Katherine Athayde Teixeira de Carvalho

Os efeitos da pressão positiva intermitente e do incentivador respiratório no pós-operatório de revascularização miocárdica

BACKGROUND Pulmonary complications are important causes of morbidity and fatalities among patients subject to cardiac surgery. The respiratory physiotherapy has been aiding in the recovery of these patient ones. OBJECTIVE To evaluate the physiotherapeutic effect of intermittent positive pressure breathing (IPPB) and incentive spirometry (IS) in patients submitted to myocardial revascularization surgery. METHODS Forty patients were divided in two groups: one was submitted to IPPB (n=20) and the other to IS (n=20). The patients were evaluated at the preoperative period and 24, 48 and 72 hours postoperatively, with the resources being applied in the postoperative period. The following parameters were analyzed: oxygen saturation (SpO2), respiratory frequency (RF), minute volume (MV), current volume (CV), maximum inspiratory pressure (Ip max) and maximum expiratory pressure (Ep max). RESULTS The groups were considered homogeneous regarding the demographic and clinical variables. In the group submitted to IPPB, an increase in SpO2 was observed 48 (p=0.007) and 72 h (p=0.0001) after surgery, when compared to the IS group. As for the RF, MV and CV variables, there were no statistically significant differences between the groups. The group submitted to IS showed a significant increase in the Epmax 24 (p=0.02) and 48 (p=0.01) h after surgery. CONCLUSION Aiming at reversing hypoxemia earlier, IPPB showed to be more efficient when compared to IS; however, IS was more effective in improving respiratory muscle strength.

Evaluation of Bone Marrow Mesenchymal Stem Cell Standard Cryopreservation Procedure Efficiency

INTRODUCTION Mesenchymal stem cells are obtained from a variety of sources, particularly bone marrow. These cells have great potential for clinical research due to their potential to regenerate tissue. As is well known, the cryopreservation process can store any cell type, particularly blood cells, for an indeterminate time. OBJECTIVE The aim of this study was to analyze the efficiency of standard cryopreservation procedures for adult mesenchymal stem cells from bone marrow. METHODS Mononuclear stem cells isolated from 10 Wistar male rats were cultivated for 4 weeks to obtain mesenchymal stem cells. The parameters considered in this study were trypan blue exclusion test and annexin V conjugated with 7-amino-actinomycin for flow cytometry before cryopreservation in liquid nitrogen vapor phase for 1 month and after thawing. RESULTS The viabilities determined by the trypan blue exclusion test were 94.76% and 90.58%, and the flow cytometry assay (annexin V conjugated with 7-amino-actinomycin) were 85.52% and 66.25%, before cryopreservation and after thawing, respectively. CONCLUSIONS Standard procedures for cryopreservation were not efficient for those cells. The flow cytometry assay was more sensitive than the trypan blue exclusion test to demonstrate nonviability.

Stem Cell Therapy in Heart Diseases: A Review of Selected New Perspectives, Practical Considerations and Clinical Applications

Degeneration of cardiac tissues is considered a major cause of mortality in the western world and is expected to be a greater problem in the forthcoming decades. Cardiac damage is associated with dysfunction and irreversible loss of cardiomyocytes. Stem cell therapy for ischemic heart failure is very promising approach in cardiovascular medicine. Initial trials have indicated the ability of cardiomyocytes to regenerate after myocardial injury. These preliminary trials aim to translate cardiac regeneration strategies into clinical practice. In spite of advances, current therapeutic strategies to ischemic heart failure remain very limited. Moreover, major obstacles still need to be solved before stem cell therapy can be fully applied. This review addresses the current state of research and experimental data regarding embryonic stem cells (ESCs), myoblast transplantation, histological and functional analysis of transplantation of co-cultured myoblasts and mesenchymal stem cells, as well as comparison between mononuclear and mesenchymal stem cells in a model of myocardium infarction. We also discuss how research with stem cell transplantation could translate to improvement of cardiac function.

Stem cell death and survival in heart regeneration and repair.

