The biological effect of contralateral forepaw stimulation in rat focal cerebral ischemia: a multispectral optical imaging study

Abstract

Our group has already published the possible neuroprotective effect of contralateral forepaw stimulation in temporary focal ischemia in a study. However, the background is still unclear. In the present study we investigated the possible mechanism by monitoring focal ischemia with multispectral [laser speckle, imaging of intrinsic signals (OIS)] imaging. Sprague-Dawley rats were prepared using 1.2% isoflurane anesthesia. The middle cerebral artery was occluded by photothrombosis (4 mW) and the common carotid artery was ligated permanently. Physiological variables were constantly monitored during the experiment. A 6 x 6 mm area centered 3 mm posterior and 4 mm lateral to Bregma was thinned for laser speckle and OIS imaging. Nine circular regions of interests (0.3 mm in diameter) were evenly spaced on the speckle contrast image for the analysis of peri-infarct flow transients, blood flow, and metabolic changes. Both the sham (n = 7) and forepaw-stimulated animals (n = 7) underwent neurological examinations 24 h after ischemia at which point all animals were sacrificed and the infarct size was determined by triphenyltetrazolium chloride. The physiological variables were in normal range and the experimental protocol did not cause significant differences between groups. Both the neurological scores (sham: 3.6 +/- 1.7, stimulated: 4.3 +/- 1.4) and the infarct volume (sham: 124 +/- 39 mm(3), stimulated: 147 +/- 47 mm(3)) did not show significant differences between groups. The forepaw stimulation did not increase the intra-ischemic flow neither over the penumbral or the peri-ischemic area. However, the hemoglobin transients related metabolic load (CMRO(2)) was significantly lower (p < 0.001) while the averaged number of hyperemic flow transients were significantly (p = 0.013) higher in the forepaw (sham: 3.5 +/- 2.2, stimulated: 7.0 +/- 2.3) stimulated animals.

Keywords: OIS; flow transients; focal cerebral ischemia; forepaw stimulation; middle cerebral artery occlusion; optical imaging; photothrombosis; speckle contrast.

Figure 1

Schematic figure of the thinned skull with the positions of the nine regions of interests (ROI). The shadowing (white line) shows the border zone of the lesion in both groups of animals (no significant difference) after mapping the infarct area with the map of ROIs. The red ellipse indicates the cortical representation of the forepaw as determined by blood oxygen-level dependent imaging (Ghosh et al., 2009) and by OIS in our laboratory.

Figure 2

Schematic of study design. Ischemia was produced by direct occlusion of the right middle cerebral artery (MCA) by photothrombosis along with right common carotid artery occlusion (rCCAO), and optical imaging was done during the acute phase of ischemia.

Figure 3

Demonstration of flow transients in relative cerebral blood flow (CBF/CBF_o) in peri-ischemia regions. (A, B) and in the ischemic core (C, D) from a typical rat not receiving forepaw stimulation during ischemia (A, C) and from a rat receiving forepaw stimulation during ischemia (B, D). The “o” subscript refers to the parameter prior to MCA occlusion. Note the significant flow transients in the peri-ischemic territory (ROIs 7–9), whereas the occurrence of the transients over the ischemic core (ROIs 1-3) was very low.

Figure 4

(A) Maps of changes in relative blood flow (rCBF), relative oxyhemoglobin (rHbO), relative deoxyhemoglobin (rHb), and relative oxygen consumption (rCMRO₂) during a transient in the peri-ischemia cortex of a exemplar control (non-stimulated) and forepaw-stimulated animal. In both animals, the transient is moving toward the right of the image (caudally). The color bar indicates fractional decreases or increases of each parameter with respect to its level just prior to the start of the transient. The time course of the parameters for the region of interest shown by the circular ROI in the rHb images is shown for the control (B) and stimulated (C) animal, with the images shown in (A) obtained at the time indicated by the arrows. Scale bar is 1 mm.