Permanent Middle Cerebral Artery MCA Occlusion Model and BMSC Transplantation

Eight-week-old male Balb/c mice were subjected to permanent middle cerebral artery (MCA) occlusion, as described before [3, 5-7]. The BMSCs harvested from the Balb/c mice were labeled with PKH-26 (2 x 10-6 M; Sigma, St. Louis, MO, USA) according to the manufacturer's protocol. The animals were anesthetized 7 days after the insult. The cranium was exposed through a midline skin incision, and the surface of the intact neocortex was exposed through a small craniectomy (3 x 5 mm) made in the right parietal region (Fig. 1A), using a dental drill under surgical microscope. The dura mater was carefully removed so as not to damage the brain surface. The animals were assigned to three groups. In group A (n = 6), 50 |l of phosphate-buffered saline [phosphate-buffered saline (PBS); vehicle] was poured onto the brain surface, and the wound was closed. In group B (n = 6), 50 |l of the PKH-26-labeled BMSC suspension (5 x 105 cells) was poured onto the brain surface, and the wound was closed. In group C (n = 6), the PKH-26-labeled BMSCs (4 x 106 cells) were embedded into 1.0 ml of TGP-culture medium mixture at 4°C, and 0.5 ml of the BMSC-TGP construct was placed into a flexible cell culture

Fig. 1. A At 7 days after the onset of permanent middle cerebral artery occlusion, a small craniectomy (3 X 5 mm; arrow) was made in the ipsilateral parietal region to expose the surface of the intact neocortex. B The bone marrow stromal cell (BMSC)-thermoreversible gelation polymer (TGP) construct (arrow) was solidified at 37°C and transplanted onto the surface of the intact neocortex in group C (arrow)

Fig. 1. A At 7 days after the onset of permanent middle cerebral artery occlusion, a small craniectomy (3 X 5 mm; arrow) was made in the ipsilateral parietal region to expose the surface of the intact neocortex. B The bone marrow stromal cell (BMSC)-thermoreversible gelation polymer (TGP) construct (arrow) was solidified at 37°C and transplanted onto the surface of the intact neocortex in group C (arrow)

chamber (flexiPERM Disc; growth area, 1.8 cm2; Greiner Bio-One, San Francisco, CA, USA). The BMSC-TGP construct was solidified at 37°C for 1 h, yielding a 0.5-mm-thick gel in each chamber. Subsequently, one-fourth of the construct, including 5 x 105 BMSCs, was mounted onto the surface of the intact neocortex (Fig. 1B).

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