Gender differences in microcirculation: Observation using the hamster cheek pouch

OBJECTIVES: Estrogen has been shown to play an important protective role in non-reproductive systems, such as the cardiovascular system. Our aim was to observe gender differences in vivo with regard to the increase in macromolecular permeability and leukocyte-endothelium interaction induced by ischemia/reperfusion as well as in microvascular reactivity to vasoactive substances using the hamster cheek pouch preparation. METHODS: Thirty-six male and 36 female hamsters, 21 weeks old, were selected for this study, and their cheek pouches were prepared for intravital microscopy. An increase in the macromolecular permeability of post-capillary venules was quantified as a leakage of intravenously injected fluorescein-labeled dextran, and the leukocyte-endothelium interaction was measured as the number of fluorescent rolling leukocytes or leukocytes adherent to the venular wall, labeled with rhodamin G, during reperfusion after 30 min of local ischemia. For microvascular reactivity, the mean internal diameter of arterioles was evaluated after the topical application of different concentrations of two vasoconstrictors, phenylephrine (α1-agonist) and endothelin-1, and two vasodilators, acetylcholine (endothelial-dependent) and sodium nitroprusside (endothelial-independent). RESULTS: The increase in macromolecular permeability induced by ischemia/reperfusion was significantly lower in females compared with males [19 (17-22) leaks/cm2 vs. 124 (123-128) leaks/cm2, respectively, p<0.001), but the number of rolling or adherent leukocytes was not different between the groups. Phenylephrine-induced arteriolar constriction was significantly lower in females compared with males [77 (73-102)% vs. 64 (55-69)%, p<0.04], but there were no detectable differences in endothelin-1-dependent vasoreactivity. Additionally, arteriolar vasodilatation elicited by acetylcholine or sodium nitroprusside did not differ between the groups. CONCLUSION: The ...

Preconditioning of the response to ischemia/ reperfusion-induced plasma leakage in hamster cheek pouch microcirculation

OBJECTIVE: Ischemic preconditioning and some drugs can protect tissues from injury by preserving microcirculation. This study evaluated vascular permeability in a hamster cheek pouch preparation using either short ischemic periods or bradykinin as preconditioning stimuli followed by 30 min of ischemia/reperfusion. METHOD: Sixty-six male hamsters were divided into 11 groups: five combinations of different ischemic frequencies and durations (one, three or five shorts periods of ischemia, separated by one or five minutes) with 10 min intervals between the ischemic periods, followed by 30 min ischemia/reperfusion; three or five 1 min ischemic periods with 10 min intervals between them followed by the topical application of histamine (2 µM); bradykinin (400 nM) followed by 30 min of ischemia/reperfusion; and three control groups (30 min of ischemia/reperfusion or histamine or bradykinin by themselves). Macromolecular permeability was assessed by injection of fluorescein-labeled dextran (FITC-dextran, MW= 150 kDa; 250 mg/Kg body weight), and the number of leaks/cm2 was counted using an intravital microscope and fluorescent light in the cheek pouch. RESULTS: Plasma leakage (number of leaks/cm²) was significantly reduced by preconditioning with three and five 1 min ischemic periods, one and three 5 min ischemic periods and by bradykinin. Histamine-induced macromolecular permeability was also reduced after three periods of 5 min of ischemia. CONCLUSION: Short ischemic periods and bradykinin can function as preconditioning stimuli of the ischemia/reperfusion response in the hamster cheek pouch microcirculation. Short ischemic periods also reduced histamineinduced macromolecular permeability.

