Yasir Abdul

Delta-Opioid Agonist SNC-121 Protects Retinal Ganglion Cell Function in a Chronic Ocular Hypertensive Rat Model

PURPOSE This study examined if the delta-opioid (δ-opioid) receptor agonist, SNC-121, can improve retinal function and retinal ganglion cell (RGC) survival during glaucomatous injury in a chronic ocular hypertensive rat model. METHODS IOP was raised in brown Norway rats by injecting hypertonic saline into the limbal venous system. Rats were treated with 1 mg/kg SNC-121 (intraperitoneally [IP]) once daily for 7 days. Pattern-electroretinograms (PERGs) were obtained in response to contrast reversal of patterned visual stimuli. RGCs were visualized by fluorogold retrograde labeling. Expression of TNF-α and p38 mitogen-activated protein (MAP) kinase was measured by immunohistochemistry and Western blotting. RESULTS PERG amplitudes in ocular hypertensive eyes were significantly reduced (14.3 ± 0.60 μvolts) when compared with healthy eyes (18.0 ± 0.62 μvolts). PERG loss in hypertensive eyes was inhibited by SNC-121 treatment (17.20 ± 0.1.3 μvolts; P < 0.05). There was a 29% loss of RGCs in the ocular hypertensive eye, which was inhibited in the presence of SNC-121. TNF-α production and activation of p38 MAP kinase in retinal sections and optic nerve samples were upregulated in ocular hypertensive eyes and inhibited in the presence of SNC-121. Furthermore, TNF-α induced increase in p38 MAP kinase activation in astrocytes was inhibited in the presence of SNC-121. CONCLUSIONS These data provide evidence that activation of δ-opioid receptors inhibited the loss of PERG amplitudes and rate of RGC loss during glaucomatous injury. Mechanistic data provided clues that TNF-α is mainly produced from glial cells and activates p38 MAP kinase, which was significantly inhibited by SNC-121 treatment. Overall, data indicate that enhancement of δ-opioidergic activity in the eye may provide retina neuroprotection against glaucoma.

Non-Analgesic Effects of Opioids: Neuroprotection in the Retina

Inadequate blood flow in the retina (ischemia) is a common cause of visual impairment and blindness. Retinal ischemia plays a pivotal role in a number of ocular degenerative diseases such as diabetic retinopathy, glaucoma, and retinal artery occlusion. The sequence of events by which ischemia leads to retinal degeneration are not completely understood, but likely involve both necrotic and apoptotic processes. A variety of diverse chemical mediators (e.g., glutamate, oxygen free-radical, nitric oxide, and proinflammatory cytokines) have been implicated as participants in ischemic retinal injury. In the eye, experimental and/or clinical evidence has suggested roles for endogenous opioids and their receptors in the regulation of iris function, aqueous humor dynamics, corneal wound healing, and retinal development and neuroprotection. In numerous vital organs, opioid receptor activation prior to ischemia or severe hypoxia is neuroprotective. Recently, activation of opioid-receptors, particularly δ-opioid-receptors (DOR), has been demonstrated to suppress several steps in the deleterious cascade of events during ischemic/hypoxic stress. In providing neuroprotection against ischemia, opioid-receptor activation appears to block proinflammatory cytokines, such as TNF-α, and glutamate excitotoxicity. Depending on duration and severity of cellular stress, DOR activation can trigger different mechanisms at multiple levels to preserve neuronal survival, including: stabilized ionic homeostasis, augmented pro-survival signaling (e.g., PKC, ERK, PI3K/Akt) and enhanced anti-oxidative capacity. This review will summarize the potential roles of opioids in protecting the viability of ocular tissues. Special emphasis will be focused on enhancing the understanding of the molecular mechanisms of opioid actions in protecting the retina against ischemic/hypoxic injury.

Delta-opioid receptors attenuate TNF-α-induced MMP-2 secretion from human ONH astrocytes.

