Adult male rats were housed in standard cages with free access to food and water on a 12-h light/dark cycle. All procedures performed on animals were approved by Columbia University’s Institutional Animal Care and Use Committee and conducted according to institutional and federal guidelines.
Pilocarpine induced status epilepticus
After premedication with scopolamine (5 mg/kg, i.p.) to prevent the effects of peripheral cholinergic stimulation, pilocarpine (330 mg/kg, i.p.) was administered to Sprague-Dawley rats (100–150 g) to induce seizures. Seizures were graded on the modified Racine scale , and only animals with grade 4–5 seizures for 2 h were used in experiments. After 2 h of continuous seizures, ketamine (80 mg/kg, i.p.) was administered to stop seizures, and a second dose (40 mg/kg, i.p.) was administered if seizures did not stop in 10 min after the first.
Kainic acid induced status epilepticus
Kainic acid dissolved in isotonic saline (pH 7.4) was given i.p. to Sprague-Dawley rats (100–150 g) at 10 mg/kg with repeated injections of the same dose over 30 min until the appearance of grade 4–5 seizures, according to the modified Racine scale. After 2 h of continuous seizures, ketamine (80 mg/kg, i.p.) was administered to stop seizures, and a second dose (40 mg/kg, i.p.) was administered if seizures did not stop in 10 min after the first.
Cortical stab wound model
Sprague-Dawley Rats (100–250 g) were anesthetized (ketamine 80 mg/kg, xylazine 8 mg/kg, i.p.) and placed in a stereotactic frame and the skull was exposed using sterile technique. After drilling the skull, a blunt 26-G needle (Hamilton) was inserted into the frontal cortex. 10 μl of solution (95% saline, 5% ethanol) was administered. After 96 h, animals were deeply anesthetized with an overdose of ketamine/xylazine, and perfused with 4% paraformaldehyde (PFA).
Stroke/middle cerebral artery occlusion (MCAO)
Wistar rats (275–300 g) were subjected to transient middle cerebral artery occlusion using a method of intraluminal vascular occlusion . The animals were anesthetized with halothane in a mix of 70% nitrous oxide/30% oxygen. The animals’ core temperatures were maintained at 37 °C throughout the entire procedure and for 60 min after reperfusion. The right common carotid artery, the right external carotid artery, and the right internal carotid artery were exposed and isolated. MCA occlusion was accomplished by advancing a 25 mm 4–0 nylon suture with a blunted silicone tip (outer diameter, 0.38 mm) through an incision in the external carotid artery until the suture was 18 mm past the carotid bifurcation. MCA occlusion was confirmed by transcranial measurements of cerebral blood flow via laser Doppler flowmetry (Periflux System 5000; Perimed, Inc., Järfälla, Sweden). After 120 min of ischemia, the occluding suture was removed, and reperfusion was confirmed by laser Doppler flowmetry. After 96 h, animals were deeply anesthetized with an overdose of ketamine/xylazine, and perfused with 4% PFA.
Histology and immunohistochemistry
After perfusion brains were removed and additionally fixed in 4% PFA in PBS for 14–18 h (40 C). 40 μm sections were prepared with a vibratome (Leica VT1000S) and stored in cryoprotectant solution at − 200 C. Standard procedure for Nissl staining with Cresyl violet was used for routine analysis of tissue.
