ABHD6 and MAGL control 2-AG levels in the PAG and allodynia in a CSD-induced periorbital model of headache.

Introduction: The high prevalence and severe symptoms of migraines in humans emphasizes the need to identify underlying mechanisms that can be targeted for therapeutic benefit. Clinical Endocannabinoid Deficiency (CED) posits that reduced endocannabinoid tone may contribute to migraine development and other neuropathic pain conditions. While strategies that increase levels of the endocannabinoid n-arachidonoylethanolamide have been tested, few studies have investigated targeting the levels of the more abundant endocannabinoid, 2-arachidonoylgycerol, as an effective migraine intervention. Methods: Cortical spreading depression was induced in female Sprague Dawley rats via KCl (potassium chloride) administration, followed by measures of endocannabinoid levels, enzyme activity, and neuroinflammatory markers. Efficacy of inhibiting 2-arachidonoylglycerol hydrolysis to mitigate periorbital allodynia was then tested using reversal and prevention paradigms. Results: We discovered reduced 2-arachidonoylglycerol levels in the periaqueductal grey associated with increased hydrolysis following headache induction. Pharmacological inhibition of the 2-arachidonoylglycerol hydrolyzing enzymes, α/ß-hydrolase domain-containing 6 and monoacylglycerol lipase reversed and prevented induced periorbital allodynia in a cannabinoid receptor-dependent manner. Discussion: Our study unravels a mechanistic link between 2-arachidonoylglycerol hydrolysis activity in the periaqueductal grey in a preclinical, rat model of migraine. Thus, 2-arachidonoylglycerol hydrolysis inhibitors represent a potential new therapeutic avenue for the treatment of headache.

Depletion of Endothelial-Derived 2-AG Reduces Blood-Endothelial Barrier Integrity via Alteration of VE-Cadherin and the Phospho-Proteome.

Mounting evidence supports the role of the endocannabinoid system in neurophysiology, including blood-brain barrier (BBB) function. Recent work has demonstrated that activation of endocannabinoid receptors can mitigate insults to the BBB during neurological disorders like traumatic brain injury, cortical spreading depression, and stroke. As alterations to the BBB are associated with worsening clinical outcomes in these conditions, studies herein sought to examine the impact of endocannabinoid depletion on BBB integrity. Barrier integrity was investigated in vitro via bEnd.3 cell monolayers to assess endocannabinoid synthesis, barrier function, calcium influx, junctional protein expression, and proteome-wide changes. Inhibition of 2-AG synthesis using DAGLα inhibition and siRNA inhibition of DAGLα led to loss of barrier integrity via altered expression of VE-cadherin, which could be partially rescued by exogenous application of 2-AG. Moreover, the deleterious effects of DAGLα inhibition on BBB integrity showed both calcium and PKC (protein kinase C)-dependency. These data indicate that disruption of 2-AG homeostasis in brain endothelial cells, in the absence of insult, is sufficient to disrupt BBB integrity thus supporting the role of the endocannabinoid system in neurovascular disorders.

Inhibition of HSP90 Preserves Blood-Brain Barrier Integrity after Cortical Spreading Depression.

Cortical spreading depression (CSD) is a pathophysiological mechanism underlying headache disorders, including migraine. Blood-brain barrier (BBB) permeability is increased during CSD. Recent papers have suggested that heat shock proteins (HSP) contribute to the integrity of the blood-brain barrier. In this study, the possible role of HSP90 in CSD-associated blood-brain barrier leak at the endothelial cell was investigated using an in vitro model, for the blood-endothelial barrier (BEB), and an in vivo model with an intact BBB. We measured barrier integrity using trans endothelial electric resistance (TEER) across a monolayer of rodent brain endothelial cells (bEnd.3), a sucrose uptake assay, and in situ brain perfusion using female Sprague Dawley rats. CSD was induced by application of 60 mM KCl for 5 min in in vitro experiments or cortical injection of KCl (1 M, 0.5 µL) through a dural cannula in vivo. HSP90 was selectively blocked by 17-AAG. Our data showed that preincubation with 17-AAG (1 µM) prevented the reduction of TEER values caused by the KCl pulse on the monolayer of bEnd.3 cells. The elevated uptake of 14C-sucrose across the same endothelial monolayer induced by the KCl pulse was significantly reduced after preincubation with HSP90 inhibitor. Pre-exposure to 17-AAG significantly mitigated the transient BBB leak after CSD induced by cortical KCl injection as determined by in situ brain perfusion in female rats. Our results demonstrated that inhibition of HSP90 with the selective agent 17-AAG reduced CSD-associated BEB/BBB paracellular leak. Overall, this novel observation supports HSP90 inhibition mitigates KCl-induced BBB permeability and suggests the development of new therapeutic approaches targeting HSP90 in headache disorders.