PURPOSE We examined the signaling mechanisms involved in δ-opioid-receptor agonist, SNC-121-mediated attenuation of TNF-α-induced matrix metalloproteinase-2 (MMP-2) secretion from human optic nerve head (ONH) astrocytes. METHODS Human ONH astrocytes were treated with SNC-121 (1 μmol/L) for 15 minutes followed by TNF-α (25 ng/mL) treatment for 6 or 24 hours. Cells were pretreated with inhibitors of p38 mitogen-activated protein (MAP) kinase (SB-203580) or NF-κB (Helenalin) prior to TNF-α treatment. Changes in phosphorylation and expression of p38 MAP kinase, IκBα, NF-κB, and MMP-2 were measured by Western blotting. Translocation of NF-κB was determined by immunocytochemistry. RESULTS TNF-α treatment increased MMP-2 secretion from ONH astrocytes to 236% ± 17% and 142% ± 8% at 6 and 24 hours, respectively; while SNC-121 treatment reduced MMP-2 secretion to 149% ± 11% and 108% ± 7% at 6 and 24 hours, respectively. The SNC-121-mediated inhibitory response was blocked by the δ-opioid-receptor antagonist naltrindole. TNF-α treatment resulted in a sustained phosphorylation of p38 MAP kinase up to 24 hours (226% ± 15% over control levels), which was reduced to 150% ± 20% by SNC-121 treatment. TNF-α treatment increased the expression of NF-κB to 179% ± 21% and 139% ± 6% at 6 and 24 hours, respectively, which was significantly blocked by SNC-121 treatment. Furthermore, TNF-α-induced MMP-2 secretion was blocked by 100% and 78% in the presence of SB-203580 and Helenalin, respectively. CONCLUSIONS Evidence is provided that SNC-121 attenuated TNF-α-induced MMP-2 secretion from ONH astrocytes. Data also supported the idea that p38 MAP kinase and NF-κB played central roles in TNF-α-induced MMP-2 secretion, and both were negatively regulated by SNC-121.

Regulation of nitric oxide production by δ-opioid receptors during glaucomatous injury.

To determine the roles of nitric oxide in glaucomatous injury and its regulation by δ-opioid-receptor activation, animals were treated with: 1) a selective inducible nitric oxide synthase (iNOS) inhibitor (aminoguanidine; AG; 25 mg/kg, i.p.); 2) δ-opioid-receptor agonist (SNC-121; 1 mg/kg, i.p.); or 3) with both drugs simultaneously for 7 days, once daily. The loss in retinal ganglion cell (RGC) numbers and their function in glaucomatous eyes were significantly improved in the presence of AG or SNC-121; however, we did not see any significant additive or synergistic effects when animals were treated with both drugs simultaneously. The levels of nitrate-nitrite were significantly increased in the glaucomatous retina when compared with normal retina (normal retina 86±9 vs. glaucomatous retina 174±10 mM/mg protein), which was reduced significantly when animals were treated either with SNC-121 (121±7 mM/mg protein; P<0.05) or AG (128±10 mM/mg protein; P<0.05). Additionally, SNC-121-mediated reduction in nitrate-nitrite levels was not only blocked by naltrindole (a δ-opioid-receptor antagonist), but naltrindole treatment potentiated the nitrate-nitrite production in glaucomatous retina (235±4 mM/mg protein; P<0.001). As expected, naltrindole treatment also fully-blocked SNC-121-mediated retina neuroprotection. The nitrotyrosine level in the glaucomatous retina was also increased, which was significantly reduced in the SNC-121-treated animals. Additionally, the expression level of iNOS was clearly increased over the control levels in the glaucomatous retina and optic nerves, which was also reduced by SNC-121 treatment. In conclusion, our data support the notion that nitric oxide plays a detrimental role during glaucomatous injury and inhibition of nitric oxide production provided RGC neuroprotection. Furthermore, δ-opioid receptor activation regulates the production of nitric oxide via inhibiting the activity of iNOS in the retina and optic nerve.

Interferon-gamma (IFN-γ)-mediated retinal ganglion cell death in human tyrosinase T cell receptor transgenic mouse.

