Browsing by Author "Krishnamoorthy, Raghu"
Now showing 1 - 17 of 17
- Results Per Page
- Sort Options
Item Assessment of WIN 55,212-2 Loaded Reconstituted High-Density Lipoprotein Nanoparticles for Ocular Delivery(2024-03-21) Petty, R. Max; Ranjan, Rajiv; Sabnis, Nirupama; Fudala, Rafal; Lacko, Andras; Gryczynski, Zygmunt; Krishnamoorthy, Raghu; Stankowska, DorotaPurpose: Overcoming challenges in glaucoma therapy, such as biological barriers and retina delivery, led us to develop innovative reconstituted high-density lipoprotein nanoparticles (rHDL NPs) for effective drug delivery. Optic nerve head astrocytes (ONHAs) are vital in maintaining retinal ganglion cell (RGC) axon integrity. This study describes the encapsulation of WIN 55,212-2 (WIN) in rHDL NPs and investigates the delivery mechanism of these nanoparticles in ONHAs. Methods: Using a novel preparation method, a stable rHDL-payload complex was created by combining lipophilic fluorescent dye IR780 or therapeutic agent WIN with phosphatidylcholine and apolipoprotein A-I (Apo A-I). Fluorescent rHDL (rHDL-IR780) was used to assess cellular uptake in human primary ONHAs in vitro. Scavenger receptor class B1 (SR-B1) expression was confirmed in retinal cell lysates by SDS-PAGE followed by western blot analysis. Receptor-mediated payload release through SR-B1 was confirmed by receptor blocking using BLT-1 as a specific SR-B1 receptor-blocking agent. Results: Fluorescent rHDL NPs exhibited an IR780 encapsulation efficiency of 68.7% (103 M), a polydispersity index (PDI) of 0.287 ± 0.013, a size of 14.01 ± 4.36 nm, and a zeta potential of -7.44 ± 0.90 mV. Additionally, drug-loaded rHDL-WIN NPs displayed a WIN encapsulation efficiency of 44.6% (341.4 M), a PDI of 0.280 ± 0.011, a size of 62.04 ± 25.06 nm, and a zeta potential of -20.13 ± 0.86 mV. Western blot analysis on human retinal lysates, ONHA lysates, and RGC lysates indicated the expression of SR-B1 (57/82 kDa (unmodified/glycosylated)). Cellular uptake studies confirmed the ability of rHDL to deliver payloads to ONHAs and RGCs. Receptor blocking with 10 nM BLT-1 highlighted the role of SR-B1 in specific cellular uptake from rHDL to ONHAs (p < 0.01). Conclusions: Our study highlights the role of SR-B1 in facilitating the delivery of rHDL payloads to ONHAs, offering the potential for targeted drug delivery in glaucoma. We anticipate that the cellular uptake by RGCs will follow the same SR-B1-mediated pathway. Successful WIN encapsulation in rHDL NPs suggests a potential avenue for targeting therapies to treat and prevent glaucomatous damage. Further studies are needed to determine the neuroprotective effects of rHDL-WIN and develop the potential of rHDL NPs to be used as an agent to target therapies in glaucoma.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 Endothelin B (ETB) receptor selective agonist endothelin 3 (ET-3) andmediated RGC death and implication for glaucoma.(2020) Kodati, Bindu; Krishnamoorthy, Raghu; Stankowska, Dorota; Harris, Payton; Wisniewski, Zoe; Krishnamoorthy, Vignesh; Beall, KallenPurpose: To determine if dietary administration of the dual ETA/ ETB receptor antagonist, macitentan, could protect retinal ganglion cells (RGCs) from cell death following an intravitreal injection of endothelin-1 (ET-1) and endothelin-3 (ET-3) in Brown Norway rats. Methods: Brown Norway rats (n=3 per group) were either untreated or treated with macitentan (5 mg/kg body weight) once a day for 3 days, followed by intravitreal injection of either 4 µl of 500 µM ET-1, ET-3 (2 nmole/eye) or vehicle in one eye. Following the injections, treatments with either macitentan or control gels without macitentan was continued for 7 days. After the treatments, retinal flat mounts were prepared and surviving RGCs were counted in a masked manner. Results: ET-1 produced 46% increase in RGC loss (p< 0.0001), compared to contralateral control eyes in rats 7 days post intravitreal administration. Macitentan treatment modestly protected from RGC loss following intravitreal ET-1 injection (27% RGC loss). ET-3 administration caused a modest increase (23%) in RGC death in this acute model, however, macitentan did not cause any further improvement in RGC survival (25% RGC loss). Conclusion: Both ET-1 and ET-3 mediate RGC loss following intravitreal administration in rats. Endothelin receptor antagonists have the potential to be developed as neuroprotective agents for the treatment of glaucoma.Item Endothelin Mediated Changes in Gene Expression Determined by RNA-sequencing of the Translatome in Primary Retinal Ganglion Cells.