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• Nrxn3 expression within the central amygdala enhances GABA release within the lateral parabrachial nucleus. • Nrxn3 expression within the central amygdala reduces VZV associated pain. • GABA release within the lateral parabrachial nucleus attenuates VZV associated pain. 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. [ABSTRACT FROM AUTHOR]

We compare nonparametric permutation method using intra-meal rate as endpoint with existing ANOVA method that uses average daily meal duration as an endpoint for detection of chronic pain in Sprague-Dawley rats. Nociception following bilateral temporomandibular joint ( TMJ) injection of high dose of Complete Freunds Adjuvant ( CFA, 250 μg/50 μL per side) could be detected in young adult male Sprague-Dawley rats using average daily meal durations as a measure of nociception for up to 19 days (Kramer, Kerins, Schneiderman & Bellinger, Physiology & Behavior, 99, 2010; 669) using ANOVA and multiple comparison range tests. In this study, we reanalyzed the data using a nonparametric permutation procedure based on absolute differences between intra-meal feeding rate curves. In addition to that experiment, we injected bilaterally the TMJ of naive rats with either a low-dose CFA (15 μg/50 μL per side, n = 6) or saline (50 μL of 0.9%, n = 4) and monitored the animals for 7 days. The permutation test of the intra-meal feeding rate detected the presence of nociception in the high-dose CFA treatment group for up to 40 days or twice as long as when using ANOVA on average daily meal durations. The permutation method also detected the low-dose CFA-induced nociception with ten times lower p-values and for several days longer than ANOVA of changes in meal durations. CFA-induced injury resulted in even reduction in intra-meal feeding rate and lengthening of the meals in both high- and low-dose CFA-injected animals. The rate analysis also showed when the rats first started a meal they were experiencing the same level of nociception as at the end of the meal. This demonstrated that intra-meal chewing itself did not alter the level of nociception. These results suggest that permutation tests based on differences in intra-meal feeding rates can be used as a sensitive test to determine and study the temporal patterns of TMJ nociception. [ABSTRACT FROM AUTHOR]

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 (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. [ABSTRACT FROM AUTHOR]

Background: Clinical studies have tested the use of an engineered herpes virus to treat pain. We hypothesized that subcutaneous injections of an engineered herpes virus that expresses enkephalin would attenuate orofacial nociception and hypersensitivity in male and female rats by a central mechanism. Methods: Herpes virus was injected subcutaneously around the mouth of male and female rats seventy-two hours before ligatures were placed on the masseter tendon, control treatment groups received either no virus or no ligature. Enkephalin expression was measured and von Frey filament testing and meal duration were utilized to measure mechanical hypersensitivity and the nociceptive response, respectively. Naloxone or naloxone methiodide was administered to rats injected with the enkephalin expressing virus to test if enkephalin was acting peripherally or centrally. Results: Ligature significantly lengthened meal duration and reduced the threshold to von Frey filaments for 18 days. Infection with the enkephalin transgene significantly decreased this response for at least 11 days but only in male rats. Virus injection significantly increased expression of enkephalin in the mental nerve that innervates the mouth region, the trigeminal ganglia and the trigeminal nucleus caudalis but no increase was observed in the masseter nerve after virus injection. Naloxone but not naloxone methiodide reversed the response to the enkephaline expressing virus. Conclusions: The data suggests that sex should be a considered when using this virus and that viral transfection of the mental nerve with an enkephalin transgene can reduce nociception and hypersensitivity through a central mechanism. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Fc[gamma] receptor III (Fc[gamma]RIII; 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 Fc[gamma]RIII 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. Fc[gamma]RIII expression in the TMJ tissue was assayed by immunocytochemistry or Western blotting. Cleavage of Fc[gamma]RIII transcript was then assayed by 5' rapid amplification of complementary DNA ends. Interleukin-1[beta] (IL-1[beta]) and IgG content was measured in the TMJ tissue by enzyme-linked immunosorbent assay. RESULTS: Injection of Fc[gamma]RIII siRNA reduced the amount of Fc[gamma]RIII in the TMJ tissues, and the transcript was cleaved in a manner consistent with an RNA interference mechanism. Moreover, injection of Fc[gamma]RIII siRNA reduced the nociceptive response of rats with an arthritic TMJ and reduced the amount of the proinflammatory cytokine IL-1[beta]. CONCLUSION: Fc[gamma]RIII contributes to the pain resulting from inflammatory arthritis of the TMJ, and siRNA has the potential to be an effective treatment for this disorder. [ABSTRACT FROM AUTHOR]

