ROS generation is ameliorated by ischemic preconditioning in organotypic hippocampal slice cultures

Introduction

Ischemic preconditioning (IPC) is an endogenous mechanism for neuroprotection against ischemia/reperfusion injury. Our earlier findings suggest that IPC protects the integrity of mitochondrial oxidative phosphorylation after cerebral ischemia ¹ . Studies in heart suggest that mitochondria generate reactive oxygen species (ROS) via the mitochondrial ATP sensitive potassium channel, which then activate downstream kinases. Employing organotypic hippocampal slice cultures, we aimed to investigate whether (1) IPC reduced ROS generation during a subsequent lethal ischemic event, and whether (2) reactive oxygen species (ROS) generated in the mitochondrial respiratory chain act as a trigger of IPC.

Methods

Hippocampal slices were obtained from 9–11 days old Sprague Dawley rats. Slices were cultured for 14–15 days before experiments. Slices were divided in 4 groups, viz. sham, ischemia (40 min of oxygen-glucose deprivation (OGD)), IPC-1 (48 h prior to ischemia, slices were exposed to IPC-15 min of OGD) and IPC-2 (represents only 15 min of OGD without subsequent ischemic insult). Individual slices were incubated with the cell-permeant dihydrorhodamine 123 (DHR) (5 ìM, 30 min), washed and transfer to an interface chamber mounted on a fluorescence microscope, where they were exposed to OGD (40/15 min). In the chamber slices were superfused with warmed (35–36 degree C) ACSF at a rate of 1 ml/min, and oxygenated with humidified 95% O₂, 5% CO₂. OGD of either 40/15 min was achieved by changing ACSF to aglycemic ACSF for 5 min followed by switching the gas composition from 95/5% O₂/CO₂ to 95/5% N₂/CO₂ for 35/10 min, respectively. The rhodamine fluorescence images were obtained every 5 min using a SPOT CCD camera and digitized using SPOT advanced software. Quantification of neuronal death in CA1 region was carried out using propidium iodide (PI) staining technique. Percentage of relative optical intensity of rhodamine/PI fluorescence was used as an index of ROS generation/cell death, respectively. Statistical significance was determined with an ANOVA test followed by a Bonferroni's post-hoc test.

Results

Slices exposed to ischemia followed by 1 h of reperfusion injury showed significant increase in ROS production. Quantification of rhodamine fluorescence demonstrated 51% (% of baseline) (n = 5) increase after ischemia as compare with 7% (n = 6) in control slices (p < 0.05). IPC to slices reduced ROS production during subsequent ischemic episode to 21% (n = 5) as compared with ischemic group (p<0.01). IPC to organotypic slices itself showed 20% (n = 3) increase in ROS, although not at the levels of the ischemic tissue. In correspondence to above mentioned results quantification of PI fluorescence in ischemic and IPC groups were 55.39 ± 4.05 % (Mean ± SD) (n = 17) and 22.65 ± 1.61 % (n = 12), respectively (p < 0.05).

Conclusion

Our study demonstrate that mild increases in ROS will be neuroprotective, whereas larger increases in ROS may induced cell death CA1 region of hippocampus in organotypic slice cultures.