Animal care and housing

Prior to initiating any research, the Thomas Jefferson University Institutional Animal Care and Use Committee (IACUC) reviewed and approved the research protocol and the use of male C57BL/6 mice. Animal care and use was monitored by the University Animal Care and Use Committee to assure compliance with the provisions of Federal Regulations and the NIH `Guide for the Care and Use of Laboratory Animals’. All mice were housed in the Thomas Jefferson University Laboratory Animal Services Facility which is accredited by the American Association for the Accreditation of Laboratory Animal Care and complies with NIH standards.

Experimental design

Seventy-four (n = 74) adult male mice at approximately 8 weeks of age (weighing 22 to 24 g), including strains of C57BL/6 wild-type mice (Charles River, Wilmington, MA, USA) or CB 2 R knockout B6.129P2-Cnr2 tm1Dgen/J mice (Taconic, Hudson, NY, USA) were randomly assigned to undergo controlled cortical impact injury (n = 66) or serve as craniotomy controls (control) (n = 8). There were three study arms to determine the effects of CB 2 R modulation on genes and proteins expression for primary vascular inflammatory markers (TNF-α, ICAM, iNOS, and BBB permeability) which included: (1) CCI injury over time compared to craniotomy, (2) CB 2 R agonists and CB 2 R antagonist compared to vehicle-treated mice, and (3) CB 2 R knockout (CB 2 R KO) CCI groups with and without JWH-133 compared to wild-type CCI (Figure 1). Endpoints for CCI time course experiments were at either 6 hours (n = 4) or 1 (n = 10), 2 (n = 7), or 3 (n = 5) days after CCI injury. Two administrations of synthetic selective CB 2 R agonists, 0-1966 (n = 8) or JWH-133 (n = 12), a selective CB 2 R antagonist, SR144528 (n = 4), or vehicle solution (n = 10) were administered to wild-type CCI mice as described below. To determine the selectivity for the CB 2 R, knockout mice lacking the CB 2 R were treated with a CB 2 R agonist JWH-133 (n = 3) and 0-1966 (n = 4) or vehicle (n = 9) and compared to wild-type CCI mice (n = 8). Controls underwent all surgical procedures including an equal time of isoflurane exposure, buprenorphine injection, incision and craniotomy but were not subjected to CCI injury, did not receive treatment or vehicle, and were euthanized at 3 days post-operatively. All surgeries and experimental post-mortem procedures were performed so that, within each cohort of mice, craniotomy or vehicle-treated control groups were run in parallel with their respective experimental groups to insure consistent environmental conditions. On an annual basis, there is a 6 to 8% mortality rate for our CCI injury model due to the formation of fatal hematomas or cerebrovascular blood clots. Controlled cortical impact injury (CCI) injury resulted in a loss of 6 of the original 66experimental CCI mice (9% mortality rate) equally distributed among groups, and final group sizes were reported for each experimental outcome (see figures).

Figure 1 Experimental design flowchart showing experimental groups, endpoints, and outcome measures under each experimental arm: (1) CCI injured groups over time compared to craniotomy (control), (2) CCI injured mice treated with vehicle, cannabinoid receptor type 2 (CB 2 R) agonist, (*0-1966 or **JWH-133 (JWH)), or CB 2 R antagonist (SR144528), and (3) wild-type CCI mice treated with vehicle or JWH, compared to CB 2 R knockout CCI injured mice with and without JWH. Full size image

Traumatic brain injury

Traumatic brain injury was induced using CCI injury, a highly reproducible non-penetrating brain injury model [31]. Mice were injured using methods previously described by our laboratory [19],[23],[32]. Anesthesia was induced with 3% isoflurane and maintained throughout the procedure at a dose of 2.5% isoflurane. Prior to the start of the procedure, all mice were injected with short-acting buprenorphine (0.01 cc subcutaneous) for acute post-operative pain control. A right-sided 4 mm craniotomy was performed at 1 mm posterior to bregma exposing the mouse somatosensory cortex. A rounded aluminum 3 mm diameter stereotaxic impactor tip (MyNeuroLab, St. Louis, MO, USA) was used to produce a cortical injury at a 1.0 mm depth, 3 m/s velocity, and 100 msec contact time. Following injury, the bone flap was sealed with permanent cyanoacrylate-based fast-acting adhesive closures and the skin was closed with 6.0 silk sutures. Post-operative care included warming with indirect heat from a heat lamp until ambulation resumed, and unlimited access to food and water. Brain and core body temperature were maintained at 37 ± 0.5 °C throughout the procedure and monitored with temporalis muscle and rectal temperature probes to avoid the neuroprotective effects of anesthesia-induced hypothermia.

