Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/30810
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Browsing Eye / Vision by Author "Krishnamoorthy, Raghu"
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Item Changes in the Expression of SMARCA4 in a Rat Model of Ocular Hypertension(2022) Worley, Josh; Stankowska, Dorota; Kodati, Bindu; Krishnamoorthy, RaghuTitle: Changes in the Expression of SMARCA4 in a Rat Model of Ocular Hypertension Purpose: SMARC4 (BRG1) is an ATP-dependent chromatin remodeling protein belonging to the SWI/SNF family of proteins involved in regulation of gene expression in numerous cell types in the body. The purpose of this study was to determine changes in the expression of SMARCA4 in the retina following intraocular pressure (IOP) elevation by the Morrison model in Brown Norway rats. We hypothesize that SMARCA4 expression may modulate the expression of the key genes involved in the neurodegenerative changes seen secondary to IOP elevation. Methods: The Morrison model of ocular hypertension (by injection of hypertonic saline through the episcleral veins) was utilized to unilaterally elevate the IOP in Brown Norway rats. IOP was elevated in the left eye of three retired breeder Brown Norway rats, with the right eye serving as the corresponding contralateral control. Rats were maintained for 2 weeks following IOP elevation and IOP measurements were carried out twice per week. Rats were subsequently euthanized, and retinal sections were obtained from both IOP-elevated and contralateral control eyes. Immunohistochemical analysis of SMARCA4 expression was carried out by immunostaining. Following confocal microscopy imaging, the intensity of immunofluorescence was quantified with the ImageJ software (NIH), and compared between IOP elevated and control eyes. Results: Immunohistochemical analysis revealed an appreciable decrease in the expression of SMARCA4 in retinal sections in two out of three rats, mainly the nerve fiber layer (by 47 to 57%), ganglion cell layer (by 18 to 40%) and inner plexiform layer (by 9 to 19%) in IOP elevated rat eyes compared to control eyes. One out of three tested rats showed a modest increase in immunostaining for SMARCA4 in the nerve fiber layer, ganglion cell layer and inner plexiform layer. Ongoing experiments will replicate these findings in order to generate statistically significant data. Conclusion: Changes in SMARCA4 expression could serve to regulate the expression of gene contributing to neurodegenerative effects due to elevated IOP. Understanding the role of SMARCA4 may allow us to better understand and address the mechanisms involved in glaucomatous neurodegeneration.Item Mechanisms of peptain-mediated neuroprotection in retinal ganglion cells(2022) Johnson, Gretchen A.; Pham, Jennifer; Kodati, Bindu; Krishnamoorthy, Raghu; Nagaraj, Ram; Stankowska, DorotaPURPOSE: To determine mechanisms underlying neuroprotective effects of the core peptide of alpha-B crystallin, peptain-1 (P1) conjugated to a cell-permeable peptide CPP (P1-CPP) in retinal ganglion cells (RGCs) in a rodent model of glaucoma. METHODS: Intraocular pressure (IOP) was elevated in Brown Norway (BN) rats and intravitreally injected with 2 µl of either P1-CPP or vehicle, once a week for a period of 2 weeks. Rats were euthanized, primary adult RGCs were isolated by the immunopanning method. Total RNA was isolated using the Trizol/column method. RNA-sequencing was performed using an Illumina platform. The resulting FASTQ files were uploaded into Galaxy for analysis with FASTQC, RNASTAR, feature counts, and finally DESeq2. The results from DESeq2 were then assessed with Qiagen's Ingenuity Pathway Analysis (IPA) to identify significantly upregulated pathways. Relative Creb-1 expression normalized to reference gene GAPDH was determined in IOP-P1-CPP and IOP-vehicle treated rat RGCs. Briefly, quantitative Polymerase Chain Reaction (qPCR) was performed using BioRad's PrimePCR Assay and SsoAdvanced Universal SYBR Green Supermix on the BioRad's CFX96 Real-Time System C1000 Touch Thermal Cycler. RESULTS: RNA-seq analysis from rat RGCs isolated following 2 weeks of IOP-elevation revealed that P1-CPP treated groups had several differentially expressed (DEGs), compared to vehicle-treated groups, including 6343 significantly upregulated and 5960 significantly downregulated. Some significantly upregulated pathways following P1-CPP treatment include phagosome formation, synaptic long-term depression, and CREB signaling in neurons. The IOP and vehicle-treated groups, when compared to the naïve group, demonstrated a decreased expression of members of the CREB signaling pathway (Creb-1, c-RAF, MEK1/2, ERK1/2, and p90RSK). This decline was prevented by P1-CPP treatment. Quantitative PCR further confirmed the RNA-seq findings of the increased expression of Creb-1 in P1-CPP treated rats compared to that of vehicle-treated group. CONCLUSIONS: Mechanism of action of P1-CPP in a rodent model of glaucoma includes the activation of the pro-survival CREB signaling pathway, phagosome formation, and long-term synaptic depression to prevent cell death and vision loss.