Primary antibodies: (1) markers of astrocytes: (i) glial fibrillary acidic protein (GFAP): mouse monoclonal (1:1000, G3893, Sigma-Aldrich, St. Louis, MO), rabbit polyclonal (1:1000, Z 0334, Dako, Carpinteria, CA), phospho-GFAP (Ser8) mouse monoclonal (1:100, NBA-115, Stressgen, Ann Arbor, MI); (ii) vimentin: monoclonal (1:500, M 0725, Dako), phospho-vimentin (Ser55): mouse monoclonal (1:300, D076–3, MBL International, Woburn, MA); (iii) nestin: rabbit polyclonal (1:500, PRB-570, Covance, Emeryville, CA); astrocyte specific glutamate transporters: (iv) GLAST: monoclonal (1: 100, clone 10D4, Novocastra Lab, Newcastle upon Tyne, UK); (v) GLT1: mouse monoclonal (1:500, 611,654, BD Transduction Lab., Franklin Lakes, NJ); (vi) calcium binding protein specific for glial cells - S100: rabbit polyclonal (1:600, A 5110, Dako); (vii) alpha-B crystallin: rabbit polyclonal (1:300, SPA-223, Stressgen, Canada); (2) marker of NG2 cells: NG2 Chondroitin Sulfate Proteoglycan (1:100, AB5320, Millipore,Temecula, CA); (3) marker of microglial cells: Iba1, rabbit polyclonal (1:500, 019–19,741,Wako, Richmond, VA); (4) markers of DNA damage: phospho-gamma-H2AX (Ser139), mouse monoclonal (1:300, KAM-CC255, Stressgen, Ann Arbor, MI); (5) chromosome markers in dividing cells: (i) Ki67: mouse monoclonal (1:100, #550609, BD Pharmingen, San Jose, CA) and rabbit polyclonal (1:200, AB9260, Millipore); (ii) PCNA: mouse monoclonal (1:100, NA03, Millipore); (ii) phospho-Histone H3(Ser10): rabbit polyclonal (1:100, #9701, Cell Signaling Technology, Inc., Danvers, MA) and phospho-Histone H3 (Ser10): mouse monoclonal (1:100, #9706, Cell Signaling); (6) centrosome markers, (i) pericentrin: rabbit polyclonal (1:500, PRB-432C, Covance); (ii) gamma-tubulin: mouse monoclonal (1:100, T6557, Sigma-Aldrich) and goat polyclonal (1:100, sc-7396, Santa Cruz Technology, Inc., Santa Cruz, CA); (7) markers of microtubules: (i) alpha-tubulin: mouse monoclonal (1:500, T6074, Sigma-Aldrich); (ii) TPX2: rabbit polyclonal (1:500, Novus Biologicals, Littleton, CO); (8) marker of nuclear envelope: Lamin A/C: rabbit polyclonal (1:100, #2032, Cell Signaling): (9) markers of proteins activated in mitosis: (i) Aurora A: mouse monoclonal (1:200, A1231, Sigma-Aldrich) and rabbit polyclonal (1:100, NB100–635, Novus); (ii) Aurora B: rabbit polyclonal (1:100, A5102, Sigma-Aldrich); (iii) Bub3: mouse monoclonal (1:100, #611730, BD Biosciences) and rabbit polyclonal (1:200, NB110–40721); (iv) BubR1: mouse monoclonal (1:200, #612503, BD Biosciences, San Jose, CA) rabbit polyclonal (1:100, NB100–55254, Novus); (v) NUMA: rabbit polyclonal (1:400, NB500–174, Novus); (vi) Survivin: rabbit polyclonal (1:100, #2808, Cell Signaling); (10) Lucifer yellow: rabbit polyclonal (1:300, AB154, Millipore).
Secondary antibodies conjugated to fluorophores: anti-mouse Alexa Fluor 488, 594, and 633, anti-rabbit Alexa Fluor 488, 594, and anti-goat Alexa Fluor 488, 594, 633; all from goat or donkey (1:300, ThermoFisher Scientific, Eugene, OR).
For double- and triple-immunofluorescence, after blocking with 10% normal goat (or donkey) serum (30 min, RT), free-floating sections were incubated in a mixture of primary antibodies raised in different species overnight (40 C). Alexa Fluor -conjugated secondary antibodies were used for 1 h at RT. For visualization of nuclei and chromosomes fluorescent Nissl reagent (NeuroTrace 640/660 deep-red, 1:150, ThermoFisher Scientific) and DAPI (5 μg /ml; D9542, Sigma-Aldrich) were applied with secondary antibodies.
Blocking serum, primary, and secondary antibodies were applied in 0.2% Triton X-100 in PBS. Sections for fluorescence microscopy were mounted on slides in Vectashield (Vector Laboratories, Burlingame, CA). To control for the specificity of immunostaining, primary antibodies were omitted and substituted with appropriate normal serum.
