Aditi Bhargava

Gliopathic Pain: When Satellite Glial Cells Go Bad

Neurons in sensory ganglia are surrounded by satellite glial cells (SGCs) that perform similar functions to the glia found in the CNS. When primary sensory neurons are injured, the surrounding SGCs undergo characteristic changes. There is good evidence that the SGCs are not just bystanders to the injury but play an active role in the initiation and maintenance of neuronal changes that underlie neuropathic pain. In this article the authors review the literature on the relationship between SGCs and nociception and present evidence that changes in SGC potassium ion buffering capacity and glutamate recycling can lead to neuropathic pain-like behavior in animal models. The role that SGCs play in the immune responses to injury is also considered. We propose the term gliopathic pain to describe those conditions in which central or peripheral glia are thought to be the principal generators of principal pain generators.

Evaluation of a polymerase chain reaction for the diagnosis of tuberculosis.

A polymerase chain reaction for the specific detection of Mycobacterium tuberculosis has been developed and evaluated for clinical applicability. Primers were designed to amplify a 240 base pair region in the MPB 64 protein coding gene (nts 460-700). From among 15 different DNA templates tested (including 10 species of mycobacteria) PCR amplified the DNA from M. tuberculosis complex only, demonstrating its exquisite specificity. Sensitivity studies using serial ten-fold dilutions of M. tuberculosis bacilli determined the limit of detectability to be 10 organisms. A total of 143 clinical specimens were analysed. This consisted of 26 known non-tuberculous specimens (control group) and 117 specimens received at the Tuberculosis Diagnostic Service of AIIMS (test group). None of the specimens in the control group was positive by PCR. Out of 117 specimens in the test group, 19 were culture positive for mycobacteria and 17 of these isolates were identified as M. tuberculosis. All the specimens from which M. tuberculosis was grown were also PCR positive. The remaining two isolates were identified as mycobacteria other than M. tuberculosis and these two specimens were PCR negative. An additional 14 culture negative specimens were PCR positive yielding an overall M. tuberculosis positivity rate of 26.5% (31/117) compared to 14.5% (17/117) by culture. The superior sensitivity of PCR over culture was more evident in non-pulmonary cases where PCR picked up 10 cases in addition to three culture positives out of 69 specimens. On the other hand, out of 48 pulmonary specimens only four cases in addition to 14 culture positives were picked up by PCR.

Evidence for a Role of Connexin 43 in Trigeminal Pain Using RNA Interference In Vivo

The importance of glial cells in the generation and maintenance of neuropathic pain is becoming widely accepted. We examined the role of glial-specific gap junctions in nociception in the rat trigeminal ganglion in nerve-injured and -uninjured states. The connexin 43 (Cx43) gap-junction subunit was found to be confined to the satellite glial cells (SGCs) that tightly envelop primary sensory neurons in the trigeminal ganglion and we therefore used Cx43 RNA interference (RNAi) to alter gap-junction function in SGCs. Using behavioral evaluation, together with immunocytochemical and Western blot monitoring, we show that Cx43 increased in the trigeminal ganglion in rats with a chronic constriction injury (CCI) of the infraorbital nerve. Reducing Cx43 expression using RNAi in CCI rats reduced painlike behavior, whereas in non-CCI rats, reducing Cx43 expression increased painlike behavior. The degree of painlike behavior in CCI rats and intact, Cx43-silenced rats was similar. Our results support previous suggestions that increases in glial gap junctions after nerve injury increases nociceptive behavior but paradoxically the reduction of gap junctions in normal ganglia also increases nociceptive behavior, possibly a reflection of the multiple functions performed by glia.

Can satellite glial cells be therapeutic targets for pain control

Satellite glial cells (SGCs) undergo phenotypic changes and divide the following injury into a peripheral nerve. Nerve injury, also elicits an immune response and several antigen-presenting cells are found in close proximity to SGCs. Silencing SCG-specific molecules involved in intercellular transport (Connexin 43) or glutamate recycling (glutamine synthase) can dramatically alter nociceptive responses of normal and nerve-injured rats. Transducing SGCs with glutamic acid decarboxylase can produce analgesia in models of trigeminal pain. Taken together these data suggest that SGCs may play a role in the genesis or maintenance of pain and open a range of new possibilities for curing neuropathic pain.

