ECM remodeling and increased stiffness thus reducing AH outflow and elevating IOP. The hypothesis of this study was that increased expression of TGF-b2 will cause increased stiffness (e.g. increased elastic modulus) of the TM. The goal of our study was to compare the stiffness of the TM cultured overnight with or without TGF-b2. Methods: Bovine eyes were obtained from an abattoir and the TM dissected from the anterior segment. The dissected TM was then cut into halves. One half was cultured in control media and the other half cultured in media containing TGF-b2 (5 ng/ml). Both TM halves were incubated at 37oC for a period of 72 hours. The TM was then loaded onto a force transducer and contraction induced with carbachol to measure stiffness. Results: The raw data obtained indicated that the TM exposed to TGF-b2 contracted with greater force than control indicating that TGF-b2 reduced the stiffness of the TM. However, using a Students T test at a p of 0.05 the results showed no statistical significance. Conclusions: The results obtained are from one experiment with an N =8 and since no statistical significance was observed, the data has to be considered inconclusive. The force transducer used was actually that designed for skeletal muscles which may not be the best system to test trabecular meshwork which has some smooth muscle components. The future goal of the study is to actually use force microscopy, the current standard for such experiments.
CASPASE-7 ABLATION PROTECTS THE T17M RHODOPSIN MICE FROM SEVERE RETINAL DEGENERATION THROUGH REPROGRAMING OF THE UPR AND INHIBITION OF TRAF2-JNK APOPTOSIS
(2013-04-12) Choudhury, Shreyasi
Purpose: We previously demonstrated that misfolded T17M rhodopsin (RHO) activates the Unfolded Protein Response (UPR) in mouse rod photoreceptor cells eventually leading to Autosomal Dominant Retinitis Pigmentosa. We also have shown that the ablation of the UPR-induced CASP-7 in T17M RHO transgenic mice slows down the rate of retinal degeneration measured by ERG and SD-OCT. Therefore, the goal of this study is to elucidate the molecular mechanisms involved in the preservation of vision in T17MRHO CASP7-/- retinas to validate new therapeutic targets Methods: In vitro and in vivo studies were conducted to elucidate the pathway by which CASP-7 ablation promotes the ADRP photoreceptor cell survival. RNA and protein extracts were obtained from the 661W cells co-transfected with either wt or T17M RHO plasmid and control or CASP-7 siRNA. Retinas were harvested from C57/BL6, T17MRHO, T17MRHO CASP7-/- mice at P30 to perform qRT-PCR and western blot analysis. Results: The study of the cellular signaling in T17MRHO CASP7-/- retina demonstrated that the preservation of the structure and function of ADRP photoreceptors is occurred via down-regulation of the UPR-induced gene and protein expression. The ATF4, pATF6, mTor and Hif1 proteins were down regulated by 55%, 57%, 31%, 77% correspondingly and the level of pAKT was elevated by 60% in T17MRHO CASP7-/- retina. In addition, the inhibition of PARP1 and TNFa proteins in T17MRHO CASP7-/- retina was observed. All together these modifications lead to diminishing the TRAF2 and pc-Jun by 31%, 50% correspondingly. In vitro study also confirmed the modulation of cellular signaling observed in T17M RHO CASP7-/- retina. Conclusions: Both in vivo and in vitro studies indicated that the ablation of CASP-7 in the T17MRHO retina prevents the deterioration of retinal function and structure through reprograming of the UPR and modulation of TRAF2-JNK-induced apoptosis. This reduction is believed to occur through the down-regulation of the mTOR and Hif1a proteins. The inhibition of the PARP1 and TNFa proteins is also found to be responsible for diminishing the TRAF2-JNK apoptosis. In both scenarios, the reduction in c-Jun apoptosis leads to ADRP photoreceptor survival. This study points out c-Jun as a potential therapeutic targets for ADRP treatment.
