1. Surgical procedures
Anesthetize adult female mice via an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Perform laminectomy at the 10th thoracic spinal vertebrae (T10), and then expose the dorsal surface of dura matter and induce SCI using a commercially available SCI device (IH impactor, Precision Systems & Instrumentation, Lexington, KY) as previously described. This device creates a reliable contusion injury by rapidly applying a force-defined impact (60 kdyn) with a stainless steel-tipped impounder. Close the muscles and the incision in layers, and place the animals in a temperature-controlled chamber until thermoregulation is reestablished. Perform manual bladder expression twice a day until reflex bladder emptying is reestablished.
2. Functional evaluation
Motor function of the hind limbs can be evaluated by the locomotor rating test on the Basso-Beattie-Bresnahan (BBB) scale, in which 21 is normal function and 0 is bilateral total paralysis of the hindlimbs. When BBB scoring is applied to mice, the size and speed of the hindpaws make it difficult to assign a precise score if the score exceeds 13 points (Ma et al., 2001), but that was not a problem in our study, because none of the mice had scores over 13 points (Jakeman et al., 2000). A team of three experienced examiners evaluated each animal for 4 min and assigned an operationally defined score for each hindlimb.
Anesthetize animals and transcardially perfuse with 4% paraformaldehyde in 0.1 M PBS. Remove the spinal cords, embed in OCT compound, and sagittally section at 20 um on a cryostat. Stain tissue sections with primary antibodies to GFAP (DAKO), cleaved caspase-3 (Cell signaling), CD11b (a marker of monocyte/macrophages and granulocytes) (MBL), BrdU (CHEMICON), Nestin (Rat 401, CHEMICON), GFP (MBL), phosphorylated form of Stat3 (Cell signaling), GSTp (BD Biosciences), serotonin (5HT) (DiaSorin Inc.), GAP43 (CHEMICOM), E-cadherin (Santa Cruz) and Hu29 (a gift from Dr.Robert Darnell, The Rockefeller University). Nuclear counterstain with Hoechst 33342 (Molecular Probes). Obtain images by fluorescence microscopy (Axioskop 2 plus; Carl Zeiss) or confocal microscopy (LSM510; Carl Zeiss).
4. In vitro migration assay
Prepare primary astrocytes from 2-d old Wt and conditional knock out mice as described. After several passages in DMEM with 10% FBS, trypsinize cells and plated in confluence on coverslips coated with poly-L-Lysiine. After they reach subconfluency, treat with 10 mg/ml mitomycin C for 2 h to avoid the proliferative effect and finally subject to wound scratch assay. Introduce a cell-free area by scratching the monolayer with a yellow pipette tip, and evaluate cell migration to cell-free area for another 24 h and 48 h. The number of migrating astrocytes should be counted after taking photographs of ten non-overlapping fields.