All experiments were approved by the Institutional Animal Care and Use Committee at the University of California, San Francisco, and carried out with the highest standards of animal care and housing according to the National Institutes of Health Guide for the care and use of laboratory animals
. Heterozygous transgenic mice carrying 200 copies of the human intracellular GPx gene (GPx1, Mirochnitchenko et al. 1995
) were bred to produce mixed litters of wildtype (WT) and GPx1-overexpressing (GPx Tg) mice. Genotype was identified from tail clippings taken on postnatal day (pnd) 14. PCR was performed on purified DNA using Platinum Supermix (Invitrogen, Carlsbad, CA) with the primer sequences ATG TGT GCT GCT CGG CTA GCG GC [5′-3′] (forward) and GCT GCA GGA ATT CGG GCG GG [5′-3′] (reverse). PCR products were separated on a 2% agarose gel containing ethidium bromide for UV visualization.
On pnd 21, male GPx Tg and WT littermates were anesthetized with 1.25% 2,2,2-tribromoethanol (Avertin, Sigma, St. Louis, MO; 0.02 mL/g body weight). During both the surgical and postoperative recovery period, normal body temperature was maintained with a circulating-water heating pad.
Each animal was placed in a stereotaxic frame (Kopf, Tujunga, CA) for surgery. After a midline scalp incision, underlying soft tissues were reflected and a circular craniotomy, 5 mm in diameter, was made with a drill in the left parietal bone between bregma and lambda with the medial edge of the craniotomy 0.5 mm lateral to midline. Animals were then subjected to controlled cortical impact injury as we have previously described (Tong et al. 2002
) using a convex impactor tip that was 3 mm in diameter and oriented perpendicular to the surface of the brain. The injury was generated using a velocity of 4 m/sec, 1 mm depth of penetration, and a sustained depression of 150 msec. Sham control animals underwent identical procedures but did not receive cortical impact. In both sham and injured animals, the scalp was closed with sutures and 1 ml of saline was injected subcutaneously to prevent dehydration during recovery. Animals were regularly observed until they had fully recovered from anesthesia.
GPx activity assay
To determine the activity of GPx1 in the brains of WT and GPx Tg after sham surgery or injury, WT (n= 5 shams; n= 6 injured) and GPx Tg (n= 4 shams; n= 3 injured) mice were anesthetized with 1 ml of 1.25% Avertin and transcardially perfused with 50 ml of isotonic saline. Brains were then dissected and separated into cortex, hippocampus, and thalamus. Tissue samples were homogenized in cold 0.1 M potassium phosphate buffer (pH 7.0) and then centrifuged at 12,000 rpm for 10 min. Ten microliters of the supernatant was used to determine the amount of protein in each sample using the Pierce BCA Protein Assay (Pierce, Rockford, IL). GPx activity was determined by spectrophotometric detection in a coupled test system in which glutathione reductase was used to regenerate oxidized glutathione (Fan et al. 2003
; Flohe and Gunzler 1984
). This reaction requires the oxidation of NADPH to NADP+ and the decreasing absorbance of NADPH was measured at an optical density of 340 nm. Activity was expressed as units per milligram of protein, where one unit is defined as 1 nmol NADPH oxidized per minute.
Markers of early neurogenesis – precursor cell proliferation and immature neurons
We have previously shown significant neuronal loss in the hippocampus at two weeks after TBI at pnd 21 (Pullela et al. 2006
). To determine if TBI also influences precursor cell function at that same time point, a second group of WT (n= 3 shams; n= 4 injured) and GPx Tg (n= 5 shams; n= 6 injured) mice was euthanized two weeks postinjury. Mice were anesthetized as previously described and transcardially perfused with 50 ml of 10% buffered formalin. Brains were then dissected and postfixed in the same formalin solution for 72 hours. After fixation, brains were placed in a Mouse Brain Matrix (Harvard Aparatus, Natick, MA), which facilitated reproducible transverse sectioning of the whole brain. A 5 mm thick section of brain containing the dorsal hippocampus was obtained and placed into a plastic tissue holder and processed for paraffin embedding. Transverse sections were cut from the rostral face of each tissue block using a rotary microtome. Beginning at the rostral-most edge of the hippocampus, a series of five consecutive 5 μm sections was collected every 300 μm until the entire dorsal hippocampus had been sampled. The caudal boundary of the dorsal hippocampus was defined as the posterior commissure. A subset of these sections was reserved for Nissl staining to qualitatively assess injury.