JNK INHIBITOR EFFECTS ON RETINA MORPHOLOGY OBSERVED BY SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHY IN MOUSE MODEL OF RETINAL ISCHEMIA/PERFUSION
(2013-04-12) Mandal, Partha
Purpose: We hypothesize that inhibition of the JNK signaling pathway will protect the retina following ischemia reperfusion injury. Methods: JNK inhibitor, SP600125 will be intraperitoneally administered prior to retinal ischemia/reperfusion injury as well as daily for 28 days. The left eyes of female C57BL6/J mice will be cannulated with a 30-gauge needle for raising intraocular pressure to 120 mmHg for 60 min, followed by removal of the needle thus allowing retinal reperfusion. At 3, 7, 14, 21 and 28 days after I/R injury, retinal layers will be monitored by SD-OCT scanning. Thickness of the whole retina and each sub-retinal layer (e.g. inner plexiform layer, inner nuclear layer and outer nuclear layer) will be measured with SD-OCT software. Subsequently, changes in layer thickness will be statistically analyzed. The experiment will utilize three groups of five mice consisting of one control group, one ischemia/reperfusion group and one ischemia/reperfusion group with JNK inhibitor. Statistical analysis will be performed by using one-way ANOVA for group comparison. Results: SD-OCT scanning demonstrated similar retinal morphology compared to traditional histological techniques. Retinal thickness was reduced in the I/R eye in comparison with its non-pressured contralateral eye (right eye) after injury. Reduced retinal degeneration was seen in SP600125 treated mice. Conclusions: 1. SD-OCT visualized retinal morphology which is highly compatible to traditional histological assessment. 2. Retinal thickness was reduced in I/R eye in comparison with its control eye. 3. SP600125 administration to I/R eye show prevention of layer thickness degeneration indicating that JNK pathway contributes to pathogenesis following I/R injury. 4. The JNK signaling pathway is implicated in the inflammatory cascade. Further investigation is warranted to examine the possibility of the JNK signaling pathway as a future pharmacological drug target for I/R injury.
DIFFERENTIAL ACTIVATION OF P38 AND C-JUN N-TERMINAL KINASE IN THE VISUAL PATHWAY FOLLOWING OPTIC NERVE CRUSH
(2013-04-12) Liu, Yang
Purpose: p38 and c-jun N-terminal kinase (JNK) are two major stress-activated signaling pathways. In this study, we compared the activation of p38 and JNK in the retina, optic nerve, and superior colliculus after optic nerve crush (ONC) in adult mice. Methods: ONC was performed unilaterally in adult mice. The damage to the retinal ganglion cell (RGC) layer and superior colliculus (SC) was determined by Nissl and black gold II staining. The activation of p38 and JNK in the retina, optic nerve, and superior colliculus was examined by immunofluorescence staining. Results: Starting from 4 weeks after ONC, there were very few RGCs remaining in the RGC layer and fewer neurons (P < 0.05) in the contralateral SC compared to the control group. The volume of the contralateral SC was smaller (P < 0.01) than that of the control group. Phosphorylated JNK (p-JNK) was observed mainly on the proximal side of the optic nerve crush site as early as 1 hour after ONC. The expression of phosphorylated c-JUN was detected in the RGC layer starting 24 hours after ONC. The activation of p38 started 3 days post-ONC and was observed on both sides of the crush site. An increase of phosphorylated p38 (p-p38) was detected in the inner nuclear layer of the retina from 3 through 28 days following ONC. Two weeks after ONC, both p-JNK and p-p38 increased in the contralateral SC; however, the p38 activation was also observed in the ipsilateral SC. Conclusions: Optic nerve crush induces damage in both the RGC layer and the superior colliculus. p38 and JNK activation are differently modulated by optic nerve crush, and might play different roles in neuronal death in the retina and superior colliculus.
COMPLEMENT AND GLIAL ACTIVITY IN THE RETINOCOLLICULAR PATHWAY OF MICE USING A NOVEL MODEL OF GLAUCOMA
(2013-04-12) Silverman, Sean
Purpose: 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.
PROTECTIVE EFFECT OF JNK INHIBITION IN RETINAL GANGLION CELLS AND IN RETINAL ISCHEMIA/REPERFUSION INJURY
(2013-04-12) Kim, Byung-Jin
Purpose: The JNK (cJUN N-terminal kinase) signaling pathway plays an important role in various neuronal pathophysiologies. Using JNK inhibitor SP600125, we examined involvement of the JNK pathway in cultured retinal ganglion cell (RGC) death and in mouse retinal ischemia/reperfusion (I/R) injury. Methods: The in vitro effects of of several JNK inhibitors were evaluated in cultured adult rat retinal cells enriched in RGCs. Cytotoxicity was induced by glutamate or trophic factor withdrawal. Survival of RGCs was assessed by counting Thy-1 postive cells. Retinal I/R was induced in female C57BL/6J mice by raising the intraocular pressure to 120 mmHg for 60 min followed by reperfusion. SP600125 (5, 15, 30 mg/kg) was administered intraperitoneally once daily for 28 days starting at 2 h prior to injury. At various time points after I/R, phosphorylation of JNK and cJun was examined by immunoblotting of retinal proteins. The thickness of retinal layers and cell numbers in ganglion cell layer (GCL) were monitored using H&E stained retinal cross sections, and retinal function was measured by scotopic ERG. Results: SP600125 dose-dependently and significantly (p<0.05) completely protected against glutamate- and trophic factor withdrawal-induced cultured RGC cell death. In the I/R model, phosphorylation of JNK and cJun in retina was significantly (p<0.05) increased at 1 h after injury. I/R injury significantly (p<0.05) decreased the thickness of retinal layers, including whole retina (-23.2 ± 5.7%), inner plexiform layer (-38.0 ± 6.7%), and inner nuclear layer (-25.1 ± 7.4 %) and cell numbers in GCL (-30.0 ± 5.6%). Importantly, administration of all three doses of SP600125 protected against all these degenerative morphological changes (p<0.05). In addition, SP600125 administration also significantly (p<0.05) protected against I/R-induced reduction in b-wave amplitude at 3, 7, 14, 21 and 28 days after injury. Conclusions: Our results demonstrated involvement of the JNK pathway in retinal degeneration in both in vitro and in vivo models and suggest that JNK inhibitors may be a useful therapeutic strategy for neuroprotection in the retina.
