The present study explored the antinociceptive effects of H3 (R-alpha-methylhistamine) and GABAB (baclofen) receptor ligands in an orofacial model of pain in rats. Orofacial pain was induced by subcutaneous injection of formalin (50 μl, 5 %) in the upper lip region, and the number of jumps and time spent face rubbing was recorded for 40 min. Formalin produced a marked biphasic pain response; first phase, 0-10 min (jumps), and second phase, 15-40 min, (rubbing). Baclofen (50 μg) injected into the rat wiskerpad 5 min before formalin administration suppressed both phases of pain whereas R-alpha-methylhistamine (12.5 μg) abolished the first phase only. Brains were taken immediately after behavioral testing was completed. HPLC/ED analysis showed that 5-hydroxytryptamine (5-HT) turnover was increased in hippocampus, thalamus, and brain stem of all formalin groups, excepting the baclofen group in which the balance of 5-HT metabolism was restored to control values. These findings demonstrate that GABAB receptors represent peripheral targets for analgesia. Consequently, locally administered baclofen may be a useful approach in treating inflammatory trigeminal pain.

Superficial layers of the dorsal horn receive a dense plexus of nerve fibers immunoreactive to pituitary adenylate cyclase activating polypeptide (PACAP). In vivo experiments were conducted in the mice to evaluate the effects of PACAP-38, herein referred to as PACAP, PACAP receptor antagonist PACAP(6-38) and PACAP-antiserum on nociceptive behaviors induced by radiant heat, intrathecally administered N-methyl-D-aspartate (NMDA) or intraplantarly administered formalin. PACAP (0.05-0.5μg) dose-dependently decreased the paw-withdrawal latencies induced by thermal stimulation and enhanced the aversive licking and biting behaviors induced by intrathecally injected NMDA. Pretreatment with the PACAP receptor antagonist PACAP(6-38) (0.5-2μg) or PACAP-antiserum (1:500-2000 dilution) dose-dependently attenuated the second phase, but not the first phase, of nociceptive responses to formalin. Next, the effects of PACAP on NMDA- and kainate-induced currents evoked in single dorsal horn neurons were studied. Whole-cell patch recordings were made from superficial dorsal horn neurons of spinal cord slices from 14- to 20-day-old mice. PACAP at the concentrations of 100 and 200nM, which caused no significant change of holding currents, increased NMDA-but not kainate-induced currents in superficial dorsal horn neurons. Our results suggest that exogenously applied PACAP sensitizes the dorsal horn neurons to formalin stimulation, and facilitates NMDA receptor-mediated nociceptive response. As a corollary, PACAP, which may be released from primary afferent fibers potentiates nociceptive transmission to the dorsal horn by interacting primarily with NMDA receptors.

In postmortem toxicology, certain cases require the examination of embalmed biological specimens to investigate the presence and potential role drugs may have played in a person’s death. Key factors, like postmortem distribution, which can be greatly affected by temperature, may alter drug concentrations in different areas of the body. In the United States, the involvement of cocaine in overdose deaths has significantly increased between 2012 and 2019 (1). The purpose of this project was to examine the stability of cocaine and its primary metabolite, benzoylecgonine, in perfused postmortem rat tissues stored at different temperatures over a one month. Twelve frozen cocaine positive rat specimens, intracardially perfused with a saline and formaldehyde solution, were received from a chronic cocaine rat study at Boston University Department of Psychological and Brain Sciences (Dr. Kathleen Kantak, Boston, MA, USA). The specimens were dissected, and the spleen, one kidney, and one lung were removed from each specimen. A fine-needle aspiration biopsy was performed on each organ to collect a time zero (T0) sample. One set of four rat specimens were stored at room temperature (20-22°C), another four were stored at refrigerator temperature (4°C), and another four were stored at freezer temperature (-20°C). A section of each organ was collected for analysis at two weeks (T1) and one month (T2). Samples underwent solid-phase extraction before liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using a QSight 220 CR Laminar Flow Triple Quadrupole Mass Spectrometer with electrospray ionization, operated in positive ion mode (PerkinElmer, Shelton, CT, USA). Simplicity 3QTM software (PerkinElmer) was used for all data collection, analysis, and quantification. All calibration curves generated for each analyte had acceptable R2 values greater than 0.98 using a weighted linear regression model (1/x). Between T1 and T2, eight samples demonstrated a 15-873% increase in cocaine concentration and four samples had a 13-45% decrease in cocaine concentration. For benzoylecgonine, nine samples demonstrated an 18-289% increase in concentration between T1 and T2 and six samples had a 3-57% decrease in concentration. In samples collected at one month, concentration values for cocaine were highest in samples stored at freezer temperature (-20°C) and lowest in samples stored at refrigerator temperature (4°C). The highest benzoylecgonine values were found in samples stored at freezer temperature (-20°C) as well, and the lowest concentrations were in samples stored at room temperature (20-22°C). Due to the variability in analyte concentration in the organs of the intracardially perfused specimens, the impact storage conditions had on analyte stability could not be determined.

