RESUMO
Transient receptor potential melastatin-4 (TRPM4) is activated by an increase in intracellular Ca2+ concentration and is expressed on smooth muscle cells (SMCs). It is implicated in the myogenic constriction of cerebral arteries. We hypothesized that TRPM4 has a general role in intracellular Ca2+ signal amplification in a wide range of blood vessels. TRPM4 function was tested with the TRPM4 antagonist 9-phenanthrol and the TRPM4 activator A23187 on the cardiovascular responses of the rat, in vivo and in isolated basilar, mesenteric, and skeletal muscle arteries. TRPM4 inhibition by 9-phenanthrol resulted in hypotension and a decreased heart rate in the rat. TRPM4 inhibition completely antagonized myogenic tone development and norepinephrine-evoked vasoconstriction, and depolarization (high extracellular KCl concentration) evoked vasoconstriction in a wide range of peripheral arteries. Vasorelaxation caused by TRPM4 inhibition was accompanied by a significant decrease in intracellular Ca2+ concentration, suggesting an inhibition of Ca2+ signal amplification. Immunohistochemistry confirmed TRPM4 expression in the smooth muscle cells of the peripheral arteries. Finally, TRPM4 activation by the Ca2+ ionophore A23187 was competitively inhibited by 9-phenanthrol. In summary, TRPM4 was identified as an essential Ca2+-amplifying channel in peripheral arteries, contributing to both myogenic tone and agonist responses. These results suggest an important role for TRPM4 in the circulation. The modulation of TRPM4 activity may be a therapeutic target for hypertension. Furthermore, the Ca2+ ionophore A23187 was identified as the first high-affinity (nanomolar) direct activator of TRPM4, acting on the 9-phenanthrol binding site.
Assuntos
Sinalização do Cálcio , Canais de Cátion TRPM/metabolismo , Vasoconstrição , Administração Intravenosa , Animais , Artérias/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Calcimicina/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Ionóforos/farmacologia , Masculino , Desenvolvimento Muscular/efeitos dos fármacos , Músculo Esquelético/irrigação sanguínea , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Norepinefrina/farmacologia , Fenantrenos/administração & dosagem , Fenantrenos/farmacologia , Cloreto de Potássio/farmacologia , Ratos Wistar , Canais de Cátion TRPM/agonistas , Vasoconstrição/efeitos dos fármacosRESUMO
During the perinatal adaptation process N2BA titin isoforms are switched for N2B titin isoforms leading to an increase in cardiomyocyte passive tension (Fpassive). Here we attempted to reveal how titin isoform composition and oxidative insults (i.e. sulfhydryl (SH)-group oxidation or carbonylation) influence Fpassive of left ventricular (LV) cardiomyocytes during rat heart development. Moreover, we also examined a hypothetical protective role for titin associated small heat shock proteins (sHSPs), Hsp27 and αB-crystallin in the above processes. Single, permeabilized LV cardiomyocytes of the rat (at various ages following birth) were exposed either to 2,2'-dithiodipyridine (DTDP) to provoke SH-oxidation or Fenton reaction reagents (iron(II), hydrogen peroxide (H2O2), ascorbic acid) to induce protein carbonylation of cardiomyocytes in vitro. Thereafter, cardiomyocyte force measurements for Fpassive determinations and Western immunoblot assays were carried out for the semiquantitative determination of oxidized SH-groups or carbonyl-groups of titin isoforms and to monitor sHSPs' expressions. DTDP or Fenton reagents increased Fpassive in 0- and 7-day-old rats to relatively higher extents than in 21-day-old and adult animals. The degrees of SH-group oxidation or carbonylation declined with cardiomyocyte age to similar extents for both titin isoforms. Moreover, the above characteristics were mirrored by increasing levels of HSP27 and αB-crystallin expressions during cardiomyocyte development. Our data implicate a gradual build-up of a protective mechanism against titin oxidation through the upregulation of HSP27 and αB-crystallin expressions during postnatal cardiomyocyte development.