EFFECT OF TRANSFORMING GROWTH FACTOR BETA-2 SIGNALING AND GREMLIN INDUCTION ON FIBRONECTIN, OCULAR HYPERTENSION, AND OPTIC NERVE DAMAGE
(2013-04-12) McDowell, Colleen
Purpose: Transforming growth factor β2 (TGFβ2) induces extracellular matrix (ECM) remodeling and alters the cytoskeleton, which likely contribute to the inefficient function of the trabecular meshwork (TM) tissue leading to glaucomatous phenotypes. Bone morphogenetic proteins (BMPs) inhibit these profibrotic effects of TGFβ2. The BMP antagonist gremlin is elevated in glaucomatous TM cells and increases intraocular pressure (IOP) in an ex vivo perfusion culture model. The purpose of this study was to determine whether TGFβ2 and gremlin regulate ECM proteins in the TM, induce ocular hypertension, and cause optic nerve damage in mice. Methods: Ad5.hTGFβ226/228 or Ad5.Gremlin (2 uL, 2 X 10^7 pfu) was injected intravitreally into one eye of A/J mice (n=7-13 mice per group), with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was assessed using the optic nerve damage score of PPD stained optic nerve cross sections. TGFβ2, fibronectin, and gremlin protein expression in the TM was determined by immunofluorescence and immunohistochemistry. Transduction of the TM with viral vector Ad5.hTGFβ2226/228 caused a prolonged, reproducible, and statistically significant IOP elevation. IOPs increased to approximately 25 mm Hg for 8 weeks (p<0.001). IOPs were stable (12-15 mm Hg) in the uninjected control eyes. The TGFP2 induced ocular hypertension also caused significant optic nerve damage with optic nerve damage scores (ONDS) > 3 (p<0.001) in the injected eye. Intraocular administration of viral vector Ad5.Gremlin also caused significant IOP elevation in A/J mice for 3 weeks (n=9, injected eye 23.2 +/- 5.6 mmmHg, uninjected eye 15.5 +/- 2.4; p<0.01). In addition, immunofluorescence and immunohistochemistry demonstrated that intraocular injection of Ad5.hTGFβ226/228 and Ad5.Gremlin increased TGFβ2 and fibronectin expression in the TM. Conclusions: These results demonstrate that intravitreal injections of Ad5.hTGFβ226/228 and Ad5.Gremlin in A/J mice elevate IOP and upregulate the ECM protein fibronectin. In addition, Ad5.hTGFβ226/228 expression induced significant optic nerve damage. These data demonstrate for the first time gremlin's role in inducing ocular hypertension in an in vivo model system and emphasize the importance of the TGFβ2 signaling pathway in ocular hypertension.