Vimentin and laminin are altered on cheek pouch microvessels of streptozotocin-induced diabetic hamsters

OBJECTIVE: Normal endothelial cells respond to shear stress by elongating and aligning in the direction of fluid flow. Hyperglycemia impairs this response and contributes to microvascular complications, which result in deleterious effects to the endothelium. This work aimed to evaluate cheek pouch microvessel morphological characteristics, reactivity, permeability, and expression of cytoskeleton and extracellular matrix components in hamsters after the induction of diabetes with streptozotocin. METHODS: Syrian golden hamsters (90-130 g) were injected with streptozotocin (50 mg/kg, i.p.) or vehicle either 6 (the diabetes mellitus 6 group) or 15 (the diabetes mellitus 15 group) days before the experiment. Vascular dimensions and density per area of vessels were determined by morphometric and stereological measurements. Changes in blood flow were measured in response to acetylcholine, and plasma extravasation was measured by the number of leakage sites. Actin, talin, α-smooth muscle actin, vimentin, type IV collagen, and laminin were detected by immunohistochemistry and assessed through a semiquantitative scoring system. RESULTS: There were no major alterations in the lumen, wall diameters, or densities of the examined vessels. Likewise, vascular reactivity and permeability were not altered by diabetes. The arterioles demonstrated increased immunoreactivity to vimentin and laminin in the diabetes mellitus 6 and diabetes mellitus 15 groups. DISCUSSION: Antibodies against laminin and vimentin inhibit branching morphogenesis in vitro. Therefore, laminin and vimentin participating in the structure of the focal adhesion may play a role in angiogenesis. CONCLUSIONS: Our results indicated the existence of changes related to cell-matrix interactions, which may contribute to the pathological remodeling that was already underway one week after induction of experimental diabetes.

A microcirculação da bolsa da bochecha do hamster sob a influência do diabetes mellitus experimental induzido por estreptozotocina: aspectos morfofuncionais / Microcirculation of hamster cheek pouch under the influence of experimental Diabetes Mellitus induced by streptozotocin: morpho-functional aspects

O Diabetes Mellitus é uma doença metabólica crônica com múltiplos fatores etiológicos (genético, viral e imunológico) que condiciona deficiência absoluta ou relativa de insulina, causando persistência de níveis elevados de glicose no sangue. Atualmente, o Diabetes Mellitus é considerado um importante problema de saúde devido a sua prevalência e alta morbimortalidade. Sua importância clínica resulta essencialmente de suas graves complicações, especialmente as microvasculares. A hiperglicemia crônica ou intermitente tem sido identificada como o fator indutor de lesão endotelial, sendo este, o agente desencadeante das complicações microvasculares. As células endoteliais, por serem influenciadas pela força hemodinâmica local, respondem com a transdução de sinais (mecanotrans dução), as quais podem ser responsáveis pelo início de processos patológicos na parede dos vasos. Desta forma, o objetivo deste estudo foi analisar a microcirculação da bolsa da bochecha do hamster sob a influência do Diabetes Mellitus tipo 1 experimental induzido por estreptozotocina, avaliando seus aspectos morfofuncionais aos 6 e 15 dias de evolução da doença. As características morfológicas de arteríolas e vênulas foram estimadas por medidas do diâmetro do lúmen e da espessura da parede; pela densidade de volume e de área destes vasos na bolsa da bochecha; pela análise imunohistoquímica da expressão de actina, talina, alfa-actina de músculo liso, vimentina, laminina e colágeno IV através da microscopia de luz com a utilização de um sistema semiquantitativo baseado em uma escala de intensidade de imunomarcação; e por microscopia eletrônica de transmissão. Também foi avaliado o relaxamento dependente do endotélio, medido pela variação do diâmetro do lúmen antes e após a aplicação de acetilcolina e a permeabilidade de vênulas pós-capilares à histamina, determinada pelo número de pontos de extravasamento plasmático. Nossos resultados mostraram que arteríolas e vênulas...