Slides were viewed using a confocal microscope (Nikon Ti Eclipse). 3D reconstructions were done from stacks of images with confocal microscope software NIS-Elements.
BrdU administration and visualization
5-Bromo-2′-deoxyuridine (BrdU, B5002, Sigma-Aldrich, St. Louis, MO) was dissolved in sterile DPBS (10 mg/ml) and given i.p. at 80 mg/kg. At different time-points (see Results) animals were deeply anesthetized with an overdose of ketamine/xylazine, and perfused with 4% PFA. Vibratome sections were used for immunohistochemical detection of BrdU with anti-BrdU mouse monoclonal (1:330, B8434, Sigma-Aldrich) and rat monoclonal (1:100, MCA2060, AbDSerotec) antibodies after DNA denaturation: 30 min treatment with 2 M HCl (RT) followed with 20 min treatment with 0.1 M sodium borate buffer (pH 8.5) at RT.
TUNEL was performed with DeadEnd™Fluorometric TUNEL system (G3250, Promega Corporation, Madison, WI) according to the manufacturer’s recommendations on vibratome sections. Regular procedures for immunohistochemical staining was performed after TUNEL.
For regular transmission EM, animals were deeply anesthetized with a ketamine/xylazine, and perfused with 2% paraformaldehyde and 2.5% glutaraldehyde in PBS. After postfixation in 2% osmium tetroxide in 0.2 PB (2 h at 4 °C) and dehydration, pieces of tissue were embedded in Epon-Araldite (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections were cut with a Reichert Ultracut E, stained with uranyl acetate and lead citrate, and examined with a JEOL 1200 electron microscope.
Lucifer yellow filling of astrocytes in situ
Coronal slices of rat forebrain (160–180 μm) were cut with a Vibratome (LEICA VT 1000S) in ice-cold oxygenated-modified artificial CSF (aCSF) (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1.5 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 Dextrose. The slices recovered at 30 °C in a chamber for at least 1 h before electrophysiological recording. The above solution was also used for whole-cell patch-clamp recording in brain slices at 30 °C. The bath solution was applied at a flow rate of 1.5 ml/min using the VC-6 perfusion valve control system (Warner Instruments) with the TC-344B temperature controller. Cells were visualized under a LEICA DMLFS microscope with a 63× water-immersion lens. The intracellular solution in the patch pipettes contained the following (in mM): 140 KCl, 1 MgCl2, 10 EGTA, 10 HEPES, 3 MgATP, 0.3 Na2ATP, pH 7.3 with KOH. Pipette resistance was ∼3–5 MΩ. Cell capacitance and series resistance were measured using the software MultiClamp 700A Commander Ver. 220.127.116.11 and pClamp 8 (Axon Instrument, Molecular Devices). Cells were initially identified morphologically based on the sizes and shapes of their somas and the architecture of their processes. For analysis of cell morphology and gap junction coupling with the surrounding cells, Lucifer yellow (LY, Sigma-Aldrich) was added to the intracellular solution (final concentration 0.1%) and filtered through a 0.2 μm PTFE filter. After the experiment, slices were fixed in 4% paraformaldehyde in PBS overnight at 4 °C. Slices were immunostained, and observed under a confocal microscope, as above.
The numbers of mitotic astrocytes visualized with Ki67 or BrdU were counted in the images (merged from stacks of 6 adjacent images with 1024 × 1024 pixel resolution in an observed area of 295 × 295 μm, captured with a confocal microscope at a distance of 0.5 μm from each other) obtained from neocortices in coronal sections (10 images from each section, 5 sections per animal).
Levels of GFAP immunofluorescence were evaluated in the images obtained as described above from coronal brain sections stained for GFAP and Ki67. Images were transferred to Image J 1.46r (public domain), grayscaled and quantified based on the optical density (OD).
Data are expressed as mean ± SEM. Continuous parameters were analyzed with Student’s t-test and one-way Anova. p < 0.05 was considered significant.