Abstract Retrograde cerebral perfusion (RCP) techniques have been adopted in aortic arch surgery for clinical use. The clear benefits of RCP are that it reduces embolic injury and prolongs the permissible period of hypothermic circulatory arrest (HCA). At the same time, however, there is a great deal of evidence according to which RCP may be associated with an increased risk of fluid sequestration and cerebral edema. In the current study intermittent RCP was compared with continuous RCP and HCA alone to clarify if periodical RCP decreases fluid sequestration (I). HCA is an effective method of cerebral protection, but is associated with long cardiopulmonary bypass times, and coagulation disturbances. We tested the hypothesis that deep hypothermic RCP could improve cerebral outcome during moderate HCA (II and III). Glutamate excitotoxicity plays an important role in the development of ischemic brain injury. The purpose of the present study was to determine the efficacy of lamotrigine, a Na+ channel blocker, to mitigate cerebral injury after HCA (IV). A chronic porcine model was used in the present series of studies. Hemodynamic, electrophysiologic, and metabolic monitoring were performed until four hours after the instigation of rewarming. S-100β was measured up to 20 hours. Daily behavioral assessment performed until death or elective sacrifice on the seventh postoperative day. After continuous RCP the median fluid sequestration volume was 145 (0–250) ml compared with -50 (-100 - 0) ml after intermittent RCP (p = 0.04). In comparison of 15°C RCP to HCA alone during moderate 25°C hypothermia, 5/6 animals in the RCP group survived seven days compared with 2/6 in the HCA group (p = 0.04). The total histopathologic scores in the RCP(15°C) group were lower than those for the RCP(25°C) group during moderate 25°C hypothermia (p = 0.04). EEG bursts were recovered better in the RCP(15°C) group at 3 hours after the start of rewarming compared to HCA group (p = 0.05). The rate of EEG burst recovery was higher in lamotrigine treated animals compared to placebo treated animals after 4 hours during the rewarming (p = 0.02). Among the animals that survived for 7 days, the median behavioral score was higher in the lamotrigine group (8) compared with controls (7) (p = 0.02). The results indicate that intermittent RCP decreases the rate of fluid sequestration after continuous RCP. The cold RCP at moderate systemic hypothermia seems to provide a better neurological outcome than that with moderate temperature RCP, a finding suggesting that enhanced cranial hypothermia is the major beneficial factor of RCP. The Na+ channel blocker lamotrigine improves neurological outcome after a prolonged period of HCA. In conclusion, two refinements in the RCP concept are to administer it at low temperatures and if longer periods of perfusion are necessary, RCP should be applied intermittently.

The Banner University Medical Center's (BUMC) renal transplant program relies on the LifePort Kidney Transporter to optimize marginal kidney organs via hypothermic machine perfusion (HMP) prior to transplantation. Hemodynamic parameters produced by the device followed over the duration of support, combined with clinical experience, guide decisions in determining the acceptability of a donor kidney for implantation. Thus far, statistical evidence supporting ideal parameters remain undefined. The purpose of this study is to create a logistic model that will ascertain the post-implant sustainability of LifePort® supported kidneys and predict clinical outcomes. My hypothesis is that the statistical models constructed based on retrospective LifePort® parameters and clinical outcome data will successfully predict donor organ vascular health for transplantation and the optimal support duration. A successful model will contribute to increased efficiencies in the kidney transplant process as well as improved patient outcomes. An overview of the institution’s success was weighed using a survival analysis, with delayed graft function (DGF) as the endpoint. A logistic regression model and forecast model were built to predict the outcome for rejecting or accepting the organ for transplant, as well as to predict the hemodynamic parameters hours after the start of infusion. Results concluded a flow greater than 80 mL/min had a 90% probability of transplantation. The forecast model was capable of predicting flow for up to five hours. The calculated flow was in a 10 mL/min range of the actual flow, when up to one hour parameters were entered into the model. The study concluded practicality in the clinical setting, in kidney transplantation.

