High number of transplanted stem cells improves myocardial recovery after AMI in a porcine model.

OBJECTIVE: The clinical data considering the bone marrow mononuclear cell (BMMNC) therapy in treatment for acute myocardial infarction (AMI) are controversial and the mechanisms remain unknown. Our objective was to study the cardiac function and changes in cytokine levels after administration of BMMNC in experimental AMI model. DESIGN: Unlabeled or Super-Paramagnetic-Iron-Oxide-labeled BMMNCs or saline was injected into myocardium of 31 pigs after circumflex artery occlusion. Ejection fraction (EF) was measured preoperatively, postoperatively and at 21 days by echocardiography. Cardiac MRI was performed postoperatively and after 21 days in 7 BMMNC animals. Serum cytokine levels were measured at baseline, 24 h and 21 days. Cellular homing was evaluated comparing MRI and histology. RESULTS: From baseline to 21 days EF decreased less in BMMNC group (EF mean control -19 SD 12 vs. BMMNC -4 SD 15 percentage points p = 0.02). Cytokine concentrations showed high variability between the animals. MRI correlated with histology in cell detection and revealed BMMNCs in the infarction area. By MRI, EF improved 11 percentage points. The improvement in EF was associated with the number of transplanted BMMNCs detected in the myocardium. CONCLUSION: BMMNC injection after AMI improved cardiac function. Quantity of transplanted BMMNCs correlated with the improvement in cardiac function after AMI.

Analysis of molecular changes after autologous cell therapy in swine myocardial infarction tissue can reveal novel targets for future therapy.

Although several studies have demonstrated a functional recovery of infarcted myocardial tissue after cell therapy, little is known about the molecular mechanisms behind it. The aim of this study was to characterize the effect of cell therapy at the molecular level to screen for novel target candidates for future therapy of infarcted myocardial tissue. We used a swine acute myocardial infarction model evoked by transient occlusion of the circumflex coronary artery. Autologous bone marrow-derived mononuclear cells (BMMCs) or saline were injected intramyocardially or into the circumflex coronary artery. Samples for protein and RNA analysis were collected from the infarction area and healthy myocardium after a 3 week recovery period and analysed by two-dimensional gel electrophoresis (2DE) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Proteomic screening detected 13 protein spots which were altered after infarction but had been restored by BMMC treatment. The identification of seven proteins by mass spectrometry revealed that five proteins with decreased expression after infarction corresponded to mitochondrial proteins involved in energy metabolism. Their restored levels after BMMC treatment indicate their involvement in the recovery of heart function. In contrast, the elevated levels of α-crystallin B chain and cathepsin D after infarction suggest an involvement in the pathological mechanisms causing a decreased heart function. This study reveals that cell therapy with BMMCs after myocardial infarction causes restoration of several altered protein levels after 3 weeks and identifies potential marker proteins involved in the pathology of infarction.

Intra-arterial bone marrow mononuclear cell distribution in experimental global brain ischaemia.

OBJECTIVES: Bone marrow mononuclear cells (BM-MNCs) can ameliorate focal ischaemic brain injury. A global ischaemic brain injury, which can occur after cardiac or thoracic surgery, could be an essential target for BM-MNCs. No studies using BM-MNCs for this indication have been conducted. DESIGN: Ten porcine underwent a global normothermic ischaemic insult, followed by an intra-arterial injection of Technetium(99m)-HMPAO-labelled BM-MNCs after 2, 4, 6, 12 or 24 hours. A whole-body scan and a SPECT/CT were performed 2 hours after the injection. Severity of the injury was assessed with EEG and tissue biopsies were analysed by scintigraphy. RESULTS: The majority of the cells appeared in the lungs and the liver. Only a minimal number of cells were located in the brain. Median distribution of cells between organs in all animals was as follows: lungs 32.7% (30.6-38.2), liver 14.2% (12.0-17.2), spleen 7.3% (3.3-11.3) and kidneys 2.5% (2.0-3.3). The transplanted cells could not be detected within the brain tissue by radionuclide imaging. CONCLUSIONS: Intra-arterially transplanted BM-MNCs did not migrate to the damaged brain tissue in significant quantity when transplanted during the first 24 hours after the global ischaemic insult, contrary to results with models of focal brain injury.

Activity of mesenchymal stem cells in a nonperfused cardiac explant model.

Stem cell therapy represents a potential novel additional therapy for acute myocardial infarction. Cardiac applications of stem cell therapy are now undergoing clinical trials though many properties, including localization, possible adhesion, and infiltration of the injected stem cells in the myocardium, have not been studied in detail even in vitro. To study these mechanisms in a controlled microenvironment, we developed a model where mesenchymal stem cells (MSCs) were transported into live, cultured cardiac explants for further co-culture. About 10×10(3) porcine MSCs were injected into freshly excised and isolated cardiac explants of the pig. The explants were present in the culture medium for up to 7 days, with the time course of viability of the myocardial tissue, and the migration and the localization of the injected MSCs were analyzed with histological and immunohistological stainings. The myocyte structure was observed to be well preserved, and proliferation of capillaries and myofibroblasts was detected at the explant periphery. There were injected MSCs localized in the capillaries and in contact with the endothelial cells. The migration range and the number of adherent MSCs increased over time, suggesting active movement of MSCs in the explant. Our results suggest that this cardiac explant culture model is a feasible method for studying the effects of stem cells in the myocardium in vitro.

