Sheep corneal endothelium morphology - evaluation with trypan blue and alizarin red

Background: The endothelium is a layer fundamental to maintaining corneal transparency. In ophthalmology, sheep eyes have been used as a model in research related to corneal transplantation. Different techniques have been used to evaluate the corneal endothelium. Concerning vital dyes, corneal endothelial cell analyses have not yet been studied in ovines. The purpose of the present study was to evaluate the morphology of endothelial cells from different regions of the cornea of sheep after staining with alizarin red and trypan blue using an optical microscope. Materials, Methods & Results: Twenty healthy eyes of 10 male sheep obtained from a licensed commercial slaughterhouse were studied. The study was approved by the Research Committee of the Faculty of Veterinary at UFRGS and followed the ethical standards of the Association for Research in Vision and Ophthalmology (ARVO). Immediately after the slaughter, the eyes were enucleated and underwent eye examination. The corneal endothelium was stained with trypan blue and alizarin red and examined and photographed using an optical microscope. The central, superior, inferior, nasal and temporal areas of the cornea were evaluated for cell morphology. Data were compared by t-tests. Differences were considered statistically significant at P < 0.05. Immediately after staining the corneal endothelium, it was possible to examine with an optical microscope, obtain images and analyse the shape of endothelial cells from all regions of the sheep cornea. Polygonal, uniform and continuous cells were observed in all samples studied. Considering all the corneas analysed, cells with 6 sides (75.11%), 5 sides (12.76%) and 4 sides (12.12%) were found. In the central region of the cornea 75.91% of cells with 6 sides, 12.6% of cells with 5 sides and 11.48% with 7 sides were found. In the superior region of the cornea 76.07% of cells with 6 sides, 13.25% with 5 sides and 10.68% with 7 sides were found. In the lower region were found 74.72% of cells with 6 sides, 13% with 5 sides and 12.27% with 7 sides. In the temporal region, 74.14% were 6-sided cells, 11.42% had 5 sides, and 14.43% had 7 sides. Furthermore, in the nasal region, 74.72% of the cells had 6 sides, 13.54% had 5 sides, and 11.73% had 7 sides. No significant differences were found between cell morphology in all corneal regions evaluated. In addition, no significant difference was found when comparing the right eye with the left eye. Discussion: Different methods are used for the analysis of corneal endothelium. For ex vivo research optical microscopy after endothelial staining is an alternative low-cost technique that allows the analysis of all regions of the cornea. Quantitative analyses must characterise the endothelial parameters of the different species. The analysis of the morphology of corneal endothelium with an optic microscope after staining with alizarin red has been described as an effective, rapid and cost-efficient method, since this dye blends with the borated cells, allowing identification. In the present study, using optical microscopy and coloration with alizarin red it was possible to explore and obtain images of the ovine endothelium of all regions of the cornea. In the current study, the endothelium had a predominance of cells will 6 sides in all regions studied. This study allowed us to obtain images of the endothelium as well as quantitative data on the morphology of the different regions of the sheep cornea. This study demonstrated that morphology did not differ between the central and peripheral regions. The findings of this study represent a further source of reproducible data that should be considered when using sheep cornea as ex vivo model for experimental research.

Proteolytic activity of Triatoma infestans saliva associated with PAR-2 activation and vasodilation

Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect and the main vector of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae). In the present study, the authors investigated whether a serine protease activity from the saliva of T. infestans has a role in vasomotor modulation, and in the insect-blood feeding by cleaving and activating protease-activated receptors (PARs). Methods T. infestans saliva was chromatographed as previously reported for purification of triapsin, a serine protease. The cleavage activity of triapsin on PAR peptides was investigated based on FRET technology. Mass spectrometry was used to analyze the sites of PAR-2 peptide cleaved by triapsin. NO measurements were performed using the DAN assay (2,3-diaminonapthalene). The vasorelaxant activity of triapsin was measured in vessels with or without functional endothelium pre-contracted with phenylephrine (3 µM). Intravital microscopy was used to assess the effect of triapsin on mouse skin microcirculation. Results Triapsin was able to induce hydrolysis of PAR peptides and showed a higher preference for cleavage of the PAR-2 peptide. Analysis by mass spectrometry confirmed a single cleavage site, which corresponds to the activation site of the PAR-2 receptor. Triapsin induced dose-dependent NO release in cultured human umbilical vein endothelial cells (HUVECs), reaching a maximum effect at 17.58 nM. Triapsin purified by gel-filtration chromatography (10-16 to 10-9 M) was applied cumulatively to mouse mesenteric artery rings and showed a potent endothelium-dependent vasodilator effect (EC30 = 10-12 M). Nitric oxide seems to be partially responsible for this vasodilator effect because L-NAME (L-NG-nitroarginine methyl ester 300 µM), a nitric oxide synthetase inhibitor, did not abrogate the vasodilation activated by triapsin. Anti-PAR-2 antibody completely inhibited vasodilation observed in the presence of triapsin activity. Triapsin activity also induced an increase in the mouse ear venular diameter. Conclusion Data from this study suggest a plausible association between triapsin activity mediated PAR-2 activation and vasodilation caused by T. infestans saliva.(AU)