OVEREXPRESSION OF POU DOMAIN TRANSCRIPTION FACTOR, BRN3B CAUSES NEURITE OUTGROWTH IN CULTURED PC 12 CELLS
(2013-04-12) Phatak, Nitasha
Purpose: Brn3b is a POU domain transcription factor shown to play key role in regulating retinal ganglion cell axon outgrowth during development of the retina. The purpose of this study was to determine if overexpression of Brn3b could promote neurite outgrowth in cultured PC 12 cells. Methods: Rat Pheochromocytoma cells ( PC 12) were seeded and grown on Poly-D-lysine coated 100 mm dish and transfected either with pCMV6-Brn3b (an expression vector encoding Brn3b cDNA) or pCMV6-MCS (empty vector). Medium changed to complete medium with NGF (100ng/ml) after 6 hours of transfection. Protein extracts were isolated from these cells and analyzed for Brn3b and GAP43, TUBA-1 protein expression in 24 hours by immunoblot analysis. In another set of experiments, PC 12 cells were seeded on Poly-D-Lysine coated 25mm cover slip and transfected with either pCMV6-Brn3b or pCMV6 -MCS. Medium changed to complete medium with NGF (100ng/ml) after 6 hours of transfection. Brn3b, GAP43 and TUBA-1 expression in 24 hours were analyzed by using immunocytochemistry in the transfected cells. Morphological changes in PC 12 cells transfected with Brn3b were studied by using confocal microscopy. Results: Immunoblot analysis showed overexpression of Brn3b in PC12 cells transfected with Brn3b cDNA. Overexpression of transcription factor Brn3b in PC12 cells produced morphological changes including increased neurite outgrowth. An increased immunostaining for Brn3b and neurite-specific GAP43, TUBA-1 were also observed in PC12 cells overexpressing Brn3b. Conclusions: The POU domain transcription factor, Brn3b, could promote neurite outgrowth in PC12 cells.
BONE MORPHOGENETIC PROTEIN 4 INDUCES ID1 AND ID3 TRANSCRIPTION FACTORS IN TRABECULAR MESHWORK CELLS.
(2013-04-12) Mody, Avani
Purpose: Bone morphogenetic protein4 (BMP4) attenuates transforming growth factor β2 (TGFβ2) mediated responses in trabecular meshwork (TM) cells. However, the overall regulatory mechanisms of this inhibition remain unclear. BMP4 regulates various cellular processes by induction of transcription factor known as inhibitors of DNA binding protein (ID1, ID3) which bind and suppress other transcription factors. The purpose of the current study is to determine whether BMP4 will induce ID1 and ID3 expression in cultured human TM cells. This study will aid in understanding whether BMP4 transcriptionally suppress TGFβ-2 responses in TM cells via induction of IDs. Methods: Transformed (GTM-3) and primary TM cells were treated with different doses of recombinant BMP-4 for various time points (0-48 hrs.) to study ID1/ID3 induction. Quantitative RT- PCR and western immunoblotting were used to study ID1 and ID3 mRNA and protein expression. Results: BMP-4 significantly induced IDl and ID3 m RNA levels in TM cells (p<0.05). Maximum IDl and ID3 induction was observed at a BMP4 dose of l0ng/ml. ID1 and ID3 mRNA induction in GTM3 cells significantly (p<0.05) peaked at 6 hr after BMP4 treatment. Maximum induction of IDl and ID3 proteins was observed at 12 hrs. after BMP-4 treatment. Similar induction of ID1 and ID3 mRNA was observed in primary TM cells. Conclusions: BMP4 induces ID1 and ID3 in cultured human trabecular meshwork cells. BMP4 induction of ID1 and/or ID3 may be responsible for the BMP4 suppression of TGFb2 activity in TM cells
INCREASED EXPRESSION OF ENDOTHELIN B RECEPTORS PRECEDES RETINAL GANGLION CELL DEATH IN A RODENT MODEL OF GLAUCOMA
(2013-04-12) Minton, Alena Z.
Purpose: Endothelin B (ETB) receptors have been shown to be involved in the pathogenesis of glaucoma. However, the precise sequence of molecular events involving ETB receptor expression and retinal ganglion cell death is not completely understood. This study was aimed at investigating whether the expression of ETB receptors precedes retinal ganglion cell loss in vivo in the Morrison's elevated intraocular pressure (IOP) model of glaucoma in rats. Methods: Intraocular pressure (IOP) was elevated in one eye of retired breeders Brown Norway rats using the Morrison's method (injection of hypertonic saline through episcleral veins), while the contralateral eye served as control. Retinas were collected after 1 and 2 weeks of IOP elevation, sectioned and stained for ETB receptor expression by immunohistochemistry. In a separate set of experiments, retired breeders Brown Norway rats were used for retrograde labeling of retinal ganglion cells (RGCs) with Fluoro-gold. Following retrograde labeling, IOP was elevated in one eye using the Morrison's method, while the contralateral eye served as control. After IOP was elevated, rats were maintained for 2 weeks and sacrificed. Fluoro-gold labeled retinas were isolated, flat-mounted, and images taken using a Zeiss LSM-510 confocal microscope. Fluoro-gold-labeled RGCs were counted in three eccentricities from the optic nerve head. In addition, optic nerves were isolated from Brown Norway rats following 2 weeks of IOP elevation, sectioned and stained using p-phenylenediamine. Confocal images were taken and morphology of optic nerve axons was compared. Results: Immunohistochemical analysis of retinal sections showed no appreciable change in ETB receptor immunostaining following 1 week of IOP elevation. However, IOP elevation for 2 weeks produced increased expression of ETB receptor in the RGCs, inner plexiform layer (IPL) and inner nuclear layer (INL). In addition, IOP elevation for 2 weeks resulted in 25% loss of RGCs in the first eccentricity, while no significant loss was seen in the second and third eccentricities. Moreover, there was no appreciable damage observed in the optic nerve axons after 2 weeks of IOP elevation. Conclusions: Early increase in expression of ETB receptors (at 2 weeks of IOP elevation) could contribute to RGC loss and damage to the optic nerve axons.