Granulation tissue is altered after intramyocardial and intracoronary bone marrow-derived cell transfer for experimental acute myocardial infarction.

BACKGROUND: Bone marrow-derived mononuclear cell (BMMC) treatment in acute myocardial infarction (AMI) has been shown to have a beneficial effect. Our objective was to study in detail the histopathological process after the cell therapy after intramyocardial (IM) or intracoronary (IC) administration of BMMCs following experimental AMI. METHODS: Twenty-fours pigs were randomized to the IM group (n=8), the IC group (n=8), and the control group (n=8).After 90 min of transient occlusion of the circumflex coronary artery, BMMCs were injected either intramyocardially or by a transfemoral catheter into the circumflex coronary artery. Echocardiography was performed preoperatively, postoperatively, and after a 21-day recovery period. The heart biopsies were examined histopathologically. Volumetric ex vivo CT scan was performed to evaluate calcification of the infarcted myocardium. RESULTS: The ejection fraction (EF) showed significant recovery in the IM group compared to the control group at Day 21 (P=.05). Despite beneficial histological changes in the infarction site in the IC group, compared to the control group, EF failed to recover. Reduction of collagen density that depicts scar formation was seen in both cell therapy groups compared to the control (P<.001). The number of mitotic cells was higher in the control group compared to the cell therapy groups (P<.001). The IC and IM groups differed significantly from each other in muscle-specific actin staining (P<.001) and smooth muscle actin staining (P<.004). The IM therapy group showed higher density for both stainings. Additionally, macrophage density was higher in the IC group compared to the IM and control groups (P<.002). Both cell therapy regimens substantially diminished tissue calcification; due to the large variation, the effect was not statistically significant. CONCLUSION: BMMC therapy launches cellular changes that affect mostly the repair process in the granulation tissue. The cell transplantation method might have some effect on the magnitude of the effect.

Acute homing of bone marrow-derived mononuclear cells in intramyocardial vs. intracoronary transplantation.

OBJECTIVES: Cell homing optimisation after transplantation is critical in myocardial infarction (MI) cell therapy. DESIGN: Eight pigs were randomized to receiving autologous purified (111)indium-labeled bone marrow mononuclear cells (BMMCs) (10(8) cells/2 ml) by intramyocardial (IM) (n=4) or by intracoronary (IC) (n=4) transplantation after 90 minutes occlusion of the CX-coronary artery. Dual isotope SPECT imaging was performed 2 and 24 hours postoperatively. Two animals were additionally analyzed on the sixth postoperative day. Tissue samples from the major organs were analyzed. RESULTS: In SPECT imaging revealed that BMMCs administered using IM injection remained in the injured area. In contrast, minor proportion of IC transplanted cells remained in the myocardium, as most of the cells showed homing in the lungs. Analysis of the biopsies showed a seven-fold greater number of cells in the myocardium for the IM method and a 10-fold greater number of cells in the lungs in the IC group (p < 0.001). CONCLUSIONS: In producing persistently high cell homing at the infarction site, the IM transplantation is superior to the IC transplantation. However, the IC administration might be more specific in targeting injured capillaries and epithelial cells within the infarcted myocardium.

Bone marrow-derived mononuclear cell transplantation improves myocardial recovery by enhancing cellular recruitment and differentiation at the infarction site.

OBJECTIVE: Stem cell therapy in myocardial infarction is under intensive investigation; however, the mechanisms of recovery and the optimal transplantation technique remain controversial. The goal of this controlled and randomized study was to test the hypothesis that locally injected bone marrow-derived mononuclear cells can focus in on the damaged myocardium and improve cardiac function by means of active participation in remodeling. METHODS: Myocardial infarction was introduced through occlusion of the circumflex coronary artery for 90 minutes in 14 piglets (24.0 +/- 4.9 kg) that were randomized to a cell-therapy group (n = 7) and a control group (n = 7). At reperfusion, autologous purified prelabeled or unlabeled cells (10(8) cells/2 mL) or saline were injected into the myocardium. Cardiac function was measured by using echocardiography preoperatively and postoperatively and at 3 weeks, when hearts were collected for histopathologic examination. RESULTS: The ejection fraction recovered in the cell-therapy group (P = .02) but failed to recover in the control group, and at 3 weeks, it remained at the lower level compared with that in the cell-therapy group (P = .067). The number of living cells in the necrotic area was significantly greater in the cell-therapy group (P < .001). Labeled cells were detected in the infarcted area, and they showed signs of myocyte differentiation. Furthermore, the proportional area of muscle actin-positive cells at the granulation area was higher in the cell-therapy group (P = .035). CONCLUSIONS: Autologous bone marrow-derived mononuclear cells at the infarcted area localize in the myocardium. The exact mechanism of recovery remains to be determined, but our findings may give new information concerning the cellular events that occur during cell therapy-enhanced recovery.