Proteolytic activity of Triatoma infestans saliva associated with PAR-2 activation and vasodilation

Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect and the main vector of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae). In the present study, the authors investigated whether a serine protease activity from the saliva of T. infestans has a role in vasomotor modulation, and in the insect-blood feeding by cleaving and activating protease-activated receptors (PARs). Methods T. infestans saliva was chromatographed as previously reported for purification of triapsin, a serine protease. The cleavage activity of triapsin on PAR peptides was investigated based on FRET technology. Mass spectrometry was used to analyze the sites of PAR-2 peptide cleaved by triapsin. NO measurements were performed using the DAN assay (2,3-diaminonapthalene). The vasorelaxant activity of triapsin was measured in vessels with or without functional endothelium pre-contracted with phenylephrine (3 µM). Intravital microscopy was used to assess the effect of triapsin on mouse skin microcirculation. Results Triapsin was able to induce hydrolysis of PAR peptides and showed a higher preference for cleavage of the PAR-2 peptide. Analysis by mass spectrometry confirmed a single cleavage site, which corresponds to the activation site of the PAR-2 receptor. Triapsin induced dose-dependent NO release in cultured human umbilical vein endothelial cells (HUVECs), reaching a maximum effect at 17.58 nM. Triapsin purified by gel-filtration chromatography (10-16 to 10-9 M) was applied cumulatively to mouse mesenteric artery rings and showed a potent endothelium-dependent vasodilator effect (EC30 = 10-12 M). Nitric oxide seems to be partially responsible for this vasodilator effect because L-NAME (L-NG-nitroarginine methyl ester 300 µM), a nitric oxide synthetase inhibitor, did not abrogate the vasodilation activated by triapsin. Anti-PAR-2 antibody completely inhibited vasodilation observed in the presence of triapsin activity. Triapsin activity also induced an increase in the mouse ear venular diameter. Conclusion Data from this study suggest a plausible association between triapsin activity mediated PAR-2 activation and vasodilation caused by T. infestans saliva.(AU)

The effect of hirudin on antagonisting thrombin induced apoptosis of human microvascular endothelial cells

Purpose:To investigate whether hirudin exerts its antithrombin action to decrease the ratio of Human Microvascular Endothelial Cells (HMVECs) apoptosis.Methods:Human microvascular endothelial cells (HMVECs) cultured in the third and fifth generations were used. HMVECs were divided into normal group, thrombin group (T group), natrual hirudin group (H group), thrombin + natrual hirudin group (T + H group), AG490 group, thrombin + AG490 group (T + AG490 group), natrual hirudin + AG490 group (H + AG490 group), thrombin + natural hirudin + AG490 (T + H + AG490 group).Apart from the normal group, the other groups were exposed to the relevant drugs for 24 hours.HMVEC apoptosis was assessed by flow cytometric and double Immunofluorescence of phosphorylation of JAK (P-JAK2) and TUNEL assay.Results:Compared with the normal group, in thrombin group the HMVECs apoptosis rate were significantly increased (P<0.05).The results indicated that the index of apoptosis and the apoptosis rate were improved in cultures treated by natural hirudin (T + H group), relative to cultures with thrombin only (T group). We found that the index of apoptosis and the apoptosis rate in the AG490 + thrombin group were higher than that in the hirudin + thrombin group (P<0.05). Double Immunofluorescence of p-JAK2 and TUNEL assays showed that cells were double positive for P-JAK2 uptake and TUNEL detection liquid binding.Conclusion:The natural hirudin and JAK2/STATs signal inhibitor AG490 could block the effects of thrombin. Natural hirudin could attenuate HMVECs apoptosis via antagonizing thrombin and it is suggested that this effect may occur by blocking the JAK2/STATs signaling pathway and this signaling pathways appears to be not the only pathway.(AU)

