Eight cyclic analogues of Substance P were made in order to investigate the conformation of the C-terminal end of the peptide. These analogues were designed to test three literature models describing the active conformation of substance P. Although the potencies of the analogues were low (in the micromolar range), our results support Cotrait's and Hospital's model (1986). Several substance P antagonists were synthesized. These compounds did not demonstrate agonistic activity nor anatagonistic activity. The tryptophan side chain is contributing to the antagonistic activity of these analogues, and not just the chirality of the α-carbon. Highly potent and selective photoaffinity ligands of H-Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE) and D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH₂ (CTP) were synthesized. These compounds will be useful in the isolation of δ and μ opioid receptors. Several new amino acids designed and synthesized to contain both the natural amino acid side chain and a thiol group which can be used to make disulfide constraints. The racemic amino acids made were as follows: (1) 2-amino-4-methyl-2- [(p-methylbenzyl)thiomethyl] pentanoic acid; (2) 2-amino-2- [(p-methylbenzyl)thiomethyl] -3-phenylpropanoic acid; (3) 2-amino-e- [(p-methylbenzyl)thio] pentanoic acid; and (4) 2-amino-3- [(p-methylbenzyl)-thio] -3-phenyl-pentanoic acid. These amino acids will be useful in the conformational restriction of peptides. To investigate the δ-opioid receptor conformation proposed for DPDPE by Hruby et al. (1988) and the μ-opioid receptor conformation proposed for Tyr-c [Abu₂,Gly,Phe,Leu] by Mierke et al. (1988), constrained phenylalanine amino acids were incorporated into H-Try-D-Pen-Gly-Phe-D-Pen-OH (DPDPE) in the four position. Our results indicate that these models are correct. And in an investigation into the physical-chemical properties of the delta opioid receptor, our results suggest that the δ receptor topochemical site for the Phe⁴ residue contains a partial positive charge on its surface and has specific steric requirements.

Gliomas malignos são tumores cerebrais primários, resistentes a vários tratamentos, como quimioterapia, radioterapia, indução de apoptose e cirurgia. Uma alternativa para o tratamento dos gliomas malignos é a terapia radionuclídea. Essa técnica utiliza moléculas radiomarcadas que se ligam seletivamente às células tumorais e nelas depositam dose citotóxica de radiação, provocando a morte das células doentes. A maioria dos protocolos de terapia radionuclídea para tumores cerebrais malignos envolve a administração de peptídeos marcados com radioisótopos emissores -. A substância P (SP) é um neuropeptídeo de 11 aminoácidos, da família das taquicininas, caracterizada pela sequência C-terminal Phe-X-Gly-Leu-Met-NH2. A SP radiomarcada com diferentes radioisótopos, inclusive Lutécio-177, tem sido descrita e proposta para tratamento in vivo de tumores. A SP é o ligante mais importante dos receptores de neuroquinina tipo 1, superexpressos em gliomas malignos. O objetivo deste trabalho foi estudar as condições de marcação de SP-DOTA com 177Lu, a estabilidade do composto marcado e suas propriedades in vitro e in vivo, a fim de desenvolver um protocolo de produção e avaliar o potencial do radiofármaco para terapia de gliomas. As condições de marcação foram otimizadas variando-se temperatura, tempo de reação, atividade de cloreto de lutécio-177 e massa de SP-DOTA e analisou-se a pureza radioquímica das preparações por meio de técnicas cromatográficas. A estabilidade da SP-DOTA-177Lu radiomarcada com baixa atividade de 177Lu foi avaliada por diferentes tempos a 2 - 8 ºC ou incubadas em soro humano a 37 ºC. A estabilidade das marcações com alta atividade de 177Lu também foi analisada na presença de ácido gentísico (6 mg/mL) adicionado após a reação de marcação. As condições de marcação em baixa e alta atividade foram submetidas à avaliação quanto à possibilidade de oxidação do resíduo de metionina, adicionando o aminoácido D-Lmetionina (6 mg/mL) ao meio de reação e posterior avaliação cromatográfica. Estudo in vitro com SP-DOTA-177Lu, radiomarcada na ausência e presença de metionina, utilizando células de glioma humano M059J e U-87 MG, verificou o efeito da