Optical imaging of disrupted functional connectivity following ischemic stroke in mice

Abstract

Recent human neuroimaging studies indicate that spontaneous fluctuations in neural activity, as measured by functional connectivity magnetic resonance imaging (fcMRI), are significantly affected following stroke. Disrupted functional connectivity is associated with behavioral deficits and has been linked to long-term recovery potential. FcMRI studies of stroke in rats have generally produced similar findings, although subacute cortical reorganization following focal ischemia appears to be more rapid than in humans. Similar studies in mice have not been published, most likely because fMRI in the small mouse brain is technically challenging. Extending functional connectivity methods to mouse models of stroke could provide a valuable tool for understanding the link between molecular mechanisms of stroke repair and human fcMRI findings at the system level. We applied functional connectivity optical intrinsic signal imaging (fcOIS) to mice before and 72 h after transient middle cerebral artery occlusion (tMCAO) to examine how graded ischemic injury affects the relationship between functional connectivity and infarct volume, stimulus-induced response, and behavior. Regional changes in functional connectivity within the MCA territory were largely proportional to infarct volume. However, subcortical damage affected functional connectivity in the somatosensory cortex as much as larger infarcts of cortex and subcortex. The extent of injury correlated with cortical activations following electrical stimulation of the affected forelimb and with functional connectivity in the somatosensory cortex. Regional homotopic functional connectivity in motor cortex correlated with behavioral deficits measured using an adhesive patch removal test. Spontaneous hemodynamic activity within the infarct exhibited altered temporal and spectral features in comparison to intact tissue; failing to account for these regional differences significantly affected apparent post-stroke functional connectivity measures. Thus, several results were strongly dependent on how the resting-state data were processed. Specifically, global signal regression alone resulted in apparently distorted functional connectivity measures in the intact hemisphere. These distortions were corrected by regressing out multiple sources of variance, as performed in human fcMRI. We conclude that fcOIS provides a sensitive imaging modality in the murine stroke model; however, it is necessary to properly account for altered hemodynamics in injured brain to obtain accurate measures of functional connectivity.

Keywords: Functional connectivity; Functional recovery; Global signal regression; Mice; Stroke.

Figure 1. Infarct volume characterization

Infarct volumes were sorted in ascending order and mice were grouped according to infarct location and volume. (A) group 1: mice with only subcortical infarcts (N=14, infarct volume ranging from 7.3 mm³ to 54.3 mm³); group 2: mice with infarcts involving subcortex and small lateral portions of cortex (N=20, infarct volume ranging from 32.9 mm³ to 71.6 mm³); group 3: mice with infarcts involving subcortex and large portions of cortex (N=12, infarct volume ranging from 90 mm³ to 169.3 mm³). (B) Direct infarct volume quantification, mean +/− S.D., # = p<0.00001. (C) Representative coronal TTC slices, and (D) image histological atlas assignments of regions of interest with rearranged brain slices depicting infarct locations within the image field.

Figure 2. Functional connectivity maps for seeds placed in the right (unaffected) hemisphere

Group-averaged functional connectivity patterns in mice before (control) and 72 hours after tMCAO using HbO. Functional connectivity maps for seeds in right (unaffected) olfactory, somatosensory, motor, retrosplenial, and visual cortices (black circles) exhibit changes in connectivity patterns commensurate with injury, indicating a loss of temporal synchrony between brain networks. Note that connectivity patterns of regions within the MCA territory (motor, somatosensory, visual) are more affected than perilesional regions (retrosplenial) and those far from the insult (olfactory).

Figure 3. Functional connectivity maps for seeds placed in the left (affected) hemisphere

Group-averaged functional connectivity patterns (calculated using HbO) in mice before (control) and 72 hours after tMCAO. Functional connectivity maps for seeds in left (affected) hemisphere for olfactory, somatosensory, motor, retrosplenial, and visual cortices (black circles) exhibit changes in connectivity patterns commensurate with injury. Note that the seed placed in left somatosensory cortex is effectively in the ischemic core and shows a near lack of correlation with any part of the brain as one would expect from dead tissue.