Isolation and characterization of the human ovarian cell population for transplantation into an artificial ovary

To support survival and growth of follicles, the transplantable artificial ovary should mimic the original organ, offering a physical (3D matrix) and biological support (cells). In order to replicate the ovarian cell populations, the aim of this study is to assess the proportions of stromal and endothelial cells in the ovarian cortex. To this end, ovarian biopsies were obtained from six women (mean age: 49 years). The epithelial layer and medulla were carefully removed. The cortex was finely minced and enzymatically digested and the isolated cells were fixed. For cell characterization, immunostaining for CD31 (for endothelial cells) and inhibin-α (for granulosa cells) was performed. Positive cells in each staining were counted and the proportion of the different cell populations was estimated from the total number of isolated cells. Since there is no specific marker for ovarian stromal cells, we estimated the proportion of these cells by performing a vimentin immunostaining and subtracting the proportions of CD31- and inhibin-α-positive cells. Immunostaining showed that 84% of isolated cells were vimentin-positive. From this pool, 3% were endothelial cells and 1% granulosa cells. Consequently, the population of ovarian stromal cells was 80%. In conclusion, our findings show that stromal cells represent the larger population of cells in the human ovarian cortex. While this ensures follicle survival and development in a normal ovary, we believe that the low proportion of endothelial cells could have a negative impact on the angiogenesis in the artificial ovary after the first days of transplantation.

Isolation and characterization of the human ovarian cell population for transplantation into an artificial ovary

To support survival and growth of follicles, the transplantable artificial ovary should mimic the original organ, offering a physical (3D matrix) and biological support (cells). In order to replicate the ovarian cell populations, the aim of this study is to assess the proportions of stromal and endothelial cells in the ovarian cortex. To this end, ovarian biopsies were obtained from six women (mean age: 49 years). The epithelial layer and medulla were carefully removed. The cortex was finely minced and enzymatically digested and the isolated cells were fixed. For cell characterization, immunostaining for CD31 (for endothelial cells) and inhibin-α (for granulosa cells) was performed. Positive cells in each staining were counted and the proportion of the different cell populations was estimated from the total number of isolated cells. Since there is no specific marker for ovarian stromal cells, we estimated the proportion of these cells by performing a vimentin immunostaining and subtracting the proportions of CD31- and inhibin-α-positive cells. Immunostaining showed that 84% of isolated cells were vimentin-positive. From this pool, 3% were endothelial cells and 1% granulosa cells. Consequently, the population of ovarian stromal cells was 80%. In conclusion, our findings show that stromal cells represent the larger population of cells in the human ovarian cortex. While this ensures follicle survival and development in a normal ovary, we believe that the low proportion of endothelial cells could have a negative impact on the angiogenesis in the artificial ovary after the first days of transplantation.(AU)

Primary diffuse mammary hemangiosarcoma in a female dog

In veterinary medicine, primary mammary hemangiosarcoma is a very rare disease and there was no previous report describing the disease in dogs. Herein, we describe necropsy findings of a female dog, which presented a diffuse mammary hemangiosarcoma affecting the mammary gland. A diffuse irregular plaque was found in all mammary gland, involving the whole mammary gland tissue. The histopathological evaluation revealed neoplastic cells with an ovoid to spindle shaped basophilic and numerous atypical neoplastic tumor cells forming vascular structures. There was no glandular proliferation and no changes in the superficial dermis and no neoplastic emboli in the lymph vessels. Immunohistochemistry for vimentin, pan-cytokeratin, CK8/18 and CD31 was conducted. Positive expression was found to the pan-cytokeratin and CK8/18 by remaining epithelial cells confirmed the luminal mammary origin. Vimentin and CD31 positive expression by neoplastic cells confirmed the endothelial origin of the neoplasia. The histopathological and immunohistochemical findings supported the diagnosis of a primary mammary hemangiosarcoma.

