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Varicella zoster virus (VZV) is responsible for chronic pain. VZV injection has similarities to herpes zoster (HZ) "shingles" pain in humans. In this study orofacial pain was induced by injecting male rats with the human VZV. The amygdala and parabrachial have been implicated to control affective/motivational orofacial pain. Recently our lab reported neurexin 3α (Nrxn3α) is expressed in the central amygdala and parabrachial. GABAergic neurons descend from the central amygdala to the lateral parabrachial region and Nrxn3α is important for presynaptic (γ-Aminobutyric acid) GABA release. Thus, we hypothesized that lateral parabrachial neuronal activity and orofacial pain are controlled by Nrxn3α within the central amygdala. To test the hypothesis Nrxn3α expression was knocked down (i.e., using short hairpin RNA or shRNA) in the central amygdala and GABA release and neuronal activity were quantitated in the parabrachial concomitant with measurement of the VZV induced pain response. Results revealed that attenuating Nrxn3 expression within the amygdala reduces GABA release in the parabrachial and increases neuronal activity within the lateral parabrachial region. Attenuating Nrxn3 expression also increases VZV associated orofacial pain. Activating GABAergic neurons within the central amygdala with opsins increase GABA release in the parabrachial and reduced the pain response after Nrxn3 shRNA treatment. These results are consistent with the idea that Nrxn3 within the central amygdala controls VZV associated pain by regulating GABA release in the lateral parabrachial that then controls the activity of ascending pain neurons.

Varicella zoster virus (VZV) induces orofacial pain and female rats show greater pain than male rats. During the proestrus phase of the estrous cycle the VZV induce pain response is attenuated in female rats. A screen of gene expression changes in diestrus and proestrus female rats indicated neurexin 3α (Nrxn3α) was elevated in the central amygdala of proestrus rats vs. diestrus rats. GABAergic neurons descend from the central amygdala to the lateral parabrachial region and Nrxn3α is important for presynaptic γ-Aminobutyric acid (GABA) release. Thus, we hypothesized that the reduced orofacial pain in male rats and proestrus female rats is the result of increased Nrxn3α within the central amygdala that increases GABA release from axon terminals within the parabrachial and inhibits ascending pain signals. To test this hypothesis Nrxn3 α expression was knocked-down by infusing shRNA constructs in the central amygdala. Then GABA release in the parabrachial was quantitated concomitant with measuring the pain response. Results revealed that knockdown of Nrxn3α expression significantly increases the pain response in both male rats and proestrus female rats vs. diestrus rats. GABA release was significantly reduced in the parabrachial of male and proestrus female rats after Nrxn3α knockdown. Neuronal activity of excitatory neurons was significantly inhibited in the parabrachial after Nrxn3α knockdown. These results are consistent with the idea that Nrxn3 within the central amygdala controls VZV associated pain by regulating GABA release in the lateral parabrachial that then modulates ascending orofacial pain signals.

Reactivation of latent varicella-zoster virus (VZV) causes herpes zoster (HZ), which is commonly accompanied by acute pain and pruritus over the time course of a zosteriform rash. Although the rash and associated pain are self-limiting, a considerable fraction of HZ cases will subsequently develop debilitating chronic pain states termed postherpetic neuralgia (PHN). How VZV causes acute pain and the mechanisms underlying the transition to PHN are far from clear. The human-specific nature of VZV has made in vivo modeling of pain following reactivation difficult to study because no single animal can reproduce reactivated VZV disease as observed in the clinic. Investigations of VZV pathogenesis following primary infection have benefited greatly from human tissues harbored in immune-deficient mice, but modeling of acute and chronic pain requires an intact nervous system with the capability of transmitting ascending and descending sensory signals. Several groups have found that subcutaneous VZV inoculation of the rat induces prolonged and measurable changes in nociceptive behavior, indicating sensitivity that partially mimics the development of mechanical allodynia and thermal hyperalgesia seen in HZ and PHN patients. Although it is not a model of reactivation, the rat is beginning to inform how VZV infection can evoke a pain response and induce long-lasting alterations to nociception. In this review, we will summarize the rat pain models from a practical perspective and discuss avenues that have opened for testing of novel treatments for both zoster-associated pain and chronic PHN conditions, which remain in critical need of effective therapies.

Herpes zoster, the result of varicella-zoster virus (VZV) reactivation, is frequently complicated by difficult-to-treat chronic pain states termed postherpetic neuralgia (PHN). While there are no animal models of VZV-induced pain following viral reactivation, subcutaneous VZV inoculation of the rat causes long-term nocifensive behaviors indicative of mechanical and thermal hypersensitivity. Previous studies using UV-inactivated VZV in the rat model suggest viral gene expression is required for the development of pain behaviors. However, it remains unclear if complete infection processes are needed for VZV to induce hypersensitivity in this host. To further assess how gene expression and replication contribute, we developed and characterized three replication-conditional VZV using a protein degron system to achieve drug-dependent stability of essential viral proteins. Each virus was then assessed for induction of hypersensitivity in rats under replication permissive and nonpermissive conditions. VZV with a degron fused to ORF9p, a late structural protein that is required for virion assembly, induced nocifensive behaviors under both replication permissive and nonpermissive conditions, indicating that complete VZV replication is dispensable for the induction of hypersensitivity. This conclusion was confirmed by showing that a genetic deletion recombinant VZV lacking DNA packaging protein ORF54p still induced prolonged hypersensitivities in the rat. In contrast, VZV with a degron fused to the essential IE4 or IE63 proteins, which are involved in early gene regulation of expression, induced nocifensive behaviors only under replication permissive conditions, indicating importance of early gene expression events for induction of hypersensitivity. These data establish that while early viral gene expression is required for the development of nocifensive behaviors in the rat, complete replication is dispensable. We postulate this model reflects events leading to clinical PHN, in which a population of ganglionic neurons become abortively infected with VZV during reactivation and survive, but host signaling becomes altered in order to transmit ongoing pain., Author summary Acute and chronic pain are common complications of herpes zoster, but the mechanisms underlying the transition to chronic pain states remain poorly understood. Varicella-zoster virus (VZV)-inoculated rats develop persistent behaviors that indicate the development of prolonged hypersensitivity that may model postherpetic neuralgia (PHN) seen in the clinic. To address the requirements of viral gene expression and replication for the induction of pain, we developed mutant VZV and applied them to rat models of PHN. Our results reveal the production of essential VZV regulatory proteins is required to induce nocifensive behaviors, but that full productive virus replication is dispensable. These data suggest a mechanism for pain induction that involves the early expression of VZV regulatory proteins in abortively infected neurons after herpes zoster that may cause aberrant host pain signaling and PHN.

Reactivation of latent varicella-zoster virus (VZV) causes herpes zoster (HZ), which is commonly accompanied by acute pain and pruritus over the time course of a zosteriform rash. Although the rash and associated pain are self-limiting, a considerable fraction of HZ cases will subsequently develop debilitating chronic pain states termed postherpetic neuralgia (PHN). How VZV causes acute pain and the mechanisms underlying the transition to PHN are far from clear. The human-specific nature of VZV has made in vivo modeling of pain following reactivation difficult to study because no single animal can reproduce reactivated VZV disease as observed in the clinic. Investigations of VZV pathogenesis following primary infection have benefited greatly from human tissues harbored in immune-deficient mice, but modeling of acute and chronic pain requires an intact nervous system with the capability of transmitting ascending and descending sensory signals. Several groups have found that subcutaneous VZV inoculation of the rat induces prolonged and measurable changes in nociceptive behavior, indicating sensitivity that partially mimics the development of mechanical allodynia and thermal hyperalgesia seen in HZ and PHN patients. Although it is not a model of reactivation, the rat is beginning to inform how VZV infection can evoke a pain response and induce long-lasting alterations to nociception. In this review, we will summarize the rat pain models from a practical perspective and discuss avenues that have opened for testing of novel treatments for both zoster-associated pain and chronic PHN conditions, which remain in critical need of effective therapies.

