Figure 2

TRP601 has neuroprotective effects in a perinatal stroke model. The 7-day-old rats underwent electrocoagulation of the left middle cerebral artery and transient homolateral common carotid artery occlusion for 50 min, followed by 48 h of recovery. (a) Pre-treatment with TRP601 confers strong cerebroprotection. Vehicle (▪; n=15), 100 μg TRP601 (□; n=24) or 100 μg TRP901 (▪; n=9) was injected intraperitoneally (i.p.) before ischemia. Histograms represent mean±S.E.M. Kruskal–Wallis (P=0.0004), post hoc Dunn's (P<0.001 for TRP601 versus vehicle). (b) Dose–response of TRP601 administered 1 h after MCAO onset (n=103, P<0.002 Kruskal–Wallis). Histograms represent mean±S.E.M. Asterisks indicate the level of significance in Dunn's post hoc. (c) Representative cresyl violet-stained coronal sections from vehicle-treated and TRP601-treated (1 mg/kg i.p.; 1 h post-ischemia) animals at 48 h post-reperfusion at the level of dorsal hippocampus and anterior commissure. Dotted lines indicate infarct area. Scale bar=500 μm. (d) Time window for treatment with TRP601 (n=274, P<0.0001 Kruskal–Wallis). Individual percentage infarct volumes at 48 h post-ischemia are shown in vehicle- (n=43) or TRP601-treated rats (1 mg/kg; i.p.) at 1 h (n=51, ^***), 2 h (n=45, ^***), 3 h (n=40, ^***), 4 h (n=48, ^*), and 6 h (n=47, ^*) post-occlusion (asterisks indicate the level of significance in Dunn's post hoc). Temperature and body weight were systematically monitored in ischemic animals (treated or not with TRP601) and were found unchanged during ischemia, at reperfusion, and up to 48 h post-ischemia. (e and f) TRP601 effects after intravenous injection. TRP601 was administered intrajugularly 1 h after the ischemic onset (n=15). (e) Representative micrographs of entire pup brains at 48 h post-stroke. Arrows indicate electrocoagulation points. Upper micrograph shows representative lesion at 48 h with visible ischemic territory. Lower micrograph is representative for low lesional score found in 33% of animals injected with 0.1 mg/kg TRP601. (f) Raustro-caudal profiles (coronal sections of ipsilateral hemisphere) after intravenous (i.v.) administration of TRP601 (1 h after ischemia) at 0.1 mg/kg (n=15) or 1 mg/kg (n=16). (g and h) TRP601 has long-term protective effects. Vehicle or 0.1 mg/kg TRP601 was i.p. injected 1 h after the ischemic onset. After 21 days of recovery, animals were killed. (g) Cresyl violet-stained coronal sections showing brain injury in a representative animal for each treatment (upper: bregma 0.7; lower: −3.3 mm). Note that TRP601 reduces the cortical cavity and tissue loss in external and internal capsule (ec, ic), caudate putamen (CPu), and amygdaloid nucleus (bregma −3.6, −4 mm; data not shown). (h) Quantification of the cavity volume (% cavitation) and hemispheric tissue loss (100−% ipsi/contra) in the presence (n=12) or absence (n=6) of TRP601. Data are mean±S.E.M. (bars) values. ^*P=0.0017; ^#P=0.0192 (Mann–Whitney). (i and j) In vivo and ex vivo cell death, at 48 h post-stroke, in the ipsilateral cortex of vehicle- and TRP601-treated ischemic animals. Propidium iodide was injected intrajugularly (10 mg/kg) into rat pups before ischemia and coronal sections were analyzed by fluorescence microscopy (i, representative micrograph; j, left histograms). Alternatively, coronal sections were subjected to in situ 3′-OH end DNA labeling (terminal deoxynucleotidyl transferase dUTP nick-end labeling , TUNEL), counterstained with Hoechst 33342, and analyzed by fluorescence microscopy (j). Data are mean±S.E.M. (bars) values (n=5). ^**P=0.0079 (Mann–Whitney). The arrows point to apoptotic nuclei. Scale bar=100 μm. (k) Representative western blot analysis of caspase-2 (Casp2) processing at 24 h post-ischemia in the presence or absence of TRP601 or TRP901 injected (1 mg/kg, i.p.) at reperfusion. Ipsilateral penumbra zone (IL) and contralateral counterpart (CL) of hemispheres were dissected and homogenized before biochemical analysis. Co., control brains (without ischemia). (l) Detection of caspase-like activities at 24 h post-stroke in ipsilateral penumbra zones. Histograms show means of VDVADase, DEVDase, and LEHDase activities of animals i.p.-treated (▪, n=5) or not (□, n=5) with TRP601 or TRP901. Note that kinetic analysis showed that the earliest and quantitatively major detected caspase-like activity is a VDVADase activity that reached 14 pmol/min/mg at t=5 h post-ischemia, whereas neither DEVDase nor LEHDase activities were detected (Supplementary Figure S5). (m) TRP601 prevents cytochrome c release in vivo. A representative western blot analysis is shown. MF: mitochondrial fraction; CF: cytosolic fraction (CF). (n) Electron microscopy analysis of the mitochondria isolated from ischemic brains. The mitochondria isolated from vehicle-treated ipsilateral (IL), contralateral (CL) hemispheres, or TRP601-treated ipsilateral (IL+TRP601) hemispheres are shown. Each panel show an example of the dominant phenotype (>60%) of the isolated mitochondria population