Protein kinase C in porcine retinal arteries and neuroretina following retinal ischemia-reperfusion

Abstract

Purpose: Identification of the intracellular signal-transduction pathways activated in retinal ischemia may be important in revealing novel pharmacological targets. To date, most studies have focused on identifying neuroprotective agents. The retinal blood vessels are key organs in circulatory failure, and this study was therefore designed to examine the retinal vasculature separately from the neuroretina.

Methods: Retinal ischemia was induced by elevating the intraocular pressure in porcine eyes, followed by 5, 12, or 20 h of reperfusion. Protein kinase C (PKC)alpha, PKCbeta1, and PKCbeta2 mRNA levels, and protein expression were determined using real-time PCR, western blot, and immunofluorescence staining techniques.

Results: The retinal arteries could easily be dissected free and studied separately from the neuroretina in this porcine model. The PKCalpha, PKCbeta1, and PKCbeta2 mRNA levels tended to be lower in ischemia-reperfused than in sham-operated eyes in both the retinal arteries and the neuroretina. This was most prominent after 5 h, and less pronounced after 12 h and 20 h of reperfusion. Likewise, the protein levels of PKCalpha, PKCbeta1, and PKCbeta2 were slightly lower following ischemia-reperfusion when compared to sham-operated eyes. PKCalpha, PKCbeta1, and PKCbeta2 immunostaining were observed in bipolar cells of the neuroretina and in endothelial cells, and to a low extent in the smooth muscle layer, of the retinal arteries.

Conclusions: Retinal ischemia followed by reperfusion results in lower levels of PKC in both the neuroretina and retinal arteries. New targets for pharmacological treatment may be found by studying the retinal vasculature so as to identify the intracellular signal-transduction pathways involved in the development of injury following retinal circulatory failure.

Figure 1

PKC mRNA levels in the retinal arteries and neuroretina. PKCα, PKCβ1, and PKCβ2 mRNA expression levels assessed by real-time PCR in (A) the retinal arteries and (B) in the neuroretina in eyes subjected to ischemia and 5 (n=6), 12 (n=14), or 20 (n=6) hours of reperfusion versus sham-operated eyes. Values are presented as mean±SEM. Statistical comparison was performed using Student’s paired ratio t-test (ischemia–reperfusion versus sham-operated) with Bonferroni correction. Exact p-values are given in the figure. Note that the levels for PKCα, PKCβ1, and PKCβ2 show similar patterns of change in the retinal arteries and the neuroretina.

Figure 2

PKC protein levels in the retinal arteries and neuroretina. Phosphorylated and total PKCα, PKCβ1, and PKCβ2 protein expression levels, assessed by western blot, in (A) retinal arteries and (B) neuroretina, in eyes subjected to ischemia and 12 (n=7) or 20 (n=5) hours of reperfusion versus sham-operated eyes. The right panels are representative examples of western blots of neuroretina and retinal arteries from animals in the 20 h of reperfusion group. Values are presented as mean values±SEM. Statistical comparison was performed using Student’s paired ratio t-test (ischemia–reperfusion versus sham) with Bonferroni correction. Exact p-values are given in the figure.

Figure 3

PKC immunoreactivity in the retinal arteries and neuroretina. Representative examples showing phosphorylated PKCα, PKCβ1, and PKCβ2 immunoreactivity in the retinal arteries and retina following ischemia and 20 h of reperfusion. A: Double staining with CD31 (also called PECAM-1), an endothelial cell marker, showed co-localization of phosphorylated PKC in the endothelium (arrows). Weak phosphorylated PKC staining could also be seen in the smooth muscle layer. B: The lower levels of PKCα, PKCβ1, and PKCβ2 observed in the neuroretina after ischemia–reperfusion, according to western blot, were reflected in the immunofluorescence staining results, showing less staining for phosphorylated PKCα and PKCβ2 in the ischemia–reperfusion eyes compared to sham-operated eyes. Furthermore, the phosphorylated PKCβ1 staining showed fewer labeled bipolar cells bodies in the eyes subject to ischemia-reperfusion compared to sham-operated eyes (see insert in the p-PKCβ1 picture). Similar results were seen in all pigs studied. Abbreviations: outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), and nerve fiber layer (NFL).

Figure 4

PKC immunoreactivity in the smooth muscle layer of the retinal arteries. Representative examples showing phosphorylated PKCα, PKCβ1, and PKCβ2 immunoreactivity in the retinal arteries following ischemia and 20 h of reperfusion. Double staining with smooth muscle actin, a smooth muscle marker, showed colocalization with phosphorylated PKC in the smooth muscle layer (arrows). Note that the colocalization was most apparent for phosphorylated PKCα.