Assuntos
Conectina/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Animais , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/toxicidade , Ferro/toxicidade , Miócitos Cardíacos/efeitos dos fármacos , Isoformas de Proteínas , RatosRESUMO
INTRODUCTION: A technique of reconstructing the inguinal ligament using pedicled fascia lata flap is described. PRESENTATION OF CASE: A 66-year-old woman was referred with massive incarcerated left inguinal hernia, following acute surgery on a femoral vein leasion and numerous attempts at repair and subsequent recurrences. There was complete absence of the left inguinal ligament. The inguinal ligament was reconstructed using a strip of fascia lata, pedicled on the anterior superior iliac spine. This was transposed to cover the external iliac vessels, and sutured to the pubic tubercle. The musculoaponeurotic abdominal wall was reconstructed with 15×13 cm sheet of polypropylene mesh, placed preperitoneal and sutured to the remaining abdominal wall muscles and to the neo-Pouoart ligament. DISCUSSION: Complete destruction of the inguinal ligament is rare but can occur following multiple operative procedures or trauma. Published reports of inguinal ligament reconstruction have been performed using synthetic mesh. The use of autologous tissue should reduce the risk of erosion into the neurovascular bundle, seroma formation, and enhance integration into surrounding tissues. CONCLUSION: This new technique for autologous reconstruction of the inguinal ligament provides a safe alternative to the use of synthetic mesh in the operative armamentarium of plastic and general surgeons. This is the first reported case in Hungary.
Assuntos
Veia Femoral/cirurgia , Hérnia Inguinal/cirurgia , Ligamentos Articulares/cirurgia , Retalhos Cirúrgicos , Parede Abdominal , Idoso , Fascia Lata , Humanos , Complicações Pós-Operatórias/etiologia , Procedimentos de Cirurgia Plástica/métodos , Reoperação , Resultado do TratamentoRESUMO
BACKGROUND: Partial or subtotal spleen resection or spleen autotransplantation can partly preserve/restore the splenic filtration function, as previous studies demonstrated. OBJECTIVE: For better evaluation and follow-up of the various spleen-preserving operative techniques' effectiveness versus splenectomy, a composite methodological approach was applied in a canine experimental model. METHODS: Beagle dogs were subjected to control (nâ=â6), splenectomy (SE, nâ=â4), partial and subtotal spleen resection (nâ=â4/each) or spleen autotransplantation groups (AU, Furka's spleen-chip method, nâ=â8). The follow-up period was 18 postoperative (p.o.) months. Erythrocyte deformability was determined in parallel by bulk filtrometry (Carat FT-1 filtrometer), slit-flow ektacytometry (RheoScan D-200) and rotational ektacytometry (LoRRca MaxSis Osmoscan). RESULTS: By filtrometry, relative cell transit time increased in the SE group (mostly in animal Nr. SE-3), showing the highest values on the 3rd, 9th and in 18th p.o. months. Elongation index values decreased in this group (both by slit-flow and rotational ektacytometers). In general, AU and two resection groups' values were lower versus control and higher than in SE. CONCLUSIONS: Forasmuch in the circulation both elongation by shear stress and filtration occur, these various erythrocyte deformability testing methods together may describe better the alterations. Considering the possible complications related to functional asplenic-hyposplenic conditions, individual analysis of cases is highly important.
Assuntos
Deformação Eritrocítica/fisiologia , Baço/cirurgia , Esplenectomia/métodos , Transplante Autólogo/métodos , Animais , Modelos Animais de Doenças , Cães , Seguimentos , Humanos , Período Pós-Operatório , Baço/patologiaRESUMO
OBJECTIVES: Corneal blindness due to limbal stem-cell deficiency can be treated by transplantation of cultivated limbal epithelial stem cells (LESCs). We examined LESC cultivation on a contact lens (CL) carrier. Our goal was to optimize explant affixation and assess the possible benefit of 3T3 feeder cells. METHODS: Human cadaver limbal and conjunctival explants were allowed to attach to CLs under the airflow of the laminar box (dried group) or affixed on CLs using suturing (sutured group) or tissue adhesives (glued group), then cultivated with or without 3T3 feeder cells. Outgrowth efficiency was statistically analyzed. CEBPδ, p63, CK3/12, and CK13 were detected by immunofluorescence in expanded cells. RESULTS: Suturing and gluing provided excellent sample attachment, whereas drying was less effective. Cell expansion was better in sutured than in dried or glued samples. Presence of 3T3 feeder resulted in significantly better cell growth (P=0.048), most importantly in dried samples (P=0.008). Stepwise regression analysis indicated that cell expansion was dependent on the affixing method (P<0.001) and the presence of feeder layer (P=0.003). Expanded cells maintained their CK expression profiles and expressed putative stem-cell markers p63 and CEBPδ. The 3T3 feeder did not influence the expression of putative LESC markers or growth rate. CONCLUSIONS: Suturing is an effective way to fasten explants to CLs. 3T3 fibroblasts are not necessary in this system, although they may enhance cell outgrowth when samples are exposed to stress. However, once cells begin to expand, neither expression of putative stem-cell markers nor growth rate is influenced by feeder cells.
