Nancy Nutile-McMenemy

Differential migration, LPS-induced cytokine, chemokine and NO expression in immortalized BV-2 and HAPI cell lines and primary microglial cultures

Microglial cells are hematopoietically derived monocytes of the CNS and serve important neuromodulatory, neurotrophic, and neuroimmune roles. Following insult to the CNS, microglia develop a reactive phenotype, migrate to the site of injury, proliferate, and release a range of proinflammatory, anti‐inflammatory, and neurotrophic factors. Isolation of primary microglial cell cultures has been an integral step in elucidating the many roles of these cells. In addition to primary microglial cells, several immortalized cell lines have been created to model primary microglia in vitro, including murine‐derived BV‐2 cells and rat derived highly aggressive proliferating immortalized (HAPI) cells. Here, we compare rat primary microglial, BV‐2, and HAPI cells in experiments assessing migration, expression of activation markers, and production and release of nitric oxide, cytokines, and chemokines. BV‐2 and HAPI cells responded similarly to primary microglia in experiments assessing migration, ionized calcium binding adaptor molecule 1 expression, and nitric oxide release. However, BV‐2 and HAPI cells did not model primary microglia in experiments assessing tumor necrosis factor‐α, interleukin‐1β, interleukin‐6, and monocyte chemoattractant protein‐1 expression and release and phospho‐extracellular signal‐regulated kinase 44/42 expression following lipopolysaccharide treatment. These results indicate that BV‐2 and HAPI cell cultures only partially model primary microglia and that their use should therefore be carefully considered.

A comparison of spinal Iba1 and GFAP expression in rodent models of acute and chronic pain

The treatment of acute and chronic pain is still deficient. The modulation of glial cells may provide novel targets to treat pain. We hypothesize that astrocytes and microglia participate in the initiation and maintenance of both, acute surgical and chronic neuropathic pain. Rats underwent paw incision, L5 nerve exposure or L5 nerve transection surgery. Behavioral mechanical allodynia was assessed using von Frey filaments. Immunohistochemistry was performed using anti-ionized calcium binding adaptor protein, Iba-1 (microglia), and anti-Glial Fibrillary Acidic Protein, GFAP (astrocytes) on day 1, 4 and 7 after surgery. Following paw incision and at spinal L5 segment GFAP expression was increased in laminae I-II and Iba1 in deep laminae on day 1, in the entire dorsal horn on day 4 and dissipated on day 7 after paw incision in parallel with the allodynia. L5 nerve transection induced mechanical allodynia from day 1 to 7 which correlated with Iba-1 increases on day 1, 4 (entire dorsal horn) and day 7 after nerve injury (deep laminae of the dorsal horn) at spinal L5 segment. Conversely, GFAP increased at later time points from day 4 (deep laminae) and on day 7 (entire dorsal horn). Our data demonstrates that astrocytes (GFAP expression) play a role in the initiation of acute pain and the maintenance of chronic pain while Iba-1 increases closely correlated with the early phase of neuropathic pain. Iba1 and GFAP increased rostrally, at L3 segment, after paw incision (day 4) and only Iba1 increased following L5 nerve transection (day 7).

Spinal microglial and perivascular cell cannabinoid receptor type 2 activation reduces behavioral hypersensitivity without tolerance after peripheral nerve injury.

Background:Cannabinoids induce analgesia by acting on cannabinoid receptor (CBR) types 1 and/or 2. However, central nervous system side effects and antinociceptive tolerance from CBR1 limit their clinical use. CBR2 exist on spinal glia and perivascular cells, suggesting an immunoregulatory role of these receptors in the central nervous system. Previously, the authors showed that spinal CBR2 activation reduces paw incision hypersensitivity and glial activation. This study tested whether CBR2 are expressed in glia and whether their activation would induce antinociception, glial inhibition, central side effects, and antinociceptive tolerance in a neuropathic rodent pain model. Methods:Rats underwent L5 spinal nerve transection or sham surgery, and CBR2 expression and cell localization were assessed by immunohistochemistry. Animals received intrathecal injections of CBR agonists and antagonists, and mechanical withdrawal thresholds and behavioral side effects were assessed. Results:Peripheral nerve transection induced hypersensitivity, increased expression of CR3/CD11b and CBR2, and reduced ED2/CD163 expression in the spinal cord. The CBR2 were localized to microglia and perivascular cells. Intrathecal JWH015 reduced peripheral nerve injury hypersensitivity and CR3/CD11b expression and increased ED2/CD163 expression in a dose-dependent fashion. These effects were prevented by intrathecal administration of the CBR2 antagonist (AM630) but not the CBR1 antagonist (AM281). JWH015 did not cause behavioral side effects. Chronic intrathecal JWH015 treatment did not induce antinociceptive tolerance. Conclusions:These data indicate that intrathecal CBR2 agonists may provide analgesia by modulating the spinal immune response and microglial function in chronic pain conditions without inducing tolerance and neurologic side effects.

