Chemotherapy-induced peripheral neuropathy (CIPN) is one of the most prevalent and dose-limiting complications in chemotherapy patients. One identified mechanism underlying CIPN is neuroinflammation. Most of this research has been conducted in only male or female rodent models, making direct comparisons regarding the role of sex differences in the neuroimmune underpinnings of CIPN limited. Moreover, most measurements have focused on the dorsal root ganglia (DRG) and/or spinal cord, while relatively few studies have been aimed at characterizing neuroinflammation in the brain, for example the periaqueductal grey (PAG). The overall goals of the present study were to determine (1) paclitaxel-associated changes in markers of inflammation in the PAG and DRG in male and female C57Bl6 mice and (2) determine the effect of prophylactic administration of an anti-inflammatory cannabinoid, cannabigerol (CBG). In Experiment 1, male and female mice were treated with paclitaxel (8–32 mg/kg/injection, Days 1, 3, 5, and 7) and mechanical sensitivity was measured using Von Frey filaments on Day 7 (Cohort 1) and Day 14 (Cohort 2). Cohorts were euthanized on Day 8 or 15, respectively, and DRG and PAG were harvested for qPCR analysis of the gene expression of markers of pain and inflammation Aig1, Gfap, Ccl2, Cxcl9, Tlr4, Il6, and Calca. In Experiment 2, male and female mice were treated with vehicle or 10 mg/kg CBG i.p. 30 min prior to each paclitaxel injection. Mechanical sensitivity was measured on Day 14. Mice were euthanized on Day 15, and PAG were harvested for qPCR analysis of the gene expression of Aig1, Gfap, Ccl2, Cxcl9, Tlr4, Il6, and Calca. Paclitaxel produced a transient increase in potency to produce mechanical sensitivity in male versus female mice. Regarding neuroinflammation, more gene expression changes were apparent earlier in the DRG and at a later time point in the PAG. Also, more changes were observed in females in the PAG than males. Overall, sex differences were observed for most markers at both time points and regions. Importantly, in both the DRG and PAG, most increases in markers of neuroinflammation and pain occurred at paclitaxel doses higher than those associated with significant changes in the mechanical threshold. Two analytes that demonstrated the most compelling sexual dimorphism and that changed more in males were Cxcl9 and Ccl2, and Tlr4 in females. Lastly, prophylactic administration of CBG protected the male and female mice from increased mechanical sensitivity and female mice from neuroinflammation in the PAG. Future studies are warranted to explore how these sex differences may shed light on the mechanisms of CIPN and how non-psychoactive cannabinoids such as CBG may engage these targets to prevent or attenuate the effects of paclitaxel and other chemotherapeutic agents on the nervous system.

Although strokes are frequent and severe, treatment options are scarce. Plasminogen activators, the only FDA-approved agents for clot treatment (tissue plasminogen activators (tPAs)), are used in a limited patient group. Moreover, there are few approaches for handling the brain’s inflammatory reactions to a stroke. The orphan G protein-coupled receptor 55 (GPR55)’s connection to inflammatory processes has been recently reported; however, its role in stroke remains to be discovered. Post-stroke neuroinflammation involves the central nervous system (CNS)’s resident microglia activation and the infiltration of leukocytes from circulation into the brain. Additionally, splenic responses have been shown to be detrimental to stroke recovery. While lymphocytes enter the brain in small numbers, they regularly emerge as a very influential leukocyte subset that causes secondary inflammatory cerebral damage. However, an understanding of how this limited lymphocyte presence profoundly impacts stroke outcomes remains largely unclear. In this study, a mouse model for transient middle cerebral artery occlusion (tMCAO) was used to mimic ischemia followed by a reperfusion (IS/R) stroke. GPR55 inactivation, with a potent GPR55-specific antagonist, ML-193, starting 6 h after tMCAO or the absence of the GPR55 in mice (GPR55 knock out (GPR55ko)) resulted in a reduced infarction volume, improved neurological outcomes, and decreased splenic responses. The inhibition of GPR55 with ML-193 diminished CD4+T-cell spleen egress and attenuated CD4+T-cell brain infiltration. Additionally, ML-193 treatment resulted in an augmented number of regulatory T cells (Tregs) in the brain post-tMCAO. Our report offers documentation and the functional evaluation of GPR55 in the brain–spleen axis and lays the foundation for refining therapeutics for patients after ischemic attacks.

