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.

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.

Remote ischemic preconditioning protects the brain against injury after hypothermic circulatory arrest.

BACKGROUND: Ischemic preconditioning (IPC) is a mechanism protecting tissues from injury during ischemia and reperfusion. Remote IPC (RIPC) can be elicited by applying brief periods of ischemia to tissues with ischemic tolerance, thus protecting vital organs more susceptible to ischemic damage. Using a porcine model, we determined whether RIPC of the limb is protective against brain injury caused by hypothermic circulatory arrest (HCA). METHODS AND RESULTS: Twelve piglets were randomized to control and RIPC groups. RIPC was induced in advance of cardiopulmonary bypass by 4 cycles of 5 minutes of ischemia of the hind limb. All animals underwent cardiopulmonary bypass followed by 60 minutes of HCA at 18°C. Brain metabolism and electroencephalographic activity were monitored for 8 hours after HCA. Assessment of neurological status was performed for a week postoperatively. Finally, brain tissue was harvested for histopathological analysis. Study groups were balanced for baseline and intraoperative parameters. Brain lactate concentration was significantly lower (P<0.0001, ANOVA) and recovery of electroencephalographic activity faster (P<0.05, ANOVA) in the RIPC group. RIPC had a beneficial effect on neurological function during the 7-day follow-up (behavioral score; P<0.0001 versus control, ANOVA). Histopathological analysis demonstrated a significant reduction in cerebral injury in RIPC animals (injury score; mean [interquartile range]: control 5.8 [3.8 to 7.5] versus RIPC 1.5 [0.5 to 2.5], P<0.001, t test). CONCLUSIONS: These data demonstrate that RIPC protects the brain against HCA-induced injury, resulting in accelerated recovery of neurological function. RIPC might be neuroprotective in patients undergoing surgery with HCA and improve long-term outcomes. Clinical trials to test this hypothesis are warranted.

Intestinal bacterial translocation and tight junction structure in acute porcine pancreatitis.

BACKGROUND/AIMS: To examine whether intestinal bacterial translocation occurs early in acute mild and severe pancreatitis and whether the intestinal expression of tight junction proteins (claudins-2, -3, -4, -5, -7), apoptosis or proliferation would explain the possible translocation. METHODOLOGY: Fifteen pigs were randomized to controls (n=5) or to develop mild edematous pancreatitis (n=5, saline infusion to pancreatic duct) or severe necrotic pancreatitis (n=5, taurocholic acid infusion). Translocation was studied by measuring bacterial cultures from portal vein blood and mesenteric lymph nodes. Immunohistochemical expression of the tight junction proteins, apoptosis rate (TUNEL) and Ki-67 were analyzed quantitatively from the epithelium of the jejunum and colon. RESULTS: There was no bacterial translocation during the 6 hours followup, nor changes in the expression of tight junction proteins claudins-2 and -5 in jejunum or colon. Saturation and proportional area of claudin-3 staining decreased in the colon, as did claudins-4 and -7 staining in the jejunum of the necrotic pancreatitis group. Increased apoptosis was found in all samples from controls and the edematous pancreatitis group but not in jejunum in the necrotic pancreatitis group. Ki-67 activity tended to increase in the upper half of the villus in edematous and necrotic pancreatitis. There were no changes in the basic histology. CONCLUSIONS: The major finding of this study was that bacterial translocation from the gut is not present at the beginning of acute pancreatitis. Tight junction proteins claudin-2 and -5 do not become altered in the early stages of pancreatitis. Claudin-3 decreases in the colon and claudins-4 and -7 in the jejunum in necrotic pancreatitis. Laparotomy itself causes increased apoptosis in the colon and the jejunum.

Two novel direct SPIO labels and in vivo MRI detection of labeled cells after acute myocardial infarct.