Pain is a common complication of herpes zoster (HZ) infection which results from reactivation of a latent varicella zoster virus (VZV). A third of HZ patients’ progress to a chronic pain state known as post herpetic neuralgia (PHN), and about a quarter of these patients’ have orofacial pain. The mechanisms controlling the pain responses are not understood. Studies suggest central pathways involving the thalamus could control pain related to HZ, and studies in our lab suggest vesicular GABA transporter (VGAT) in the lateral thalamus influences orofacial pain. We hypothesized that thalamic VGAT functions, in part, to reduce pain, particularly orofacial pain, associated with VZV. To address this hypothesis VZV was injected into the whisker pad. Affective and motivational aspects of pain were measured using the Place Escape/Avoidance Paradigm. Thalamic neuronal activity was modulated after injecting an adeno-associated virus (AAV) expressing an engineered acetylcholine Gi-protein coupled receptor. This receptor inhibits neuronal firing when bound by clozapine-n-oxide (CNO). VGAT expression was attenuated in the thalamus by injecting an AAV construct that expressed a VGAT silencing shRNA. VZV induced nociception was significantly decreased after administering CNO in male rats. Nociception significantly increased concomitant with increased thalamic c-fos expression after attenuating thalamic VGAT expression. These data establish that the lateral thalamus (posterior, ventral posteromedial, ventral posterolateral and/or reticular thalamic nucleus) controls VZV induced nociception in the orofacial region, and that GABA in this region appears to reduce the response to VZV induced nociception possibly by gating facial pain input.

Within the reticular thalamic nucleus neurons express gamma aminobutyric acid (GABA) and these cells project to the ventral posteromedial thalamic nucleus. When GABA activity decreases the activity of excitatory cells in the ventral posteromedial nucleus would be expected to increase. In this study, we addressed the hypothesis that attenuating GABAergic cells in the reticular thalamic nucleus increases excitatory activity in the ventral posteromedial nucleus increasing varicella zoster virus (VZV) associated pain in the orofacial region. Adeno-associated virus (AAV) was infused in the reticular thalamic nucleus of Gad1-Cre rats. This virus transduced a G inhibitory designer receptor exclusively activated by designer drugs (DREADD) gene that was Cre dependent. A dose of estradiol that was previously shown to reduce VZV pain and increase GABAergic activity was administered to castrated and ovariectomized rats. Previous studies suggest that estradiol attenuates herpes zoster pain by increasing the activity of inhibitory neurons and decreasing the activity of excitatory cells within the lateral thalamic region. The ventral posteromedial nucleus was infused with AAV containing a GCaMP6f expression construct. A glass lens was implanted for miniscope imaging. Our results show that the activity of GABA cells within the reticular thalamic region decreased with clozapine N-oxide treatment concomitant with increased calcium activity of excitatory cells in the ventral posteromedial nucleus and an increased orofacial pain response. The results suggest that estradiol attenuates herpes zoster pain by increasing the activity of inhibitory neurons within the reticular thalamus that then inhibit excitatory activity in ventral posteromedial nucleus causing a reduction in orofacial pain.

Optogenetic manipulation is uniquely useful in unraveling the functional organization of neuronal circuits in the central nervous system by enabling reversible gain- or loss-of-function of discrete populations of neurons within restricted brain regions. This state-of-the-art technology can produce circuit-specific neuromodulation by overexpressing light-sensitive proteins (opsins) in particular cell types of interest. Here, we discuss the principle of optogenetic manipulation and its application in pain research using animal models, and we also discuss how to potentially use optogenetic stimulation in the treatment of migraine headache in the future.

Neuropathic pain represents a challenge to clinicians because it is resistant to commonly prescribed analgesics due to its largely unknown mechanisms. Here, we investigated a descending dopaminergic pathway-mediated modulation of trigeminal neuropathic pain. We performed chronic constriction injury of the infraorbital nerve from the maxillary branch of trigeminal nerve to induce trigeminal neuropathic pain in mice. Our retrograde tracing showed that the descending dopaminergic projection from hypothalamic A11 nucleus to spinal trigeminal nucleus caudalis is bilateral. Optogenetic/chemogenetic manipulation of dopamine receptors D1 and D2 in the spinal trigeminal nucleus caudalis produced opposite effects on the nerve injury-induced trigeminal neuropathic pain. Specific excitation of dopaminergic neurons in the A11 nucleus attenuated the trigeminal neuropathic pain through the activation of D2 receptors in the spinal trigeminal nucleus caudalis. Conversely, specific ablation of the A11 dopaminergic neurons exacerbated such pain. Our results suggest that the descending A11-spinal trigeminal nucleus caudalis dopaminergic projection is critical for the modulation of trigeminal neuropathic pain and could be manipulated to treat such pain.

Herpes zoster or shingles is the result of varicella zoster virus (VZV) infection and often results in chronic pain that lasts for months after visible symptoms subside. Testosterone often attenuates pain in males. Previous work demonstrates ovarian estrogen effects γ-aminobutyric acid (GABA) signaling in the thalamus, reducing pain but the role of testosterone within the thalamus is currently unknown. Because aromatase affects pain and is present in the thalamus we tested a hypothesis that testosterone converted to estrogen in the thalamus attenuates herpes zoster induced pain. To address this hypothesis, male Sprague-Dawley rats received whisker pad injection of either MeWo cells or MeWo cells containing VZV. To reduce aromatase derived estrogen in these animals we injected aromatase inhibitor letrozole systemically or infused it into the thalamus. To test if estrogen was working through the estrogen receptor (ER) agonist, 4, 4′, 4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) was infused concomitant with letrozole. Motivational and affective pain was measured after letrozole and/or PPT treatment. Vesicular GABA transporter (VGAT) is important in pain signaling. Because estrogen effects VGAT expression we measured its transcript and protein levels after letrozole treatment. Virus injection and letrozole significantly increased the pain response but thalamic infusion of PPT reduced zoster pain. Letrozole increased the number of thalamic neurons staining for phosphorylated ERK (pERK) but decreased VGAT expression. The results suggest in male rats aromatase derived estradiol interacts with the ER to increase VGAT expression and increase neuronal inhibition in the thalamus to attenuate VZV induced pain.

Pain can vary over the estrous cycle as a result of changes in estradiol concentration but the mechanism causing this variation is unclear. Because the thalamus is important in pain control, gene expression in the lateral thalamus (ventral posteromedial, ventral posterolateral, reticular thalamic nuclei) was screened at different phases of the estrous cycle. Gene expression changes in Sprague–Dawley rats were further analyzed by real-time PCR and ELISA and plasma estradiol levels were measured by RIAs at different phases of the estrous cycle. Our results indicated that both the RNA and protein expression of glutamate decarboxylase 1 and 2 (GAD1, GAD2), GABA(A) receptor-associated protein like 1 (GABARAPL1), and vesicular GABA transporter (VGAT) significantly increased in the lateral thalamus when plasma estradiol levels were elevated. Estradiol levels were elevated during the proestrus and estrus phases of the estrous cycle. Estrogen receptor α (ERα) was observed to be co-localized in thalamic cells and thalamic infusion of an ERα antagonist significantly reduced GAD1 and VGAT transcript. GAD1, GAD2, GABARAPL1, and VGAT have been shown to effect neuronal responses suggesting that attenuation of pain during the estrous cycle can be dependent, in part, through estradiol induced changes in thalamic gene expression. J. Cell. Physiol. 231: 1057–1064, 2016. © 2015 Wiley Periodicals, Inc.