Despite advancements in dental pain management, one of the most common reasons for emergency dental care is orofacial pain. Our study aimed to determine the effects of non-psychoactive Cannabis constituents in the treatment of dental pain and related inflammation. We tested the therapeutic potential of two non-psychoactive Cannabis constituents, cannabidiol (CBD) and β-caryophyllene (β-CP), in a rodent model of orofacial pain associated with pulp exposure. Sham or left mandibular molar pulp exposures were performed on Sprague Dawley rats treated with either vehicle, the phytocannabinoid CBD (5 mg/kg i.p.) or the sesquiterpene β-CP (30 mg/kg i.p.) administered 1 h pre-exposure and on days 1, 3, 7, and 10 post-exposure. Orofacial mechanical allodynia was evaluated at baseline and post-pulp exposure. Trigeminal ganglia were harvested for histological evaluation at day 15. Pulp exposure was associated with significant orofacial sensitivity and neuroinflammation in the ipsilateral orofacial region and trigeminal ganglion. β-CP but not CBD produced a significant reduction in orofacial sensitivity. β-CP also significantly reduced the expression of the inflammatory markers AIF and CCL2, while CBD only decreased AIF expression. These data represent the first preclinical evidence that non-psychoactive cannabinoid-based pharmacotherapy may provide a therapeutic benefit for the treatment of orofacial pain associated with pulp exposure.

Current treatments for stroke, which account for 6.5 million global deaths annually, remain insufficient for treatment of disability and mortality. One targetable hallmark of stroke is the inflammatory response following infarct, which leads to significant damage post-infarct. Cannabinoids and their endogenous targets within the CNS have emerged as potential treatments for neuroinflammatory indications. We and others have previously shown that synthetic agonists of the cannabinoid CB2 receptor reduce infarct size and microglial activation in rodent models of stroke. The non-cannabinoid receptor mediated effects of the phytocannabinoid cannabidiol (CBD) have also shown effectiveness in these models. The present aim was to determine the single and combined effects of the cannabis-derived sesquiterpene and putative CB2 receptor agonist β-caryophyllene (BCP) and CBD on permanent ischemia without reperfusion using a mouse model of photothrombosis. Because BCP and CBD likely work through different sites of action but share common mechanisms of action, we sought to determine whether combinations of BCP and CBD were more potent than either compound alone. Therefore we determined the effect of BCP (3–30 mg/kg IP) and CBD (3–30 mg/kg IP), given alone or in combination (30:3, 30:10, and 30:30 BCP:CBD), on infarct size, microglial activation, and motor performance.

The molecular substrates underlying cocaine reinforcement and addiction have been studied for decades, with a primary focus on signaling molecules involved in modulation of neuronal communication. Brain-derived neurotrophic factor (BDNF) is an important signaling molecule involved in neuronal dendrite and spine modulation. Methyl CpG binding protein 2 (MeCP2) binds to the promoter region of BDNF to negatively regulate its expression and cocaine can recruit MeCP2 to alter the expression of genes such as BDNF that are involved in synaptic plasticity. For several decades, BDNF has been implicated in mediating synaptic plasticity associated with cocaine abuse, and most studies report that neurons are the primary source for BDNF production in the brain. The current study assessed the effects of intravenous cocaine self-administration on microglial activation, and MeCP2 and BDNF expression in reward regions of the brain in vivo, as well as determined specific effects of cocaine exposure on MeCP2 and BDNF expression in human primary neurons and microglia. The results from this study highlight a distinct molecular pathway in microglia through which cocaine increases BDNF, including the phosphorylation of MeCP2 its subsequent translocation from the nucleus to the cytosol, which frees the BDNF promoter and permits its transcriptional activation. Results from these studies show for the first time that cocaine self-administration increases microglial activation, and that microglial MeCP2 is a sensitive target of cocaine resulting in increased release of BDNF from microglia, and possibly contributing to cocaine-induced synaptic plasticity.

We evaluated the effects of the non-psychoactive cannabinoid cannabidiol (CBD) on the inflammatory response and recovery of function following spinal cord injury (SCI). Female C57Bl/6 mice were exposed to spinal cord contusion injury (T9-10) and received vehicle or CBD (1.5 mg/kg IP) injections for 10 weeks following injury. The effect of SCI and CBD treatment on inflammation was assessed via microarray, qRT-PCR and flow cytometry. Locomotor and bladder function and changes in thermal and mechanical hind paw sensitivity were also evaluated. There was a significant decrease in pro-inflammatory cytokines and chemokines associated with T-cell differentiation and invasion in the SCI-CBD group as well as a decrease in T cell invasion into the injured cord. A higher percentage of SCI mice in the vehicle-treated group (SCI-VEH) went on to develop moderate to severe (0-65.9% baseline thermal threshold) thermal sensitivity as compared with CBD-treated (SCI-CBD) mice. CBD did not affect recovery of locomotor or bladder function following SCI. Taken together, CBD treatment attenuated the development of thermal sensitivity following spinal cord injury and this effect may be related to protection against pathological T-cell invasion.