oxidação da metionina sobre a ligação às células. Estudos de biodistribuição foram realizados em camundongos Nude com modelo tumoral e em camundongos Balb-c sadios. Obteve-se a maior pureza radioquímica (> 95 %) associada à maior atividade específica de SP-DOTA-177Lu quando o tempo de reação foi de 30 minutos, temperatura de 90 ºC, massa de SP-DOTA de 10 g e a atividade do 177Lu de 185 MBq. A SP radiomarcada em condições otimizadas manteve-se estável a 2 - 8 ºC e em soro humano por 4 horas. Os estudos in vitro demonstraram ligação aos receptores celulares e essa ligação mostrou-se reduzida quando o peptídeo apresenta-se em sua forma oxidada. A adição de metionina combinada com ácido gentísico preveniu a oxidação peptídica e assegurou-se a estabilidade do composto marcado, principalmente com alta atividade de 177Lu, quando se utilizou maior massa de SP-DOTA. Nos estudos in vivo, os resultados mostraram uma cinética de biodistribuição favorável do composto e capacidade de ligação às células tumorais. SP-DOTA-177Lu pode ser uma ferramenta útil para estudos in vivo devido à facilidade de preparação, alta estabilidade e afinidade pelas células tumorais. / Malignant gliomas are primary brain tumors, resistant to various treatments, as chemotherapy, radiotherapy, induction of apoptosis and surgery. An alternative for the treatment of malignant gliomas is the radionuclide therapy. This technique apply radiolabeled molecules that selectively bind to tumor cells producing cytotoxic effect by dose irradiation, and resulting in death of tumor cells. Most protocols for radionuclide therapy of malignant brain tumors involve the administration of peptides labeled with - emitting radioisotopes. The Substance P (SP) is an 11- amino acid neuropeptide, characterized by the C-terminal sequence Phe-X-Gly-Leu-Met-NH2. The use of SP labeled with different radionuclides including 177Lu, have been proposed for in vivo treatment of tumors. SP is the most important target of neurokinin 1 receptors, overexpressed in malignant gliomas. The objective of this work was to study conditions of radiolabeling DOTA-SP with 177Lu, the stability of labeled compound and in vivo and in vitro, to develop a protocol production and evaluate the potential of the radiopharmaceutical in the therapy of gliomas. The labeling conditions were optimized varying the temperature, reaction time, activity of lutetium-177 chloride and mass of DOTA-SP. The radiochemical purity of preparations were analyzed by chromatographic techniques. The stability of 177Lu -DOTA- SP radiolabeled with low activity of 177Lu was evaluated for different time at 2-8 °C or incubated in human serum. The stability of the labeled with high activity of 177Lu was also analyzed in the presence of gentisic acid (6 mg / mL) added after the labeling reaction. The labeled conditions in low and high activity were subjected to evaluation for the ability to cause oxidation of methionine residue, adding the D-L- methionine amino acid to the reaction medium (6 mg / mL) and subsequent chromatographic evaluation. In vitro study with 177Lu-DOTA-SP, radiolabeled in the absence and presence of methionine, using human M059J U-87 MG glioma cells and, showed the effect of oxidation of methionine on the cells binding. Biodistribution studies were performed in Nude mice with tumor model and Balb-c mice. Highest radiochemical purity (> 95%) associated with the highest specific activity of 177Lu-DOTA-SP when the reaction time was 30 minutes, temperature of 90 °C, 10 g of DOTA-SP, and the activity of 177Lu of 185 MBq. The radiolabeled SP in optimized conditions remained stable at 2-8 °C and in human serum for 4 hours. In vitro studies showed the binding to cell receptors and this binding was reduced when the peptide was presented in its oxidized form. The addition of methionine combined with gentisic acid prevented the oxidation of peptide and increased the stability of the labeled compound, particularly with high activity of 177Lu, when using larger mass of DOTA-SP. In vivo studies results showed a favorable biodistribution kinetics of the compound and ability to bind to tumor cells. 177Lu-DOTA-SP can be a useful tool for in vivo studies due to ease preparation, high stability and affinity for tumor cells.

