Thesis (M.Sc.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Pharmacy and Pharmaceutical Sciences. Title from pdf file main screen (viewed on July 31, 2009). Includes bibliographical references.

As reabsorções radiculares externas são conseqüências decorrentes de lesões traumáticas severas como a avulsão e a intrusão. O estudo de medicamentos que possam auxiliar no controle e prevenção desses processos é de grande interesse. O objetivo desse estudo foi avaliar a citotoxicidade do alendronato de sódio e da calcitonina, aplicados em forma de pasta em cultura de fibroblastos humanos de gengiva. Foi utilizado para o experimento meio condicionado pelas substâncias a serem testadas, divididas em quatro grupos: alendronato de sódio (ALN) a 10-6M; ALN a 10-7M; ALN a 10-8M; e calcitonina (CAL). Como grupos controle usou-se meio de cultura não condicionado e meio de cultura condicionado apenas pelo veículo das pastas (talco farmacêutico e solução salina). A sobrevivência celular foi analisada pelo método de contagem de células viáveis nos tempos experimentais de 1 e 24 horas. Os resultados foram analisados estatisticamente e mostraram que o veículo empregado e as pastas de ALN nas concentrações de 10-7 e 10-8 M não apresentaram diferença significante do grupo controle. Conclui-se que os produtos oriundos das pastas formuladas com ALN a 10-6 M e com CAL a 0,002% apresentaram citotoxicidade para fibroblastos de gengiva humana na forma e período empregados. / External root resorption is a common consequence of dental trauma injuries, mainly in dental avulsion and extrusion. Doubtful prognoses awake the interesting in studying medications, which could help in controlling and preventing these resorption processes. The aim of this study was to evaluate the citotoxicity of sodium alendronate (ALN) and calcitonin, used in a paste formulation in human gingival fibroblasts. Products leached from the pastes were divided into four experimental groups and had conditioned cellular medium: ALN 10-6 M, ALN 10-7 M, ALN 10-8 M and calcitonin 0,002%. Such groups were taking under: fresh media and conditioned media with vehicle (talcum powder and saline). On experimental times, 1 and 24 hours, citotoxicity analyses were performed using the Trypan blue dye exclusion assay. The results were statistically analyzed and showed that the vehicle used and ALN concentrations 10-7 M and 10-8 M did not lead to differences from the control group with fresh media. In contrast, the calcitonin and the ALN pastes in a higher concentration, 10-6 M, demonstrated cell growth reduction. In conclusion, this research showed that products leached from pastes with calcitonin and ALN 10-6 were cytotocic for human gingival fibroblasts in vitro. Meanwhile, substances leached from ALN 10-7 M and 10-8 M were biocompatible.

Orientador: Jose Ricardo Albergaria Barbosa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-08T02:07:16Z (GMT). No. of bitstreams: 1 Luna_AnibalHenriqueBarbosa_D.pdf: 1133521 bytes, checksum: 0b56f9a0d7a3c458d4ca3675a6467a94 (MD5) Previous issue date: 2006 / Resumo: O granuloma central de células gigantes (GCCG) é uma lesão benigna que acomete tanto a maxila como a mandíbula, representando menos de 7% de todos os tumores benignos dos maxilares. A sua etiologia é incerta, sendo implicados fatores genéticos. O GCCG pode manifestar-se como lesões de grandes dimensões com características de agressividade ¿ como crescimento rápido, reabsorções radiculares ou parestesia e tendência à recidiva, ou como lesões pequenas, uniloculares, sem aspectos de agressividade. A modalidade de tratamento mais empregada é a curetagem, associada ou não a ostectomia periférica. No entanto são relatadas outras modalidades de