Kimura's disease (KD) is a rare condition, with only a few cases reported to date, mainly in Asian patients. We herein present the case of a 48-year-old man with KD who presented with recurrent masses in the right parotid gland and neck region over a 15-year period. The masses were not accompanied by pain, or significant functional or neurosensory dysfunction. The results of the laboratory tests revealed an increased eosinophil count and markedly elevated serum IgE levels. On magnetic resonance imaging examination, a widespread abnormal signal was detected in the area of the lesions; the contrast-enhanced scan revealed inhomogeneous enhancement, with partial involvement of the sternocleidomastoid muscle and the parotid gland. The patient underwent surgical resection of the right parotid and neck masses, and the postoperative pathological examination revealed eosinophilic hyperplastic lymphogranuloma, also referred to as KD. This presented case and review of the relevant literature aim to improve our understanding of KD in order to increase the accuracy of diagnosis, reduce the misdiagnosis rate and ensure proper treatment of this rare disease.

Modulation of the endocannabinoid system has been shown to have a significant impact on outcomes in animal models of stroke. We have previously reported a protective effect of the CB1 antagonist, SR141716A, in a transient reperfusion mouse model of cerebral ischemia. This protective effect was in part mediated by activation of the 5HT1A receptor. Here we have examined its effect in a mouse model of permanent ischemia induced by photoinjury. The CB1 antagonist was found to be protective in this model. As was the case following transient ischemia reperfusion, SR141716A (5mg/kg) resulted in smaller infarct fractions and stroke volumes when utilized both as a pretreatment and as a post-treatment. In contrast to the effect in a transient ischemia model, the pretreatment effect did not depend on the 5HT1A receptor. Neurological function correlated favorably to the reduction in stroke size when SR141716A was given as a pretreatment. With the incidence of stroke predicted to rise in parallel with an ever aging population, understanding mechanisms underlying ischemia and therapeutics remains a paramount goal of research.

Epi‐illuminescence intravital fluorescence microscopy has been employed to study leukocyte‐endothelial interactions in a number of brain pathologies. Historically, dyes such as Rhodamine 6G have been injected intravenously. However, intravenous injections can predispose experimental animals to a multitude of complications and requires a high degree of technical skill. Here, we study the efficacy of injecting Rhodamine 6G into the peritoneum (IP) for the purpose of analyzing leukocyte‐endothelial interactions through a cranial window during real time intravital microscopy. After examining the number of rolling and adherent leukocytes through a cranial window, we found no advantage to the intravenous injection (IV). Additionally, we tested blood from both routes of injection by flow cytometry to gain a very precise picture of the two methods. The two routes of administration failed to show any difference in the ability to detect cells. The study supports the notion that IP Rhodamine 6G works as efficaciously as IV and should be considered a viable alternative in experimental design for investigations employing intravital microscopy. Facilitated intravital studies will allow for more exploration into cerebral pathologies and allow for more rapid translation from the laboratory to the patient with less chance of experimental error from failed IV access. Microsc. Res. Tech. 78:894–899, 2015. © 2015 Wiley Periodicals, Inc.

Abstract CB2 agonists have beneficial effects in experimental autoimmune encephalomyelitis (EAE). Our previous studies showed that the CB2 selective agonist O-1966 inhibited EAE development and leukocyte rolling and adhesion to CNS microvasculature. Here we report that Gp1a, a highly selective CB2 agonist, inhibits EAE development following early treatment and improves recovery following late treatment. Gp1a treatment resulted in lower levels of demyelination, axonal loss, and immune cell infiltration. Our results indicate that early Gp1a treatment inhibits Th1 and Th17 and promotes Th2 and Treg differentiation both in vivo and in vitro. We confirmed that Gp1a inhibited Th1 and Th17 differentiation both in non-polarizing and polarizing conditions. Following later treatment, Gp1a reduced the accumulation of pathogenic immune cells in the CNS, which was associated with reductions in the expression of CNS chemokines and adhesion molecules and of chemokine receptors in T cells. We concluded that at least two mechanisms were involved in the beneficial effects of Gp1a in EAE, an early effect on Th1/Th17 differentiation and a later effect on the accumulation of pathogenic immune cells in the CNS. The combined effect of CB2 agonists on Th17 differentiation and immune cell accumulation into the CNS, suggest the CB2 receptors are potential therapeutic targets in neuroinflammation.