EFFECT OF CELLULAR AND PLASMA FIBRONECTIN ISOFORMS ON NORMAL HUMAN TRABECULAR MESHWORK CELLS
(2013-04-12) Medina-Ortiz, Wanda E.
Purpose: The expression of cellular (cFN) and plasma (pFN) fibronectin isoforms are induced by TGF-β2 in human trabecular meshwork (HTM) cultured cells. Expression of specific FN isoforms can alter ECM homeostasis, ECM-cell interactions, and gene expression. Our purpose is to determine cFN levels in HTM tissues and to explore the impact of FN isoforms on HTM cells by studying changes in adhesion, cytoskeletal organization and gene expression. Methods: Differences between cFN levels in normal (NTM) and glaucomatous (GTM) tissues were obtained by immunohistochemistry. NTM cell strains were cultured for 24-48 hrs on surfaces coated with cFN or pFN, and the responses were compared to PBS controls. Changes in formation and redistribution of F-actin fibers and adhesion proteins were analyzed by phalloidin staining, Western immunoblots, and immunocytochemistry. Gene expression changes were analyzed using PCR arrays. Results: GTM tissues exhibited significantly greater cFN levels (1.7-fold, p<0.05). NTM strains exposed to both FN isoforms showed increased F-actin formation and redistribution; however, the F-actin pattern and distribution was different between cFN and pFN. Similarly, adhesion molecules such as talin, vinculin, paxillin and integrin beta 1 were increased and redistributed. Both FN isoforms changed gene expression, including alpha-smooth muscle actin-2, metalloproteases and their inhibitors, inflammatory cytokines, and TGF-β related genes. Conclusions: Our results show that GTM tissues expressed more cFN and that NTM cells respond differently depending on the FN isoform. The relationship between TGF-β2 modulation of FN isoform expression and the effect of FN isoforms on NTM cells suggests that this type of ECM remodeling may contribute to the TM changes associated with glaucoma.
ROLE OF NEURITIN 1 IN RESPONSE TO OPTIC NERVE CRUSH
(2013-04-12) Sharma, Tasneem
Purpose: Glaucoma is a progressive optic neuropathy characterized by axonal injury, retinal ganglion cell (RGC) loss, and visual field defects. Neuritin1 (Nrn1) is an extracellular, GPI-linked protein, which can be secreted as a soluble form. It stimulates axonal plasticity, dendritic arborization, and synapse maturation in the CNS. The purpose of this study was to evaluate the expression of Nrn1 in an in vivo optic nerve crush (ONC) mouse model that mimics features of glaucoma axonopathy. Methods: Unilateral ONC was performed on 8-10-week-old BALB/cJ eyes using the Nickell's technique. Retinas (N=8/group) and ONs (N=6/group) were harvested at six different time points (0, 3, 7, 14, 21, and 28 days post crush) (dpc). Real time PCR and immunohistochemistry (IHC) was performed to spatially and temporally evaluate Nrn1 expression in the retina and ON. Results: After ONC, Nrnl gene expression in the retina significantly decreased by 7 dpc compared to control eyes, with slight recovery by 14 dpc and then a significant reduction by 21 dpc (p<0.05, fold change of 1.5). In contrast, we observed basal expression until 21 dpc in the ON, with a significant increase at 28 dpc (p<0.05, fold change of 1.5). Biphasic expression patterns were observed in both retina and ON IHC sections. Conclusions: Axonopathy and degeneration of neurons and axonopathy are classical hallmarks of glaucoma neuropathy and also occur in other neurodegenerative diseases such as Parkinson and Alzheimer's. Nrn1, a vital player in neuronal plasticity, exhibited a biphasic expression pattern after ONC suggesting that modifications in regenerative ability of neurons lead to RGC and ON axonal injury. Future studies will determine whether Nrn1 gene therapy can prevent the loss of neurons by reviving regeneration after optic nerve axonopathy.

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