Cytopathology plays a significant role in cancer diagnostics. One of the ancillary methods used for prognostication is immunocytochemistry (ICC). Formalin pre-fixed tissue blocks are often used as validated immunohistochemical controls for cytology samples. However, those are not treated as clinical cytology specimen, which are furthermore commonly fixed in alcohol. As cell structure might be affected by added fixatives and block materials, the aim of this project was to compare the effects of fixation in formalin and PreservCyt®, a methanol-based fixative, and the two cell block materials, plasma-thrombin and agar, on the ICC results. The material used in the study consisted of several cell lines provided by the Technological University Dublin, which were chosen to be representative of the clinical cytology samples. The cells were fixed and put into cell blocks for each cell type or as a mixture of different cell types – a composite cell block. All cell blocks were then stained with suitable antibodies. Composite cell blocks could be tested as simultaneously positive and negative ICC control material. The results showed that in general, PreservCyt®-fixed cell blocks preserved sharper cellular morphology compared to those fixed in formalin. However, PreservCyt® also gave unexpected negative results for some antibodies, including some nuclear markers. Plasma-thrombin cell block method showed to have more advantages than the agar method.This study suggests, that with further optimization and validation of both fixative methods and cell blocks, composite cell block has good potential to be used as source of control blocks for cytology samples.

La recherche dans le domaine de la prise en charge de la douleur, notamment chronique, a un besoind’innovation car les traitements disponibles à l’heure actuelle sont pour la plupart anciens et souventliés à des effets indésirables. Il est maintenant admis que les études précliniques de la douleur ont denombreuses limites : pertinence des modèles, utilisation d’une stimulation douloureuse surajoutée,détermination d’un simple seuil ou délai, prise en compte de la seule composante sensoridiscriminative…De ce fait, certaines molécules efficaces chez l’animal et donc prometteuses, n’ontpas eu les effets escomptés chez l’homme. La base de notre travail de recherche s’attache donc àproposer de nouvelles méthodes d’appréciation de la douleur chronique chez l’animal en prenant encompte ses aspects multidimensionnels. De nombreuses études ont mis en évidence une altération dela qualité de vie chez des patients atteints de douleur chronique. Cette altération se caractérisenotamment par des perturbations émotionnelles et cognitives. Ces paramètres ne sont pas toujours prisen compte chez l’animal dans l’évaluation de traitements antalgiques mais pourrdouleurnt amener denouvelles possibilités et perspectives précliniques. Notre travail a consisté à étudier l’impact de ladouleur sur les comportements spontanés (automatisation du test au formol), la composanteémotionnelle et les capacités cognitives chez le rongeur. Il a été complété par l’exploration du rôle del’amygdale dans les mécanismes impliqués dans ces modifications comportementales.L’amélioration du test au formol a été réalisée dans le but de visualiser au mieux les comportementsspécifiques observés lors d’une douleur aiguë de type inflammatoire. Notre adaptation a permis, chezles mêmes animaux, de pouvoir dissocier l’effet antalgique et l’effet sédatif d’une molécule à l’aided’une méthode automatisée plus rapide et moins subjective.Parallèlement, nous avons apprécié l’impact de la douleur chronique sur la composante émotionnelleet les performances cognitives dans deux modèles de douleur chronique (inflammatoire etneuropathique). Les animaux souffrant de douleur chronique inflammatoire présentent desperturbations plus importantes que les animaux neuropathiques, perturbations pouvant être amélioréespar un traitement pharmacologique. Des études mécanistiques utilisant des micro-injections demorphine au niveau de l’amygdale ont souligné une implication importante du complexe basolatéraldans ces composantes émotionnelles et cognitives de la douleur.Ces nouvelles approches comportementales pourrdouleurnt permettre de mieux caractériser l’impact globalde la douleur chronique chez l’animal et de compléter la batterie de tests couramment utilisés enpréclinique. Ceci pourrait déboucher sur une transposition plus réaliste des résultats obtenus chezl’animal à l’homme, et donc conduire à une meilleure prédictibilité clinique de l’efficacité destraitements. Enfin, la mise en évidence de nouvelles cibles thérapeutiques innovantes implique l’étudedes mécanismes responsables de ces altérations comportementales. / Research in the field of pain management, including chronic pain management, needs innovationbecause available treatments are mostly old and often associated with many side effects. It is now wellrecognized that preclinical studies on pain have many limitations: