Mice were subjected randomly to sham injury (n = 38) or central fluid percussion brain injury (cFPI; n = 78). Based on previous results in TBI models showing early oligodendrocyte death and transient OPC proliferation with a peak at seven days post-injury,10 (link),16 (link) three survival end-points were used; two days post-injury (dpi), seven dpi, and 14 dpi. The cFPI protocol was adapted to mice and used in the present report as described previously.16 (link),30 (link) Anesthesia (4% isoflurane in air) was induced in a chamber, and the animals were then moved to a stereotaxic frame where anesthesia was maintained through a nosecone delivering isoflurane 1.2% and N2O/O2 70/30% and spontaneous breathing. The scalp was shaved and cleaned with ethanol, and bupivacaine 2.5 mg/mL (Marcaine®, AstraZeneca, Sweden) was applied subcutaneously. A midline incision was made and the skull exposed.
A 3.0 mm diameter craniotomy was made in the midline between bregma and lambda sutures, carefully leaving the underlying dura intact. A dural breach was an exclusion criterion. A plastic cap was attached over the craniotomy using dental cement (HeraeusKulzer GmbH, Hanau, Germany). Saline was added into the cap, and any signs of saline leakage were noted. If the seal was intact, the saline cap was attached to the Luer-Lock on the fluid percussion device (VCU Biomedical Engineering Facility, Richmond, VA). The injury was produced by releasing the fluid percussion pendulum striking a saline-filled cylinder creating a pressure wave transmitted into the closed cranial cavity. A transducer measured the pressure pulse and the pressure peak noted in pressure by square inch (psi).
Immediately after the injury, an apnea was noted, and when spontaneous breathing resumed, anesthesia was resumed. An apnea >60 sec was used as an exclusion criterion. The cement and cap were removed and the bone flap replaced over the craniotomy and the skin sutured using resorbable sutures. The animal was placed in a separate cage until fully recovered from anesthesia. Animal weight was recorded for a minimum of three days after the injury. If weight loss more than 10% was noted, the animal was sacrificed and excluded from the study. Sham-injured animals were subjected to an identical procedure as the cFPI animals except that the pendulum was not released.
All animals were sacrificed by an intraperitoneal (ip) overdose of sodium pentobarbital (pentobarbital natrium 60 mg/mL, VET ATL, Apoteket, Sweden; 200 mg/kg. Cardiac perfusion was performed using 4% formaldehyde (formaldehyde 4% Fosfatbuffrad, HistoLab Products AB, Gothenburg), and the brains were removed rapidly from the cranium, placed in formaldehyde for 24 h, and then placed in 30% sucrose solution for 72 h until snap frozen in isopentane and stored at −70°C until sectioned.
A 3.0 mm diameter craniotomy was made in the midline between bregma and lambda sutures, carefully leaving the underlying dura intact. A dural breach was an exclusion criterion. A plastic cap was attached over the craniotomy using dental cement (HeraeusKulzer GmbH, Hanau, Germany). Saline was added into the cap, and any signs of saline leakage were noted. If the seal was intact, the saline cap was attached to the Luer-Lock on the fluid percussion device (VCU Biomedical Engineering Facility, Richmond, VA). The injury was produced by releasing the fluid percussion pendulum striking a saline-filled cylinder creating a pressure wave transmitted into the closed cranial cavity. A transducer measured the pressure pulse and the pressure peak noted in pressure by square inch (psi).
Immediately after the injury, an apnea was noted, and when spontaneous breathing resumed, anesthesia was resumed. An apnea >60 sec was used as an exclusion criterion. The cement and cap were removed and the bone flap replaced over the craniotomy and the skin sutured using resorbable sutures. The animal was placed in a separate cage until fully recovered from anesthesia. Animal weight was recorded for a minimum of three days after the injury. If weight loss more than 10% was noted, the animal was sacrificed and excluded from the study. Sham-injured animals were subjected to an identical procedure as the cFPI animals except that the pendulum was not released.
All animals were sacrificed by an intraperitoneal (ip) overdose of sodium pentobarbital (pentobarbital natrium 60 mg/mL, VET ATL, Apoteket, Sweden; 200 mg/kg. Cardiac perfusion was performed using 4% formaldehyde (formaldehyde 4% Fosfatbuffrad, HistoLab Products AB, Gothenburg), and the brains were removed rapidly from the cranium, placed in formaldehyde for 24 h, and then placed in 30% sucrose solution for 72 h until snap frozen in isopentane and stored at −70°C until sectioned.
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