The article focuses on a study which tested the hypothesis that a reduction in Fcγ receptor III (FcγRIII) expression in temporomandibular joint (TMJ) will reduce nociceptive and inflammatory response in an inflamed joint. Study authors injected small interfering RNA (siRNA), naked or complexed with linear polyethyleneimine, into the superior joint space of TMJ in rats. They found that FcγRIII siRNA reduces the amount of FcγRIII in TMJ tissues.

Abstract: 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 19days and also decreased meal frequency for 42days. Interestingly, the meal duration varied significantly from day to day within the 19day period. TMJ interleukin-1β (IL-1β) 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 10min. 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-1β. Seven days post-injection, both IL-6 and IL-1β 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-1β, 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. [Copyright &y& Elsevier]

Abstract: Objective: Reducing the force between the implant and the bone by recapitulating a similar matrix has the potential to reduce implant failure. To begin to pursue the goal of creating a periodontal ligament interface between a dental implant and bone, the mechanism of cellular attachment to dental implant surfaces must be characterized. Methods: In this study we examined the role of integrin receptors in the attachment of periodontal ligament fibroblasts to titanium surfaces utilized on dental implants; those surfaces included smooth polished titanium, acid pickled titanium, ground titanium, sandblasted and acid etched titanium, non-oxidized titanium that has been sandblasted and acid etched, hydroxyapatite coated titanium, titanium plasma sprayed or uncoated titanium. For these studies integrin mediated fibroblast attachment was blocked by the integrin blocking peptide GRGDSP or anti-integrin β1 antibody or a combination of the two. Quantitation of periodontal ligament fibroblast attachment was completed by counting cells on the various implant surfaces after culturing in vitro for 24h with and without the integrin receptor blockers. Results: Antibody and peptide treatment significantly reduced the number of fibroblasts cells attached to the various implant surfaces but this effect varied significantly depending on the surface. Moreover, increased levels of peptide further decreased fibroblasts attachment in a dose dependent manner. Significance: Blocking studies suggest first, that integrin receptors function in periodontal ligament attachment to titanium surfaces and second, that different integrin subunits are important in attachment to a particular surface. [Copyright &y& Elsevier]

Abstract: Temporomandibular joint (TMJ) disorders are a heterogeneous group of diseases that cause progressive joint degeneration leading to chronic pain and reduced quality of life. Both effective pain reduction and restoration of TMJ function remain unmet challenges. Intra-articular injections of corticosteroids and hyaluronic acid are currently used to treat chronic pain, but these methods require multiple injections that increase the risk of iatrogenic joint damage and other complications. The small and emerging field of TMJ tissue engineering aims to reduce pain and disability through novel strategies that induce joint tissue regeneration. Development of methods for sustained, intra-articular release of growth factors and other pro-regenerative signals will be critical for the success of TMJ tissue engineering strategies. This review discusses methods of intra-articular drug delivery to the TMJ, as well as emerging injectable controlled release systems with potential to improve TMJ drug delivery, to encourage further research in the development of sustained release systems for both long-term pain management and to enhance tissue engineering strategies for TMJ regeneration. [Copyright &y& Elsevier]