Cardiovascular diseases are major causes of mortality and morbidity. Cardiomyocyte apoptosis disrupts cardiac function and leads to cardiac decompensation and terminal heart failure. Delineating the regulatory signaling pathways that orchestrate cell survival in the heart has significant therapeutic implications. Cardiac tissue has limited capacity to regenerate and repair. Stem cell therapy is a successful approach for repairing and regenerating ischemic cardiac tissue; however, transplanted cells display very high death percentage, a problem that affects success of tissue regeneration. Stem cells display multipotency or pluripotency and undergo self-renewal, however these events are negatively influenced by upregulation of cell death machinery that induces the significant decrease in survival and differentiation signals upon cardiovascular injury. While efforts to identify cell types and molecular pathways that promote cardiac tissue regeneration have been productive, studies that focus on blocking the extensive cell death after transplantation are limited. The control of cell death includes multiple networks rather than one crucial pathway, which underlies the challenge of identifying the interaction between various cellular and biochemical components. This review is aimed at exploiting the molecular mechanisms by which stem cells resist death signals to develop into mature and healthy cardiac cells. Specifically, we focus on a number of factors that control death and survival of stem cells upon transplantation and ultimately affect cardiac regeneration. We also discuss potential survival enhancing strategies and how they could be meaningful in the design of targeted therapies that improve cardiac function.

O transplante em conjunto de células mioblásticas esqueléticas e mesenquimais (cocultivadas) na disfunção ventricular pós-infarto do miocárdio

OBJECTIVE: Cell therapy in the myocardium has been mainly performed with satisfactory results using 2 cell types: skeletal myoblasts (myogenic) and mesenchymal cells (angiogenic). This study assessed the combined transplantation of those 2 cell types (SMM) into infarcted rats. METHODS: Myocardial infarction was induced by ligature of the left coronary artery in 26 Wistar rats. After one week, the animals underwent echocardiography for assessing ejection fraction (EF%) and left ventricular end-diastolic and systolic volumes (EDV, ESV, mL). After 2 days, the animals were reoperated on and divided into 2 groups: 1) control (n=10), which received 0.15 mL of culture medium; and 2) SMM (n=16), which received 7.5x106 heterologous skeletal myoblasts and mesenchymal cells in the infarcted region. The cells were obtained from puncture of the iliac crest and biopsy of skeletal muscle, and were cultured in vitro. After one month, the animals underwent a new echocardiography. RESULTS: No significant difference in EF, EDV, and ESV was observed between the 2 groups on baseline echocardiographic values. One month after transplantation, the following was observed: a reduction in EF in the control group (29.31 ± 5.6% to 23.54 ± 6.51%; P=0.048); and an increase in EF in the SMM group (24.03 ± 8.68% to 31.77 ± 9.06%; P=0.011). The presence of neovascularization and muscle fibers was identified in the regions of myocardial fibrosis in the SMM group. CONCLUSION: Cocultivation of skeletal myoblasts and mesenchymal cells is functionally effective.

Controversies About the Chromosomal Stability of Cultivated Mesenchymal Stem Cells: Their Clinical Use is it Safe?

The usefulness of adult stem cells in research and therapeutic applications highly relies on their genomic integrity and stability. Many laboratories including ours have addressed this concern using methods such as karyotyping, Qbanding, fluorescent in situ hybridization, array CGH, flow cytometry and Pap test to evaluate number and structure of chromosomes and cellular phenotype. This review attempts to summarize the findings reported so far for the studies on chromosomal aberrations in adult stem cells and warrant to perform certain basic tests before transplantation to avoid any adverse reactions, which will thus aid in better therapeutic output after cellular transplantation in the treatment of various diseases.

Effect of exercise associated with stem cell transplantation on ventricular function in rats after acute myocardial infarction

OBJECTIVE To analyze the functional and anatomical-pathological effect of transplantation of bone marrow mononuclear cells associated to aquatic physical activity after myocardial infarction in rats. METHODS Twenty-one rats were induced by myocardial infarction, through left coronary artery ligation. After a week, the animals were subjected to echocardiography for evaluation of left ventricle ejection fraction (LVEF, %) and dyastolic and end systolic volume of the left ventricle (EDV, ESV, ml), randomized and the transplantation of mononuclear stem cells. The animals were divided into four groups: sedentary group without cells (n=5), sedentary with cells (n=5), trained without cells (n=5) and trained with cells (n=6). The physical training was started 30 days after infarction and held in swimming during 30 days. At the beginning and at the end of the physical training protocol were held assay of lactate. The animals have been subjected to new echocardiography after 60 days of myocardial infarction. RESULTS Two months after the transplant, were observed decrease in FE in the control group (35.2 to 23.54 P=0.022) and addition of LVEF and stabilization of ventricular remodeling in the group trained with cells (29.85 to 33.43% P=0.062 and 0.71 to 0.73 ml, P=0.776, respectively). Identified the reduction of collagen fibers, myocardial fibrosis regions in the group trained with and without cells. CONCLUSION The group trained with cells improves ventricular function compared to the control group, suggesting the benefit of associated cell therapy will physical activity.

Pap Test as the First Step in Screening Genetic Stability in Cell-BasedTherapy

The possibility of cell modifications compromises the safety of stem cell therapy under standardized conditions. The aim of this study was to analyze adipose tissue-derived mesenchymal stem cells (AT-MSCs) using the Pap test as a first screening step to evaluate genetic stability. Methods: Human adipose tissue from six healthy female donors was obtained from elective liposuction procedures. The cells were isolated, cultivated at P2/P3, characterized by flow cytometric analysis, and differentiation induced. The AT-MSCs were stained by Papanicolaou (Pap) staining and analyzed according to the Bethesda classification, and viability-apoptosis relationships were evaluated. Results: The Pap test for Sample I indicated high-grade alterations consistent with genetic instability; for Samples II-V, atypical cells of undetermined significance; and for Sample VI, normal cells. Conclusions: These results demonstrate the potential of using the Pap test as an initial screening step to evaluate the genetic stability of cultured AT-MSCs as well for other adherent stem cells.

Cellular transplant: functional, immunocytochemical and histopathologic analysis in an experimental model of ischemic heat disease using different cells

OBJECTIVE: To present the functional, immunocytochemical and histopathologic results (in vitro or in heart specimens) after isolation, culture and co-culture of mesenchymal stem cells and skeletal myoblast cells transplanted and co-transplanted in experimental animals with ischemic heart disease and left ventricular ejection fractions lower than 40%. METHOD: We utilized 72 Wistar rats, divided into four groups according to the culture media or injected cells: control group into which only culture media was injected (22 rats); mesenchymal stem cell group (17 rats); myoblast skeletal cell group (16 rats) and co-culture group (17 rats). In the immunohistochemical studies, the cells were stained with anti-vimentin, anti-desmin and anti-myosin. In the histopathologic analysis, slides were stained with Gomori Trichrome, and neo-vessels and muscle tissues were identified. In the functional analysis the left ventricle ejection fraction was analyzed one week after myocardial infarction and one month after the injection. RESULTS: The initial left ventricle ejection fraction (control echo) was not statistically significant between the four groups (P=0.276), but was significantly different in the follow-up examination (P=0.001). This difference was seen between the control and the myoblast skeletal cells groups (P=0.037), between the control and the co-culture groups (P<0.001), and between the mesenchymal stem cell and co-culture groups (P=0.025). When the initial and final echocardiograms in each group were compared, the control group deteriorated (P=0.005) and the co-culture group improved (P=0.006). With the immunocytochemical in vitro analysis, mesenchymal stem cells were identified when stained with anti-vimentin and muscle cells when stained with anti-desmin. In the heart specimens, muscle tissue, stained with anti-desmin and skeletal myoblasts cells, stained with fast anti-miosin were identified. In the histopathologic analysis, new vessels were observed in the mesenchymal stem cell and skeletal myoblast groups, and muscular tissue, angiogenesis and myogenesis in the co-culture group. CONCLUSION: The left ventricle ejection fraction improved in the group in which muscle cells were injected and more strikingly in the co-culture group. The immunohistochemical findings in the culture and co-culture groups evidenced the corresponding cells. In the heart specimens, muscle and skeletal myoblast cells were found. In the histopathologic examination, new vessels and muscle tissue were found in the mesenchymal stem cell, skeletal myoblast cell and co-culture groups.

Avaliação ecocardiográfica evolutiva do infarto do miocárdio em ratos jovens e adultos

BACKGROUND: The regeneration of cardiomyocytes after a myocardial infarction (MI) is more evident in young animals; however, it is not known whether it is associated with functional improvement. OBJECTIVE: To perform the functional analysis by echocardiography (echo) of young adult rats submitted to MI. METHODS: Seventy-two animals were included in the study: 35 young rats (group Y) that were 28 days old and 37 adult rats (group A) that were 153 days old. The rats were subdivided in two subgroups: infarcted (YI and AI) and control (YC and AC). The animals were assessed by echocardiogram on the 7thand 30th postoperative days for the analysis of the ejection fraction (EF) and the final systolic (FSV) and diastolic volume (FDV) of the left ventricle. Only animals with EF < 40% were included in the study. RESULTS: The comparison of the FDV and FSV between infarcted and control animals showed that there was a significant increase in infarcted adult animals at the two analyzed phases. Among young animals only the FSV was significantly higher on the 7th day. The intragroup evolution analysis showed an increase in FDV and FSV in the two young subgroups, which was proportional to growth and only increase in FDV in the infarcted adult group. There was an improvement in EF in young rats, whereas EF remained decreased in adult rats when compared to controls. CONCLUSION: The infarcted young rats presented improvement in the systolic function and ventricular volumes 30 days after the infarction, whereas the adult rats presented increased FDV with no improvement in systolic function.