Gliomas malignos são tumores cerebrais primários, resistentes a vários tratamentos, como quimioterapia, radioterapia, indução de apoptose e cirurgia. Uma alternativa para o tratamento dos gliomas malignos é a terapia radionuclídea. Essa técnica utiliza moléculas radiomarcadas que se ligam seletivamente às células tumorais e nelas depositam dose citotóxica de radiação, provocando a morte das células doentes. A maioria dos protocolos de terapia radionuclídea para tumores cerebrais malignos envolve a administração de peptídeos marcados com radioisótopos emissores -. A substância P (SP) é um neuropeptídeo de 11 aminoácidos, da família das taquicininas, caracterizada pela sequência C-terminal Phe-X-Gly-Leu-Met-NH2. A SP radiomarcada com diferentes radioisótopos, inclusive Lutécio-177, tem sido descrita e proposta para tratamento in vivo de tumores. A SP é o ligante mais importante dos receptores de neuroquinina tipo 1, superexpressos em gliomas malignos. O objetivo deste trabalho foi estudar as condições de marcação de SP-DOTA com 177Lu, a estabilidade do composto marcado e suas propriedades in vitro e in vivo, a fim de desenvolver um protocolo de produção e avaliar o potencial do radiofármaco para terapia de gliomas. As condições de marcação foram otimizadas variando-se temperatura, tempo de reação, atividade de cloreto de lutécio-177 e massa de SP-DOTA e analisou-se a pureza radioquímica das preparações por meio de técnicas cromatográficas. A estabilidade da SP-DOTA-177Lu radiomarcada com baixa atividade de 177Lu foi avaliada por diferentes tempos a 2 - 8 ºC ou incubadas em soro humano a 37 ºC. A estabilidade das marcações com alta atividade de 177Lu também foi analisada na presença de ácido gentísico (6 mg/mL) adicionado após a reação de marcação. As condições de marcação em baixa e alta atividade foram submetidas à avaliação quanto à possibilidade de oxidação do resíduo de metionina, adicionando o aminoácido D-Lmetionina (6 mg/mL) ao meio de reação e posterior avaliação cromatográfica. Estudo in vitro com SP-DOTA-177Lu, radiomarcada na ausência e presença de metionina, utilizando células de glioma humano M059J e U-87 MG, verificou o efeito da oxidação da metionina sobre a ligação às células. Estudos de biodistribuição foram realizados em camundongos Nude com modelo tumoral e em camundongos Balb-c sadios. Obteve-se a maior pureza radioquímica (> 95 %) associada à maior atividade específica de SP-DOTA-177Lu quando o tempo de reação foi de 30 minutos, temperatura de 90 ºC, massa de SP-DOTA de 10 g e a atividade do 177Lu de 185 MBq. A SP radiomarcada em condições otimizadas manteve-se estável a 2 - 8 ºC e em soro humano por 4 horas. Os estudos in vitro demonstraram ligação aos receptores celulares e essa ligação mostrou-se reduzida quando o peptídeo apresenta-se em sua forma oxidada. A adição de metionina combinada com ácido gentísico preveniu a oxidação peptídica e assegurou-se a estabilidade do composto marcado, principalmente com alta atividade de 177Lu, quando se utilizou maior massa de SP-DOTA. Nos estudos in vivo, os resultados mostraram uma cinética de biodistribuição favorável do composto e capacidade de ligação às células tumorais. SP-DOTA-177Lu pode ser uma ferramenta útil para estudos in vivo devido à facilidade de preparação, alta estabilidade e afinidade pelas células tumorais. / Malignant gliomas are primary brain tumors, resistant to various treatments, as chemotherapy, radiotherapy, induction of apoptosis and surgery. An alternative for the treatment of malignant gliomas is the radionuclide therapy. This technique apply radiolabeled molecules that selectively bind to tumor cells producing cytotoxic effect by dose irradiation, and resulting in death of tumor cells. Most protocols for radionuclide therapy of malignant brain tumors involve the administration of peptides labeled with - emitting radioisotopes. The Substance P (SP) is an 11- amino acid neuropeptide, characterized by the C-terminal sequence Phe-X-Gly-Leu-Met-NH2. The use of SP labeled with different radionuclides including 177Lu, have been proposed for in vivo treatment of tumors. SP is the most important target of neurokinin 1 receptors, overexpressed in malignant gliomas. The objective of this work was to study conditions of radiolabeling DOTA-SP with 177Lu, the stability of labeled compound and in vivo and in vitro, to develop a protocol production and evaluate the potential of the radiopharmaceutical in the therapy of gliomas. The labeling conditions