Abstract: Experimental methods targeting molecules or drugs to specific neuronal tissue(s) can be important in determining function. In this study we focused on blockade of the small channel or aqueduct connecting the third and fourth ventricles of the rat brain. A cannula was placed into the aqueduct between the third and fourth ventricle. A second cannula was placed into the third or fourth ventricle. An aqueous dispersion of hydrogel nanoparticles, that maintains a liquid state at temperatures below 33 °C and solidifies near body temperature (35 °C), was infused into the aqueduct. Two interpenetrating polymer networks (IPN) of hydrogel nanoparticles with polymer concentrations at 2% by weight and 3% by weight were separately infused into the aqueduct to block cerebrospinal fluid (CSF) flow. Following infusion of hydrogel CSF was isolated to a particular ventricle as shown by the lack of dye movement between the ventricles. In addition, stress hormone, corticosterone, feeding behavior and blood glucose levels were measured. Results show upon reaching the aqueduct the hydrogel dispersion solidified and restricted the flow of CSF. A higher concentration of dispersion (3% wt.) was more effective in blocking the aqueduct and isolating the third from the fourth ventricle. Over the period of measurement, infusion of the dispersion had no measurable detrimental physiological effects on the animal. We conclude that isolation of ventricles in the brain can be completed for 48-h by using dispersions of hydrogel nanoparticles and the effects of drugs on certain brain tissues can be determined with this method. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Infection with varicella zoster virus (VZV) results in chicken pox and reactivation of VZV results in herpes zoster (HZ) or what is often referred to as shingles. Patients with HZ experience decreased motivation and increased emotional distress consistent with functions of the ventral tegmental area (VTA) of the brain. In addition, activity within the ventral tegmental area is altered in patients with HZ. HZ primarily affects individuals that are older and the VTA changes with age. To begin to determine if the VTA has a role in HZ symptoms, a screen of 10,000 genes within the VTA in young and old male rats was completed after injecting the whisker pad with VZV. The two genes that had maximal change were membrane progesterone receptors PAQR8 (mPRβ) and PAQR9 (mPRε). Neurons and non-neuronal cells expressed both PAQR8 and PAQR9. PAQR8 and PAQR9 protein expression was significantly reduced after VZV injection of young males. In old rats PAQR9 protein expression was significantly increased after VZV injection and PAQR9 protein expression was reduced in aged male rats versus young rats. Consistent with previous results, pain significantly increased after VZV injection of the whisker pad and aged animals showed significantly more pain than young animals. Our data suggests that PAQR8 and PAQR9 expression is altered by VZV injection and that these changes are affected by age. [ABSTRACT FROM AUTHOR]

• Fam20c is expressed in the neuronal cells in mouse brain. • Global and brain-specific deletion of Fam20c results in brain calcification in mice. • Intracranial calcification is due to local loss of FAM20C function in the brain. FAM20C (family with sequence similarity 20-member C) is a protein kinase that phosphorylates secretory proteins, including the proteins that are essential to the formation and mineralization of calcified tissues. FAM20C loss-of-function mutations cause Raine syndrome in humans, characterized by generalized osteosclerosis, distinctive craniofacial dysmorphism, along with extensive intracranial calcification. Our previous studies revealed that inactivation of Fam20c in mice led to hypophosphatemic rickets. In this study, we examined the expression of Fam20c in the mouse brain and investigated brain calcification in Fam20c -deficient mice. Reverse transcription polymerase chain reaction (RT-PCR), Western-blotting and in situ hybridization analyses demonstrated the broad expression of Fam20c in the mouse brain tissue. X-ray and histological analyses showed that the global deletion of Fam20c (mediated by Sox2-cre) resulted in brain calcification in mice after postnatal 3 months and that the calcifications were bilaterally distributed within the brain. There was mild perifocal microgliosis as well as astrogliosis around calcospherites. The calcifications were first observed in the thalamus, and later in the forebrain and hindbrain. Furthermore, brain-specific deletion (mediated by Nestin-cre) of Fam20c in mice also led to cerebral calcification at an older age (postnatal 6 months), but no obvious skeletal or dental defects. Our results suggest that the local loss of FAM20C function in the brain may directly account for intracranial calcification. We propose that FAM20C plays an essential role in maintaining normal brain homeostasis and preventing ectopic brain calcification. [ABSTRACT FROM AUTHOR]

Intermittent administration of nicotine (NIC) decreases food intake (FI) and body weight (BW). Infusion of the NIC antagonist mecamylamine into the 4th ventricle reduced this effect, suggesting NIC is acting on neurons adjacent to the fourth ventricle to reduce FI and BW. Norepinephrine (NE) [A2, A4, A6] and epinephrine (EPI) [C2, C3] cell bodies, adjacent to the fourth ventricle, directly innervate feeding areas in the hypothalamus including the perifornical hypothalamus (PFH). In the PFH NE release stimulates FI, while EPI release inhibits intake. NE and EPI fibers were eliminated (i.e. lesioned) in the PFH using 6-OHDA hydrobromide. Bupropion HCl was also administered to protect dopamine (DA) neurons from damage. Lesioning significantly reduced PFH NE and EPI levels, while DA levels remained unchanged. Lesioned animals FI and BW were recorded by computer before and after intermittent NIC injections. PFH lesioned rats injected with NIC did not show the typical drop in FI and BW. We conclude that normally NIC normally attenuates FI, in part, by releasing EPI in the PFH. Remember EPI is a FI inhibitor and PFH lesioning will cause removal of the EPI fibers thus, leading to the observation of an attenuated effect of NIC on FI and BW suppression. The release of PFH DA can also suppress feeding in the PFH, but since PFH DA levels were unchanged in the lesioned rats NIC can not be suppressing FI through a DA mechanism. [ABSTRACT FROM AUTHOR]