Assuntos
Túnica Conjuntiva/patologia , Lentes de Contato , Doenças da Córnea/patologia , Transplante de Córnea , Células Epiteliais/patologia , Rejeição de Enxerto/prevenção & controle , Limbo da Córnea/patologia , Idoso , Cadáver , Contagem de Células , Técnicas de Cultura de Células , Proliferação de Células , Doenças da Córnea/cirurgia , Células Alimentadoras , Humanos , Células-Tronco/patologiaRESUMO
INTRODUCTION: The purposes of this study were to measure the 3-dimensional parameters of the posed smile and to see whether there are any correlations with vertical cephalometric skeletal measurements. METHODS: Pretreatment records from a sample of 110 white girls between the ages of 12 and 18 years were gathered. The measurements of SN-GoGn, anterior facial height, and lower and upper facial height percentages were obtained from tracing lateral cephalograms. Superimposing the repose and the posed smile facial scans allowed for measurements to be obtained showing the movements in the x, y, and z dimensions of the upper and lower lips, the commissures, and the Cupid's bows. Correlations and multiple linear regression analyses were run to check for associations and predictive relationships between the cephalometric skeletal measurements and soft tissue changes. RESULTS: We found significant moderate correlations and weak correlations. Significant multiple regression models were found for intercommissural width, smile index, and lower lip in the y and z dimensions. CONCLUSIONS: There were moderate correlations showing that as SN-GoGn and anterior facial height increased, the interlabial gap increased as the smile index decreased. Significant relationships were found between certain hard tissue cephalometric measurements and the width of the smile as well as the movements of the lower lip.
Assuntos
Cefalometria/métodos , Sorriso , Dimensão Vertical , Adolescente , Pontos de Referência Anatômicos , Criança , Estética Dentária , Feminino , Humanos , Imageamento TridimensionalRESUMO
Testicular torsion may lead to serious ischemia, and the viability depends on the duration of torsion and the effect of ischemia-reperfusion. Testicular decompression and tunica vaginalis flap application technique were introduced in 2008 by Kutikov et al. We aimed to examine the impact of this method on the testicular microcirculation and hemorheological parameters in a rat model. Six adult rats underwent bilateral scrotal exploration. Intravaginal torsion of the testis was created by 720° rotation on both sides for 2 h. After detorsion, the right testes underwent tunica albuginea incision and tunica vaginalis flap application. Testicular microcirculation was monitored and hematological parameters, erythrocyte deformability, and aggregation were determined. Measurements were performed before and after torsion, directly after detorsion, on the 1 st -2 nd and 8 th postoperative day. After the last sampling, testicles were removed to determine their volume for histological examinations. The microcirculatory parameters demonstrated slight differences between testicles. Apical zone of the left (nondecompressed) testicles had elevated compared to the middle zone (P < 0.05). On the 2 nd and 8 th day, the microcirculation of the testes normalized but not equally. The erythrocyte aggregation and deformability decreased by the 8 th day. Both testicles underwent atrophy and epithelial necrosis, but the volume of the decompressed ones was lower (1.07 ± 0.08 vs 1.25 ± 0.31). Histologically, there was no significant difference in epithelial damage score between decompressed and nondecompressed testes. In conclusion, 2-h ischemia led to alteration in testicular microcirculation, reduction in volume, changes in hemorheological parameters and serious epithelial necrosis both in decompressed and nondecompressed testicles without remarkable differences.
