This study was approved by the German animal protection committee (Landesuntersuchungsamt Rheinland-Pfalz, protocol number G-13-1-074). Experiments were performed in accordance with all national animal welfare guidelines and ARRIVE guidelines for reporting animal research.45 The study was conducted using 90 male C57Bl6/N mice (Charles River Laboratories, Sulzfeld, Germany; weight: 22–28 g). Two mice had to be excluded from the study before the end of the trial.
Ribonuclease-1 (RNase1, Sigma, Germany) was dissolved in sterile normal saline by a third person not involved in the experiments or analysis, numbered and prepared for intraperitoneal (i.p.) injection. Mice were randomly allocated to receive either one or two i.p. injections of 20, 60, or 180 µg/kg RNase1 or vehicle at 30 min and 12 h post-TBI. The experiments and the processing of the material was performed in blinded fashion. These doses are at least tenfold lower than required to induce adverse side effects in rodents.3,19,28 To minimize the total number of animals used in accordance with ARRIVE guidelines45, no additional sham group was established for most experiments. Also, after the initial dose–response study, subsequent investigations of brain water accumulation, tissue IgG changes, and 120-h survival used only the most effective (low) dose of RNase1, with sham animals examined for comparison only when necessary.
Traumatic brain injury
Traumatic brain injury was induced by CCI using an electromagnetic impact device (Leica Impact OneTM Stereotaxic Impactor, Richmond, IL; tip: 3 mm; velocity: 6 m/s; duration: 200 ms; displacement: 1.5 mm). Animals were anesthetized by intraperitoneal injection of 5 mg/kg midazolam, 0.05 mg/kg fentanyl, and 0.5 mg/kg medetomidine, and body temperature controlled at 37°C based on feedback from a rectal thermometer. After craniotomy, trauma was induced on the right cortex by an experimenter blinded to treatment group allocation.46–48 Thereafter, the craniotomy was closed and anesthesia was antagonized using 0.5 mg/kg Flumazenil and 2.5 mg/kg Atepamezol hydrochloride. Animals were allowed to recover for 1.5 h in a neonatal incubator (IC8000, Draeger, Luebeck, Germany) with controlled air temperature (35°C) and ambient humidity (35%).
Neurological outcome and Rotarod test
Before CCI (1 h) and at 24 h after CCI, an investigator blinded to experimental group assigned a neurologic severity score ranging from 0 (healthy) to 15 (severely impaired) (24 h groups: N = 40).49 An accelerating rotarod test (Rotarod Treadmill, MED Associates, INC, St Albans, VT) was conducted in the long-time surviving groups one day before and at 24 and 120 h post-CCI (N = 20). Briefly, each mouse was placed on an accelerating rotating cylinder, and the time and maximum speed at which the animal fell off (27 cm fall height) were recorded.50,51 Briefly, four rotarod tasks were conducted before CCI and the mean value of each animal was defined as 100% “starting value”. 24 h and 120 h following injury, animals were tested in two trials per time point, averaged and evaluated relative to pre-injury latencies to correct for individual pre-injury performance.
