Comparative analysis of spreading depolarizations in brain slices exposed to osmotic or metabolic stress

Abstract

Background Recurrent spreading depolarizations (SDs) occur in stroke and traumatic brain injury and are considered as a hallmark of injury progression. The complexity of conditions associated with SD in the living brain encouraged researchers to study SD in live brain slice preparations, yet methodological differences among laboratories complicate integrative data interpretation. Here we provide a comparative evaluation of SD evolution in live brain slices, in response to selected SD triggers and in various media, under otherwise standardized experimental conditions. Methods Rat live coronal brain slices (350 μm) were prepared (n = 51). Hypo-osmotic medium (Na+ content reduced from 130 to 60 mM, HM) or oxygen-glucose deprivation (OGD) were applied to cause osmotic or ischemic challenge. Brain slices superfused with artificial cerebrospinal fluid (aCSF) served as control. SDs were evoked in the control condition with pressure injection of KCl or electric stimulation. Local field potential (LFP) was recorded via an intracortical glass capillary electrode, or intrinsic optical signal imaging was conducted at white light illumination to characterize SDs. TTC and hematoxylin-eosin staining were used to assess tissue damage. Results Severe osmotic stress or OGD provoked a spontaneous SD. In contrast with SDs triggered in aCSF, these spontaneous depolarizations were characterized by incomplete repolarization and prolonged duration. Further, cortical SDs under HM or OGD propagated over the entire cortex and occassionally invaded the striatum, while SDs in aCSF covered a significantly smaller cortical area before coming to a halt, and never spread to the striatum. SDs in HM displayed the greatest amplitude and the most rapid propagation velocity. Finally, spontaneous SD in HM and especially under OGD was followed by tissue injury. Conclusions While the failure of Na+/K+ ATP-ase is thought to impair tissue recovery from OGD-related SD, the tissue swelling-related hyper excitability and the exhaustion of astrocyte buffering capacity are suggested to promote SD evolution under osmotic stress. In contrast with OGD, SD propagating under hypo-osmotic condition is not terminal, yet it is associated with irreversible tissue injury. Further investigation is required to understand the mechanistic similarities or differences between the evolution of SDs spontaneously occurring in HM and under OGD.

Malignant astrocyte swelling and impaired glutamate clearance drive the expansion of injurious spreading depolarization foci

“…The ex vivo brain slice preparations here were exposed to severe osmotic stress achieved by the lowering of [Na + ] of the incubation medium to 100 or 60 mM. 46 This was considered as a model system to study the mechanisms of SiD, rather than the faithful reproduction of a complex disease state. There is no proof that osmotic stress of such severity may occur focused to a discrete site of injury in the human brain.…”

New Insights and Methods for Recording and Imaging Spontaneous Spreading Depolarizations and Seizure-Like Events in Mouse Hippocampal Slices

“…Use of 0 Mg 2+ /5 K + aCSF is also useful as a comparison to osmotic stress or oxygen and glucose deprivation. A very comprehensive recent study compared osmotic stress, oxygen/glucose deprivation, and focal methods using normal aCSF (Frank et al, 2021). Both osmotic stress and oxygen/glucose deprivation led to spontaneous SD, and SD had incomplete recovery and injury compared to SD induced by focal methods in normal aCSF.…”

The evolution of spreading depolarization in response to osmotic or ischemic challenge in rat brain slices

“…Individual slices were placed in an interface type recording tissue chamber (Brain Slice Chamber BSC1, Scientific Systems Design Inc., Ontario, Canada) and continuously perfused with carbogenated aCSF at a rate of 2.5 ml/min. Chamber temperature was kept at 32°C using a dedicated proportional temperature controller unit (PTC03, Scientific Systems Design Inc., Ontario, Canada) (61,65) .…”

“…Brain slices were exposed to severe osmotic stress achieved by the lowering of [Na + ] of the incubation medium to 100 or 60 mM (65) . SiD in our study occurred invariably superimposed on tissue edema, a condition to affect astrocytes first.…”