the relevance of the models, the useof imposed painful stimulations, determination of simple thresholds or delays, taking into account thesensory-discriminative component of pain alone… Indeed, some molecules that are efficient inanimals and that are considered as promising, didn’t have the desired effect in humans. Therefore, thebasis of our research aims to propose new methods to assess chronic pain in animals taking intoaccount its multidimensional aspects. Many studies have shown impaired quality of life in patientssuffering from chronic pain. This alteration is characterized by emotional and cognitive disturbances.These components of pain are not always taken into account in animal when studying analgesictreatments, but could bring new preclinical possibilities and perspectives. Our work consisted instudying the impact of pain on spontaneous behaviours (automated formalin test), emotionalcomponent and cognitive capacities in rodents. This work has been completed by the exploration ofthe role of the amygdala in the mechanisms underlying those behavioural modifications.Improvement of the formalin test was conducted in order to better visualize the specific behaviorsobserved during an acute inflammatory pain. Our adaptation has allowed dissociating the analgesicand sedative effect of a molecule in a same animal, using an automated method which is faster and lesssubjective than the manual method.In the meantime, we assessed the impact of chronic pain on the emotional and cognitive performancesin two models of chronic pain (inflammatory and neuropathic). Animals suffering from chronicinflammatory pain have more important impairments than animal suffering from neuropathic pain,impairments that can be improved with a pharmacological treatment. Mechanistic studies using microinjectionsof morphine in the amygdala have emphasized an important involvement of the basolateralcomplex in these emotional and cognitive components of pain.These new behavioural approaches may help better characterize the overall impact of chronic pain inanimals and complete the battery of tests commonly used in preclinical studies. This could lead to amore realistic transposition of the results obtained from animals to humans, and thus lead to betterpredictability for the clinical efficacy of treatments. Finally, the identification of new targets forinnovative therapies involves the study of mechanisms responsible for these behavioral impairments.

An ultra-low dose of a drug is approximately 1000-fold lower than the dose range traditionally used to induce a therapeutic effect. The purpose of the present thesis was to broaden the knowledge of the ultra-low dose effect, that was previously identified in the opioid receptor system, by looking at whether opioids and cannabinoids interact at the ultra-low dose level, whether cannabinoid receptors themselves demonstrate the ultra-low dose antagonist effect, and whether the opioid ultra-low dose effect is maintained in a model of persistent, unavoidable pain. For experiment 1, separate groups of Long Evans rats were tested for antinociception following an injection of vehicle, the cannabinoid agonist WIN 55 212-2 (WIN), the opioid antagonist naltrexone (an ultra-low or a high dose), or a combination of WIN and naltrexone doses. Ultra-low dose naltrexone elevated WIN-induced tail flick thresholds without extending its duration of action. In experiment 2, antinociception was tested in rats following either acute or sub-chronic (7 days) injections of vehicle, WIN, ultra-low doses of the CB1 receptor antagonist rimonabant (SR 141716), or a combination of WIN and ultra-low dose rimonabant. Following the chronic experiment, striatal tissue was rapidly extracted and subjected to co-immunoprecipitation to analyse CB1 receptor coupling to G-protein subtypes. Ultra-low dose rimonabant extended the duration of WIN-induced antinociception, and attenuated the development of WIN-induced tolerance. Animals chronically treated with WIN alone had CB1 receptors predominately coupling to Gs proteins, whereas all other groups had CB1 receptors predominately coupling to Gi proteins. For experiment 3, all animals were subjected to the formalin test following either acute or sub-chronic injections of vehicle, the opiate morphine, ultra-low doses naltrexone, or a combination of morphine and ultra-low dose naltrexone. Ultra-low dose naltrexone had no significant effect on morphine-induced pain ratings in either the acute, or sub-chronic drug treatments. This thesis provides evidence that the ultra-low dose effect, including the agonist-induced G-protein coupling switch, extends to another receptor type. This effect may, therefore, be part of a generalized principle that applies to many G-protein coupled receptors. / Thesis (Ph.D, Psychology) -- Queen's University, 2007-11-05 09:31:30.162 / A portion of this research was supported by a Canadian Institutes of Health Research (CIHR) Proof of Principle Grant to M.C. Olmstead and J.J. Paquette.