Assuntos
Isquemia/patologia , Microcirculação , Torção do Cordão Espermático/patologia , Testículo/patologia , Animais , Atrofia , Descompressão Cirúrgica , Índices de Eritrócitos , Masculino , Necrose , Tamanho do Órgão , Ratos , Torção do Cordão Espermático/cirurgia , Retalhos Cirúrgicos , Testículo/irrigação sanguíneaRESUMO
Laboratory investigations often require centrifugation of blood samples for various erythrocyte tests. Although there is a lack of data about the effect of centrifugation at various g force levels on erythrocyte rheological properties. We aimed to investigate the effect of a 10-minute centrifugation at 500, 1000 or 1500âg at 15°C of rat, dog, pig and human venous (K3-EDTA, 1.5âmg/ml) blood samples. Hematological parameters, erythrocyte deformability, cell membrane stability, osmotic gradient ektacytometry (osmoscan) and erythrocyte aggregation were determined. Hematological and erythrocyte deformability parameters showed interspecies differences, centrifugation caused no significant alterations. Cell membrane stability for human erythrocytes centrifuged at higher g level showed less decrease in deformability. Osmoscan O min parameter showed slight elevation in dog centrifuged aliquots. Erythrocyte aggregation parameters changed unexpectedly. Rat and dog erythrocyte aggregation indices significantly dropped in centrifuged aliquots. Pig erythrocyte aggregation indices increased significantly after centrifugation. Human erythrocyte aggregation was the most stable one among the investigated species. The used centrifugation protocols caused the largest alterations in erythrocyte aggregation in a controversial way among the investigated species. On the other hand, erythrocyte deformability parameters were stable, cell membrane stability and osmoscan data show minor shifts.
Assuntos
Centrifugação , Agregação Eritrocítica , Deformação Eritrocítica , Índices de Eritrócitos , Eritrócitos/citologia , Hemorreologia , Adulto , Animais , Cães , Membrana Eritrocítica/metabolismo , Feminino , Gravitação , Testes Hematológicos/métodos , Humanos , Masculino , Osmose , Ratos , Especificidade da Espécie , SuínosRESUMO
PURPOSE: To examine how the ischemia-reperfusion injury of latissimus dorsi-cutaneous maximus (LDCM) musculocutaneous flap affects the microcirculatory (flap's skin surface) and hemorheological parameters, and whether an intraoperative deterioration would predictively suggest flap failure in the postoperative period. METHODS: Ten healthy male rats were subjected to the study. In Group I the left flap was sutured back after 2-hour, while the contralateral side was right after its elevation. In Group II the same technique was applied, but the pedicle of the left flap was atraumatically clamped for 2-hour. The contralateral side was left intact. On the flap skin surface laser Doppler tissue flowmetry measurements were done before and after and during the protocols applied in the groups. Microcirculatory and hemorheological examinations were done postoperatively. RESULTS: The microcirculatory parameters significantly decreased during immobilization and ischemia. Afterwards, all the regions showed normalization. In the retrospective analysis there was a prominent difference between the microcirculatory parameters of necrotic and survived flap during the early postoperative days (1-3) in Group II. Erythrocyte aggregation and deformability showed only slight differences. CONCLUSIONS: Two-hour ischemia and reperfusion caused deterioration in latissimus dorsi-cutaneous maximus flap microcirculation. Predicting the possible postoperative complication, the intraoperative laser Doppler measurement can be informative.
Assuntos
Hemorreologia/fisiologia , Microcirculação/fisiologia , Retalho Miocutâneo/irrigação sanguínea , Traumatismo por Reperfusão/fisiopatologia , Pele/irrigação sanguínea , Músculos Superficiais do Dorso/irrigação sanguínea , Animais , Procedimentos Cirúrgicos Dermatológicos , Modelos Animais de Doenças , Período Intraoperatório , Fluxometria por Laser-Doppler , Masculino , Retalho Miocutâneo/patologia , Período Pós-Operatório , Distribuição Aleatória , Ratos , Pele/patologia , Transplante de Pele/métodos , Músculos Superficiais do Dorso/patologia , Fatores de TempoRESUMO
PURPOSE:To examine how the ischemia-reperfusion injury of latissimus dorsi-cutaneous maximus (LDCM) musculocutaneous flap affects the microcirculatory (flap's skin surface) and hemorheological parameters, and whether an intraoperative deterioration would predictively suggest flap failure in the postoperative period.METHODS: Ten healthy male rats were subjected to the study. In Group I the left flap was sutured back after 2-hour, while the contralateral side was right after its elevation. In Group II the same technique was applied, but the pedicle of the left flap was atraumatically clamped for 2-hour. The contralateral side was left intact. On the flap skin surface laser Doppler tissue flowmetry measurements were done before and after and during the protocols applied in the groups. Microcirculatory and hemorheological examinations were done postoperatively.RESULTS: The microcirculatory parameters significantly decreased during immobilization and ischemia. Afterwards, all the regions showed normalization. In the retrospective analysis there was a prominent difference between the microcirculatory parameters of necrotic and survived flap during the early postoperative days (1-3) in Group II. Erythrocyte aggregation and deformability showed only slight differences.CONCLUSIONS: Two-hour ischemia and reperfusion caused deterioration in latissimus dorsi-cutaneous maximus flap microcirculation. Predicting the possible postoperative complication, the intraoperative laser Doppler measurement can be informative.