tratamento, como a administração de corticosteróides, calcitonina ou a-interferon. Os índices de recidiva podem ser altos, variando de 0% a 49%. A ocorrência de recidiva parece depender do comportamento clínico da lesão, da localização anatômica e da modalidade de tratamento instituída. O presente estudo retrospectivo analisou o tratamento de GCCG no período de janeiro de 1996 a julho de 2006 atendidos pela Área de Cirurgia Buco-Maxilo-Facial da FOP ¿ Unicamp, correlacionando seus aspectos clínicos. Foram analisados 14 casos (9M; 5F) com uma média de idade de 18,5 (variando de 5 ¿ 59) anos, sendo a maxila o osso mais acometido. Do total, 5 casos foram tratados cirurgicamente por meio de curetagem associada a ostectomia periférica, e 9 foram tratados clinicamente. A administração intralesional de corticosteróides foi iniciada nestes casos, sendo o tratamento com calcitonina instituído na ausência de uma resposta clínica satisfatória. O tempo médio de tratamento com corticosteróides foi de 3,84 (±3,87) meses, sendo que em dois casos foi instituída a administração de calcitonina. O tempo médio de tratamento com calcitonina foi de 18,8 (±7,94) meses, sendo que em um caso não foi observada boa evolução clínica. Nenhum caso de recidiva foi observado após um acompanhamento de 38,22 (variando de 3 ¿ 174) meses / Abstract: The central giant cell granuloma is a benign lesion of the jaws, accounting for less than 7% of all benign lesions of the jaws. Its origin is unknown, but it has been suggested that genetic factors may be implicated. The central giant cell granuloma demonstrates a variable clinical behavior, ranging from slowly growing painless swelling to rapidly expanding aggressive tumors, characterized by pain, local destruction of bone, root displacement or resorption and a significantly high recurrence rate. Surgical treatment represented by curettage with peripheral ostectomy or not is the most widely used procedure. However, other treatment options such as intralesional corticosteroids, daily calcitonin administration or a-interferon are advocated. The recurrence rate may be high (ranging form 0% to 49%), and it seems to depend on the clinical behavior, the treatment employed, and anatomic site envolved. The aim of this study was to report the results of long-term follow up of the management of central giant cell granulomas. A retrospective analysis was conducted from January 1996 to July 2006, analyzing all cases of the Oral and Maxillofacial Area, Piracicaba Dental School. The sample was represented by 14 patients (9 M; 5 F) with a mean age of 18.5 (ranging from 5 ¿ 59) years, and the maxilla was involved in most of the cases. Regarding the treatment modality, 5 cases were treated by curettage with peripheral ostectomy, and a medical treatment was instituted in the others. In these cases, intralesional injections with corticosteroids were initiated, and the treatment with calcitonin was employed only if proper resolution was not achieved. The mean time of treatment with corticosteroids was 3.84 (±3.87) months, but in two cases calcitonin daily administration was initiated. The mean time of treatment with calcitonin was 18.8 (±7.94) months, but in one case calcitonin did not seem to be effective. No case of recurrence was observed after a mean follow-up of 38.22 (ranging from 3 ¿ 174) months / Doutorado / Cirurgia e Traumatologia Buco-Maxilo-Faciais / Doutor em Clínica Odontológica