(2017-03-14) Krishnamoorthy, Raghu; Stankowska, Dorota; Chaphalkar, Renuka M.Purpose: Endothelin treatment has been shown to produce increased cell death in primary RGCs, however the underlying changes in gene expression are not completely understood. The purpose of the study was to assess endothelin-mediated changes in mRNA expression that occur at the translational level. Methods: Primary RGCs were isolated from post-natal day 5 rat pups by immunopanning with an antibody to Thy1.1. RGCs obtained were allowed to attach and maintained for 7 days for neurite outgrowth to occur. The RGCs were either untreated or treated with endothelin-1 (100 nM) for 24 h in trophic factor-free medium. Following brief incubation with cycloheximide to inhibit protein synthesis, total polysomes were isolated by magnesium precipitation and polysomal RNA was extracted using the Trizol reagent. Libraries for RNA-Seq were prepared with KAPA Stranded RNA-Seq Kit. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina Hiseq3000/4000 for a pair end 150 run. The reads were first mapped to the latest UCSC transcript set using STAR version 2.4.1d and the gene expression level was quantified to annotation model (Partek E/M). Differentially expressed genes were identified using differential gene expression (GSA) algorithm in Partek. Genes showing altered expression with p Results: Analysis of gene ontology of the changes in gene expression revealed a significant increase in expression of several mitochondrial genes including mitochondrial intermediate peptidase (MIPEP) (3.3-fold), cytochrome c oxidase assembly factor (SURF1) (8.7-fold) and Apolipoprotein O Like (APOOL) (7.5-fold). On the other hand, a decrease in expression of mitochondrial genes cytochrome c oxidase subunit 4I2 (8-fold), cytochrome c oxidase 6B2 (8-fold), and DNA polymerase gamma 2 (POLG2) (7-fold) was observed. Conclusions: Analysis of the translatome offers a glimpse into de novo protein synthesis which is an important manifestation of changes in gene expression. Endothelin treatment produced changes in several key regulators of mitochondrial metabolism and bioenergetics which could be indicative of their involvement in neurodegeneration in glaucoma.Item Endothelin receptors are targets for neuroprotection in a rat model of glaucoma(2017-03-14) Stankowska, Dorotoa; Rendon, Caitlin; Krishnamoorthy, Raghu; McGrady, NolanPurpose: The endothelin system has been shown to play a causative role in the neurodegenerative effects seen in animal models of glaucoma. However, the underlying mechanisms are not completely understood. The goal of this study was to investigate the interaction between the ETA and ETB receptors and the involvement of the MAP kinase pathways in endothelin mediated cell death and determine if the dual ETA/ETB receptor antagonist, macitentan could attenuate neurodegenerative changes following IOP elevation in Brown Norway rats. Methods: Cultured transformed 661W cells were transiently transfected with a plasmid DNA encoding the ETA receptor and treated with ET-1 or ET-3 for 24 h. Phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) was determined by immunoblotting. Wild type and ETB-deficient Wistar-Kyoto rats were subjected to IOP elevation by the Morrison’s method and maintained for 2 weeks. Retinal sections obtained from the rats were subjected to immunohistochemical analysis of ERK1/2 and JNK and their phosphorylation. Brown Norway rats subjected to IOP elevation in one eye were either untreated or treated with macitentan (10 mg/kg body wt) for 1 month. Retinal flatmounts obtained from these rats were used to determine RGC counts following staining with the RBPMS antibody. Results: Cell culture experiments showed an appreciable upregulation of pERK1/2, while pJNK levels were not appreciably altered, following overexpression of the ETA receptor in 661W