Varicella zoster virus (VZV) infects the face and can result in chronic, debilitating pain. The mechanism for this pain is unknown and current treatment is often not effective, thus investigations into the pain pathway become vital. Pain itself is multidimensional, consisting of sensory and affective experiences. One of the primary brain substrates for transmitting sensory signals in the face is the ventral posterior medial/posterior lateral thalamus (VPM/VPL). In addition, the anterior cingulate cortex (ACC) has been shown to be vital in the affective experience of pain, so investigating both of these areas in freely behaving animals was completed to address the role of the brain in VZV-induced pain. Our lab has developed a place escape avoidance paradigm (PEAP) to measure VZV-induced affective pain in the orofacial region of the rat. Using this assay as a measure of the affective pain experience a significant response was observed after VZV injection into the whisker pad and after VZV infusion into the trigeminal ganglion. Local field potentials (LFPs) are the summed electrical current from a group of neurons. LFP in both the VPM/VPL and ACC was attenuated in VZV injected rats after inhibition of neuronal activity. This inhibition of VPM/VPL neurons was accomplished using a designer receptor exclusively activated by a designer drug (DREADD). Immunostaining showed that cells within the VPM/VPL expressed thalamic glutamatergic vesicle transporter-2, NeuN and DREADD suggesting inhibition occurred primarily in excitable neurons. From these results we conclude: (1) that VZV associated pain does not involve a mechanism exclusive to the peripheral nerve terminals, and (2) can be controlled, in part, by excitatory neurons within the VPM/VPL that potentially modulate the affective experience by altering activity in the ACC.

Introduction: Calcium aluminate cements have shown little affinity for bacterial growth, low toxicity, and immunogenicity when used as a restoration material, but calcium aluminate cements have not been tested in vivoinpulpotomy procedures.Methods:Toaddress this question, a calcium aluminosilicate cement (QuickSet) was tested along with 2 mineral trioxide aggregates, ProRoot MTA and MTA Plus. These cements were used as a capping agent after pulpotomy. Control rats had no pulpotomy, or the pulpotomy was not capped. Proinflammatory cytokines interleukin (IL)-1b and IL-1a were measured, and histology was performed at 30 and 60 days after capping. The nociceptive response was determined by measuring the lengthening of the rat’s meal duration. Results: and Conclusions: IL-1b and IL-1a concentrations were reduced in the capped teeth, but no differences were observed among the 3 cements. Dentinal bridging could be detected at both 30 and 60 days with each of the 3 cements, and the pulps were still vital 60 days after capping. Meal duration significantly shortened after placement ofthe 3 different cements, indicating a nociceptive response, but there were no differences among the materials. Calcium aluminosilicate cement had similar properties to mineral trioxide aggregates and is a viable option for pulpotomy procedures. (J Endod 2014;40:1429‐1434)

Temporomandibular arthritis will lengthen a rodent’s meal duration. We hypothesized that meal duration would also lengthen after tooth pulp exposure, suggesting that this behavior could be used to measure tooth nociception. To test this hypothesis, we placed rats in feeding units and subjected 4 anterior mandibular molars to pulp exposure, with and without pre-treatment with the analgesic buprenorphine-HCl. In the first study, male Sprague-Dawley rats were placed in computerized sound-attenuated feeding modules, the pulp of 4 molars on the mandible were exposed, and meal duration was measured for 13 days. In a second study, rats were injected with either the analgesic buprenorphine-HCl or saline every 12 hrs; injections were started one day before pulp exposure. Meal duration was determined before and after treatment. In the first study, pulp exposure significantly increased daily meal duration for 8 days. In the second study, pulp exposure lengthened daily meal duration, but the group that was treated with buprenorphine-HCl showed no significant difference compared with control rats without pulp exposure. Evidence supports that a lengthening in meal duration is a response to tooth nociception and that this nociception can be measured for over a week.

Background Previous studies have shown 17β-estradiol will reduce temporomandibular joint (TMJ) inflammation and hypersensitivity in female rats. Although male rats contain significant amounts of oestradiol, it was unknown whether a physiological concentration of 17β-estradiol would attenuate male TMJ inflammation and nociception. Methods Intact and castrated rats were given a physiological concentration of oestradiol to examine first, if oestradiol will affect male TMJ nociception/inflammation and, second, if administration of oestradiol would act synergistically with endogenous male hormones to attenuate TMJ nociception. The hormonally treated rats were given TMJ injections of complete Freund's adjuvant (CFA) and then nociception was measured using a validated method in which a lengthening in meal duration is directly correlated to the intensity of deep TMJ nociception. Inflammation was assayed by quantitating pro-inflammatory gene expression. Results Meal duration was significantly lengthened after TMJ CFA injection and this lengthening was significantly attenuated in the castrated but not intact males after administering a physiological concentration of oestradiol. A physiological concentration of 17β-estradiol also significantly increased IL-6 expression in the inflamed TMJ of castrated males while 17β-estradiol did not alter IL-1β, CXCL2 and CCL20 expression. Castration increased pro-inflammatory mediators IL-6, IL-1β and CXCL2 suggesting male sex hormones were anti-inflammatory. Calcitonin gene-related peptide in the trigeminal ganglia was unchanged. Conclusions Similar to females, male rats with TMJ inflammation showed a reduced nociceptive response after treatment with a physiological concentration of oestradiol suggesting the effects of oestradiol treatment were not constrained by organizational processes in the males.

Trigeminal ganglia neurons express the GABA(A) receptor subunit alpha 6 (Gabrα6) but the role of this particular subunit in orofacial hypersensitivity is unknown. In this report the function of Gabrα6 was tested by reducing its expression in the trigeminal ganglia and measuring the effect of this reduction on inflammatory temporomandibular joint (TMJ) hypersensitivity. Gabrα6 expression was reduced by infusing the trigeminal ganglia of male Sprague Dawley rats with small interfering RNA (siRNA) having homology to either the Gabrα6 gene (Gabrα6 siRNA) or no known gene (control siRNA). Sixty hours after siRNA infusion the rats received a bilateral TMJ injection of complete Freund's adjuvant to induce an inflammatory response. Hypersensitivity was then quantitated by measuring meal duration, which lengthens when hypersensitivity increases. Neuronal activity in the trigeminal ganglia was also measured by quantitating the amount of phosphorylated ERK. Rats in a different group that did not have TMJ inflammation had an electrode placed in the spinal cord at the level of C1 sixty hours after siRNA infusion to record extracellular electrical activity of neurons that responded to TMJ stimulation. Our results show that Gabrα6 was expressed in both neurons and satellite glia of the trigeminal ganglia and that Gabrα6 positive neurons within the trigeminal ganglia have afferents in the TMJ. Gabrα6 siRNA infusion reduced Gabrα6 gene expression by 30% and significantly lengthened meal duration in rats with TMJ inflammation. Gabrα6 siRNA infusion also significantly increased p-ERK expression in the trigeminal ganglia of rats with TMJ inflammation and increased electrical activity in the spinal cord of rats without TMJ inflammation. These results suggest that maintaining Gabrα6 expression was necessary to inhibit primary sensory afferents in the trigeminal pathway and reduce inflammatory orofacial nociception.

Females report temporomandibular joint (TMJ) pain more than men and studies suggest estrogen modulates this pain response. Our goal in this study was to determine genes that are modulated by physiological levels of 17β-estradiol that could have a role in TMJ pain. To complete this goal, saline or complete Freund’s adjuvant was injected in the TMJ when plasma 17β-estradiol was low or when it was at a high proestrus level. TMJ, trigeminal ganglion and trigeminal subnucleus caudalis/upper cervical cord junction (Vc/C1–2) tissues were isolated from the treated rats and expression of 184 genes was quantitated in each tissue using real time PCR. Significant changes in the amount of specific transcripts were observed in the TMJ tissues, trigeminal ganglia and Vc/C1–2 region when comparing rats with high and low estrogen. GABA A receptor subunit α6 (Gabra6) and the glycine receptor α2 (Glra2) were two genes of interest because of their direct function in neuronal activity and a greater than 29 fold increase in the trigeminal ganglia was observed in proestrus rats with TMJ inflammation. Immunohistochemical studies showed that Gabrα6 and Glrα2 neuronal and not glial expression increased when comparing rats with high and low estrogen. Estrogen receptors α and β are present in neurons of the trigeminal ganglia, whereby 17β-estradiol can alter expression of Gabrα6 and Glrα2. Also, estrogen receptor α (ERα) but not ERβ was observed in satellite glial cells of the trigeminal ganglia. These results demonstrate that genes associated with neurogenic inflammation or neuronal excitability were altered by changes in the concentration of 17β-estradiol.