Die Definition von Calcitonin-Referenzintervallen in der Pädiatrie ist für den behandelnden Arzt und den betroffenen Patienten von hohem Stellwert, um die Thyreoidektomie beim medullären Schilddrüsenkarzinom zeitgerecht einzuleiten. Die Leitlinien der „American Thyroid Association“ empfehlen die Operation bei Anstieg des Hormons über den Referenzbereich. Es existiert jedoch kein Referenzbereich für Kinder- und Jugendliche für die sensitivste Messmethode (elektrochemischer Lumineszenzimmunoassay - ECLIA) zum Nachweis von Calcitonin in der Patientenversorgung. Mit Hilfe der LIFE-Child-Kohorte war es möglich, ein phänotypisch gesundes Probandenkollektiv (Probanden: 2639, Messungen: 6090) zu rekrutieren, das zur Referenzwerterstellung hinzugezogen wurde. Die Blutproben konnten standardisiert im Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik (Universitätsklinikum Leipzig) weiterverarbeitet werden. Probanden mit möglichen Einflussfaktoren auf den Calcitonin-Spiegel wurden ausgeschlossen. Statistisch wurden die Referenzbereiche nach einem neuen Ansatz berechnet. Die Validation der Referenzbereiche erfolgte anhand zweier klinischer Patientenkohorten: Kinder und Jugendliche mit Multipler Endokriner Neoplasie Typ 2 (MEN 2) und mit Schilddrüsenerkrankungen. Wir konnten erstmalig nachweisen, dass Calcitonin in Kindern nicht nur alters- sondern auch geschlechtsabhängig konzentriert ist. Weiterhin unbekannt war, dass während der Pubertät der Calcitonin-Spiegel ein zweites Mal verstärkt abfällt. Die erstellten Referenzintervalle wurden erfolgreich an zwei Patientenkohorten validiert. Bei den MEN 2 erkrankten Kindern war es möglich zwischen gesunden, erkrankten und wiedererkrankten Patienten zu differenzieren. Während bei Erwachsenen Kontroverse bezüglich des Einflussfaktors von Schilddrüsenerkrankungen auf die Calcitonin-Spiegel besteht, wurde bei Kindern kein Effekt auf die Höhe des Hormons im Serum nachgewiesen. Es lässt sich schlussfolgern, dass Calcitonin-Messungen bei Säuglingen, Kleinkindern, Kindern und Adoleszenten alters- und geschlechtsabhängig interpretiert werden müssen. Der ECLIA ist in der Lage, Diagnose- und Therapiemonitoring beim medullären Schilddrüsenkarzinom von pädiatrischen Patienten optimal zu unterstützen und den Zeitpunkt einer Schilddrüsenentfernung bedarfsgerecht zu planen. Dabei scheinen Schilddrüsenerkrankungen einen geringeren Einfluss auf die Calcitonin-Spiegel von Kindern als von Erwachsenen zu haben.:1. Abkürzungsverzeichnis III 2. Bibliographische Beschreibung IV 3. Einführung 1 3.1 Calcitoninwirkung und Calciumhomöostase 1 3.2 Spontanes versus heriditär auftretendes medulläres Schilddrüsenkarzinom 2 3.3 Diagnostik des medullären Schilddrüsenkarzinoms bei Kindern und Jugendlichen 3 3.3.1 RET-Protoonkogen 4 3.3.2 Calcitonin 5 3.3.3 Carcinoembryonales Antigen, Procalcitonin und Chromogranin A 10 3.4 Etablierung von Referenzwerten für Calcitonin bei Kindern und Jugendlichen 11 3.5 Aufgabenstellungen 12 4. Publikationsmanuskript 13 5. Zusammenfassung 25 6. Literaturverzeichnis VI 7. Addendum XVII 7.1 Darstellung des eigenen Beitrages XVII 7.2 Selbstständigkeitserklärung XIX 7.3 Curriculum vitae XX 7.4 Publikationen XXI 7.5 Danksagungen XXII

Die Definition von Calcitonin-Referenzintervallen in der Pädiatrie ist für den behandelnden Arzt und den betroffenen Patienten von hohem Stellwert, um die Thyreoidektomie beim medullären Schilddrüsenkarzinom zeitgerecht einzuleiten. Die Leitlinien der „American Thyroid Association“ empfehlen die Operation bei Anstieg des Hormons über den Referenzbereich. Es existiert jedoch kein Referenzbereich für Kinder- und Jugendliche für die sensitivste Messmethode (elektrochemischer Lumineszenzimmunoassay - ECLIA) zum Nachweis von Calcitonin in der Patientenversorgung. Mit Hilfe der LIFE-Child-Kohorte war es möglich, ein phänotypisch gesundes Probandenkollektiv (Probanden: 2639, Messungen: 6090) zu rekrutieren, das zur Referenzwerterstellung hinzugezogen wurde. Die Blutproben konnten standardisiert im Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik (Universitätsklinikum Leipzig) weiterverarbeitet werden. Probanden mit möglichen Einflussfaktoren auf den Calcitonin-Spiegel wurden ausgeschlossen. Statistisch wurden die Referenzbereiche nach einem neuen Ansatz berechnet. Die Validation der Referenzbereiche erfolgte anhand zweier klinischer Patientenkohorten: Kinder und Jugendliche mit Multipler Endokriner Neoplasie Typ 2 (MEN 2) und mit Schilddrüsenerkrankungen. Wir konnten erstmalig nachweisen, dass Calcitonin in Kindern nicht nur alters- sondern auch geschlechtsabhängig konzentriert ist. Weiterhin unbekannt war, dass während der Pubertät der Calcitonin-Spiegel ein zweites Mal verstärkt abfällt. Die erstellten Referenzintervalle wurden erfolgreich an zwei Patientenkohorten validiert. Bei den MEN 2 erkrankten Kindern war es möglich zwischen gesunden, erkrankten und wiedererkrankten Patienten zu differenzieren. Während bei Erwachsenen Kontroverse bezüglich des Einflussfaktors von Schilddrüsenerkrankungen auf die Calcitonin-Spiegel besteht, wurde bei Kindern kein Effekt auf die Höhe des Hormons im Serum nachgewiesen. Es lässt sich schlussfolgern, dass Calcitonin-Messungen bei Säuglingen, Kleinkindern, Kindern und Adoleszenten alters- und geschlechtsabhängig interpretiert werden müssen. Der ECLIA ist in der Lage, Diagnose- und Therapiemonitoring beim medullären Schilddrüsenkarzinom von pädiatrischen Patienten optimal zu unterstützen und den Zeitpunkt einer Schilddrüsenentfernung bedarfsgerecht zu planen. Dabei scheinen Schilddrüsenerkrankungen einen geringeren Einfluss auf die Calcitonin-Spiegel von Kindern als von Erwachsenen zu haben.:1. Abkürzungsverzeichnis III 2. Bibliographische Beschreibung IV 3. Einführung 1 3.1 Calcitoninwirkung und Calciumhomöostase 1 3.2 Spontanes versus heriditär auftretendes medulläres Schilddrüsenkarzinom 2 3.3 Diagnostik des medullären Schilddrüsenkarzinoms bei Kindern und Jugendlichen 3 3.3.1 RET-Protoonkogen 4 3.3.2 Calcitonin 5 3.3.3 Carcinoembryonales Antigen, Procalcitonin und Chromogranin A 10 3.4 Etablierung von Referenzwerten für Calcitonin bei Kindern und Jugendlichen 11 3.5 Aufgabenstellungen 12 4. Publikationsmanuskript 13 5. Zusammenfassung 25 6. Literaturverzeichnis VI 7. Addendum XVII 7.1 Darstellung des eigenen Beitrages XVII 7.2 Selbstständigkeitserklärung XIX 7.3 Curriculum vitae XX 7.4 Publikationen XXI 7.5 Danksagungen XXII

The purpose of this study was to determine if endogenous tachykinins can cause bradycardia in the isolated perfused guinea pig heart through stimulation of cholinergic neurons. Capsaicin was used to stimulate release of tachykinins and calcitonin gene-related peptide (CGRP) from cardiac afferents. A bolus injection of 100 nmol capsaicin increased heart rate by 26 ± 7% from a baseline of 257 ± 14 beats/min (n = 6, P < 0.01). This positive chronotropic response was converted to a minor bradycardic effect in hearts with 1 μM CGRP (8-37) present to block CGRP receptors. The negative chronotropic response to capsaicin was markedly potentiated in another group of hearts with the further addition of 0.5 μM neostigmine to inhibit cholinesterases. In this group, capsaicin decreased heart rate by 30 ± 10% from a baseline of 214 ± 6 beats/min (n = 8, P < 0.05). This large bradycardic response to capsaicin was inhibited by 1) infusion of neurokinin A to desensitize tachykinin receptors or 2) treatment with 1 μM atropine to block muscarinic receptors. The latter observations implicate tachykinins and acetylcholine, respectively, as mediators of the bradycardia. These findings support the hypothesis that endogenous tachykinins could mediate axon reflexes to stimulate cholinergic neurons of the intrinsic cardiac ganglia.

