Chronic abdominal pain is a major cause of patient morbidity in inflammatory bowel diseases (IBD). A balance of pro- and anti-nociceptive factors regulating colonic dorsal root ganglion (DRG) neurons, which synapse onto second order dorsal horn neurons, are known to regulate chronic pain but the mechanisms are poorly understood. This thesis examined whether neuroanatomical remodeling of DRG central nerve terminals underlies pro-nociceptive signaling and whether subsets of immune cells source the anti-nociceptive factor, β-endorphin. To examine pro-nociceptive mechanisms, acute and chronic dextran sulfate sodium (DSS) mouse models of colitis were established and substance P (SP; marker of nociceptor terminals) immunohistochemistry used to investigate changes in immunoreactivity of DRG terminals in the thoracic dorsal horn (segments T9-T13). SP immunoreactivity was increased in the dorsal horn (4 fold; P < 0.001) and central canal (P < 0.001) following chronic colitis. In contrast, SP immunoreactivity was unchanged in acute colitis. However, five weeks later SP immunoreactivity was increased both in the dorsal horn (4 fold; P < 0.01) and central canal (P < 0.001). In the cervical spinal cord, SP immunoreactivity was not increased following colitis, suggesting that changes seen in the thoracic level were specific to signaling from colonic DRG neurons. Immunoreactivity for the SP NK1 receptor on second order neurons was also examined and a significant increase in immunoreactivity was observed on post-synaptic second order cell bodies following chronic DSS. This could provide an additional mechanism for enhanced SP neurotransmission centrally. ii The source of the anti-nociceptive mediator, β-endorphin, during chronic DSS colitis was investigated using magnetic cell sorting and flow cytometry. The number of β- endorphin expressing CD4+ (2.4 fold; P < 0.05) and CD11b+ (2.6 fold; P < 0.05) cells in mice increased following chronic colitis. These findings suggest that during colitis there is a time-dependent increase of SP immunoreactivity in thoracic DRG central terminals, which could play a role in pro- nociceptive signaling in chronic inflammation. These actions may be balanced by anti- nociceptive factors such as β-endorphin which are found in subsets of immune cells. / Thesis (Master, Physiology) -- Queen's University, 2012-08-29 16:28:41.166

Les principaux analgésiques utilisés en clinique ciblent majoritairement le récepteur opioïdergique mu (MOPR). Or, l'activation de MOPR engendre d'importants effets secondaires. Les agonistes sélectifs au récepteur opioïdergique delta (DOPR) représentent une cible thérapeutique intéressante, puisqu'ils engendrent moins d'effets secondaires comparativement à l'activation du récepteur opioïdergique mu (MOPR). Dans la littérature, 2 hypothèses s'opposent concernant à la fois la distribution de DOPR et de MOPR au sein des sous-populations neuronales ainsi que leur implication fonctionnelle dans certaines modalités de douleurs. D'une part, des études, dont certaines basées sur l'utilisation d'anticorps, ont suggéré une colocalisation de ces 2 récepteurs au sein des mêmes sous-populations neuronales. Cette colocalisation suggère que l'activation de MOPR ou DOPR soulage les mêmes modalités de douleurs. D'autre part, une étude a remis en doute la spécificité des anticorps commerciaux ciblant DOPR, remettant ainsi en question les travaux réalisés avec cet outil. Par ailleurs, les auteurs de cette même étude ont aussi observé une ségrégation physique et fonctionnelle entre DOPR et MOPR : non seulement DOPR et MOPR seraient