Primary diffuse mammary hemangiosarcoma in a female dog

In veterinary medicine, primary mammary hemangiosarcoma is a very rare disease and there was no previous report describing the disease in dogs. Herein, we describe necropsy findings of a female dog, which presented a diffuse mammary hemangiosarcoma affecting the mammary gland. A diffuse irregular plaque was found in all mammary gland, involving the whole mammary gland tissue. The histopathological evaluation revealed neoplastic cells with an ovoid to spindle shaped basophilic and numerous atypical neoplastic tumor cells forming vascular structures. There was no glandular proliferation and no changes in the superficial dermis and no neoplastic emboli in the lymph vessels. Immunohistochemistry for vimentin, pan-cytokeratin, CK8/18 and CD31 was conducted. Positive expression was found to the pan-cytokeratin and CK8/18 by remaining epithelial cells confirmed the luminal mammary origin. Vimentin and CD31 positive expression by neoplastic cells confirmed the endothelial origin of the neoplasia. The histopathological and immunohistochemical findings supported the diagnosis of a primary mammary hemangiosarcoma.(AU)

Heparin modulates the expression of genes encoding pro and anti-apoptotic proteins in endothelial cells exposed to intestinal ischemia and reperfusion in rats

PURPOSE: To investigate if expression of genes encoding pro and anti-apoptotic proteins in the rat enteric endothelial cells stimulated by intestinal ischemia followed by reperfusion (IR) can be modified by treatment with heparin (HP). METHODS: Eighteen adult Wistar rats were divided in three groups: sham group submitted to laparotomy only (SG), ischemia followed by reperfusion group (IRG); ischemia followed by reperfusion plus pretreatment with HP 100 mg.kg-1 (IRG+HP). Ischemia was performed by clamping of the superior mesenteric artery. After 60 min of ischemia, metal clamps were removed for reperfusion for 120 min. Gene expression of encoding pro (Casp1, Casp6, Casp3, Cflar, Fas and Pgl) and anti-apoptotic (Bcl2, Bcl2l1 and Naip2) proteins in rat enteric endothelial cells was evaluated by PCR microarray method. RESULTS: Compared to rat endothelial cells of SG, the expression of pro-apoptotic genes was up-regulated in IRG while anti-apoptotic genes were down-regulated. In contrast, the expression of anti-apoptotic genes in IRG+HP was up-regulated while pro-apoptotic genes was down-regulated compared to SG. CONCLUSION: The attenuation by heparin of intestinal ischemia-reperfusion previously demonstrated in rodents could be related with ability of this drug to stimulate and reduce gene expression of encoding anti and pro-apoptotic proteins, respectively. (AU)

Babesia bovis: expression of adhesion molecules in bovine umbilical endothelial cells stimulated with plasma from infected cattle / Babesia bovis: expressão de moléculas de adesão em células endoteliais de cordão umbilical de bovinos estimuladas com plasma de bovinos infectados

Ten male, 12-month-old Jersey with intact spleens, serologically and parasitologically free from Babesia were housed individually in an arthropod-free isolation system from birth and throughout entire experiment. The animals were randomly divided into two groups. Five animals (group A) were intravenously inoculated with 6.6 X10(7) red blood cells parasitized with pathogenic sample of Babesia bovis (passage 7 BboUFV-1), for the subsequent "ex vivo" determination of the expression of adhesion molecules. Five non-inoculated animals (group B) were used as the negative control. The expression of the adhesion molecules ICAM-1, VCAM, PECAM-1 E-selectin and thrombospondin (TSP) was measured in bovine umbilical vein endothelial cells (BUVECs). The endothelial cells stimulated with a pool of plasma from animals infected with the BboUFV-1 7th passage sample had a much more intense immunostaining of ICAM-1, VCAM, PECAM-1 E-selectin and TSP, compared to the cells which did not received the stimulus. The results suggest that proinflammatory cytokines released in the acute phase of babesiosis may be involved in the expression of adhesion molecules thereby implicating them in the pathophysiology of babesiosis caused by B. bovis.(AU)

Dez bezerros machos, da raça Jersey, com 1 ano de idade com baços "in situ", sorológica e parasitologicamente livres de Babesia, foram mantidos em baias individuais no isolamento a prova de artrópodes do Depto de Veterinária desde o nascimento e ao longo de toda a experimentação. Os animais foram divididos aleatoriamente em dois grupos. Cinco animais (grupo A) foram inoculados por via intravenosa com 6,6 x10(7) hemácias parasitados com amostra patogênica de Babesia bovis (BboUFV - 1 7ª passagem) , para a determinação subseqüente "ex vivo" da expressão de moléculas de adesão . Cinco animais não inoculados (Grupo B ) foram utilizados como controlo negativo . A expressão de moléculas de adesão ICAM - 1, VCAM , PECAM - 1, E - selectina e trombospondina ( TSP ) foi medida em células endoteliais da veia umbilical de bovinos (BUVECs). As células endoteliais estimuladas com um pool de plasma proveniente de animais infectados com BboUFV - 1 7ª passagem tinham uma imunocoloração muito mais intensa de ICAM - 1 , VCAM , PECAM - 1 de E - selectina e de TSP , em comparação com as células que não receberam o estímulo . Os resultados sugerem que as citocinas pró-inflamatórias liberados na fase aguda da babesiose pode estar envolvida na expressão de moléculas de adesão , implicando , assim, elas na fisiopatologia da babesiose causada por B. bovis.(AU)