BACKGROUND: Acute myocardial infarction (AMI) is a leading cause of morbidity and mortality worldwide. Cellular decay due hypoxia requires rapid and validated methods for possible therapeutic cell transplantation. PURPOSE: To develop direct and rapid superparamagnetic iron oxide (SPIO) cell label for a large-animal model and to assess in vivo cell targeting by magnetic resonance imaging (MRI) in an experimental AMI model. MATERIAL AND METHODS: Bone marrow mononuclear cells (BMMNCs) were labeled with SPIO particles using two novel direct labeling methods (rotating incubation method and electroporation). Labeling, iron incorporation in cells and label distribution, cellular viability, and proliferation were validated in vitro. An AMI porcine model was used to evaluate the direct labeling method (rotating incubation method) by examining targeting of labeled BMMNCs using MRI and histology. RESULTS: Labeling (1 h) did not alter either cellular differentiation potential or viability of cells in vitro. Cellular relaxation values at 9.4 T correlated with label concentration and MRI at 1.5 T showing 89 ± 4% signal reduction compared with non-labeled cells in vitro. In vivo, a high spatial correlation between MRI and histology was observed. The extent of macroscopic pathological myocardial changes (hemorrhage) correlated with altered function detected on MRI. CONCLUSION: We demonstrated two novel direct SPIO labeling methods and demonstrated the feasibility of clinical MRI for monitoring targeting of the labeled cells in animal models of AMI.

Bone Marrow Mononuclear Cell Transplantation Restores Inflammatory Balance of Cytokines after ST Segment Elevation Myocardial Infarction.

BACKGROUND: Acute myocardial infarction (AMI) launches an inflammatory response and a repair process to compensate cardiac function. During this process, the balance between proinflammatory and anti-inflammatory cytokines is important for optimal cardiac repair. Stem cell transplantation after AMI improves tissue repair and increases the ventricular ejection fraction. Here, we studied in detail the acute effect of bone marrow mononuclear cell (BMMNC) transplantation on proinflammatory and anti-inflammatory cytokines in patients with ST segment elevation myocardial infarction (STEMI). METHODS: Patients with STEMI treated with thrombolysis followed by percutaneous coronary intervention (PCI) were randomly assigned to receive either BMMNC or saline as an intracoronary injection. Cardiac function was evaluated by left ventricle angiogram during the PCI and again after 6 months. The concentrations of 27 cytokines were measured from plasma samples up to 4 days after the PCI and the intracoronary injection. RESULTS: Twenty-six patients (control group, n = 12; BMMNC group, n = 14) from the previously reported FINCELL study (n = 80) were included to this study. At day 2, the change in the proinflammatory cytokines correlated with the change in the anti-inflammatory cytokines in both groups (Kendall's tau, control 0.6; BMMNC 0.7). At day 4, the correlation had completely disappeared in the control group but was preserved in the BMMNC group (Kendall's tau, control 0.3; BMMNC 0.7). CONCLUSIONS: BMMNC transplantation is associated with preserved balance between pro- and anti-inflammatory cytokines after STEMI in PCI-treated patients. This may partly explain the favorable effect of stem cell transplantation after AMI.

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.

Improved cerebral recovery from hypothermic circulatory arrest after remote ischemic preconditioning.

BACKGROUND: Remote ischemic preconditioning is a novel method of reducing ischemia-reperfusion injury in which a transient ischemic period of the limb provides systemic protection against a prolonged ischemic insult. This method of preconditioning has shown some potential in ameliorating ischemia-related injury in various organs and experimental settings. We hypothesized that remote ischemic preconditioning might also improve the recovery from hypothermic circulatory arrest (HCA). METHODS: Twenty-four juvenile pigs underwent 60 minutes of HCA at 18 degrees C with either transient right hind leg ischemic preconditioning or no ischemic preconditioning. Preconditioning was induced by four cycles of 5-minute ischemia periods with three 5-minute reperfusion periods in between. Microdialysis and electroencephalography (EEG) data were recorded to detect any possible changes during the recovery phase. RESULTS: The EEG data showed that the remote ischemic preconditioning group had significantly better EEG recovery time and a lower burst suppression ratio throughout the follow-up period. Cerebral extracellular glucose and glycerol content rose significantly immediately after HCA in the control group compared with the remote ischemic preconditioning group, and significantly higher lactate concentrations were measured in the control group at 5 and 6 hours after reperfusion, indicating a difference in cerebral metabolism. CONCLUSIONS: Our data imply that remote ischemic preconditioning improves the recovery from HCA. It provides a faster recovery of cortical neuronal activity and protection against potential oxygen radical-mediated ischemia damage during and after HCA. In addition, it seems to protect from a late phase lactate and pyruvate burst, mitigating possible damage from an anaerobic metabolism phase.