Figure 4. Homotopic functional connectivity within the ipsilesional hemisphere

Group-averaged correlation maps of interhemispheric homotopic functional connectivity for all pixels within our field of view reveal regional differences in observed connectivity after focal ischemia that depend on stroke severity. Note the overall trend that the brain becomes gradually less connected (connectivity goes to zero) as the injury becomes larger. Functional connectivity values are Fisher z-transformed Pearson correlations.

Figure 5. Functional connectivity vs. degree of injury

Post-stroke homotopic functional connectivity correlated strongly with infarct volume in motor and retrosplenial cortices. Homotopic functional connectivity in somatosensory and visual regions, and left intrahemispheric somatomotor functional connectivity was much more uniformly depressed in mice with infarcts of all sizes. Homotopic functional connectivity in olfactory cortex was nearly unaffected by infarcts and maintained values similar to those in pre-stroke mice. Functional connectivity values are Fisher z-transformed Pearson correlations. Control mice are not included in this figure. See Figure 9A for comparison of infarcted mice vs. controls.”

Figure 6. Reduced activations in affected forepaw

Cortical activation maps for electrical forelimb stimulation of left (unaffected) forepaw (top row) and right (injured) forepaw (bottom row). The right limb exhibits reduced response amplitude as a function of stroke severity (see Fig. S3), while the induced response in the unaffected left limb is similar to the control group. Pixels having a response amplitude within 75% of maximum response are overlaid on a representative white light image of the brain.

Figure 7. Sensorimotor performance in affected limb is significantly correlated with homotopic functional connectivity in motor and retrosplenial cortices

(A) During the 5 days before tMCAO, all mice were trained to remove pieces of adhesive tape from the right and lefts paws within 5 seconds. Post-stroke removal times (which represent the average adhesive removal times at 48 and 72 hrs.) are incrementally disrupted, mean +/− S.D., * = p<0.05, ** = p<0.01 calculated using an unpaired, one tailed t-test and (B) strongly correlate with functional connectivity in motor and retrosplenial cortex. Functional connectivity values are Fisher z-transformed Pearson correlations.

Figure 8. Altered hemodynamics and homotopic temporal coherence following ischemic stroke

(A) Representative segmentation for mice with cortical infarcts. All pixel time traces within infarct or non-infarct regions were averaged to create the two regressors in Eqn. (6) (B) Spontaneous activity occurring within the infarct (red) and non-infarct (black) tissue exhibits marked differences in hemodynamic fluctuations (representative traces from mice in Group 3). (C) Power spectra of spontaneous activity within infarct (red) tissue for all mice in Group 3 show significant attenuation in all frequencies above 0.03 Hz compared with non-infarct tissue (black). (D) Time shifts in the ipsilesional hemisphere were estimated by cross-correlating every pixel in the left hemisphere with its contralateral homologue and measuring the time shift associated with peak correlation. Across all three groups, time-to-peak correlation between homotopic brain pixels is gradually delayed within the MCA territory and surrounding areas.

Figure 9. Global signal regression overestimates functional connectivity after stroke

Seed-to-seed homotopic connectivity in olfactory, somatosensory, motor, retrosplenial, and visual cortices and intrahemispheric connectivity between somatosensory and motor cortices in the right and left hemisphere for (A) multiple signal and (B) global signal regression methods quantified as Fisher Z scores. With increasing stroke severity, homotopic functional connectivity switches sign using global regression and increases, but decreases using multiple signal regression. Intrahemispheric somatomotor connectivity does not increase with larger infarcts in either hemisphere using multiple signal regression. Values represent mean +/− S.D., * = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001; # = p<0.00001; n.s.=not significant. Functional connectivity values are Fisher z-transformed Pearson correlations.

Figure 10. Measured functional connectivity following ischemic stroke depends on regression method (seeds in affected hemisphere)

Functional connectivity patterns for seeds (black circles) in left (affected) hemisphere show graded decline in inter- and intra-hemispheric functional connectivity using multiple signal regression (MSR). Global signal regression (GSR) results in the largest changes in connectivity, with contralateral regions becoming increasingly anticorrelated in groups 2 and 3. This increased connectivity (both positive and negative correlations) following GSR is a result of the temporal delay between infarct and non-infarct tissue and is an artifact. Note with MSR that correlations with dead tissue (seed in left somatosensory cortex) approach zero as one might expect.