Materials and methods Neuropathological studies were undertaken in two

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

10 groups of animals: 1 AxCAhGDNF 5 ml, 1310 pfu 10 2.1. Adenovirus preparation ml-treated group and 2 AxCALacZ 5 ml, 1310 pfu ml-treated group had 18 and 14 gerbils, respectively. Generation of the replication-defective recombinant Ad Frozen sections of six two animals of each model of the carrying human GDNF cDNA AxCAhGDNF and bac- AxCAhGDNF-treated group and two animals of 4 days terial b-galactosidase gene AxCALacZ have been de- model of the AxCALacZ-treated group were used for scribed elsewhere [48,61]. Briefly, the human GDNF hGDNF staining and in situ hybridization, and the other cDNA was derived from cultured human fetal astrocytes. animals in both groups were subjected to paraffin section This cDNA placed into a cassette cosmid carrying an for other antibody staining. Animals were anesthetized adenovirus type-5 genome lacking the E1A, E1B, and E3 with a lethal dose of pentobarbital sodium and transcardial- region, which has Swa I cloning site flanked by the CAG ly perfused with normal saline followed by 4 paraformal- cytomegalovirus-enhancer-chicken b-actin hybrid pro- dehyde in 0.1 M phosphate buffer, pH 7.4 PB. The brain moter on the 59 end and a rabbit globin poly A sequence tissue was dissected and immersion fixed in the same on the 39 end. These Ads were generated by in vivo fixative. For paraffin section, the brain tissues were fixed homologous recombination in 293 cells. The recombinant for at least 24 h at 48C, dehydrated, embedded in paraffin Ad was propagated and isolated from 293 cells, and wax, sliced coronally at the hippocampal levels into 3-mm- purified by two rounds of CsCl centrifugation [23]. Bioas- thick sections and collected on glass slides coated with say of AxCAhGDNF has been described elsewhere [61]. 3-aminopropyl-triethoxy-silane Silan. After deparaffiniz- ing and rehydrating, sections were pretreated with 0.3 2.2. Animals and surgical procedures H O in phosphate-buffered saline PBS, rinsed in PBS 2 2 three times, and incubated with PBS containing 10 Adult Mongolian gerbils weighing about 70 g were normal goat serum NGS for 1 h at room temperature T . Yagi et al. Brain Research 885 2000 273 –282 275 RT, for blocking nonspecific binding. Sections were Auto Wash Research Genetics, counterstained with He- incubated overnight at 48C with a mouse monoclonal matoxylin Research Genetics and coverslipped with antibody to b-tubulin Promega or glial fibrillary acidic Pristine Mount Research Genetics. protein GFAP DAKO at a dilution of 1:300 or 1:500, respectively. They were then incubated with biotinylated 2.5. Tunel assay anti-mouse IgG, at a dilution of 1:200, ABC reagent Vector and visualized by 3,39-diaminobenzidine tetrahy- Histochemical staining for TUNEL at hippocampus 7 drochloride DAB-H O solution. Griffonia simplicifolia days after the operation was performed with a kit 2 2  B 4 isolectin B4–lectin Sigma staining was performed ApopTag Peroxidase In Situ Apoptosis Detection Kit by a method described elsewhere [52]. These sections were [ S7100, Intergen. After a detection of double-strand counterstained with Hematoxylin. For histopathological breaks in genomic DNA with DAB-H O solution, the 2 2 analysis, Hematoxylin–Eosin or Toluidine Blue TB was sections were counterstained with Methyl Green according performed on some sections of each animal. to the protocol in the kit. For frozen section, the brain tissues were fixed for 2 h at 48C, cryoprotected in 30 sucrose in 0.1 M PB and serial 2.6. Statistical analysis sections 10-mm-thick at hippocampal levels were made by cryostat and collected on glass slides coated with Silan. The number of neurons in the bilateral CA1 area was For immunostaining against hGDNF, sections were pre- counted on paraffin sections at two points each side from treated with 0.3 H O in PBS, followed by preincubation each animal and expressed as neuronal cell density per 2 2 with 0.3 Triton X-100 in PBS T-PBS for 30 min at RT, millimeter linear length. Hence, the four numbers of rinsed in PBS three times and preincubated in 3 NGS in neurons were made from one animal. Sections obtained T-PBS for 1 h at RT. Sections were incubated overnight at from control animals AxCALacZ-treated group were also 48C with a rabbit polyclonal antibody to hGDNF Santa examined. Results are expressed as the mean6S.D. from