Experimental Animals
This study used 51 adult male C57BL/6 mice (weighing 19.7–26.4 g; Charles River Laboratory, Sulzfeld, Germany). Animal care before and during experiments adhered strictly to the guidelines of the Johannes Gutenberg University, Mainz, Germany. The mice remained in their home cages with constant access to food and water. The Animal Ethics Committee approved all experiments of the Landesuntersuchungsamt Rheinland-Pfalz, Germany (protocol number 23 177-07/ G 12-1-010) in accordance with the ARRIVE guidelines. We made every effort to minimize the number of animals used and their suffering.
Experimental TBI
Animals were placed on a temperature-controlled heating pad to maintain body temperature at 37°C during the surgical procedure. General anesthesia was induced with 4 vol% isoflurane (AbbVie, Wiesbaden, Germany) and maintained with 1.5–2 vol% isoflurane through a face mask. Trauma was induced by controlled cortical impact (CCI) as previously described [13]. Briefly, after craniotomy on the right rostrocaudal plane, a mechanical lesion was induced on the right parieto-temporal cortex using a custom-fabricated impactor (L. Kopacz, Germany) and with the following parameters: tip diameter of 3 mm, 1.5 mm brain penetration, impact duration of 150 ms, and impact velocity of 8 m/s. After trauma, the craniotomy was immediately closed using histoacrylic glue (B Braun Melsungen AG, Melsungen, Germany), and wounds were closed with sutures. Mice were returned to their cages and placed in an incubator (33°C, 35% humidity; IC8000, Draeger, Germany) for 2 h. Animals regained consciousness within 10 min after the induction of trauma.
Treatment and Drugs
All drugs were administered 24 h after CCI by intravenous injection into the tail vein. Animals were randomized to four different treatment regimens as follows: midazolam solution (Midazolam hydrochloride, hameln pharma plus GmbH, Hameln, Germany) was administered at a dose of 0.5 mg/kg body weight (low-dose group, LD) or 5 mg/kg body weight (high-dose group, HD). One group received midazolam 5 mg/kg body weight (high dose) plus flumazenil (Flumazenil-hameln, hameln pharma plus, Hameln, Germany) 0.5 mg/kg body weight. All drugs were dissolved or diluted in normal saline (0.9% NaCl) and injected at a volume of 10 mL/kg body weight. The vehicle group received 10 mL/kg body weight normal saline (0.9% NaCl). A blinded observer scored the mice for loss of righting reflex, as described previously [14, 15].
Histological Evaluation of Brain Damage
Animals were euthanized under isoflurane anesthesia, after which their brains were carefully removed, snap-frozen in powdered dry ice, and stored at −20°C. Brains were cut in the coronal plane using a cryostat (HM 560 Cryo Star; Thermo Fisher Scientific, Walldorf, Germany). Sections (10 µm in thickness) were collected at 500-µm intervals and stained with cresyl violet. The area of both hemispheres and contused brain tissue, defined as the region lacking cresyl violet staining, was determined. The contusion volume was calculated using a computerized image system (Delta Pix Insight; Delta Pix, Maalov, Denmark) by an investigator blinded to the experimental group randomization. Lesion volumes were calculated by multiplying the lesion areas obtained from 16 consecutive sections with a 500-µm distance interval [0.5 × (A1 + A2+ A3 + … + An)] [16].
Motor Coordination
Motor coordination was analyzed by the rotarod test as described previously and by an investigator blinded to the group allocation [17]. After 3 days of TBI, mice were tested twice before euthanasia. The mean maximum speed and the mean latency time to balance until fall from the rod were recorded using a five-lane accelerating rotarod device (Panlab Rota Rod, Harvard Apparatus, Holliston, MA). The rotarod speed was increased linearly from 4 to 40 rpm over 5 min. The investigation was completed when the mice fell off the rods.
Neurological Severity Score
An investigator blinded to the group allocation evaluated the mice using a modified neurological severity score consisting of 10 different tasks for evaluating motor ability, alertness, balancing, and general behavior [18]. A 15-point scale was used where 0 indicates no neurological impairment and 15 indicates severe neurological dysfunction, as described previously [19].
Gene Expression Analysis
Tissue samples of the right upper quadrant from the brain sections of injured brains were collected during histological processing and snap-frozen in liquid nitrogen. Samples were stored at −80°C until processing. Quantification of mRNA was performed using a real-time polymerase chain reaction. Absolute copy numbers of target genes were normalized against the housekeeping gene cyclophilin A (PPIA) [20]. Samples were homogenized in QIAzol® reagent (Qiagen). RNA isolation was performed using the RNeasy® Lipid Tissue Mini kit (Qiagen) per the manufacturer’s instructions. Table 1 shows the primer sequences. The same amounts of cDNA were amplified in duplicates using Absolute Fast SYBR Green Mix (Thermo Fisher Scientific) for PPIA and iNOS, Absolute Blue SYBR Green for TNF-α, and Light-Cycler 480 Probes Master (Thermo Fisher Scientific) for IL-1β and IL-6 according to the manufacturer’s instructions.
Statistical Analysis
Data were analyzed using the Sigma Plot 13 Statistical Software package (Systat Software, Inc., San José, USA). Exact Wilcoxon–Mann–Whitney tests were performed. Values were adjusted for multiple comparisons using the Holm–Bonferroni method. Data are expressed as mean ± SD. A P level of ≤0.05 was considered statistically significant.
The group size was planned as follows: n = 11 animals were planned for the vehicle group, the low-dose midazolam group, and the group of animals that received midazolam plus flumazenil low-dose and n = 12 animals for the group that received high-dose midazolam solution. During sedation, mice were persistently responsive to pain stimuli by pinprick. Three mice in the high-dose midazolam group did not lose their righting reflex, and one mouse that received midazolam plus flumazenil did lose righting reflex. Furthermore, one mouse in the midazolam plus flumazenil group was found dead in the cage one day after trauma. These animals were excluded from further analysis. After exclusion, the group size was n = 11 each for the vehicle and the low-dose midazolam group and n = 9 each for the group of animals that received high-dose midazolam or midazolam plus flumazenil.