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Neuroprotection of Rodent and Human Retinal Ganglion Cells In Vitro/Ex Vivo by the Hybrid Small Molecule SA-2.

ABSTRACT: The mechanisms underlying the neuroprotective effects of the hybrid antioxidant-nitric oxide donating compound SA-2 in retinal ganglion cell (RGC) degeneration models were evaluated. The in vitro trophic factor (TF) deprivation model in primary rat RGCs and ex vivo human retinal explants were used to mimic glaucomatous neurodegeneration. Cell survival was assessed after treatment with vehicle or SA-2. In separate experiments, tert-Butyl hydroperoxide (TBHP) and endothelin-3 (ET-3) were used in ex vivo rat retinal explants and primary rat RGCs, respectively, to induce oxidative damage. Mitochondrial and intracellular reactive oxygen species (ROS) were assessed following treatments. In the TF deprivation model, SA-2 treatment produced a significant decrease in apoptotic and dead cell counts in primary RGCs and a significant increase in RGC survival in ex vivo human retinal explants. In the oxidative stress-induced models, a significant decrease in the production of ROS was observed in the SA-2-treated group compared to the vehicle-treated group. Compound SA-2 was neuroprotective against various glaucomatous insults in the rat and human RGCs by reducing apoptosis and decreasing ROS levels. Amelioration of mitochondrial and cellular oxidative stress by SA-2 may be a potential therapeutic strategy for preventing neurodegeneration in glaucomatous RGCs.

SUBMITTER: Pham JH

PROVIDER: S-EPMC9735605 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Neuroprotection of Rodent and Human Retinal Ganglion Cells In Vitro/Ex Vivo by the Hybrid Small Molecule SA-2.

Pham Jennifer H JH Johnson Gretchen A GA Rangan Rajiv S RS Amankwa Charles E CE Acharya Suchismita S Stankowska Dorota L DL

Cells 20221123 23

The mechanisms underlying the neuroprotective effects of the hybrid antioxidant-nitric oxide donating compound SA-2 in retinal ganglion cell (RGC) degeneration models were evaluated. The in vitro trophic factor (TF) deprivation model in primary rat RGCs and ex vivo human retinal explants were used to mimic glaucomatous neurodegeneration. Cell survival was assessed after treatment with vehicle or SA-2. In separate experiments, <i>tert</i>-Butyl hydroperoxide (TBHP) and endothelin-3 (ET-3) were us ...[more]

PMID: 36497005

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Project description:PurposeRetinal ischemia induces apoptosis leading to neurodegeneration and vision impairment. Carbon monoxide (CO) in gaseous form showed cell-protective and anti-inflammatory effects after retinal ischemia-reperfusion-injury (IRI). These effects were also demonstrated for the intravenously administered CO-releasing molecule (CORM) ALF-186. This article summarizes the results of intravitreally released CO to assess its suitability as a neuroprotective and neuroregenerative agent.MethodsWater-soluble CORM ALF-186 (25 μg), PBS, or inactivated ALF (iALF) (all 5 μl) were intravitreally applied into the left eyes of rats directly after retinal IRI for 1 h. Their right eyes remained unaffected and were used for comparison. Retinal tissue was harvested 24 h after intervention to analyze mRNA or protein expression of Caspase-3, pERK1/2, p38, HSP70/90, NF-kappaB, AIF-1 (allograft inflammatory factor), TNF-α, and GAP-43. Densities of fluorogold-prelabeled retinal ganglion cells (RGC) were examined in flat-mounted retinae seven days after IRI and were expressed as mean/mm2. The ability of RGC to regenerate their axon was evaluated two and seven days after IRI using retinal explants in laminin-1-coated cultures. Immunohistochemistry was used to analyze the different cell types growing out of the retinal explants.ResultsCompared to the RGC-density in the contralateral right eyes (2804±214 RGC/mm2; data are mean±SD), IRI+PBS injection resulted in a remarkable loss of RGC (1554±159 RGC/mm2), p<0.001. Intravitreally injected ALF-186 immediately after IRI provided RGC protection and reduced the extent of RGC-damage (IRI+PBS 1554±159 vs. IRI+ALF 2179±286, p<0.001). ALF-186 increased the IRI-mediated phosphorylation of MAP-kinase p38. Anti-apoptotic and anti-inflammatory effects were detectable as Caspase-3, NF-kappaB, TNF-α, and AIF-1 expression were significantly reduced after IRI+ALF in comparison to IRI+PBS or IRI+iALF. Gap-43 expression was significantly increased after IRI+ALF. iALF showed effects similar to PBS. The intrinsic regenerative potential of RGC-axons was induced to nearly identical levels after IRI and ALF or iALF-treatment under growth-permissive conditions, although RGC viability differed significantly in both groups. Intravitreal CO further increased the IRI-induced migration of GFAP-positive cells out of retinal explants and their transdifferentiation, which was detected by re-expression of beta-III tubulin and nestin.ConclusionIntravitreal CORM ALF-186 protected RGC after IRI and stimulated their axons to regenerate in vitro. ALF conveyed anti-apoptotic, anti-inflammatory, and growth-associated signaling after IRI. CO's role in neuroregeneration and its effect on retinal glial cells needs further investigation.