cells. ETB-deficient rats showed increased immunostaining for pERK1/2 in the nerve fiber layer (NFL), ganglion cell layer (GCL) and inner plexiform layer (IPL), compared to wild type rats. Following IOP elevation, ERK1/2 phosphorylation was greatly reduced in wild type rats, while ETB-deficient rats showed better preservation of pERK1/2 levels. Conversely, immunostaining for pJNK in wild type rats was increased in the NFL following IOP elevation, but was attenuated in ETB-deficient rats. Rats fed with macitentan displayed increased RGC survival by 25 to 42% following IOP elevation, compared to untreated rats. Conclusions: There is a substantial body of evidence for the pro-survival role of ERKs, and the pro-death role of JNKs. The current study points to an involvement of ERK1/2 and JNK signaling with endothelin receptor expression following IOP elevation. Blocking both ETA and ETB receptors has neuroprotective effects on RGCs during ocular hypertension.Item Endothelin-1 Mediated Decrease in Expression of Mitochondrial Proteins ATP5H and COX17 in Retinal Ganglion Cells.(2019-03-05) Stankowska, Dorota; He, Shaoqing; Kodati, Bindu; Krishnamoorthy, Raghu; Chaphalkar, Renuka M.; Chaphalkar, Renuka M.TITLE: Endothelin-1 Mediated Decrease in Expression of Mitochondrial Proteins ATP5H and COX17 in Retinal Ganglion Cells. Purpose: Endothelin-1 (ET-1) treatment has been shown to promote apoptosis of retinal ganglion cells (RGCs), however, the precise mechanisms underlying these effects are still unknown. The purpose of the study was to assess the changes in gene expression at the level of the translatome, occurring during ET-1 mediated neurodegeneration of RGCs. Methods: Primary RGCs isolated from post-natal day 5 rat pups were treated with ET-1 (100 nM) for 24 h in trophic factor-free medium. Polysomal RNA was isolated and libraries for RNA-Seq were prepared. Trimmed mean of M-values (TMM) was used to normalize the gene expression. Genes with expression changes more than 1.5 fold with p Results: STRING network analysis revealed 156 differentially expressed genes, of which 23 genes were identified with known or predicted mitochondrial function. Immunostaining of primary RGCs showed an appreciable decline in expression of COX17, while ATP5H expression was modestly decreased. A decreasing trend (three out of four rats) in immunostaining for ATP5H as well as COX17 was found in retinas of rats intravitreally injected with ET-1 (n=4). Conclusions: ET-1 treatment produced a decrease in expression of key components of mitochondrial electron transport chain. A compromise in bioenergetics could be one mechanism by which ET-1 promotes neurodegeneration of RGCs in glaucoma.Item ETA receptor upregulation may be associated with ERK signaling in a rat model of glaucoma.(2016-03-23) Krishnamoorthy, Raghu; McGrady, NolanPurpose: The endothelin system has been shown to play a causative role in the neurodegenerative effects seen in animal models of glaucoma. However, the mechanisms leading to neurodegeneration need to be examined further. The goal of this study was to investigate the endothelin signaling pathway to determine the contribution of extracellular signal-regulated kinases 1 and 2 (ERK1/2) to endothelin-mediated cell death. Methods: Male retired breeder Brown Norway rats were subjected to IOP elevation by the Morrison’s method and maintained for 2 and 4 weeks. Retinal sections obtained from the rats were subjected to immunohistochemical analysis of ETA receptor expression. In a separate set of experiments, western blots were performed on transformed 661W cells transiently transfected with either the ETA receptor or ETB receptor cDNA expression vector. Another set of experiments was performed with stable clones overexpressing the ETA receptor. The cells were grown on 100 mm dishes and treated for 24 hr with 100nM endothelin-1 (ET-1) or endothelin-3 (ET-3). Immunoblot analysis of levels of endothelin receptor and ERK1/2 phosphorylation was carried out. Results: An increase in immunostaining for ETA receptors was observed mainly in the inner plexiform layer and a modest increase was also observed in the RGC layer which was significant at 4 weeks of IOP elevation. Cell culture experiments showed