Key clinical features of cumulative trauma disorders include pain, muscle weakness, and tissue fibrosis, although the etiology is still under investigation. Therefore, we first sought to characterize the temporal pattern of altered sensorimotor behaviors and inflammatory and fibrogenic processes occurring in forearm muscles and serum of young adult, female rats performing an operant, high repetition high force (HRHF) reaching and grasping task for 6, 12, or 18 weeks. Palmar mechanical sensitivity, cold temperature avoidance and spontaneous behavioral changes increased, while grip strength declined, in 18-week HRHF rats, compared to controls. Flexor digitorum muscles had increased MCP-1 levels after training and increased TNFα in 6-week HRHF rats. Serum had increased IL-1β, IL-10 and IP-10 after training. Yet both muscle and serum inflammation resolved by week 18. In contrast, IFNg increased at week 18 in both muscle and serum. Given the anti-fibrotic role of IFNg, and to identify a mechanism for the continued grip strength losses and behavioral sensitivities, we evaluated the fibrogenic proteins CCN2, collagen type I and TGFß-1, as well as the nociceptive/fibrogenic peptide substance P. Each increased in and around flexor digitorum muscles and extracellular matrix in the mid-forearm, and in nerves of the forepaw at 18 weeks. CCN2 was also increased in serum at week 18. At a time when inflammation had subsided, increases in fibrogenic proteins correlated with sensorimotor declines. Thus, muscle and nerve fibrosis may be critical components of chronic work-related musculoskeletal disorders. CCN2 and substance P may serve as potential targets for therapeutic intervention, and CCN2 as a serum biomarker of fibrosis progression. TGFß-1 and CCN2 are important mediators of tissue fibrosis by their stimulatory effect on extracellular matrix deposition, with CCN2 functions as a downstream mediator of TGFß-1. Substance P (SubP), a nociceptor-related neuropeptide, has also been linked to tissue fibrosis, although little work has been done to understand whether SubP directly causes fibrotic responses in tenocytes. Therefore, we sought to determine if SubP induces fibroblast proliferation and collagen production via CCN2 signaling directly or through the TGFß-1/CCN2 signaling pathway. We hypothesized that SubP may act directly through CCN2, independently from the TGFß-1/CCN2 signaling pathway, to increase fibroblast proliferation and fibrogenic and extracellular matrix protein production in vitro. To examine this question, we assayed cell proliferation and production of CCN2, TGFB1 and collagen type 1 in vitro using primary tendon fibroblasts (tenocytes) isolated from flexor digitorum tendons, and using rat dermal fibroblasts (RDF). We observed that cells isolated from flexor digitorum tendons that express proteins characteristic of tenocytes (vimentin and tenomodulin) underwent increased proliferation in a dose dependent manner after TGFß-1 treatment, but not SubP treatment, as did RDF cells. TGFß-1 treatment increased CCN2 production in both tenocytes and RDF cells, while SubP induced CCN2 production only in rat tenocytes. Expectedly, TGFß-1 treatment increased collagen expression in each cell type, as did SubP treatment alone using In-cell Western analysis. Interestingly, preliminary data that needs to be repeated showed that SubP treatment of each cell type enhanced TGFß-1 expression, assayed using In-cell Western and traditional western blot analyses. Our findings suggest that both SubP and TGFß-1 have distinct fibrogenic actions on tenocytes and dermal fibroblast and that both may be involved in tendinosis observed in animal models and patients with fibrosis. Inflammatory pain, muscle weakness, and tissue fibrosis are key clinical features of work-related musculoskeletal disorders. So, lastly, we evaluated the effects of therapeutic interventions on behavioral and cytokine changes in muscle, tendon and serum of HRHF rats that performed the reaching and grasping task for 11 weeks. We compared sensorimotor behavioral changes, and flexor digitorum tissue inflammation and fibrosis in rats receiving anti-TNFα therapy prophylactically during the initial training, or anti-TNFα therapy with or without rest as secondary interventions during the HRHF work task. Untreated or saline only treated animals at the end of the initial training period had decreased grip strength, increased mechanical sensitivity, and increased serum and tissue inflammatory cytokines (TNFα, IL-1ß, IL-6 and VEGF), changes prevented by prophylactic anti-TNFα treatment. Regarding the secondary interventions, four weeks of anti-TNFα therapy with or