CB2, the cannabinoid receptor expressed primarily on hematopoietic cells and activated microglia, mediates the immunoregulatory functions of cannabinoids. The involvement of CB2 in EAE has been demonstrated by using both endogenous and exogenous ligands. We showed previously that CB2 selective agonists inhibit leukocyte rolling and adhesion to CNS microvasculature and ameliorate clinical symptom in both chronic and remitting-relapsing EAE models. Here we showed that Gp1a, a highly selective CB2 agonist, with a four log higher affinity for CB2 than CB1, reduced clinical scores and facilitated recovery in EAE in conjunction with long term reduction in demyelination and axonal loss. We also established that Gp1a affected EAE through at least two different mechanisms, i.e. an early effect on Th1/Th17 differentiation in peripheral immune organs, and a later effect on the accumulation of pathogenic immune cells in the CNS, associated with reductions in the expression of CNS and T cell chemokine receptors, chemokines and adhesion molecules. This is the first report on the in vivo CB2-mediated Gp1a inhibition of Th17/Th1 differentiation. We also confirmed the Gp1a-induced inhibition of Th17/Th1 differentiation in vitro, both in non-polarizing and polarizing conditions. The CB2-induced inhibition of Th17 differentiation is highly relevant in view of recent studies emphasizing the importance of pathogenic self-reactive Th17 cells in EAE/MS. In addition, the combined effect on Th17 differentiation and immune cell accumulation into the CNS, emphasize the relevance of CB2 selective ligands as potential therapeutic agents in neuroinflammation.

Coagulation factor XI (FXI) plays an important part in both venous and arterial thrombosis, rendering FXIa a potential target for the development of antithrombotic therapy. The kunitz protease inhibitor (KPI) domain of protease nexin-2 (PN2) is a potent, highly specific inhibitor of FXIa, suggesting its possible role in the inhibition of FXI-dependent thrombosis in vivo. Therefore, we examined the effect of PN2KPI on thrombosis in the murine carotid artery and the middle cerebral artery. Intravenous administration of PN2KPI prolonged the clotting time of both human and murine plasma, and PN2KPI inhibited FXIa activity in both human and murine plasma in vitro. The intravenous administration of PN2KPI into WT mice dramatically decreased the progress of FeCl3-induced thrombus formation in the carotid artery. After a similar initial rate of thrombus formation with and without PN2KPI treatment, the propagation of thrombus formation after 10 minutes and the amount of thrombus formed were significantly decreased in mice treated with PN2KPI injection compared with untreated mice. In the middle cerebral artery occlusion model, the volume and fraction of ischemic brain tissue were significantly decreased in PN2KPI-treated compared with untreated mice. Thus, inhibition of FXIa by PN2KPI is a promising approach to antithrombotic therapy.

We reported previously that both a cannabinoid receptor 2 (CB2R) agonist and a cannabinoid receptor 1 (CB1R) antagonist were protective in the treatment of transient middle cerebral artery occlusion/reperfusion injury (MCAO/R) and that they acted in a synergistic manner when administered in combination. The goal of the current study was to determine which of the potential cannabinoid receptors participate in the protective effects of this drug combination in a mouse model of MCAO/R. The effects of administration of the CB2R agonist/CB1R antagonist combination on infarct size and cerebral blood flow during a 1-h occlusion were tested in CB1R-deficient animals, CB2R-deficient animals, and animals treated with capsazepine, the antagonist for the vanilloid receptor type I (TRPV1) and WAY100135, the antagonist for the hydroxytryptamine1A receptor (5-HT1A). The protective effect of the CB2R agonist/CB1R antagonist combination on infarct size was not influenced by the absence of the CB1R nor by blocking the TRPV1 receptor, but was attenuated by the absence of CB2R and by blocking the 5-HT1A receptor. Increases in cerebral blood flow and arteriolar diameter were also found to be independent of the CB1R and TRPV1 receptor. In conclusion, administration of the CB2R agonist/CB1R antagonist combination causes a significant reduction in infarct size in the MCAO/R model. The protective effect involves both the CB2R and the 5-HT1A receptor. Neither the CB1R nor the TRPV1 receptors appear to participate in this response.

