Translational Research for axona regeneration by using Stroke model of minature pig

• Project/Area Number: 17591499
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Cerebral neurosurgery
• Research Institution: Gunma University
• Principal Investigator: IMAI Hideaki Gunma University, Faculty of Medicine, Research Associate, 医学部, 助手 (70359587)
• Co-Investigator(Kenkyū-buntansha): SAITO Nobuhito Tokyo University, Hospital, Professor, 医学部附属病院, 教授 (60262002) ; ISHIZAKI Yashuki Gunma University, Graduate School of Medicine, Professor, 大学院医学系研究科, 教授 (90183003) ; UCHIDA Takayuki Gunma University, Graduate School of Medicine, Research Associate, 大学院医学系研究科, 助手 (80334093) ; KAZAMA Ken Gunma University, Graduate School of Medicine, Research Associate, 大学院医学系研究科, 助手 (30396626) ; KONNO Kenjiro Jichi Medical University, Faculty of Medicine, Assistant Professor, 医学部, 講師 (30323348)
• Project Period (FY): 2005 – 2006
• Project Status: Completed (Fiscal Year 2006)
• Budget Amount: ¥3,400,000 (Direct Cost: ¥3,400,000); Fiscal Year 2006: ¥900,000 (Direct Cost: ¥900,000); Fiscal Year 2005: ¥2,500,000 (Direct Cost: ¥2,500,000)
• Keywords: stroke model / miniature pig / translational research / MRI / SVZ / stem cell / transplantation / axonal regeneration / ミニブタ脳梗塞モデル / 再現性 / 低侵襲性 / 神経細胞塊

Research Abstract

Background and Purpose The first purpose of the present set of studies was to introduce a reproducible stroke model in the gyrencephalic brain of miniature pig with minimum invasiveness. The paper provides information on both the surgical technique and the methods of quantification of ischemic damage to both grey and white matter in the miniature pig. The second purpose was to apply this new stroke model for the translational research for the neuronal regeneration by using thestem cell trasplantation into the ischemic brain. Methods Sixteen male miniature pigs were randomly divided into three groups and underwent transcranial surgery, a frontotemporal approach with orbital rim osteotomy, for : permanent middle cerebral artery occlusion (MCAO) (n=5), permanent internal carotid artery occlusion (ICAO) (n=6) and sham operation (n=5). Histological mapping and magnetic resonance (MR) imaging were used to delineate the areas of ischemia. Subventricular zone (SVZ) cells isolated from the mini ature pig were cultured in Iscove's modified Dulbecco's medium in the presence of basic fibroblast growth factor for 8 days. We stereotaxically transplanted subventricular zone (SVZ) cells labeled by ferromagnetic particles into stroked miniature pig. The transplanted cells were non-invasively tracked using magnetic resonance imaging (MRI). To detect ferromagnetic-labeled SVZ cells in the host brain, we stained sagittal or coronal sections for iron using Prussian blue reaction. Results The volumes of the infarction measured directly from MR images were 16.2 ±1.1, 1.5±0.5 and 0.0±0.0 cm^3 (mean±SD) in the MCAO, ICAO and sham-operation groups, respectively. The histological and MR imaging volumes showed a good correlation (r^2=0.86, p <0.0001) although the infarct volume measured by quantitative histopathology was smaller than that measured by MRI. Immunohistochemical staining using amyloid precursor protein antibody (APP) allowed us to evaluate the axonal damage, providing total APP score of 43.8±3.5 (mean±SD) in the MCAO. MRI signals detected the tranplanted cells with labeling at the lesion, and these signals were detected 1 and 2 weeks after transplantation. Prussian blue-positive cells were also detected in the same lesion where the cells were transplanted. The transplanted cells were likely to be just homing in the host brain. We have neither confirmed the construction of the network between the transplanted cells and host brain. Conclusions This paper describes a simple and reproducible method for the induction of white and grey matter damage in focal cerebral ischemia with minimum invasiveness. This miniature pig stroke model has utility for studying the pathophysiology of ischemia in gyrencephalic brain and for the assessing the therapeutic efficacy of drugs or stem cell transplants, prior to initiating human clinical trials.