This study describes the in vivo biocompatibility of intra-articular poly(DL-lactic- co-glycolic acid) (PLGA) microparticle (MP) formulations in the rat temporomandibular joint (TMJ). To our knowledge, this is the first intra-articular microparticle-based drug delivery system for the TMJ. The impact of PLGA MP concentration on rat TMJ function was quantified via computerized meal pattern analysis; in this non-invasive technique, previously validated markers of TMJ pain or nociception (specifically, meal duration and food intake) were recorded by computer-monitored pellet feeders. Bilateral intra-articular injection of 15, 30, or 50 mg/mL PLGA MPs had no impact on meal duration or food intake over 6 days, compared with controls that did not receive injections. Histological analysis showed that the MPs were retained within the synovial lining. These findings indicate that the PLGA MPs described herein are biocompatible and suitable for intra-articular delivery to the rat TMJ, a finding that has significant implications for the improvement of TMJ therapeutics.

Objectives This study investigated the cytotoxicity of elemental ions contained in four fixed prosthodontic materials (gold, nickel–chromium, stainless-steel alloys and CAD–CAM ceramics). Materials and methods According to the determination of elements released from prosthodontic materials by using inductively coupled plasma mass spectroscopy, similar amounts of elements Pd, Ag, Zn, Cu, Ni, Cr, Mo, Be, Fe, Al, and K were prepared as salt solutions. Wells with a tenfold higher concentration of the tested elements were used as positive controls, while a well without any tested element was used as a negative control. These salt solutions were tested for cytotoxicity by culturing mouse L-929 fibroblasts in the salt solutions for a 7-day period of incubation. Then, the percentage of viable cells for each element was measured using trypan blue exclusion assay. The data ( n = 5) were statistically analyzed by ANOVA/Tukey test ( p Results The results showed a statistically significant difference for the cytotoxic effect of the tested elements salt solutions. For the released element concentrations the lowest percentage of viable cells (mean ± SD) was evident with Zn, Cu or Ni indicating that they are the highly toxic elements. Be and Ag were found to be intermediate in cytotoxic effect. Fe, Cr, Mo, Al, Pd or K were found to be the least cytotoxic elements. Significance Zn and Cu released from gold alloys, and Ni released from nickel–chromium alloys, which are commonly used as fixed prosthodontic restorations, show evidence of a high cytotoxic effect on fibroblast cell cultures.

A greater incidence of temporomandibular joint (TMJ) pain is reported in females, suggesting that gonadal hormones may play a role in this condition. However, the exact roles of 17β-estradiol (E2) and progesterone (P4) in TMJ pain are not completely known. Two experiments were performed to determine the separate roles of E2 and P4 in TMJ nociception at various stages of the estrous cycle. Ovariectomized (OVX) rats were cycled with physiological concentrations of E2 or P4. The E2-cycled rats then received bilateral TMJ injections of saline (SAL) or complete Freund’s adjuvant (CFA) on the morning of diestrus-2 (low E2 condition) or proestrus (high E2 condition). As a control, OVX rats (no ovarian E2 and no replacement) were injected with SAL or CFA. The TMJ nociception was measured using a validated novel method in which an increase in meal duration directly correlated to the intensity of deep TMJ nociception. In the E2 experiment, CFA injection, but not SAL, increased TMJ nociception in the OVX group, but the effect was less pronounced in diestrus-2 and even less in proestrus. In the P4 experiment, the rats receiving TMJ CFA in diestrus-2 (end of minor P4 surge) did not show an increase in TMJ nociception, whereas the rats injected in proestrus (major P4 surge), estrus (low P4), and metestrus (low P4) had similar increases in TMJ nociception. The hormones’ concentration did not affect TMJ IL-1β, IL-6, C-C motif ligand 20, or C-X-C motif ligand 2 or the trigeminal ganglia calcitonin gene-related peptide. The high physiological concentrations of E2 observed at proestrus and the low P4 concentrations observed at diestrus-2 attenuated or eliminated CFA-induced TMJ nociception. The results suggest that the cyclic estrous cycle concentrations of E2 and P4 can influence CFA-induced TMJ nociception in the rat.

Mesenchymal stem cells (MSC) and multipotent adult progenitor cells (MAPC) copurify from the bone marrow. Past studies have shown that bone marrow-derived cells transplanted into the periodontium obtain characteristics of several periodontal cells types, such as alveolar bone, periodontal fibroblasts, and cementoblasts. Bone marrow-derived cell populations are heterogeneous mixtures of different cell types. Characterization of each individual cell type in the bone marrow has not been completed. In this study, the MAPC were isolated from the bone marrow of 3-week-old rats by selecting attached cells that did not express leukocyte (CD45), erythroid (HIS49), or the MSC marker CD44. DiI-labeled male MAPC were injected into the periodontal ligament (PDL) of female adult rats. After 3 weeks, the morphology of the DiI-labeled cells was determined. Periodontal tissues sections that included alveolar bone, PDL, cementum, and dentin were also immunostained for collagen I and III after 3 days, 3 weeks, and 6 weeks following injection of male MAPC into the female PDL. Male and female cells were differentiated by fluorescent in situ hybridization tagging of the X and Y chromosomes. Our results show that DiI-labeled MAPC were spherical in shape before injection but after 3 weeks a portion of the injected MAPC obtained the characteristic spindle shape of PDL cells with an orientation parallel to the surrounding PDL cells. Consistent with the DiI results, male MAPC had a spherical shape on immunostained sections 3 days after injection and expressed collagen I and III at significantly reduced levels as compared with the endogenous female PDL. Six weeks after injection, the MAPC expressed both collagen I and collagen III at levels similar to the endogenous surrounding PDL and obtain a morphology characteristic of PDL. In conclusion, MAPC obtain PDL-like characteristics over a 6-week period after injection into the PDL.

– The ability of storage media to preserve periodontal ligament (PDL) cell vitality has been previously evaluated. However, the mechanisms by which different storage conditions alter the functional status of PDL cells have not been determined. The purpose of the present study was to investigate, in vitro, the level of programed cell death or apoptosis in a population of PDL cells following storage under different conditions. Primary human PDL cells were plated into 24-well-culture plates and allowed to attach for 24 h. Cells were then exposed for 1 h to milk, Hank's balanced salt solution (HBSS), Soft Wear contact lens solution or Gatorade at room temperature or on ice. Culture medium was used as a negative control. Apoptosis was evaluated at 24, 48, and 72 h after treatment on quadruplicate samples by using the ST 160 ApopTag Fluorescein Direct In Situ Detection Kit. The total number of cells and the total number of apoptotic cells were counted. The results indicated that at 24 and 72 h, PDL treated with Gatorade and the contact lens solution displayed the highest percentages of apoptotic cells when compared with the other treatment groups at room temperature. Overall, cells treated on ice showed significantly lower levels of apoptosis when compared with treatments at room temperature. In conclusion, the results indicated that apoptosis plays a major role in cell death in cells treated with Gatorade and contact lens solutions in comparison to other storage solutions and that storage on ice can inhibit programed cell death.

Previously we showed that intermittent administration of nicotine (NIC) in the dark phase decreased food intake and body weight and this could be blocked when the NIC receptor antagonist mecamylamine was infused into the fourth ventricle. Catecholaminergic neurons adjacent to the fourth ventricle contain NIC receptors and directly innervate the perifornical hypothalamus (PFH) which has been shown to be involved in regulation of feeding. This study explored whether NIC regulates feeding behavior by modulating catecholaminergic input to the PFH. Epinephrine and norepinephrine neuronal input was ablated within the PFH by infusion of 6-hydroxydopamine hydrobromide (6-OHDA), while bupropion was infused to protect dopaminergic neurons. After recovery of body weights to pre-surgery levels, food intake, meal size, meal number and body weight were measured after intermittent NIC injections. The results showed the PFH lesioned animals did not exhibit the typical prolonged drop in food intake, meal size and body weight normally associated with NIC administration. High performance liquid chromatography analyses demonstrated that compared to control rats, 6-OHDA administration significantly reduced PFH norepinephrine and epinephrine levels, but not dopamine levels. These results are consistent with NIC reducing food intake in part by acting through catecholaminergic neurons within or extending through the PFH.