Propentofylline-Induced Astrocyte Modulation Leads to Alterations in Glial Glutamate Promoter Activation Following Spinal Nerve Transection

We have previously shown that the atypical methylxanthine, propentofylline, reduces mechanical allodynia after peripheral nerve transection in a rodent model of neuropathy. In the present study, we sought to determine whether propentofylline-induced glial modulation alters spinal glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in vivo, which may contribute to reduced behavioral hypersensitivity after nerve injury. In order to specifically examine the expression of the spinal glutamate transporters, a novel line of double transgenic GLT-1-enhanced green fluorescent protein (eGFP)/GLAST-Discosoma Red (DsRed) promoter mice was used. Adult mice received propentofylline (10 mg/kg) or saline via i.p. injection starting 1 h prior to L5-spinal nerve transection and then daily for 12 days. Mice receiving saline exhibited punctate expression of both eGFP (GLT-1 promoter activation) and DsRed (GLAST promoter activation) in the dorsal horn of the spinal cord, which was decreased ipsilateral to nerve injury on day 12. Propentofylline administration reinstated promoter activation on the injured side as evidenced by an equal number of eGFP (GLT-1) and DsRed (GLAST) puncta in both dorsal horns. As demonstrated in previous studies, propentofylline induced a concomitant reversal of L5 spinal nerve transection-induced expression of glial fibrillary acidic protein (GFAP). The ability of propentofylline to alter glial glutamate transporters highlights the importance of controlling aberrant glial activation in neuropathic pain and suggests one possible mechanism for the anti-allodynic action of this drug.

Induction of astrocyte differentiation by propentofylline increases glutamate transporter expression in vitro: Heterogeneity of the quiescent phenotype

Reactive astrocytes display decreased glutamate transporters, such as GLT‐1, and as a result synaptic glutamate clearance is impaired. In addition, these activated astrocytes are immunocompetent and release algesic mediators that can sensitize neurons in the spinal cord. Currently, we evaluated the effect of propentofylline (PPF), an experimental antiallodynic agent, on the phenotype and glutamate transporter expression of astrocytes. Primary astrocyte cultures, which represent an activated phenotype with a polygonal morphology and low GLT‐1 expression, were treated for 3 or 7 days with 10, 100, or 1,000 μM PPF or dibutyryl‐cAMP (db‐cAMP), a known inducer of GLT‐1 expression. PPF dose‐dependently induced astrocytes to display a mature phenotype, with elongated processes and a stellate shape, as well as increased GLT‐1 and GLAST immunoreactivity, similar to that seen with db‐cAMP. Real time RT‐PCR and Western blot analysis clearly demonstrated that PPF caused a potent dose‐dependent induction of GLT‐1 and GLAST mRNA and protein in these astrocytes. Importantly, the observed increase in glutamate transporters was found to have a functional effect, with significantly enhanced glutamate uptake in astrocytes treated with 100 or 1,000 μM PPF that was sensitive to dihydrokainate inhibition, suggesting it is GLT‐1 mediated. Finally, the effect of PPF on lipopolysaccaride‐induced chemokine release was investigated. Interestingly, PPF was able to dampen both MCP‐1 (CCL2) and MIP‐2 (CXCL2) release from astrocytes while db‐cAMP significantly enhanced this chemokine expression. These findings suggest that PPF is capable of differentiating astrocytes to a homeostatic, mature phenotype, competent for glutamate clearance and distinct from that induced by db‐cAMP.

Role of astrocytic S100β in behavioral hypersensitivity in rodent models of neuropathic pain

S100beta is a calcium-binding peptide produced mainly by astrocytes that exerts paracrine and autocrine effects on neurons and glia. We have previously shown that S100beta is markedly elevated at the mRNA level in the spinal cord following peripheral inflammation, intraplantar administration of complete Freunds adjuvant in the rat. The purpose of the present study was to further investigate the role of astrocytic S100beta in mediating behavioral hypersensitivity in rodent models of persistent pain. First, we assessed the lumbar spinal cord expression of S100beta at the mRNA and protein level using real-time RT-PCR, Western blot and immunohistochemistry analysis following L5 spinal nerve transection in rats, a rodent model of neuropathic pain. Second, we assessed behavioral hypersensitivity (mechanical allodynia) in wild type and genetically modified mice lacking or overexpressing S100beta following L5 spinal nerve transection. Third, we assessed the expression level of S100beta protein in the CD1 wild type mice after nerve injury. We report that lumbar spinal S100beta mRNA steadily increased from days 4-28 after nerve injury. S100beta protein in the lumbar spinal cord was significantly increased in both rats and mice at day 14 following nerve injury as compared with sham control groups. S100beta genetically deficient mice displayed significantly increased tactile thresholds (reduced response to non-noxious stimuli) after nerve injury as compared with the wild type group. S100beta overexpressing mice displayed significantly decreased tactile threshold responses (enhanced response to non-noxious stimuli). Together, these results from both series of experiments using a peripheral nerve injury model in two different species implicate the involvement of glial-derived S100beta in the pathophysiology of neuropathic pain.

