Patterns and dynamics of subventricular zone neuroblast migration in the ischemic striatum of the adult mouse.

All experiments procedures were approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital.

DCX-eGFP Transgenic Mice

A pair of breeding colony DCX-eGFP transgenic mice (DCX-GFP/bacterial artificial chromosome (BAC)) was purchased from the Mutant Mouse Regional Resource Center, Davis, CA, USA. Adult DCX-eGFP animals (3 months old) were identified by PCR analysis using tail DNA with primers to GFP (450 bp): forward 5′-TGCAGTGCTTCAGCCGCTACC-3′; reverse 5′-ATGTGATCGCGCTTCTCGTTG-3′.

Animal Model of Middle Cerebral Artery Occlusion Male

DCX-eGFP mice (3 months old) were subjected to permanent focal cerebral ischemia by inserting a 6-0 nylon filament (9 to 9.5 mm in length) into the right middle cerebral artery (MCA) ( Zhang et al , 1997 b ). Occlusion of the right MCA for 7 days induces the ischemic lesion in the territory supplied by the MCA ( Supplementary Figure 1 ) and evokes a peak increase of neurogenesis (Zhang et al , 2001 , 2004 a ). Therefore, all mice were killed 7 days after MCA occlusion. Nonischemic DCX-eGFP mice (3 months old) were used as a control group.

Organotypic Brain Slice Culture

Nonischemic mice ( n = 4) and 7-day ischemic mice ( n =9) were killed. The procedure used for the organotypic slice culture assay is based on published protocols ( Miyata et al , 2002 ; Zhang et al , 2007 ). When removed from the skull, the brain was immediately immersed in ice-cold Hank’s balanced salt solution (Gibco, Carlsbad, CA, USA) at pH 7.2 for 3 mins. A 4-mm-thick coronal brain section between the level of bregma (2.7 mm) and the bregma (-1.3 mm) was used for the organotypic brain slice culture ( Franklin and Paxinos, 1997 ), which includes the entire territory supplied by the MCA ( Zhang et al , 1997 a ). The coronal brain section was cut into 300- μ m-thick sections using a vibratome, and the coronal slices were placed in a six-well plate with one slice per well. In some cases, sagittal sections at the striatal SVZ plane were cut. Brain tissue was kept in ice-cold Hank’s balanced salt solution during these procedures. The brain slice was then embedded in the Cellmatrix gel (200 μ l, Type 1-A Nitta Gelatin, Osaka, Japan) and incubated for 1 h with the brain slice media containing Dulbecco’s modified Eagle’s medium/F-12, insulin (25 μ g/mL), APO-transferrin (100 μ g/mL), progesterone (20 nmol/L), sodium selenate (30 nmol/L), putrescine (60 μ mol/L), epidermal growth factor (10 ng/mL), basic fibroblast growth factor (10 ng/mL), horse serum (5%), fetal calf serum (5%), penicillin—streptomycin (1%), sodium bicarbonate (1%), and HEPES ((4-(2-hydroxyethyl)-1-piper-azineethanesulfonic acid), 0.33%) ( Miyata et al , 2002 ). Thereafter, the brain slices were ready for time-lapse microscopy studies.

Time-Lapse Microscopy

Organotypic brain slices were incubated in a stage top chamber with 5% CO 2 and 37°C (LiveCell Control Unit), which was placed on the stage of a Nikon TE2000-U inverted microscope (Nikon, Tokyo, Japan) equipped with a motorized Z-stage. A × 10 objective with 1.5× electronic zoom was used for taking images. Fluorescent field images were taken for the brain slices with excitation and emission filters at 546 and 585 nm, respectively, at 0.5 msecs exposure times. A stack of images (36 images with a 5- μ m step in Z axes) was acquired at 15-min intervals during 4 to 10 h using a CCD camera (CoolSnap, Tuscon, AZ, USA) and MetaView Software (Universal Imaging, Dowiningtown, PA, USA).

Quantification of Cell Motility

Cell migration speed and distances, and lengths of cell processes were analyzed offline by tracing individual cells with the best focus plane from a Z-stack at different times using Universal Imaging MetaMorph Software. Subsequently, migratory values were statistically analyzed.

Immunohistochemistry

To examine whether DCX-eGFP-expressing cells are DCX positive in the transgenic mouse, nonischemic mice ( n =4) and mice subjected to 7 days of stroke ( n = 6) were killed and their brains were fixed for immunostaining. Immunostaining for fixed brain coronal sections were carried out according to a published protocol ( Zhang et al , 2004 b ; Robin et al , 2006 ). The following primary antibodies were used in this study: rabbit anti-glial fibrillary acidic protein (1:500; Dako Cytomation California, Carpinteria, CA, USA), goat anti-DCX (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and chicken anti-GPF (1:500, Aves Labs, Tigard, OR, USA). Double immunofluorescent images were taken using a Zeiss confocal microscope (Zeiss LSM 510 NLO, Thornwood, NY, USA).

Statistical Analysis

The measurements of migration distance and speed were presented as mean±s.e. Significant differences between the SVZ and chains were analyzed using Student’s t -test. Statistical significance was set at P < 0.05.

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