Computerized meal pattern analysis, and more specifically meal duration, has recently been used as a non-invasive biological marker of nociception in the temporomandibular joint (TMJ). Cells responsible for the nociceptive response in the inflamed TMJ may include capsaicin (CAP) sensitive neurons. To test the role of CAP sensitive neurons in acute nociceptive responses first, male and female rats were treated neonatally with vehicle or CAP, an agent known to destroy a majority of C fibers. Second, after 56 days the rats were divided into four groups: neonatal vehicle-injected and treated with and without complete Freund's adjuvant (CFA). Treatment groups included neonatal non-CAP vehicle treated and TMJ not-injected (CON); vehicle treated and TMJ CFA injected (CFA); CAP-treated and not-injected (CAP); and CAP-treated and CFA injected (CAP+CFA). Meal patterns were analyzed for two days after injection. CFA-injection in non-CAP-treated rats lengthened meal duration on the first and second day after treatment in the males, but only on the first day in the females. CAP treatment in male and female rats prevented significant lengthening of meal duration induced by CFA. CAP treatment attenuated the CFA-induced increase in calcitonin gene-related peptide expression in the trigeminal ganglia similarly in males and females. The data suggests CAP-sensitive neurons are responsible, in part, for transmission of acute nociceptive responses associated with CFA administration and suggest gender can affect nociception in the inflamed TMJ region.

In previous studies food intake and meal size significantly decreased in rats two days after injecting 4 mg/kg/day nicotine tartrate. Food intake returned to normal after nine days of continued nicotine treatment, when reduced meal size is countered by an increase in meal number. Nicotine also reduced body weight after nicotine injection and body weight remained low after nine days. To begin characterizing the mechanism that modulates these changes in feeding behavior and/or body weight during nicotine exposure the transcript levels for agouti related protein (AGRP), cocaine-amphetamine-regulated transcript (CART), corticotropin releasing hormone receptor one (CRH-R1), melanocortin receptors three and four (MC3R/4R), neuropeptide Y (NPY), NPY Y1 and Y5 receptors and/or pro-opiomelanocortin (POMC) were analyzed in the arcuate (ARC), dorsomedial (DMN) and paraventricular (PVN)/periventricular (PE) hypothalamic nuclei on the second and ninth day of saline or nicotine treatment. Results show that the transcript levels of the anorexigenic molecule CART increased in the PVN and/or PE two days after nicotine treatment but after nine days CART levels equalize. In contrast, nine days of nicotine treatment reduced CART levels in the DMN as compared to saline controls. To investigate CART's role in regulating feeding, infusion of CART (55-102) into the third ventricle reduced food intake and meal size. These results are consistent with nicotine modulating feeding behavior and body weight, in part, by affecting CART transcript levels in the DMN, PVN and/or PE.

Adult stem cells play a role in remodeling and regeneration of tissues and organs throughout the life of an organism. Data from adult stem cell studies suggested these cells produce a limited subset of mature cell types; in other words, the cells have limited potency. However, recent findings suggest that these adult stem cells may be capable of giving rise to multiple cell types produced from different germ layers. Adult stem cells have been identified in the craniofacial complex including stem cells from craniofacial bone, dental pulp, periodontal ligament, and developing tooth bud. Utilization of adult stem cells has shown these cells to express markers consistent with differentiated tissues and cell types present within the oral cavity. Currently, studies are using adult stem cells to fabricate new tissues for replacement and regeneration of lost tissues caused by trauma or disease. Additional focus is currently on the mechanism by which these stem cells produce differentiated cells expressing protein markers and having function similar to tissues in which the stem cells were placed. This review discusses discoveries delineating stem cell potency, the critical roles of adult stem cells in the postnatal growth/development as well as remodeling, and recent studies utilizing adult stem cells to repair and/or regenerate craniofacial bone, teeth, periodontium, and temporomandibular joint in clinical protocols.

Analyzing feeding behavior, and in particular meal duration, can be used as a biological marker for temporomandibular joint (TMJ) inflammation/pain. The present study determined the specificity of meal duration as a measure of TMJ inflammation/pain in a rodent model. The model was also used to test the efficacy of dexamethasone (DEX) as a treatment for TMJ inflammation/pain that was induced by TMJ injection of complete Freund's adjuvant (CFA). In the first study, anesthetized male Sprague-Dawley rats housed in computerized feeding modules received bilateral intra-articular knee injections of CFA or saline. The next day, CFA-injected rats had significant knee swelling and impaired mobility. Food intake in the CFA-injected group was reduced over the next two days and this was due to reduced meal number with no change in meal size. Notably, meal duration was normal in both the CFA and saline knee-injected groups. In the second study, male rats were assigned to one of four groups: Group 1, no CFA and no DEX treatment; Group 2, no CFA and treatment with DEX (0.4 mg/kg i.m. once daily); Group 3, bilateral TMJ CFA injection and no DEX treatment; and Group 4, bilateral TMJ CFA injection and treatment with DEX. CFA significantly increased TMJ swelling and stress-induced chromodacryorrhea in Group 3, but treatment with DEX attenuated these effects in Group 4. Compared to the controls, meal duration was significantly lengthened 24 and 48 h post-CFA injection in Group 3, whereas DEX treatment attenuated TMJ swelling, chromodacryorrhea and normalized meal duration. The data demonstrate that meal pattern analysis, and in particular meal duration, can be used as a non-invasive specific measure of TMJ inflammation/pain and can be used as a marker of DEX treatment efficacy.

Objective Macrophages release cytokines, such as tumor necrosis factor α (TNFα), interleukin-1 (IL-1), and IL-6, which modulate the symptoms of rheumatoid arthritis (RA). Macrophage release of these cytokines can be modulated by estrogen. Fcγ receptor type IIIA (CD16a) is a receptor expressed on macrophages that selectively binds IgG molecules, an important rheumatoid factor in RA. Binding of CD16 by anti-CD16 monoclonal antibodies stimulates macrophage cytokine release. We undertook this study to test the hypothesis that decreased concentrations of estrogen (17β-estradiol) directly cause an increase in CD16 expression, resulting in increased release of proinflammatory cytokines from monocytes and/or macrophages upon receptor binding. Methods THP-1 cells and female human primary monocytes and monocyte-derived macrophages were treated with no 17β-estradiol, physiologic levels (1 × 10−8M) of 17β-estradiol, or 1 × 10−8M 17β-estradiol followed by withdrawal of 17β-estradiol. Surface expression of CD16 and CD16 messenger RNA was measured using fluorescence-activated cell sorting (FACS) and semiquantitative reverse transcription–polymerase chain reaction, respectively. Cytokine release from 17β-estradiol–treated or untreated monocytes was then quantitated by enzyme-linked immunosorbent assay and FACS after crosslinking the receptor with anti-CD16 antibodies. Results CD16 transcript significantly increased in macrophage-like THP-1 cells and in primary, peripheral blood macrophages in the absence of 17β-estradiol, and the observed increase in message was dependent on transcription. CD16 receptor levels on CD14+, transforming growth factor β–treated primary monocytes also increased in cells deprived of 17β-estradiol. Analysis of the cytokines released showed that CD16 crosslinking stimulated significant increases in TNFα, IL-1β, and IL-6 due to the absence of estrogen. Conclusion Estrogen can modulate proinflammatory cytokine release from activated monocytes and/or macrophages, in part through modulation of CD16 expression.