exprimés par des populations neuronales différentes, mais DOPR régulerait préférentiellement les douleurs d'origine mécanique, alors que MOPR serait plutôt impliqué dans le soulagement des douleurs d'origine thermique. Afin de répondre à cette controverse, il a d'abord été nécessaire de trouver un outil alternatif à l'utilisation des anticorps ciblant DOPR. Idéalement, cet outil doit permettre la visualisation de la distribution de ce récepteur autant au niveau de cellules en culture que des tissus. Pour ce faire, les propriétés pharmacologiques de 2 ligands biotinylés, le TIPP-biotine (Tyr-Tic-Phe-Phe(para-bromoacétamide)-Asp-desthiobiotine) et la deltorphine-biotine, ont été déterminées. Les résultats obtenus ont démontré que le TIPP-biotine possède des caractéristiques de liaison sur DOPR fort intéressantes autant sur des extraits cellulaires que sur du tissu animal, alors que la deltorphine-biotine nécessite encore des modifications afin d'optimiser ses propriétés de liaison à DOPR. Dans un second temps, à l'aide d'études d'électrophysiologie unitaire extracellulaire in vivo et d'immunohistochimies du récepteur NK? (récepteur de la substance P), l'implication de MOPR dans le soulagement des douleurs thermiques et mécaniques, et celle de DOPR dans le soulagement des douleurs mécaniques ont été mises en évidence. [symboles non conformes]

The interactions between the immune and nervous systems play an important role in immune and inflammatory conditions. Substance P (SP), the unidecapeptide RPKPQQFFGLM-NH2, is known to upregulate the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α. We report here that 5 (Acetylamino) 4 oxo-6-phenyl-2-hexenoic acid methyl ester (AOPHA-Me) and 4 phenyl 3 butenoic acid (PBA), two anti-inflammatory compounds developed in our laboratory, reduce SP stimulated TNF-α expression in RAW 264.7 macrophages. We also show that AOPHA Me and PBA both inhibit SP stimulated phosphorylation of JNK and p38 MAPK. Furthermore, molecular modeling studies indicate that both AOPHA Me and PBA dock at the ATP binding site of apoptosis signal regulating kinase 1 (ASK1) with predicted docking energies of -7.0 kcal/mol and 5.9 kcal/mol, respectively; this binding overlaps with that of staurosporine, a known inhibitor of ASK1. Taken together, these findings support the conclusion that AOPHA Me and PBA inhibition of TNF-α expression in SP-stimulated RAW 264.7 macrophages is a consequence of the inhibition JNK and p38 MAPK phosphorylation. We have previously shown that AOPHA-Me and PBA inhibit the amidative bioactivation of SP, which also would be expected to decrease formation of pro-inflammatory cytokines. It is conceivable that this dual action of inhibiting amidation and MAPK phosphorylation may be of some advantage in enhancing the anti-inflammatory activity of a therapeutic molecule. We also encapsulated AOPHA-Me separately in polyketal and poly(lactic co glycolic acid) microparticles. The in-vitro release profiles of AOPHA-Me from these particles were characterized. We have also shown that AOPHA-Me, when encapsulated in PCADK microparticles, is an effective treatment for edema induced by adjuvant arthritis in rats. In separate work, it was determined that myo inositol 1,2,3,4,5,6 hexakisphosphate is an inhibitor to early-stage Loblolly pine somatic embryo growth. In addition, it was determined that muco inositol 1,2,3,4,5,6 hexakisphosphate is not an inhibitor to early-stage Loblolly pine somatic embryo growth. These experiments demonstrate the stereochemical dependence of myo inositol 1,2,3,4,5,6 hexakisphosphates inhibitory activity.

