Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart.

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α(2)-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α(2)-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α(2)-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α(2)-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α(2)-adrenergic receptor stimulation.

Phospholipase C gamma and ERK1/2 mitogen activated kinase pathways are differentially modulated by Trypanosoma cruzi during tissue invasion in human placenta.

Chagas' disease is caused by the haemophlagelated protozoan Trypanosoma cruzi (T. cruzi). During congenital transmission the parasite breaks down the placental barrier, however studies about the physiopathology of this process are scarce. Different signal transduction pathways are involved during cell invasion of the parasite. However, the possible role of those processes during tissue infection has not been studied. In the present study we analyzed the modulation of two signal transduction pathways, PLC-γ and ERK1/2 MAPK, during ex vivo infection of human placental chorionic villi explants. Chorionic villi from healthy woman placentas were incubated in the presence or absence of 10(5) or 10(6)T. cruzi trypomastigotes (DM28c strain) with or without specific inhibitors for each pathway. Effective infection was tested determining parasite DNA by PCR. The activation of PLC-γ and ERK1/2 MAPK signaling pathways was determined by western blotting and immunofluorescence. The low concentration of T. cruzi trypomastigotes activates both signaling pathways; however, the high concentration of parasite induces a modest activation of the PLC-γ pathway and impairs the ERK1/2 MAPK pathway activation. Interestingly, inhibition of any of those signaling pathways did not prevent parasite infection, as it was previously shown in cell cultures. We conclude that both signal transduction pathways are modulated during ex vivo T. cruzi infection of human placental chorionic villi explants.

Protection of vascular endothelium by aspirin in a murine model of chronic Chagas' disease.

Chronic Chagas' disease affects 10-30 % of patients infected with Trypanosoma cruzi, and it mainly manifests as cardiomyopathy. Important pathophysiological mechanisms involved in the cardiac lesions include activation of the endothelium and induced microvascular alterations. These processes involve the production of endothelial adhesion molecules and thromboxane A2, which are involved in inflammatory cell recruitment and platelet aggregation, respectively. Cyclooxygenase inhibitors such as aspirin decrease thromboxane production and alter the course of Chagas' disease, both in the acute and chronic phases. We studied the effects of the administration of low and high doses of aspirin during the early phase of T. cruzi infection, following microvascular damage in the context of a chronic murine model of Chagas' disease. The effects of both schedules were assessed at 24 and 90 days postinfection by evaluating parasitemia, mortality, and cardiac histopathological changes as well as the expression of ICAM, VCAM, and E-selectin in cardiac tissue. Thromboxane A2, soluble ICAM, and E-selectin blood levels were also measured. While aspirin did not affect parasitemia or mortality in the infected mice, it decreased both cardiac inflammatory infiltrates and thromboxane levels. Additionally, at 90 days postinfection, aspirin normalized sICAM and sE-selectin levels. Considering the improved endothelial function induced by aspirin, we propose the possibility of including this drug in clinical therapy to treat chronic Chagas' disease.

Trypanosoma cruzi infectivity assessment in "in vitro" culture systems by automated cell counting.

Chagas disease is an endemic, neglected tropical disease in Latin America that is caused by the protozoan parasite Trypanosoma cruzi. In vitro models constitute the first experimental approach to study the physiopathology of the disease and to assay potential new trypanocidal agents. Here, we report and describe clearly the use of commercial software (MATLAB(®)) to quantify T. cruzi amastigotes and infected mammalian cells (BeWo) and compared this analysis with the manual one. There was no statistically significant difference between the manual and the automatic quantification of the parasite; the two methods showed a correlation analysis r(2) value of 0.9159. The most significant advantage of the automatic quantification was the efficiency of the analysis. The drawback of this automated cell counting method was that some parasites were assigned to the wrong BeWo cell, however this data did not exceed 5% when adequate experimental conditions were chosen. We conclude that this quantification method constitutes an excellent tool for evaluating the parasite load in cells and therefore constitutes an easy and reliable ways to study parasite infectivity.

Toxic and therapeutic effects of Nifurtimox and Benznidazol on Trypanosoma cruzi ex vivo infection of human placental chorionic villi explants.

Nifurtimox (Nfx) and Benznidazole (Bnz) are the only available drugs in use for the treatment of Chagas disease. These drugs are recommended but not fully validated in evidence-based medicine and reports about the differential toxicity of both drugs are controversial. Here, we evaluated the toxic and therapeutic effects of Nfx and Bnz on human placental chorionic villi explants (HPCVE) during ex vivo infection of Trypanosoma cruzi, performing histopathological, histochemical, immunohistochemical as well as immunofluorescence analysis of the tissue. Additionally, we determined the effect of both drugs on parasite load by real time PCR. Bnz prevents the parasite induced tissue damage in ex vivo infected HPCVE compared to Nfx, which is toxic per se. The presence of T. cruzi antigens and DNA in infected explants suggests that these drugs do not impair parasite invasion into the HPCVE. Additionally, our results confirm reports suggesting that Bnz is less toxic than Nfx and support the need for the development of more effective and better-tolerated drugs.

Benznidazole prevents endothelial damage in an experimental model of Chagas disease.

OBJECTIVES: To evaluate the effect of benznidazole on endothelial activation in a murine model of Chagas disease. METHODS: A low (30mg/kg/day) and a high (100mg/kg/day) dose of benznidazole were administered to mice infected with Trypanosoma cruzi during the early phases of the infection. The effects of the treatments were assessed at 24 and 90 days postinfection by evaluating the parasitaemia, mortality, histopathological changes and expression of ICAM in the cardiac tissue. The blood levels of thromboxane A2, soluble ICAM and E-selectin were also measured. T. cruzi clearance was assessed by the detection of parasite DNA in the heart tissue of infected mice. RESULTS: Benznidazole decreased the cardiac damage induced by the parasite, and amastigote nests disappeared at 90 days postinfection. Both doses cleared the parasite from the cardiac tissue at 24 and 90 days postinfection. In addition, benznidazole decreased the thromboxane levels and normalized the plasma sICAM and sE-selectin levels by 90 days postinfection. CONCLUSIONS: Early administration of benznidazole at a dose as low as 30mg/kg eradicates T. cruzi from cardiac tissue. Additionally, benznidazole prevents cardiac damage and modulates endothelial activation as part of its antichagasic activity.

Reorganization of extracellular matrix in placentas from women with asymptomatic chagas disease: mechanism of parasite invasion or local placental defense?

Chagas disease, produced by the protozoan Trypanosoma cruzi (T. cruzi), is one of the most frequent endemic diseases in Latin America. In spite the fact that in the past few years T. cruzi congenital transmission has become of epidemiological importance, studies about this mechanism of infection are scarce. In order to explore some morphological aspects of this infection in the placenta, we analyzed placentas from T. cruzi-infected mothers by immunohistochemical and histochemical methods. Infection in mothers, newborns, and placentas was confirmed by PCR and by immunofluorescence in the placenta. T. cruzi-infected placentas present destruction of the syncytiotrophoblast and villous stroma, selective disorganization of the basal lamina, and disorganization of collagen I in villous stroma. Our results suggest that the parasite induces reorganization of this tissue component and in this way may regulate both inflammatory and immune responses in the host. Changes in the ECM of placental tissues, together with the immunological status of mother and fetus, and parasite load may determine the probability of congenital transmission of T. cruzi.