Mesenchymal stem cells differentiate into multiple types of cells derived from mesenchyme. Periodontal ligament cells are primarily derived from mesenchyme; thus, we expected mesenchymal stem cells to differentiate into periodontal ligament. Using a combination of immunohistochemistry and in situ hybridization on co-cultures of mesenchymal stem cells and periodontal ligament, we observed a significant increase in mesenchymal stem cells’ expression of osteocalcin and osteopontin and a significant decrease in expression of bone sialoprotein, characteristics of periodontal ligament in vivo. Increased osteopontin and osteocalcin and decreased bone sialoprotein expression was detected within 7 days and maintained through 21 days of co-culture. We conclude that contact or factors from periodontal ligament induced mesenchymal stem cells to obtain periodontal-ligament-like characteristics. Importantly, analysis of the data suggests the feasibility of utilizing mesenchymal stem cells in clinical applications for repairing and/or regenerating periodontal tissue.

Oestrogen receptor (ER) regulation of gene transcription in neurosecretory and pituitary cells has been proposed as an important mechanism for increased hypothalamic-pituitary-adrenal (HPA) axis responses in females of several mammalian species, including humans. Inbred female Fischer (F344/N) and Lewis (LEW/N) rats have similar oestrogen levels, although Fischer rats exhibit hyper- and Lewis rats hypo-HPA axis responses. The blunted HPA axis response of Lewis rats has been associated with their blunted hypothalamic corticotropin releasing hormone (CRH) expression. To determine if the female CRH expression deficiency in Lewis rats is associated with defective ER expression and regulation, hypothalamic paraventricular nucleus (PVN) transcript levels of CRH and ER were determined under basal conditions and after immune challenge. Microdissected PVN were obtained from control and lipopolysaccharide (LPS) treated Lewis and Fischer rats and CRH, ERalpha and beta mRNA levels were determined by semiquantitative reverse-transcriptase-polymerase chain reaction. In addition, ERalpha and beta protein levels were determined by semiquantitative Western blots. ERalpha and beta mRNA and protein levels in the PVN of control Fischer rats were significantly higher than in control Lewis rats. ERalpha and beta mRNA and protein levels in Fischer rats were reduced by LPS administration at the time of maximal CRH mRNA levels but did not change in Lewis rats, an effect independent of oestrogen levels. These data indicate that defective neuroendocrine HPA axis responses are associated with defective ER expression and regulation in Lewis PVN despite oestrogen concentrations.

Macrophage activation and cholesterol processing can be affected by changes in estrogen concentrations. However, there is a paucity of information about the genes and mechanisms regulating this estrogen effect. In primary monocyte-derived macrophages we detected transcript and protein for estrogen receptor beta (ERbeta). Determination of genes regulated by estrogen was completed using cDNA arrays and semiquantitative RT-PCR on RNA isolated from macrophages cultured in serum free media containing (5-10) x 10(-9)M 17-beta-estradiol and subsequently deprived of estrogen for a 24h period. The data indicate that the transcript levels of interleukin 1 receptor antagonist (IL-1ra), beta 2-microglobulin, annexin XI and the LXR(alpha) receptor significantly increased and that Ly-GDI transcript levels significantly decreased after estrogen withdrawal; data congruent with estrogen depletion regulating macrophage inflammatory and biochemical processes. Treatment of THP-1 cells with phorbol 12-myristate-13-acetate in the presence or absence of estrogen indicate that differentiation to a macrophage-like cell type was a prerequisite for production of the estrogen response. In addition, experiments using cycloheximide treatment, that blocks nascent protein synthesis, indicated that estrogen withdrawal affected the transcript levels of LXR(alpha) and IL-1ra through dissimilar pathways.

Women report TMJ pain more often than men and epidemiological evidence suggests the pain response varies between men and women and over the menstrual cycle. Recent studies show TMJ responsive neuro...

A lengthening in meal duration can be used to measure an increase in orofacial mechanical hyperalgesia having similarities to the guarding behavior of humans with orofacial pain. To measure meal duration unrestrained rats are continuously kept in sound attenuated, computerized feeding modules for days to weeks to record feeding behavior. These sound-attenuated chambers are equipped with chow pellet dispensers. The dispenser has a pellet trough with a photobeam placed at the bottom of the trough and when a rodent removes a pellet from the feeder trough this beam is no longer blocked, signaling the computer to drop another pellet. The computer records the date and time when the pellets were taken from the trough and from this data the experimenter can calculate the meal parameters. When calculating meal parameters a meal was defined based on previous work and was set at 10 min (in other words when the animal does not eat for 10 min that would be the end of the animal's meal) also the minimum meal size was set at 3 pellets. The meal duration, meal number, food intake, meal size and inter-meal interval can then be calculated by the software for any time period that the operator desires. Of the feeding parameters that can be calculated meal duration has been shown to be a continuous noninvasive biological marker of orofacial nociception in male rats and mice and female rats. Meal duration measurements are quantitative, require no training or animal manipulation, require cortical participation, and do not compete with other experimentally induced behaviors. These factors distinguish this assay from other operant or reflex methods for recording orofacial nociception.

A novel protein (NELF) was identified screening embryonic luteinizing hormone releasing hormone (LHRH) neurons at different migrational states. Experiments in vitro revealed that NELF functions in olfactory axon outgrowth and subsequently alters LHRH neuronal migration. NELF was not restricted to LHRH neurons in the developing rodent. Multiple CNS and PNS tissues expressed this gene. To characterize the specific regions that express NELF in situ hybridization histochemistry was performed. Within the CNS, cells in the cortex, hippocampus, thalamus and olfactory regions express NELF pre- and postnatally.

Neurons differentiating into the luteinizing hormone-releasing hormone (LHRH) neuroendocrine phenotype are derived from the nasal placode. Cells within the vomeronasal organ anlage that turn on LHRH gene and peptide expression subsequently migrate into the forebrain where they influence reproductive function. The molecular and cellular cues regulating differentiation and migration of these cells are unknown. Discovery of developmental markers can indicate proteins directing or associated with differentiation. Analysis of such markers after manipulation of external cues can elucidate important extracellular differentiation signals. Embryonic LHRH neurons were examined in vivo for Mash-1 and nestin, two factors that delineate precursor populations in PNS and forebrain CNS cells. Nestin, but not Mash-1, was detected in early expressing LHRH cells in the vomeronasal organ anlage. These results were duplicated in LHRH neurons maintained in vitro in nasal explants. Such LHRH cells expressed nestin mRNA but not Mash-1 mRNA and were also negative for three other olfactory epithelial developmental transcription factors, Math4A, Math4C/neurogenin1, and NeuroD mRNA. Experimental manipulation of nasal explants revealed dual expression of nestin protein and LHRH in cells proximal to the vomeronasal organ anlage that was dependent upon midline cartilaginous/mesenchymal tissues. Prolonged nestin expression in LHRH cells after midline removal is consistent with nasal midline tissues modulating differentiation of LHRH neurons from the nasal placode.

Although a variety of cues have been implicated in axonal targeting during embryogenesis and regeneration, the precise mechanisms guiding olfactory axons remain unclear. Appropriate olfactory axon pathfinding is essential for functional chemoreceptive and pheromone receptive systems. Olfactory axon pathfinding is also necessary for establishment of the neuroendocrine LHRH system, cells critical for reproductive function. LHRH cells exhibit neurophilic migration moving from the nasal region along olfactory axons into the brain. Factors involved in the migration of these neuroendocrine cells are as yet unresolved. We report identification of a novel factor termed nasal embryonic LHRH factor (NELF) that was discovered in a differential screen of migrating versus nonmigrating primary LHRH neurons. NELF is expressed in PNS and CNS tissues during embryonic development, including olfactory sensory cells and LHRH cells. NELF antisense experiments indicate that a reduction in NELF expression decreases olfactory axon outgrowth and the number of LHRH neurons that migrate out of the nasal tissue. These results demonstrate that NELF plays a role as a common guidance molecule for olfactory axon projections and subsequently, either directly or indirectly, in the neurophilic migration of LHRH cells.

