Obesity is pandemic. Pharmacological treatment development depends on modeling the regulation of feeding, particularly by free fatty acids (FFA). Most models have been employed in the rat in vivo, and show FFA-stimulated intestinal satiety signals are dependent on the fat’s acyl chain-length, involve cholecystokinin (CCK) secretion, and are mediated by vagal afferents. I hypothesized that an in vitro mouse model could be employed, with sensitivity to measure afferent responses to nutrient stimuli. Male C57BL/6N mice were killed, the intestine harvested en bloc, and a jejunal section dissected with neurovascular mesenteric arcade emanating centrally. The tissue was placed in a Krebs-superfused chamber, the lumen cannulated with the outlet open to drain, and Krebs or other mediators were continuously perfused intraluminally. The dissected afferent nerve was placed in a suction electrode for extracellular recording. Afferent responses to distension and the perfusion of mediators (e.g. CCK or FFA) were tested. Preparations from normal mice (no surgery), or from mice following chronic subdiaphragmatic vagotomy or sham operation, were used to assess vagal afferent contributions. Luminally-perfused CCK (100 nM) increased afferent firing. This response was abolished with the CCK-1 receptor antagonist lorglumide (10 µM). The short-chain fatty acid (SCFA) sodium butyrate (30 mM) potentiated firing. The long-chain fatty acid (LCFA) sodium oleate (1-300 mM) activated concentration-dependent firing (EC50=25.35 mM) that was significantly greater at 30 mM than that evoked by butyrate. Lorglumide (30 µM) abolished the oleate (30 mM) response. The L-type Ca2+ channel (LTCC) inhibitor nicardipine (3 µM), intraluminally, potentiated the oleate response, while bath application abolished it. Vagotomy attenuated the oleate response. Vagotomy abolished the intraluminal CCK (100 nM) response, and attenuated the response to bath-superfused CCK. These findings support FFA chain-length-dependent mesenteric afferent activation and CCK involvement in oleate-induced firing, and suggest LTCC mediation of excitatory and inhibitory oleate response transduction pathways. The murine oleate response was shown to be mostly vagally-mediated, with some spinal contribution, and both vagal and spinal contributions to CCK responses were suggested. These data provide a basis for further investigation in vitro of cellular and molecular mechanisms of afferent satiety signals, and ultimately of obesity pathogenesis. / Thesis (Master, Physiology) -- Queen's University, 2010-06-29 15:56:08.387

A reconstrução microcirúrgica de faringe e esôfago com jejuno é uma das opções para a reparação de defeitos resultantes de faringolaringectomias. Suas principais vantagens são: o diâmetro da alça jejunal é compatível com o diâmetro das bocas faríngea e esofágica, apresenta menos estenose do que reconstruções cutâneas e há menos contaminação do que quando se emprega o cólon. Entretanto, o pedículo vascular é, por vezes, curto; além disso, as paredes flácidas do intestino delgado e sua secreção mucosa dificultam a adaptação de prótese fonatórias. Finalmente, é necessária uma laparotomia para a obtenção do segmento jejunal, o que aumenta a potencial morbidade operatória. O objetivo deste trabalho foi avaliar de forma retrospectiva os aspectos técnicos, mobi-mortalidade e resultados funcionais de uma série de doentes submetidos a este método reconstrutivo, numa única instituição. No período de 1989 a 2000, 35 pacientes do sexo masculino, com média de idade de 55 anos, foram submetidos à reconstrução faringoesofágica com retalho microcirúrgico de jejuno, no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Trinta e quatro doentes eram portadores de tumores malignos do trato aerodigestivo alto, e um sofreu um ferimento cervical por arma de fogo. Onze casos foram previamente submetidos à radioterapia. A reconstrução foi imediata na maioria dos casos (85,7%). Através de laparotomia mediana supra-umbilical, escolheu-se segmento de alça jejunal de tamanho compatível, situado de 30 a 50 cm do ângulo do Treitz e nutrido por ramos longos dos vasos mesentéricos superiores, atentando-se para preservar a continuidade da arcada vascular primária em todo o segmento a ser transplantado. Este foi transposto para o seu leito definitivo sempre em posição isoperistáltica. Obteve-se um restabelecimento do trânsito digestivo alto em 84,0% dos casos. Houve perda do retalho em 14%, e a taxa de mortalidade foi de 2,9%, ocasionada por abdome agudo obstrutivo. O resultado funcional foi avaliado através de escala de pontuação de Schechter, incluindo