Mild sensory stimulation reestablishes cortical function during the acute phase of ischemia

Abstract

When delivered within 1 and in most cases 2 h of permanent middle cerebral artery occlusion (pMCAO), mild sensory stimulation (intermittent single whisker stimulation) was shown to be completely neuroprotective 24 h after pMCAO in a rodent model of ischemic stroke, according to assessment with multiple techniques (Lay et al., 2010). The acute effect of stimulation treatment on the ischemic cortex, however, has yet to be reported. Here we characterize cortical function and perfusion during the 120 min whisker stimulation period in four experimental groups with treatment initiated 0, 1, 2 (protected groups), or 3 h (unprotected group) post-pMCAO using multiple techniques. According to functional imaging, a gradual return of evoked whisker functional representation to baseline levels was initiated with treatment onset and completed within the treatment period. Evoked neuronal activity and reperfusion to the ischemic area also showed a gradual recovery in protected animals. Surprisingly, a similar recovery profile was observed in response to treatment in all protected animals, regardless of treatment onset time. Nonstimulated pMCAO control group data demonstrate that reperfusion is not spontaneous. This makes the complete protection observed in the majority of animals stimulated at 2 h post-pMCAO even more surprising, as these animals recovered despite having been in a severely ischemic state for two full hours. In summary, when delivered within a 2 h window post-pMCAO, whisker stimulation treatment initiated reperfusion and a gradual recovery of cortical function that was completed or nearly completed within the treatment period.

Figure 1.

pMCAO produces irreversible damage in animals that receive treatment 3 h postocclusion (+3 h animals), while return of cortical function is evident in +0 h, +1 h, and +2 h groups during protective stimulation treatment. Data is from representative animals that underwent pMCAO and received whisker stimulation treatment 3 h postocclusion. A, Linearly color-scaled LSI images taken at baseline and following pMCAO in a +3 h animal. The vessel traversing these images is a representative segment of the analyzed MCA ROI in a branch of MCA above somatosensory cortex, distal to pMCAO. Scale bar, 1 mm. B, Intrinsic signal optical imaging was conducted before pMCAO and during the postocclusion treatment period. Whisker functional representation was completely absent throughout the entire postocclusion stimulus period in +3 h animals. Linear grayscale bar indicates intrinsic signal strength ×10⁻⁴. Black and white streaks correspond to large surface blood vessels. Scale bar, 5 mm. C, Coronal section taken from TTC assay for infarct in a +3 h animal. Note that the area devoid of staining (arrow) within the +3 h subject's cortex indicates ischemic infarct due to pMCAO and late stimulation treatment. Scale bar, 5 mm. D, Representative data from intrinsic signal optical imaging of the initial dip for each group (+0 h, +1 h, +2 h, and +3 h) were arranged according to time of treatment initiation for comparison (treatment time is included in white in the top left corner of each image). Groups +0 h, +1 h, and +2 h all regained evoked functional response comparable to baseline after 90 min of whisker stimulation, while +3 h animals never demonstrated any post-pMCAO cortical activity.

Figure 2.

Whisker functional representation returns gradually upon stimulation onset in all protected animals throughout the treatment period. A, B, Group baseline is plotted with 120 min of postocclusion stimulation period data. Means and SEs are provided for the area (left) and amplitude (right) of the ipsi-ischemic C2 initial dip (A) and overshoot (B) phases of whisker functional representation before and after pMCAO. +3 h animals had no response to stimulation at any time point post-pMCAO. For all other groups (sham-pMCAO, +0 h, +1 h, and +2 h), asterisks indicate a significant difference from baseline: *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3.

Vital parameters remain stable throughout the treatment period in protected subjects. In each plot, means and SEs are provided for heart rate, breath rate, arterial saturation, and pulse distension (blood pressure) before and after pMCAO in +0 h subjects (n = 10); all four parameters remained stable throughout the duration of the experiment.

Figure 4.

Contra-ischemic ISOI reveals that changes in cortical function are limited to the ischemic hemisphere. The initial dip and overshoot phases of the contra-ischemic whisker functional representation remain constant throughout experimentation. A, B, Group baseline is paired with postocclusion stimulus data. Means and SEs are provided for the area (left) and amplitude (right) of the contra-ischemic C2 initial dip (A) and overshoot (B) phases of whisker functional representation before and immediately after stimulation treatment. ANOVA confirmed that there were no differences before and after pMCAO occlusion for either the area (+0 h: F_(1,6) = 0.0004, p > 0.05; +1 h: F_(1,6) = 0.078, p > 0.05; +3 h: F_(1,6) = 0.007, p > 0.05) and amplitude (+0 h: F_(1,6) = 0.009, p > 0.05; +1 h: F_(1,6) = 0.275, p > 0.05; +3 h: F_(1,6) = 0.132, p > 0.05) of the initial dip, or the area (+0 h: F_(1,6) = 0.044, p > 0.05; +1 h: F_(1,6) = 0.215, p > 0.05; +3 h: F_(1,6) = 1.252, p > 0.05) and amplitude (+0 h: F_(1,6) = 0.027, p > 0.05; +1 h: F_(1,6) = 0.729, p > 0.05; +3 h: F_(1,6) = 0.912, p > 0.05) of the overshoot phase. FC, Fractional change.

Figure 5.

Evoked neuronal activity was suppressed following pMCAO and reestablished during whisker stimulation treatment in +0 h animals. A, B, Representative +0 h animals' LFP (A) and MUA (B) responses at baseline and during the stimulus period following pMCAO. Stepping function indicates stimulus delivery. C, D, F, Evoked LFP (C), evoked MUA (D), and spontaneous spiking activity (F) mean and SE is plotted preocclusion and postocclusion. Asterisks indicated a significant difference from baseline: **p < 0.01 and ***p < 0.001. E, Representative multiunit raster plot recorded ∼21 min before and after pMCAO (∼21 min are required to collect one full 64 stimulus trial block). x-Axis represents 1000 ms of spontaneous activity, 1000 ms of evoked activity (whisker stimulation indicated by step function), and 500 ms of spontaneous activity. Arrow indicates the approximate point at which evoked MUA responses begin to return.

Figure 6.

LSI experiments demonstrate that post-pMCAO blood flow return in MCA is induced by whisker stimulation treatment. Insets, Representative cases of no-stimulus control and +0 h animals presented as linearly gray-scaled LSI images taken at baseline and following pMCAO during treatment at ∼30 min intervals. Scale bar, 1 mm. The dark vessel diagonally traversing the image in each case is a cortical branch of MCA (most clear in preocclusion baseline images; other vessels such as dural vessels are also visible, but much less apparent than MCA at baseline). Representative segments of MCA branch ROIs above the region of somatosensory cortex, distal to pMCAO are shown [(for an in depth description regarding criteria for MCA regions of interest analyzed, see the Methods section of Lay et al. (2010)]. Means and SEs for MCA for no-stimulus and +0 h animals at baseline, following pMCAO, and during whisker stimulation. **p < 0.01, significant increase in +0 h flow compared with the no-stimulus control group.