LHRH is the neuropeptide responsible for reproductive function. Prenatally, LHRH expression begins when neurons are in the olfactory pit and continues as these cells migrate into the brain. Thus, LHRH neurons maintain neuropeptide expression through very distinct environments. The regulatory interactions that control onset and continued expression of the LHRH phenotype are unknown. To begin to address this question primary LHRH neurons were removed from nasal explants at different ages. A complementary DNA (cDNA) subtraction screen was performed comparing a 3.5-days in vitro LHRH neuron [approximately embryonic day 15 (E15) in vivo] to two 10.5-days in vitro LHRH neurons (approximately postnatal day 1 in vivo). The transcription factor activator protein-2 (AP-2alpha) was differentially expressed and was present in the developmentally younger LHRH neuron. In vivo analysis revealed that LHRH neurons expressed AP-2 as they migrated across the cribriform plate and into the forebrain beginning on E13.5, but that coexpression of LHRH and AP-2 was no longer detected in postnatal day 1 animals. This suggested a regulatory role for AP-2 in LHRH neurons. Analysis of animals lacking AP-2alpha revealed a dramatic decrease in forebrain LHRH neurons between E13.5 and E14.5, correlating with normal onset of AP-2 expression in LHRH neurons as they entered the central nervous system. Nasal cells robustly expressing LHRH were still present on E 14.5. The continued presence of forebrain LHRH cells is proposed based on a second marker, galanin, and lack of increased apoptotic/necrotic cells in this region. A decrease in LHRH messenger RNA in forebrain neurons indicates regulation of LHRH occurred at the transcriptional or posttranscriptional level in mutant animals. These results indicate a developmentally restricted involvement of the transcription factor AP-2 in LHRH expression once the LHRH neurons have migrated into the forebrain, but before establishment of an adult-like distribution.

Unrestrained DNA supercoiling was measured using a Me3-psoralen photobinding assay within a transcriptionally active hygromycin B phosphotransferase (hph) gene integrated into different chromosomal locations in five transformed human fibrosarcoma cell lines. The level of unrestrained supercoiling in the hph gene varied, from high to low levels, in different chromosomal locations in living human cells. In one cell line, the hph gene contained no unrestrained supercoiling. Consequently, supercoiling was not dictated by the DNA sequence of the active hph gene. The addition of alpha-amanitin, which can inhibit transcription, reduced unrestrained supercoiling by 75% at one chromosomal location, by 50% at two other locations, and had little, if any, effect at two other chromosomal locations. Different levels of supercoiling in separate regions of the chromosome require that the chromosome be organized into independent topological domains in vivo. Evidence for independent topological domains in living cells is presented. From analysis of the relaxation of supercoiling as a function of the number of breaks introduced into the chromosome, the in vivo topological domain size for the human ribosomal RNA genes was estimated between 30,000 and 45,000 kb.

At least nine human genetic diseases, including myotonic dystrophy (DM) and fragile X syndrome have been associated with the expansion of CTG or CGG trinucleotide repeats within the disease loci. Little is known about the molecular mechanisms or the genetic control of the expansion of triplet repeats. Mutations in human mismatch repair genes are associated with the increased polymorphism of many microsatellites, including dinucleotide repeats. The effect of mutations in two mismatch repair genes on the size of trinucleotide repeats in the DM and FRAXA loci has been analyzed. PCR and Southern analysis of the triplet repeat regions of the DM and fragile X mental retardation (FRAXA) loci in cell lines HTC116 and LoVo, which contain mutations in both alleles of the hMLH1 and hMSH2 genes, respectively, indicated that the size of the endogenous (CTG)n and (CGG)n tracts fall within the range observed in the normal population. This suggests that mutations in hMLH1 or hMSH2 do not result in the instability of CTG or CGG tracts to the levels observed in individuals with myotonic dystrophy or fragile X syndrome.

Activation of the GABAA receptor results in inhibition of neuronal activity. One subunit of this multi-subunit receptor termed alpha 6 (Gabrα6) contributed to inflammatory temporomandibular joint (TMJ) nociception but TMJ disorders often include myofascial pain. To address Gabrα6 role in myofascial pain we hypothesized that Gabrα6 has an inhibitory role in myofascial nociceptive responses similar to inflammatory TMJ arthritis. To test this hypothesis a, myofascial nociceptive response was induced by placing a ligature bilaterally on the tendon attachment of the anterior superficial part of a male rat's masseter muscle. Four days after ligature placement Gabrα6 expression was reduced by infusing the trigeminal ganglia (TG) with small interfering RNA (siRNA) having homology to either the Gabrα6 gene (Gabrα6 siRNA) or no known gene (control siRNA). After siRNA infusion nociceptive behavioral responses were measured, i.e., feeding behavior and head withdrawal after pressing upon the region above the ligature with von Frey filaments. Neuronal activity in the TG and trigeminal nucleus caudalis and upper cervical region (Vc-C1) was measured by quantitating the amount of phosphorylated extracellular signal-regulated kinase (p-ERK). Total Gabrα6 and GABAA receptor contents in the TG and Vc-C1 were determined. Gabrα6 siRNA infusion reduced Gabrα6 and GABAA receptor expression and significantly increased the nociceptive response in both nociceptive assays. Gabrα6 siRNA infusion also significantly increased TG p-ERK expression of the ligated rats. From these results we conclude GABAA receptors consisting of the Gabrα6 subunit inhibit TG nociceptive sensory afferents in the trigeminal pathway and have an important role in the regulation of myofascial nociception.

We investigated the in vivo therapeutic efficacy of an intra-articular controlled release system consisting of biodegradable poly(DL-lactic-co-glycolic acid) (PLGA) microparticles (MPs) encapsulating anti-inflammatory small interfering RNA (siRNA), together with branched poly(ethylenimine) (PEI) as a transfecting agent, in a rat model of painful temporomandibular joint (TMJ) inflammation. The in vivo effects of PLGA MP dose and siRNA-PEI polyplex delivery were examined via non-invasive meal pattern analysis and by quantifying the protein level of the siRNA target as well as of several downstream inflammatory cytokines. Controlled release of siRNA-PEI from PLGA MPs significantly reduced inflammation-induced changes in meal patterns compared to untreated rats with inflamed TMJs. These changes correlated to decreases in tissue-level protein expression of the siRNA target to 20–50% of the amount present in the corresponding control groups. Similar reductions were also observed in the expression of downstream inflammatory cytokines, e.g., interleukin-6 (IL-6), whose tissue levels in the siRNA-PEI PLGA MP groups were 50% of the values for the corresponding controls. This intra-articular sustained release system has significant implications for the treatment of severe TMJ pain, and also has the potential to be readily adapted and applied to mitigate painful, chronic inflammation in a variety of conditions.