Assuntos
Animais , Masculino , Ratos , Hemorreologia/fisiologia , Microcirculação/fisiologia , Retalho Miocutâneo/irrigação sanguínea , Traumatismo por Reperfusão/fisiopatologia , Pele/irrigação sanguínea , Músculos Superficiais do Dorso/irrigação sanguínea , Procedimentos Cirúrgicos Dermatológicos , Modelos Animais de Doenças , Período Intraoperatório , Fluxometria por Laser-Doppler , Retalho Miocutâneo/patologia , Período Pós-Operatório , Distribuição Aleatória , Transplante de Pele/métodos , Pele/patologia , Músculos Superficiais do Dorso/patologia , Fatores de TempoRESUMO
AIM: To assess how ovarian-derived sex hormones (in particular progesterone) modify the effects of single acute stress on the mechanical and biochemical properties of left ventricular cardiomyocytes in the rat. METHODS: Non-ovariectomized (control, n=8) and ovariectomized (OVX, n=8) female rats were kept under normal conditions or were exposed to stress (control-S, n=8 and OVX-S, n=8). Serum progesterone levels were measured using a chemiluminescent immunoassay. Left ventricular myocardial samples were used for isometric force measurements and protein analysis. Ca(2+)-dependent active force (Factive), Ca(2+)-independent passive force (Fpassive), and Ca(2+)-sensitivity of force production were determined in single, mechanically isolated, permeabilized cardiomyocytes. Stress- and ovariectomy-induced alterations in myofilament proteins (myosin-binding protein C [MyBP-C], troponin I [TnI], and titin) were analyzed by sodium dodecyl sulfate gel electrophoresis using protein and phosphoprotein stainings. RESULTS: Serum progesterone levels were significantly increased in stressed rats (control-S, 35.6±4.8 ng/mL and OVX-S, 21.9±4.0 ng/mL) compared to control (10±2.9 ng/mL) and OVX (2.8±0.5 ng/mL) groups. Factive was higher in the OVX groups (OVX, 25.9±3.4 kN/m(2) and OVX-S, 26.3±3.0 kN/m(2)) than in control groups (control, 16.4±1.2 kN/m(2) and control-S, 14.4±0.9 kN/m(2)). Regarding the potential molecular mechanisms, Factive correlated with MyBP-C phosphorylation, while myofilament Ca(2+)-sensitivity inversely correlated with serum progesterone levels when the mean values were plotted for all animal groups. Fpassive was unaffected by any treatment. CONCLUSION: Stress increases ovary-independent synthesis and release of progesterone, which may regulate Ca(2+)-sensitivity of force production in left ventricular cardiomyocytes. Stress and female hormones differently alter Ca(2+)-dependent cardiomyocyte contractile force production, which may have pathophysiological importance during stress conditions affecting postmenopausal women.
Assuntos
Estrogênios/sangue , Miócitos Cardíacos/fisiologia , Ovariectomia , Ovário/fisiologia , Progesterona/sangue , Estresse Fisiológico , Animais , Proteínas de Transporte/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Ventrículos do Coração , Humanos , Medições Luminescentes , Fosforilação , Ratos , Ratos Sprague-Dawley , Troponina I/metabolismoRESUMO
PURPOSE: The failure of small-caliber vascular grafts still means a serious problem. Concerning the early postoperative complications we aimed to investigate the hemostaseological and hemorheological aspects of this issue in a canine model. METHODS: In the Control group only anesthesia was induced. In the Grafted group under general anesthesia a 3.5-cm segment was resected unilaterally from the femoral artery and replaced with a PTFE graft (diameter: 3 mm). On the 1st-3rd-5th-7th and 14th postoperative days the skin temperature of both hind limbs was measured, and blood sampling occurred for hematological, hemostaseological and hemorheological tests. RESULTS: The skin temperature of the operated versus intact limbs did not differ. In the Grafted group leukocyte count was elevated by the 1st postoperative day, while platelet count increased over the entire follow-up period. Fibrinogen concentration rose on the 1st-5th days, activated partial thromboplastin time increased on the 3rd-7th days. Erythrocyte aggregation was enhanced significantly on the 1st-5th days. In specimens taken on the 14th day, histologically we found matured thrombus narrowing the graft lumen. CONCLUSIONS: Small-caliber PTFE graft implantation into the femoral artery caused significant changes in several hemostaseological and hemorheological parameters. However, better clarifying the factors leading to early thrombosis of these grafts needs further studies.
