Abstract
BackgroundReceptor-interacting protein 3 (RIP3), a member of RIP family proteins, has been shown to participate in programmed necrosis or necroptosis in cell biology studies. Evidence suggests that necroptosis may be a mode of neuronal death in the retina.ResultsIn the present study we determined the expression of RIP3 in normal rat retina and its changes following acute high intraocular pressure (aHIOP). RIP3 immunoreactivity (IR) was largely present in the inner retinal layers, localized to subsets of cells expressing neuron-specific nuclear antigen (NeuN), parvalbumin and calbindin in the ganglion cell layer (GCL) and inner nuclear layer (INL). No double labeling was detected for RIP3 with PKC-α or rhodopsin. RIP3 immunoreactivity was increased in the GCL at 6 hr and 12 hr, but reduced at 24 hr in the retina, without apparent alteration in laminar or cellular distribution pattern. Western blot analysis confirmed the above time-dependent alteration in RIP3 protein expression. RIP3 expressing cells frequently co-localized with propidium iodide (PI). A few co-localized cells were observed between RIP3 and Bax or cleaved caspase-3 in the GCL in 12 hr following aHIOP.ConclusionsThe results indicate that RIP3 is expressed differentially in retinal neurons in adult rats, including subsets of ganglion cells, amacrine and horizontal cells. RIP3 protein levels are elevated rapidly following aHIOP. RIP3 labeling co-localized with PI, Bax or cleaved caspase-3 among cells in the ganglion cell layer following aHIOP, which suggest its involvement of RIP3 in neuronal responses to acute ischemic insults.
Highlights
Receptor-interacting protein 3 (RIP3), a member of RIP family proteins, has been shown to participate in programmed necrosis or necroptosis in cell biology studies
A strong cellular labeling was notable in the ganglion cell layer (GCL), with some labeled somata seen in the inner nuclear layer (INL)
Diffuse and moderately intense neuropillike labeling was present in the inner plexiform layer (IPL)
Summary
Overall laminar distribution of RIP3 immunoreactivity in retina The RIP3 antibody detected a monoband migrated approximately at 57 kDa, consistent with the previous reports regarding the molecular weight of the protein [8]. Changes of RIP3 expression following aHIOP The immunofluorescence staining result showed that RIP3 was mainly present in ganglion cell layer (GCL), nerve fiber layer (NFL), inner plexiform layer (IPL) and inner nuclear layer(INL), but weakly staining occurs in other layers in retina (Figure 5), no difference. Double labeling for RIP3 and PI in retina following aHIOP at 12 hr PI-positive cells were largely detected in the ganglion cell layer (GCL) and inner nuclear layer (INL) in 12 hr group (Figure 7). Some RIP3 strong expression cells are not exhibit Bax or cleaved caspase-3 reactivities This finding suggests that RIP3 regulation may be potentially involved in the process of neuronal injury or death. Because there is no specific marker for necroptosis, future studies are required to determine the precise role of early RIP3upregulation in retinal injury and diseases [28,31,32]
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