Mild sensory stimulation completely protects the adult rodent cortex from ischemic stroke

Abstract

Despite progress in reducing ischemic stroke damage, complete protection remains elusive. Here we demonstrate that, after permanent occlusion of a major cortical artery (middle cerebral artery; MCA), single whisker stimulation can induce complete protection of the adult rat cortex, but only if administered within a critical time window. Animals that receive early treatment are histologically and behaviorally equivalent to healthy controls and have normal neuronal function. Protection of the cortex clearly requires reperfusion to the ischemic area despite permanent occlusion. Using blood flow imaging and other techniques we found evidence of reversed blood flow into MCA branches from an alternate arterial source via collateral vessels (inter-arterial connections), a potential mechanism for reperfusion. These findings suggest that the cortex is capable of extensive blood flow reorganization and more importantly that mild sensory stimulation can provide complete protection from impending stroke given early intervention. Such non-invasive, non-pharmacological intervention has clear translational potential.

Figure 1. Schematic of the Experimental Design with Constituent Experimental and Control Groups.

For all groups (except sham pMCAO controls that never undergo MCA occlusion) the red vertical line indicates time of permanent occlusion of the MCA (pMCAO), the x-axis at bottom demarcates the chronology of events post-occlusion, and the black dotted line indicates the initiation of all second day experimentation. (Note, however, that the behavior animals are examined for the first time at one week post-pMCAO). (A–L) Experimental paradigms and chronology of events are listed for animals undergoing either functional imaging or electrophysiology (M–O). Chronology of events for animals undergoing behavioral assessment one week post-occlusion (P-R). Experimental paradigms and chronology of events are listed for animals undergoing blood flow imaging. N-sizes for each treatment condition are as follows: (A) n = 7; (B) n = 3; (C) n = 7; (D) n = 8; (E) n = 7; (F) n = 5; (G) n = 2; (H) n = 3; (I) n = 3; (J) n = 3; (K) n = 3;(L) n = 21 (M) n = 22; (N) n = 22; (O) n = 22; (P) n = 13; (Q) n = 6;(R) n = 6.

Figure 2. Experimental Infarct Volumes.

The total volume of infarct tissue sustained by animals which underwent: pMCAO (no-stimulation control animals, n = 10), pMCAO and immediate whisker stimulation (+0 h, n = 38), or pMCAO and whisker stimulation initiated three hour post-occlusion (+3 h, n = 38) as assessed via 2,3,5-Triphenyltetrazolium Chloride (TTC) assay for infarct. No-stimulation control animals never received whisker stimulation, yet underwent identical anesthetic and surgical procedures (including pMCAO) as +0 h and +3 h groups, were only assessed using TTC. Smaller subdivisions of the larger experimental +0 h and +3 h groups were assessed using various other techniques as fully described in the remainder of this article. Asterisk indicates a significant difference in infarct volume between the no-stimulation controls and +3 h subjects. Note that multiple groups did not sustain infarct are included here for the sake of comparison.

Figure 3. +0 h animals maintained whisker functional representation and sustained no infarct.

A: Schematic of the ISOI experiments. The vertical arrow denotes manipulations which were performed between the two functional imaging sessions. B, left half: +3 h and +0 h subjects' images of the ipsi-ischemic C2 whisker functional representation collected before and 24 hours after pMCAO. Scale bar indicates 5 mm. Linear grayscale bar indicates intrinsic signal strength ×10⁻⁴. Black and white streaks correspond to large surface blood vessels. B, right half: Representative coronal sections taken from experimental and control animal's TTC assay for infarct. Note that the area devoid of staining (arrows) within the +3 h subject's and control subject's left cortex indicate ischemic infarct due to an occlusion of the left MCA. +0 h no stimulation controls and +3 h no stimulation control rats were anesthetized for the same duration as their respective experimental group and underwent the same pMCAO surgical procedure, but never received whisker stimulation. Scale bar indicates 5 mm.

Figure 4. +0 h animals maintained baseline level (or stronger) whisker representation and sustained no infarct.

A: In each plot, group baseline is paired with +24 hour data. Means and standard errors are provided for the area (first row) and amplitude (second row) of the ipsi-ischemic C2 whisker functional representation before and 24 hours after pMCAO. A value of zero indicates no response to whisker stimulation. Asterisks indicate significant differences between baseline and 24 hour values (** p = 0.0086, **** p = 0.00082, ***** p = 0.000001). B, C: Location and extent of infarct due to ischemia present in the +3h and +0h experimental groups (B), and +3h and +0h No-stimulation controls (C) according to TTC analysis. Color coding indicates area of infarct common to all subjects (dark blue), and maximum observed infarct (medium blue). Atlas legend includes six vertical lines which correspond to each coronal section at 2 mm intervals from bregma. Brain map adapted from .

Figure 5. Normal evoked neuronal activity was maintained in all +0 h animals.

