Volume 12, Issue 1, Pages 47-62 (July 2005) Angiogenesis and stem cell transplantation as potential treatments of cerebral ischemic stroke  Ling Wei, Christine L. Keogh, Vivian Riley Whitaker, Michelle Hedrick Theus, Shan Ping Yu  Pathophysiology  Volume 12, Issue 1, Pages 47-62 (July 2005) DOI: 10.1016/j.pathophys.2004.11.002 Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 TUNEL positive cells of different morphology in the barrel cortex and thalamus. (A) TUNEL positive cells (green) 24h after ischemia in the ischemic barrel cortex and the non-ischemic thalamus; most cells in the cortex still have intact nuclei (named as Type I TUNEL positive cells). On the other hand, TUNEL positive cells in the thalamus showed nuclear condensation and fragmentation (named as Type II TUNEL positive cells). The Hoechst staining (blue) labeled all cells. (B) H&E staining of damaged cortex and thalamus cells 10 days after the cortex ischemia, showing different morphology of nuclei in these cells (arrows point to typical cells in the cortex and thalamus). (C) High magnification of TUNEL positive cells in the ischemic cortex and non-ischemic thalamus 10 days after ischemia. In striking contrast to Type I cell in the cortex, Type II TUNEL positive cells in the thalamus showed distinct feature of nuclear condensation and fragmentation. Bar=100μm in A, 20μm in B and C. From Fig. 4 in [12]. Pathophysiology 2005 12, 47-62DOI: (10.1016/j.pathophys.2004.11.002) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Electron microscopy of cell injury in the cortex and thalamus. Electron micrographs show ultrastructural alterations in individual neurons in the ischemic barrel cortex and ipsilateral thalamus. (A) The control cortical neuron had a large nucleus, relatively small cytoplasm volume, and intact mitochondria and other organelles. (B) Primarily necrotic alterations appeared in a damaged cortical neuron 3 days after ischemia, featured by swollen cytoplasm, disrupted organelles, formation of vacuoles (*), and deteriorated membranes (arrow). Note that no nuclear condensation took place. (C) The injured thalamic neuron 7 days after the cortex ischemia showed both apoptotic and necrotic features; there was apoptotic nuclear shrinkage, chromatin condensation, and necrotic chaotic injuries in the cytoplasm including volume increase, formation of vacuoles (*), swollen organelles and deteriorated membranes (arrow). Bar=5μm. From Fig. 5 in [12]. Pathophysiology 2005 12, 47-62DOI: (10.1016/j.pathophys.2004.11.002) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Angiogenesis after focal ischemia in the whisker–barrel cortex. The whisker–barrel cortex ischemic stroke in rats was induced by selective occlusion to branches of the MCA [191]. (A) Before ischemia, optical signals (IOS) upon stimulating large whiskers on the left face. Activated area represents larger whiskers (arrow, warm colors; midline is up and rostral to the right). Bar=0.5mm (applies to B, C, E, and G). (B) IOS 30 days later. The area activated by stimulation of the large whiskers was smaller and rostral to that in panel A (arrow). There is no activation of infarcted cortex (blue; compare with panel G). (C) Arterial phase after FITC injection before ligations. Sites for the arterial time courses (a) and parenchymal fields in the central (*) or the peripheral (†) ischemic border. (D) Time courses of transits after FITC at the three locations. Arrowheads indicate half-maximal dye intensity values that were reached at short latencies after the dye first appeared in the arteriole (a) (<0.5s). FITC recirculates after 4s in the artery and the parenchyma. (E) Transit 3min after ligations (arrowheads) were placed. Collaterals provide delayed, less robust blood flow to the central ischemic border. (F) FITC transit in the central ischemic border, which was considerably delayed and diminished acutely after ligations compared with that at the peripheral ischemic border, which was significantly slowed (compare with panel D). (G) FITC transit 30 days later. Collaterals have increased in diameter, are more tortuous, and have more prominent branches. The infarct is at the lower left. (H) FITC transit after 30 days, which was faster in the central ischemic border but delayed compared with that in the peripheral ischemic border. Transits were still slower than immediately before the ligations (D). From Fig. 2 in [38]. Pathophysiology 2005 12, 47-62DOI: (10.1016/j.pathophys.2004.11.002) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Angiogenesis after whisker–barrel cortex ischemia. The whisker–barrel cortex stroke in rats was induced by MCA branch ligations [191]. Angiogenesis was indicated by specific labeling for DNA synthesis (BrdU), integrins (αvβ3), and capillary endothelium (GLUT-1). (A) At 5 days after a cranial window without arteriolar ligations (sham-operated control), no microvessels (arrows) or surface vessels in the cortex were labeled with BrdU. (B) BrdU-labeled nuclei in the ischemic border 5 days after ligations are shown. A surface arteriole at the upper right (*) shows labeled nuclei in presumed perivascular smooth muscle (arrowheads). A radial intraparenchymal vessel below it has several labeled nuclei (arrows) that are likely endothelial. Other labeled nuclei are not associated with vessels. (C) Endothelium of an intraparenchymal microvessel in a sham-operated control animal is labeled lightly for GLUT-1 (green). (D) Double labeling of BrdU (yellow) and GLUT-1 demonstrates endothelial proliferation in an intraparenchymal capillary in the ischemic border. (E) Antibody staining for αvβ3 shows a sharp division between the ischemic border (lower left) and normal unstained cortex (upper right) 7 days after ligation. A large and a small vessel are labeled (arrowheads). (F) Higher power image of the field outlined in panel E shows association of αvβ3 with vessels in the ischemic border (bis-benzimide counterstain). Above the dashed line, there is no vascular staining for αvβ3 in the normal cortex (*). (G) αvβ3 in the ischemic border 10 days after arteriolar ligation outlines vessels (arrowheads). Bars=100μm (A, B, and E), 10μm (C), and 50μm (F and G). From Fig. 3 in [38]. Pathophysiology 2005 12, 47-62DOI: (10.1016/j.pathophys.2004.11.002) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 ES cell transplantation after focal ischemic stroke. The whisker–barrel cortex ischemic stroke in rats was induced by selective occlusion to branches of the MCA [191]. (A) Cerebral blood flow measured by 14C-IAP autoradiography was substantially reduced in a discrete cortical region 10min after ligation of MCA branches and occlusion of the ipsilateral common cerebral artery. (B) Infarct formed in the ischemic core revealed by TTC stain. (C) TUNEL positive cells (yellow) in the ischemic cortex, especial layer IV, 24h after ischemia. Red denotes GFAP positive cells (astrocytes). (D) Activated caspase-3 positive cells (arrow) co-localization with PI staining in the ischemia cortex. (E) ES cells pre-labeled with Hoechst (blue) in the ischemic cortex 7 days after transplantation. Green represents GFAP positive cells and red NeuN positive neurons. (F–H) Enlarged images from E. (F) Native neurons (NeuN positive, red) and astrocytes (GFAP positive, green) in the non-ischemia region (frame in D). (G) Differentiated neurons derived from ES cells (NeuN positive, red) and astrocytes (GFAP positive, green) in the transplantation site of ischemic core. The overlap of these markers with pre-labeled Hoechst (blue) demonstrates their ES cell origin. (H) Confocal triple labeling demonstrates NeuN-positive neurons (red), GFAP-positive astroglia (green), and Hoechst (blue) labeled nuclei indicating ES cells origin. Modified from Fig. 2 in [184]. Pathophysiology 2005 12, 47-62DOI: (10.1016/j.pathophys.2004.11.002) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions