Mice received a lethal dose of sodium pentobarbital (1.6 mg/g IP) 2 days postinjury. After loss of pain reflexes mice were transcardially perfused, first with heparinized (10 units/ml) saline for 1 min then 4% paraformaldehyde in Millonig’s buffer pH 7.4 for 20 min. Brains were dissected and then sectioned coronally at 60 μm using a vibratome (Leica VT1000S). Sections directly below the craniectomy (bregma level −0.6 to −2.4 mm) were collected in 24-well plates filled with Millonig’s buffer pH 7.4. To quantitatively assess for intrinsic AIS and extrinsic/synaptic perisomatic structural plasticity, for each animal, we labeled ankG, NaV1.6 and double-labeled glutamate decarboxylase-67 (GAD67) with PV in sections taken from a randomly selected and two adjacent wells, respectively, containing caudal S1BF (bregma level −1.5 to −2.0). This was done because of the consistency with which cFPI generates DAI within this well characterized area of neocortex, which was also the rational for choosing this region-of-interest (ROI) in our previously published electrophysiological studies (Greer et al., 2012 (link); Hånell et al., 2015a (link); Sun and Jacobs, 2016 (link)).
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