More than 15 million people worldwide will suffer a stroke each year two thirds will die or be left permanently disabled. Accordingly, stroke represents an enormous financial burden on the community, due to the cost of hospitalisation, treatment and rehabilitation of stroke patients. Despite the significance of this public health problem, a safe and widely applicable stroke therapeutic remains elusive. Cerebral oedema is widely recognised as a common and often fatal complication of stroke that is associated with worsened outcome. However, the exact mechanisms of oedema formation remain unclear, with current therapies largely ineffective in addressing the mechanisms of cerebral swelling, and also being associated with their own negative side-effect profile. This thesis characterises the role of neurogenic inflammation and the neuropeptide, substance P (SP), in mediating the development of blood brain barrier breakdown, cerebral oedema and resultant functional deficits following stroke, using a rodent model of reversible cerebral ischaemia. The findings of this thesis demonstrate that increased SP immunoreactivity, particularly of the penumbral tissue vasculature, is a feature of tissue perfusion following stroke, but not in non-reperfused infarcts. The central role for SP in the breakdown of the BBB following stroke and the associated deleterious effects of such breakdown was confirmed by studies using an NK₁ receptor antagonist. These antagonists conferred a profound attenuation of BBB breakdown, cerebral oedema formation, neuronal death and injury, and the associated development of functional deficits following reversible stroke. Similarly, depletion of all neuropeptides by capsaicin pre-treatment also reduced both histological abnormalities and functional deficits following stroke, confirming the central role of neuropeptides in the secondary injury process after stroke. The NK₁ receptor antagonist was able to be safely combined with the currently approved treatment for stroke, tPA, producing a synergistic effect of greater protection from the ischaemic insult. In particular, histological and functional outcome were markedly improved, as well as a reduction in the risk of intracerebral haemorrhage and death. Furthermore, the NK₁ receptor antagonist was effective even when administered up to 8 h following the onset of ischaemia, and in a variety of stroke severities. We conclude that SP plays a central role in the secondary injury that occurs following stroke, in particular, the genesis of BBB breakdown and cerebral oedema. Accordingly, combination therapy of tPA and an NK₁ receptor antagonist may offer a novel therapeutic strategy for the clinical management of ischaemic stroke of varying severity. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1298280 / Thesis (Ph.D.) -- The University of Adelaide, School of Medical Sciences, 2007

Copies of author's previously published articles inserted. / Bibliography: leaves 89-126. / vii, 126, [88] leaves, [1] leaf of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Finds that the metabolism of substance P (SP) and the subsequent production of SP-(7-11) is important in regulating the biological activity of SP on chondrycytes in the synoviol joint. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1996?

More than 15 million people worldwide will suffer a stroke each year two thirds will die or be left permanently disabled. Accordingly, stroke represents an enormous financial burden on the community, due to the cost of hospitalisation, treatment and rehabilitation of stroke patients. Despite the significance of this public health problem, a safe and widely applicable stroke therapeutic remains elusive. Cerebral oedema is widely recognised as a common and often fatal complication of stroke that is associated with worsened outcome. However, the exact mechanisms of oedema formation remain unclear, with current therapies largely ineffective in addressing the mechanisms of cerebral swelling, and also being associated with their own negative side-effect profile. This thesis characterises the role of neurogenic inflammation and the neuropeptide, substance P (SP), in mediating the development of blood brain barrier breakdown, cerebral oedema and resultant functional deficits following stroke, using a rodent model of reversible cerebral ischaemia. The findings of this thesis demonstrate that increased SP immunoreactivity, particularly of the penumbral tissue vasculature, is a feature of tissue perfusion following stroke, but not in non-reperfused infarcts. The central role for SP in the breakdown of the BBB following stroke and the associated deleterious effects of such breakdown was confirmed by studies using an NK₁ receptor antagonist. These antagonists conferred a profound attenuation of BBB breakdown, cerebral oedema formation, neuronal death and injury, and the associated development of functional deficits following reversible stroke. Similarly, depletion of all neuropeptides by capsaicin pre-treatment also reduced both histological abnormalities and functional deficits following stroke, confirming the central role of neuropeptides in the secondary injury process after stroke. The NK₁ receptor antagonist was able to be safely combined with the currently approved treatment for stroke, tPA, producing a synergistic effect of greater protection from the ischaemic insult. In particular, histological and functional outcome were markedly improved, as well as a reduction in the risk of intracerebral haemorrhage and death. Furthermore, the NK₁ receptor antagonist was effective even when administered up to 8 h following the onset of ischaemia, and in a variety of stroke severities. We conclude that SP plays a central role in the secondary injury that occurs following stroke, in particular, the genesis of BBB breakdown and cerebral oedema. Accordingly, combination therapy of tPA and an NK₁ receptor antagonist may offer a novel therapeutic strategy for the clinical management of ischaemic stroke of varying severity. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1298280 / Thesis (Ph.D.) -- The University of Adelaide, School of Medical Sciences, 2007

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 7 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 6 uppsatser.