Infection with varicella zoster virus (VZV) results in chicken pox and reactivation of VZV results in herpes zoster (HZ) or what is often referred to as shingles. Patients with HZ experience decreased motivation and increased emotional distress consistent with functions of the ventral tegmental area (VTA) of the brain. In addition, activity within the ventral tegmental area is altered in patients with HZ. HZ primarily affects individuals that are older and the VTA changes with age. To begin to determine if the VTA has a role in HZ symptoms, a screen of 10,000 genes within the VTA in young and old male rats was completed after injecting the whisker pad with VZV. The two genes that had maximal change were membrane progesterone receptors PAQR8 (mPRß) and PAQR9 (mPRe). Neurons and non-neuronal cells expressed both PAQR8 and PAQR9. PAQR8 and PAQR9 protein expression was significantly reduced after VZV injection of young males. In old rats PAQR9 protein expression was significantly increased after VZV injection and PAQR9 protein expression was reduced in aged male rats versus young rats. Consistent with previous results, pain significantly increased after VZV injection of the whisker pad and aged animals showed significantly more pain than young animals. Our data suggests that PAQR8 and PAQR9 expression is altered by VZV injection and that these changes are affected by age. [ABSTRACT FROM AUTHOR]

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 >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. J. Cell. Physiol. 226: 3169-3180, 2011. © 2011 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Sigalas E, Regan JD, Kramer PR, Witherspoon DE, Opperman LA. Survival of human periodontal ligament cells in media proposed for transport of avulsed teeth. Dent Traumatol 2004; 20: 21–28. © Blackwell Munksgaard, 2004. Many solutions have been examined as possible storage media for avulsed teeth. In this report, human periodontal ligament (PDL) cells were exposed for 1 h to culture medium, milk, Hanks Balanced Salt Solution (HBSS), Soft Wear, Opti Free, and Solo Care contact lens solutions, Gatorade®, and tap water, at room temperature and on ice. The number of viable cells was counted using the trypan blue exclusion technique, immediately after exposure (0 h) and at 24 and 48 h, to test the proliferative capacity of the cells after treatment. The results indicated that a significantly higher number of cells survived and proliferated when the exposures were performed at 0°C. Water had a detrimental effect on the cells, whereas culture medium and HBSS preserved significantly more viable cells than the other experimental solutions. Within the parameters of this study, it appears that HBSS is the optimal storage medium for avulsed teeth. Low-fat milk could serve as an alternative if ice is available. Contact lens solutions or Gatorade® on ice could serve as short-term (1 h) storage media if the other solutions are not readily available. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Varicella zoster virus (VZV) results in chicken pox and herpes zoster. Female rats show a higher level of herpes zoster associated pain than males, consistent with human studies. In this study, we addressed the novel hypothesis that sex difference in herpes zoster associated pain is due, in part, to estradiol modulating activity in the thalamus. To test this hypothesis a high and low physiological dose of estradiol was administered to castrated and ovariectomized rats and the affective pain response was measured after injection of VZV into the whisker pad. Thalamic infusion of the estrogen receptor antagonist ICI 182,780 concomitant with a high dose of estradiol addressed the role of estradiol binding to its receptor to effect pain. Phosphorylated extracellular signal-regulated protein kinase (pERK) positive cells were measured in excitatory (glutaminase positive) and inhibitory (glutamate decarboxylase 67 positive) cells of the lateral thalamic region. Our results show that a high dose of estradiol significantly reduced the pain response in both males and females. pERK significantly increased in excitatory cells after treatment with a low dose of estradiol and increased in inhibitory cells after treatment with a high dose of estradiol. Administration of ICI 182,780 significantly increased the pain response, reduced expression of GABA related genes in the thalamic region and significantly reduced the number of inhibitory cells expressing pERK. The results suggest that estradiol attenuates herpes zoster pain by increasing the activity of inhibitory neurons within the thalamus and that this reduction includes an estrogen receptor dependent mechanism. • Estradiol decreases VZV induced orofacial pain. • Blocking estrogen receptor in thalamus increases pain. • Estradiol increases GABA cell activity in thalamus. • Blocking estrogen receptor decreases GABA cell activity. [ABSTRACT FROM AUTHOR]

