ABSTRACT
Title
Microglia and neuropathic pain: molecular and cellular aspects
Authors
M. Barbierato
Dept. of Pharmacology and Anesthesiology, University of Padua, Largo E. Meneghetti, 2, 35131 Padua
Doctoral (PhD) School of Pharmacological Sciences, course in Molecular and Cellular Pharmacology
Dept. of Pharmacology and Anesthesiology, University of Padua, Largo E. Meneghetti, 2, 35131 Padua
Doctoral (PhD) School of Pharmacological Sciences, course in Molecular and Cellular Pharmacology
Abstract
Neuropathic pain is initiated or caused by a primary lesion or dysfunction or transitory perturbation in the peripheral or central nervous systems [1]. Many common diseases, such as cancer, stroke, multiple sclerosis, syringomyelia, diabetes, and herpes zoster may produce neuropathic pain. Specific syndromes include postherpetic neuralgia, root avulsions, painful traumatic mononeuropathy and polyneuropathy (as in diabetes), post-surgical pain syndromes (e.g. phantom pain) and post-stroke pain [2].
It has been increasingly recognized that central nervous system glial cells, such as microglia and astrocytes, play an important role in the development and maintenance of neuropathic pain. Indeed, astrocytes can release inflammatory cytokines and chemokines. Indeed during neuropathic pain, after a early activation of microglia, astrocytes respond with a more prolonged release of inflammatory factors. In addition, like astrocytes and neurons, microglia express various neurotransmitter receptors, and during pain they can release factors that activate microglia, astrocytes, and neurons [3]. The genesis and maintenance of neuropathic pain involves several factors. The Toll-like receptor (TLR) family plays an important role in activation and maintenance of pain, and microglia and astrocytes express several members, including TLR2 and TLR4 [4-6].
In order to understand the molecular mechanism involved in activation and modulation of the microglial inflammatory response, we set up cortical and spinal microglia and astrocyte primary culture from 2 day newborn rats, cultured in Dulbecco’s modified Eagle’s medium with 10% foetal bovine serum and antibiotics. Cultures where treated with lipopolysaccaride (LPS) for 6-24 h. Immunofluorescent staining was performed for Iba1 (microglia), glial fibrillary acidic protein (astrocytes), galactocerebroside (oligodendrocytes); mRNA was quantified by real time RT-PCR, and ELISA and Griess reaction where performed to detect proteins and nitric oxide, respectively, in the cell culture medium.
The mixed glial cell cultures where separated into purified (³99%) microglia and astrocyte-enriched (³95%) populations, as shown by immunostaining. Cells were then plated in 96 wells. Treatment of microglia with 1 ng/ml LPS strongly up-regulated interleukin-1β, interleukin-6, tumor necrosis factor-α and nitric oxide synthase 2 mRNAs after 6 h, while astrocytes showed a weaker response at 10-fold higher concentration of LPS. Unlike microglia, the astrocyte response was more protracted and lasted for a at least 24 h. Calcitonin gene related peptide, substance P, and vasoactive intestinal peptide did not elicit any responses from microglia or astrocytes per se, nor did they affect the cells’ response to LPS (up to 500 ng/ml) – in contrast to literature reports that these neuropeptides promote or mitigate the inflammatory response.
In conclusion, we have set up a glial cell culture system to evaluate the response to a classic endotoxin, LPS. The results confirm that, after LPS treatment, microglia and astrocytes display different time-courses to reach maximum activation, and respond in a concentration-dependent manner. Neuropeptides involved in pain stimuli failed to affect cortical microglia and astrocyte up-regulation of inflammatory factors. Further studies will examine the actions of opioids and antidepressants against an inflammatory stimulus to cortical and spinal cord-derived glia.
It has been increasingly recognized that central nervous system glial cells, such as microglia and astrocytes, play an important role in the development and maintenance of neuropathic pain. Indeed, astrocytes can release inflammatory cytokines and chemokines. Indeed during neuropathic pain, after a early activation of microglia, astrocytes respond with a more prolonged release of inflammatory factors. In addition, like astrocytes and neurons, microglia express various neurotransmitter receptors, and during pain they can release factors that activate microglia, astrocytes, and neurons [3]. The genesis and maintenance of neuropathic pain involves several factors. The Toll-like receptor (TLR) family plays an important role in activation and maintenance of pain, and microglia and astrocytes express several members, including TLR2 and TLR4 [4-6].
In order to understand the molecular mechanism involved in activation and modulation of the microglial inflammatory response, we set up cortical and spinal microglia and astrocyte primary culture from 2 day newborn rats, cultured in Dulbecco’s modified Eagle’s medium with 10% foetal bovine serum and antibiotics. Cultures where treated with lipopolysaccaride (LPS) for 6-24 h. Immunofluorescent staining was performed for Iba1 (microglia), glial fibrillary acidic protein (astrocytes), galactocerebroside (oligodendrocytes); mRNA was quantified by real time RT-PCR, and ELISA and Griess reaction where performed to detect proteins and nitric oxide, respectively, in the cell culture medium.
The mixed glial cell cultures where separated into purified (³99%) microglia and astrocyte-enriched (³95%) populations, as shown by immunostaining. Cells were then plated in 96 wells. Treatment of microglia with 1 ng/ml LPS strongly up-regulated interleukin-1β, interleukin-6, tumor necrosis factor-α and nitric oxide synthase 2 mRNAs after 6 h, while astrocytes showed a weaker response at 10-fold higher concentration of LPS. Unlike microglia, the astrocyte response was more protracted and lasted for a at least 24 h. Calcitonin gene related peptide, substance P, and vasoactive intestinal peptide did not elicit any responses from microglia or astrocytes per se, nor did they affect the cells’ response to LPS (up to 500 ng/ml) – in contrast to literature reports that these neuropeptides promote or mitigate the inflammatory response.
In conclusion, we have set up a glial cell culture system to evaluate the response to a classic endotoxin, LPS. The results confirm that, after LPS treatment, microglia and astrocytes display different time-courses to reach maximum activation, and respond in a concentration-dependent manner. Neuropeptides involved in pain stimuli failed to affect cortical microglia and astrocyte up-regulation of inflammatory factors. Further studies will examine the actions of opioids and antidepressants against an inflammatory stimulus to cortical and spinal cord-derived glia.
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