parâmetros como deglutição, voz e peso corpóreo. Em 45% dos casos, observou-se uma pontuação entre 5 e 6, evidenciando uma boa qualidade de reabilitação. Em virtude da gravidade dos doentes e da magnitude dos atos operatórios, concluiu-se que a reconstrução faringoesofágica com retalho microcirúrgico jejunal foi um método exeqüível em nosso meio, oferecendo uma boa qualidade de reabilitação funcional, com morbi-mortalidade aceitável / Microsurgical reconstruction of the esophagus and pharynx with a jejunal segment is one of the current options available for repairing defects caused by pharyngolaryngectomies. Main advantages of this technique are: compatible diameters of the jejunal segment with the pharyngeal and esophageal openings, lower incidence of stenosis when compared to cutaneous reconstructions, and less contamination in relation to techniques using colonic fragments. Nevertheless, the vascular pedicle is sometimes too short and the flaccid walls of the jejunum associated with its mucous secretion render adaptation to phonatory prosthesis more difficult. Finally, operative morbidity may be increased due to the need for laparotomy in order to obtain the jejunal segment. The aim of this work was to evaluate, in a retrospective fashion, the technical aspects, morbi-mortality and functional results of a series of patients submitted to this reconstructive method at a single institution. During the period of 1989 to 2000 a total of 35 male patients with an average age of 55 years received a microsurgical flap of the jejunum for pharyngoesophageal reconstruction, at the Hospital das Clínicas of São Paulo University Medical School. Thirty four patients had malignant tumors of the upper aerodigestive tract and one had a injury. Eleven cases had been previously submitted to radiotherapy. The majority of patients (85.7%) underwent reconstruction immediately following ablative surgery. By means of median supraumbilical laparotomy an intestinal segment located 30 to 50 cm away from the angle of Treitz was chosen taking into note that it had to be nourished by long branches of the superior mesenteric vessels and to also maintain its continuity to the primary vascular arcade throughout the segment to be transplanted. The segment was transposed to its definitive vascular bed always respecting an isoperistaltic position. Functional effective restoration of the higher digestive transit was possible in 84.0% of cases. Graft loss occurred in 14%, and the mortality rate was of 2.9%, caused by obstructive acute abdomen. Functional results were evaluated according to the Schechter scoring scale, where parameters such as swallowing, voice and weight are taken into account. In 45% of the cases the scores were between 5 and 6, representing good repairing quality. Considering the degree of severity of these patients and the magnitude of the surgical procedures, we concluded that pharyngoesophageal reconstruction utilizing microsurgical jejunal flaps is a feasible method with good functional rehabilitation results and acceptable morbidity and mortality rates for our patient population

Background: The use of stapling equipment for intestinal surgery in cats is rarely reported, and appropriate staple sizes for cat intestine are unknown. Objective: To determine staple cartridge sizes for thoracoabdominal (TA) and endoscopic gastrointestinal anastomosis (EndoGIA) that will simultaneously prevent leakage of small intestinal contents while also allowing for sufficient vascular permeability past the staple lines for intestinal healing. Methods: Two sizes of EndoGIA cartridges (2.0/2.5/3.0mm and 3.0/3.5/4.0mm) and two sizes of TA cartridges (2.5mm and 3.5mm), applied in a transverse manner across fresh cadaveric cat jejunum, were evaluated via intestinal burst pressure testing for maximum intraluminal pressure prior to leaking, and via infusion of an intravascular dye at normal arterial pressures to determine percentage of vascular patency past the staple lines. Vascular patency was compared not only from pre-and post-staple segments of the same intestinal sample, but also EndoGIA vascular patency was evaluated against TA vascular patency. Results: Two cats met study criteria. All samples had intraluminal burst pressures over twice the chosen minimum (of 30mmHg). Vascular patency post- staple line ranged from 0-90.8%, with the most consistently high numbers noted with the TA 3.5mm cartridges. No EndoGIA cartridge had a post- staple line vascular patency higher than 31.1%, and no intravascular dye was noted in any post- staple line sample in the EndoGIA 2.0/2.5/3.0mm group. Conclusions: While statistical analysis of the dataset was unable to be performed due to low numbers of samples for comparison, both intestinal intraluminal burst pressure trends and intravascular dye patterns suggested both the TA 3.5mm cartridge and (to a lesser extent) the 3.0/3.5/4.0mm EndoGIA cartridge could provide the ideal combination of intraluminal seal without restriction of vascular access for healing. The intravascular dye infusion technique, developed during this research, shows promise as a future instrument to determine vascular patterns around intestinal implants in cadaveric cat specimens. / Master of Science / Despite the regularity of feline small intestinal surgery, few reports exist of stapled anastomoses in cats, in part due to stapler size limitations. However, the recently developed endoscopic gastrointestinal anastomosis (EndoGIA) stapler shows promise as a future surgical tool for cats because it fits into cat intestine. In dogs, 3.5mm staples are often chosen for intestinal surgery; however, dog intestine is considerably thicker than cat intestine. The study goal was to evaluate not only intestinal burst pressures (the pressure at which repaired intestine leaks), but also the ability of fluids to flow through blood vessels that cross the staple lines of four stapler cartridge types from two staple lineages (EndoGIA 2.0/2.5/3.0mm, EndoGIA 3.0/3.5/4.0mm, TA 2.5mm, and TA 3.5mm). The central hypothesis was twofold. First, smaller stapler cartridge sizes (the EndoGIA 2.0/2.5/3.0mm and TA 2.5mm) would have higher intraluminal burst pressures when compared to the larger sizes (the EndoGIA 3.0/3.5/4.0mm). Second, larger stapler cartridges (the EndoGIA 3.0/3.5/4.0mm and the TA 3.5mm) would allow for increased flow of fluids in blood vessels past the stapler lines compared to the smaller cartridges (the EndoGIA 2.0/2.5/3.0mm and the TA 2.5mm). Two cats were included in the study. Trends in the data suggested that all components of the hypothesis might be proven with further data. However, due to the low number of cats acquired during the study period, the hypotheses could not be verified with statistics. The dye infusion technique to evaluate flow of fluids in blood vessels, developed during this research, shows promise as a future instrument to determine vascular patterns around intestinal implants. Future research should focus on acquiring more cats to have the ability able to perform statistical analyses (and prove the hypothesis), before proceeding with additional related studies.

As lesões térmicas corporais (LTC) são um sério problema de saúde, atingindo principalmente crianças. A extensão e a profundidade da lesão são fatores que alteram várias estruturas. Alterações gastrintestinais também são relatadas, sendo a principal delas, a atrofia das mucosas, provocando ulcerações e a perda da barreira seletiva. Na presente pesquisa avaliou-se através de técnicas histoquímicas, imunohitoquimicas e de microscopia de luz, as alterações ocorridas nos componentes do plexo mioentérico e na espessura da mucosa do jejuno em três porções: oral (O), média (M) e aboral (A), de ratos adultos com 30% da superfície corpórea exposta ao escaldamento, 4 dias (q4) e 10 dias (q10) após a LTC. Verificou-se em q10 o não restabelecimento da massa corpórea, a diminuição da área do jejuno, bem como espessura de sua mucosa. No plexo mioentérico, a área média do perfil celular dos neurônios NADPH não variou, tendo estes uma menor densidade em q10, sendo estes corpos altamente reativos em q4 e q10. Varicosidades grandes destacaram-se em q4 e q10, quando pela SP e VIP. / Burn is a determinant factor to alter body structures as the striated muscle. It also determines gastrintestinal mucosal atrophy what produce loss of selective barrier. With histochemical, immunohistochemical and light microscopy methods the myenteric plexus (MP) of the jejunum was evaluated in rats submitted to burn injury. The scalding was performed in 30% of the body surface. The MP and the mucosa of the oral (O), middle (M) and aboral (A) parts of the jejunum were analyzed four (q4) and ten (q10) days post-lesion. The loss of weight due the burn is not recovered in q10 where the jejunal surface area and the thickness of the mucosa decreased. The neuronal profile of nitregic neurons was similar in q4, q10. The density of nitregic neurons was lower in q10 showing that the time post injury is an important factor able to alter this parameter. The q4 and q10 groups exhibited neuronal bodies highly reactive to NADPH. The immunoreactivity to SP and VIP in q4 and q10 was expressed mainly in large varicosities.