Ischemic preconditioning and the gene expression of enteric endothelial cell biology of rats submitted to intestinal ischemia and reperfusion

PURPOSE: To investigate the effects of ischemic preconditioning (IPC) on the expression of pro and anti-apoptotic genes in rat endothelial cells undergoing enteric ischemia (I) and reperfusion (R). METHODS: Thirty rats underwent clamping of the superior mesenteric vessels. Sham group (GS) laparotomy only; Ischemia (GI): intestinal ischemia (60 min); Ischemia and Reperfusion (GIR): ischemia (60 min) and reperfusion (120 min); Ischemia and intestinal ischemic preconditioning (GI + IPC) : 5 minutes of ischemia followed by 10 min of reperfusion before sustained ischemia (60 min) ischemia and reperfusion and IPC (GIR + IPC): 5 min ischemia followed by 10 min of reperfusion before sustained ischemia (60min) and reperfusion (120 min). Rat Endothelial Cell Biology (PCR array) to determine the expression of genes related to endothelial cell biology. RESULTS: Gene expression of pro-apoptotic markers (Casp1, Casp6, Cflar, Fas, and Pgl) was down regulated in GI+IPC and in GIR + IPC. In contrast, the expression of anti-apoptotic genes (Bcl2 and Naip2), was up-regulated in GI + IPC and in GIR + IPC. CONCLUSION: Ischemic preconditioning may protect against cell death caused by ischemia and reperfusion.(AU)

Aspectos celulares da interação de células endoteliais da veia do cordão umbilical humano e Toxoplasma gondii

O Toxoplasma gondii, um protozoário intracelular obrigatório, com distribuição cosmopolita é de grande relevância médica e veterinária. Toxoplasmose congênita representa uma das mais sérias consequências da infecção aguda da gestante podendo acarretar: transmissão vertical (19%); aborto (9%) ou má formação do feto (30%). Durante a transmissão congênita, as células endoteliais da veia do cordão umbilical (HUVEC) estão inseridas na principal rota de transmissão da infecção vertical. No entanto, esse modelo celular tem sido pouco explorado no contexto da toxoplasmose experimental. O principal objetivo dessa dissertação foi analisar aspectos celulares da interação de T. gondii e HUVEC in vitro. Assim, as culturas foram caracterizadas morfologicamente por microscopia óptica e eletrônica de transmissão e quanto a interação parasito-célula os seguintes parâmetros avaliados: cinética da susceptibilidade de HUVEC frente à infecção; comparação da capacidade infectiva de taquizoítos obtidos de diferentes fontes e de duas cepas polares (ME-49 WT e GFP e RH) e ainda, o destino intracelular do parasito. Culturas de HUVEC mantiveram as características morfológicas e funcionais preservadas como no sistema in vivo. As análises quantitativas mostraram que T. gondii é capaz de invadir e se multiplicar ativamente nessas células; taquizoítos da cepa RH são mais infectivos do que taquizoítos da cepa ME-49 WT e, que o método de obtenção dos parasitos pode influenciar na capacidade infectiva de taquizoítos provenientes do cultivo celular. As análises qualitativas realizadas por microscopia eletrônica de transmissão e citoquímica indicaram que a infecção de HUVEC gera ativação celular e altera a dinâmica de suas organelas em resposta à infecção, dentre elas, os corpúsculos de Weibel-Palade. Pela primeira vez, a conversão taquizoítos- bradizoítos e o estabelecimento da cistogênese em HUVEC foram demonstradas. O encistamento, nesta importante rota de infecção para o feto, pode atuar como repositório de parasitos ao longo da gestação, potencializando, por meio do rompimento dos cistos, re-infecções. Esse trabalho abre novas perspectivas para o estudo da interação de T. gondii e HUVEC in vitro e com isso potencialmente elucidar os mecanismos moleculares envolvidos na transmissão congênita

