Optogenetic Functional Activation Is Detrimental During Acute Ischemic Stroke in Mice

Abstract

Background: Functional activation of the focal ischemic brain has been reported to improve outcomes by augmenting collateral blood flow. However, functional activation also increases metabolic demand and might thereby worsen outcomes. Indeed, preclinical and clinical reports have been conflicting. Here, we tested the effect of functional activation during acute ischemic stroke using distal middle cerebral artery occlusion in anesthetized mice.

Methods: Using transgenic mice expressing channelrhodopsin-2 in neurons, we delivered functional activation using physiological levels of transcranial optogenetic stimulation of the moderately ischemic cortex (ie, penumbra), identified using real-time full-field laser speckle perfusion imaging during a 1-hour distal microvascular clip of the middle cerebral artery. Neuronal activation was confirmed using evoked field potentials, and infarct volumes were measured in tissue slices 48 hours later.

Results: Optogenetic stimulation of the penumbra was associated with more than 2-fold larger infarcts than stimulation of the contralateral homotopic region and the sham stimulation group (n=10, 7, and 9; 11.0±5.6 versus 5.1±4.3 versus 4.1±3.7 mm³; P=0.008, 1-way ANOVA). Identical stimulation in wild-type mice that do not express channelrhodopsin-2 did not have an effect. Optogenetic stimulation was associated with a small increase in penumbral perfusion that did not explain enlarged infarcts.

Conclusions: Our data suggest that increased neuronal activity during acute focal arterial occlusions can be detrimental, presumably due to increased metabolic demand, and may have implications for the clinical management of hyperacute stroke patients.

Keywords: ischemia; mice; middle cerebral artery; optogenetics; perfusion imaging.

Figure 1.. Optogenetic functional activation of the peri-infarct tissue during distal middle cerebral artery occlusion worsens the outcome.

(A) Timeline shows the experimental protocol. Laser speckle flowmetry (LSF) and optical intrinsic signal (OIS) imaging started before and continued during 1-hour distal middle cerebral artery occlusion (MCAO, shaded area). We delivered low-intensity optogenetic stimulation (8 mW/mm², 6 ms pulse, at 8 Hz, for 15 seconds) over the ipsilesional penumbra or contralesional homotopic cortex through the intact skull. Stimulation started posteriorly and moved 0.5 mm along the perimeter every 15 seconds guided by real-time LSF. When the anterior edge of the perfusion defect was reached, the stimulus probe was moved back to the most posterior location, and the cycle repeated 10 times. Infarcts were assessed 48 hours after distal MCAO using topical TTC staining where whole brains were dipped in TTC solution. After taking a dorsal image of the TTC-stained brain, 1 mm-thick coronal slices were cut, and infarct areas at each coronal level were measured (middle panels in B and C) and integrated to calculate the total infarct volume (left panels in B and C). In addition, the distance between the midline and medial infarct edge and between the midline and the lateral edge of the hemisphere were measured and plotted to recreate infarct contours on dorsal view (right panels in B and C). (B) and (C) Infarct volume, infarct areas on coronal slices, and infarct contours on dorsal view (left, middle, and right panels, respectively) are shown for transgenic mice expressing channelrhodopsin-2 in neurons (ChR2+) and non-transgenic littermates (ChR2−). Total Functional activation of ipsilesional peri-infarct cortex slightly increase the SD numbers, as shown on the infarct volume graph under each group. Stimulated (Opto) and non-stimulated (Control) groups were compared using one-way ANOVA and unpaired t-test (infarct volume), Kruskal-Wallis and Mann Whitney tests (SD numbers), two-way repeated-measures ANOVA (infarct areas and contours). ChR2+ control 5 male, 4 female; ipsilesional stimulation 6 male, 4 female; contralesional stimulation 3 male, 4 female. ChR2− all male.

Figure 2.. Effects of optogenetic functional activation on peri-infarct cerebral blood flow in the distal middle cerebral artery occlusion model.

(A) Diagram shows the floating optogenetic stimulus (470 nm, 8 mW/mm², 6 ms, 8hz, moved 0.5 mm every 15 sec), its sweep path, and the region of interest within which CBF changes were quantified (red outlined periinfarct tissue) during distal middle cerebral artery occlusion (MCAO). (B) A representative tracing of CBF during optogenetic stimulation in a ChR2+ mouse shows the baseline, stimulus, and reperfusion time segments (shaded) averaged to calculate CBF changes shown in panel C.

(C) Average CBF changes during baseline, stimulation, and reperfusion in ChR2+ (and ChR2− mice were compared using two-way ANOVA. P values represent group effect. ChR2+ control 5 male, 3 female; ipsilesional 6 male, 4 female; contralesional 2 male, 4 female. ChR2− all male.

Figure 3.. Electrophysiological confirmation of peri-infarct optogenetic functional activation using epicranial evoked potentials.

(A) Timeline shows the experimental protocol. Laser speckle flowmetry (LSF) and optical intrinsic signal (OIS) imaging started before and continued during 1-hour distal middle cerebral artery occlusion (MCAO, shaded area). Single pulses (8 mW/mm², 6 msec) were delivered every 5 seconds over the medial margin of the perfusion defect guided by LSF, as well as the contralesional homotopic region starting 10 minutes after MCAO. For each region, 20 evoked potentials were averaged. (B) and (C) Epicranial optogenetic evoked potentials (EP) were recorded from the stimulated region as well as the contralesional homotopic region (transcallosal) simultaneously using two Ag/AgCl ball electrodes during MCAO. Upper panels show local and transcallosal EP amplitudes for ipsilesional penumbra (left) and contralesional homotopic cortex stimulation (right) in MCAO (n=5) and sham-operated mice (n=5). Lower panels show the same for stimulation of the ischemic core (only MCAO shown, n=5). Representative tracings are from an MCAO experiment. Unpaired t-test. Sham 1 male, 4 female; MCAO 1 male, 4 female.