Introduction ability to infect nerve cells, which are not its natural target,

Brain Research 885 2000 273–282 www.elsevier.com locate bres Research report Rescue of ischemic brain injury by adenoviral gene transfer of glial cell line-derived neurotrophic factor after transient global ischemia in gerbils a,e , a b c d Takashi Yagi , Ikuyo Jikihara , Masayuki Fukumura , Kazuhiko Watabe , Toya Ohashi , d e a Yoshikatsu Eto , Mitsuhiro Hara , Mitsuyo Maeda a First Department of Anatomy , Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan b DNAVEC Research Inc ., Tsukuba, Japan c Department of Molecular Neuropathology , Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan d Department of Pediatrics and Institute of DNA Medicine , Jikei University School of Medicine, Tokyo, Japan e Department of Neurosurgery , Osaka City University Medical School, Osaka, Japan Accepted 12 September 2000 Abstract Glial cell line-derived neurotrophic factor GDNF, a member of the transforming growth factor TGF–b superfamily, is one of the most potent neurotrophic factors and promotes survival of many populations of cells. We examined neuroprotective effect of an adenoviral vector encoding glial cell line-derived neurotrophic factor AxCAhGDNF on the transient global ischemia. Gerbils received administration of AxCAhGDNF or an adenoviral vector encoding bacterial b-galactosidase gene AxCALacZ through the lateral ventricle. Two days later, occluding bilateral common carotid arteries for 5 min using aneurysm clips produced the transient global forebrain ischemia. Animals showed intense immunolabeling for GDNF in ependymal cells on 2, 4 and 7 days after the operation. The exogenous gene transducted by adenovirus in the same cells was detected by in situ hybridization. The treatment with AxCAhGDNF significantly prevented the loss of hippocampal CA1 pyramidal neurons 2 to 7 days after the operation, as compared to AxCALacZ treatment. Also terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling TUNEL staining was markedly reduced in the case with AxCAhGDNF treatment at 7 days after the operation. These results indicated that the adenovirus-mediated gene transfer of GDNF might prevent the delayed neuronal death of stroke and other disorders of the cerebral vasculature.  2000 Elsevier Science B.V. All rights reserved. Theme : Disorders of the nervous system Topic : Ischemia Keywords : GDNF; Adenoviral vector; Delayed neuronal death; Transient global ischemia

1. Introduction ability to infect nerve cells, which are not its natural target,

and that the gene transfer and expression of Ad are highly Numerous studies in recent years have reported that the efficient and maintain long-term period both in vitro and in adenovirus Ad expression vector is useful for application vivo. In addition, its genome can accommodate foreign to gene therapy [7,11–13,17,29,31,33,44,61]. The advan- genes of up to 7.5 kb and high titers of the virus can be tage for its use is that Ad vector is the replication-deficient obtained easily [23]. recombinant adenovirus, which has low pathogenicity, due Glial cell line-derived neurotrophic factor GDNF, a to lacking the E1A, E1B and E3 regions [40]. It has the member of the transforming growth factor TGF–b superfamily [35], is one of the most potent neurotrophic factors and promotes survival of many populations of cells, Corresponding author. Tel.: 181-6-6645-3701; fax: 181-6-6645- including brain tissues under the ischemic condition 3702. E-mail address : m3377173med.osaka-cu.ac.jp T. Yagi. [1,8,25,26,60], peripheral sensory neurons [46], hippocam- 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 5 6 - 5 274 T pal pyramidal neurons induced by kainic acid-mediated used. The animals were maintained at constant temperature seizures [39], spinal motor neurons [4,5,17– and 12:12 h light:dark cycle and allowed free access to 19,34,45,61,64,66], mesencephalic dopaminergic neurons food and water until the investigations. The animals were [6,7,10,12–14,22,29,31,38,50,53,54], and axotomized reti- anesthetized with intraperitoneal injection of chloral hy- nal ganglion cells [27,28]. GDNF is a target-derived drate 300 mg kg and positioned in a stereotaxy frame. neurotrophic factor [18,53] with a molecular weight of 15 These animals received a unilateral injection of either 10 kDa, forming a naturally occurring dimer and inducing AxCAhGDNF 5 ml, 1310 pfu ml, n518 or 10 glycosylation [32,35]. GDNF signaling is mediated AxCALacZ 5 ml, 1310 pfu ml, n514 over a 6 min through a two-component system consisting of the Ret period into the left lateral ventricle using a 33-gauge 10 ml tyrosine kinase and a glycosyl-phosphatidyl-inositol-linked Hamilton syringe. The Hamilton syringe was left in place protein termed GFRa-1 or GFRa-2, which complexes with for 3 min before removal. Injection coordinates relative to GDNF and binds to and activates the tyrosine kinase bregma were 1.5 mm posteriorly and 2.0 mm laterally to receptor Ret [3,16,21,49,55,56]. In previous investigations, the left side at a depth of 1.2 mm from cortical surface. the Ret phosphorylation in response to GDNF results in After the injection, the animals were again allowed free activation of the mitogen-activated protein kinase MAPK access to food and water. Two days later, injected animals through the Ras-GTP activation [43,63] and of phospha- were reanesthetized with intraperitoneal injection of chlor- tidylinositol PI-3 kinase indirectly or directly, through al hydrate 300 mg kg. The bilateral CCA were exposed the Ras-GTP activation or not [15,47,57,65]. It has been through the midline skin incision in the neck and occluded demonstrated in vitro that, PI-3 kinase activation can for 5 min using aneurysm clips to produce transient prevent apoptosis in rat oligodendrocytes and their pre- forebrain global ischemia. Rectal temperature was kept as cursors [58], rat pheochromocytoma PC-12 [65] and close as possible to 388C during and up to 30 min after fibroblasts [24]. Wang [60] and Abe [1] have demonstrated ischemia with the aid of a heating blanket and overhead that GDNF can prevent ischemia-induced injury in cerebral lamp [36,37]. After these procedures and their wounds cortex, which is mainly a necrotic lesion. However, it is closed, the animals were allowed to recover. The animals not known whether GDNF can prevent the delayed neuro- were sacrificed at 2 six animals in hGDNF model and four nal death induced by transient ischemia. In the present animals in LacZ model, 4 six animals in both models, study, we investigated whether the treatment of intraven- and 7 days six animals in hGDNF model and four animals tricular injection with Ad vector encoding GDNF cDNA in LacZ model after the operation as described above. The can prevent the delayed neuronal death in hippocampal experimental protocol and procedures conformed to that of CA1 pyramidal neurons induced by occluding bilateral the Animal Committee of the Osaka City University common carotid arteries CCA for 5 min in Mongolian School of Medicine. gerbils. 2.3. Immunohistochemistry

2. Materials and methods Neuropathological studies were undertaken in two