gondii, an obligate intracellular protozoan with cosmopolitan distribution is of great medical and veterinary relevance. Congenital toxoplasmosis represents one of the most serious consequences of the acute infection of the pregnant woman, which can lead to: vertical transmission (19%); abortion (9%) or malformation of the fetus (30%). During congenital transmission, the endothelial cells of the umbilical vein (HUVEC) are inserted in the main route of vertical transmission infection. However, this cellular model has been little explored in the context of experimental toxoplasmosis. The main objective of this dissertation was to analyze cellular aspects of the interaction of T. gondii and HUVEC in vitro. Thus, cultures were characterized morphologically by optical and transmission electron microscopy and as for the parasite-cell interaction the following parameters evaluated: kinetics of HUVEC susceptibility to infection; comparation of the infective capacity of tachyzoites obtained from different sources and from two polar strains (ME-49 WT and GFP and RH) and the intracellular fate of the parasite. Cultures of HUVEC maintained the morphological and functional characteristics preserved as in vivo. Quantitative analyzes showed that T. gondii is able to invade and multiply actively in these cells; tachyzoites of the RH strain are more infective than tachyzoites of the ME-49 WT strain and that the method of obtaining the parasites may influence the infective capacity of tachyzoites from the cell culture. Qualitative analyzes performed by transmission electron microscopy and cytochemistry indicated that HUVEC infection generates cellular activation and changes the dynamics of its organelles in response to infection, among them Weibel-Palade corpuscles. For the first time, tachyzoites-bradyzoites conversion and establishment of cystogenesis in HUVEC was demonstrated. Cysts formation, in this important route of infection to the fetus, can act as a repository of parasites throughout gestation, potentializing, through the rupture of the cysts, re-infections. This work opens new perspectives for the study of the interaction of T. gondii and HUVEC in vitro and thus can elucidate the molecular mechanisms involved in congenital transmission.

MORFOLOGIA DAS CÉLULAS DO ENDOTÉLIO DE DIFERENTES REGIÕES DA CÓRNEA DE CÃES (Canis familiaris) UTILIZANDO A MICROSCOPIA ÓPTICA

preservação da integridade funcional do endotélio da córnea após procedimentos cirúrgicos intraoculares é preconizada para a manutenção da sua transparência. Dessa maneira, o conhecimento e a avaliação da forma do endotélio nas diferentes regiões da córnea é de suma importância, pois os estudos a respeito da córnea de cães limitam-se a região central. Objetivou-se avaliar a forma das células endoteliais de cães (Canis familiaris) de diferentes regiões da córnea. Foram estudados 20 olhos hígidos de cães, machos ou fêmeas de diferentes faixas etárias, separados entre olhos direito e esquerdo. A morfologia do endotélio da córnea das regiões central, superior, inferior, temporal e nasal foi avaliada após a coloração com o vermelho de alizarina. As imagens do endotélio foram obtidas usando um microscópio óptico. O percentual médio de células hexagonais na região central da córnea foi de 80,6 ±4,3% para o olho esquerdo e de 78,8 ±4,7% para o direito; na região superior foi de 78,5 ±4,4% para o olho esquerdo e de 79,4 ±4,9% para o direito; na região inferior foi de 80,2 ±6,0% para o olho esquerdo e de 80,1 ±4,4% para o direito; na região temporal foi de 79,7 ±5,0% para o olho esquerdo e de 78 ±2,9% para o olho direito e, na região nasal, foi de 79,5 ±5,7% para o olho esquerdo e de 76,7 ±6,0% para o direito. O percentual médio de células com cinco lados, na região central da córnea, olho esquerdo, foi 11,9 ±3,0% e para o direito 13,2 ±3,8%; na região superior, no olho esquerdo, 13,6 ±1,8% e para o direito, 13 ±4,1%; na região inferior foi de 11,7 ±3,1% para o olho esquerdo e de 11,7 ±3,5% para o direito; na região temporal, olho esquerdo, 11,9 ±3,8% e para o direito 13,2 ±2,3% e na região nasal, olho esquerdo, 12,6 ±13,1% e, para o direito, 14,1 ±4,0%. O percentual médio de células com sete lados, na região central, olho esquerdo, foi 7,5 ±2,3% e para o direito 8 ±2,2%; na região superior, no olho esquerdo, 7,9 ±4% e para o direito, 7,6 ±2,3%; na região inferior foi de 7,9 ±3,4% para o olho esquerdo e de 8,2 ±3,2% para o direito; na região temporal, olho esquerdo, 8,4 ±2,0%, para o direito 8,8 ±1,3% e na região nasal, olho esquerdo, 8,6 ±2,6% e para o direito 9,2 ±2,9%. Os dados foram submetidos à análise de variância de fator duplo com medidas repetidas. Não houve diferença com relação a forma das células do endotélio nas diferentes regiões da córnea de cães.