Silencing the Kir4.1 potassium channel subunit in satellite glial cells of the rat trigeminal ganglion results in pain-like behavior in the absence of nerve injury

Growing evidence suggests that changes in the ion buffering capacity of glial cells can give rise to neuropathic pain. In the CNS, potassium ion (K+) buffering is dependent on the glia-specific inward rectifying K+ channel Kir4.1. We recently reported that the satellite glial cells that surround primary sensory neurons located in sensory ganglia of the peripheral nervous system also express Kir4.1, whereas the neurons do not. In the present study, we show that, in the rat trigeminal ganglion, the location of the primary sensory neurons for face sensation, specific silencing of Kir4.1 using RNA interference leads to spontaneous and evoked facial pain-like behavior in freely moving rats. We also show that Kir4.1 in the trigeminal ganglion is reduced after chronic constriction injury of the infraorbital nerve. These findings suggests that neuropathic pain can result from a change in expression of a single K+ channel in peripheral glial cells, raising the possibility of targeting Kir4.1 to treat pain in general and particularly neuropathic pain that occurs in the absence of nerve injury.

Satellite glial cells in the trigeminal ganglion as a determinant of orofacial neuropathic pain.

Satellite glial cells (SGCs) tightly envelop the perikarya of primary sensory neurons in peripheral ganglion and are identified by their morphology and the presence of proteins not found in ganglion neurons. These SGC-unique proteins include the inwardly rectifying K(+) channel Kir4.1, the connexin-43 (Cx43) subunit of gap junctions, the purinergic receptor P2Y4 and soluble guanylate cyclase. We also present evidence that the small-conductance Ca(2+)-activated K(+) channel SK3 is present only in SGCs and that SGCs divide following nerve injury. All the above proteins are involved, either directly or indirectly, in potassium ion (K(+)) buffering and, thus, can influence the level of neuronal excitability, which, in turn, has been associated with neuropathic pain conditions. We used in vivo RNA interference to reduce the expression of Cx43 (present only in SGCs) in the rat trigeminal ganglion and show that this results in the development of spontaneous pain behavior. The pain behavior is present only when Cx43 is reduced and returns to normal when Cx43 concentrations are restored. This finding shows that perturbation of a single SGC-specific protein is sufficient to induce pain responses and demonstrates the importance of PNS glial cell activity in the pathophysiology of neuropathic pain.

Intermittent Morphine Administration Induces Dependence and is a Chronic Stressor in Rats

Although constant treatment with morphine (implanted pellets) does not activate the hypothalamic–pituitary–adrenal (HPA) axis, intermittent injections of morphine may constitute a chronic stressor in rats. To test this hypothesis, we compared the effects of morphine in escalating doses (10–40 mg/kg, s.c.) or saline injected twice daily for 4 days on energy balance, hormones, HPA responses to novel restraint and central corticotropin-releasing factor (CRF) mRNA 12 h and 8 days after the last morphine injection in adult male Sprague–Dawley rats. Weight gain stopped at the onset of morphine, weight loss was marked 36 h postmorphine; thereafter, body weight gain paralleled saline controls. At 12 h, insulin, leptin, and testosterone concentrations were reduced but normalized by 8 days. Restraint and tail nicks caused facilitated ACTH responses at 12 h, under-responsiveness at 8 days. CRF mRNA, measured only at 12 h, was increased in the paraventricular (PVN) and Barringtons nuclei (BAR), decreased in the bed nuclei of the stria terminalis (BNST) and unchanged in the amygdala (CeA) in morphine-treated rats. After stress, CRF mRNA increased in PVN in both groups, increased in BAR and decreased in BNST in saline but not morphine groups, and was unchanged in CeA in both groups. Results from all variables characterize intermittent morphine injections as a chronic stressor. In contrast to constant treatment, injected morphine probably allows some withdrawal during each 12 h interval, causing repeated stress. Drug addicts treat themselves intermittently, and stress causes relapse after withdrawal. Thus, intermittent morphine, itself, may promote relapse.