Tissue preparation, Nissl, Iba-1 and CD45 staining
Animals were anesthetized and brains excised, frozen in powdered dry ice, and stored at -20°C. Coronal sections (10-µm thick) were cut at 500-µm intervals (24 h: n = 40 mice, 120 h: n = 20 mice) and subjected to cresyl violet staining. Brain lesion volume was measured using a computerized image system (DeltaPix InSight, Smorum, Denmark).52 In brief, to control for the effect of brain edema, the area of uninjured brain tissue and the total area of the contralateral hemisphere were quantified in each section. Afterwards the injured area was calculated by subtraction of “normal” area in the injured hemisphere from total contralateral. Other slices were immunostained for the activated microglial marker Iba-1 using an anti-rabbit Iba-1 antibody (1:1500, Wako Pure Chemical Industries, Osaka, Japan) and a biotinylated anti-rabbit IgG (Vector Laboratories Inc., Burlingame, CA). Signals were detected using ABC Complex (Vector) and DAB (Thermo-Fischer, Waltham, MA). The appearance of Iba-1+/CD45+ cells 120 h after lesion induction was quantified by dual staining using rat anti-CD45 (1:500, Thermo-Fisher) with Alexa Fluor 568-conjugated goat anti-rat IgG (1:500, Thermo-Fisher, Waltham, MA) and anti-rabbit Iba-1 antibody (1:500, Wako) with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500, Thermo-Fisher). Sections were then counterstained with DAPI (1:10000, Thermo-Fisher).53 Images were acquired at 20× magnification, and cells were counted in the perilesional and corresponding, non-injured contralateral regions of two serial sections in the zone with the largest lesion (bregma -1.64 mm and bregma -1.82 mm, www.mbl.org, ROI: 2.55 mm²).51 In the predefined areas (border zone at lower outside corner the lesion and the corresponding contralateral side) double-immunolabeled cells were counted by an investigator blinded to treatment using ImageJ (U.S. National Institutes of Health, Bethesda, MD).
RNA isolation and quantitative polymerase chain reaction (qPCR)
Samples from the contused brain region were collected (n = 40) as described,22 frozen in liquid nitrogen, and stored at -80°C. RNA extraction, reverse transcription, and mRNA quantification by real-time RT-PCR were performed as described.49,52 Absolute copy numbers of target genes were normalized to the reference gene cyclophilin A (PPIA).
Brain water content
Brains were isolated 24 h after lesion and the hemispheres cut along the interhemispheric plane (n = 30). The hemispheres were weighed (wet weight, WW), dried in a vacuum-centrifuge (Univapo 100H, UniEquip, Planegg, Germany) for 48 h, and reweighed (dry weight, DW). Brain water content was calculated according to the equation water content (%) = (WW - DW)/WW ×100.44
Brain tissue samples (n = 30 mice) were lysed in RIPA buffer, and total cellular proteins separated by SDS-PAGE, transferred to nitrocellulose membranes, immunolabeled (IgG; 1 : 10 000; Li-cor), and visualized by a near-infrared laser scanning system (LI-COR Odyssey) essentially as described.54 An antibody against GAPDH (Acris, clone 6C5) was used as the reference and quantification was performed using ImageJ (NIH, MD).
All experiments were randomized and performed by blinded investigators (computer-based randomization). The experimental groups are presented in Figure 4. To determine the required sample size, an a priori power analysis was performed using G∗Power55 based on lesion volume data from previous studies. A priori power analysis for an effect size of 0.7 suggested that a standard statistical power (1-β) of Pβ = 0.95 and significance level (α) of 0.05 can be obtained for expected lesion volumes using 10 subjects per group (4 groups) and for expected brain water content using 10 subjects per group (2 groups). GraphPad Prism 9 statistical software (GraphPad Software Inc., La Jolla, CA, USA) was used to perform statistical analysis. Prior to analysis, we checked the test assumptions. Due to the limited power in small samples, we did not perform formal goodness-of-fit tests prior to the t test or ANOVA, but instead relied on the graphical assessment of distribution characteristics.56 Normality was checked by inspecting the unimodality and symmetry of histograms, as well as by Q-Q plots. The equality of variances was checked by inspecting histograms and standard deviations. For comparison of multiple independent groups, Brown-Forsythe and Welch ANOVA with post-hoc Dunnett T3 multiple comparisons test (comparisons between all groups) was employed. To evaluate group differences in repeated-measurements from the same animals (rotarod), RM two-way ANOVA was applied (factors: treatment and time), followed by Šidáks multiple comparisons test. Comparisons between two independent groups were carried out by the Welch’s t test. Values of p < 0.05 were considered significant. Data are presented as the mean and standard deviation (mean ± SD).