The preservation of the functional integrity of the corneal endothelium after intraocular surgical procedures is recommended to maintain its transparency. Thus, assessment and knowledge of corneal endothelial morphology in dog has great importance, because the studies are limited the central areas of the cornea of dogs. This study aimed to evaluate the endothelial cells shape of dogs (Canis familiaris) in to different corneal areas. Twenty healthy dogs eyes were studied, males and females of different age groups, separated between right and left eyes. The corneal endothelium morphology of the central, superior, inferior, temporal and nasal areas was assessed by alizarin red staining 0.2%. The endothelium images were obtained using an optic microscope. The average percentage of hexagonal cells in the central area of the cornea, was 80.6 ±4.3% for the left eye and 78.8 ±4.7% for the right; in the superior area was 78.5 ±4.4% for the left eye and 79.4 ±4.9% for the right; in the inferior area was 80.2 ±6.0% for the left eye and 80.1 ±4.4% for the right; in the temporal area was 79.7 ±5.0% for the left eye and 78 ±2.9% for the right eye and nasal area was 79.5 ±5.7% for the left eye and 767 ±6.0% for the right. The average percentage of cells with five sides, in the central area of the cornea, left eye, was 11.9 ±3.0% and to the right 13.2 ±3.8%; in the superior area in the left eye, 13.6 ±1.8% and to the right, 13 ±4.1%; in the inferior area was 11.7 ±3.1% for the left eye and 11.7 ±3.5% for the right; in the temporal area, left eye, 11.9 ±3.8% and to the right 13.2 ±2.3%; in the nasal area, left eye, 12.6 ±13.1% and 14.1 ±4.0% right. The average percentage of cells with seven sides, central, left eye, was 7.5 ±2.3% and to the right 8 ±2.2%; in the superior area in the left eye, 7.9 ±4% and to the right, 7.6 ±2.3%; in the inferior area was 7.9 ±3.4% for the left eye and 8.2 ±3.2% for the right; in the temporal area, left eye, 8.4 ±2.0% and 8.8 ±1.3% right; in the nasal area, left eye, 8.6 ± 2.6% and to the right 9.2 ±2.9%. The data were subjected to two-way analysis of variance. There was no difference in endothelial cells shape in the different corneal areas of dog.

rACLF, a recombinant snake venom metalloprotease, activates endothelial cells in vitro

Snake venom metalloproteases (SVMPs) comprise a family of snake venom toxins responsible for most of local and systemic effects observed during envenomation by snakes from the Viperidae family. The vascular system and more specifically the endothelium seem to be the preferential targets of these proteins. This work describes the effects of rACLF, a recombinant SVMP from Agkistrodon contortrix laticinctus on human umbilical vein endothelial cells (HUVECs) in vitro. Our results showed that rACLF activates HUVECs by the release of mediators involved in inflammation and hemostasis such as prostacyclin and interleukin-8. We also demonstrated that rACLF increased the expression of ICAM-I and decay accelerating factor (DAF). Moreover, rACLF protects the HUVECs against apoptosis induced by serum deprivation. These results suggest that the endothelial cell activation induced by SVMPs may have a significant role in the development of the local inflammatory lesion observed in Viperidae envenomation.

ExoU contributes to late killing of Pseudomonas aeruginosa - infected endothelial cells

To ascertain the role of ExoU in late P. aeruginosa cytotoxicity, endothelial cells (EC) were exposed to wild type PA103, PA103deltaexoU and PA103::exsA for 1h and to gentamicin in culture medium. After 24h, the viability of PA103-infected cells (33.7 ± 14.3%) was significantly lower than the viability of PA103deltaexoU- (77.7 ± 6.3%) or PA103::exsA- (79.5 ± 23.3%) infected EC. P. aeruginosa cytotoxicity did not depend on the bacterial ability to interact with EC because the percentage of cells with associated PA103 (35.9 ± 15.8%) was similar to the percentage in PA103deltaexoU- (34.2 ± 16.0%) and lower than the percentage in PA103::exsA-infected cultures (82.9 ± 18.9%). Cell treatment with cytochalasin D reduced the PA103 internalization by EC but did not interfere with its ability to kill host cells.

