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Effects of intravitreal injection of siRNA against caspase-2 on retinal and optic nerve degeneration in air blast induced ocular trauma.

ABSTRACT: Ocular repeated air blast injuries occur from low overpressure blast wave exposure, which are often repeated and in quick succession. We have shown that caspase-2 caused the death of retinal ganglion cells (RGC) after blunt ocular trauma. Here, we investigated if caspase-2 also mediates RGC apoptosis in a mouse model of air blast induced indirect traumatic optic neuropathy (b-ITON). C57BL/6 mice were exposed to repeated blasts of overpressure air (3 × 2 × 15 psi) and intravitreal injections of siRNA against caspase-2 (siCASP2) or against a control enhanced green fluorescent protein (siEGFP) at either 5 h after the first 2 × 15 psi ("post-blast") or 48 h before the first blast exposure ("pre-blast") and repeated every 7 days. RGC counts were unaffected by the b-ITON or intravitreal injections, despite increased degenerating ON axons, even in siCASP2 "post-blast" injection groups. Degenerating ON axons remained at sham levels after b-ITON and intravitreal siCASP2 "pre-blast" injections, but with less degenerating axons in siCASP2 compared to siEGFP-treated eyes. Intravitreal injections "post-blast" caused greater vitreous inflammation, potentiated by siCASP2, with less in "pre-blast" injected eyes, which was abrogated by siCASP2. We conclude that intravitreal injection timing after ocular trauma induced variable retinal and ON pathology, undermining our candidate neuroprotective therapy, siCASP2.

SUBMITTER: Thomas CN

PROVIDER: S-EPMC8377143 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The glial protein S100B, which belongs to a calcium binding protein family, is up-regulated in neurological diseases, like multiple sclerosis or glaucoma. In previous studies, S100B immunization led to retinal ganglion cell (RGC) loss in an experimental autoimmune glaucoma (EAG) model. Now, the direct degenerative impact of S100B on the retina and optic nerve was evaluated. Therefore, 2 ?l of S100B was intravitreally injected in two concentrations (0.2 and 0.5 ?g/?l). At day 3, 14 and 21, retinal neurons, such as RGCs, amacrine and bipolar cells, as well as apoptotic mechanisms were analyzed. Furthermore, neurofilaments, myelin fibers and axons of optic nerves were evaluated. In addition, retinal function and immunoglobulin G (IgG) level in the serum were measured. At day 3, RGCs were unaffected in the S100B groups, when compared to the PBS group. Later, at days 14 and 21, the RGC number as well as the ?-III tubulin protein level was reduced in the S100B groups. Only at day 14, active apoptotic mechanisms were noted. The number of amacrine cells was first affected at day 21, while the bipolar cell amount remained comparable to the PBS group. Also, the optic nerve neurofilament structure was damaged from day 3 on. At day 14, numerous swollen axons were observed. The intraocular injection of S100B is a new model for a glaucoma like degeneration. Although the application site was the eye, the optic nerve degenerated first, already at day 3. From day 14 on, retinal damage and loss of function was noted. The RGCs in the middle part of the retina were first affected. At day 21, the damage expanded and RGCs had degenerated in all areas of the retina as well as amacrine cells. Furthermore, elevated IgG levels in the serum were measured at day 21, which could be a sign of a late and S100B independet immune response. In summary, S100B had a direct destroying impact on the axons of the optic nerve. The damage of the retinal cell bodies seems to be a consequence of this axon loss.

Project description:PurposeTo investigate anterograde degenerative changes along the visual pathway in a rat model of optic nerve axotomy.MethodsOptic nerve transection was performed in adult Sprague-Dawley rats. Animals were sacrificed at regular time intervals and tissues harvested. Immunoblotting followed by densitometric analysis was used to determine the phosphorylation profile of Akt in the dorsal lateral geniculate nucleus (dLGN) and the primary visual cortex (V1). The neuronal cell size and cell density were measured in the dLGN and the V1 using Nissl staining. The prevalence of apoptosis was characterized by terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end labelling (TUNEL) histochemistry. Caspase-3 antibodies were also used to identify apoptotic cells. Neurons and astrocytes were detected using NeuN and glial fibrillary acidic protein (GFAP), respectively.ResultsAn early and sustained loss of Akt phosphorylation was observed after optic nerve transection in both dLGN and V1. At week one, a decrease in the neuronal cell size (50.5±4.9 vs 60.3±5.0 µm(2), P = 0.042) and an increase of TUNEL positive cells (7.9±0.6 vs 1.4±0.5 ×10(2) cells/mm(2), P<0.001) were evident in the dLGN but not in V1. A significant decline in neuronal cell number (14.5±0.1 vs 17.4±1.3 ×10(2) cells/mm(2), P = 0.048), cell size (42.5±4.3 vs 62.1±4.7 µm(2), P = 0.001) and an increase in apoptotic cells (5.6±0.5 vs 2.0±0.4 ×10(2) cells/mm(2), P<0.001) appeared in V1 initially at one month post-transection. The changes in the visual pathway continued through two months. Both neuronal cells and GFAP-positive glial cells were affected in this anterograde degeneration along the visual pathway.ConclusionsAnterograde degeneration along the visual pathway takes place in target relay (LGN) and visual cortex following the optic nerve injury. Apoptosis was observed in both neural and adjacent glial cells. Reduction of Akt phosphorylation preceded cellular and apoptotic changes.

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Effects of intravitreal injection of siRNA against caspase-2 on retinal and optic nerve degeneration in air blast induced ocular trauma.

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