Immunohistochemistry and Western blot analysis
At 16 d after CHI, a subgroup of injured and sham-operated mice were anesthetized with a lethal dose of anesthetic and perfused via the ascending aorta with PBS followed by cold 4% paraformaldehyde in PBS. Brain tissue was postfixed in the same fixative for 24 h at 4°C and embedded in paraffin, thereafter. Coronal brain sections (6 m thick) were serially taken at 200
m intervals throughout the neuraxis, between –1.06 mm and
2.30 mm from bregma (38). Correspondent slides with ad-
hered sections were selected for various histological techniques, thereafter, in the following order: immunohistochemistry for astrocytes, microglia, BDNF, and synaptophysin.
Adjacent sections were double stained for BDNF and synapthopysin. In brief, antigen retrieval was performed in citrate buffer (pH.6) and the endogenous peroxidase was blocked with H2O2 (0.3% in PBS). Sections were then incu- bated in blocking buffer for 1h. Following incubation over- night at 4°C with anti-BDNF antibody (N-20, Santa Cruz),
DCS IMPROVES FUNCTIONAL RECOVERY AND LTP IN CHI MICE
Figure 1. DCS facilitates motor and cognitive functions after CHI. A) Neurological Severity Score tests reflexes, alertness, coordination, and motor abilities. One point is awarded for failure to perform a particular task; thus a normal mouse scores 0. The extent of recovery is calculated as the difference between NSS at 1 h post-injury and that at any other time:
NSS NSS (1 h) – NSS (t). NSS at 1 h in the
range of 8 –10 reflects severe injury. Mice (n18) were subjected to moderate-severe CHI (NSS1h6.80.4). After 24 h, they were treated with either DCS (10 mg/kg, intraperi- toneally) or vehicle (n9/group). NSS was assessed during 22 d thereafter. NSS NSS (1 h) – NSS (t) represents functional recovery. *P 0.05; #P 0.02 vs. vehicle-treated mice, Mann-Whitney test. B) Object recognition (OR) was evaluated on the same mice (n9/ group), on days 3, 9, and 16. Four hours after baseline evaluation (baseline) the mice were reintroduced in the same cages to a novel object that replaced the “familiar” one, and the time spent near each of the objects was monitored (test). Memory impairment of the CHIvehicle-treated mice is shown by the inability to distinguish between the novel and “familiar” object. The results of naive animals on day 3 were also included as control. DCS- treated mice regained the ability to explore the novel object from day 9 and on. *P 0.02; vs. vehicle-treated mice, t test.
sections were incubated with goat anti-rabbit IgG, Rhodam- ine-conjugated (Jackson, ImmunoResearch Laboratories, West Grove, PA USA), as a secondary antibody. Following intensive washes, sections were incubated overnight at 4°C with antisynaptophysin mouse monoclonal antibody (Clone SY38, DakoCytomation, Denmark) and goat anti-mouse IgG, FITC – conjugated (Jackson, ImmunoResearch Laborato- ries), as a secondary antibody, thereafter. In a number of sections, either the first or the second primary antibody was omitted for the detection of non-specific binding of either secondary antibody used.
A series of adjacent sections were then treated with primary antibody against glial fibrillary acidic protein (GFAP) (Dako- Cytomation, Denmark) and then with goat anti-rabbit (Vec- tor, Burlingame, CA, USA) as secondary antibody. Immuno- reactions were visualized with the avidin–biotin complex (Vectastain) and the peroxidase reaction was visualized with diaminobenzidine (DAB) (Vector) as chromogen. In adja- cent sections, endogenous biotin blocking was performed by