without rest, provided in HRHF task weeks 4-7, was more effective than rest alone for restoring grip strength; no treatments rescued forepaw mechanical sensitivity. Effectiveness of the 4-week anti-TNFα therapy extended to week 11, despite no further drug treatment after week 7, for maintenance of grip strength. Tissue cytokine analysis in week 11 showed that HRHF rats treated with saline had increased IL-18 in serum, muscle and tendon, and trends for increased muscle CCN2. Each treatment, particularly anti-TNF with or without rest, decreased serum and tendon IL-18 and IL-1alpha. Rats receiving combined rest and anti-TNFα therapy also had increased serum IL-10. Thus, similar short-term anti-TNFα therapy may be a potential intervention in WMSDs. These results demonstrate that both Substance P and CCN2 play important roles in the development of fibrosis in muscle and tendon in WMSDs based on our model of repetition reaching and grasping. Using in vitro methods, it was demonstrated that substance P is capable of inducing CCN2 in isolated tenocytes and rat dermal fibroblasts, independent of TGFß-1 signaling, a novel discovery that make suggest new treatments for fibrotic disorders. Finally, anti-TNFalpha treatment successfully prevented behavioral declines and increases in IL-18 in serum and tissues in our rat model when provided during the course of HRHF task performance. Key clinical features of cumulative trauma disorders include pain, muscle weakness, and tissue fibrosis, although the etiology is still under investigation. Therefore, we first sought to characterize the temporal pattern of altered sensorimotor behaviors and inflammatory and fibrogenic processes occurring in forearm muscles and serum of young adult, female rats performing an operant, high repetition high force (HRHF) reaching and grasping task for 6, 12, or 18 weeks. Palmar mechanical sensitivity, cold temperature avoidance and spontaneous behavioral changes increased, while grip strength declined, in 18-week HRHF rats, compared to controls. Flexor digitorum muscles had increased MCP-1 levels after training and increased TNFα in 6-week HRHF rats. Serum had increased IL-1β, IL-10 and IP-10 after training. Yet both muscle and serum inflammation resolved by week 18. In contrast, IFNg increased at week 18 in both muscle and serum. Given the anti-fibrotic role of IFNg, and to identify a mechanism for the continued grip strength losses and behavioral sensitivities, we evaluated the fibrogenic proteins CCN2, collagen type I and TGFß-1, as well as the nociceptive/fibrogenic peptide substance P. Each increased in and around flexor digitorum muscles and extracellular matrix in the mid-forearm, and in nerves of the forepaw at 18 weeks. CCN2 was also increased in serum at week 18. At a time when inflammation had subsided, increases in fibrogenic proteins correlated with sensorimotor declines. Thus, muscle and nerve fibrosis may be critical components of chronic work-related musculoskeletal disorders. CCN2 and substance P may serve as potential targets for therapeutic intervention, and CCN2 as a serum biomarker of fibrosis progression. TGFß-1 and CCN2 are important mediators of tissue fibrosis by their stimulatory effect on extracellular matrix deposition, with CCN2 functions as a downstream mediator of TGFß-1. Substance P (SubP), a nociceptor-related neuropeptide, has also been linked to tissue fibrosis, although little work has been done to understand whether SubP directly causes fibrotic responses in tenocytes. Therefore, we sought to determine if SubP induces fibroblast proliferation and collagen production via CCN2 signaling directly or through the TGFß-1/CCN2 signaling pathway. We hypothesized that SubP may act directly through CCN2, independently from the TGFß-1/CCN2 signaling pathway, to increase fibroblast proliferation and fibrogenic and extracellular matrix protein production in vitro. To examine this question, we assayed cell proliferation and production of CCN2, TGFB1 and collagen type 1 in vitro using primary tendon fibroblasts (tenocytes) isolated from flexor digitorum tendons, and using rat dermal fibroblasts (RDF). We observed that cells isolated from flexor digitorum tendons that express proteins characteristic of tenocytes (vimentin and tenomodulin) underwent increased proliferation in a dose dependent manner after TGFß-1 treatment, but not SubP treatment, as did RDF cells. TGFß-1 treatment increased CCN2 production in both tenocytes and RDF cells, while SubP induced CCN2 production only in rat tenocytes. Expectedly, TGFß-1 treatment increased collagen expression in each cell type, as did SubP treatment alone using In-cell Western