Previous studies showed that cannabinoids have anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE), but the mechanism is elusive. In the present study, we investigated the effects of Gp1a (a new CB2 selective agonist) in EAE. EAE developed in a similar way in both vehicle and Gp1a treatment group, but mice treated with Gp1a recovered faster than the vehicle group. Cellular analysis was performed in spleen, brain and spinal cord. In spleen, there was no difference in numbers of CD3+ T cells, Th1 and Th17 cells, or macrophages. In contrast, CNS mononuclear cell analysis showed fewer CD3+ T cells, Th1 and Th17 cells, macrophages and activated microglia in spinal cord but not in brainin the Gp1a treatment group. Accordingly, for the Gp1a-treated group, histology studies also showed fewer CD3+ T cells, CD11b+ macrophages/microglia and less Iba-1 expression in spinal cord but not in brain. In vivo cell proliferation as measured through BrdU incorporation in CD3+CD45hi, CD11b+CD45hi and CD11b+CD45int mononuclear cells was not affected by Gp1a treatment. We hypothesize that Gp1a inhibits cell migration to the CNS. We also analyzed gene expression in brain and spinal cord following EAE development and found lower levels of VCAM-1, CXCL-10 and MMP9 in spinal cord. We also found that Gp1a inhibits MMP9 and TNFα production in BV.2 cells (a microglia cell line). We conclude that Gp1a improves EAE recovery through inhibiting immune cell migration into CNS.

The kunitz protease inhibitor domain of PN2 (PN2KPI) is a potent and specific inhibitor (K-i 0.5-2 nM) of factor XIa (FXIa) and inhibits cerebrovascular thrombosis in mice. To determine whether the antithrombotic properties of PN2KPI arise from its FXIa-inhibitory activity, we have now prepared mutant forms of PN2KPI. Mutations at the P1 (Arg(15)) site in combination with P2' (Met(17)) mutations profoundly affect inhibition of FXIa, plasmin, kallikrein, factor Xa and thrombin. The mutant proteins PN2KPI-(RK)-K-15, -(MK)-K-17, -(RK)-K-15,(MK)-K-17 and -(RK)-K-15,(MR)-R-17 lost inhibitory activity against FXIa (K-i 34, 94, 3081 and 707 nM, respectively) and kallikrein (no inhibition) and gained inhibitory activity against plasmin (K-i 108, 7, 8 and 8 nM, respectively). The intravenous administration of rPN2KPI into mice dramatically decreased thrombus formation in a murine model of FeCl3-induced carotid injury, whereas rPN2KPI-(RK)-K-15,(MK)-K-17 failed to inhibit thrombus formation. Molecular modelling studies showed that fine structural variations explain the observed functional differences in FXIa and plasmin inhibition. PN2KPI has potent antithrombotic activity due to its specific FXIa anticoagulant activity, whereas PN2KPI-(RK)-K-15,(MK)-K-17 and PN2KPI-(RK)-K-15,(MR)-R-17 have potent antifibrinolytic (antiplasmin) activity without anticoagulant or antithrombotic activity.

The goal of the current investigation was to evaluate the mechanisms through which administration of a selective cannabinoid-2 (CB2) agonist (O-1966) modifies inflammatory responses and helps to improve function following spinal cord injury. A comparison of motor function, autonomic function, and inflammatory responses was made between animals treated with O-1966 (5 mg/kg IP) and animals treated with vehicle 1 h and 24 h following contusion injury to the spinal cord. Motor function was significantly improved in the treated animals at each time point during the 14 days of evaluation. The percentage of animals able to spontaneously void their bladder was also greater over the entire study period in the group treated with the selective CB2 agonist. Seven days following injury there was a significant reduction in both hematopoietic and myeloid cell invasion of the spinal cord, and a reduction in the number of immunoreactive microglia. The results of the evaluation of chemokine/cytokine expression and inflammatory cell invasion also demonstrated a significant effect of treatment on inflammatory reactions following injury. Two days after injury, animals treated with O-1966 had significant reductions in CXCL-9 and CXCL-11, and dramatic reductions in IL-23p19 expression and its receptor IL-23r. Treatment with O-1966 also caused inhibition of toll-like receptor expression (TLR1, TLR4, TLR6 and TLR7) following injury. These results demonstrate that the improvement in motor and autonomic function resulting from treatment with a selective CB2 agonist is associated with a significant effect on inflammatory responses in the spinal cord following injury.