A majority of individuals reporting temporomandibular joint (TMJ) disorders have joint damage, inflammation or arthritis (Manfredini, Chiappe & Bosco, 2006; Plesh, Sinisi, Crawford & Gansky, 2005). The joint will show loss of extra-cellular matrix components in the articular cartilage and subchondral bone resulting in destruction of cartilage and bone, such processes lead to inflammation and exacerbation of joint tissue catabolism (Tanaka, Detamore & Mercuri, 2008). Matrix metalloproteinases 1 and 9 (MMP-1 and MMP-9) are two major enzymes that contribute to tissue catabolism and have been observed in patients with TMJ disorders (Kanyama, Kuboki, Kojima, Fujisawa, Hattori, Takigawa & Yamashita, 2000; Srinivas, Sorsa, Tjaderhane, Niemi, Raustia, Pernu, Teronen & Salo, 2001; Yoshida, Takatsuka, Hatada, Nakamura, Tanaka, Ueki, Nakagawa, Okada, Yamamoto & Fukuda, 2006). Reversal of these disease processes and treatment of the joint to reduce pain are effective in the early stages of the disease, but treatment often fails to alleviate the severe, chronic pain caused by advanced joint degeneration (Gerwin, Hops & Lucke, 2006; Tanaka, Detamore & Mercuri, 2008). Treatment of TMJ osteoarthritis with intra-articular injections of non-steroidal antiinflammatory drugs (NSAIDs) and opiates into the superior joint space have shown efficacy (Bryant, Harrison, Hopper & Harris, 1999; Swift, Roszkowski, Alton & Hargreaves, 1998; Zuniga, Ibanez & Kozacko, 2007). Intra-articular administration versus systemic administration of NSAID or opiates would be advantageous for treatment of TMJ inflammation and pain because local administration avoids the ectopic effects seen with NSAIDs like rofecoxib (i.e., Vioxx) (Lin, Weisdorf, Solovey & Hebbel, 2000) or opiates. For example, nonselective NSAIDS can cause intestinal bleeding, whereas some selective cyclooxygenase-2 inhibitors have significant cardiovascular and renal safety risks (Davies & Jamali, 2004; Mukherjee, Nissen & Topol, 2001). Moreover, opioids frequently cause constipation, sedation, nausea, vomiting, and respiratory depression (Mercadante, 1999). Intra-articular injection remains controversial in light of decades of mixed reports demonstrating intra-articular injections either accelerate or trigger destruction of tissues within the TMJ and the surrounding area (Bjornland, Rorvik, Haanaes & Teige, 1994; Sugisaki, Ikai & Tanabe, 1995; Westesson, Eriksson & Liedberg, 1986). Recent reports

Objective Fcγ receptor III (FcγRIII; CD16) is a receptor expressed on immune cells that selectively binds IgG molecules. IgG binding results in cellular activation and cytokine release. IgG is an important factor in arthritis and can be found in the arthritic temporomandibular joint (TMJ). We undertook this study to test the hypothesis that a reduction in FcγRIII expression in TMJ tissues would reduce the nociceptive and inflammatory responses in an inflamed joint. Methods Small interfering RNA (siRNA), either naked or complexed with linear polyethyleneimine, was injected into the superior joint space of the TMJ in rats. After administration of siRNA the joint was injected with saline or with Freund's complete adjuvant to induce arthritis. Nociceptive responses were quantitated in the rat by measuring the animal's meal duration. FcγRIII expression in the TMJ tissue was assayed by immunocytochemistry or Western blotting. Cleavage of FcγRIII transcript was then assayed by 5′ rapid amplification of complementary DNA ends. Interleukin-1β (IL-1β) and IgG content was measured in the TMJ tissue by enzyme-linked immunosorbent assay. Results Injection of FcγRIII siRNA reduced the amount of FcγRIII in the TMJ tissues, and the transcript was cleaved in a manner consistent with an RNA interference mechanism. Moreover, injection of FcγRIII siRNA reduced the nociceptive response of rats with an arthritic TMJ and reduced the amount of the proinflammatory cytokine IL-1β. Conclusion FcγRIII contributes to the pain resulting from inflammatory arthritis of the TMJ, and siRNA has the potential to be an effective treatment for this disorder.

Temporomandibular joint (TMJ) pain has been reported to last for prolonged periods in humans. In rodents a variety of methods have been used to measure TMJ nociception, but for most of these methods the period of measurement has been minutes to a couple of hours. In addition, most measurement protocols required restraint or training of the animal. Previous studies from our laboratory demonstrated that feeding behavior, particularly meal duration, was an indicator of TMJ nociception in unrestrained and untrained male and female Sprague-Dawley rats for up to two days. In this study, we first found that injection of complete Freund's adjuvant (CFA) into the TMJ of rats significantly lengthened meal duration for 19 days and also decreased meal frequency for 42 days. Interestingly, the meal duration varied significantly from day to day within the 19 day period. TMJ interleukin-1 beta (IL-1 beta) and calcitonin gene-related peptide (CGRP) were significantly elevated in the TMJ tissues of CFA-injected animals and the level of these markers was attenuated as the meal duration decreased with time. Control animals injected with saline into the TMJ or CFA into the knee did not show a significant lengthening in meal duration but did show a decrease in meal frequency. In a second study, DBA/1LacJ mice given TMJ CFA injections showed a significantly lengthened meal duration on four of the seven days measured using end-of-the meal definition of 5 or 10 min. No other meal pattern changed significantly. Two days post-CFA injection, the DBA/1LacJ mice showed significantly elevated interleukin-6 (IL-6), but not elevated IL-1 beta. Seven days post-injection, both IL-6 and IL-1 beta were significantly elevated. No change in CGRP was detected. In this study C57Bl/6 mice also received TMJ CFA injections, but they did not show a lengthening in any meal pattern or significant increases in IL-1 beta, IL-6 or CGRP. Our data show, for the first time, that meal duration can be used to measure CFA-induced nociception in the TMJ over the course of several weeks in unrestrained rats and for up to seven days in the DBA/1LacJ mouse strain. In addition, C57Bl/6 mice are resistant to CFA-induced TMJ nociception at the same dose used in the DBA/1LacJ mice.

BackgroundEstrogen is known to play role in temporomandibular joint (TMJ) disorders and estrogen effects can be mediated by estrogen receptor (ER) alpha present in the TMJ. Cells expressing the estrogen receptor ERalpha are present in the temporomandibular joint (TMJ) but changes in expression due to estrogen and inflammation have not been characterized. In this study, ERalpha protein content and the number of cells expressing ERalpha was measured in 17 beta-estradiol-treated rats after inflammation was induced in the TMJ.MethodsSixteen ovariectomized female rats were divided into two groups such that one group received 17 beta estradiol (E2) and the other was given vehicle (VEH). Groups were then subdivided further, one received injections of saline and the other received Complete Freund's adjuvant (CFA) within the superior joint space of the TMJ. Thus the four groups include no E2/saline, E2/saline, no E2/CFA and E2/CFA. After treatment, the rats were sacrificed, and the TMJ anterior, disc, retrodiscal and synovial tissues were analyzed by western blot and immunocytochemistry. Positive stained cells were counted using a Nikon epifluorescent microscope.ResultsThe western blot showed that ERalpha protein significantly decreased with inflammation. The number of ERalpha-positive cells in the TMJ was not affected by inflammation or 17 beta-estradiol with exception of the retrodiscal tissue. In the retrodiscal tissue 17 beta-estradiol significantly decreased the number of ERalpha-positive cells but only in a non-inflamed joint.ConclusionsIn conclusion, inflammation and 17 beta-estradiol can modulate ERalpha expression in the TMJ but the effects are tissue specific.

It is unclear what contribution food intake and metabolism have in causing weight loss after administering a dose of nicotine equivalent to smoking one to three packs of cigarettes per day because previous studies have been of a very short duration. To address this question, male Sprague Dawley rats were housed in computerized food intake modules and fed 45 mg pellets: Group 1 [nicotine injected with 1.4 mg/kg/day (free base), fed ad libitum]; and Group 2 [saline injected and pair-fed by computer with Group 2]; and Group 3 [saline injected (i.p.), fed ad libitum]. The rats received 4 equally spaced injections over the dark phase. Treatment consisted of: Phase 1 (nicotine or saline for 14 days), Phase 2 (all rats saline for 8 days and Phase 3 (pair-fed group "unyoked" for 6 days)). Nicotine inhibited food intake over the first 6 days. On termination of nicotine, there was no compensatory hyperphagia in either Groups 1 or 2; and their body weight was reduced starting on day 5 until day 28. In another study, rats were housed in an indirect calorimetry system. Saline or nicotine was injected for 14 days, as noted above; then all rats were injected with saline for 4 days and then no injections for 10 days to follow changes in body weight. Energy expenditure (Kcal/Kg(0.75)) was measured for 18 days. Nicotine significantly reduced food intake on 7 of 14 days of nicotine injections. The body weight of the nicotine injected rats was significantly reduced starting on day 3 until day 25. There were no differences in energy expenditures of the groups, which suggested that a decrease in food intake and not an increase in metabolism was the reason the rats lost weight after administering nicotine.