Research Products

• [Journal Article] Experimental Model of Lacunar Infarction in the Gyrencephalic Brain of the Miniature Pig : Neurological Assessment and Histological, Immunohistochemical, and Physiological Evaluation of Dynamic Corticospinal Tract Deformation (2007)
  • Author(s): Y Tanaka, H Imai, K Konno, T Miyagishima, C Kubota, S Puentes, T Aoki, H Hata, K Takata, Y Yoshimoto, N Saito
  • Journal Title: Stroke (in press)
• [Journal Article] A New Model of Focal Cerebral Ischemia in the Miniature Pig (2006)
  • Author(s): H Imai, K Konno, M Nakamura, T Shimizu, C Kubota, K Seki, F Honda, S Tomizawa, Y Tanaka, H Hata, N Saito.
  • Journal Title: J Neurosurg (2 Suppl Pediatrics) 104 Pages: 123-132
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Cilostazol Attenuates Both Gray and White Matter Damage in a Rodent Model of Focal Cerebral Ischemia. (2006)
  • Author(s): F Honda, H Imai, M Ishikawa, C Kubota, T Shimizu, M Fukunaga, N Saito.
  • Journal Title: Stroke 37 Pages: 223-228
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A New Model of Focal Cerebral Ischemia in the Miniature Pig. (2006)
  • Author(s): H Imai, K Konno, M Nakamura, T Shimizu, C Kubota, K Seki, F Honda, S Tomizawa, Y Tanaka, H Hata, N Saito.
  • Journal Title: J Neurosurg (2 Suppl Pediatrics) 104 Pages: 123-132
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Nestin expression in vascular malformations : a novel marker for proliferative endothelium. (2006)
  • Author(s): Shimizu T, Sugawara K, Tosaka M, Imai H, Hoya K, Takeuchi T, Sasaki T, Saito N.
  • Journal Title: Neurol Med Chir (Tokyo) 46(3) Pages: 111-117
  • NAID: 110004656790
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] New Model of Focal Cerebral Ischemia in the Miniature Pig.. (2006)
  • Author(s): H Imai, K Konno, M Nakamura, T Shimizu, C Kubota, K Seki, F Honda, S Tomizawa, Y Tanaka, H Hata, N Saito. A
  • Journal Title: J Neurosurg (2 Suppl Pediatrics) 104 Pages: 123-132
• [Journal Article] New Model of Focal Cerebral Ischemia in the Miniature Pig. (2006)
  • Author(s): H Imai, K Konno, M Nakamura, T Shimizu, C Kubota, K Seki, F Honda, S Tomizawa, Y Tanaka, H Hata, N Saito.A
  • Journal Title: J Neeurosurg(2 Suppl Pediatrics) 104 Pages: 123-132
• [Journal Article] Cyclophilin C-associated protein and cyclophilin C mRNA are up- regulated in penumbral neurons and microglia after focal cerebral ischemia. (2005)
  • Author(s): T Shimizu, H Imai, K Seki, S Tomizawa, M Nakamura, F Honda, N Kawahara, N Saito.
  • Journal Title: J Cereb Blood Flow Metab 25(3) Pages: 325-337
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] The detection and quantification of highly reactive oxygen species using the novel HPF fluorescence probe in a rat model of focal cerebral ischemia. (2005)
  • Author(s): S Tomizawa, H Imai, S Tsukada, T Simizu, F Honda, M Nakamura, T Nagano, Y Urano, Y Matsuoka, N Fukasaku, N Saito.
  • Journal Title: Neuroscience Research 53 Pages: 304-313
  • NAID: 10017131014
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Evolution of GADD34 expression after focal cerebral ischaemia. (2005)
  • Author(s): McCaig D, Imai H, Gallagher L, Graham DI, Harland J, Moira Brown S, Mhairi Macrae I
  • Journal Title: Brain Res 1034 Pages: 51-61
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Evolution of GADD34 expression after focal cerebral ischaemia. (2005)
  • Author(s): McCaig D, Imai H, Gallagher L, Graham DI, Harland J, Moira Brown S, Mhairi Macrae I.
  • Journal Title: Brain Res. 1034 Pages: 51-61
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Cyclophilin C-associated protein and cyclophilin C mRNA are up-regulated in penumbral neurons and microglia after focal cerebral ischemia. (2005)
  • Author(s): T Shimizu, H Imai, K Seki, S Tomizawa, M Nakamura, F Honda, N Kawahara, N Saito.
  • Journal Title: J Cereb Blood Flow Metab. 25(3) Pages: 325-337
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] The detection and quantification of highly reactive oxygen species using the novel HPF fluorescence probe in a rat model of focal cerebral ischemia. (2005)
  • Author(s): S Tomizawa, H Imai, S Tsukada, T Simizu, F Honda, M Nakamura, T Nagano, Y Urano, Y Matsuoka, N Fukasaku, N Saito.
  • Journal Title: Neuroscience 53 Pages: 304-313
  • NAID: 10017131014
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Cyclophilin C-associated protein and cyclophilin C mRNA are up-regulated in penumbral neurons and microglia after focal cerebral ischemia. (2005)
  • Author(s): T Shimizu, H Imai, K Seki, S Tomizawa, M Nakamura, F Honda, N Kawahara, N Saito.
  • Journal Title: J Cereb Blood Flow Metab 25(3) Pages: 325-337
• [Journal Article] The detection a d quantification of highly reactive oxygen species using the novel HPF fluorescence probe in a rat model of focal cerebral ischemia. (2005)
  • Author(s): S Tomizawa, H Imai, S Tsukada, T Shimizu, F Honda, M Nakamura, T Nagano, Y Ureno, Y Matsuoka, N Fukasaku, N Saito.
  • Journal Title: Neuroscience Research 53 Pages: 304-313
• [Journal Article] Evolution of GADD34 expression after focal cerebral ischaemia. (2005)
  • Author(s): McCaig D, Imai H, Gallagher L, Graham DI, Harland J, Moria Brown S, Mhairi Macrae I
  • Journal Title: Brain Res 1034 Pages: 51-61
• [Journal Article] Focal Cerebral ischemia increases type2 iodothyronine deiodinase. (2005)
  • Author(s): K Seki, H Imai, T Shimizu, K Tsunekawa, T Kasahara, T Ogiwara, N Saito, M Murakami.
  • Journal Title: J Cereb Blood Flow Metab. 25
• [Journal Article] Vasorelaxing effect of the Rho-kinase inhibitor, Y-27632, in isolated canine basilar arteries.
  • Author(s): Y Hashiba, M Tosaka, N Saito, H Imai, T Shimizu, T Sasaki.
  • Journal Title: Neurological Research (in press)
  • Description: 「研究成果報告書概要(欧文)」より