an appreciable upregulation of ETB receptors following overexpression of ETA receptors and a reciprocal upregulation of ETA receptors following overexpression of ETB receptors. A 1.8-fold increase in ERK1/2 phosphorylation was observed in stable clones overexpressing ETA receptors, which was further elevated 2-3 fold after treating cells with either endothelin-1 or endothelin-3, compared to empty vector transfected cells. Conclusions: While the two endothelin receptors may have distinct functions, there is a significant overlap of the ETA and ETB receptor mediated signal transduction pathways and there appears to be some level of cross-talk between the two receptors. While there is a substantial body of evidence for the pro-survival role of ERKs, prolonged activation of ERK1/2 has been shown to be associated with cell death. While the mechanisms are not completely clear, the current study points to an association of ERK1/2 with cell death following overexpression of ETA and ETB receptors.Item Intravitreal Endothelin-1 (ET-1) Injection Reduces Mitophagy in Retinal Ganglion Cells in MitoQC Mice(2023) Brooks, Calvin D.; Kodati, Bindu; Inman, Denise; Stankowska, Dorota; Krishnamoorthy, RaghuPurpose: The peptide endothelin-1 (ET-1), and its receptors are upregulated in the aqueous humor and retina in animal models of experimentally induced ocular hypertension, and have been shown to have a causative role in retinal ganglion cell (RGC) neurodegeneration. The purpose of this experiment was to assess the role of mitophagy in RGC neurodegeneration following intravitreal ET-1 administration in MitoQC mice. Methods: MitoQC mice (Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl on a C57BL/6 background) at the age of 3 months were used for the study. The mitochondria in these mice display both red and green fluorescence due to expression of a mCherry-GFP tag fused to the mitochondrial targeting sequence of an outer mitochondrial membrane protein, FIS1. When these mitochondria are trafficked to the lysosome for degradation, the green fluorescence is quenched, leaving only the red fluorescence. The MitoQC mice were intravitreally injected in both eyes with either ET-1 (1 nmole) or vehicle (water), and 72 hours following the injections the mice eyes were enucleated and retinal flat mounts were live-imaged using a Zeiss LSM 880 super resolution confocal microscope. Z-stack imaging was used to image the ganglion cell layer. For each Z layer, a threshold algorithm was used to define a region of interest (ROI) that included only areas with red fluorescence, after which red and green fluorescence were quantified for that ROI. Red/green fluorescence intensity was calculated and averaged per image. A red/green ratio larger than 1 is indicative of active mitophagy. This ratio was compared between ET-1 and vehicle-injected mice using a Mann-Whitney test (n=4 eyes per group). Results: At 72 hours after injection with ET-1, the average red/green fluorescence ratio in the RGCs was 0.86, while the vehicle-injected mice had an average red/green ratio of 1.29. These ratios were significantly different (P=0.0003), and the smaller red/green ratio in the ET-1 group indicates lesser mitophagy than the vehicle group. Conclusion: Mitophagy is known to be an important quality control mechanism for neuronal cell survival, and this study provides evidence that mitophagy is impaired by ET-1. The finding indicates that a decline in mitophagy may be associated with endothelin-mediated neurodegeneration in RGCs.Item Involvement of the c-Jun N-terminus kinase (JNK) pathway in Endothelin (ET-1) mediated neurodegeneration of retinal ganglion cells(2021) Kodati, Bindu; Stankowska, Dorota; Krishnamoorthy, Vignesh; Krishnamoorthy, RaghuPURPOSE: Endothelins contribute to neurodegeneration in glaucoma, however, the mechanisms are not completely understood. The goal of this study was to determine if JNK2 plays a causative role in endothelin-1 (ET-1)-mediated loss of RGCs in mice. METHODS: JNK2-/- and wild type (C57BL6) mice (n=4) were intravitreally injected in one eye with 2 nmoles of ET-1, while the contralateral eye was injected with 2 µl of vehicle. The mice were euthanized at 2 h and 24 h post-injection. Retinal