The present study was done to characterize the effects of endogenous tachykinins on heart rate in urethane-anesthetized guinea pigs. Intravenous injection of capsaicin (32 nmol/kg) was used to evoke release of tachykinins and calcitonin gene-related peptide (CGRP) from cardiac sensory nerve fibers. Such injections caused a brief decrease in heart rate (- 37 ± 7 beats/min, n = 6) that was followed by a more prolonged increase (+ 44 ± 10 beats/min). Blood pressure was lowered by - 11 ± 2 mmHg. Bilateral vagotomy did not affect the chronotropic or depressor responses to capsaicin, but atropine (1 μmol/kg) nearly abolished the bradycardic response (- 8 ± 3 beats/min, n = 7). Combined blockade of NK2 and NK3 receptors, with SR48968 and SR14801 respectively, also caused a significant reduction of capsaicin-evoked bradycardia (- 14 ± 3 beats/min, n = 4) but did not affect bradycardia evoked by vagal nerve stimulation. Blockade of CGRP receptors eliminated capsaicin-evoked tachycardia and prolonged the capsaicin-evoked bradycardia. These findings suggest that capsaicin-evoked bradycardia in the anesthetized guinea pig is mediated by tachykinins that stimulate cardiac cholinergic neurons. This effect appears to be truncated by the positive chronotropic action of CGRP that is also released from cardiac afferents by capsaicin.

Calcitonin gene-related peptide (CGRP) is a nonadrenergic/noncholinergic (NANC) peptide with vasodilatative/inotropic action that may benefit the failing heart. However, precise mechanisms for its in vivo inotropic action remain unclear. To assess this, dogs with normal or failing (sustained tachypacing) hearts were instrumented for pressure-dimension analysis. In control hearts, CGRP (20 pmol/kg per minute) enhanced cardiac contractility (eg, +33±4.2% in end-systolic elastance) and lowered afterload (-14.2±2% in systemic resistance, both P<0.001). The inotropic response was markedly blunted by heart failure (+6.5±2%; P<0.001 versus control), whereas arterial dilation remained unaltered (-19.3±5%). CGRP-positive inotropy was not attributable to reflex activation because similar changes were observed in the presence of a ganglionic blocker. However, it was fully prevented by the β-receptor antagonist (timolol), identifying a dominant role of sympatho-stimulatory signaling. In control hearts, myocardial interstitial norepinephrine assessed by microdialysis almost doubled in response to CGRP infusion, whereas systemic plasma levels were unchanged. In addition, CGRP receptors were not observed in ventricular myocardium but were prominent in coronary arteries and the stellate ganglia. Ventricular myocytes isolated from normal and failing hearts displayed no inotropic response to CGRP, further supporting indirect sympatho-stimulation as the primary in vivo mechanism. In contrast, the peripheral vasodilatative capacity of CGRP was similar in femoral vascular rings from normal and failing hearts in dogs. Thus, CGRP-mediated positive inotropy is load-independent but indirect and attributable to myocardial sympathetic activation rather than receptor-coupled stimulation in canine hearts. This mechanism is suppressed in heart failure, so that afterload reduction accounts for CGRP-enhanced function in this setting.