Assuntos
Coagulação Sanguínea/fisiologia , Agregação Eritrocítica/fisiologia , Artéria Femoral/transplante , Modelos Animais , Politetrafluoretileno/uso terapêutico , Enxerto Vascular/métodos , Anastomose Cirúrgica , Animais , Contagem de Células Sanguíneas , Prótese Vascular , Cães , Fibrinogênio/análise , Tempo de Tromboplastina Parcial , Complicações Pós-Operatórias , Período Pós-Operatório , Tempo de Protrombina , Fatores de Tempo , Resultado do TratamentoRESUMO
PURPOSE: The failure of small-caliber vascular grafts still means a serious problem. Concerning the early postoperative complications we aimed to investigate the hemostaseological and hemorheological aspects of this issue in a canine model. METHODS: In the Control group only anesthesia was induced. In the Grafted group under general anesthesia a 3.5-cm segment was resected unilaterally from the femoral artery and replaced with a PTFE graft (diameter: 3 mm). On the 1st-3rd-5th-7th and 14th postoperative days the skin temperature of both hind limbs was measured, and blood sampling occurred for hematological, hemostaseological and hemorheological tests. RESULTS: The skin temperature of the operated versus intact limbs did not differ. In the Grafted group leukocyte count was elevated by the 1st postoperative day, while platelet count increased over the entire follow-up period. Fibrinogen concentration rose on the 1st-5th days, activated partial thromboplastin time increased on the 3rd-7th days. Erythrocyte aggregation was enhanced significantly on the 1st-5th days. In specimens taken on the 14th day, histologically we found matured thrombus narrowing the graft lumen. CONCLUSIONS: Small-caliber PTFE graft implantation into the femoral artery caused significant changes in several hemostaseological and hemorheological parameters. However, better clarifying the factors leading to early thrombosis of these grafts needs further studies. .
Assuntos
Animais , Cães , Coagulação Sanguínea/fisiologia , Agregação Eritrocítica/fisiologia , Artéria Femoral/transplante , Modelos Animais , Politetrafluoretileno/uso terapêutico , Enxerto Vascular/métodos , Anastomose Cirúrgica , Contagem de Células Sanguíneas , Prótese Vascular , Fibrinogênio/análise , Tempo de Tromboplastina Parcial , Complicações Pós-Operatórias , Período Pós-Operatório , Tempo de Protrombina , Fatores de Tempo , Resultado do TratamentoRESUMO
We aimed to investigate hemodynamic, microcirculatory and hemorheological consequence of infrarenal or suprarenal aortic cross-clamping (IRAXC, SRAXC) in the rat. We hypothesized that the magnitude of the changes are different. Twenty-one male rats were randomized into Control, IRAXC or SRAXC groups. Under anesthesia the right carotid artery was cannulated for monitoring heart rate and mean arterial pressure, then median laparotomy was performed. In AXC groups the abdominal aorta and the caudal caval vein were atraumatically clamped for 60 minutes below or above the renal vessels. Before and just after the ischemia, in the 30th and 60th minutes of the reperfusion besides hemodynamic test, laser Doppler flowmetry was used on the liver's, small-intestine's and the kidney's surface, then arterial (cannulated carotid artery) and venous (lateral tail vein) blood samples were taken for determining hematological, acid-base, erythrocytes' deformability, osmoscan and aggregation parameters. We found that when hemodynamic changes were prominent, microcirculatory or hemorheological parameters did not show such large differences. However, every parameter changed in various manners, showing more or less differences between IRAXC and SRAXC groups. Although the largest deviations were observable in SRAXC group, the acid-base and hemodynamic alterations were much more expressed than the micro-rheological ones. Further investigations of in vivo relations-correlations of changes in hemodynamic, microcirculatory, metabolic and hemorheological factors need further studies providing simultaneous monitoring possibilities.