As lesões térmicas corporais (LTC) são um sério problema de saúde, atingindo principalmente crianças. A extensão e a profundidade da lesão são fatores que alteram várias estruturas. Alterações gastrintestinais também são relatadas, sendo a principal delas, a atrofia das mucosas, provocando ulcerações e a perda da barreira seletiva. Na presente pesquisa avaliou-se através de técnicas histoquímicas, imunohitoquimicas e de microscopia de luz, as alterações ocorridas nos componentes do plexo mioentérico e na espessura da mucosa do jejuno em três porções: oral (O), média (M) e aboral (A), de ratos adultos com 30% da superfície corpórea exposta ao escaldamento, 4 dias (q4) e 10 dias (q10) após a LTC. Verificou-se em q10 o não restabelecimento da massa corpórea, a diminuição da área do jejuno, bem como espessura de sua mucosa. No plexo mioentérico, a área média do perfil celular dos neurônios NADPH não variou, tendo estes uma menor densidade em q10, sendo estes corpos altamente reativos em q4 e q10. Varicosidades grandes destacaram-se em q4 e q10, quando pela SP e VIP. / Burn is a determinant factor to alter body structures as the striated muscle. It also determines gastrintestinal mucosal atrophy what produce loss of selective barrier. With histochemical, immunohistochemical and light microscopy methods the myenteric plexus (MP) of the jejunum was evaluated in rats submitted to burn injury. The scalding was performed in 30% of the body surface. The MP and the mucosa of the oral (O), middle (M) and aboral (A) parts of the jejunum were analyzed four (q4) and ten (q10) days post-lesion. The loss of weight due the burn is not recovered in q10 where the jejunal surface area and the thickness of the mucosa decreased. The neuronal profile of nitregic neurons was similar in q4, q10. The density of nitregic neurons was lower in q10 showing that the time post injury is an important factor able to alter this parameter. The q4 and q10 groups exhibited neuronal bodies highly reactive to NADPH. The immunoreactivity to SP and VIP in q4 and q10 was expressed mainly in large varicosities.

Background: Intestinal inflammation elicited by environmental determinants including dietary proteins and microbes is implicated in type 1 diabetes (T1D) pathogenesis. Also, intrinsic pancreatic abnormalities could precede classic insulitis, contributing to T1D. Materials and Methods: Spontaneous rat T1D models were used for in situ analyses of gut and pancreas to explore novel disease pathways using immunohistochemistry and detailed morphometry, gene expression studies, and molecular screening analyses. Results: In BBdp rats, feeding a cereal diet stimulated T1D under germ-free or specific pathogen-free (SPF) conditions compared with a protective hydrolyzed casein (HC) diet. Cereal-induced T1D was paralleled by increased gut T cell infiltration and TH1-associated pro-inflammatory transcription. HC-fed rats displayed an increased number of anti-inflammatory CD163+ M2 macrophages compared with cereal-fed rats. Cereal-associated promotion of T1D in Lewis diabetes-prone (LEW-DP) rats, a different rat model, similarly featured gut T cell infiltration in conjunction with decreased immunoregulation. The Camp gene was induced in diet-protected HC-fed BBdp rats. Camp encodes the cathelicidin antimicrobial peptide (CAMP), a pleiotropic immunomodulatory host defence factor. Intestinal CAMP was enriched in CD163+ M2 macrophages and could represent a novel marker of these tolerogenic innate immune cells. CAMP expression was also discovered in pancreatic lymph nodes (PLN) and islets, indicating a novel role for this factor in target tissue homeostasis. There was a positive correlation between pancreatic CAMP and total islet number. Also, islet-associated CAMP+ cells were increased in rats with islet inflammation, suggesting upregulation in parallel with insulitis. Exogenous CAMP/LL-37 injections increased the abundance of T1D-protective probiotic bacteria and promoted islet neogenesis in BBdp rats. A prospective partial pancreatectomy (PPx) study was performed to obtain pre-diabetic pancreas biopsies from iii pre-insulitic BBdp rats. The number of endothelium-associated CD68+ macrophages was increased in pre-diabetic pancreata, indicating that perivascular inflammation was an early lesion in the animals. In addition, pre-diabetic pancreata featured enhanced regenerative Reg3a and Reg3b gene expression, indicating abnormal islet expansion preceding insulitis. Conclusions: Small intestinal inflammation paired with deficits in local immunoregulation parallels T1D development. CAMP represents a novel factor in T1D that could have several pleiotropic functions including regulation of commensal microbes, intestinal homeostasis, and pancreatic homeostasis. In addition, target tissue abnormalities precede insulitis and T1D. This research focused on the integrative biology of T1D pathogenesis in spontaneous rat models. This work provides a novel working model that incorporates key roles for gut lumen antigens, intestinal immunity, and the role of islets and altered regenerative capacity in T1D. This research could lead to new therapeutic opportunities for T1D treatment.