Temporomandibular Disorder (TMD) is a dysfunction that affects the temporomandibular joint (TMJ), characterized primarily by the presence of pain. It has been shown that low level laser therapy (LLLT) presents analgesic and anti-inflammatory effects in cases of TMD, but still experimental studies are scarce in order to understand the mechanisms involved in this biological activity. Therefore, the purpose of this work was to evaluate the ability of LLLT to control pain and inflammation induced by different chemical agents injected in the rat s TMJ. A study was carried out in two steps. At first induction of nociception was performed with 2.5% formalin in 25 Wistar rats ATM of 25 (300 ± 50 g), which they were randomly divided into 05 groups (n=5): CRG (no treatment); DFN (diclofenac sodium); LST1 (LLLT 12,5 J/cm2); LST2 (LLLT 52.5 J/cm2) and LST3 (LLLT 87.5 J/cm2). The LLLT was performed in a single application, with the apparatus laser with 780 nm (GaAlAs diode laser). The animals were evaluated considering the nociceptive behaviors characterized by rubbing the orofacial (RO) region and flinching the head (FH) quickly, analyzed separately and summed the behaviors. Data were analyzed through Anova test (one way) and Tukey s test. In the second stage, inflammation was induced in the TMJ of 45 Wistar rats (300 ± 50 g) with carrageenan to 1%. Then, the animals were divided into 03 main groups (n=15): CRG (no treatment); DFN (diclofenac sodium) and LST3 (LLLT 52.5 J/cm2). The LLLT was performed every 48 h, with the laser device described above. At specific times (24 h, 3 days and 7 days) five animals from each group were randomly sacrificed and their TMJ specimens were processed and stained with hematoxylin/eosyn (HE). The data were submitted to descriptive analysis regarding to semiquantitative and morphostrutural changes and mphasizing the inflammatory aspects and vascular neoformation. Data were analyzed through Kruskal-Wallis test and Dunn s post-hoc test. The results showed that DFN, LST2 and LST3 significantly reduced nociceptive responses characterized by the sum of the behaviors of FH and RO (p<0.001). DFN was also able to reduce the behaviors of FH (p< 0.05) and CO (p< 0.01) when assessed separately, as well as it was observed in LST2 (FH and RO, p<0.001). The LST1 did not elicit nociceptive effect. In the histological analysis, an inflammatory infiltration was observed significantly more intense in CRG than in DFN and LST2 in 24 h (P<0.05 e p<0.001), 3 days (p<0.01 e p<0.001) and 7 days (p<0.001 e p<0.01). In addition, the contingent of neutrophils was more conspicuous in CRG than in other groups in 24h. The vascular neoformation was less expressive in CRG than DFN (p<0.001) and LST2 (p<0.001) in 24 h, while in 3 days, exhibited greater quantitative LST2 blood vessels than CRG (p<0.001) and DFN (p<0.001). In 7 days, there was no difference in vascular content between experimental groups (p>0.05). Based on the results, one might conclude that the LLLT showed anti-inflammatory and antinociceptive effect in the inflammation induced in the TMJ of animals. / A Desordem temporomandibular (DTM) é uma disfunção que afeta a articulação temporomandibular (ATM), sendo caracterizada principalmente pela presença de dor. Tem sido demonstrado que fototerapia laser de baixa potência (FLBP) apresenta efeitos analgésicos e anti-inflamatórios em casos de DTM, mas ainda são escassos os estudos experimentais que permitam compreender os mecanismos envolvidos nesta atividade biológica. Sendo assim, o propósito desse trabalho foi avaliar a capacidade da FLBP em controlar a dor e a inflamação induzidas na ATM de ratos com diferentes agentes químicos. Para isso, foi realizado um estudo em duas etapas. Na primeira, foi induzida nocicepção com formalina a 2,5% na ATM de 25 ratos Wistar (325 ± 25 g), que foram então distribuídos aleatoriamente em 05 grupos (n=5): CRG (sem tratamento); DFN (tratados com diclofenaco sódico); LST1 (tratado com FLBP 12,5 J/cm2); LST2 (tratado com FLBP 52,5 J/cm2) e LST3 (tratado com FLBP 87,5 J/cm2). A FLBP foi realizada em única aplicação, com laser diodo GaAlAs a 780 nm. Os animais foram avaliados em relação aos comportamentos de levantar a cabeça (LC), coçar a face (CO), analisados separadamente e somados os comportamentos. Os dados foram analisados por meio de teste Anova (uma via) e teste de Tukey. Na segunda etapa, foi induzida inflamação na ATM de 45 ratos Wistar (325 ± 25 g) com carragenina a 1%. Posteriormente, os animais foram divididos em 03 grupos principais (n=15): CRG (sem tratamento), DFN (tratados com diclofenaco sódico) e LST2 (tratado com FLBP 52,5 J/cm2). A FLBP foi realizada a cada 48 h, com laser diodo GaAlAs a 780 nm. Em 24 h, 3 e 7 dias, cinco animais de cada grupo foram randomicamente eutanasiados e os espécimes de ATM foram processados e corados com hematoxilina/eosina (HE). Foi realizada análise descritiva das alterações morfoestruturais e semiquantitativa da intensidade da resposta inflamatória e neoformação vascular. Os dados foram analisados pelo teste Kruskal-Wallis e extensão posthoc de Dunn. Os resultados demonstraram que o DFN, o LST2 e o LST3 significativamente reduziram as respostas nociceptivas caracterizadas pela soma dos comportamentos de LC e CO (p<0.001). DFN também foi capaz de reduzir os comportamentos de LC (p<0.05) e CO (p<0.01) quando avaliados separadamente, da mesma forma que o LST2 (LC e CO, p<0.001). O LST1 não apresentou efeito antinociceptivo. Na análise histológica, foi observada uma infiltração inflamatória significativamente mais intensa in CRG do que em DFN e LST2 em 24 h (P<0,05 e p<0,001), 3 dias (p<0,01 e p<0,001) e 7 dias (p<0,001 e p<0,01). Além disso, o contingente de neutrófilos foi mais conspícuo em CRG do que nos demais grupos em 24 h. A neoformação vascular se apresentou menos expressiva em CRG do que DFN (p<0,001) e LST2 (p<0,001) em 24 h, enquanto que em 3 dias, LST2 exibiu maior quantitativo de vasos sanguíneos que CRG (p<0,001) e DFN (p<0,001). Em 7 dias, não houve diferença no conteúdo vascular entre os grupos (p>0,05). Baseado nos resultado pôde-se concluir que a FLBP apresentou atividade antinociceptiva e anti-inflamatória na indução realizada na ATM de animais experimentais.