analysis. Interestingly, preliminary data that needs to be repeated showed that SubP treatment of each cell type enhanced TGFß-1 expression, assayed using In-cell Western and traditional western blot analyses. Our findings suggest that both SubP and TGFß-1 have distinct fibrogenic actions on tenocytes and dermal fibroblast and that both may be involved in tendinosis observed in animal models and patients with fibrosis. Inflammatory pain, muscle weakness, and tissue fibrosis are key clinical features of work-related musculoskeletal disorders. So, lastly, we evaluated the effects of therapeutic interventions on behavioral and cytokine changes in muscle, tendon and serum of HRHF rats that performed the reaching and grasping task for 11 weeks. We compared sensorimotor behavioral changes, and flexor digitorum tissue inflammation and fibrosis in rats receiving anti-TNFα therapy prophylactically during the initial training, or anti-TNFα therapy with or without rest as secondary interventions during the HRHF work task. Untreated or saline only treated animals at the end of the initial training period had decreased grip strength, increased mechanical sensitivity, and increased serum and tissue inflammatory cytokines (TNFα, IL-1ß, IL-6 and VEGF), changes prevented by prophylactic anti-TNFα treatment. Regarding the secondary interventions, four weeks of anti-TNFα therapy with or without rest, provided in HRHF task weeks 4-7, was more effective than rest alone for restoring grip strength; no treatments rescued forepaw mechanical sensitivity. Effectiveness of the 4-week anti-TNFα therapy extended to week 11, despite no further drug treatment after week 7, for maintenance of grip strength. Tissue cytokine analysis in week 11 showed that HRHF rats treated with saline had increased IL-18 in serum, muscle and tendon, and trends for increased muscle CCN2. Each treatment, particularly anti-TNF with or without rest, decreased serum and tendon IL-18 and IL-1alpha. Rats receiving combined rest and anti-TNFα therapy also had increased serum IL-10. Thus, similar short-term anti-TNFα therapy may be a potential intervention in WMSDs. These results demonstrate that both Substance P and CCN2 play important roles in the development of fibrosis in muscle and tendon in WMSDs based on our model of repetition reaching and grasping. Using in vitro methods, it was demonstrated that substance P is capable of inducing CCN2 in isolated tenocytes and rat dermal fibroblasts, independent of TGFß-1 signaling, a novel discovery that make suggest new treatments for fibrotic disorders. Finally, anti-TNFalpha treatment successfully prevented behavioral declines and increases in IL-18 in serum and tissues in our rat model when provided during the course of HRHF task performance. / Cell Biology

Le globus pallidus (GP) des primates reçoit une innervation massive des neurones GABAergiques du striatum qui co-libèrent la substance P (SP). Afin d'approfondir notre connaissance de l'interaction de la SP au niveau pallidal, nous avons étudié la localisation cellulaire et subcellulaire de ce peptide et de ses récepteurs à hautes affinités neurokinine-1 (NK-lR) et neurokinine-3 (NK-3R) dans le GP de singes écureuils. Un grand nombre de neurones et de fibres dans le segment externe (GPe) et interne (GPi) du GP exprimaient de l'immunoréactivité pour NK-lR ou NK-3R en position pré- et postsynaptique. Les NK-lR et NK-3R étaient principalement localisés dans le cytoplasme ou sur la membrane plasmique, mais en dehors des jonctions synaptiques. Certaines terminaisons axonaies immunoréactives pour la SP exprimaient préférentiellement NK-3R. Ces données suggèrent que la SP peut influencer de manière significative le traitement de l'information neuronale voyageant à travers les ganglions de la base. / The primate globus pallidus (GP) receives a massive innervation from GABAergic striatal neurons that co-release substance P (SP). We used single and double antigen staining retrieval methods to study cellular and subcellular localization of SP and its high affinity receptors NK-IR and NK-3R in GP of squirrel monkeys. A large number of neurons and fibers in GPe and GPi expressed NK-IR or NK-3R. NK-IR and NK-3R were mainly associated with intracellular sites or located at extrasynaptic positions on the plasma membrane. SP+ axon terminals preferentially expressed NK-3R. Distribution of NK-IR and NK-3R indicates that SP effects at pallidal level are mediated through postsynaptic receptor as well as presynaptic autoreceptors and heteroreceptors. These data suggest that SP may influence in a significant manner the treatment of neural information that flows through the basal ganglia.