Para determinar o papel de ExoU na citotoxicidade tardia de P. aeruginosa, células endoteliais (CE) foram expostas às cepas PA103, PA103deltaxoU e PA103::exsA por 1h e à gentamicina em meio de cultura. Após 24h, a viabilidade das CE infectadas com PA103 (33.7 ± 14.3%) foi inferior à de CE infectadas com PA103deltaexoU (77.7 ± 6.3%) e PA103::exsA (79.5 ± 23.3%). A citotoxicidade não dependeu da capacidade de interagir com as CE porque o percentual de células com bactérias associadas em culturas expostas a PA103 foi semelhante ao percentual em culturas expostas a PA103deltaexoU e inferior em culturas expostas a PA103::exsA. O tratamento das CE com citocalasina D reduziu a internalização de PA103, mas não interferiu em sua citotoxicidade.

ExoU contributes to late killing of Pseudomonas aeruginosa - infected endothelial cells

To ascertain the role of ExoU in late P. aeruginosa cytotoxicity, endothelial cells (EC) were exposed to wild type PA103, PA103deltaexoU and PA103::exsA for 1h and to gentamicin in culture medium. After 24h, the viability of PA103-infected cells (33.7 ± 14.3%) was significantly lower than the viability of PA103deltaexoU- (77.7 ± 6.3%) or PA103::exsA- (79.5 ± 23.3%) infected EC. P. aeruginosa cytotoxicity did not depend on the bacterial ability to interact with EC because the percentage of cells with associated PA103 (35.9 ± 15.8%) was similar to the percentage in PA103deltaexoU- (34.2 ± 16.0%) and lower than the percentage in PA103::exsA-infected cultures (82.9 ± 18.9%). Cell treatment with cytochalasin D reduced the PA103 internalization by EC but did not interfere with its ability to kill host cells.

Para determinar o papel de ExoU na citotoxicidade tardia de P. aeruginosa, células endoteliais (CE) foram expostas às cepas PA103, PA103deltaxoU e PA103::exsA por 1h e à gentamicina em meio de cultura. Após 24h, a viabilidade das CE infectadas com PA103 (33.7 ± 14.3%) foi inferior à de CE infectadas com PA103deltaexoU (77.7 ± 6.3%) e PA103::exsA (79.5 ± 23.3%). A citotoxicidade não dependeu da capacidade de interagir com as CE porque o percentual de células com bactérias associadas em culturas expostas a PA103 foi semelhante ao percentual em culturas expostas a PA103deltaexoU e inferior em culturas expostas a PA103::exsA. O tratamento das CE com citocalasina D reduziu a internalização de PA103, mas não interferiu em sua citotoxicidade.

ExoU contributes to late killing of Pseudomonas aeruginosa - infected endothelial cells

To ascertain the role of ExoU in late P. aeruginosa cytotoxicity, endothelial cells (EC) were exposed to wild type PA103, PA103deltaexoU and PA103::exsA for 1h and to gentamicin in culture medium. After 24h, the viability of PA103-infected cells (33.7 ± 14.3%) was significantly lower than the viability of PA103deltaexoU- (77.7 ± 6.3%) or PA103::exsA- (79.5 ± 23.3%) infected EC. P. aeruginosa cytotoxicity did not depend on the bacterial ability to interact with EC because the percentage of cells with associated PA103 (35.9 ± 15.8%) was similar to the percentage in PA103deltaexoU- (34.2 ± 16.0%) and lower than the percentage in PA103::exsA-infected cultures (82.9 ± 18.9%). Cell treatment with cytochalasin D reduced the PA103 internalization by EC but did not interfere with its ability to kill host cells.

Para determinar o papel de ExoU na citotoxicidade tardia de P. aeruginosa, células endoteliais (CE) foram expostas às cepas PA103, PA103deltaxoU e PA103::exsA por 1h e à gentamicina em meio de cultura. Após 24h, a viabilidade das CE infectadas com PA103 (33.7 ± 14.3%) foi inferior à de CE infectadas com PA103deltaexoU (77.7 ± 6.3%) e PA103::exsA (79.5 ± 23.3%). A citotoxicidade não dependeu da capacidade de interagir com as CE porque o percentual de células com bactérias associadas em culturas expostas a PA103 foi semelhante ao percentual em culturas expostas a PA103deltaexoU e inferior em culturas expostas a PA103::exsA. O tratamento das CE com citocalasina D reduziu a internalização de PA103, mas não interferiu em sua citotoxicidade.