sections from the JNK2-/- and wild type (C57BL6) mice were used for immunohistochemical analysis of the phosphorylation of JNK substrate, c-Jun. In a separate set of experiments, JNK2 -/- and wild type mice (n=6) were intravitreally injected with either 2 nmoles of ET-1 or vehicle, and euthanized 7 days post-injection. RGC counts and axonal degeneration were assessed. RESULTS: Intravitreal ET-1 administration produced a significant increase in immunostaining for phospho c-Jun in wild type mice which was appreciably lower in the JNK2 -/- mice. Following ET-1 administration, a significant (p< 0.05) 26% loss of RGCs was found in wild type mice, while JNK2-/- mice showed no significant (p=0.36) loss of RGCs. A significant decrease in the axonal counts and an increase in the collapsed axons was found in ET-1 injected eyes in wild type mice. CONCLUSION: JNK2 appears to play a major role in ET-1 mediated loss of RGCs in mice.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.Item Neuroprotective effects of an endothelin receptor antagonist in a rat model of ocular hypertension.(2017-03-14) McGrady, Nolan; Krishnamoorthy, Raghu; Jefferies, HaydenPurpose: Endothelin 1 is elevated in both patients and in animal models of glaucoma and has been shown to contribute to neurodegeneration. Since endothelin 1 acts through two receptors, endothelin A receptor and endothelin B receptor, the purpose of this study is to determine if antagonism of both endothelin receptors by Macitentan can promote neuroprotection during intraocular pressure (IOP) elevation in rats. Methods: IOP was elevated in the left eye of adult male retired breeder Brown Norway rats using Morrison’s model of ocular hypertension (injection of hypertonic saline through episcleral veins) and maintained for 4 weeks. Contralateral eyes served as the corresponding contralateral controls. Rats were then separated into treated and untreated groups, with the treated group receiving Macitentan (10 mg/kg) 3 times per week in the diet (administered in gel packs). After one month of treatment, rats were sacrificed and retinal flat mounts were prepared for both IOP elevated and contralateral eyes of both the treated and untreated rats. The flat mounts were immunostained for the retinal ganglion cell-specific marker Brn3a, imaged in a confocal microscope and masked cell counts were performed. Results: IOP elevation in untreated rats showed a dramatic decline in retinal ganglion cell counts compared to the contralateral controls, whereas rats treated with Macitentan showed significant preservation of retinal ganglion cell numbers. Conclusion: Currently, therapies for the treatment of glaucoma are solely focused on the reduction of IOP. The study described herewith demonstrates the ability of a dual endothelin receptor antagonist, Macitentan, to promote retinal ganglion cell survival independent of IOP. Due to its IOP independent action, this therapy could be developed for neuroprotection either independent or concurrent with available glaucoma therapies.Item Neuroprotective effects of SB203580 (p38 MAP kinase inhibitor) against Endothelin-1-induced retinal ganglion cell death(2020) He, Shaoqing; Chaphalkar, Renuka; Kodati, Bindu; Krishnamoorthy, Raghu; Krishnamoorthy, VigneshPurpose: Endothelin-1 (ET-1) is a vasoactive peptide contributing to neurodegeneration in glaucoma, however, the underlying mechanisms are not completely understood. The current study tested the involvement of the p38 MAP kinase in ET-1-induced cell death in primary rat retinal ganglion cells (RGCs). Methods: Primary RGCs were treated for 24 hours with 100nM ET-1 either in the presence or absence of the p38 MAP kinase inhibitor SB203580. A Live/Dead assay was used to determine cell viability and the number of surviving and dying/dead cells were quantitated. Results: ET-1 induced RGC death following a 24 hour treatment (as seen by an increase in the dead/live ratio from 0.446 to 0.621), compared to untreated controls. Interestingly, compared to the untreated controls, an appreciable decrease in cell death was found in cells treated with SB203580 alone (a decrease in the dead/live ratio from 0.446 to 0.243). Following treatment with a combination of ET-1 