We have recently demonstrated the characterization of human tyrosinase TCR bearing h3T-A2 transgenic mouse model, which exhibits spontaneous autoimmune vitiligo and retinal dysfunction. The purpose of current study was to determine the role of T cells and IFN-γ in retina dysfunction and retinal ganglion cell (RGC) death using this model. RGC function was measured by pattern electroretinograms (ERGs) in response to contrast reversal of patterned visual stimuli. RGCs were visualized by fluorogold retrograde-labeling. Expression of CD3, IFN-γ, GFAP, and caspases was measured by immunohistochemistry and Western blotting. All functional and structural changes were measured in 12-month-old h3T-A2 mice and compared with age-matched HLA-A2 wild-type mice. Both pattern-ERGs (42%, p = 0.03) and RGC numbers (37%, p = 0.0001) were reduced in h3T-A2 mice when compared with wild-type mice. The level of CD3 expression was increased in h3T-A2 mice (h3T-A2: 174±27% vs. HLA-A2: 100%; p = 0.04). The levels of effector cytokine IFN-γ were also increased significantly in h3T-A2 mice (h3T-A2: 189±11% vs. HLA-A2: 100%; p = 0.023). Both CD3 and IFN-γ immunostaining were increased in nerve fiber (NF) and RGC layers of h3T-A2 mice. In addition, we have seen a robust increase in GFAP staining in h3T-A2 mice (mainly localized to NF layer), which was substantially reduced in IFN-γ (-/-) knockout h3T-A2 mice. We also have seen an up-regulation of caspase-3 and -9 in h3T-A2 mice. Based on our data we conclude that h3T-A2 transgenic mice exhibit visual defects that are mostly associated with the inner retinal layers and RGC function. This novel h3T-A2 transgenic mouse model provides opportunity to understand RGC pathology and test neuroprotective strategies to rescue RGCs.

PI3K/Akt Pathway: A Role in δ-Opioid Receptor–Mediated RGC Neuroprotection

Purpose This study examines the role of PI3K/Akt pathway in δ-opioid receptor agonist (SNC-121)-induced RGC neuroprotection in a chronic glaucoma rat model. Methods Injecting hypertonic saline into the limbal veins of Brown Norway rats elevated IOP. Rats were treated either with 1 mg/kg SNC-121 or 3 mg/kg 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY-294002; PI3K/Akt inhibitor) plus SNC-121 once daily for 7 days. Pattern ERGs were recorded in response to contrast reversal of patterned visual stimuli. Retinal ganglion cells (RGC) were visualized by Fluorogold retrograde labeling. Optic nerve head (ONH) astrocytes were pretreated with PI3K/Akt inhibitors for 30 minutes followed by 1-μM SNC-121 treatment. Changes in matrix metalloproteinases (MMP-1, -2, and -3) production and PI3K/Akt activation in optic nerve and TNF-α treated ONH astrocytes were measured by immunohistochemistry and Western blotting. Results SNC-121 activates the PI3K/Akt pathway in ONH astrocytes and the retina. In ONH astrocytes, SNC-121–induced Akt activation was fully inhibited by PI3K/Akt inhibitors. A sustained decline (7–42 days post injury) in Akt activation was seen in the ocular-hypertensive retina and optic nerve. This decline is reversed to normal levels by 1-mg/kg intraperitoneally (i.p.) SNC-121 treatment. Both pattern ERG amplitudes and RGC numbers were reduced in ocular hypertensive eyes, which were significantly increased in SNC-121–treated animals. Interestingly, SNC-121–induced increase in pattern-ERG amplitudes and RGC numbers were inhibited in LY-294002 pretreated animals. Additionally, SNC-121 treatment inhibited MMP-1, -2, and -3 production from the optic nerve of ocular hypertensive rats and TNF-α–treated ONH astrocytes. Conclusions PI3K/Akt pathway plays a crucial role in SNC-121–mediated RGC neuroprotection against glaucomatous injury.

Impact of p53 arg72pro SNP on Breast Cancer Risk in North Indian Population

Background: Genetic changes in p53 gene contribute to breast cancer susceptibility. Objective and Methods: A case-control study and a meta-analysis were performed to investigate the role of p53 codon72 SNP with breast cancer susceptibility in Indian women. Results: p53 heterozygous arginine variant was associated with decreased risk of breast cancer in total cohort. In meta-analysis, Allelic and GG vs. CC genetic comparison model were found to be associated with breast cancer risk. Moreover, recessive comparison model indicated a protective correlation with breast cancer occurrence. Conclusion: The findings of our case-control study and meta-analysis suggest a significant association between p53 Arg72Pro polymorphism and an increased risk of breast cancer in Indian population.