Reappraisal of anoxic spreading depolarization as a terminal event during oxygen\u2013glucose deprivation in brain slices in vitro

Elvira Juzekaeva,Marat Mukhtarov,Roustem Khazipov,Azat Gainutdinov

doi:10.1038/s41598-020-75975-w

Abstract

Anoxic spreading depolarization (aSD) has been hypothesized as a terminal event during oxygen–glucose deprivation (OGD) in submerged cortical slices in vitro. However, mechanical artifacts caused by aSD-triggered edema may introduce error in the assessment of neuronal viability. Here, using continuous patch-clamp recordings from submerged rat cortical slices, we first confirmed that vast majority of L4 neurons permanently lost their membrane potential during OGD-induced aSD. In some recordings, spontaneous transition from whole-cell to out-side out configuration occurred during or after aSD, and only a small fraction of neurons survived aSD with reperfusion started shortly after aSD. Secondly, to minimize artifacts caused by OGD-induced edema, cells were short-term patched following OGD episodes of various duration. Nearly half of L4 cells maintained membrane potential and showed the ability to spike-fire if reperfusion started less than 10 min after aSD. The probability of finding live neurons progressively decreased at longer reperfusion delays at a rate of about 2% per minute. We also found that neurons in L2/3 show nearly threefold higher resistance to OGD than neurons in L4. Our results suggest that in the OGD ischemia model, aSD is not a terminal event, and that the “commitment point” of irreversible damage occurs at variable delays, in the range of tens of minutes, after OGD-induced aSD in submerged cortical slices.

Highlights

Anoxic spreading depolarization has been hypothesized as a terminal event during oxygen– glucose deprivation (OGD) in submerged cortical slices in vitro
In the present study we explored neuronal survival during OGD in slices of the barrel cortex using whole-cell patch clamp recordings and concomitant extracellular recordings of local field potential (LFP), and optical intrinsic signals (OIS) imaging
The main conclusion of our study is that Anoxic spreading depolarization (aSD) is not a terminal event during OGD in submerged cortical slices, and that the “commitment point” of irreversible damage in this model occurs at variable, in the range of tens of minutes, delays after aSD

Summary

Introduction

Anoxic spreading depolarization (aSD) has been hypothesized as a terminal event during oxygen– glucose deprivation (OGD) in submerged cortical slices in vitro. Whole-cell recordings from neocortex and hippocampus in vitro showed that even when oxygen and glucose were reintroduced to the brain slice immediately or few minutes after aSD, the membrane potential was irreversibly lost[12,16,18,21] Along with these findings, extracellular recordings of responses evoked by afferents electrical stimulation were permanently lost during OGD even if reperfusion with oxygenated ACSF started shortly after aSD both in hippocampus and neocortex[12,13,20,21,22]. We provide evidence that aSD is not a terminal event during OGD in submerged cortical slices, as previously thought, and that the “commitment point” of irreversible loss of membrane potential and neuronal function occurs at variable and much longer delays, in the range of tens of minutes, after aSD, in the OGD model of ischemia in vitro. Our findings suggest that the OGD model of ischemia in vitro could be useful in further explorations of neuroprotective interventions that could stop the progression of neuronal damage initiated by aSD during the post-aSD period

Methods

Results

Conclusion