and SB203580, cell counts showed a decrease in cell death when compared with cells treated with ET-1 alone (a change in the dead/live ratio from 0.621 to 0.333). Conclusions: The p38 MAP kinase pathway is known to be involved in signaling mechanisms underlying numerous pathways related to cellular stress. The current study suggests that p38 MAP kinase contributes to ET-1-mediated RGC death. Elucidation of endothelin-mediated signaling pathways will help understand mechanisms by which endothelins promote neurodegeneration in glaucoma.Item Novel Peptain for Neuroprotection in Glaucoma(2020) Stankowska, Dorota; Krishnamoorthy, Vignesh; Krishnamoorthy, Raghu; Chaphalkar, Renuka; Nagaraj, Ram; Nahomi, Rooban; Nam, Mi-hyun; Beall, Kallen; Brodrick, Ashley; Kodati, BinduPurpose: To determine if intravitreal administration of the core peptide of [alpha]-B crystallin, peptain-1 (P1) conjugated to a cell permeable peptide (CPP) (named P1-CPP) could inhibit retinal ganglion cell (RGC) death and functional decline in a rodent model of glaucoma. Methods: Primary RGCs were treated with endothelin-3 (ET-3) in the presence of either P1-CPP (12.5 µg/ml) or vehicle, following which RGC survival was assessed. In a different set of experiments, intraocular pressure (IOP) was elevated in one eye of Brown Norway (BN) rats and intravitreally injected with 2 µl of either P1-CPP or vehicle, once in a week for 6 weeks. RGC function was assessed by the pattern electroretinogram (PERG) amplitude. Retinal flat mounts were imaged and surviving RGCs were counted. Results: ET-3 treatment lead to 24% of RGCs loss compared to vehicle treated cells (p< 0.0001). P1-CPP treatment significantly lowered the ET-3-mediated cell loss (7% cell death, p< 0.001). IOP elevation in vehicle injected animals produced 11% and 27% loss of RGCs, in central and peripheral retina respectively, which was significantly lower in P1-CPP treated rats (7% loss in both eccentricities, **p< 0.01). P1-CPP treatment also promoted axonal protection during IOP elevation. IOP elevation caused 63% decline in the PERG amplitude (*p< 0.03) in comparison with naïve rats, which was sustained by P1-CPP treatment. Conclusion:The intravitreally injected P1-CPP provides cellular as well as functional protection of RGCs, which could facilitate neuroprotection against glaucomatous insults.Item P1-CPP promotes Foxo1 and Creb signaling and reduces apoptosis in Neurotrophic Factor-Deprived Primary Retinal Ganglion Cells(2023) Johnson, Gretchen; Pham, Jennifer; Krishnamoorthy, Raghu; Nagaraj, Ram; Stankowska, DorotaPurpose: To elucidate the intracellular mechanisms underlying neuroprotective effects of the core peptide of a-B crystallin, peptain-1 (P1) conjugated to a cell-permeable peptide CPP (P1-CPP) in primary retinal ganglion cells (RGCs). Targets of the investigation were limited to Creb1, Bak1/Bad, and Foxo1, based upon RNA sequencing data obtained from RGCs of IOP-elevated rats treated with P1-CPP in comparison with the vehicle. Methods: Primary RGCs isolated from Sprague Dawley rat pups were deprived of neurotrophic factors (NT) namely, BDNF, CNTF, and Forskolin for 48 hours, either in the presence or absence of P1-CPP (4µM). After the treatments, RNA isolation was carried out using Trizol reagent. Subsequently, cDNA synthesis and qPCR analysis of the target genes expression, including Creb1 (n=2), Foxo1 (n=3), and Bak1 (n=3), was performed. Another set of RGCs subjected to the same treatments was fixed with 4% paraformaldehyde for 20 minutes and used for immunocytochemical analyses of p-CREB (n=3), FOXO1 (n=3), and BAD (n=3) protein expression. Immunostaining with an RBPMS antibody was used as an RGC marker. N indicates experimental repeats. Results: Following NT deprivation, there was an increase in mRNA expression of Creb1 (2-fold) in RGCs treated with P1-CPP, compared to the vehicle-treated RGCs. Moreover, the phosphorylated (active) form, p-CREB, was increased (by 102%; p=0.04) in primary RGCs treated with P1-CPP, compared to the vehicle-treated group. Pro-apoptotic Bak1 mRNA expression was not changed in the P1-CPP-treated RGCs compared to the vehicle-treated group. Primary RGCs stained for BAD protein showed a decrease (by 