Sex differences in the expression of the endocannabinoid system within V1M cortex and PAG of Sprague Dawley rats.

BACKGROUND: Several chronic pain disorders, such as migraine and fibromyalgia, have an increased prevalence in the female population. The underlying mechanisms of this sex-biased prevalence have yet to be thoroughly documented, but could be related to endogenous differences in neuromodulators in pain networks, including the endocannabinoid system. The cellular endocannabinoid system comprises the endogenous lipid signals 2-AG (2-arachidonoylglycerol) and AEA (anandamide); the enzymes that synthesize and degrade them; and the cannabinoid receptors. The relative prevalence of different components of the endocannabinoid system in specific brain regions may alter responses to endogenous and exogenous ligands. METHODS: Brain tissue from naïve male and estrous staged female Sprague Dawley rats was harvested from V1M cortex, periaqueductal gray, trigeminal nerve, and trigeminal nucleus caudalis. Tissue was analyzed for relative levels of endocannabinoid enzymes, ligands, and receptors via mass spectrometry, unlabeled quantitative proteomic analysis, and immunohistochemistry. RESULTS: Mass spectrometry revealed significant differences in 2-AG and AEA concentrations between males and females, as well as between female estrous cycle stages. Specifically, 2-AG concentration was lower within female PAG as compared to male PAG (*p = 0.0077); female 2-AG concentration within the PAG did not demonstrate estrous stage dependence. Immunohistochemistry followed by proteomics confirmed the prevalence of 2-AG-endocannabinoid system enzymes in the female PAG. CONCLUSIONS: Our results suggest that sex differences exist in the endocannabinoid system in two CNS regions relevant to cortical spreading depression (V1M cortex) and descending modulatory networks in pain/anxiety (PAG). These basal differences in endogenous endocannabinoid mechanisms may facilitate the development of chronic pain conditions and may also underlie sex differences in response to therapeutic intervention.

Cannabinoid-2 Agonism with AM2301 Mitigates Morphine-Induced Respiratory Depression.

Introduction: An escalating number of fatalities resulting from accidental opioid overdoses typically attributed to respiratory depression continue to define the opioid epidemic. Opioid respiratory depression results from a decrease in reflexive inspiration within the preBötzinger complex in the brainstem. Objective: Cannabinoid receptor agonism is reported to enhance opioid analgesia, yet whether cannabinoids enhance or inhibit opioid-induced respiratory depression is unknown. Methods: Studies herein sought to define the roles of cannabinoid-1 receptor (CB1R) and cannabinoid-2 receptor (CB2R) on respiratory depression using selective agonists alone and in combination with morphine in male mice. Results: Using whole body plethysmography, the nonselective CB1R and CB2R agonist (Δ9-tetrahydrocannabinol) and the CB1R synthetic cannabinoid, AM356, induced respiratory depression, whereas the well-published selective CB2 agonist, JWH 133, and the novel CB2 agonist (AM2301) did not. Moreover, a selective CB2R agonist (AM2301) significantly attenuated morphine sulfate-induced respiratory depression. Conclusion: Notably, findings suggest that attenuation of opioid-induced respiratory depression relies on CB2R activation, supporting selective CB2R agonism as an opioid adjunct therapy.

An underrepresented majority: A systematic review utilizing allodynic criteria to examine the present scarcity of discrete animal models for episodic migraine.

