A Pre-Existing Myogenic Temporomandibular Disorder Increases Trigeminal Calcitonin Gene-Related Peptide and Enhances Nitroglycerin-Induced Hypersensitivity in Mice.

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.

Estradiol Acts in Lateral Thalamic Region to Attenuate Varicella Zoster Virus Associated Affective Pain.

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.

Aromatase Derived Estradiol Within the Thalamus Modulates Pain Induced by Varicella Zoster Virus.

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.

Gi protein functions in thalamic neurons to decrease orofacial nociceptive response.

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 Gi. 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 Gi with clozapine-n-oxide significantly reduced LFP in the VP and reduced the orofacial nociceptive response. Because DREADD silencing can result from Gi-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.

Effect of Pregnancy on TMJ Nociception in Rats.

OBJECTIVE: Does TMJ nociception, as measured by a behavioral assay, differ during pregnancy, potentially due to the effect of sex steroids? METHODS: Thirty - two Sprague Dawlcy rats were randomly allocated to either mid- (EH 11) or late- (EH 17) pregnancy groups. The animals within each group were further allocated to a saline or CFA treatment. At EH 11 or EH 17, the animals were injected bilaterally into TMJ with either saline (15 µL.) or 1 µg/µL CFA (15 µL). Nociception was measured with a feeding assay (pellet recording) and analyzed by comparing in-tra-meal rates using a distance-based permutation method. RESULTS: CFA injection resulted in the animals eating longer in both the mid - pregnant and late-pregnant groups. Plasma estradiol was higher in the late - pregnant group versus the mid - pregnant group. Importantly, the CFA injected late-pregnant group ate for a shorter amount of time (i. e., shorter meal rate) than the CFA injected rats at mid - pregnancy. CONCLUSION: The reduced nociceptive response (shorter meal rate) in the CFA injected late - pregnant group may be due to greater estradiol versus the CFA injected mid - pregnancy rats. Thus, one potential reason why women report fewer TMJ symptoms during pregnancy is the higher level of circulating estradiol.

Corrigendum: Aromatase Derived Estradiol Within the Thalamus Modulates Pain Induced by Varicella Zoster Virus.

[This corrects the article DOI: 10.3389/fnint.2018.00046.].

Distance-based permutation of inter-meal differences as a sensitive test of temporomandibular joint nociception in rats.

PURPOSE: Compare non-parametric permutation method using intr-meal rate as endpoint to existing ANOVA method that uses average daily meal duration as an endpoint for detection of chronic pain in Sprague-Dawley rats. METHODS: 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, 2010) using ANOVA and multiple comparisons range tests. In this study, we reanalyzed the data using a non-parametric 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. RESULTS: 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 of 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. CONCLUSIONS: 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.

Role for the Ventral Posterior Medial/Posterior Lateral Thalamus and Anterior Cingulate Cortex in Affective/Motivation Pain Induced by Varicella Zoster Virus.

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.

Sex differences underlying orofacial varicella zoster associated pain in rats.

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.

Lateral thalamic control of nociceptive response after whisker pad injection of varicella zoster virus.

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.

Genes in the GABA Pathway Increase in the Lateral Thalamus of Sprague-Dawley Rats During the Proestrus/Estrus Phase.

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.

Preliminary study of light-cured hydrogel for endodontic drug delivery vehicle.

AIM: Direct pulp capping is the treatment of an exposed vital pulp with a dental material to facilitate the formation of reparative dentin and maintenance of vital pulp. A bioengineered drug delivery vehicle has the potential to increase the success rate of pulp capping. The aim of this study was to develop an injectable and light-curing drug delivery vehicle for endodontic treatment including direct pulp capping. METHODS: Polyethylene glycol-maleate-citrate (PEGMC) hydrogel was synthesized as a drug delivery vehicle that is composed of PEGMC (45% w/v), acrylic acid (AA) (5% w/v), 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) (0.1% w/v), and deionized water. The association between prehydrogel-solution volume and visible light-curing was examined. The cytotoxicity of the hydrogel was tested using L929 cells in a cell culture system. Ca(2+) release from the hydrogel was determined using calcium hydroxide as the incorporated medicine. RESULTS: The results showed that the light-curing time for hydrogel is comparable to composite resin. The hydrogel had cell toxicity similar to adhesive systems. Moreover, controlled Ca(2+) release was obtained from the calcium hydroxide incorporated hydrogel. CONCLUSIONS: The data suggest that hydrogel should be explored further as a promising drug delivery vehicle for vital pulp therapy and regenerative endodontics.

Attenuation of myogenic orofacial nociception and mechanical hypersensitivity by viral mediated enkephalin overproduction in male and female rats.

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.

Meal duration as a measure of orofacial nociceptive responses in rodents.

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.

Cytotoxicity evaluation of methacrylate-based resins for clinical endodontics in vitro.

This study examines the cytotoxicity of Super-Bond C&B (SB-C&B), Super-Bond RC Sealer (SB-RC), MetaSEAL (Meta), and AH Plus Sealer (AH+). Freshly mixed and set materials (100 mg) were prepared in vitro and placed in cell culture medium (1 mL) for the working time and for 6 h, respectively. L929 cells seeded into 96-well plates at 5,000 cells/well were incubated with the eluted medium (200 µL) for 24 h. Cells cultured with medium alone served as the control. Cytotoxicity was evaluated by MTS assay and analyzed with ANOVA. In the freshly mixed group, the average ± SD (%) for cell viability were 66.0 ± 13.6, 55.5 ± 15.6, 10.6 ± 0.7, and 8.9 ± 2.2 for SB-C&B, SB-RC, Meta, and AH+, respectively. In the set group, the average ± SD (%) for cell viability were 100 ± 21.9, 81.8 ± 38.5, 24.9 ± 7.9, and 23.6 ± 10.0 for SB-C&B, SB-RC, Meta, and AH+, respectively. SB-C&B and SB-RC are less cytotoxic than are Meta and AH+.

Estrogen in cycling rats alters gene expression in the temporomandibular joint, trigeminal ganglia and trigeminal subnucleus caudalis/upper cervical cord junction.

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 17ß-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 17ß-estradiol was low or when it was at a high proestrus level. TMJ, trigeminal ganglion, and trigeminal subnucleus caudalis/upper cervical cord junction (Vc/C(1-2) ) 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/C(1-2) 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 ß are present in neurons of the trigeminal ganglia, whereby 17ß-estradiol can alter expression of Gabrα6 and Glrα2. Also, estrogen receptor α (ERα) but not ERß 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 17ß-estradiol.

Knockdown of Fcγ receptor III in an arthritic temporomandibular joint reduces the nociceptive response in rats.

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-1ß (IL-1ß) 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-1ß. 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.

Measuring persistent temporomandibular joint nociception in rats and two mice strains.

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.

Estrogen and inflammation modulate estrogen receptor alpha expression in specific tissues of the temporomandibular joint.

BACKGROUND: Estrogen 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. METHODS: Sixteen 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. RESULTS: The 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. CONCLUSIONS: In conclusion, inflammation and 17 beta-estradiol can modulate ERalpha expression in the TMJ but the effects are tissue specific.

Selective blockade of the rat brain aqueduct with thermogelling hydrogel nanoparticle dispersion.

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 degrees C and solidifies near body temperature (35 degrees 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.