62%; p=0.08) in the P1-CPP treated group compared to the vehicle-treated RGCs. Foxo1 mRNA levels were increased by more than 2-fold in the P1-CPP treated RGCs, compared to the vehicle-treated RGCs. FOXO1 protein was also elevated in primary RGCs treated with P1-CPP compared to the vehicle group (by 59%). Conclusions: P1-CPP is neuroprotective against neurotrophic factor deprivation through multiple mechanisms, including early changes in the expression of mitochondrial homeostasis regulator Foxo1, activation of the pro-survival CREB pathway, and inhibition of pro-apoptotic members of the BCL-2 family of proteins.Item Quality Assurance Training: Will a New Training Intervention Improve Data Collection of the Texas Emergency Medicine Research Associate Program (TEMRAP)?(2018-12) Saldana, Miguel Antonio; Hodge, Lisa; Pierce, Ava; Krishnamoorthy, RaghuIntroduction: Data collection is vital for the success of a clinical research project. The purpose of this practicum was to address the inadequate data collection by the Texas Emergency Medicine Research Associate Program (TEMRAP) research associates (RAs). The primary goal was to incorporate a more efficient training method to reduce the RAs' error rate in the documentation. The secondary aim of this experiment was to determine if RAs' knowledge of clinical research studies and/or their self-confidence when enrolling a patient had an effect on quality of data collection and if these variables could be improved by a new training method. Methods: A randomized clinical trial was used to evaluate the efficacy of simulated clinical research enrollment training as a teaching and/or learning method to reduce the error rate in submitted research packets by RAs. The returning RAs were randomized into an intervention group with new training (simulations) and a control group with current training (didactic presentations). A self-confidence survey and a knowledge questionnaire were completed by RAs pre/post-training and one-month follow-up. Quality of data collection was measured by comparing the error rates of data collection in completed clinical research enrollment packets submitted by the RAs in the intervention group versus the control group. Results: Results showed no statistically significant difference in the level of knowledge, confidence or error rates between the patient enrollment simulation (intervention) group and the didactic presentations (control) group after their respective training (p [greater than] .05). However, there was a statistically significant increase in knowledge and confidence post-training in patient simulations group. A significant association was present between confidence and error rate but not between knowledge and error rate for research associates in either training group. Conclusion: Clinical simulation training was not a significantly more effecting training method compared to current TEMRAP didactic presentation training. Even though knowledge and confidence did increase post-training there was no significant difference between the two types of training. Future experiments should explore the possibility of combining the two types of training and observing other potential variables affecting the quality of data, such as research associates' motivation. Additionally, the need for a larger sample size and enrolling participants with no prior research experience should be explored for significant results.Item Temporal Dynamics of Mitochondrial Dysfunction and Retinal Ganglion Cell Degeneration Following Endothelin-1 Exposure(2024-03-21) Brooks, Calvin; Kodati, Bindu; Stankowska, Dorota; Krishnamoorthy, RaghuPurpose: Endothelin-1 (ET-1) and its receptors have been identified as upregulated factors in the aqueous humor and retina of animal models with glaucoma, implicating them in the pathogenesis of glaucomatous neurodegeneration. This study aims to investigate the effects of ET-1 on mitochondrial morphology, a key aspect of cellular health. Methods: C57BL/6J mice (13 weeks old) received intravitreal injections of either ET-1 or vehicle (water) in both eyes 24 hours, 72 hours, or 7 days before collection. One eye was used for retinal flat mounts stained with RBPMS to quantify retinal ganglion cells (RGCs), while