Neuregulin 1 is a pronociceptive cytokine that is regulated by progesterone in the spinal cord: implications for sex specific pain modulation.

Sex differences in the magnitude of response to thermal and tactile stimuli have been demonstrated in both clinical and animal studies. Females typically display lower threshold responses to painful stimuli as compared to males. We have previously observed sexually dimorphic expression of the growth factor, neuregulin 1 (NRG1) following L5 nerve root ligation (LR) in male and female rats. In the present study, we sought to determine which gonadal hormones were involved in regulating NRG1 expression following L5 nerve root ligation. We observed that expression of NRG1 mRNA and the neuregulin receptors, ErbB2 and ErbB4 in the lumbar spinal cord was facilitated by the presence of progesterone in female rats following L5 nerve root ligation. An increase in NRG1 protein and NRG1 immunoreactivity was also observed in the ipsilateral spinal cord of progesterone treated female rats as compared to ovariectomized female rats and male rats at day 14 following LR. NRG1 immunoreactivity was equally colocalized with either the astrocytic marker, GFAP, and with NeuN labeled neurons 14days following L5 nerve root ligation. Intrathecal administration of recombinant NRG1‐β1 protein significantly decreased the hindpaw tactile withdrawal threshold in male rats, ovariectomized female rats, and progesterone treated female rats. These results demonstrate a role for progesterone‐dependent regulation of glial and/or neuronal neuregulin 1 in female rats in mediating sex differences in nociception. Furthermore, our results suggest that NRG1 may be involved in central sensitization during the maintenance phase, but not in the initiation of persistent pain in female rats.

Progesterone mediates gonadal hormone differences in tactile and thermal hypersensitivity following L5 nerve root ligation in female rats

Sex differences in the magnitude of response to thermal and tactile stimuli have been demonstrated in both clinical and animal studies. Female rats typically display lower thresholds to painful stimuli and display more robust responses following nerve injury as compared with males. There is a body of evidence implicating the sex hormones in mediating this sex difference. In the present study, we sought to determine which gonadal hormones were involved in mediating the observed female hypersensitivity in female rats both prior to and following experimental nerve root injury using a chronic hormone replacement paradigm. Female rats were ovariectomized and hormone pellets containing 17beta-estradiol, progesterone (P), 17beta-estradiol+progesterone or placebo were implanted s.c. Our results demonstrate that only the group of female rats that received progesterone alone maintained the hypersensitive phenotype following ovariectomy, compared with gonadally intact male rats. This result was observed both in response to thermal stimuli in non-injured female rats and to thermal and tactile stimuli following L5 nerve root ligation, a model of low back pain associated with lumbar radiculopathy. Postmortem analysis of serum gonadal hormone concentrations demonstrates that the hormonal manipulations were successful and the exogenous hormones were similar to physiological levels observed in the sham-ovariectomized controls. Taken together, these results demonstrate the critical role for progesterone in mediating enhanced female tactile and thermal hypersensitivity following L5 nerve root ligation.

Differential regulation of neuregulin 1 expression by progesterone in astrocytes and neurons

Glial-neuronal interactions are crucial processes in neuromodulation and synaptic plasticity. The neuregulin 1 family of growth and differentiation factors have been implicated as bidirectional signaling molecules that are involved in mediating some of these interactions. We have shown previously that neuregulin 1 expression is regulated by the gonadal hormones progesterone and 17beta-estradiol in the CNS, which might represent a novel, indirect mechanism of the neuromodulatory actions of these gonadal hormones. In the present study, we sought to determine the effects of progesterone and 17beta-estradiol on neuregulin 1 expression in rat cortical astrocytes and neurons in vitro. We observed that progesterone increased the expression of neuregulin 1 mRNA and protein in a dose-dependent manner in cultured astrocytes, which was blocked by the progesterone receptor antagonist RU-486. In contrast, 17beta-estradiol did not increase either neuregulin 1 mRNA or protein in astrocytes. We observed no effect of either progesterone or 17beta-estradiol on neuregulin 1 mRNA and protein in rat cortical neurons in vitro. Finally, we observed that treatment of cortical neurons with recombinant NRG1-beta1 caused PSD-95 to localize in puncta similar to that observed following treatment with astrocyte-conditioned medium. These results demonstrate that progesterone regulates neuregulin 1 expression, principally in astrocytes. This might represent a novel mechanism of progesterone-mediated modulation of neurotransmission through the regulation of astrocyte-derived neuregulin 1.

Localization of acyl coenzyme A:cholesterol acyltransferase gene to human chromosome 1q25

Acyl coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the formation of cholesterol esters from cholesterol and long-chain fatty acyl-coenzyme A. It is believed that ACAT plays a key role in lipoprotein metabolism and atherogenesis. Recently our laboratory succeeded in molecular cloning and functional expression of human macrophage ACAT cDNA. We have now mapped the ACAT gene to chromosome 1, band q25 by using fluorescence in situ hybridization to metaphase chromosomes, and by Southern blotting analysis of human-hamster somatic cell hybrid panels.