Mild hypothermic conditions (30ºC to 33ºC) have previously been shown to increase cell specific productivity (Qp) of recombinant proteins from mammalian cells. However, this is often associated with a lower growth rate which off-sets any potential advantage of higher product titres. This thesis describes the isolation of a novel population of Chinese Hamster Ovary (CHO) cells that have been adapted to low temperature growth by continuous subculture at low temperature for a duration of 400 days. This adapted cell population achieved a growth rate 2-fold greater than non-adapted cells under low temperature conditions (32ºC) while maintaining an elevated level of cell specific expression of recombinant beta-interferon. The volumetric titre of beta-interferon was enhanced by 70% in stationary cultures and by more than 2-fold by application of a temperature-shift strategy involving a growth to production phase. However, the low temperature-adapted cells were fragile and demonstrated an increased sensitivity to hydrodynamic stress in agitated cultures. This problem, caused by a weakened vimentin intermediate filament network, was resolved by the use of macroporous microcarriers which were demonstrated to entrap and protect the cold-adapted cells. Cold-adapted microcarrier cultures were able to achieve high cell densities (greater than 5x10^6 nuclei/mL) cultures under hypothermic conditions. This resulted in a 3-fold enhancement of volumetric titre of monomeric beta-interferon compared to the original control culture at 37ºC.

Mild hypothermic conditions (30ºC to 33ºC) have previously been shown to increase cell specific productivity (Qp) of recombinant proteins from mammalian cells. However, this is often associated with a lower growth rate which off-sets any potential advantage of higher product titres. This thesis describes the isolation of a novel population of Chinese Hamster Ovary (CHO) cells that have been adapted to low temperature growth by continuous subculture at low temperature for a duration of 400 days. This adapted cell population achieved a growth rate 2-fold greater than non-adapted cells under low temperature conditions (32ºC) while maintaining an elevated level of cell specific expression of recombinant beta-interferon. The volumetric titre of beta-interferon was enhanced by 70% in stationary cultures and by more than 2-fold by application of a temperature-shift strategy involving a growth to production phase. However, the low temperature-adapted cells were fragile and demonstrated an increased sensitivity to hydrodynamic stress in agitated cultures. This problem, caused by a weakened vimentin intermediate filament network, was resolved by the use of macroporous microcarriers which were demonstrated to entrap and protect the cold-adapted cells. Cold-adapted microcarrier cultures were able to achieve high cell densities (greater than 5x10^6 nuclei/mL) cultures under hypothermic conditions. This resulted in a 3-fold enhancement of volumetric titre of monomeric beta-interferon compared to the original control culture at 37ºC.

When grown under hypothermic conditions, Chinese Hamster Ovary (CHO) cells become growth arrested in the G₀/G₁ phase of the cell cycle and also often exhibit increased recombinant protein production. In this study, we have validated this hypothesis by stimulating hypothermic growth using basic fibroblast growth factor and fetal bovine serum supplementation. This method led to 7.7- and 4.9-fold increase in total production compared to the 37°C and 32°C control cultures, respectively. This proof-of-concept study will motivate the creation of cell lines capable of growing at low temperatures for use in industrial processes. / Singapore-MIT Alliance (SMA)

Abstract Cessation of the blood circulation for some time during surgery of the aortic arch and repair of congenital heart defects is normally required to allow a bloodless operation field. Hypothermia is the most important mechanism for end-organ protection, particularly the brain, during such operations. Cardiopulmonary bypass is used for core cooling before total hypothermic circulatory arrest (HCA) or selective cerebral perfusion (SCP) are initiated. During hypothermic cardiopulmonary bypass, pH can be managed according to either pH- or alpha-stat principles. In the present work, the optimal pH management strategy for operations requiring HCA or SCP was explored. An experimental porcine model was used. Firstly, outcome was evaluated in a HCA model using either the α- or pH-stat perfusion strategy (I). Secondly, we sought to determine which acid-base management is more effective in attenuating ischemic brain injury during combined HCA and embolization conditions (II). In the third study, the impact of propofol anesthesia and α-stat perfusion strategy on outcome was explored (III). Finally, the acute effects of perfusion strategies in a SCP porcine were compared (IV). Hemodynamics, temperature, EEG (I-III), brain microdialysis, intracranial pressure (I-III), brain tissue oxygen partial pressure (I-III), and intravital microscopy (IV) were monitored intraoperatively. In the chronic studies, survival, postoperative neurologic recovery and brain histopathologic examination were evaluated (I-III). pH-stat strategy was associated with superior outcome compared to the α-stat strategy during a 75-minute period of deep HCA (I). In addition, despite the pH-stat strategy-related cerebral vasodilatation, this method provided better neuroprotection in a setting of cerebral particle embolization prior to a 25-minute period of deep HCA (II). Propofol anesthesia combined with α-stat perfusion strategy was observed to deteriorate the brain injury during HCA evaluated by key brain microdialysis parameters (III). Finally, when employing moderately hypothermic SCP, the differences between pH- and α-stat strategies in cerebral metabolism and microcirculation were minimal. These findings are clinically relevant since α-stat perfusion strategy is still the most commonly used acid-base perfusion strategy during hypothermic cardiopulmonary bypass in adults, and propofol one of the most used anesthetics in clinical practice. It is also noteworthy that the pH-stat strategy is not currently used in adults because of the perceived increased risk of atherosclerotic embolization. However, the advantage of pH-stat strategy over α-stat strategy could not be observed when employing SCP.