<p>Anabolic-androgenic steroids (AAS) have been used in clinics for decades. The misuse of AAS has previously been attributed merely to sport athletes, taking AAS with intentions to increase muscle mass, enhance physical performance and to improve results in competitions. Today, the misuse of AAS has spread to adolescents and young adults not connected to sports. Alarmingly, many reports are pointing at severe psychiatric adverse effects among AAS abusers, which include mood swings, mania, anxiety, depression and aggression. Numerous examples of severe and often unprovoked violence and brutal crimes have been connected to AAS abuse and there is a strong need for a better understanding of the underlying biochemical events that might account for the adverse behaviors induced by AAS. The general aim of this thesis was to study the effect of chronic AAS administration on neuropeptide circuits in the rat brain associated with the regulation of rewarding effects, memory, anxiety, depression and aggression, using nandrolone decanoate as a prototype AAS.</p><p>Results demonstrated that daily administration of AAS to rats in doses comparable to those taken by AAS abusers, in certain brain structures significantly affected, <i>a</i>) the levels of the opioid peptides dynorphin B and Met-enkephalin-Arg⁶Phe⁷, <i>b</i>) the levels of the tachykinin substance P (SP), <i>c</i>) the density of the SP neurokinin 1 (NK1) receptor, <i>d</i>) the level of the SP metabolite SP_1-7that frequently exerts opposite effects to SP, <i>e</i>) the SP_1-7generating enzyme substance P endopeptidase (SPE) and finally, <i>f</i>) the levels of the neuropeptide calcitonin gene-related peptide (CGRP) often co-localized with SP. The alterations seen in the levels and activities of these neurochemical components are in many aspects compatible with behaviors typified among AAS abusers.</p>

Anabolic-androgenic steroids (AAS) have been used in clinics for decades. The misuse of AAS has previously been attributed merely to sport athletes, taking AAS with intentions to increase muscle mass, enhance physical performance and to improve results in competitions. Today, the misuse of AAS has spread to adolescents and young adults not connected to sports. Alarmingly, many reports are pointing at severe psychiatric adverse effects among AAS abusers, which include mood swings, mania, anxiety, depression and aggression. Numerous examples of severe and often unprovoked violence and brutal crimes have been connected to AAS abuse and there is a strong need for a better understanding of the underlying biochemical events that might account for the adverse behaviors induced by AAS. The general aim of this thesis was to study the effect of chronic AAS administration on neuropeptide circuits in the rat brain associated with the regulation of rewarding effects, memory, anxiety, depression and aggression, using nandrolone decanoate as a prototype AAS. Results demonstrated that daily administration of AAS to rats in doses comparable to those taken by AAS abusers, in certain brain structures significantly affected, a) the levels of the opioid peptides dynorphin B and Met-enkephalin-Arg6Phe7, b) the levels of the tachykinin substance P (SP), c) the density of the SP neurokinin 1 (NK1) receptor, d) the level of the SP metabolite SP1-7 that frequently exerts opposite effects to SP, e) the SP1-7 generating enzyme substance P endopeptidase (SPE) and finally, f) the levels of the neuropeptide calcitonin gene-related peptide (CGRP) often co-localized with SP. The alterations seen in the levels and activities of these neurochemical components are in many aspects compatible with behaviors typified among AAS abusers.

A substância P (SP) é um neuropeptídeo da família das taquicininas que regula numerosas funções biológicas por meio da ligação ao seu receptor altamente específico neuroquinina-1 (NK-1R). Este complexo SP/NK-1R está envolvido em diversos processos relacionados à oncogênese, como a mitogênese, angiogênese, migração celular e metástase. O objetivo deste trabalho foi investigar a expressão de substância P e de seu receptor NK-1 e sua correlação com o índice de proliferação celular em 73 pacientes portadores de 90 carcinomas espinocelulares de boca, diagnosticados e tratados no Hospital General e Hospital de La Princesa, Jaen, Espanha, durante o período de 1995 a 2008. Todos os tumores foram corados pela técnica imunoistoquímica da estreptavidina-biotina-peroxidase com os anticorpos anti-SP, anti-NK-1R e anti-Ki-67. As alterações celulares epiteliais das margens cirúrgicas livres de doença também foram registradas. A expressão imunoistoquímica da substância P e do seu receptor neurokinina-1 foi avaliada na membrana, no citoplasma e no núcleo das células epiteliais malignas e do epitélio da mucosa bucal adjacente ao tumor, nos linfócitos