Project description:BackgroundVascular dysregulation is one of the major risk factors of glaucoma, and endothelin-1 (ET-1) may have a role in the pathogenesis of vascular-related glaucoma. Fruit extract from Lycium Barbarum (LB) exhibits anti-ageing and multitarget mechanisms in protecting retinal ganglion cells (RGC) in various animal models. To investigate the therapeutic efficacy of LB glycoproteins (LbGP) in ET-1 induced RGC degeneration, LbGP was applied under pre- and posttreatment conditions to an ET-1 mouse model. Retina structural and functional outcomes were characterised using clinical-based techniques.MethodsAdult C57BL/6 mice were randomly allocated into four experimental groups, namely vehicle control (n = 9), LbGP-Pretreatment (n = 8), LbGP-Posttreatment (day 1) (n = 8) and LbGP-Posttreatment (day 5) (n = 7). Oral administration of LbGP 1 mg/Kg or PBS for vehicle control was given once daily. Pre- and posttreatment (day 1 or 5) were commenced at 1 week before and 1 or 5 days after intravitreal injections, respectively, and were continued until postinjection day 28. Effects of treatment on retinal structure and functions were evaluated using optical coherence tomography (OCT), doppler OCT and electroretinogram measurements at baseline, post-injection days 10 and 28. RGC survival was evaluated by using RBPMS immunostaining on retinal wholemounts.ResultsET-1 injection in vehicle control induced transient reductions in arterial flow and retinal functions, leading to significant RNFL thinning and RGC loss at day 28. Although ET-1 induced a transient loss in blood flow or retinal functions in all LbGP groups, LbGP treatments facilitated better restoration of retinal flow and retinal functions as compared with the vehicle control. Also, all three LbGP treatment groups (i.e. pre- and posttreatments from days 1 or 5) significantly preserved thRNFL thickness and RGC densities. No significant difference in protective effects was observed among the three LbGP treatment groups.ConclusionLbGP demonstrated neuroprotective effects in a mouse model of ET-1 induced RGC degeneration, with treatment applied either as a pretreatment, immediate or delayed posttreatment. LbGP treatment promoted a better restoration of retinal blood flow, and protected the RNFL, RGC density and retinal functions. This study showed the translational potential of LB as complementary treatment for glaucoma management.

Dataset Information

Neuroprotection of Rodent and Human Retinal Ganglion Cells In Vitro/Ex Vivo by the Hybrid Small Molecule SA-2.

Publications

Neuroprotection of Rodent and Human Retinal Ganglion Cells In Vitro/Ex Vivo by the Hybrid Small Molecule SA-2.

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