Cannabinoid receptor type-2 stimulation, blockade, and deletion alter the vascular inflammatory responses to traumatic brain injury

Abstract

Background: Immunomodulatory therapies have been identified as interventions for secondary injury after traumatic brain injury (TBI). The cannabinoid receptor type-2 (CB2R) is proposed to play an important, endogenous role in regulating inflammation. The effects of CB2R stimulation, blockade, and deletion on the neurovascular inflammatory responses to TBI were assessed.

Methods: Wild-type C57BL/6 or CB2R knockout mice were randomly assigned to controlled cortical impact (CCI) injury or to craniotomy control groups. The effects of treatment with synthetic, selective CB2R agonists (0-1966 and JWH-133), a selective CB2R antagonist, or vehicle solution administered to CCI groups were assessed at 1-day after injury. Changes in TNF-α, intracellular adhesion molecule (ICAM-1), inducible nitric oxide synthase (iNOS), macrophage/microglial ionized calcium-binding adaptor molecule, and blood-brain-barrier (BBB) permeability were assessed using ELISA, quantitative RT-PCR, immunohistochemistry, and fluorometric analysis of sodium fluorescein uptake. CB2R knockouts and wild-type mice with CCI injury were treated with a CB2R agonist or vehicle treatment.

Results: TNF-α mRNA increased at 6 hours and 1 to 3 days after CCI; a CB2R antagonist and genetic knockout of the CB2R exacerbated TNF-α mRNA expression. Treatment with a CB2R agonist attenuated TNF-α protein levels indicating post-transcriptional mechanisms. Intracellular adhesion molecule (ICAM-1) mRNA was increased at 6 hours, and at 1 to 2 days after CCI, reduced in mice treated with a CB2R agonist, and increased in CB2R knockout mice with CCI. Sodium fluorescein uptake was increased in CB2R knockouts after CCI, with and without a CB2R agonist. iNOS mRNA expression peaked early (6 hours) and remained increased from 1 to 3 days after injury. Treatment with a CB2R agonist attenuated increases in iNOS mRNA expression, while genetic deletion of the CB2R resulted in substantial increases in iNOS expression. Double label immunohistochemistry confirmed that iNOS was expressed by macrophage/microglia in the injured cortex.

Conclusion: Findings demonstrate that the endogenous cannabinoid system and CB2R play an important role in regulating inflammation and neurovascular responses in the traumatically injured brain. CB2R stimulation with two agonists (0-1966 and JWH-133) dampened post-traumatic inflammation, while blockade or deletion of the CB2R worsened inflammation. Findings support previous evidence that modulating the CB2R alters infiltrating macrophages and activated resident microglia. Further investigation into the role of the CB2R on specific immune cell populations in the injured brain is warranted.

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.

Figure 2

TNF-α mRNA in the injured cortex measured using quantitative real-time PCR (A-C) and TNF-α protein concentration using an enzyme-linked immunosorbent assay (D). (A) TNF-α mRNA at 6 hours, 2 and 3 days after CCI injury compared to craniotomy (control), *P < 0.05 and ***P < 0.001. (B) TNF-α mRNA at 1 day after CCI injury in wild-type mice treated with a vehicle solution (vehicle), cannabinoid receptor type-2 (CB₂) agonist (0-1966) or CB₂ receptor antagonist (SR144528) compared to vehicle control, ***P < 0.001. (C) TNF-α mRNA at 1 day after CCI injury in CB₂ knockout (CB₂ KO) and wild-type mice compared to control, ***P < 0.001, and ##P < 0.01 compared to wild-type mice. (D) TNF-α protein concentration at 1 day post-CCI in vehicle-treated and CB₂ agonist-treated (JWH-133) mice compared to control and vehicle, respectively, **P < 0.01.

Figure 3

Intracellular adhesion molecule (ICAM) mRNA expression in the injured cortex measured using quantitative real-time PCR. (A) ICAM mRNA at 6 hours, 2 and 3 days after CCI injury compared to control, *P < 0.05. (B) ICAM mRNA at 1 day after CCI injury in wild-type mice treated with a cannabinoid type-2 (CB₂) agonist (0-1966) and CB₂ antagonist (SR144528) compared to vehicle-treated CCI controls, **P < 0.01. (C) ICAM mRNA at 1 day after CCI injury in CB₂ knockout (CB₂ −/−) mice and wild-type mice compared to control, **P < 0.01 and ***P < 0.001; ICAM mRNA post-CCI in CB₂ knockout (CB₂ −/−) mice compared to wild-type mice, #P < 0.05.

Figure 4

Blood-brain-barrier permeability assessment using sodium fluorescein (NaF) uptake in the injured cortex. NaF uptake for wild-type CCI injured mice treated with vehicle compared to JWH-133 (JWH), *P < 0.05 and cannabinoid type-2 knockout CCI mice (CB₂R KO) treated with vehicle or JWH, **P < 0.01 compared to wild-type vehicle-treated CCI mice (vehicle).

Figure 5

Inducible nitric oxide synthase (iNOS) mRNA in the injured cortex. (A) iNOS mRNA at 6 hours, 1 and 3 days after CCI injury compared to control, *P < 0.05, **P < 0.01, and ***P < 0.001. (B) iNOS mRNA at 1 day after CCI injury in mice compared to control, P < 0.001, and CCI mice treated with JWH-133 (JWH) compared to vehicle, ###P < 0.001. (C) iNOS mRNA in CB₂ knockout (CB₂ KO) mice compared to wild-type mice at 1 day after CCI injury, ***P < 0.001, and CB₂ KO treated with JWH and 0-1966 (1966) compared to CB₂ KO mice ###P < 0.001.

Figure 6

Macrophage/microglial marker, Iba-1, and inducible nitric oxide synthase (iNOS) immunofluorescence in the remaining cortical area adjacent to the contusion. Image shows (A) Iba-1 positive cells (green), (B) iNOS positive cells (red), and (C) merged image showing Iba-1 co-localized with iNOS positive cells with a retracted, amoeboid morphology (yellow).