Orofacial pain includes neuronal pathways that project from the trigeminal nucleus to and through the thalamus. What role the ventroposterior thalamic complex (VP) has on orofacial pain transmission is not understood. To begin to address this question an inhibitory G protein (Gi) designer receptor exclusively activated by a designer drug (DREADD) was transfected in cells of the VP using adeno-associated virus isotype 8. Virus infected cells were identified by a fluorescent tag and immunostaining. Cells were silenced after injecting the designer drug clozapine-n-oxide, which binds the designer receptor activating G i . Facial rubbing and local field potentials (LFP) in the VP were then recorded in awake, free moving Sprague Dawley rats after formalin injection of the masseter muscle to induce nociception. Formalin injection significantly increased LFP and the nociceptive behavioral response. Activation of DREADD G i with clozapine-n-oxide significantly reduced LFP in the VP and reduced the orofacial nociceptive response. Because DREADD silencing can result from G i -coupled inwardly-rectifying potassium channels (GIRK), the GIRK channel blocker tertiapin-Q was injected. Injection of GIRK blocker resulted in an increase in the nociceptive response and increased LFP activity. Immunostaining of the VP for glutamate vesicular transporter (VGLUT2) and gamma-aminobutyric acid vesicular transporter (VGAT) indicated a majority of the virally transfected cells were excitatory (VGLUT2 positive) and a minority were inhibitory (VGAT positive). We conclude first, that inhibition of the excitatory neurons within the VP reduced electrical activity and the orofacial nociceptive response and that the effect on excitatory neurons overwhelmed any change resulting from inhibitor neurons. Second, inhibition of LFP and nociception was due, in part, to GIRK activation. [ABSTRACT FROM AUTHOR]

Background: Most people are initially infected with varicella zoster virus (VZV) at a young age and this infection results in chickenpox. VZV then becomes latent and reactivates later in life resulting in herpes zoster (HZ) or "shingles". Often VZV infects neurons of the trigeminal ganglia to cause ocular problems, orofacial disease and occasionally a chronic pain condition termed post-herpetic neuralgia (PHN). To date, no model has been developed to study orofacial pain related to varicella zoster. Importantly, the incidence of zoster associated pain and PHN is known to be higher in women, although reasons for this sex difference remain unclear. Prior to this work, no animal model was available to study these sex-differences. Our goal was to develop an orofacial animal model for zoster associated pain which could be utilized to study the mechanisms contributing to this sex difference.Methods: To develop this model VZV was injected into the whisker pad of rats resulting in IE62 protein expression in the trigeminal ganglia; IE62 is an immediate early gene in the VZV replication program.Results: Similar to PHN patients, rats showed retraction of neurites after VZV infection. Treatment of rats with gabapentin, an agent often used to combat PHN, ameliorated the pain response after whisker pad injection. Aversive behavior was significantly greater for up to 7 weeks in VZV injected rats over control inoculated rats. Sex differences were also seen such that ovariectomized and intact female rats given the lower dose of VZV showed a longer affective response than male rats. The phase of the estrous cycle also affected the aversive response suggesting a role for sex steroids in modulating VZV pain.Conclusions: These results suggest that this rat model can be utilized to study the mechanisms of 1) orofacial zoster associated pain and 2) the sex differences underlying zoster associated pain. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Clinical presentation of temporomandibular joint (TMJ) disorders are more common in women and changes in the female hormone estrogen affect the level of swelling, pro-inflammatory cytokine release and pain in animal models of TMJ arthritis. Estrogen also modulates the expression of the CD16 receptor in vitro. This alters pro-inflammatory cytokine release in monocytes/macrophages when auto-antigens and arthritic factors bind the CD16 receptor. This study investigated the effects of various levels of estrogen on the intensity of inflammation and CD16 expression in a TMJ arthritic animal model. The experiments included rats that were intact or ovariectomized (OVX), eliminating the major source of estrogen output. A portion of the OVX animals had estrogen replaced with 17-β estradiol (E2) using Alzet® pumps. In OVX animals E2 levels were administered for 10 days to create an artificial estrus cycle or to simulate pregnancy. Following E2 treatment the rats were given an intra-articular TMJ injection of saline or complete Freund''s adjuvant (CFA). CFA injection significantly increased TMJ swelling, stress induced chromodacryorrhea and attenuated food intake, thus indicating the adjuvant induced TMJ pain/inflammation. Removing endogenous E2 through OVX reduced CFA induced TMJ inflammation, whereas CFA increased the number of TMJ monocytes expressing the CD14 receptor equally in all groups irrespective of plasma E2 levels. Paradoxically, higher levels of E2 reduced the number of TNF-α positive, CD16+ and double labeled CD14+/CD16+ cells. The findings indicate that reduced plasma E2 levels attenuated CFA induced TMJ inflammation, whereas increasing E2 levels enhanced TMJ swelling in a dose dependent manner. Estrogenic group differences in CFA induced swelling were independent of TMJ CD14+, CD14+/CD16+ or CD16+ cell numbers suggesting E2 action on the CFA immune response primarily excluded CD16 receptor action. [Copyright &y& Elsevier]