Made available in DSpace on 2016-06-02T19:22:51Z (GMT). No. of bitstreams: 1 2475.pdf: 1572607 bytes, checksum: 64043b30c1df210d393c4540c2d88933 (MD5) Previous issue date: 2009-05-29 / Universidade Federal de Minas Gerais / The locus coeruleus (LC) has been suggested as a CO2 chemoreceptor site in mammals. Substance P (SP) has been used as a marker of respiratory neurons and it plays an important role in compensatory responses to hypercapnia in several sites of the central nervous system. Neurokinin-1 (NK-1) receptor immunoreactive (NK1Rir) neurons and processes are widely distributed within the LC. Thus, the present study assessed the role of NK-1 receptors in the LC in the cardiorespiratory and thermal responses to hypercapnia. To this end, substance P-saporin conjugate (SPSAP; 2&#956;M) was injected in the LC to kill NK1R-ir neurons, or IgG-SAP as a control in male Wistar rats. The animals that the drug reached the fourth ventricle (4ºV) were considered as a 4ºV group. Pulmonary ventilation (VE, body plethysmograph), mean arterial pressure (MAP), heart rate (HR) and body core temperature were measured followed by 60 min of hypercapnic exposure (7% CO2). To verify the correct placement and effectiveness of the chemical lesions, immunohistochemistry for NK1R was performed. In addition, tyrosine hydroxylase (TH) immunoreactivity was performed to verify if noradrenergic neurons were eliminated. Fluoro-Jade technique was used to evaluate neuronal degeneration. A reduced NK1R (72% of reduction) and TH immunoreactivity (66% of reduction) was observed seven days after the injections of SP-SAP in the LC and an intense Fluoro-Jade staining, showing the effectiveness of the lesion. Focal lesions of NK1R-ir did not affect basal ventilation in the SP-SAP in LC and SP-SAP in 4ºV groups. Hypercapnia caused an increase in pulmonary ventilation in all groups, which was a result of increases in respiratory frequency (fR) and tidal volume (VT), SP-SAP treatment in the LC and in the fourth ventricle attenuated the hypercapnic ventilatory response (30% and 20%, respectivally), due to a reduction in the VT. SP-SAP in the LC and SP SAP in the 4ºV 11 lesion did not affect MAP, but caused an increase in HR in both groups. The results suggest that NK1R-ir neurons in the LC modulate hypercapnic ventilatory response but play no role in breathing control under resting conditions. Additionally, NK1R-ir neurons seem to play no role in body temperature and MAP regulation in resting conditions and during hypercapnia, but modulate HR during CO2 exposure. This modulation may be due to a change in the noradrenaline release. / O locus coeruleus (LC) é considerado uma região quimiorreceptora a CO2/pH em mamíferos. A substância P (SP) tem sido usada como marcador de neurônios respiratórios, pois possui importante função nas respostas compensatórias a hipercapnia em muitas áreas do sistema nervoso central. Neurônios e processos imunorreativos a receptores neurocinina 1 (NK-1) estão amplamente distribuídos dentro do LC. Portanto, o presente estudo teve por objetivo avaliar a participação de receptores NK-1 no LC nas respostas cardiorrespiratórias e termorreguladoras à hipercapnia. Para este fim, foi injetado o conjugado SP-Saporina (SP-SAP; 2&#956;M) no LC de ratos Wistar para lesar neurônios que expressam esses receptores, ou IgGSAP como controle. Os animais em que as injeções atingiram o quarto ventrículo (4ºV) foram considerados como grupo 4ºV. A ventilação pulmonar (VE, pletismografia de corpo inteiro), pressão arterial média (PAM), freqüência cardíaca (FC) e temperatura corporal (Tc) foram medidas por 60 min de exposição à hipercapnia (7% CO2). Para verificar