e nos vasos sanguíneos dos tumores. O índice de proliferação celular tumoral foi determinado pela expressão imunoistoquímica de Ki-67 identificada no núcleo das células neoplásicas. As correlações entre as diversas localizações da SP, de seu receptor NK-1R e do índice de proliferação tumoral determinado pelo Ki-67 foram determinadas estatísticamente utilizando-se o Crosstab, Regress e Descript de SUDAAN. A expressão de SP foi identificada no estroma de 77% dos tumores, na membrana de 71% das células malignas e no citoplasma de 81,2% dos tumores. A maioria dos tumores apresentou altas taxas de proliferação das células neoplásicas com mais de 50% das células imunopositivas para o Ki-67. Ao analisar as margens cirúrgicas livres de doença, observou-se expressão da SP, sobretudo no terço inferior e médio, tanto no núcleo, como no citoplasma e na membrana celular. A expressão concomitante de substância P e do receptor neurokinina-1 no citoplasma das células neoplásicas ocorreu mais frequentemente nas células tumorais em proliferação. Verificou-se que expressão de SP no câncer bucal ocorre juntamente com o aumento da expressão de NK-1R, sugerindo que as células neoplásicas epiteliais bucais podem utilizar esta via para tornarem-se mais susceptíveis aos estímulos mediados pela SP. A expressão de substância P nos linfócitos do infiltrado inflamatório e vasos sanguíneos intratumorais e peritumorais se associaram a tumores de menor tamanho, menor estádio clínico e com menor frequência metástase ganglionar. Concluiu-se que as células neoplásicas epiteliais bucais podem utilizar a via substância P/NK-1R para tornarem-se mais susceptíveis aos estímulos mediados pela SP, particularmente aqueles associados à proliferação celular. Além disso, a expressão epitelial, citoplasmática e nuclear da substância P é um evento precoce na carcinogênese bucal podendo ser considerado um marcador da presença e intensidade de displasia epitelial. / The substance P (SP) is a neuropeptide of the tachykinin family that regulates multiple biological functions by binding to the highly specific receptor neurokinin-1. This complex SP/NK-1 is involved in several processes related to oncogenesis, such as mitogenesis, angiogenesis, cell migration and metastasis. This study investigated the expression of substance P and its receptor NK-1 and its correlation with the cell proliferation index in 73 patients with oral squamous cell carcinoma, diagnosed and treated at the General Hospital and Princess Hospital at Jaen, Spain, during the period 1995 to 2008. All tumors were stained immunohistochemically by the streptavidin-biotin-peroxidase technique using the antibodies anti-SP, anti-NK-1R and anti-Ki-67. The epithelial cell alterations on the disease-free surgical margins were registered. The immunohistochemical expression of SP and its receptor neurokinin-1 were evaluated on the membrane, cytoplasm and nucleus of malignant epithelial cells and cells of healthy oral mucosa adjacent to the tumor, as well as on the infiltrating lymphocytes and peritumoral or intratumoral blood vessels. The tumor cell proliferation index was determined by the immunohistochemical expression of Ki- 67 identified on the malignant cell nucleus. The correlations between the distinct localizations of SP, its receptor NK-1 and the proliferation index Ki-67 were statistically analyzed using the Sudaan Crosstab, Regress and Descript tests. The SP expression was identified on the stroma of 77% of tumors, on the membrane of 71% of malignant cells and cytoplasm of 81.2% of tumors. Most tumors presented high proliferation rates of neoplastic cells, with more than 50% of cells immunopositive for Ki-67. Analysis of the disease-free surgical margins revealed SP expression especially on the lower and medium third, both on the nucleus, cytoplasm and cell membrane. The simultaneous expression of substance P and its receptor NK-1 on the cytoplasm of neoplastic cells occurred more frequently in proliferating malignant cells. The expression of SP in oral cancer occurred simultaneously to an increased expression of NK-1R, suggesting that the oral malignant epithelial cells might use this pathway to become more susceptible to the stimuli mediated by the SP. The substance P expression on infiltrating lymphocytes and intratumoral or peritumoral blood vessels was associated with tumors of small size, lower clinical stage and less frequent node metastasis. It was concluded that the oral neoplastic epithelial cells may use the pathway SP/NK-1R to become more susceptible to the stimuli mediated by the SP, particularly those associated with cell proliferation. Additionally, the epithelial, cytoplasmic and nuclear expression of substance P is an early event in oral carcinogenesis and may be considered a marker of the presence and intensity of epithelial dysplasia.