Abstract: Previously we observed in male rats that intermittent administration of nicotine (NIC) during the dark phase reduces food intake (FI) by initially decreasing only dark phase meal size. This was followed several days later by an increase in dark phase meal frequency such that FI returned to normal, while body weight remained suppressed. Termination of NIC treatment resulted in a modest dark phase hyperphagia. Since some human females use NIC as a weight control drug, the present study investigated changes in FI and body weight regulation in adult female rats treated for five estrous cycles with saline or a 1.40 mg/kg/day (free base) dose of NIC, which was given in four equal i.p. doses during the dark phase. The rats were followed for 15 days after cessation of NIC. Initially both dark and light phase FI were reduced and this was caused by an immediate decrease in dark and light phase meal size; the attenuation of meal size continued after cessation of NIC. On day 7 of NIC, the rats compensated by significantly increasing the number of dark, but not light, phase meals they took. This resulted in a normal 24-h FI, which was caused by a dark phase increase in FI coupled with a continued decrease in light phase FI. Importantly, these changes in meal patterns persisted for some time after termination of NIC. Upon NIC cessation, the NIC group showed no hyperphagia even though their body weight was significantly decreased. These results document that administration of NIC during the dark phase resulted in a reorganization of the microstructure of FI in females rats that resembles, but does not exactly duplicate, that observed in male rats. Like males, long lasting alterations in the microstructure of FI (e.g., meal size and meal number), were noted in female rats for up to 2 weeks after cessation of NIC. These results differ from studies in which NIC was given continuously 24-h per day and indicate that dark phase NIC administration in rats may represent an appropriate model to study the impact of NIC on meal patterns. [Copyright &y& Elsevier]

Previous studies have shown nicotine (NIC) administration leads to decreased food intake, while other investigations have reported that NIC stimulates c-Fos expression in the brainstem. Whether there is a causal relationship between NIC effects on ingestion and its effect on brainstem neurons is uncertain, however we hypothesized that blocking NIC action in the brainstem would prevent, to some extent, the hypophagic effects of NIC. In the present study, cannulas were placed in the fourth ventricle of rats. A dose of NIC or saline was injected i.p. in four equal injections during the dark phase for four days. At the start of the second day of injections the NIC receptor antagonist mecamylamine (MEC) or artificial cerebrospinal fluid (a-CSF) was infused intracerebroventricularly (i.c.v.). Thus, four experimental groups were examined: a-CSF + SAL; a-CSF + NIC; MEC + SAL; MEC + NIC. Meal patterns were recorded using a computerized system and water intake and body weight were measured daily. Peripheral NIC injections suppressed food intake by decreasing meal size, whereas infusion of the NIC receptor antagonist MEC (4 μg) into the fourth ventricle blocked the NIC suppression of food intake. Moreover, the MEC effect was due primarily to an increase in dark phase meal size, which suggests neurons localized to the brainstem transmit NIC signals that regulate feeding behavior by affecting meal size. [Copyright &y& Elsevier]