To assess proliferating precursor cells and their progeny (immature neurons), sections were stained with antibodies against Ki-67 (DakoCytomation, Carpinteria, CA), a nuclear antigen expressed in all phases of the cell cycle except G0
(Kee et al. 2002
), or doublecortin (DCx, Santa Cruz Biotechnology, Santa Cruz, CA), a tubulin-associated protein expressed in migrating neuroblasts (Englund et al. 2002
). After deparaffinization and quenching of endogenous peroxidases (30 minutes in 0.3% hydrogen peroxide in 70% ethanol), sections were incubated in preheated 10 mM sodium citrate butter (pH 6.0) and boiled for 10 minutes using a microwave oven. Sections were then incubated in the sodium citrate buffer for an additional 20 minutes, washed in phosphate-buffered saline (PBS), and blocked for 30 minutes. Blocking was done with PBS containing 2% normal rabbit serum (NRS) for Ki-67 and 5% normal horse serum (NHS) for DCx. Sections were then incubated overnight at 4° C with the primary antibody (Ki-67 diluted 1:100 in PBS with 2% NRS and DCx diluted 1:500 in PBS with 5% NHS). After washing, sections were incubated for 60 minutes at room temperature with biotinylated secondary antibodies. Rabbit anti-rat IgG (Vector, Burlingame, CA) diluted 1:200 in PBS with 2% NRS was used for Ki-67 while anti-goat IgG (Vector) diluted 1:500 in 5% NHS was used for DCx. Biotinylated secondary antibodies were then detected using an ABC system (Vector) and diaminobenzidine as the chromogen. Sections were then counterstained with Gill's hematoxylin, dehydrated, and mounted.
The number of proliferating precursor cells within the SGZ and immature neurons within the GCL were scored blinded to genotype and treatment. For each animal, counts were done on a series of consecutive sections, spaced 300 μm apart, beginning with the rostral-most section containing both the infra- and suprapyramidal blades of the dentate gyrus and ending once the posterior commissure was reached. Sections were visualized using a Nikon brightfield microscope and a 40× objective. The data are reported as the total sum of Ki-67- or DCx-positive cells in each animal.
To determine the effects of TBI on the survival and neuronal differentiation of newly born cells in the SGZ, a third group of WT (n= 4 shams; n= 6 injured) and GPx Tg (n= 6 shams; n= 7 injured) mice was administered 5-bromo-2′-deoxyuridine (BrdU, Sigma), a thymidine analog that is incorporated into the DNA of dividing cells and thus allows dividing cells and their progeny to be tracked. Beginning two weeks after injury (the same time point when Ki-67 and DCx were analyzed), mice were given seven consecutive daily intraperitoneal injections of BrdU (50 mg/kg diluted in saline). Three weeks after the last injection (six weeks postinjury), mice were anesthetized as described above and transcardially perfused with 50 ml of 4% paraformaldehyde. After postfixation in 4% paraformaldehyde for six hours, brains were cryoprotected in 30% sucrose for 48 hours and then sectioned into 50 μm sections using a sliding microtome. Floating sections were then double-stained with antibodies against BrdU and NeuN (a marker of mature neurons). Blocking steps were done in tris-buffered saline (TBS) containing 0.3% Triton X-100 (Sigma) and 3% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) while all antibody incubations were done in TBS with 0.3% Triton-X and 1% normal donkey serum. After washing in PBS, sections were blocked for 30 minutes at room temperature. Sections were then incubated overnight at 4° C with monoclonal mouse anti-NeuN (Chemikon, Temecula, CA, diluted 1:200). After washing, sections were then incubated for six hours at room temperature with the appropriate secondary fluorescent antibodies diluted at 1:200. Following this, sections were washed in TBS, postfixed for 10 minutes in 4% paraformaldehyde, washed for 10 minutes in saline, and then incubated in 3N hydrochloric acid for 30 minutes at 37°C. After further washing in TBS, sections were incubated with rat anti-BrdU (Oxford Biotechnology, Kidlington, Oxford, UK) diluted 1:100. Incubation with a secondary fluorescent antibody was done as described in the preceding step.
Confocal microscopy with a Nikon C1 confocal microscope was used to quantify surviving newly born cells (BrdU+) and newly born neurons (BrdU+/NeuN+). The observer was blinded to genotype and treatment. A z-stack at 60× magnification was obtained for every sixth section throughout the dorsal hippocampus (as defined above). Appropriate gain and black-level settings were obtained on control tissues stained with secondary antibodies alone. Upper and lower thresholds were set using a range indicator function to minimize data loss due to saturation. The total number of BrdU-positive cells within the dentate gyrus was scored. To quantify double-labeled cells, each BrdU-positive cell was manually examined in its full ‘z’ dimension and only those cells that were unambiguously associated with NeuN were scored as positive. Final counts were reported as the total sum of BrdU-positive or double-labeled cells in each animal.
Data were analyzed using two-way analysis of variance (ANOVA) with genotype (WT or GPx Tg) and treatment (sham or injury) as factors. Two-way ANOVA allowed us to test three null hypotheses for each experiment. The primary null hypothesis for each analysis was that there was no interaction between the two factors (genotype and treatment). When that null hypothesis proved correct (ie, the interaction between genotype and treatment was not significant), we could then individually test the null hypotheses that there was no effect of genotype and no effect of treatment on the study population. Significance was set at p<0.05.