were optimized varying the temperature, reaction time, activity of lutetium-177 chloride and mass of DOTA-SP. The radiochemical purity of preparations were analyzed by chromatographic techniques. The stability of 177Lu -DOTA- SP radiolabeled with low activity of 177Lu was evaluated for different time at 2-8 °C or incubated in human serum. The stability of the labeled with high activity of 177Lu was also analyzed in the presence of gentisic acid (6 mg / mL) added after the labeling reaction. The labeled conditions in low and high activity were subjected to evaluation for the ability to cause oxidation of methionine residue, adding the D-L- methionine amino acid to the reaction medium (6 mg / mL) and subsequent chromatographic evaluation. In vitro study with 177Lu-DOTA-SP, radiolabeled in the absence and presence of methionine, using human M059J U-87 MG glioma cells and, showed the effect of oxidation of methionine on the cells binding. Biodistribution studies were performed in Nude mice with tumor model and Balb-c mice. Highest radiochemical purity (> 95%) associated with the highest specific activity of 177Lu-DOTA-SP when the reaction time was 30 minutes, temperature of 90 °C, 10 g of DOTA-SP, and the activity of 177Lu of 185 MBq. The radiolabeled SP in optimized conditions remained stable at 2-8 °C and in human serum for 4 hours. In vitro studies showed the binding to cell receptors and this binding was reduced when the peptide was presented in its oxidized form. The addition of methionine combined with gentisic acid prevented the oxidation of peptide and increased the stability of the labeled compound, particularly with high activity of 177Lu, when using larger mass of DOTA-SP. In vivo studies results showed a favorable biodistribution kinetics of the compound and ability to bind to tumor cells. 177Lu-DOTA-SP can be a useful tool for in vivo studies due to ease preparation, high stability and affinity for tumor cells.

Doctor of Philosophy (PhD) / Little is known about the role that tachykinins, such as substance P and its receptor, the neurokinin-1 receptor, play in the generation of sympathetic nerve activity and the integration within the ventrolateral medulla (VLM) of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The studies described in this thesis investigate these autonomic functions and the role of tachykinins through physiological (response to hypercapnoea, chapter 3), anatomical (neurokinin-1 receptor immunohistochemistry, chapter 4) and microinjection (neurokinin-1 receptor activation and blockade, chapters 5 and 6) experiments. In the first series of experiments (chapter 3) the effects of chemoreceptor activation with hyperoxic hypercapnoea (5%, 10% or 15% CO2 in O2) on splanchnic sympathetic nerve activity and sympathetic reflexes such as the baroreflex and somato-sympathetic reflex were examined in anaesthetized rats. Hypercapnoea resulted in sympatho-excitation in all groups and a small increase in arterial blood pressure in the 10 % CO2 group. Phrenic nerve amplitude and phrenic frequency were also increased, with the frequency adapting back to baseline during the CO2 exposure. Hypercapnoea selectively attenuated (5% CO2) or abolished (10% and 15% CO2) the somato-sympathetic reflex while leaving the baroreflex unaffected. This selective inhibition of the somato-sympathetic reflex while leaving the baroreflex unaffected was also seen following neurokinin-1 receptor activation in the rostral ventrolateral medulla (RVLM) (see below). Microinjection of substance P analogues into the RVLM results in a pressor response, however the anatomical basis for this response is unknown. In the second series of experiments (chapter 4), the distribution of the neurokinin-1 receptor in the RVLM was investigated in relation to catecholaminergic (putative sympatho-excitatory “C1”) and bulbospinal neurons. The neurokinin-1 receptor was demonstrated on a small percentage (5.3%) of C1 neurons, and a small percentage (4.7%) of RVLM C1 neurons also receive close appositions from neurokinin-1 receptor immunoreactive terminals. This provides a mechanism for the pressor response seen with RVLM microinjection of substance P analogues. Neurokinin-1 receptor immunoreactivity was also seen a region overlapping the preBötzinger complex (the putative respiratory rhythm generation region), however at this level a large percentage of these neurons are bulbospinal, contradicting previous work suggesting that the neurokinin-1 receptor is