BACKGROUND: Despite increasing evidence differentiating episodic and chronic migraine, little work has determined how currently utilized animal models of migraine best represent each distinct disease state. AIM: In this review, we seek to characterize accepted preclinical models of migraine-like headache by their ability to recapitulate the clinical allodynic features of either episodic or chronic migraine. METHODS: From a search of the Pu bMed database for "animal models of migraine", "headache models" and "preclinical migraine", we identified approximately 80 recent (within the past 20 years) publications that utilized one of 10 different models for migraine research. Models reviewed fit into one of the following categories: Dural KCl application, direct electrical stimulation, nitroglycerin administration, inflammatory soup injection, CGRP injection, medication overuse, monogenic animals, post-traumatic headache, specific channel activation, and hormone manipulation. Recapitulation of clinical features including cephalic and extracephalic hypersensitivity were evaluated for each and compared. DISCUSSION: Episodic migraineurs comprise over half of the migraine population, yet the vast majority of current animal models of migraine appear to best represent chronic migraine states. While some of these models can be modified to reflect episodic migraine, there remains a need for non-invasive, validated models of episodic migraine to enhance the clinical translation of migraine research.

DAGLα Inhibition as a Non-invasive and Translational Model of Episodic Headache.

Recent findings suggested that Clinical Endocannabinoid Deficiency underlies the pathophysiology of pain disorders, including migraine and headache. In models of medication overuse headache induced by sustained administration of sumatriptan or morphine, 2-AG levels were selectively depleted in the periaqueductal gray (PAG) and anandamide (AEA) increased in the cortex suggesting distinct regulation of the endocannabinoid system during headache pain. These results led to the hypothesis that blockade of DAGL, to reduce 2-AG levels would induce headache-like behaviors as a new, translationally relevant model of episodic headache. Our study investigated whether non-selective and selective blockade of DAGL, the main biosynthetic enzyme for 2-AG, induced periorbital and hind-paw allodynia, photophobia, anxiety-like behaviors, responsivity to abortive anti-migraine agents, and 2-AG/AEA levels. Injection of non-selective DAGL (DH376, 10 mg/kg, IP) and selective DAGLα (LEI106, 20 mg/kg, IP) inhibitors, but not DAGLß agents, induced facial sensitivity in 100% and ∼60% of female and male rats, respectively, without induction of peripheral sensitivity. Notably, male rats showed significantly less sensitivity than female rats after DAGLα inhibition, suggesting sexual dimorphism in this mechanism. Importantly, LEI106 induced periorbital allodynia was attenuated by administration of the clinically available abortive antimigraine agents, sumatriptan and olcegepant. Selective DAGLα inhibition induced significant photophobia as measured by the light-dark box, without anxiety like behaviors or changes in voluntary movement. Analysis of AEA and 2-AG levels at the time of peak pain sensitivity revealed reductions in 2-AG in the visual cortex and periaqueductal gray (PAG), without altering anandamide or significantly increasing diacylglycerol levels. These results provide foundational evidence for DAGL-2AG in the induction of headache-like pain and photophobia without extracephalic allodynia, thus modeling the clinical episodic migraine. Mechanistically, behavioral measures of headache sensitivity after DAGL inhibition suggests that reduced 2-AG signaling in the cortex and PAG, but not the trigeminal nucleus caudalis or trigeminal ganglia, drives headache initiation. Therefore, episodic DAGL inhibition, which reduces the time, cost, and invasiveness of currently accepted models of headache, may fill the need for episodic migraine/headache models mirroring clinical presentation. Moreover, use of this approach may provide an avenue to study the transition from episodic to chronic headache.

Renal and hematologic side effects of long-term intravenous immunoglobulin therapy in patients with neurologic disorders.

INTRODUCTION: For patients receiving intravenous immunoglobulin (IVIg), renal and hemolytic side effects are well recognized. However, there are very few data on the effects of chronic IVIg therapy. METHODS: We retrospectively analyzed laboratory data on 166 patients who received IVIg for 12 months with a dose range of 0.441-2.58 g/kg/month, measuring changes in hematocrit and glomerular filtration (GFR) rates at 6 and 12 months. RESULTS: Of the 2,232 infusions, there were no incidents of clinical hemolysis. However, after 12 months of treatment, 21% of patients had a ≥3-g/dl decline in hematocrit and 10% had a ≥20% decline in GFR. DISCUSSION: No clinically significant hemolysis was observed in patients receiving chronic IVIg therapy. However, a significant number of patients had a decline in hematocrit and/or GFR while on therapy. This emphasizes the need for observation of hematologic and renal function in patients treated with chronic IVIg. Muscle Nerve 56: 1173-1176, 2017.