Mechanisms of mineralocorticoid action: determinants of receptor specificity and actions of regulated gene products

The mineralocorticoid receptor (MR) and its close cousin, the glucocorticoid receptor (GR), share considerable structural and functional similarity, including indistinguishable DNA binding properties, yet they mediate distinct physiological responses in some tissues. Specificity is determined by their distinct interactions with other protein factors and modification by peptides, including the small ubiquitin modifier SUMO1. Serum and glucocorticoid-regulated kinase 1 (sgk1) is one key target gene of both MR and GR, and encodes a serine-threonine kinase that stimulates the apical membrane localization of the epithelial sodium channel ENaC. Sgk1 exerts its effects, at least in part, by inhibiting an isoform of the ENaC inhibitory ubiquitin ligase Nedd4-2. This review briefly summarizes two areas of mineralocorticoid research: molecular determinants of MR specificity, and the role of Sgk1 in mediating the effects of aldosterone on epithelial Na(+) transport.

Evidence for Glutamate as a Neuroglial Transmitter within Sensory Ganglia

This study examines key elements of glutamatergic transmission within sensory ganglia of the rat. We show that the soma of primary sensory neurons release glutamate when depolarized. Using acute dissociated mixed neuronal/glia cultures of dorsal root ganglia (DRG) or trigeminal ganglia and a colorimetric assay, we show that when glutamate uptake by satellite glial cells (SGCs) is inhibited, KCl stimulation leads to simultaneous increase of glutamate in the culture medium. With calcium imaging we see that the soma of primary sensory neurons and SGCs respond to AMPA, NMDA, kainate and mGluR agonists, and selective antagonists block this response. Using whole cell patch-clamp technique, inward currents were recorded from small diameter (<30 µm) DRG neurons from intact DRGs (ex-vivo whole ganglion preparation) in response to local application of the above glutamate receptor agonists. Following a chronic constriction injury (CCI) of either the inferior orbital nerve or the sciatic nerve, glutamate expression increases in the trigeminal ganglia and DRG respectively. This increase occurs in neurons of all diameters and is present in the somata of neurons with injured axons as well as in somata of neighboring uninjured neurons. These data provides additional evidence that glutamate can be released within the sensory ganglion, and that the somata of primary sensory neurons as well as SGCs express functional glutamate receptors at their surface. These findings, together with our previous gene knockdown data, suggest that glutamatergic transmission within the ganglion could impact nociceptive threshold.

c-Jun NH2-terminal kinase-2 mediates osmotic stress-induced tight junction disruption in the intestinal epithelium

Gastrointestinal epithelium faces osmotic stress, both at physiological and pathophysiological conditions. JNK activation is an immediate cellular response to osmotic stress. We investigated the effect of osmotic stress on intestinal epithelial barrier function and delineated the role of JNK2 in osmotic stress-induced tight junction (TJ) regulation in Caco-2 cell monolayers and ileum of Jnk(-/-) and Jnk2(-/-) mice. The role of JNK activation in osmotic stress-induced TJ disruption was evaluated using JNK-specific inhibitor and antisense oligonucleotides. Furthermore, the effect of cold restraint stress in vivo on TJ integrity was determined in rats. Osmotic stress disrupted TJs and barrier function in Caco-2 cell monolayers without affecting cell viability. Osmotic stress activated JNK1 and JNK2 and the inhibition of JNK by SP600125 attenuated osmotic stress-induced TJ disruption. TJ disruption and barrier dysfunction by osmotic stress was associated with JNK-dependent remodeling of actin cytoskeleton. Knockdown of JNK2 accelerated TJ assembly and attenuated osmotic stress-induced TJ disruption in Caco-2 cell monolayers. In mouse ileum in vitro, osmotic stress increased paracellular permeability, which was attenuated by SP600125. Osmotic stress disrupted actin cytoskeleton and TJs and increased paracellular permeability in the ileum of wild-type and JNK1(-/-) mice, but not in JNK2(-/-) mouse ileum. Cold restraint stress activated JNK in rat ileum and caused JNK-dependent remodeling of actin cytoskeleton and redistribution of occludin and zona occluden-1 from the intercellular junctions. These results reveal the role of JNK2 in the mechanism of osmotic stress-induced TJ disruption in the intestinal epithelium.