Moderate hypothermia with remote ischaemic preconditioning improves cerebral protection compared to deep hypothermia: a study using a surviving porcine model.

OBJECTIVES: The optimal temperature management of hypothermic circulatory arrest is still controversial. Moderate hypothermia preserves cerebral autoregulation and shortens cardiopulmonary bypass (CPB) duration. However, moderate hypothermia alone has inferior organ protection to deep hypothermia, so adjuncts that increase the ischaemic tolerance are needed. Thus, we hypothesized that a combination of remote ischaemic preconditioning (RIPC) and moderate hypothermia would be superior to deep hypothermia alone. METHODS: Sixteen pigs were randomized to either RIPC or control groups (8 + 8). The RIPC group underwent 4 cycles of transient hind limb ischaemia. The RIPC group underwent cooling with CPB to 24°C, and the control group underwent cooling with CPB to 18°C, followed by a 30-min arrest period and subsequent rewarming to 36°C. Measurements of cerebral metabolism were made from sagittal sinus blood samples and common carotid artery blood flow. The permissible periods of hypothermic circulatory arrest were calculated based on these measurements. Neurological recovery was evaluated daily during a 7-day follow-up, and the brain was harvested for histopathological analysis. RESULTS: Six pigs in the RIPC group reached normal neurological function, but none in the control group reached normal neurological function (P = 0.007). The composite neurological score of all postoperative days was higher in the RIPC group than in the control group [55 (52-58) vs 45 (39-51), P = 0.026]. At 24°C, the estimated permissible periods of hypothermic circulatory arrest were 21 (17-25) min in the RIPC group and 11 (9-13) min in the control group (P = 0.007). CONCLUSIONS: RIPC combined with moderate hypothermia provides superior cerebral protection.

Remote ischaemic preconditioning may prolong permissible period of hypothermic circulatory arrest in a porcine model.

Objectives. The hypothermic circulatory arrest (HCA) is still of paramount importance in aortic arch surgery, but the safe period of the arrest is limited. Remote ischaemic preconditioning (RIPC) prepares the cerebral tissue for ischaemic insult. Prolongation of the permissible period of HCA with RIPC may have a major impact on the outcome of aortic operations requiring cessation of blood flow by decreasing the rate of neurological deficits. Design. Twenty pigs were randomised into the RIPC group (n = 10) and the control group (n = 10). The RIPC group underwent four cycles of transient hind limb ischaemia. Both groups underwent cooling with cardiopulmonary bypass to 11 °C followed by a 45-minute HCA and re-warming to 36 °C. Cerebral blood flow was measured with a transit time ultrasonic flowmeter from the right common carotid artery, and the arteriovenous oxygen difference was calculated from sagittal sinus and arterial blood samples. Measurements were taken at several time points during cooling and warming. Temperature coefficient (Q10) was calculated to determine estimated permissible periods of HCA. Results. The Q10 was 2.27 (1.98-2.58) for the RIPC group and 1.87 (1.61-2.25) for the control group. The permissible period of HCA at 18 °C was 26 minutes (20-33) in the RIPC group and 17 minutes (13-25) in the control group (p = .063)(Data expressed in medians and interquartile ranges). Conclusions. RIPC tends to suppress cerebral metabolism during cooling with cardiopulmonary bypass and may prolong estimated permissible period of HCA.

Safety of direct true lumen cannulation after venous exsanguination: a study in a surviving porcine model†.

OBJECTIVES: Type A aortic dissection requires immediate surgery. Traditional cannulation methods such as the central aortic cannulation with the Seldinger technique and axillary cannulation are primary choices. Yet in the presence of tamponade or severe cardiogenic shock, these can be too time-consuming to complete. Direct true lumen cannulation after venous exsanguination not only avoids this issue but also leads to transient global ischaemia. We studied the safety of direct true lumen cannulation from the aspect of global ischaemia in a surviving porcine model. METHODS: Twelve pigs were randomized to either control or intervention groups (6 + 6). The intervention group underwent simulated direct true lumen cannulation by exsanguination and circulatory arrest for 5 min at 35°C before cardiopulmonary bypass (CPB). Both groups underwent CPB cooling to 25°C followed by a 25-min arrest period and subsequent warming to 36°C. Neuron-specific enolase levels were measured at 6 time-points from blood samples. Near-infrared spectroscopy was used to determine brain oxygenation. The neurological recovery was evaluated daily during a 7-day follow-up, and the brain was harvested for a histopathological analysis after euthanization. RESULTS: All pigs recovered their normal neurological behaviour. The neurobehavioural total score on postoperative day 2 reached borderline statistical significance, thus favouring the intervention group [(9 (8.75-9) vs 6.5 (5.5-9), P = 0.06]. Near-infrared spectroscopy values and neuron-specific enolase levels slightly favoured the control group during the cooling period, but the difference was not clinically significant. The histopathological analysis showed no difference between the groups. CONCLUSIONS: A 5-min period of normothermic global ischaemia before CPB does not impair the neurological outcome following hypothermic circulatory arrest in a surviving porcine model.

Exploring Spinal Cord Protection by Remote Ischemic Preconditioning: An Experimental Study.