Diabetes Mellitus is a chronic metabolic disease with multiple etiologic factors (genetic, viral and immunological) that results in absolute or relative insulin deficiency, causing persistent elevated blood glucose levels. Nowadays, Diabetes Mellitus is considered as an important health concern due to its increasing prevalence and high morbimortality. Its clinical importance comes from the complications, especially harm inductor factor, being this the first outcome of microvascular complications. Endothelial cells, under local hemodynamic strength, produce signal transduction (mechanotransduction), which can be responsible for the beginning of patholgical events in vessels wall. In this regard, the objective of this study was to analyze hamster cheek pouch microcirculation under the influence of type 1 diabetes mellitus induced by streptozotocin, evaluationg its morpho-functional aspects at 6 and 15 days of diseases evolution. Morphological characteristics of arterioles and venules were estimated by the measurement of lumen diameter and wall thickness; the volume density and area of these vessels from cheek pouch; immunohistochemistry of the expression of actin, talin, smooth muscle alpha-actin, vimentin, laminin and type IV collagen through light microscopy with the utilization of a semi-quantitative score system based on the intensity of the immunostaining; and transmission electron microscopy. It was also evaluated the endothelium dependent relaxation, measured by the variation of lumen diameter before and after acetylcholine administration and post-capillary venules permeability to histamine, determined by number of points of plasma extravasation. Our results reveal that arterioles and venules do not show differences between the groups concerning wall thickness, luminal diameter, density per area and volume density. Vascular permeability, after 2 minutes of histamine administration, was reduced significantly in diabetic groups...

Keloid heterograft in the hamster (mesocricetus auratus) cheek pouch

OBJETIVO: Investigar a integração do transplante heterólogo de quelóide na bolsa jugal do hamster (Mesocricetus auratus). MÉTODOS: A amostragem consiste de 18 hamsters machos, heterogênicos, com 10 a 14 semanas de idade. Fragmentos de quelóide foram obtidos de cicatrizes queloidianas da região mamária de paciente adulta parda. Cada hamster foi enxertado em ambas as bolsas com fragmentos de quelóide, totalizando 36 fragmentos enxertados. Os animais foram distribuídos em 6 grupos para exame dos fragmentos enxertados, com 5, 12, 21, 42, 84 e 168 dias. Uma avaliação macroscópica é realizada comparando a bolsa contendo o fragmento enxertado em cada período com a mesma bolsa no pós-operatório imediato, mediante a comparação de fotografias padronizadas. À microscopia, considera-se a presença de vasos sangüíneos no tecido conjuntivo do fragmento enxertado como critério de integração do mesmo. Outros eventos, como secreção de queratina, presença de infiltrados celulares e aspecto do epitélio e das fibras colágenas do quelóide, também são observados. RESULTADOS: A macroscopia revela intensa vascularização na bolsa até 12 dias de enxertia, e a presença constante de pigmentação castanho-escura nos fragmentos de quelóide enxertados. Na microscopia constata-se a integração dos fragmentos de quelóide pela presença de capilares sangüíneos no tecido conjuntivo. Observa-se, também, a presença de intenso infiltrado celular do tipo inflamatório até 12 dias, a permanência do epitélio do quelóide até 21 dias, e o aparecimento de melanócitos a partir de 42 dias. CONCLUSÃO: A bolsa jugal do hamster representa, a priori, modelo experimental para investigação do quelóide.

Effects of hypertonic NaCl solution on the hamster cheek pouch microcirculation in normo- and hypovolemia

The aim of this study was evaluate the effects of a single, 10 min, intravenous infusion of a hyperonic NaCl solution [2.400 mOsm/l; infused volume 0.35 ñ 0.03 ml (SEM) on the hamster cheek pouch microcirculation during normovolemia and after acute bleeding to a hypotension level of about 40 mmHg. Upon bleeding, the arterial pressure dropped to 39 µ 1 mmHg, arterioles > 40 -m constricted 12 ñ 3% (from their control value), arterioles < 40 µm dilated 6 ñ 2%, venules stayed largely unchanged, while RBC velocity and volume flow decreased 57 ñ 7% in all vessels. Durign the subsequent hypertonic NaCl infusion, the arterial pressure increased rapidly to a new steady state level of 66 ñ 3 mmHg. After the infusion, the large arterioles stayed constricted 11 ñ 1% and the small arterioles dilated 7 ñ 1% for a 1-h observation period. The venules constricted iniatially by 6 ñ 2% and returned to control diameter in 30-40 min. RBC velocity and calculated volume flow returned to the pre-hemorrhage cotrol values in about 10 min for the arterioles and in 40 min for the venules. An identical hypertonic infusion given to normovolemic hamsters caused no significant alterations of the measured variables