Assuntos
Aorta Abdominal/cirurgia , Hemodinâmica , Microcirculação , Traumatismo por Reperfusão/sangue , Animais , Artérias Carótidas/cirurgia , Cateterismo , Constrição , Agregação Eritrocítica , Deformação Eritrocítica , Laparotomia , Masculino , Distribuição Aleatória , Ratos Sprague-Dawley , Reologia , Veias Cavas/cirurgiaRESUMO
PURPOSE: To investigate the intraoperative microcirculatory changes of the affected organs (small bowel, liver and kidney) during the making of a modified selective portacaval (PC) shunt. METHODS: On ten anaesthetized Sprague-Dawley rats the selective end-to-side mesocaval anastomosis was performed, where only the rostral mesenteric vein is utilized and the portal vein with the splenic vein are left intact. Morphometric and microcirculatory investigations using a LDF device determining flux units (BFU) were carried out. RESULTS: After completing the shunts the microcirculatory flux values did not recover in the same manner on the surface of the small intestine, the liver or the kidney. BFU values showed deterioration in the small intestine and in the liver (p<0.001). During the reperfusion the BFU values improved, but not in the same manner. The small intestine values left behind the kidney and liver data. CONCLUSIONS: Technically, the advantages of the models include the selective characteristic, the mesocaval localization and the relatively easy access to those vessels. However, its major disadvantage is the time needed for positioning the vessels without coiling or definitive stretching. Intraoperative LDF may provide useful data on the microcirculatory affection of the organs suffering from hypoperfusion or ischemia during creating the shunts.
Assuntos
Microcirculação/fisiologia , Microcirurgia/métodos , Derivação Portocava Cirúrgica/métodos , Veia Porta/cirurgia , Veia Cava Inferior/cirurgia , Anastomose Cirúrgica , Animais , Período Intraoperatório , Veias Mesentéricas/anatomia & histologia , Modelos Animais , Veia Porta/anatomia & histologia , Ratos , Ratos Sprague-Dawley , Valores de Referência , Reprodutibilidade dos TestesRESUMO
PURPOSE: To investigate the intraoperative microcirculatory changes of the affected organs (small bowel, liver and kidney) during the making of a modified selective portacaval (PC) shunt. METHODS: On ten anaesthetized Sprague-Dawley rats the selective end-to-side mesocaval anastomosis was performed, where only the rostral mesenteric vein is utilized and the portal vein with the splenic vein are left intact. Morphometric and microcirculatory investigations using a LDF device determining flux units (BFU) were carried out. RESULTS: After completing the shunts the microcirculatory flux values did not recover in the same manner on the surface of the small intestine, the liver or the kidney. BFU values showed deterioration in the small intestine and in the liver (p<0.001). During the reperfusion the BFU values improved, but not in the same manner. The small intestine values left behind the kidney and liver data. CONCLUSIONS: Technically, the advantages of the models include the selective characteristic, the mesocaval localization and the relatively easy access to those vessels. However, its major disadvantage is the time needed for positioning the vessels without coiling or definitive stretching. Intraoperative LDF may provide useful data on the microcirculatory affection of the organs suffering from hypoperfusion or ischemia during creating the shunts.
Assuntos
Animais , Ratos , Microcirculação/fisiologia , Microcirurgia/métodos , Derivação Portocava Cirúrgica/métodos , Veia Porta/cirurgia , Veia Cava Inferior/cirurgia , Anastomose Cirúrgica , Período Intraoperatório , Modelos Animais , Veias Mesentéricas/anatomia & histologia , Veia Porta/anatomia & histologia , Ratos Sprague-Dawley , Valores de Referência , Reprodutibilidade dos TestesRESUMO
PURPOSE: The aim of this study was to identify differentially expressed genes in the human limbal epithelium by microarray analysis. METHODS: Total RNA isolates of human limbal and central corneal epithelia were used after transcription for hybridization on whole human genome expression microarrays. A set of differentially expressed genes detected by both microarrays was established. In the case of eight selected molecules, microarray results were confirmed by qRT-PCR, and protein expression in the cornea was examined by confocal immunofluorescence microscopy. Colocalization with the putative stem cell marker C/EBPδ was also examined. RESULTS: The authors established a database of 126 limbal overexpressed genes. qRT-PCR confirmed microarray results in all examined cases (SPON1, IFITM1, ITM2A, PHLDA1, CXCR4, FZD7, DCT, DKK4). Limbal localization of the protein product of SPON1, IFITM1, ITM2A, CXCR4, and DKK4 was shown with confocal immunofluorescence microscopy. SPON1, IFITM1, and ITM2A signals mostly colocalized with C/EBPδ-positive putative resting limbal stem cells. CONCLUSIONS: By detecting several new differentially expressed genes in the human corneal limbus, this study further expands current knowledge on the molecular signature of limbal epithelial stem cells. Plasma membrane localization of IFITM1 and ITM2A suggests their potential usefulness as targets to select stem cell-enriched populations from the limbal epithelium.