an exclusive anatomical marker for the propriobulbar rhythm generating neurons of the preBötzinger complex. The third series of experiments (chapter 5) investigated the effects of neurokinin-1 receptor activation and blockade in the RVLM on splanchnic sympathetic nerve activity, arterial blood pressure, and autonomic reflexes such as the baroreflex, somato-sympathetic reflex, and sympathetic chemoreflex. Activation of RVLM neurokinin-1 receptors resulted in sympatho-excitation, a pressor response, and abolition of phrenic nerve activity, all of which were blocked by RVLM pre-treatment with a neurokinin-1 receptor antagonist. As seen with hypercapnoea, RVLM neurokinin-1 receptor activation significantly attenuated the somato-sympathetic reflex but did not affect the sympathetic baroreflex. Further, blockade of RVLM neurokinin-1 receptors significantly attenuated the sympathetic chemoreflex, suggesting a role for RVLM substance P release in this pathway. The fourth series of experiments (chapter 6) investigated the role of neurokinin-1 receptors in the RVLM, caudal ventrolateral medulla (CVLM), and nucleus tractus solitarius (NTS) on regional cerebral blood flow (rCBF) and tail blood flow (TBF). Activation of RVLM neurokinin-1 receptors increased rCBF associated with a decrease in cerebral vascular resistance (CVR). Activation of CVLM neurokinin-1 receptors decreased rCBF, however no change in CVR was seen. In the NTS, activation of neurokinin-1 receptors resulted in a biphasic response in both arterial blood pressure and rCBF, but no significant change in CVR. These findings suggest that in the RVLM substance P and the neurokinin-1 receptor play a role in the regulation of cerebral blood flow, and that changes in rCBF evoked in the CVLM and NTS are most likely secondary to changes in arterial blood pressure. Substance P and neurokinin-1 receptors in the RVLM, CVLM and NTS do not appear to play a role in the brainstem regulation of tail blood flow. In the final chapter (chapter 7), a model is proposed for the role of tachykinins in the brainstem integration of the sympathetic baroreflex, sympathetic chemoreflex, cerebral vascular tone, and the sympatho-excitation seen following hypercapnoea. A further model for the somato-sympathetic reflex is proposed, providing a mechanism for the selective inhibition of this reflex seen with hypercapnoea (chapter 3) and RVLM neurokinin-1 receptor activation (chapter 5). In summary, the ventral medulla is essential for the generation of basal sympathetic tone and the integration of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The tachykinin substance P, and its receptor, the neurokinin-1 receptor, have a role to play in many of these vital autonomic functions. This role is predominantly neuromodulatory.

Neurogenic inflammatory responses can be induced by antidromic electrical stimulation or intravenous capsaicin injection. These responses were thought to be caused by neuropeptides released from the sensory axon of C-fiber nerve endings. The relation of tachykinins, reactive oxygen species (ROS) and reactive nitrogen species (RNS) on electrical stimulation of thoracic vagus nerve (TVNS) or capsaicin-evoked neurogenic inflammation in respiratory tract of atropine-treated rats was not clear. In the present studies, the role of ROS and RNS on neurogenic inflammation were investigated in TVNS and capsaicin injected rats. The experiments were divided into two parts. In the first part, TVNS was performed by thoracotomy, non-cholinergic regulation of neurogenic plasma extravasation in the trachea and bronchi were examined, and whether TVNS via NK receptor facilitates neurogenic inflammation by nuclear factor-kappaB (NF-£eB) activation and ROS production were expored. Our results in this part showed that TVNS evoked substance P release, hypotension, bronchoconstriction (as shown by increases in smooth muscle electromyographic activity and total pulmonary resistance), trachea plasma extravasation as well as increases in blood O2- and H2O2 ROS amount in a frequency-dependent manner. Histopathological examination demonstrated silver-stained leaky venules, India-ink labeled plasma extravasation, and accumulations of inflammatory cells in the right lower trachea after TVNS. L-732138 (NK1 receptor antagonist), SR-48968 (NK2 receptor antagonist), dimethylthiourea (H2O2 scavenger) or catechins (O2- and H2O2 scavenger) pretreatment reduced TVNS-enhanced hypotension, bronchoconstriction, and plasma