BACKGROUND: Paraplegia is one of the most severe complications occurring after the repair of thoracic and thoracoabdominal aortic aneurysms. Remote ischemic preconditioning (RIPC) has been shown to mitigate neurologic damage, and this study assessed its efficacy in preventing spinal cord ischemia. METHODS: The study randomized 16 female pigs into an RIPC group (n = 8) and a control group (n = 8). The RIPC group underwent four cycles of 5-minute ischemia-reperfusion episodes by intermittent occlusion of the left iliac artery. All animals underwent systematic closure of the left subclavian artery and segmental arteries of the descending thoracic aorta to the level of diaphragm. Motor-evoked potential monitoring was performed in both hind limbs. Continuous electrocardiogram and hemodynamics were monitored, and pulmonary artery blood samples were collected. A neurologic assessment was performed 6 hours after the procedure. The thoracic and lumbar portions of the spinal cord were collected for histologic and immunohistochemical analysis. RESULTS: The bilateral motor-evoked potential amplitude responses were higher in the RIPC group (p < 0.05) than in the control group; the difference was detected already before spinal cord ischemia. Paraplegia occurred in 1 control animal. Immunohistochemical total scores of antioxidant response regulator nuclear factor erythroid 2-related factor 2 were better in the RIPC group (11.0; range, 8.5 to 14.0) than in the control group (5.2; range, 1.0 to 9.0; p = 0.023). CONCLUSIONS: RIPC induces electrophysiologic changes in the central nervous system that may confer spinal cord protection extending the resistance to ischemia. The significantly higher nuclear factor erythroid 2-related factor 2 scores suggest better neuronal cell protection against oxidative stress in the RIPC group.

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.

Safety and biodistribution study of bone marrow-derived mesenchymal stromal cells and mononuclear cells and the impact of the administration route in an intact porcine model.

BACKGROUND AIMS: Bone marrow mononuclear cells (BM-MNCs) and bone marrow-derived mesenchymal stem stromal cells (BM-MSCs) could have therapeutic potential for numerous conditions, including ischemia-related injury. Cells transplanted intravascularly may become entrapped in the lungs, which potentially decreases their therapeutic effect and increases the risk for embolism. METHODS: Twelve pigs were divided into groups of 3 and received (99m)Tc- hydroxymethyl-propylene-amine-oxime-labeled autologous BM-MNCs or allogeneic BM-MSCs by either intravenous (IV) or intra-arterial (IA) transplantation. A whole body scan and single photon emission computed tomography/computed tomography (SPECT/CT) were performed 8 h later, and tissue biopsies were collected for gamma counting. A helical CT scan was also performed on 4 pigs to detect possible pulmonary embolism, 2 after IV BM-MSC injection and 2 after saline injection. RESULTS: The transplantation route had a greater impact on the biodistribution of the BM-MSCs than the BM-MNCs. The BM-MNCs accumulated in the spleen and bones, irrespective of the administration route. The BM-MSCs had relatively higher uptake in the kidneys. The IA transplantation decreased the deposition of BM-MSCs in the lungs and increased uptake in other organs, especially in the liver. Lung atelectases were frequent due to mechanical ventilation and attracted transplanted cells. CT did not reveal any pulmonary embolism. CONCLUSIONS: Both administration routes were found to be safe, but iatrogenic atelectasis might be an issue when cells accumulate in the lungs. The IA administration is effective in avoiding pulmonary entrapment of BM-MSCs. The cell type and administration method both have a major impact on the acute homing.

Transient proteolytic modification of mesenchymal stromal cells increases lung clearance rate and targeting to injured tissue.

Systemic infusion of therapeutic cells would be the most practical and least invasive method of administration in many cellular therapies. One of the main obstacles especially in intravenous delivery of cells is a massive cell retention in the lungs, which impairs homing to the target tissue and may decrease the therapeutic outcome. In this study we showed that an alternative cell detachment of mesenchymal stromal/stem cells (MSCs) with pronase instead of trypsin significantly accelerated the lung clearance of the cells and, importantly, increased their targeting to an area of injury. Cell detachment with pronase transiently altered the MSC surface protein profile without compromising cell viability, multipotent cell characteristics, or immunomodulative and angiogenic potential. The transient modification of the cell surface protein profile was sufficient to produce effective changes in cell rolling behavior in vitro and, importantly, in the in vivo biodistribution of the cells in mouse, rat, and porcine models. In conclusion, pronase detachment could be used as a method to improve the MSC lung clearance and targeting in vivo. This may have a major impact on the bioavailability of MSCs in future therapeutic regimes.

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.

Remote ischemic precondition preserves cerebral oxygen tension during hypothermic circulatory arrest.

OBJECTIVES: Remote ischemic preconditioning (RIPC) is a novel and promising method of mitigating neurological injury. In previous animal studies, RIPC has provided substantial neuroprotective effects. We hypothesized that the promising neuroprotective properties were a consequence of a better oxygen consumption profile during hypothermic circulatory arrest (HCA). DESIGN: Six 7-week-old female pigs were randomly assigned to undergo the 60 minutes of HCA with the right hind leg receiving transient RIPC preoperatively and six animals were assigned to a control group that underwent 60 minutes of HCA without any preconditioning. A combined temperature/oxygen-tension probe was inserted into the parietal cortex of each animal to monitor cerebral oxygen tension during experiments. RESULTS: The RIPC group had significantly higher cerebral oxygen tension readings throughout the HCA. Statistically significant differences were measured from the 20 minute time point onwards in every time point up to the 60 minute time point. CONCLUSIONS: This study shows that RIPC performed before HCA conserves the cerebral oxygen tension during a circulatory arrest. RIPC could possibly prolong the safe operating time during HCA as cerebral oxygen content is preserved throughout circulatory arrest.

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.

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.

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.