Assuntos
Epitélio Corneano/citologia , Epitélio Corneano/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Limbo da Córnea/citologia , Células-Tronco/citologia , Biomarcadores/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas do Olho/genética , Humanos , Microscopia Confocal , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismoRESUMO
HER2-positive breast cancers represent a distinct phenotype and are intrinsically more aggressive than HER2-negative tumors. Although HER2-targeted therapies have been rationally developed, resistance to these treatments represents a process understood poorly. There are few experimental models that allow studying the molecular mechanism of resistance. Our aim was to characterize a trastuzumab resistant breast cancer cell line (B585) that was established from an invasive ductal carcinoma. B585 grows only in immunodeficient mice as a xenograft. CGH and FISH were used to define cytogenetic alterations, gene-expression analysis and immunohistochemistry were applied to detect RNA and protein expression. By array-CGH focused amplifications were identified for C-MYC, EGFR, ErbB2, CCND1 and TOP2-A oncogenes. ErbB2 was co-amplified with TOP2-A. mRNA overexpression was detected for the amplified genes. ErbB2 protein was overexpressed and showed heterogeneous distribution. In summary, molecular cytogenetic analysis and expression profiling of B585 revealed several new alterations. Based on the experiments performed in SCID mice and the genotypic/phenotypic characteristics, this new in vivo breast cancer xenograft is a valuable model to investigate molecular mechanism of trastuzumab resistance.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Carcinoma Ductal de Mama/tratamento farmacológico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Animais , Anticorpos Monoclonais Humanizados , Hibridização Genômica Comparativa , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Camundongos , Camundongos SCID , Oncogenes/genética , Receptor ErbB-2/metabolismo , TrastuzumabRESUMO
Pathomechanisms leading to ocular surface diseases are difficult to influence, and conservative treatment efforts are inefficient in most cases. However, the development of microsurgical techniques, the clinical application of molecular biologic methods and the localisation and identification of the function of limbal stem cells made it possible to achieve visual improvement and quality of life in these cases. The authors review the methods of tissue harvesting, cell culturing, transplantation techniques and indications. They provide a detailed survey on the most recommended modern surgical procedures, especially focusing on amniotic membrane and limbal stem cell transplantation.
Assuntos
Doenças da Córnea/cirurgia , Epitélio Corneano/cirurgia , Limbo da Córnea/citologia , Procedimentos Cirúrgicos Oftalmológicos/métodos , Transplante de Células-Tronco , Transtornos da Visão/cirurgia , Técnicas de Cultura de Células , Células Cultivadas , Transplante de Córnea , Sobrevivência de Enxerto , Humanos , Limbo da Córnea/cirurgia , Microcirurgia , Qualidade de Vida , Células-Tronco , Resultado do TratamentoRESUMO
The cornea is a major protective shield of the interior of the eye and represents two thirds of its refractive power. It is made up of three tissue layers that have different developmental origins: the outer, epithelial layer develops from the ectoderm overlying the lens vesicle, whereas the stroma and the endothelium have mesenchymal origin. In the adult organism, the outermost corneal epithelium is the most exposed to environmental damage, and its constant renewal is assured by the epithelial stem cells that reside in the limbus, the circular border of the cornea. Cell turnover in the stromal layer is very slow and the endothelial cells probably do not reproduce in the adult organism. However, recent experimental evidence indicates that stem cells may be found in these layers. Damage to any of the corneal layers leads to loss of transparency and low vision. Corneal limbal stem cell deficiency results in severe ocular surface disease and its treatment by transplantating ex vivo expanded limbal epithelial cells is becoming widely accepted today. Stromal and endothelial stem cells are potential tools of tissue engineering and regenerative therapies of corneal ulcers and endothelial cell loss. In the past few years, intensive research has focused on corneal stem cells aiming to improve the outcomes of the current corneal stem cell transplantation techniques. This review summarizes the current state of knowledge on corneal epithelial, stromal and endothelial stem cells. Special emphasis is placed on the molecular markers that may help to identify these cells, and the recently revealed mechanisms that could maintain their "stemness" or drive their differentiation. The techniques for isolating and culturing/expanding these cells are also described.