(A) Representative +3 h, +0 h, and sham pMCAO control subjects' local field potential (LFP, first row) and multi-unit activity (MUA, second row) responses 24 hours following pMCAO. Stepping function indicates stimulus delivery. Representative coronal section taken from experimental and control animal's 2,3,5-Triphenyltetrazolium Chloride (TTC) assay for infarct. Area devoid of staining (arrows) within the +3 h subject's left cortex is indicative of ischemic infarct due to an occlusion of the left MCA. Scale bar indicates 5 mm. (B) LFP (left) and MUA (right) normal distributions based on sham pMCAO control data are plotted as shaded curves. Superimposed on these distributions are the individual values of +0 h subjects (blue circles) and +3 h subjects (red circles). Provided below each graph are the z-scores (as derived in relation to the normal distribution of sham pMCAO controls), and corresponding p-values.

Figure 6. Normal sensorimotor-related behaviors were maintained in +0 h animals.

All +3 h animals (n = 22) exhibited behavioral deficits 7 days after the onset of ischemia (time of pMCAO), while +0 h animals (n = 22) were indistinguishable from controls (animals which underwent surgical preparation with no MCA occlusion, n = 22). Asterisks indicate significant differences between bracketed groups and N.S. indicates no difference between bracketed groups. (A) Neurological scores according to the Bederson Scale. Each color portion of each bar represents the percentage of animals within each group with the corresponding neurological score (see color key). (B) Use of left (ipsi-ischemic) versus right (contra-ischemic) forelimbs expressed as an asymmetry score during forepaw guided exploration. Forepaw asymmetry was calculated as total number of explorations initiated with the right forepaw minus total number of explorations initiated with the left forepaw. Horizontal line indicates ‘0’, or no asymmetry. (All three spent equivalent amounts of time exploring with their paws). (C) Use of left side (ipsi-ischemic) versus right side (contra-ischemic) whisker sets expressed as whisker asymmetry during whisker guided exploration. Whisker asymmetry was calculated as total time spent scanning with the right whisker set minus total time spent scanning with the left whisker set. Both +0 h and controls used their left and right whisker sets equally. Horizontal line indicates ‘0’, or no asymmetry. All three groups explored with their whiskers for equivalent amounts of time.

Figure 7. Distal occlusion experiments demonstrate that patent distal branches of MCA are necessary for protective plasticity.

(A) Schematics of relative positions of barrel cortex, MCA, and occlusion(s) for +0 h (n = 7; left), +0 h distal (n = 3; middle), +0 h sham distal control (n = 3; right) groups. All three groups underwent identical protocols aside from the additional occlusions of distal branches of MCA performed in the +0 h distal group, and unsecured sutures over the same distal MCA branches in the +0 h sham distal control group. (B, C): Graphs Baseline and 24 hour means and standard errors for area (B) and amplitude (C) of ipsi-ischemic whisker C2 representation for +0 h group, and individual values for +0 h distal and +0 h sham distal control animals. (D): Below each set of graphs is a representative TTC stained coronal slice for each group. The area devoid of staining (arrows) within the +0 h distal subject's left cortex is indicative of ischemic infarct due to an occlusion of the left MCA. Scale bar indicates 5 mm.

Figure 8. LSI experiments demonstrate that blood flow in MCA is re-established on the following day if whisker stimulation is administered immediately post-pMCAO.

Insets, Representative linearly color scaled LSI images taken at (A) pre-pMCAO baseline and immediately post-pMCAO, or (B) 24 hours post-pMCAO for an animal from each of +0 h and +3 h groups. Scale bar indicates 0.25 mm. Graphs, the x-axis crosses the y at the mean noise level, or, ‘no flow’ level as determined by averaging values recorded from a group of euthanized animals (n = 6) at approximately 5 minutes after cessation of heart beat (MCA was thus no longer flowing in these animals hence the designation ‘no flow’ for equivalent values). To establish flow values in the absence of flow (noise) we collected data from a group of euthanized animals 5 minutes after the cessation of heart beat. We analyzed an MCA ROI as we had in live subjects and determined an average noise or ‘no-flow’ value (106,595±12,481SE). (A): Inset, note the large reduction in MCA flow. Graph, means and standard errors for MCA flow at pre-pMCAO baseline and immediately post-pMCAO. * indicates a significant difference between baseline and immediately post-pMCAO flow values (B): Inset, note the minimal blood flow apparent in the +3 h subject and the substantial blood flow in the +0 h subject despite the fact that the same double suture and complete transection of MCA's base was performed in both animals 24 hours prior. Graph, means and standard errors for MCA in +3 h and +0 h subjects 24 hours post-pMCAO. Note that the minimal flow values in +3 h group animals at 24 hours post-pMCAO are similar to levels seen immediately post-pMCAO in animals in panel A and that the flow values in +0 h animals are similar to baseline flow levels in panel A. * indicates a significant difference between +0 h and +3 h flow values at 24 hours post-pMCAO (*p = 0.000005). Note that pMCAO was performed by tightening two suture knots around the base of MCA and then completely transecting the artery between these two knots.