Abstract The purpose of this study was to investigate the clinical implications of a combination of a peripheral opioid, paracetamol (APAP) and ketoprofen (KTP) on the intensity of acute postoperative pain by focusing on tonsillectomy (TE) and third molar extraction. A second focus in the study was to assess the utility of the surgical ultrasonically activated scalpel (HS) technique for TE. In Study I, TE was performed on one side using the HS and on the contralateral side using a “blunt dissection technique”. The first TE study (I) demonstrated that - based on NRS pain scores during the first 10 postoperative hours - intra-operative blood loss and need for haemostasis were greater on the blunt dissection side than on the HS side. Pain scores were higher on the HS side than on the cold dissection side during the second postoperative week. Study III assessed the analgesic effect of a peripheral dose of 4 mg morphine. The peritonsillar infiltration of morphine locally did not significantly decrease pain compared to the control side. Studies (II and IV) included patients who were scheduled for third molar extraction. In Study II, patients received 1000 mg APAP or 100 mg KTP or both or a placebo to evaluate pain relief after third molar extraction. This study demonstrated that the mean sum of pain intensity differences scores up to the 1.5 h mark and the mean time to onset of pain relief at rest and on swallowing were favoured in the combination group more than in the APAP, KTP, and placebo groups. In Study IV, patients were assigned for a submucosal injection of 2 mg morphine or NaCl into either the non-inflamed (Trial I) or the inflamed (Trial II) peridental tissue, while the active control group received the same drugs in reverse order intramuscular (IM). Postoperative pain intensity at rest and on swallowing was assessed in all studies using the numerical rating scale (NRS). Pain scores in the peripheral morphine group at rest (Trials I and II) and on swallowing (Trial I) were not associated with any further pain reduction. Pain scores on swallowing during the 2–6 hours postoperative period (Trial II) were greater in the IM morphine group. HS TE was associated with decreased pain in the early postoperative period, but there was increased pain and otalgia during the second postoperative week. Locally administered peripheral morphine was not associated with any benefit during the postoperative period after TE. The multimodal analgesia combination of a single dose of KTP and APAP demonstrated the same benefit during the early postoperative period without an increase in side effects. Locally administered peripheral morphine produced significant analgesia on swallowing during the early postoperative stage in inflamed tissue after third molar extraction. / Tiivistelmä Hyvä leikkauksen jälkeinen kivunhoito on yksilöllisesti suunniteltua, turvallista, helppokäyttöistä ja taloudellista. Nykyään pyritään kivunlievityksessä hyödyn-tämään eri vaikutuspaikkoihin kohdistuvia hoitoja eli multimodaalista kivun¬hoitoa. Tämän työn tarkoituksena oli selvittää eri kivunlievitysmenetelmien tehoa ja turvallisuutta kahdessa eri toimenpiteessä: nielurisojen poistoleikkauksen (TE) tai viisaudenhampaan poistoleikkauksen jälkeen. Tutkimuskokonaisuus käsittelee leikkaustekniikan (Ultraääniveitsi), lääke-ainekombinaatioiden ja perifeerisesti annostellun morfiinin vaikutusta post-operatiiviseen kipuun. Tutkimusaineisto koostuu neljästä tutkimuksesta. Kaikki työt olivat satunnaistettuja ja kaksoissokkoutettuja. Kipu mitattiin numeerista asteikolla (Numerical Rating Scale, NRS) sekä levossa että nielemisen aikana enintään 2 viikon ajan. Ultraääniveitsen käytön vaikutusta postoperatiiviseen kipuun verrattiin perinteiseen leikkaustekniikkaan. Potilailta toinen nielurisa poistettiin ultraääni¬veistä käyttäen ja toinen tylpästi irrotellen kylmiä instrumentteja käyttäen. Kipu oli perinteisellä tekniikalla leikatulla puolella voimakkaampi kuin ultraääni¬veitsellä leikatulla puolella leikkauspäivänä. Toisen leikkauksen jälkeisen viikon aikana kipu oli kuitenkin voimakkaampaa ultraääniveitsillä leikatulla puolella. Parasetamolin (APAP), ketoprofeenin (KTP) tuottamaa kivunlievitystä ja näiden yhteisvaikutusta verrattiin viisaudenhampaan poistoleikkauksen jälkeen. KTP ja APAP kombinaatio antoi tehokkaamman kivunlievityksen ja nopeamman hoitovasteen kuin kumpikaan lääke yksin annettuna. Perifeerisesti infiltroidun morfiinin vaikutusta kipuun tutkittiin TE sekä viisaudenhampaan poistoleikkauksen jälkeen. TE jälkeen toiselle puolelle infiltroitiin nielurisan taakse 4 mg morfiinia ja toiselle puolelle fysiologista suolaliuosta. Viisaudenhampaan poistoleikkauksessa paikallisesti infiltroitua 2 mg morfiinia verrattiin lihakseen annettuun samaa lääkkeeseen kahdessa eri tilanteessa, joko tulehtuneeseen tai tulehtumattomaan kudokseen annosteltuna. Paikallisesti infiltroidulla morfiinilla ei todettu kipua lievittävää vaikutusta TE jälkeen. Tulehtuneeseen kudokseen infiltroitu morfiini lievensi leikkauksen jälkeistä nielemiskipua 2–6 tuntia leikkauksesta. Tulehtumattomaan kudokseen infiltroidulla morfiinilla ei saatu lisäetua. Yhteenvetona voidaan todeta, että TE ja viisaudenhampaanpoistoleikkauksen jälkeen kivunhoitoa voidaan optimoida multimodaalisin kivunhoidon keinoin. Tutkimustulokset auttavat potilaskohtaisen yksilöllisen kivunhoidon suunnittelussa.