Effects of pH management during selective antegrade cerebral perfusion on cerebral microcirculation and metabolism: alpha-stat versus pH-stat.

BACKGROUND: Selective cerebral perfusion (SCP) is used for extending the period during which surgical procedures can be safely performed. We sought to determine the direct effects of pH management on cerebral microcirculation and metabolism during SCP. METHODS: An experimental SCP porcine model was created by selectively allowing cold perfusate only into the bicarotid brachiocephalic trunk during the SCP period. Twenty-four piglets (6 to 8 weeks; mean weight, 26.1 +/- 4.1 kg) underwent 15-minute normothermic cardiopulmonary bypass, 45-minute cooling cardiopulmonary bypass, 60-minute SCP at 25 degrees C, and 45-minute rewarming cardiopulmonary bypass with either alpha-stat or pH-stat perfusion strategy randomly assigned. A cranial window was created over the parietal cortex for visualization of the cerebral vessels with intravital microscopy. Rhodamine-stained leukocytes were observed in cerebral postcapillary venules for adhesion and rolling. Microdialysis analysis was used for determination of brain metabolism. RESULTS: Brain concentration of lactate was significantly higher in the alpha-stat group at 45 minutes of SCP, and at 15- and 45-minute rewarming intervals (p = 0.03; p = 0.003; and p = 0.05; respectively), reaching borderline statistical significance when assessed throughout the experiment (p = 0.06 for differences between groups). Further, at the end of cooling, the oxygen delivery tended to be higher in the pH-stat group (p = 0.07), whereas at the 30-minute rewarming interval, the oxygen extraction tended to be higher in the alpha-stat group (p = 0.06). There were no statistically significant differences between the groups in leukocyte-endothelial interaction, arterial diameter, or tissue oxygenation. CONCLUSIONS: The higher concentration of brain lactate and the tendency to higher oxygen extraction levels during rewarming with alpha-stat strategy suggests anaerobic metabolism occurred during SCP. No major differences between pH management strategies in cerebral microcirculation could be shown during SCP.

Leukocyte filtration to decrease the number of adherent leukocytes in the cerebral microcirculation after a period of deep hypothermic circulatory arrest.

OBJECTIVE: Cardiopulmonary bypass and hypothermic circulatory arrest induce a systemic inflammatory response, including a cascade of leukocyte and endothelial cell activity, during the postischemic reperfusion phase. Accumulation of leukocytes in the brain can lead to neurologic problems after cardiac surgery. The beneficial effects of a leukocyte-depleting filter have been documented, but because of contradictory results the underlying function of the filter remains unclear. METHODS: Twenty-two juvenile piglets (6 to 8 weeks) were randomly assigned to undergo cardiopulmonary bypass with or without a leukocyte-depleting filter 60 minutes before and 60 minutes after a 75-minute hypothermic circulatory arrest at 18 degrees C. The cerebral vessels were visualized with intravital microscopy through a cranial window placed over the parietal cortex. Rhodamine staining was used to observe adherent and rolling leukocytes in the cerebral postcapillary venules. The animals were electively killed 1 hour after weaning from cardiopulmonary bypass. RESULTS: There were no significant differences between the study groups regarding hemodynamic data. Numbers of adherent activated leukocytes were lower in the leukocyte filtration group, reaching borderline statistical significance when assessed throughout the experiment (between-groups P = .069) and actual statistical significance when assessed during the rewarming period (between-groups P = .029). CONCLUSION: The leukocyte-depleting filter succeeded in reducing the number of adherent leukocytes during the reperfusion period in an experimental operation with deep hypothermic circulatory arrest. Such a filter thus could mitigate cerebral reperfusion injury after cardiac surgery.

Propofol is associated with impaired brain metabolism during hypothermic circulatory arrest: an experimental microdialysis study.

BACKGROUND: Propofol is a widely used anesthetic in cardiac surgery. It has been shown to increase cerebrovascular resistance resulting in decreased cerebral blood flow. Efficient brain perfusion and tissue oxygenation during cardiopulmonary bypass (CPB) is essential in surgery requiring hypothermic circulatory arrest (HCA). The effects of propofol on brain metabolism are reported in a surviving porcine model of HCA. METHODS: Twenty female juvenile pigs undergoing 75 minutes of HCA at a brain temperature of 18 degrees C were assigned to either propofol- or isoflurane anesthesia combined with alpha-stat perfusion strategy during CPB cooling and rewarming. Brain microdialysis analysis was used for determination of brain metabolism, and tissue oxygen partial pressure and intracranial pressures were also followed-up until 8 hours postoperatively. RESULTS: Brain concentrations of glutamate and glycerol were significantly higher in the propofol group throughout the experiment (P < .01 and P < .01, respectively). The lactate/pyruvate ratio was significantly higher in the propofol group at 6-, 7-, and 8-hour intervals (P < .05, P < .01, and P < .05, respectively). The intracranial pressure was significantly higher at the 8-hour postoperative interval (P < .05) in the propofol group. A trend toward higher brain oxygen concentrations was observed in the isoflurane group. CONCLUSIONS: Anesthesia with propofol as compared with isoflurane is associated with impaired brain metabolism during experimental HCA.