Abstract In the repair of complex congenital heart defects or in surgery of the aortic arch, normal circulation may be temporarily halted to ensure a clean, bloodless operation field. The brain is the organ most vulnerable to ischemic injury during this no-flow period, and the mortality and morbidity of these procedures today consists mostly of neurological complications. Hypothermia decreases the need for oxygen and other metabolites, and cooling the patient with an extracorporeal heart-lung machine can provide enough time to perform the necessary surgical procedures during a circulatory standstill. This procedure is referred to as hypothermic circulatory arrest (HCA). Sometimes the cerebral circulation can be maintained even if the rest of the body undergoes circulatory arrest, and this strategy, involving separate catheterization of brain-destined vessels, is referred to as selective cerebral perfusion (SCP). In this work, four separate brain protection strategies were evaluated. Two studies were performed on a surviving porcine model (I, II) to evaluate neurological recovery as well as cerebral metabolism and histopathology, and two were acute in design (III, IV), employing the modern technology of intravital microscopy to examine cerebral microcirculation. The first study (I) showed that the administration of hypertonic saline dextran (HSD) led to a decrease in intracranial pressure, improved brain metabolism, better neurological recovery and less histopathological injury of the brain tissue in association with HCA. In the second study (II) a novel pharmacological molecule, levosimendan, reduced the intracranial pressure during the operation, but no improvement in terms of cerebral metabolism, neurological recovery or histopathological brain injury was observed after HCA. In the third study (III), real-time intravital microscopy showed that in association with HCA, a leukocyte depleting filter (LDF) attached to the cardiopulmonary bypass circuit reduces the number of activated leukocytes in cerebral microcirculation. In the fourth study (IV), cerebral metabolism and microcirculation were similar during SCP independent of the acid-base management strategy. The results of this work suggest that HSD could be assessed in human trials, that levosimendan needs further studies to optimize its potential, that the LDF functions as designed and that the differences between the α- and the pH-stat acid-base management strategies with SCP did not differ in moderate hypothermia.

No description available.

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Abstract During surgery of the aortic arch and pediatric heart surgery, the blood flow to the brain has to be interrupted at times to allow a bloodless operation field and adequate conditions for surgical repair. During this no-flow period the brain is exposed to a high risk of ischaemic injury, as it will become irreversibly damaged after 5 minutes of circulatory arrest at 37°C. Additional time can be gained by cooling the patient with an extracorporeal heart-lung machine, as hypothermia reduces the cerebral metabolic rate and allows longer safe periods of circulatory standstill. This method of cerebral protection, called hypothermic circulatory arrest (HCA), is widely used in clinical practice. Thus the brain becomes susceptible to ischaemic injury after 30 minutes of HCA at 15°C. Lower temperatures than this are not practicable, however, as they require longer periods of cardiopulmonary bypass, which may further aggravate cerebral injury. To ensure a better outcome for patients undergoing these operations, additional ways of protecting the brain are required. The present work focuses on neuroprotective biochemical and fluid therapy methods for use during HCA, employing a surviving porcine model. Fructose-1,6-bisphosphate (FDP), a high-energy intermediate of glycolysis, was examined for potential neuroprotective properties in two cerebral injury settings associated with HCA. First, FDP was administered before and after a 75-minute period of HCA at a brain temperature of 18°C. This led to better survival, neurological recovery and brain histopathological findings and had supportive effects on brain metabolism (I). Second, a 25-minute period of HCA along with an iatrogenic embolic load produced by microsphere injection was used to generate a massive ischaemic injury to the brain. In this setting FDP did not affect the neurological outcome but had a clear supportive impact on cerebral metabolism (II). In addition, cerebral histopathological samples taken during the first study were analysed by electron microscopy, which revealed significant preservation of the ultrastructure in the FDP-treated animals (III). Hypertonic saline dextran (HSD) is a novel fluid therapy method which has been shown to enhance the outcome after hypovolaemic shock with or without head injury and is potentially very effective in reducing ischaemia-reperfusion injury. Its administration led to a decrease in intracranial pressure, improved brain metabolism, faster and better recovery and less histopathologically observable morphological damage (IV). The findings indicate that both FDP and HSD have significant neuroprotective properties and should be assessed in humans as well.