a correta localização e efetividade da lesão química realizou-se a imunohistoquímica para receptores NK-1. Além disso, imunohistoquímica para tirosina hidroxilase (TH) foi realizada para averigüar se neurônios noradrenérgicos foram lesados. A técnica de Fluoro-Jade foi também utilizada para avaliar a neurodegeneração. Observou-se a redução da imunorreatividade para receptores NK-1 (72% de lesão dos neurônios) e redução da imunorreatividade para neurônios noradrenérgicos (66% dos neurônios noradrenérgicos) sete dias após injeções de SP-SAP no LC e intensa marcação na técnica de Fluoro-Jade mostrando a efetividade da lesão. Lesões seletivas de neurônios que expressam receptores NK-1 no LC não afetaram a ventilação basal, o mesmo foi observado com os animais em que a injeção atingiu o 4ºV. A hipercapnia causou aumento da ventilação pulmonar 9 em todos os grupos decorrente do aumento da freqüência respiratória (fR) e volume corrente (VC). Entretanto, o tratamento com SP-SAP no LC e no 4ºV promoveu atenuação da resposta ventilatória (30% e 20%, respectivamente), devido à diminuição do VC. A lesão com SP-SAP no LC e no 4ºV não afetou a PAM, entretanto promoveu aumento na FC em ambos grupos. Os resultados sugerem que os neurônios que expressam receptores NK-1 no LC modulam a resposta ventilatória à hipercapnia, porém não possuem papel tônico na ventilação em condições basais. Além disso, esses neurônios não participam da regulação da temperatura e da PAM em normocapnia e hipercapnia, mas modulam FC durante exposição ao CO2. Essa modulação pode ser devida a alteração na liberação de noradrenalina.

Nossa hipótese é de que os efeitos pró-inflamatórios iniciais da ativação do receptor ativado por protease 2 (PAR2) na articulação temporomandibular (ATM) sejam mediados por mecanismos neurogênicos. A análise por imunofluorescência revelou um alto grau de imunorreatividade ao PAR2 em aferentes primários trigeminais da ATM. Além do mais, a imunorreatividade ao PAR2 também foi observada na camada íntima da sinóvia, além de co-localizar com o marcador neuronal PGP9.5 e o neuropeptídeo substância P. A injeção intra-articular de agonistas PAR2 na ATM induziu um aumento dependente da dose no extravasamento plasmático, influxo de neutrófilos e indução de alodinia mecânica. O bloqueio farmacológico de receptors NK1 inibiu o aumento no extravasamento plasmático, influxo de neutrófilos e alodinia induzido pela ativação do PAR2. Em conclusão, a ativação do PAR2 é pró-inflamatório na ATM, via mecanismos neurogênicos envolvendo receptores NK1, sugerindo que o PAR2 é um importante componente da resposta imunológica inata na ATM. / We hypothesised that the early pro-inflammatory effects of proteinase-activated receptor 2 (PAR2) activation in the temporomandibular joint (TMJ) are mediated by neurogenic mechanisms. Immunofluorescence analysis revealed a high degree of neurons expressing PAR2 in retrogradely labelled trigeminal ganglion neurons. Furthermore, PAR2 immunoreactivity was observed in the lining layer of the TMJ, co-localizing with the neuronal marker PGP9.5 and substance P-containing peripheral sensory nerve fibres. The intra-articular injection of PAR2 agonists into the TMJ triggered a dose-dependent increase in plasma extravasation, neutrophil influx and induction of mechanical allodynia. The pharmacological blockade of NK1 receptors abolished PAR2-induced plasma extravasation and inhibited neutrophil influx and mechanical allodynia. We conclude that PAR2 activation is pro-inflammatory in the TMJ, through a neurogenic mechanism involving NK1 receptors. This suggests that PAR2 is an important component of innate neuro-immune response in the TMJ.