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Objective: To investigate cellular changes in protein expression in the trigeminal ganglion in an established preclinical chronic model of temporomandibular joint disorder (TMD) in response to grape seed extract (GSE) supplementation based on its beneficial use in preclinical chronic orofacial pain models., Design: Three experimental conditions included female Sprague-Dawley rats as naïve controls, and animals subjected to neck muscle inflammation and prolonged jaw opening with and without daily supplementation of GSE in the drinking water prior to inflammation. Changes were evaluated in mechanical sensitivity to von Frey filaments and protein expression in the trigeminal ganglion of animals 14 days post jaw opening., Results: Calcitonin-gene related peptide and protein kinase A, proteins positively associated with peripheral sensitization and enhanced nociception, did not show elevated expression at day 14 in the model compared to naïve or GSE supplemented animals. However, neuronal levels of glutamate decarboxylase (GAD) 65/67, which are enzymes responsible for the synthesis of the inhibitory neurotransmitter GABA that functions to suppress neuronal excitability, were significantly decreased on day 14 post jaw opening. Similarly, a significant decrease in neuronal expression of the GABA receptor subunits GABAB1 and GABAB2, but not GABAA, was observed in the TMD model. Importantly, GSE prevented suppression of GAD 65/67 and GABAB subunits, maintaining levels similar to naïve animals., Conclusion: Results from our study provide evidence of the downregulation of inhibitory GABAergic proteins in trigeminal ganglion neurons in a preclinical chronic TMD model and the benefits of GSE supplementation in preventing their suppression and maintaining normal levels., Competing Interests: Declaration of Competing Interest The authors report no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose of Review: To provide information from preclinical and clinical studies on the biological activity and health benefits of dietary inclusion of nutraceuticals as a safe, effective, non-pharmacological approach for the treatment of migraine., Recent Findings: There is emerging evidence of the therapeutic benefit of nutraceuticals to inhibit oxidative stress, suppress inflammation, and prevent changes in the normal gut microbiome, which are implicated in migraine pathology. Nutraceuticals can be enriched in polyphenols, which act as molecular scavengers to reduce the harmful effects of reactive oxygen species and phytosterols that suppress inflammation. Nutraceuticals also function to inhibit dysbiosis and to maintain the commensal intestinal bacteria that produce anti-inflammatory molecules including short-chain fatty acids that can act systemically to maintain a healthy nervous system. Dietary inclusion of nutraceuticals that exhibit antioxidant, anti-inflammatory, and anti-nociceptive properties and maintain the gut microbiota provides a complementary and integrative therapeutic strategy for migraine., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Primary neuronal cultures are used to elucidate cellular and molecular mechanisms involved in disease pathology and modulation by pharmaceuticals and nutraceuticals, and to identify novel therapeutic targets. However, preparation of primary neuronal cultures from rodent embryos is labor-intensive, and it can be difficult to produce high-quality consistent cultures. To overcome these issues, cryopreservation can be used to obtain standardized, high-quality stocks of neuronal cultures., New Method: In this study, we present a simplified cryopreservation method for rodent primary trigeminal ganglion neurons and glia from Sprague-Dawley neonates, using a 90:10 (v/v) fetal bovine serum/dimethyl sulfoxide cell freezing medium., Results: Cryopreserved trigeminal ganglion cells stored for up to one year in liquid nitrogen exhibited similar neuronal and glial cell morphology to fresh cultures and retained high cell viability. Proteins implicated in inflammation and pain signaling were expressed in agreement with the reported subcellular localization. Additionally, both neurons and glial cells exhibited an increase in intracellular calcium levels in response to a depolarizing stimulus. Cryopreserved cells were also transiently transfected with reporter genes., Comparison With Existing Methods: Our method is simple, does not require special reagents or equipment, will save time and money, increase flexibility in study design, and produce consistent cultures., Conclusions: This method for the preparation and cryopreservation of trigeminal ganglia results in primary cultures of neurons and glia similar in viability and morphology to fresh preparations that could be utilized for biochemical, cellular, and molecular studies, increase reproducibility, and save laboratory resources., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Migraine is associated with neuroimaging differences in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). However, it is unknown if migraine-related disability (MRD) or if calcitonin gene-related peptide (CGRP), a vasoactive peptide important in migraine pathology, have radiographic implications. The aims of this study were to identify whether MRD or interictal serum CGRP levels impacted neuroimaging findings for those with CADASIL., Materials and Methods: A cross-sectional analysis was performed. The primary outcomes were neuroimaging differences associated with MRD among those with migraine or interictal serum CGRP levels of those with and without migraine. MRD was defined by 2 migraine disability scales (Migraine Disability Assessment, Headache Impact Test-6). Retrospective brain magnetic resonance imaging was reviewed (average 1.7 ± 2.0 y before enrollment). Rank-sum and χ 2 tests were used., Results: Those with migraine (n=25, vs. n=14 without) were younger [median 49 (25 to 82) y vs. 60 (31 to 82) y, P <0.007], had fewer cerebral microbleeds (0 to 31 vs. 0 to 50, P =0.02) and less frequently had anterior temporal lobe T2 hyperintensities [68% (17/25) vs 100% (14/14), P =0.02]. MRD scale outcomes had no significant radiographic associations. Interictal serum CGRP did not differ (migraine: n=18, 27.0±9.6 pg/mL vs. no migraine: n=10, 26.8±15.7 pg/mL, P =0.965)., Conclusions: Migraine may forestall microangiopathy in CADASIL, though possibly independent of severity as measured by MRD. Interictal serum CGRP did not differ in our cohort suggesting CGRP may not be vital to migraine pathophysiology in CADASIL. Larger studies are needed to account for age differences., Competing Interests: E.D.G. received support from the NH-NINDS U24NS107228; compensation from Medlink. A.d.H. who reports support by the NIH-NINDS K23NS105924; consultation for Integra; investigator-initiated support from Regeneron, Amgen, and AMAG pharmaceuticals. PG, PLD, JFH, MPL, HB reports no disclosures. JJM receives support from the NH-NINDS U24NS107228; associate editor, Stroke. J.F.M. receives funding from the NINDS for work related to the CREST2 trial and the DISCOVERY trial. The remaining authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