extravasation. TVNS upregulated the expression of NF-£eB in nuclear protein and intercellular adhesion molecule-1 (ICAM-1) in total protein of the lower respiratory tract tissue in a frequency-dependent manner. The upregulation of NF-£eB and ICAM-1 was attenuated by NK receptor antagonist and antioxidants. In the second part, the contribution of nitric oxide (NO) to capsaicin-evoked airway responses was investigated in rats. The measurement of plasma NO level, airway dynamics, airway smooth muscle electromyogram, and plasma extravasation by India ink and Evans blue leakage technique was adapted. Our results in this part showed that capsaicin injection evoked hypotension, bronchoconstriction, trachea plasma extravasation as well as increases in plasma NO level in a dose-dependent manner. L-732138 or SR-48968 pretreatment reduced capsaicin-enhanced hypotension, bronchoconstriction, plasma extravasation, and plasma NO level. Inhibition of a non-selective NO synthase (NOS) inhibitor (NG-nitro-L-Arginine methyl ester, L-NAME), or a selective inducible NO synthase (iNOS) inhibitor (aminoguanidine), reduced capsaicin-induced increases in plasma NO level and protected against capsaicin-induced plasma extravasation, whereas L-arginine (a NO precursor), enhances capsaicin-evoked plasma NO level and plasma extravasation. L-Arginine pretreatment ameliorated capsaicin-induced bronchoconstriction, whereas L-NAME and aminoguanidine exaggerated capsaicin-induced bronchoconstriction. In summary, both TVNS and capsaicin injection may increase oxidative stress responses. TVNS enhances proinflammatory NF-£eB and ICAM-1 expression, increases the production of O2- and H2O2 activity in the respiratory tract of atropine-treated rats. Pretreatment with antioxidants and selective NK receptor antagonists attenuate TVNS evoked airway hyperactivity, proinflammatory response, and oxidative stress. Capsaicin injection stimulates the release of tachykinins, which act on NK1 and NK2 receptors located on the smooth muscles of airways and blood vessels. The interaction of NK receptors with tachykinin enhances furtherly the NO formation, bronchoconstriction, vasodilation, and plasma extravasation in the trachea. The released tachykinins also increase the production of NO via iNOS, and iNOS -evoked NO counteracts tachykinin-mediated bronchoconstriction, but exacerbates tachykinin-mediated plasma extravasation.

Stimulation of C-fiber sensory neurons innervating the respiratory tract with electricity or capsaicin leads to the liberation of substance P, CGRP and other neuropeptides from the nerve terminals. Substance P (SP) binds to the NK-receptors on the membrane of vascular endothelial cells and elicits neurogenic inflammatory responses. These inflammatory responses include plasma leakage and the subsequent edema formation (Lundberg and Saria, 1983¡FMcDonald et al., 1988). Evans blue is a hydrophilic dye and is often used as a tracer of plasma leakage due to its¡¦ high affinity to the plasma proteins. Plasma leakage causes Evans blue extravasates from the blood vessels and remains in the tissues. The more plasma leaks from the blood vessels, the more Evans blue will extravasate into the tissues. Measuring extravasated Evans blue dye that is extracted from tissues, is useful for evaluation of the amount of plasma leakage. Potassium channel openers can inhibit neurogenic plasma leakage in the airways and urinary bladder (Hollywood et al., 1998). Evans blue directly stimulates large-conductance Ca2+-activated K+ channels in cultured endothelial cells of human umbilical vein (Wu et al., 1999). This suggests that it may influence the permeability of the microvessels in vivo. A previous study shows that Evans blue dye blocks capsaicin-induced cough and bronchospasm in the guinea pig (Bolaer et al., 1995). We postulated that pretreatment with Evans blue may influence the extent of neurogenic inflammation in the rat airways induced by the application of either SP or capsaicin. India ink was used as a colloidal tracer dye to label the leaky vessels. The present study investigated whether different concentration of Evans blue (0, 3, 15 and 30 mg/ml/kg) pretreatment could affect the plasma leakage and edema formation in rat lower airways in response to intravenous injection of either SP or capsaicin. The amount of plasma leakage was expressed by the area density of India ink-labeled leaky blood vessels. We also investigated whether Evans blue influenced the ultra-structural