Ketamine reduces deleterious consequences of spreading depolarizations

Abstract

Recent work has implicated spreading depolarization (SD) as a key contributor the progression of acute brain injuries, however development of interventions selectively targeting SD has lagged behind. Initial clinical intervention efforts have focused on observations that relatively high doses of the sedative agent ketamine can completely suppress SD. However, blocking propagation of SD could theoretically prevent beneficial effects of SD in surrounding brain regions. Selective targeting of deleterious consequences of SD (rather than abolition) could be a useful adjunct approach, and be achieved with lower ketamine concentrations. We utilized a brain slice model to test whether deleterious consequences of SD could be prevented by ketamine, using concentrations that did not prevent the initiation and propagation of SD. Studies were conducted using murine brain slices, with focal KCl as an SD stimulus. Consequences of SD were assessed with electrophysiological and imaging measures of ionic and synaptic recovery. Under control conditions, ketamine (up to 30 μM) did not prevent SD, but significantly reduced neuronal Ca²⁺ loading and the duration of associated extracellular potential shifts. Recovery of postsynaptic potentials after SD was also significantly accelerated. When SD was evoked on a background of mild metabolic compromise, neuronal recovery was substantially impaired. Under compromised conditions, the same concentrations of ketamine reduced ionic and metabolic loading during SD, sufficient to preserve functional recovery after repetitive SDs. These results suggest that lower concentrations of ketamine could be utilized to prevent damaging consequences of SD, while not blocking them outright and thereby preserving potentially protective effects of SD.

Keywords: Brain slice; Calcium loading; Excitatory postsynaptic potentials; Excitotoxicity; Metabolic compromise; NMDA receptor; Neuronal injury; Spreading depression.

Figure 1.. Basal extracellular K⁺ influences sensitivity to ketamine.

A: Representative intrinsic optical signals showing SD propagation through the hippocampal CA1 region of a brain slice. SD was triggered by micro injection of KCl from a micropipette on the left (labeled “KCl”) and SD is visualized as a slowly-propagating wave of increased light transmission. The location of the advancing wavefront is marked by white arrowheads and a second microelectrode (labeled “DC”) was used to confirm electrical responses of SD coincident with arrival of the optical signal (not shown). The upper right-hand values indicate time, in seconds, relative to the triggering of the KCl stimulus pulse. B: Effect of ketamine exposures on SD incidence under two different recording conditions (3mM vs 8mM bathing K⁺). Ketamine more potently prevented SD incidence in the lower basal K⁺ recording conditions. Values in parenthesis indicate number of preparations.

Figure 2.. Ketamine reduced SD propagation rate and DC shift duration.

A: Representative example of effect of ketamine wash-in, during a series of repetitive SDs. Top panels show intrinsic optical signal changes to track SD propagation (as described in Figure 1) in control conditions (left), and during the third SD evoked in the presence of 30 μM ketamine (right). Note the delayed propagation of the SD wavefront in ketamine. The traces show DC potential recordings during this sequence of SDs in the same slice. Black arrowheads indicate DC shift onset, and dashed lines represent 15 minutes recovery between stimulations. The duration of the DC shift of SD was progressively reduced, and then recovered after ketamine washout. B: Summary data from 9 preparations as shown in A, demonstrating progressive decreases in both propagation rate and DC duration with partial recovery upon ketamine wash out in 6 experiments (gray bar). *P<.05, **P<0.01, ***P<0.001, **** P<0.0001.

Figure 3.. Ketamine reduces neuronal intracellular Ca²⁺ accumulation during SD.

A: Top left panel: Transmitted light image, showing stratum oriens (so), stratum pyramidale (sp), stratum radiatum (sr) in area CA1. Pseudo colored images show GCaMP5G fluorescence collected during SD in control conditions. Numbers in each frame indicate time (in seconds) in relation to peak Ca²⁺ during SD, and black circles are regions of interest surrounding predominately pyramidal cell bodies or dendrites in stratum pyramidale (ROI_sp )or radiatum (ROI_sr), respectively. Scale bar = 100μm. B: Data extracted from ROI_sp and ROI_sr show that Ca²⁺ transients during SD in ketamine (blue) recover faster than control (black), and are reversible after ketamine wash out (dashed). Black arrowheads indicate SD onset. C: Summary data (n=5), show that ketamine reversibly reduces total neuronal Ca²⁺ accumulation in both ROIs (integrals of 120s transients; see Methods). *P<0.05, **P<0.01

Figure 4.. Ketamine accelerates recovery of evoked postsynaptic potentials after SD.

A: Representative example of suppression and recovery of evoked excitatory postsynaptic potentials (EPSPs) after SD. Control EPSPs (a) were abolished after SD (b,c), and slowly recovered to baseline amplitudes after ~12 min (d). The asterisk above control (a) trace indicates the bipolar stimulus artifact. The full time course of EPSP suppression and recovery in this same slice is plotted below (black circles). SD onset indicated by black arrowhead. Ketamine (30 μM) did not prevent EPSP suppression, but significantly accelerated recovery rate (lower set of traces, and white circles in plot). B: Summary data from 6 such experiments. The effect of ketamine on DC shift duration in this data set was consistent with prior observations in Figure 2 (45.9 ± 2.3 vs. 35.7 ± 1.1 s for control and ketamine, respectively; P = 0.0026). *** P<0.001

Figure 5.. Ketamine improves recovery of neuronal Ca²⁺ loading and promotes functional recovery after SD in vulnerable brain slices.

A: Top montage: GCaMP5G imaging in vulnerable brain slices show considerably prolonged Ca²⁺ elevations compared to control conditions (compare with Figure 3). Lower montage shows reduced intracellular Ca²⁺ after SD in ketamine. Scale bar =100μm. B: Plots show Ca²⁺ transients from pyramidal cell bodies and dendrites (ROI_sp and ROI_sr, white circles in A) during SD in control (black), vulnerable (red), and vulnerable + ketamine (dashed). Summary data of Ca²⁺ transient integrals (200s after SD) from a set of such experiments confirm beneficial effects of ketamine (control, n=8; vulnerable, n=6; vulnerable + ketamine, n=6). C: Data from experiments in B, showing vulnerable slices exposed to ketamine recovered the ability to generate a second SD. Values in parentheses indicate number of preparations. D: Summary data of EPSP amplitude suppression and recovery after SD in control (black, n=5), vulnerable (red, n=10), and vulnerable with ketamine (white, n=9). Black arrowhead indicates SD onset and loss of postsynaptic responses. *P<0.05, **P<0.01,***P<0.001, ****P<0.0001.

Figure 6.. Ketamine protects against IOS decreases after SD in vulnerable slices.

A: Left hand panel shows the arrangement of recording electrodes on a transmitted light image. The white box outlines the imaging area shown in panels on the right; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; LM, stratum lacunosum moleculare; M, molecular layer of dentate gyrus. The set of images on the right show representative intrinsic optical (IOS, light transmittance) changes at baseline, during SD, and 10 minutes after SD in brain slices recorded under three different conditions: control, vulnerable, and vulnerable + ketamine. Note the difference in IOS seen at the 10-minute time point after SD. White arrowheads indicate the wave front of SD. Scale bar = 250 μm. B: Traces (left) show signals extracted from regions in stratum radiatum (dotted white box in A) during the three representative experiments shown in A. Summary data (n= 6–7, right hand panel) confirm substantially decreased light transmittance 10 minutes after the SD wavefront in vulnerable slices, and prevention by ketamine. ****P<0.0001