Trigeminal Satellite Glial Cells Research Articles

Aquaporin 4 expression on trigeminal satellite glial cells under normal and inflammatory conditions

Abstract Aims Limited information is currently available for the expression and role of Aquaporin 4 (AQP4) (AQ4) in the peripheral nervous system (PNS). It has been demonstrated that AQP4 is expressed in sensory ganglia. Immunohistochemistry has revealed that satellite glial cells (SGCs) surrounding the cell bodies of the primary afferent sensory neurons in these sensory ganglia exclusively express AQP4 at a considerably lower level than what is seen in astrocytes. The pathophysiological relevance of AQP4 in peripheral nociception; however, remains unclear. Hence, this study aimed at investigating AQP4 expression in trigeminal neurons and SGCs under normal and inflammatory conditions relevant to craniofacial pain conditions. Methods Rat trigeminal ganglia (TG) were isolated from adult male Sprague-Dawley rats subjected to a model of trigeminal inflammation evoked by unilateral complete Freund’s adjuvant (CFA) injection in temporomandibular joint. Immunohistochemistry was performed on TG sections of CFA-treated animals. NeuN and GS markers were used for identification of neurons and SGCs, respectively. AQP4 expression was investigated in both ipsilateral and contralateral TG sections. The study protocol was approved by the local ethics committee. Results Co-localization of NeuN-AQP4 and GS-AQP4 were identified in both ipsi and contralateral trigeminal ganglia of the CFA-treated rats. However, we did not detect any difference between the ipsi- and contralateral side in terms of alteration in AQP4 receptor expression. Conclusions AQP4 was expressed both on trigeminal neurons and SGCs and CFA did not cause a remarkable change in AQP4 expression, when ipsilateral and contralateral TG of the test animals was compared. Previously, it has been shown that in a neuropathic pain model no difference is detectable between wild type and AQP4-deficient mice, for mechanical and thermal perception; however, in formalin pain model AQP4-deficient mice have higher thermal pain thresholds. Further investigation is required to clarify role of AQP4 in pain.

Cell-based platform for studying trigeminal satellite glial cells under normal and inflammatory conditions

Abstract Aims Satellite glial cells (SGCs) in sensory ganglia contribute to the pathogenesis of chronic pain. In vitro, providing enough fresh primary SGCs poses some practical limitations; hence, frozen stocks of primary cells for culture could be an attractive alternative for cell-based studies or drug screening. This study was designed to investigate the morphology and marker expression of frozen and freshly isolated trigeminal SGCs under normal and inflammatory conditions. Methods SGCs from trigeminal ganglia of three male Sprague–Dawley rats and three frozen (sub cultured and passaged) batches of stored primary SGCs were cultured. Their morphology was observed by phase microscopy and the phenotype was characterized by immunocytochemistry of glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP). Lipopolysaccharide (LPS) was used to simulate a state of neurogenic inflammation in vivo. A pilot test was performed to determine the optimal concentration of LPS to activate SGCs based on GFAP expression. A long-term activation of the SGCs with 50 ng/mL LPS was chosen for further characterization. Results The fresh and frozen primary SGCs elicited similar phenotypes based on GS marker expression. However, frozen primary SGCs differed in terms of size and morphology. GFAP was constantly expressed in frozen primary SGCs regardless of LPS stimulation. Activation of primary fresh SGCs with LPS spread the GFAP expression from around the cell body throughout the longer processes and activation was only seen in the LPS treatment. Conclusions The phenotypic marker, GS was independent of culture conditions. There was no difference in upregulation of GFAP in thawed SGCs regardless of LPS stimulation. This indicates that freeze-thawing might activate SGCs and therefore frozen and passaged cells cannot be suitable for use in cell-based models for inflammation. Fresh primary cells are therefore optimal for studying SGCs under normal and inflammatory conditions.

Glutamate attenuates nitric oxide release from isolated trigeminal ganglion satellite glial cells

Abstract Background/aims Elevated concentrations of nitric oxide (NO) and glutamate (Glu) in the trigeminal ganglion (TG) may contribute to the development and maintenance of migraine headache. The role of satellite glial cells (SGC) and their pattern of substance release in relation to the pathophysiology of migraine are currently under investigation. In the present study, we investigated the release of NO from isolated trigeminal SGCs and its modulation by Glu. Methods SGCs from the TG of adult male Sprague-Dawley rats (n = 8) were isolated and maintained in culture until used. SGCs were treated with graded concentrations of Glu (0, 10, 100, 1000 μM) and samples were withdrawn after 48 h of incubation. In subsequent experiments, SGCs were treated with vehicle medium, 10 μM forskolin (FSK) alone, or 10 μM FSK in conjunction with 100 μM Glu and incubated for 48 h. The NO concentration was determined using the Griess Reagent System and data was subjected to a one-way repeated measures ANOVA analysis, where p <0.05 was considered statistically significant. All experimental procedures were performed at minimum in triplicate. Results The concentration of NO was 3.59 ± 0.04 M under baseline conditions. Application of 10 or 100 μM Glu resulted in a significant drop in NO concentration (2.92 ± 0.017 μM and 2.83 ± 0.012 M, respectively) compared to baseline, whereas treatment with 1000 μM Glu did not significantly alter NO release. Treatment of SGCs with 10 μM FSK significantly increased NO release (to 125.94 ± 3.90% of baseline) compared to baseline. Coapplication of 10 M FSK with 100 M Glu significantly decreased FSK-induced NO release (83.73 ± 2.29% of baseline), compared to both FSK-mediated NO release and baseline levels. Conclusion These findings suggest that one mechanism by which SGCs protect the TG from elevated Glu concentrations that may occur in response to prolonged noxious stimulation is to reduce the release of NO.