No description available.

Opioids such as morphine are frequently used in the clinic to treat pain. However, the perennial bane of chronic opioid use is the rapid development of tolerance to the analgesic effects but delayed development of tolerance to the respiratory depressant and constipating effects. As constipation is one of the most common opioid-related adverse effects in humans, it is important to delineate mechanisms that drive opioid tolerance in the ileum and lack of it in the colon. The overarching goal of this thesis was to investigate mechanisms of morphine tolerance in the ileum by comparing the mechanism of morphine tolerance in ileum myenteric plexus neurons and dorsal root ganglia (DRG) neurons. Myenteric plexus neurons are integral to the motor function of the ileum, whereas DRG neurons are important components of peripheral nociceptive sensation. We also examined the mechanism of morphine tolerance development to small intestinal transit and to antinociception at the systemic level in male and female mice. Studies presented in this dissertation demonstrate that the mechanism of morphine tolerance in the mouse ileum is not contingent on b-arrestin 2. In fact, tolerance in the small intestine might be mediated by a b-arrestin 2-independent mechanism following protein kinase C-induced phosphorylation of the m-opioid receptor. We also demonstrate that morphine tolerance to antinociception is not solely dependent on b-arrestin 2, and is mediated by b-arrestin 2-dependent and-independent mechanisms. Lastly, we have shown how sex of the animal can influence mechanisms underlying the development of morphine tolerance. Collectively, the findings presented here increase our understanding of the mechanisms by which morphine tolerance develops in the ileum and to antinociception.

Drug addiction, opiates respectively, is a social problem which seriousness is currently on the rise. One of key elements causing addiction is tolerance to increasing doses of drug causing abstinence syndrome during withdrawal and craving. Opioid receptors are members of a large group of receptors coupled with heterotrimeric G-proteins (GPCR), whose properties can be investigated using agonist- stimulated binding [35 S] GTPγS. Many extracellular signals are transferred into a cell through GPCR. Opioid receptor agonists inhibit the activity of adenylyl cyclase and are coupled with G-protein group Gi/Go. This work is devoted to the study of changes in isolated plasma membranes of rat forebrain containing opioid receptors of healthy subjects with membranes acquired from morphine addicted subjects. The rats were long-term morphine treated in increasing doses, to develop the dependency. The comparison is done firstly by binding of [3 H]ouabain to Na,K-ATPase, which proves to be a negative standard of changes, secondly by binding [35 S]GTPγS to G-proteins, thereby providing the functional activity of G-protein in stimulating the binding by the agonist of δ-opioid receptors DADLE or agonist of µ-opioid receptors DAMGO. Furthermore, it has been studied the influence of prostaglandin E1 on binding [35...