the other eye was processed for transmission electron microscopy after paraffin embedding. Optic nerves were sectioned, and ten images were captured per nerve section. Mitochondria within optic nerve axons were enumerated and graded based on cristae appearance (with grades ranging from 1 to 5). Comparisons between vehicle and ET-1 treated eyes at each time point were performed for mitochondrial scores and counts (Mann-Whitney test, n=4 per group). Cell counts from retinal flat mounts were compared across groups and time points (Student’s T Test). Results: Significant RGC loss was observed in the peripheral and mid-peripheral retina 24 hours post ET-1 injection (P=0.01 and 0.04 respectively), with the disparity between vehicle and ET-1 treatments diminishing at later time points. While a trend towards reduced mitochondrial numbers in optic nerve axons post ET-1 injection was noted across all time points, significance was not reached. Mitochondrial health scores were notably diminished at 24 hours post-injection in both vehicle and ET-1 treated groups, with ET-1 demonstrating appreciable damage to mitochondria at 72 hours compared to vehicle-injected animals. However, mitochondrial health improved over time in the ET-1 treated group, with no discernible differences observed at 7 days. Conclusion: The acute decline in mitochondrial morphology following intravitreal injection of both ET-1 and vehicle suggests induced cellular stress. However, ET-1 injection led to immediate cell death not observed in the vehicle-injected cohort. While mitochondrial morphology swiftly recovered in the vehicle-injected group, ET-1 administration prolonged mitochondrial damage. These alterations in mitochondrial morphology may impair mitochondrial energy production efficiency, potentially contributing to retinal ganglion cell vulnerability in glaucoma.Item The Endothelin Receptor Antagonist Macitentan Ameliorates Endothelin-Mediated Vasoconstriction and Promotes Neuroprotection of Retinal Ganglion Cells in Rats.(2019-03-05) Krishnamoorthy, Vignesh; wei, Zhang; Kodati, Bindu; Chavala, Sai; Krishnamoorthy, Raghu; Stankowska, Dorota; Harris, PaytonThe Endothelin Receptor Antagonist Macitentan Ameliorates Endothelin-Mediated Vasoconstriction and Promotes Neuroprotection of Retinal Ganglion Cells in Rats. Payton Harris Vignesh Krishnamoorthy Wei Zhang Bindu Kodati Sai Chavala Raghu Krishnamoorthy Dorota L. Stankowska 1. Texas College of Osteopathic Medicine, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth TX 76107 2. Department of Pharmacology and Neuroscience, North Texas Eye Research Institute, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth TX 76107 Purpose: To determine if dietary administration of the dual ETA/ETB receptor antagonist, macitentan, could protect retinal ganglion cells (RGCs) following endothelin-1 mediated vasoconstriction in Brown Norway rats. Methods: Adult male and female Brown Norway rats were either untreated or treated with macitentan (5 mg/kg body weight) once a day for 3 days followed by intravitreal injection of either 4 µl of 500 mM ET-1 or vehicle in one eye. Imaging of the retinal vasculature using fluorescein angiography was carried out a various time points including 2, 5, 10 and 20 minutes. Following the imaging of the vasculature, treatment of rats was continued for 1 week with either macitentan (5 mg/kg/body weight) in dietary gels or untreated control gels. After euthanizing the rats, retinal flat mounts from the rats were prepared, immunostained for RGC marker Brn3a, imaged and surviving RGCs were counted in a masked manner. Results: Vasoconstrictive effects following intravitreal ET-1 injection were greatly reduced in rats administered with macitentan in the diet prior to the ET-1 administration. ET-1 intravitreal injection produced a 45% loss of RGCs which was significantly reduced in macitentan-treated rats and RGC counts were similar to that observed in control retinas. Conclusions: The endothelin receptor antagonist, macitentan, has neuroprotective effects in retinas of Brown Norway rats that occurs through different mechanisms, including, enhancement of RGC survival and reduction ET-1 mediated vasoconstriction preventing ischemia.