• First time imaging calcium activity in vivo in the rat ventral posteromedial thalamic nucleus. • Inhibition of reticular thalamic neuronal activity increased ventral posteromedial thalamic activity. • Increased calcium activity in the ventral posteromedial thalamic nucleus was concomitant with an increase in pain. 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. [ABSTRACT FROM AUTHOR]

The orofacial pain pathway projects to the parabrachial and amygdala, and sex steroids have been shown to affect neuronal activity in these regions. GABA positive cells in the amygdala are influenced by sex steroid metabolites to affect pain, and sex steroids have been shown to alter the expression of genes in the parabrachial, changing neuronal excitability. Mechanisms by which sex steroids affect amygdala and parabrachial signaling are unclear. The expression of genes in the parabrachial and amygdala in diestrus (low estradiol) and proestrus (high estradiol) female rats were evaluated in this study. First, varicella zoster virus was injected into the whisker pad of female rats to induce a pain response. Second, gene expression was quantitated using RNA-seq one week after injection. Genes that had the greatest change in expression and known to function in pain signaling were selected for the quantitation of protein content. Protein expression of four genes in the parabrachial and seven genes in the amygdala were quantitated by ELISA. In the parabrachial, neurexin 3 (Nrnx3) was elevated at proestrus. Nrnx3 has a role in AMPA receptor and GABA signaling. Neuronatin (Nnat) and protein phosphatase, Mg2+/Mn2+ dependent 1E (Ppm1e) were elevated in the parabrachial of diestrus animals both genes having a role in pain signaling. Epoxide hydroxylase (Ephx2) was elevated in the parabrachial at proestrus and the vitamin D receptor (Vdr) was elevated in the amygdala. Ephx2 antagonists and vitamin D have been used to treat neuropathic pain. In conclusion, sex steroids regulate genes in the parabrachial and amygdala that might result in the greater pain response observed during diestrus. [ABSTRACT FROM AUTHOR]

Migraine is commonly reported among patients with temporomandibular disorders (TMDs), especially myogenic TMD. The pathophysiologic mechanisms related to the comorbidity of the two conditions remain elusive. In the present study, we combined masseter muscle tendon ligation (MMTL)-produced myogenic TMD with systemic injection of nitroglycerin (NTG)-induced migraine-like hypersensitivity in mice. Facial mechanical allodynia, functional allodynia, and light-aversive behavior were evaluated. Sumatriptan, an FDA-approved medication for migraine, was used to validate migraine-like hypersensitivity. Additionally, we examined the protein level of calcitonin gene-related peptide (CGRP) in the spinal trigeminal nucleus caudalis using immunohistochemistry. We observed that mice with MMTL pretreatment have a prolonged NTG-induced migraine-like hypersensitivity, and MMTL also enabled a non-sensitizing dose of NTG to trigger migraine-like hypersensitivity. Systemic injection of sumatriptan inhibited the MMTL-enhanced migraine-like hypersensitivity. MMTL pretreatment significantly upregulated the protein level of CGRP in the spinal trigeminal nucleus caudalis after NTG injection. Our results indicate that a pre-existing myogenic TMD can upregulate NTG-induced trigeminal CGRP and enhance migraine-like hypersensitivity. [ABSTRACT FROM AUTHOR]

Purpose: It has become more apparent that studies focusing on the effect of sex steroids on orofacial pain in humans and animals require careful attention to the methods of hormone replacement. As a result, the purpose of this review is to highlight differences in estrogen concentration and its effects on temporomandibular joint (TMJ) pain and behavior. Methods: This literature review searched keywords TMJ, orofacial, pain, nociception sex steroids, estrous cycle, menstrual cycle estradiol, and progesterone. Results: In the case of estrogen, the change in concentration was a consideration, as a rise in estrogen can lead to protective effects and a decrease in estrogen appears to exacerbate the pain response. The dosage and timing was important when administering sex steroids as many orofacial pain studies conducted on animals have used large pharmacologic dosages and/or have given the hormone in a nonphysiologic way that does not mimic natural secretion patterns. Conclusions: The results demonstrate that when performing studies or experiments these factors can result in alteration of the pain or behavioral response, as well as, joint structure and inflammatory response. Therefore, these factors must be considered when designing experiments focused on determining the mechanisms of action for sex steroids. [ABSTRACT FROM AUTHOR]