It has recently been discovered that the effect of morphine can significantly reduce the tissue damage that occurs during myocardial ischemia. The molecular mechanisms by which morphine acts on the heart are still little understood. The aim of this thesis was to monitor the effect of chronic 27-day and 10-day administration of low (1 mg/kg/day) and high (10 mg/kg/ day) doses of morphine on the expression of selected G-protein-coupled receptors (GPCR) and on the expression and activity of adenylyl cyclase (AC). Chronic (27 days) morphine treatment reduced the expression of к-opioids receptors, but 10-day morphine exposure did not influence the expression of these receptors. Assessment of β1- and β2-AR by immunoblot technique did not show any significant change in the expression, but the more accurate determination of β-AR expression using the saturation binding studies revealed that 27-day treatment with high doses of morphine appreciable increased the total number of these receptors. Administration of high doses of morphine led to marked up-regulation of adenylyl cyclase (AC) isoforms V/VI, and the amount of AC decreased proportionally with the time of discontinuation of morphine administration. Low doses of morphine up- regulated AC only during 27-day administration. Chronic morphine exposure did...

Acute and chronic morphine administration can significantly reduce ischemia- reperfusion injury of the rat heart. However, the molecular mechanisms mediating the protective effect of morphine are not yet fully elucidated. Concurrently, there is a lack of information about the effects of the long-term action of morphine on heart tissue. Therefore, in the first part of the project, we studied the effect of long-term administration of high doses of morphine (10 mg/kg/day, 10 days) on rat heart tissue. In the second part of the project, we investigated the effect of 1 mM morphine on viability and redox state of rat cardiomyoblast cell line H9c2 that was influenced by oxidative stress elicited by exposure to 300 μM tert-butyl hydroperoxide (t-BHP). Our experiments have shown that long-term morphine administration affected neither the amount nor the affinity of myocardial β-adrenergic receptors (β-AR), but almost doubled the number of the dominant isoforms of myocardial adenylyl cyclase (AC) V/VI and led to supersensitization of AC. At the same time, proteomic analyses revealed that long-term morphine administration was associated with significant changes in the left ventricular proteome. In particular, there was an increase in the expression of heat shock proteins (HSP). Increased expression of HSP27...

Increases in basolateral amygdala (BLA) activity drive avoidance-seeking behavior that may be associated with stress induced drug seeking. Activity of BLA pyramidal neurons is regulated by local and paracapsular gamma aminobutyric acid (GABA) interneurons. The lateral paracapsular interneurons (LPCs) border the external capsule, receive dense cortical/thalamic input and provide feed-forward inhibition onto BLA principle neurons. The GABAergic LPCs also express high concentrations of g-protein coupled µ-opioid receptors (MORs). Therefore, the effects of opiates on LPC activity and local GABA release were examined. Fluorescently double labeled LPCs were observed in glutamate decarboxylase (GAD) 65-mcherry/GAD67-green fluorescent protein (GFP) transgenic mice. Whole-cell electrophysiology experiments demonstrated that acute exposure to [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; a synthetic selective MOR agonist), reduced LPC firing and spontaneous inhibitory postsynaptic current (sIPSC) frequency in LPCs, with no apparent effect on spontaneous excitatory currents (sEPSCs). Current injection induced firing in LPC neurons, but less effectively than in saline controls. Morphine-exposed mice (10mg/kg/day, across 5 days, 1-2 days off) had increased sIPSCs compared to saline-injected controls, as well as enhanced adenylyl cyclase (AC) activity. Together these data show that LPC neurons are a highly sensitive targets for opiate-induced inhibition, and that long-term opiate exposure results in impaired LPC excitability, possibly contributing to anxiety observed during opiate withdrawal.

The exact role of opioid receptors in drug addiction and modulatory mechanism of action of monovalent cations on these receptors are still not fully understood. Our results support the view that the mechanism of addiction to morphine is primarily based on desensitization of μ- and δ-opioid receptors. Desenzitization of agonist response proceeds already at the level of G protein functional activity. Long-term exposure of rats to morphine resulted in increase of number of δ-opioid receptors and change of their sensitivity to sodium ions. Analysis of the effect of different monovalent ions on agonist binding in δ-OR- Gi1α (Cys351 -Ile351 )-HEK293 cell line confirmed the preferential sensitivity of δ-opioid receptor to sodium ions. We have distinguished the high- and low-affinity Na+ sites. Biophysical analysis of interaction of lithium, sodium, potassium and cesium ions with plasma membranes isolated from HEK293 cells with the help of fluorescent probes indicated that monovalent ions interact, in low-affinity manner, with the polar, membrane-water interface of membrane bilayer. Key words: morphine, forebrain cortex, opioid receptors, G proteins, monovalent ions, plasma membrane, fluorescence spectroscopy.