The trigeminal ganglion is implicated in the underlying pathology of migraine and temporomandibular joint disorders (TMD), which are orofacial pain conditions involving peripheral and central sensitization. The neuropeptide calcitonin gene-related peptide (CGRP) is synthesized in some trigeminal ganglion neurons, and its release promotes inflammation, peripheral and central sensitization, and pain signaling. Recent studies in preclinical migraine and TMD models provide evidence that dietary supplementation with grape seed extract (GSE) inhibits trigeminal pain signaling. The goal of this study was to investigate the cellular mechanisms by which GSE modulates primary trigeminal ganglion cultures. The effect of GSE on CGRP secretion was determined by radioimmunoassay. To determine if GSE effects involved modulation of CGRP or the GABAergic system, expression of CGRP, GAD 65 and 67, GABAA receptor, and GABAB1 and GABAB2 receptor subunits were investigated by immunocytochemistry. GSE significantly inhibited basal CGRP secretion but did not alter neuronal CGRP expression. GAD 65 and 67 expression levels in neurons were significantly increased in response to GSE. While GSE did not cause a change in the neuronal expression of GABAA, GSE significantly increased GABAB1 expression in neurons, satellite glial cells, and Schwann cells. GABAB2 expression was significantly elevated in satellite glia and Schwann cells. These findings support the notion that GSE inhibition of basal CGRP secretion involves increased neuronal GAD 65 and 67 and GABAB receptor expression. GSE repression of CGRP release coupled with increased GABAB1 and GABAB2 glial cell expression would be neuroprotective by suppressing neuronal and glial excitability in the trigeminal ganglion., (© 2022 The Authors.)

Background: Temporomandibular joint disorder is a prevalent orofacial pain condition involving sensitization and activation of trigeminal nociceptive neurons. Dietary supplementation with a proanthocyanin-enriched grape seed extract (GSE) was found to inhibit trigeminal nociception in a chronic TMD model. In this study, the cellular mechanisms by which GSE inhibits sustained trigeminal nociception in male and female Sprague Dawley rats were investigated. Methods: Some animals were supplemented with 0.5% GSE dissolved in their water one week prior to neck muscle inflammation induced by injection of complete Freund's adjuvant into the trapezius. To investigate the mechanism of GSE, some animals were injected intracisternally with antagonists of 5-HT3, 5-HT7, GABAA, or GABAB, receptor prior to jaw opening. Results: In males and females, trapezius inflammation prior to jaw opening resulted in sustained mechanical hypersensitivity of trigeminal nociceptors that was significantly inhibited by GSE. Further, GSE beginning 14 days post jaw opening also inhibited trigeminal nociception. Intracisternal injection of antagonists of the 5-HT3/7 and GABAB, but not GABAA receptors reduced the anti-nocifensive effect of GSE in both sexes. Neuronal expression of GABAB protein and mRNA in the spinal cord and trigeminal ganglion were detected. Conclusions: The inhibitory effect of GSE is mediated via activation of 5-HT3/7 receptors and GABAB to enhance central descending inhibitory pain pathways and suppress ongoing trigeminal nociception. Further, our findings support the use of GSE as a dietary supplement in the management of pain associated with TMD and other orofacial pain conditions involving central sensitization and dysfunction of descending pain modulation.

Migraine is associated with peripheral and central sensitization of the trigeminal system and dysfunction of descending pain modulation pathways. Recently, dietary inclusion of grape seed extract (GSE) was shown to inhibit mechanical nociception in a preclinical model of chronic temporomandibular joint disorder, a condition often comorbid with migraine, with the antinociceptive effect mediated, in part, by activation of 5-HT3/7 and GABAB receptors. This study further investigated the mechanisms by which GSE inhibits mechanical nociception in a preclinical model of episodic migraine. Hyperalgesic priming of female and male Sprague Dawley rats was induced by three consecutive daily two-hour episodes of restraint stress. Seven days after the final restraint stress, rats were exposed to pungent odors from an oil extract that contains the compound umbellulone, which stimulates CGRP release and induces migraine-like pain. Some animals received dietary supplementation of GSE in their drinking water beginning one week prior to restraint stress. Changes in mechanical sensitivity in the orofacial region and hindpaw were determined using von Frey filaments. To investigate the role of the endocannabinoid receptors in the effect of GSE, some animals were injected intracisternally with the CB1 antagonist AM 251 or the CB2 antagonist AM 630 prior to odor inhalation. Changes in CGRP expression in the spinal trigeminal nucleus (STN) in response to stress, odor and GSE supplementation were studied using immunohistochemistry. Exposure of stress-primed animals to the odor caused a significant increase in the average number of withdrawal responses to mechanical stimulation in both the orofacial region and hindpaw, and the effect was significantly suppressed by daily supplementation with GSE. The anti-nociceptive effect of GSE was inhibited by intracisternal administration of antagonists of CB1 and CB2 receptors. GSE supplementation inhibited odor-mediated stimulation of CGRP expression in the STN in sensitized animals. These results demonstrate that GSE supplementation inhibits trigeminal pain signaling in an injury-free model of migraine-like pain via activation of endocannabinoid receptors and repression of CGRP expression centrally. Hence, we propose that GSE may be beneficial as a complementary migraine therapeutic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Woodman, Antonopoulos and Durham.)