Calcitonin gene-related peptide (CGRP) is a neurotransmitter that is released within the heart during myocardial ischemia. The present study was done to determine the regional localization and abundance of CGRP receptors in the guinea pig heart. CGRP binding sites in 20 μm frozen sections of heart were labeled using [125I]CGRP. Non-specific binding was determined in the presence of 1 μM unlabeled CGRP or CGRP8-37. Significant amounts of specific CGRP binding were identified in atrial and ventricular myocardium, all portions of the conducting system, coronary arteries, the aorta and pulmonary trunk and intracardiac ganglia. Specific binding of CGRP to the left atrium was two-fold higher than binding to the right atrium (0.667±0.052 v 0.340 ± 0.029 fmol/mg tissue, n = 5, CGRPs8-37 group). In contrast to the atria, a lower and uniform density of CGRP receptors occurred in contractile tissue of the ventricular myocardium (e.g. 0.239 ± 0.013 fmol/mg left ventricle, n = 5). The highest concentration of CGRP receptors in guinea pig cardiac tissue occurred at the bundle of His and the bundle branches (0.752 ± 0.087 and 0.71.3 ± 0.138 fmol/mg tissue, respectively, n = 5). CGRP receptors were localized to coronary vessels throughout the heart and to the ascending aorta and pulmonary trunk. Lastly, intracardiac ganglia exhibited moderate levels of specific [125I]CGRP binding (0.475 ± 0.043 fmol/mg, n = 5). These findings support the concept that CGRP can have direct effects on atrial and ventricular function as well as coronary flow. The high density of CGRP receptors in the distal conducting system and the presence of CGRP receptors in intracardiac ganglia further suggest that CGRP could have important effects on cardiac conduction velocity and parasympathetic regulation of the heart.

Degeneration of nociceptive afferent axons and terminals in the heart is associated with painless sudden cardiac death. However, innervation, distribution and morphological structures of sympathetic cardiac nociceptive afferent axons and terminals have not yet been fully characterized. The aim of the present study is to characterize the density, arrangement, and structural features of differentiated sympathetic afferent axons and terminals in whole-mount FVB mouse atria. FVB mice (3-6 months old) were perfused and the tissues were fixed. The right and left atria were processed with immunohistochemistry. Calcitonin gene-related peptide (CGRP) and substance P (SP) are two neuropeptides which have been widely used to label sympathetic nociceptive afferent axons in many tissues. CGRP (rabbit anti-CGRP) and SP (Goat anti-SP) primary antibodies were applied, followed by Alexa Fluor 594 and 660 conjugated secondary antibodies. Whole-mount preparations of right and left atria were examined using a laser scanning confocal microscope. We found that 1) CGRP immunoreactive (IR) axon bundles innervated the right and left atria including the auricle and entrance area of the superior vena cava, the inferior vena cava, left precaval vein and pulmonary veins. Large axon bundles entered the area from the major veins and bifurcated into smaller axon bundles and single axon fibers to form terminal end-nets and free endings in the epicardium at each region with a similar pattern. In the atrial muscle layer, varicose CGRP-IR axons had close contacts with muscle fibers. In addition, CGRP-IR axons iv terminated in the intrinsic cardiac ganglia (ICGs) with varicosities surrounding individual ganglionic principle neurons (PNs). In the aortic arch, the CGRP-IR fibers exhibited similar terminal structures to those seen in the atria. 2) SP-IR axons also projected to the right and left atria and aorta. Similar to CGRP-IR axons, these SP-IR axons also formed end-nets and free endings in these areas. In cardiac ganglia, SP-IR axons formed varicose endings around many individual PNs. However, a salient difference was found: There appeared to be fewer SPIR axons and terminals than CGRP-IR axons and terminals in the atria. 3) None of the cardiac PNs in ICG were CGRP-IR or SP-IR. 4) Many SP-IR axon terminals around PNs within ICGs and atrial muscles were found to have colocalized expression of CGRP-IR. Collectively, our data for the first time documented the distribution patterns and morphology of sympathetic afferent axons and terminals in each region of the atria in the mouse model. This will provide a foundation for future analysis of the pathological changes of sympathetic afferent nerves in the atria in different disease models (e.g., diabetes, sleep apnea, and aging). This study was supported by NIH R01 HL- 79636.