The exact role of opioid receptors in drug addiction and modulatory mechanism of action of monovalent cations on these receptors are still not fully understood. Our results support the view that the mechanism of addiction to morphine is primarily based on desensitization of μ- and δ-opioid receptors. Desenzitization of agonist response proceeds already at the level of G protein functional activity. Long-term exposure of rats to morphine resulted in increase of number of δ-opioid receptors and change of their sensitivity to sodium ions. Analysis of the effect of different monovalent ions on agonist binding in δ-OR- Gi1α (Cys351 -Ile351 )-HEK293 cell line confirmed the preferential sensitivity of δ-opioid receptor to sodium ions. We have distinguished the high- and low-affinity Na+ sites. Biophysical analysis of interaction of lithium, sodium, potassium and cesium ions with plasma membranes isolated from HEK293 cells with the help of fluorescent probes indicated that monovalent ions interact, in low-affinity manner, with the polar, membrane-water interface of membrane bilayer. Key words: morphine, forebrain cortex, opioid receptors, G proteins, monovalent ions, plasma membrane, fluorescence spectroscopy.

L’addiction est un désordre psychobiologique caractérisé par des prises de drogue répétées, une incapacité à en contrôler la consommation et une tendance chronique à la rechute. Dans le cas des opiacés (morphine, héroïne), l’arrêt de la consommation de drogue induit un syndrome de sevrage qui peut être associé de manière forte et durable à l’environnement dans lequel il est vécu. Cette association est telle que même après une longue période d’abstinence, la simple réexposition à cet environnement peut faire émerger un état émotionnel aversif qui pourrait favoriser la rechute. Dans le cadre de la dépendance aux opiacés, il est de plus en plus clair que la réactivation des mémoires affectives associées au sevrage joue un rôle dans la motivation à rechercher de la drogue. Au plan neurobiologique, il a été montré au laboratoire que le processus de plasticité synaptique se met en place lors du conditionnement des stimuli conditionnés au sevrage des opiacés, au sein de structures limbiques impliquées à différents titres dans le processus d’apprentissage associatif. Il a été proposé que les effets de la réexposition aux stimuli conditionnés au sevrage soient dus à la réactivation spécifique de ces structures limbiques. Dans ces travaux, les stimuli environnementaux étaient associés à la fois à l’état aversif précoce du sevrage et à des symptômes somatiques, ce qui permet une première avancée dans la compréhension des processus cellulaires impliqués dans la formation et le rappel de la mémoire du sevrage. Cependant afin de mieux comprendre comment cette mémoire pourrait exercer un rôle dans la rechute, il est nécessaire d’analyser les substrats neuronaux mis en jeu de manière plus spécifique dans les effets conditionnés de la seule composante aversive précoce du sevrage. En effet, cette composante dite « motivationnelle » joue un rôle majeur chez l’individu dépendant dans le besoin de continuer à consommer la drogue, et potentiellement chez l’individu abstinent dans sa vulnérabilité à la rechute.L’objectif de mon travail de thèse a consisté à préciser les substrats neurobiologiques impliqués dans le sevrage des opiacés et dans le rappel des mémoires aversives associées notamment à la composante motivationnelle du sevrage.Dans un premier temps, nous avons développé une approche d’hybridation in situ fluorescente (catFISH) dont le principal avantage est de préciser la dynamique des activations neuronales induites par une stimulation. Nous avons validé l’utilisation du catFISH en caractérisant la dynamique d’activation neuronale dans le cortex préfrontal (CPF), le noyau accumbens (Nac), le noyau central (CeA) et basolatéral (BLA) de l’amygdale lors de la précipitation d’un syndrome de sevrage des opiacés. Nos résultats montrent que le catFISH permet de révéler des activations neuronales durables et que le CeA et le Nac présentent une dynamique d’activation différente en réponse à la précipitation du sevrage des opiacés.Dans une deuxième partie nous avons étudié les substrats neuronaux impliqués lorsque le rappel des mémoires du sevrage des opiacés exerce un effet sur un comportement opérant dirigé vers la nourriture, et ceci en fonction de l’intensité du sevrage. L’utilisation du catFISH nous a permis de différencier les activations neuronales induites par la réexposition au contexte du sevrage ou par la présentation du stimulus conditionné au sevrage. Nos résultats montrent que le CPF et le Nac shell sont impliqués dans le rappel des mémoires contextuelles du sevrage et que le CPF ainsi que le Nac core et le BLA sont activés par le rappel du stimulus conditionné au sevrage.Enfin, nous avons analysé dans un protocole d’aversion de place conditionnée les substrats neurobiologiques recrutés par le rappel des mémoires associées au syndrome de sevrage motivationnel des opiacés. Nos résultats indiquent que le Nac shell et le BLA sont les deux structures cérébrales les plus sensibles au rappel des mémoires du sevrage. L’ensemble de ce travail a permis de