Orientadores: Claudia Herrera Tambeli, Maria Cecilia F. A. Veiga / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-03T22:56:59Z (GMT). No. of bitstreams: 1 Clemente-Napimoga_JulianaTrindade_M.pdf: 904782 bytes, checksum: 55d51f81e2ba465f145751f287246951 (MD5) Previous issue date: 2004 / Resumo: Este estudo avaliou as diferenças sexuais na resposta nociceptiva induzida pela administração de formalina na articulação temporomandibular (ATM) com ou sem a co-administração do U50,488 (agonista do receptor opióide capa). As fases do ciclo estral das fêmeas foram citologicamente determinadas e apenas aquelas que apresentavam-se na fase diestro ou proestro, e machos foram incluídos. A formalina induziu um comportamento nociceptivo maior nas fêmeas em diestro do que nas fêmeas em proestro ou machos. O U50,488 reduziu significativamente as respostas nociceptivas induzidas pela formalina, e esta redução foi maior nas fêmeas, especialmente nas fêmeas da fase diestro do ciclo estral. A injeção do U50,488 na ATM contralateral não afetou na magnitude do comportamento induzido pela formalina, e o pré-tratamento com o antagonista seletivo do receptor opióide capa nor-binaltorphimine (norBNI) na ATM ipsilateral reduziu os efeitos antinociceptivos do U50,488. Estes resultados demonstram a ação dos receptores opióides capa periféricos na modulação da dor inflamatória. Além disso, considerando que os níveis plasmáticos dos hormônios ovarianos são baixos durante a fase diestro, estes resultados são consistentes com a hipótese de que os hormônios sexuais femininos podem ter uma ação analgésica na redução da dor inflamatória induzida pela formalina, assim como, também ter uma ação anti-analgésica nos efeitos mediados pelos receptores opióides capa / Abstract: This study examined sex differences in nociceptive responses induced by intra-temporomandibular joint (TMJ) formalin with and without co-administration of the ?-opioid receptor agonist U50,488. The estrous phase of females was cytologically determined; only those in either proestrus or diestrus, and males, were included. Formalin elicited significantly greater nociceptive behavior in diestrus females than in either proestrus females or males. U50,488 significantly reduced formalin nociceptive responses, and this reduction was significantly greater in females, especially in the diestrus phase of the estrous cycle. U50,488 injection into the contralateral TMJ failed to affect the magnitude of formalininduced behavior, and preinjection of the selective kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI) into the ipsilateral TMJ significantly reduced the antinociceptive effect of U50,488. These findings support a role for peripheral kappa-opioid receptors in the modulation of inflammatory pain. Furthermore, since plasma levels of ovarian hormones are lowest during diestrus, these findings are consistent with the suggestion that female sex hormones may play an analgesic role in reducing formalin-induced inflammatory pain, and may also play an anti-analgesic role, at least in ?-mediated effects / Mestrado / Fisiologia Oral / Mestre em Odontologia

Philosophiae Doctor - PhD / Tumour-specific protein markers are usually present at elevated concentrations in patient biopsy tissue; therefore tumour tissue is an ideal biological material for studying cancer proteomics and biomarker discovery studies. To understand and elucidate cancer pathogenesis and its mechanisms at the molecular level, the collection and characterisation of a large number of individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardised methods of formalin fixation and paraffin embedment, these archived, FFPE tissues are important collections of pathology material, often accompanied by important metadata, such as patient medical history and treatments. FFPE tissue blocks are conveniently stored under ambient conditions for decades, while retaining cellular morphology due to the modifications induced by formalin. / 2022

no / The identification of proteins involved in tumour progression or which permit enhanced or novel therapeutic targeting is essential for cancer research. Direct MALDI analysis of tissue sections is rapidly demonstrating its potential for protein imaging and profiling in the investigation of a range of disease states including cancer. MALDI-mass spectrometry imaging (MALDI-MSI) has been used here for direct visualisation and in situ characterisation of proteins in breast tumour tissue section samples. Frozen MCF7 breast tumour xenograft and human formalin-fixed paraffin-embedded breast cancer tissue sections were used. An improved protocol for on-tissue trypsin digestion is described incorporating the use of a detergent, which increases the yield of tryptic peptides for both fresh frozen and formalin-fixed paraffin-embedded tumour tissue sections. A novel approach combining MALDI-MSI and ion mobility separation MALDI-tandem mass spectrometry imaging for improving the detection of low-abundance proteins that are difficult to detect by direct MALDI-MSI analysis is described. In situ protein identification was carried out directly from the tissue section by MALDI-MSI. Numerous protein signals were detected and some proteins including histone H3, H4 and Grp75 that were abundant in the tumour region were identified