Dibucaine mitigates spreading depolarization in human neocortical slices and prevents acute dendritic injury in the ischemic rodent neocortex

Abstract

Background: Spreading depolarizations that occur in patients with malignant stroke, subarachnoid/intracranial hemorrhage, and traumatic brain injury are known to facilitate neuronal damage in metabolically compromised brain tissue. The dramatic failure of brain ion homeostasis caused by propagating spreading depolarizations results in neuronal and astroglial swelling. In essence, swelling is the initial response and a sign of the acute neuronal injury that follows if energy deprivation is maintained. Choosing spreading depolarizations as a target for therapeutic intervention, we have used human brain slices and in vivo real-time two-photon laser scanning microscopy in the mouse neocortex to study potentially useful therapeutics against spreading depolarization-induced injury.

Methodology/principal findings: We have shown that anoxic or terminal depolarization, a spreading depolarization wave ignited in the ischemic core where neurons cannot repolarize, can be evoked in human slices from pediatric brains during simulated ischemia induced by oxygen/glucose deprivation or by exposure to ouabain. Changes in light transmittance (LT) tracked terminal depolarization in time and space. Though spreading depolarizations are notoriously difficult to block, terminal depolarization onset was delayed by dibucaine, a local amide anesthetic and sodium channel blocker. Remarkably, the occurrence of ouabain-induced terminal depolarization was delayed at a concentration of 1 µM that preserves synaptic function. Moreover, in vivo two-photon imaging in the penumbra revealed that, though spreading depolarizations did still occur, spreading depolarization-induced dendritic injury was inhibited by dibucaine administered intravenously at 2.5 mg/kg in a mouse stroke model.

Conclusions/significance: Dibucaine mitigated the effects of spreading depolarization at a concentration that could be well-tolerated therapeutically. Hence, dibucaine is a promising candidate to protect the brain from ischemic injury with an approach that does not rely on the complete abolishment of spreading depolarizations.

Figure 1. Global monitoring of terminal depolarization (TD) in human and mouse brain slices during dibucaine exposure.

A, A slice is transilluminated and transmitted light is collected by CCD camera to create the digital bright field image (left). Following OGD at 0 min, the gray matter displays a propagating wave of elevated LT (signifying terminal depolarization), represented by pseudocoloring according to changes in pixel values of the image (color scale: below, right). In this example, terminal depolarization is ignited within 13 minutes of OGD and spreads in all directions (arrows). Scale bar, 500 µm. B, fEPSPs recorded from CA1 of st. radiatum in mouse hippocampal slices are not noticeably affected by 1 hour of superfusion with ACSF containing 1 µM dibucaine. 10 µM dibucaine partially inhibited the evoked synaptic response, while 100 µM dibucaine completely abolished synaptic activity. Each row represents traces recorded from a single slice. C, Summary of synaptic responses during dibucaine exposure. Following 1 hour of control recording in standard ACSF, slices were superfused with ACSF containing 1 µM (left panel; n = 3 slices) or 10 µM dibucaine (right panel; n = 3 slices). At the end of 1 hour treatment with 1 µM dibucaine, ∼82% of the control fEPSP slope was preserved; 10 µM dibucaine reduced the fEPSP slope to ∼14% of control values (p<0.0001 for both; one-way ANOVA). D, Input/output graphs showing mean fEPSP slope at various stimulus intensities before and after 1 hour treatment with either 1 µM (left) or 10 µM (right) dibucaine (n = 3 slices for each concentration; *p<0.05, **p<0.001; two-way ANOVA).

Figure 2. Dibucaine pretreatment delays terminal depolarization in human neocortical slices.

A, Changes in LT (ΔLT) are utilized to track terminal depolarization in real time. The first image in each row shows bright field image of the slice. A control untreated slice incubated in standard ACSF (top row) undergoes terminal depolarization within 5 minutes of superfusion with 100 µM ouabain. Pseudocolored elevated ΔLT indicates cell swelling. In slices pretreated with either 1 or 10 µM dibucaine for 60 min (middle and bottom rows, respectively), ouabain-induced terminal depolarization is delayed by 2.1 and 4.8 min respectively and has less impact in terms of cell swelling. All three slices were from the same patient (#7; see Patient Table S1). Scale bar, 1 mm. B, Pretreatment for 1 hour with dibucaine significantly increases the latency to terminal depolarization onset induced by 100 µM ouabain or OGD when compared to untreated control slices from the same patient. C, Peak cell swelling during terminal depolarization in the same slices pretreated with dibucaine. The neuroprotective anti-inflammatory drug minocycline had no effect on terminal depolarization. Numbers of slices in each condition are indicated within each bar. Values are shown as percent of control (untreated slices from the same patient). Asterisks indicate significant differences from control (*p<0.05, **p<0.001, two-way ANOVA).

Figure 3. Dibucaine lessens dendritic beading induced by spontaneous spreading depolarizations (SDs) in penumbra.

A, In vivo 2PLSM image sequence of dendrites (green) and a nearby flowing blood vessel (red; blood flow is indicated by stripes within vessel) from intact mouse somatosensory cortex. The images were taken from the center of the penumbra-like “area at risk”, surrounded by partially occluded blood vessels . The dendrites bead (asterisks) during the initial spreading depolarization induced by cortical photothrombosis. The dendrites rapidly (<3 min) recover but become beaded again by the next spontaneous spreading depolarization even in the presence of a flowing blood vessel before recovering once more. Scale bar, 20 µm. B, Similar sequence to that shown in A, except dibucaine (2.5 mg/kg) was injected into the tail vein immediately after the onset of induced spreading depolarization. As in A, the dendrite rapidly recovers from beading (asterisks) induced by the initial spreading depolarization, but unlike the dendrites shown in A, the dendrite in the dibucaine-treated animal does not bead during a spontaneous spreading depolarization. Scale bar, 20 µm. C, Summary showing significantly decreased occurrence of dendritic beading (*p<0.05; Chi-Square Test) when dibucaine is administered i.v. immediately following onset of the initial spreading depolarization. D, Bead density during spontaneous spreading depolarizations is significantly decreased with dibucaine while E, spine density remains unaffected. Densities were assessed from random 30 µm segments of 3 dendrites in each imaging field as observed in maximum intensity projections of three-dimensional stacks in 5 control and 8 dibucaine treated mice. Data points (representing averages taken from individual dendrites) were only used when an image was taken near the peak of a recorded spreading depolarization. Values are shown as percentages of bead (D) or spine (E) density immediately after induced spreading depolarization (*p<0.05; two-way RM ANOVA).