A substância P (SP) é um neuropeptídeo da família das taquicininas que regula numerosas funções biológicas por meio da ligação ao seu receptor altamente específico neuroquinina-1 (NK-1R). Este complexo SP/NK-1R está envolvido em diversos processos relacionados à oncogênese, como a mitogênese, angiogênese, migração celular e metástase. O objetivo deste trabalho foi investigar a expressão de substância P e de seu receptor NK-1 e sua correlação com o índice de proliferação celular em 73 pacientes portadores de 90 carcinomas espinocelulares de boca, diagnosticados e tratados no Hospital General e Hospital de La Princesa, Jaen, Espanha, durante o período de 1995 a 2008. Todos os tumores foram corados pela técnica imunoistoquímica da estreptavidina-biotina-peroxidase com os anticorpos anti-SP, anti-NK-1R e anti-Ki-67. As alterações celulares epiteliais das margens cirúrgicas livres de doença também foram registradas. A expressão imunoistoquímica da substância P e do seu receptor neurokinina-1 foi avaliada na membrana, no citoplasma e no núcleo das células epiteliais malignas e do epitélio da mucosa bucal adjacente ao tumor, nos linfócitos e nos vasos sanguíneos dos tumores. O índice de proliferação celular tumoral foi determinado pela expressão imunoistoquímica de Ki-67 identificada no núcleo das células neoplásicas. As correlações entre as diversas localizações da SP, de seu receptor NK-1R e do índice de proliferação tumoral determinado pelo Ki-67 foram determinadas estatísticamente utilizando-se o Crosstab, Regress e Descript de SUDAAN. A expressão de SP foi identificada no estroma de 77% dos tumores, na membrana de 71% das células malignas e no citoplasma de 81,2% dos tumores. A maioria dos tumores apresentou altas taxas de proliferação das células neoplásicas com mais de 50% das células imunopositivas para o Ki-67. Ao analisar as margens cirúrgicas livres de doença, observou-se expressão da SP, sobretudo no terço inferior e médio, tanto no núcleo, como no citoplasma e na membrana celular. A expressão concomitante de substância P e do receptor neurokinina-1 no citoplasma das células neoplásicas ocorreu mais frequentemente nas células tumorais em proliferação. Verificou-se que expressão de SP no câncer bucal ocorre juntamente com o aumento da expressão de NK-1R, sugerindo que as células neoplásicas epiteliais bucais podem utilizar esta via para tornarem-se mais susceptíveis aos estímulos mediados pela SP. A expressão de substância P nos linfócitos do infiltrado inflamatório e vasos sanguíneos intratumorais e peritumorais se associaram a tumores de menor tamanho, menor estádio clínico e com menor frequência metástase ganglionar. Concluiu-se que as células neoplásicas epiteliais bucais podem utilizar a via substância P/NK-1R para tornarem-se mais susceptíveis aos estímulos mediados pela SP, particularmente aqueles associados à proliferação celular. Além disso, a expressão epitelial, citoplasmática e nuclear da substância P é um evento precoce na carcinogênese bucal podendo ser considerado um marcador da presença e intensidade de displasia epitelial. / The substance P (SP) is a neuropeptide of the tachykinin family that regulates multiple biological functions by binding to the highly specific receptor neurokinin-1. This complex SP/NK-1 is involved in several processes related to oncogenesis, such as mitogenesis, angiogenesis, cell migration and metastasis. This study investigated the expression of substance P and its receptor NK-1 and its correlation with the cell proliferation index in 73 patients with oral squamous cell carcinoma, diagnosed and treated at the General Hospital and Princess Hospital at Jaen, Spain, during the period 1995 to 2008. All tumors were stained immunohistochemically by the streptavidin-biotin-peroxidase technique using the antibodies anti-SP, anti-NK-1R and anti-Ki-67. The epithelial cell alterations on the disease-free surgical margins were registered. The immunohistochemical expression of SP and its receptor neurokinin-1 were evaluated on the membrane, cytoplasm and nucleus of malignant epithelial cells and cells of healthy oral mucosa adjacent to the tumor, as well as on the infiltrating lymphocytes and peritumoral or intratumoral blood vessels. The tumor cell proliferation index was determined by the immunohistochemical expression of Ki- 67 identified on the malignant cell nucleus. The correlations between the distinct localizations of SP, its receptor NK-1 and the proliferation index Ki-67 were statistically analyzed using the Sudaan Crosstab, Regress and Descript tests. The SP expression was identified on the stroma of 77% of tumors, on the membrane of 71% of malignant cells and cytoplasm of 81.2% of tumors. Most tumors presented high proliferation rates of neoplastic cells, with more than 50% of cells immunopositive for Ki-67. Analysis of the disease-free surgical margins revealed SP expression especially on the lower and medium third, both on the nucleus, cytoplasm and cell membrane. The simultaneous expression of substance P and its receptor NK-1 on the cytoplasm of neoplastic cells occurred more frequently in proliferating malignant cells. The expression of SP in oral cancer occurred simultaneously to an increased expression of NK-1R, suggesting that the oral malignant epithelial cells might use this pathway to become more susceptible to the stimuli mediated by the SP. The substance P expression on infiltrating lymphocytes and intratumoral or peritumoral blood vessels was associated with tumors of small size, lower clinical stage and less frequent node metastasis. It was concluded that the oral neoplastic epithelial cells may use the pathway SP/NK-1R to become more susceptible to the stimuli mediated by the SP, particularly those associated with cell proliferation. Additionally, the epithelial, cytoplasmic and nuclear expression of substance P is an early event in oral carcinogenesis and may be considered a marker of the presence and intensity of epithelial dysplasia.