Treatment administration

Stimulation of the CB 2 receptor was performed using agonists 0-1966 (Organix Inc., Woburn, MA, USA) or JWH-133 (Tocris Bioscience, Minneapolis, MN, USA). 0-1966 was used for the TNF and ICAM PCR experiments, while JWH-133 was used in all other experiments (Figure 1). The CB 2 R agonist was switched to JWH-133 in the later experiments in this study because it had the same selectivity profile for CB 2 R as 0-1966 but with better solubility, was easier to administer, and was commercially available as a solution. 0-1966 was dissolved in a pure ethanol:emulphor:normal saline solution (1:1:18) resulting in a final concentration of 0.5 mg/mL. The CB 2 R agonist, 0-1966, also known as 0-1966A, is an analog of bicyclic resorcinols (dimethoxy-resorcinol-dimethylheptyl) and structurally similar to cannabidiol as described by Wiley et al. [33]. 0-1966 demonstrates 225-fold higher selectivity for the CB 2 R (Ki = 23 ± 2.1 nM) compared to CB 1 R (Ki = 5,055 ± 984 nM) [33]. JWH-133 selective CB 2 R agonist (Ki = 3.4 nM), in water-soluble emulsion Tocrisolve TM 100 (Tocris Catalog Number 1684) has appoximately 200-fold selectivity over CB1 receptors. The times for repeated intraperitoneal (ip) injections of 0-1966 (5 mg/kg) and JWH-133 (1 mg/kg) for the one-day endpoint were at either 2 or 18 hours post-CCI. The timing of treatment administrations were based on our previous studies [19]. Dosages were based both on preliminary dosing studies performed by our laboratory for our TBI model as well as on previous studies on models of stroke and spinal cord injury [19]-[22],[34]. Vehicle solution was prepared in an identical manner to include 0.2 mL of pure ethanol:emulphor:normal saline solution (1:1:18) minus the cannabinoid and administered at the same time points as 0-1966. The selective CB 2 R antagonist, SR144528, (Cayman Chemical, Ann Arbor, MI, USA) was dissolved in DMSO:emulphor:normal saline solution (1:1:18) and injected at 5 mg/kg at 2 and 18 hours post-CCI.

BBB assessment

Fluid-phase BBB permeability was assessed using sodium fluorescein (NaF) at 1 day post-CCI in wild-type, CB 2 R KO mice with and without a CB 2 R agonist (JWH-133) or controls as previously described by our laboratories [8]. NaF uptake assay was performed for 20 mice randomly divided into CCI subgroups euthanized at either 1 day (n = 236) or serving as controls (n = 4). CCI subgroups consisted of wild-type treated with vehicle or JWH-133, and CB 2 R KO receiving vehicle or JWH-133. Brain samples were run in duplicate experiments. We selected NaF to evaluate changes in BBB permeability due to its low molecular weight (376 Da) compared to others probes that bind to albumin such as Evans Blue, horseradish peroxidase (HRP), or IgG (≥67,000 Da). Thus, NaF is a more sensitive probe that allows for detection of smaller leaks in the barrier. Mice were injected ip with 100 μL of 10% NaF in PBS and the NaF was allowed to circulate for 10 minutes. Following administration of a lethal dose of sodium pentobarbital, cardiac blood was collected followed by transcardial perfusion with 15 mL of heparin (1,000 U/L) in PBS. Brains were sectioned into a left and right hemisphere and micro-dissected to separate the cerebral cortex, and processed immediately. To determine BBB permeability, tissues were weighed, homogenized in 1/10 dilution in PBS and centrifuged at 14,000 × g for 2 minutes. Five-hundred microliters of the clarified supernatant was transferred into 500 μL of 15% trichloroacetic acid and centrifuged at 10,000 × g for 10 minutes while the pellet was retained for RNA isolation. One hundred and twenty-five microliters of 5 N NaOH was added to 500 μL of the supernatant, and the amount of fluorescein for each sample was determined using standards ranging from 125 to 4,000 × g on a Cytofluor II fluorometer (PerSeptive Biosystems, Framingham, MA, USA). Serum levels of sodium fluorescein were assessed as previously described so that signals in CNS tissue samples could be normalized against the amount present in the circulation. NaF uptake into each brain region of interest is expressed as (ug/g NaF in cortex)/(μg NaF in serum).

RT-PCR

The pellet isolated during the BBB assessment outlined above was subsequently used for RNA isolation. Total RNA was extracted with the RNeasy Miniprep Kit (Qiagen, Valencia, CA, USA), reverse transcribed into cDNA with MMLV reverse transcriptase (Promega, Madison, WI, USA) and then measured by quantitative real-time PCR with gene-specific primers and probes [35]. IQ supermix and the iCycler iQ real-time detection system were also used for quantification (Bio-Rad Laboratories, Hercules, CA, USA). All samples were run in duplicate and compared to cDNA gene standards to determine copy numbers, which were normalized to the copy number of each sample’s housekeeping gene L13. Levels of mRNA are reported as the fold change in gene expression of normalized to the endogenous reference gene L13 and relative to the untreated, craniotomy controls.

Immunohistochemistry

Mice were administered a lethal dose of sodium pentobarbital (120 mg/kg, ip) and underwent cardiac perfusion with heparinized saline followed by 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde for 24 hours, then transferred to 30% sucrose for storage. Frozen sections were cut coronally with a cryostat at -24 °C (20 μm thick), and air dried overnight. Tissues were incubated in 10% NGS in O.3% Triton-100. Coronal brain sections were labeled using the following primary antibodies overnight at room temperature: (1) rabbit anti-mouse iNOS (1:200; Enzo Life Science, Farmingdale, NY, USA) and (2) rabbit anti-mouse ionized calcium-binding adaptor molecule-1 (Iba-1) (1:250; Wako Pure Chemical Industries, Richmond, VA, USA). Fluorescent secondary antibodies DyLight 488- or 549-conjugated AffiniPure Goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) were applied for 2 hours at room temperature. Negative control staining was performed by omitting the primary antibodies.

Statistical analyses

All statistical analyses were performed using the GraphPad Prism 5 software program (La Jolla, CA, USA). To determine differences between CCI injury and controls at 6 hour, 2 day and 3 day time points, differences between wild-type and knockout mice, and and differences between vehicle-treated, agonist-treated, and antagonist-treated groups, statistical comparisons were performed using a one-way ANOVA followed by Bonferroni post hoc analysis for experimental groups compared to control. Significance levels were set at P < 0.05 for all statistical analyses and results are reported as the mean and SEM.