Protein kinase C subtypes and retinal ischemic preconditioning

Abstract

The purpose of our study was to determine the specific subtypes of protein kinase C involved in the neuroprotection afforded by retinal ischemic preconditioning (IPC), their relationship to the opening of mitochondrial KATP (mKATP) channels, and their role in apoptosis after preconditioning and ischemia. Rats were subjected to retinal ischemia after IPC, or retinas were rendered ischemic after pharmacological opening of mKATP channels. Using immunohistochemistry and image analysis, we determined cellular localization of PKC subtypes. We blocked PKC-δ and -ε to study the effect on protection with IPC or with IPC-mimicking by the opening of mKATP channels. PKC subtypes were inhibited pharmacologically or with interfering RNA. Electroretinography assessed functional recovery after ischemia. IPC was effectively mimicked by injection of diazoxide to open the mKATP channel. IPC and/or its mimicking were attenuated by the PKC-δ inhibitor rottlerin and by interfering RNA targeting PKC-δ or -ε. Using TUNEL staining and Western blotting for caspase-3 and fodrin breakdown we assessed apoptosis. The injection of interfering RNA to PKC-δ and -ε before preconditioning significantly enhanced TUNEL staining as well as the cleavage of caspase-3 and fodrin after ischemia. In summary, our experiments have shown that both PKC-δ and -ε subtypes are involved in the cellular signaling that results in neuroprotection from IPC and that both are downstream of the opening of mKATP channels.

Mitogen-activated protein kinase p38α and retinal ischemic preconditioning

“…Procedures (Roth et al 2006; Dreixler et al 2008) conformed to the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research and were approved by our Animal Care Committee. Sprague-Dawley rats (200-250 gm) from Harlan (Indianapolis, IN) were maintained on a 12 h on/12 h off light cycle.…”

Delayed post-ischemic conditioning significantly improves the outcome after retinal ischemia

“…Procedures (Roth et al 2006; Dreixler et al 2008) conformed to the ARVO Resolution on the Use of Animals in Research and were approved by our Animal Care Committee. For retinal ischemia, rats were anesthetized with chloral hydrate, 275 mg/kg i.p., and intraocular pressure (IOP) increased to 130 – 135 mm Hg for 55 min using a pressurized bag of sterile ocular irrigating solution (BSS: Alcon, Fort Worth, TX) connected to a 27-g needle positioned in the center of the anterior chamber.…”

“…ERG data were corrected for daily variation in the normal eye and for simultaneous reference to the baseline in the ischemic eye, as previously described (Roth et al 2006; Dreixler et al 2008; Dreixler et al 2009a; Dreixler et al 2009b). The a- and b-waves, SUM RMS of the OPs, and P2 from ischemic eyes 7 days after post conditioning in the groups for comparison were expressed as stimulus-intensity plots (intensity, log cd.s/m 2 on the x-axis), and % recovery of baseline on the y-axis (Dreixler et al 2008; Dreixler et al 2011a).…”

“…The a- and b-waves, SUM RMS of the OPs, and P2 from ischemic eyes 7 days after post conditioning in the groups for comparison were expressed as stimulus-intensity plots (intensity, log cd.s/m 2 on the x-axis), and % recovery of baseline on the y-axis (Dreixler et al 2008; Dreixler et al 2011a). The data were normally distributed as confirmed using normality plots and skewness/kurtosis in Stata version 10.0 (College Station, TX).…”

Abrogated cardio protective effect of ischemic preconditioning in hyperhomocysteinemia and hypertrophy

“…35 PKC e as well as PKC d has been demonstrated to mimic preconditioning due to opening of mitoK ATP . 36 As potassium enters the mitochondria, it causes them to release free radicals, known as reactive oxygen species (ROS). 37 ROS generation during preconditioning also activates PKC.…”