change in tracheal serous cells induced by intravenous injection of SP. Our results showed that pretreatment with high concentration of Evans blue reduced more than seven tenths of the area density of plasma leakage in the trachea caused by SP application (P<0.01); reduced more than seven tenths in the left main bronchus (P<0.01) and reduced about seven tenths in the right main bronchus (P<0.01), compared to the control group that received saline prior to SP. However, no statistical significance was observed in edema ratio between any two groups (P>0.05). In the neurogenic inflammation of the airways caused by injection of capsaicin, pretreatment with high concentration of Evans blue reduced more than seven tenths of the area density of plasma leakage in the trachea (P<0.01); reduced more than seven tenths in the left main bronchus (P<0.01) and reduced about seven tenths in the right main bronchus (P<0.01), respectively, compared to the control group that received saline prior to capsaicin. Pretreatment with high concentration of Evans blue prior to capsaicin also reduced more than eight-tenth in edema ratio (P<0.01). In the ultra-structure change of serous cells and the stastical analysis of the number of active serous per 1000£gm2 of tracheal epithelium, Evans blue pretreatment prior to SP significantly reduced the number of active serous cells by seven tenths (P<0.01) as compared to control group that received saline prior to SP. Therefore, we concluded that pretreatment with high concentration of Evans blue exerted its¡¦ effect by opening large-conductance Ca2+-activated K+ channels and inhibited the plasma leakage induced by SP or capsaicin. But no significant inhibition was observed in edema formation induced by SP application. Low concentration of Evans blue might enhance the neurogenic inflammation of the airway. Under the observation with SEM, we found that SP activated serous cells in airway epithelium, and high concentration of Evans blue pretreatment lowered the secretory activity of serous cells. Therefore Evans blue might inhibit the activation of serous cells.

¡iAbstract¡j Neurogenic inflammation is an acute inflammatory tissue response, that is mediated by sensory axon reflex. Accompanied with neurogenic inflammation, plasma extravasation, occurs in the eyes, esophagus, bladder, joints, the tip of tongue, and the respiratory tract of the mammal. Recently, many studies have investigated the neurogenic inflammation by electrical stimulation of nerves and intravascular injection of irritants. Upon stimulation, the sensory nerve endings in mucosa can release neuropeptides such as substance P, that causes formation of the venular endothelial gaps, plasma extravasation and tissue edema in various organs. Substance P also cause smooth muscle contraction and mucus secretion in the respiratory tract. Neurogenic plasma extravasation has been studied extensively in the trachea, and bronchi, but rarely in the esophagus. It is known that a plexus of substance P-immunoreactive axons exists in the mucosal and submucosal layers. They play an important role in releasing substance P to act on the receptors of the venular endothelium through diffusion. Based on plasma extravasation and other studies related to the respiratory tract, the purpose of the present study was to investigate neurogenic inflammatory response in the esophagus of the digestive tract. In this study, capsaicin (90 &#x00B5;g/ml/kg) and substance P (3 &#x00B5;g/ml/kg) were used as the irritant and inflammatory mediator, respectively to reduce neurogenic inflammation in the esophagus. India ink was used to label the affected venules. The magnitude of the neurogenic inflammation was expressed as area density of India ink-labeled leaky venules. Histopathological changes in the esophageal tissue were studied under the light microscope. The result of this study indicated that capsaicin at the dose of (90 &#x00B5;g/ml/kg) and substance P at the dose of (3 &#x00B5;g/ml/kg) caused similar magnitude of inflammation in the esophagus. India ink-labeled venules distributed like a network in the mucosal tissue and in connective tissue of the submucosal layer. The upper, middle and lower parts of esophagus exhibited the same degree of inflammatory response, that was similar to that in the lower respiratory tract as the previous studies reported. These results suggest that nerve branches from the vagal trunk send sensory axons to innervate both the esophagus and airways.