faire ressortir le rôle crucial du Nac shell et du BLA au sein du réseau de substrats neuronaux impliqués dans le traitement des mémoires émotionnelles aversives associées au sevrage des opiacés. Ces structures pourraient représenter les substrats communs au traitement des mémoires émotionnelles associées aux effets de drogues d’abus. L’ensemble de ces résultats devra être mis en perspective avec des travaux débutés lors de ma thèse en électrophysiologie in vivo sur animal se comportant. Ces travaux consisteront à étudier de façon longitudinale les dynamiques du réseau CPF/Nac/BLA lors de la formation et le rappel des mémoires du sevrage et permettront de mieux définir les rôles spécifiques que jouent les substrats neurobiologiques que nous avons étudiés dans le traitement des mémoires du sevrage des opiacés. / Addiction is a psychobiological disorder that is characterized by repeated drug intakes, inability to control its consumption and a chronic tendency to relapse. Concerning opiate addiction (heroin, morphine), cessation of drug consumption induces a withdrawal syndrome, which can be strongly and persistently associated with the environment in which it is experimented. This association is so tight that a single re-exposure to this specific environment is enough to provoke a negative emotional state, which may promote drug relapse. In opiate dependence, it becomes clearer and clearer that reactivation of the affective memories associated with drug withdrawal play a major role in drug seeking. In terms of neurobiological processes, previous works conducted in the lab have shown that synaptic plasticity takes place during the conditioning of stimuli to opiate withdrawal, in limbic structures known to be involved in associative learning. It has been suggested that the consequences of the re-exposition to withdrawal conditioned stimuli are due to the reactivation of these specific limbic regions. In theses studies, environmental stimuli were both associated to the early aversive state of withdrawal and to somatic symptoms. This represents a first step in the understanding of the cellular processes involved in the formation and retrieval of withdrawal memories. However, in order to better understand how these memories could play a role in relapse, it is necessary to analyze the neuronal substrates involved in the conditioned effects of the sole early aversive motivational component of opiate withdrawal. Indeed, this motivational component is considered as exerting a strong influence on the maintaining of drug consumption, and eventually on the vulnerability to relapse in abstinent addicts. The aim of my work was to specify the neurobiological substrates involved in opiate withdrawal and in the retrieval of the aversive memories especially the memories associated with the motivational component of withdrawal. We first developed an in situ hybridization approach (catFISH) whose main advantage is to add a dynamical dimension to the neuronal activations induced by a stimulation. We validated the use of the catFISH method by studying the dynamics of neuronal activations in the prefrontal cortex (PFC), the nucleus accumbens (Nac), the central (CeA) and basolateral (BLA) nucleus of the amygdala as a consequence of the precipitation of opiate withdrawal. Our results show that catFISH allows determining persistent neuronal activations and that the CeA and the Nac have a different dynamics of activation in response to opiate withdrawal. In the second part, we studied the neuronal substrates involved when the retrieval of opiate withdrawal memories modifies an operant goal-directed behaviour, according to the withdrawal intensity. The use of catFISH allowed us to differentiate the neuronal activations induced by the re-exposition to the withdrawal context or to the conditioned stimuli. Our results show that the PFC and the Nac shell are involved in the retrieval of contextual memories of withdrawal and that PFC, Nac core and BLA are activated by the retrieval of more specific conditioned stimuli.Lastly, we analysed, using a conditioned place aversion protocol, the neuronal structures recruited by the retrieval of the memories associated with the motivational component of opiate withdrawal. Our results suggest that the Nac shell and the BLA are the brain structures that are the most sensible to the retrieval of the memories of opiate withdrawal.Overall, our work emphasized the crucial role played by the Nac shell and the BLA within a network of neuronal substrates involved in the processing of aversive emotional memories associated with opiate withdrawal. These structures could be considered as the common substrates to the processing of emotional memories associated with the effects of drugs of abuse. These results will be compared with an in vivo electrophysiology on behaving animals’ approach that we initiated during my PhD. This study will consist of detailing longitudinally the dynamics of the PFC/Nac/BLA network during the formation and the retrieval of the memories of opiate withdrawal. This study will also provide more details on the specific functions of the previously studied neuronal substrates in the processing of opiate withdrawal memories.