<p>The purification and biochemical characterization of the substance P (SP) hydrolyzing enzyme, substance P endopeptidase (SPE), have been carried out; with subsequent orientation in neurobiological fundamental processes involved in opioid dependence, withdrawal, and heat-stress.</p><p>SPE was purified from rat spinal cord, human spinal cord and cerebrospinal fluid (CSF), rat ventral tegemental area (VTA), and rat hippocampus. The enzyme activity was found to release the biologically active fragments SP(1-7) and SP(1-8) as major products. The purified enzymes were characterized with regard to their biochemical and kinetic properties. The typical SPE is neither inhibited by phosphoramidon nor captopril nor phenylmethanesulfonylflourid (PMSF). In comparison to other known proteases SPE differed in characteristics regarding substrate specificity, inhibition-profile, cleavage pattern, and other kinetic parameters. The technically very delicate approach of micro purification of SPE from the rat ventral tegemental area (VTA) (this is a very small tissue), turned out to be possible with the ÄKTA™-purifier system. Studies revealed a crucial role of SPE in a series of clinically important neuropathological conditions, such as opioid tolerance, and withdrawal (SPE, increased); and heat-stress (SPE, increased). These findings emerged from assessment of enzyme activity in hypothalamus, nucleus accumbens (NAc) periaqueductal gray (PAG), pituitary, striatum, substantia nigra (SN), VTA, spinal cord. Viewing the role of SPE in morphine tolerance, it was possible to note regional differences with a decrease in PAG, and striatum, whereas an increase was seen in SN, and VTA. After heat-stress treatment, SPE was raised in several regions (cerebral cortex, hippocampus, diencephalon, cerebellum, spinal cord), and the most precise observation of this was located to the hippocampus structure.</p>

The purification and biochemical characterization of the substance P (SP) hydrolyzing enzyme, substance P endopeptidase (SPE), have been carried out; with subsequent orientation in neurobiological fundamental processes involved in opioid dependence, withdrawal, and heat-stress. SPE was purified from rat spinal cord, human spinal cord and cerebrospinal fluid (CSF), rat ventral tegemental area (VTA), and rat hippocampus. The enzyme activity was found to release the biologically active fragments SP(1-7) and SP(1-8) as major products. The purified enzymes were characterized with regard to their biochemical and kinetic properties. The typical SPE is neither inhibited by phosphoramidon nor captopril nor phenylmethanesulfonylflourid (PMSF). In comparison to other known proteases SPE differed in characteristics regarding substrate specificity, inhibition-profile, cleavage pattern, and other kinetic parameters. The technically very delicate approach of micro purification of SPE from the rat ventral tegemental area (VTA) (this is a very small tissue), turned out to be possible with the ÄKTA™-purifier system. Studies revealed a crucial role of SPE in a series of clinically important neuropathological conditions, such as opioid tolerance, and withdrawal (SPE, increased); and heat-stress (SPE, increased). These findings emerged from assessment of enzyme activity in hypothalamus, nucleus accumbens (NAc) periaqueductal gray (PAG), pituitary, striatum, substantia nigra (SN), VTA, spinal cord. Viewing the role of SPE in morphine tolerance, it was possible to note regional differences with a decrease in PAG, and striatum, whereas an increase was seen in SN, and VTA. After heat-stress treatment, SPE was raised in several regions (cerebral cortex, hippocampus, diencephalon, cerebellum, spinal cord), and the most precise observation of this was located to the hippocampus structure.