Thèse (M.Sc.)--Université Laval, 2008. / Bibliogr.: f. [69]-77. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Background: The cornea is the outermost transparent layer of the eye and it is responsible for the majorityof the eye’s total focusing power. Keratocytes are the resident cells of the corneal stroma and their function isto produce extracellular matrix components and to take part in corneal healing after injury, which may occurdue to trauma, infection or surgery. The process of corneal wound healing is complex. Shortly, keratocytesadjacent to the corneal wound undergo apoptosis and remaining cells start the process of proliferation andmigration in order to close the wound. Next, an influx of inflammatory cells such as macrophages andneutrophils occurs in order to clear the cornea from cellular debris. The final stage of the healing processrestores the quiescent state of keratocytes and remodels any disordered extracellular matrix components,leading to a healthy, transparent cornea. However, when the process of corneal wound healing is incompleteor disturbed, corneal scarring may occur, which can lead to significantly impaired vision. Despite extensiveresearch on corneal wound healing, corneal scarring remains a major cause of preventable blindness. Thehealing process is dependent on various cytokines and growth factors. However, it is possible that also othersignal substances are involved. Substance P (SP) is a neuropeptide well known for its role in pain perception.It has been shown that SP can also be produced by non-neuronal cells, including cells of the cornea, and thatit can have vast effects on physiological functions, including immune cell activity, and cellular processes, suchas cell migration, proliferation, and production of proinflammatory cytokines. Similarly, acetylcholine (ACh),a classical neurotransmitter, has also been reported to be produced by non-neuronal cells, including cornealepithelium, and to be involved in cell proliferation, angiogenesis, cell migration, apoptosis, and collagen geneexpression. In the studies of this thesis, it is hypothesized that neuropeptides and neurotransmitters areproduced by human keratocytes and that this production is increased in response to corneal injury. Moreover,it is hypothesized that the non-neuronal SP and ACh produced by injured keratocytes participate in cornealwound healing by enhancing keratocyte migration and proliferation, and/or by decreasing keratocyteapoptosis. The aims of this thesis project were to test these hypotheses and to study the underlying inter- andintracellular mechanisms of the effects of SP and ACh on keratocytes.Results: Cultured primary cells of the human corneal stroma expressed keratocyte markers (keratocan,lumican, CD34, and ALDH), the tachykinins SP and NKA, catecholamines (adrenaline, noradrenaline anddopamine), ACh, and glutamate. Moreover, the cells expressed neurokinin-1 and -2 receptors (NK-1R andNK-2R), dopamine receptor D2, muscarinic ACh receptors (mAChRs) M1, M3, M4 and M5, and NDMAR1glutamate receptor. Significant differences were observed between expression profiles in cultured keratocytesobtained from central and peripheral cornea. Such differences could also be seen between keratocytescultured under various serum concentrations. Expression and secretion of SP in cultured keratocytes wasincreased in response to injury in vitro. SP enhanced migration of cultured keratocytes through stimulation ofits preferred receptor, the NK-1R, and activation of the phosphatidylinositide 3-kinase and Rac1/RhoApathway and subsequent actin cytoskeleton reorganization and formation of focal adhesion points. Moreover,SP stimulation led to upregulated expression of the proinflammatory and chemotactic cytokine interleukin-8(IL-8), which also contributed significantly to SP-enhanced keratocyte migration and to attractingneutrophils. ACh enhanced keratocyte proliferation in vitro at low concentrations and this stimulation wasmediated through activation of mAChRs and activation of MAPK signalling. Moreover, ACh stimulation led toupregulation of two proliferation markers: PCNA and Ki-67. ACh was also able to protect cultured keratocytesfrom Fas-induced apoptosis, even at low concentrations. Activation of mAChRs was necessary for this latterprocess to occur. ACh reduced caspases 3/7 activation in Fas-treated keratocytes. Inhibition of the PKB/Aktpathway revealed that its activation is essential for mediating the anti-apoptotic effect of ACh in keratocytes.Conclusions: This thesis shows that human keratocytes express an array of neuropeptides (SP, NKA) andneurotransmitters (ACh, adrenaline, noradrenaline, dopamine and glutamate), and their receptors, and thatstimulation of NK-1R by SP and stimulation of mAChRs by ACh lead to keratocyte cellular processes that areknown to be involved in corneal wound healing. Specifically, SP enhances keratocyte migration throughupregulation of IL-8, ACh enhances keratocyte proliferation through activation of the MAPK signallingpathway, and ACh is able to protect keratocytes from apoptosis by activation of the PKB/Akt pathway. Takentogether, these findings suggest that both SP and ACh, if entered at the proper stage, could be beneficial forcorneal wound healing.

Copies of author's previously published articles inserted. / Amendments (2 leaves) in front cover pocket. / Bibliography: leaves 227-259. / xiii, 259 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the contribution of tachykinins, in particular Substance P (SP), to the processes of inflammation. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1993