This thesis is on chronic neck pain after a rear impact car injury, so called whiplash associated disorder (WAD). Three empirical studies using positron emission tomography (PET) with different radioligands have been performed. The first study evaluated resting state regional cerebral blood flow (rCBF) in WAD patients and in healthy, pain-free controls, by use of oxygen-15 labeled water. Patients had heightened resting rCBF bilaterally in the posterior parahippocampal and the posterior cingulate gyri, in the right thalamus and in the right medial prefrontal gyrus. Attenuated tempero-occipital blood flow was also observed in the patient group as compared to healthy controls. Alterations in rCBF were related to patients’ neck disability ratings. Study I suggests an involvement of the posterior cingulate, the parahippocampal and the medial prefrontal gyri in WAD. This altered resting state neural activity may be linked to an increased self-relevant evaluation of pain and stress. The second study evaluated central expression of the neurokinin-1 (NK1) receptor in WAD patients and healthy controls. Using a carbon-11 labeled specific NK1 antagonist, the receptor availability was measured. Patients displayed lowered NK1 receptor availability in the insula, anterior cingulate, frontal lobe, hippocampus, amygdala and in the periaqueductal gray matter, consistent with results from animal models of chronic pain. NK1 receptor availability was most reduced in the ventromedial orbitofrontal cortex, where attenuations were linearly related to patients fear and avoidance of movement. Thirdly, carbon-11 labeled D-deprenyl was used to investigate the presence of locally inflamed soft tissue in the cervical neck in WAD patients. Although the retention mechanism of [11C]D-deprenyl is not known, the results suggest that WAD patients have chronic inflammatory processes in the neck, most commonly in the adipose tissue at the spineous process of the second vertebra. In summary, this thesis provides evidence for altered central blood flow and receptor characteristics in WAD patients. Further, WAD patients may also have signs of persistent peripheral tissue damage. Both central and peripheral pain mechanisms have been demonstrated and visualized in patients with whiplash associated disorder.

Neuropeptides, such as substance P (SP) and bombesin/gastrin-releasing peptide (BN/GRP), and neurotrophins are involved in neuro-immunomodulatory processes and have marked trophic, growth-promoting and inflammation-modulating properties. The impact of these modulators in rheumatoid arthritis (RA) is, however, unclear. An involvement of the innervation, including the peptidergic innervation, is frequently proposed as an important factor for arthritic disease. Many patients with RA, but not all, benefit from treatment with anti-TNF medications. The studies presented here aimed to investigate the roles of neuropeptides, with an emphasis on BN/GRP and SP, and neurotrophins, especially with attention to brain-derived neurotrophic factor (BDNF), in human and murine knee joint tissue. The expression patterns of these substances and their receptors in synovial tissue from patients with either RA or osteoarthritis (OA) were studied in parallel with the levels of these factors in blood and synovial fluid from patients with RA and from healthy controls. Correlation studies were also performed comparing the levels of neuropeptides with those of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6)]. Furthermore, the impact of anti-TNF treatment on the levels of BDNF in blood was investigated. In a murine model of RA, the expression of these substances on articular chondrocytes along with their expression in synovial tissue was investigated. The expression of BN/GRP in human synovial tissue was confined to fibroblast-like and mononuclear-like cells whereas SP was detected in nerve-related structures. Receptors for these neuropeptides (GRP-R and NK-1R) were frequently present in blood vessel walls, and on fibroblast-like and mononuclear-like cells. The expression of BDNF and its receptors, p75 neurotrophin receptor and TrkB, was mainly confined to nerve structures. The levels of SP, and particularly those of BN/GRP, in synovial fluid and peripheral blood correlated with the levels of pro-inflammatory cytokines. There were clearly more correlations between SP-BN/GRP and inflammatory parameters than between BDNF and these factors. Plasma levels of BDNF were decreased following anti-TNF-treatment. In the joints of the murine model, there was a marked expression of neurotrophins, neurotrophin receptors and NK-1R/GRP-R in the articular chondrocytes. The expression was down-regulated in the arthritic animals. A neurotrophin system was found to develop in the inflammatory infiltrates of the synovium in the arthritic mice. The results presented suggest that there is a local, and not nerve-related, supply of BN/GRP in the human synovial tissue. Furthermore, BN/GRP and SP have marked effects in the synovial tissue of patients with RA, i.e., there were abundant receptor expressions, and these neuropeptides are, together with cytokines, likely to be involved in the neuro-immunomodulation that occurs in arthritis. The observations do on the whole suggest that the neuropeptides, rather than BDNF, are related to inflammatory processes in the human knee joint. A new effect of anti-TNF treatment; i.e., lowering plasma levels of BDNF, was observed. Severe arthritis, as in the murine model, lead to a decrease in the levels of neurotrophin, and neurotrophin and neuropeptide receptor expressions in the articular cartilage. This fact might be a drawback for the function of the chondrocytes. Certain differences between the expression patterns in the synovial tissue of the murine model and those of human arthritic synovial tissue were noted. It is obvious that local productions in the synovial tissue, nerve-related supply in this tissue and productions in chondrocytes to different extents occur for the investigated substances.