Paraplegia remains one of the most devastating complications of thoracoabdominal aortic surgery and is associated with a significant increase in both morbidity and mortality. Modern aortic repair techniques use many modalities aimed at reducing the risk of spinal cord ischemia inherent with surgical management. One of these modalities that acts via optimizing spinal cord blood flow is lumbar cerebrospinal fluid (CSF) drainage. Either alone or in combination with other interventions, CSF drainage remains one of the most frequently used spinal cord protection techniques. Despite no definitive proof of efficacy for reducing spinal cord injury, there are compelling data supporting its use. However, the potential benefit of CSF drainage must be balanced against the risks associated with its use, including nerve injury during insertion, compressive neuraxial hematoma formation, intracranial hemorrhage due to excessive drainage, and infection. The optimal benefit to risk ratio can be achieved by understanding the rationale for its use and following practical management guidelines.

Cryopreservation of islets adds great flexibility to clinical islet transplant programs. Methods of islet cryopreservation have traditionally utilized permeating cryoprotectants contained within isotonic solutions without specifically addressing issues of ionic balances, buffering capacity, or oxygen free radicals that occur during hypothermic stresses. These factors may become significant issues during low-temperature storage and during the freezing and thawing process. Since its development in the early 1980s, the University of Wisconsin (UW) organ preservation solution has become the standard vascular flush and preservation solution. Recently, Hypothermosol preservation solution (HTS) was developed as a hypothermic blood substitute. The unique characteristics and composition of these preservation solutions may be important when developing solutions specific for the cryopreservation of cells and tissues. It was the aim of this study to evaluate these two hypothermic preservation solutions as the media used in cryopreservation of islets. Groups of canine islets [5000 islet equivalents (IE)/group] were cryopreserved using the standard protocol of stepwise addition of dimethyl sulfoxide (DMSO) to 2 M, controlled nucleation, slow cooling (0.25°C/min), and rapid thawing (200°C/min). The cryopreservation solutions were made with 1) UW solution, 2) HTS solution, or 3) Medium 199 solution with 10% fetal calf serum (FCS). Additional control groups included islets cryopreserved using 4) HTS, 5) UW solution, and 6) Medium 199 alone, without DMSO. Recovery of islets immediately following thawing was equivalent between the groups with the exception of the islets cryopreserved without DMSO (groups 4-6, p < 0.05). After 48 h of postcryopreservation tissue culture, islet recovery was highest in the groups frozen with UW and HTS (mean ± SEM) (79.8 ± 1.9% and 82.5 ± 1.5%, p < 0.05 vs. group 3, 69.1 ± 3.3%, p < 0.05, ANOVA). Less than 15% of the islets were recovered when they were cryopreserved without the cryoprotectant DMSO (groups 4-6). Functional viability was assessed by measuring the glucose-stimulated insulin secretion during static incubation after 48-h culture. The stimulation indexes were 4.6 ± 1.0, 4.2 ± 0.8, 3.6 ± 1.2, 0.6 ± 0.5, and 0.4 ± 0.2 for islets in groups 1-5, respectively. This study demonstrates that postcryopreservation survival can be improved using intracellular-based preservation solutions, including UW or HTS, in conjunction with DMSO.

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The development of effective protocols for the low-temperature banking of pancreatic islets is an important step in islet transplantation for the treatment of type I diabetes mellitus. We have been exploring the use of islets from the newborn pig as an alternative source of tissue for transplantation. Current cryopreservation protocols are empirically derived, but may be optimized by modeling osmotic responses during the cryopreservation process. This study determined the osmotic and cryoprotectant permeability parameters of cells isolated from the pancreas of newborn pigs. Key parameters are: the osmotically inactive fraction of cell volume, hydraulic conductivity, the permeability coefficients of dimethyl sulfoxide (DMSO) and ethylene glycol (EG) at varying temperatures, and the activation energies of these transport processes. Newborn pig islets were dispersed into single cells and kinetic and equilibrium cell volumes were recorded during osmotic excursions using an electronic particle counter interfaced to a computer. Data were fitted to theoretical descriptions of the osmotic responses of cells, based on the Kedem-Katchalsky approach. The hydraulic conductivity (L p ) in the absence of cryoprotectant was calculated as 0.050 ± 0.005, 0.071 ± 0.006, and 0.300 ± 0.016 µm/min/atm at 4°C, 10°C, and 22°C, respectively (mean ± SEM, n = 7, 6, or 9). These values give an activation energy value of 16.69 kcal/mol when put into an Arrhenius plot. The solute permeability (P s ) values for 1 M DMSO were 0.89 ± 0.12, 1.86 ± 0.28, and 5.33 ± 0.26 µm/min at 4°C, 10°C, and 22°C, respectively (n = 11, 8, or 10) giving an activation energy of 15.98 kcal/mol. The L p values for cells exposed to 1 M DMSO were 0.071 ± 0.006, 0.084 ± 0.008, and 0.185 ± 0.014 µm/min/atm at 4°C, 10°C, and 22°C, respectively. The activation energy for these values was 8.95 kcal/mol. The P s values for 2 M DMSO were 1.11 ± 0.13, 1.74 ± 0.19, and 7.68 ± 0.12 µm/min for the same temperatures, with a calculated activation energy of 17.89 kcal/mol. The L p values in the presence of 2 M DMSO were 0.070 ± 0.006, 0.085 ± 0.008, and 0.192 ± 0.009 µm/min/atm at 4°C, 10°C, and 22°C, respectively, with an activation energy of 9.40 kcal/ mol. Solutions of 1 M EG gave P s values of 1.01 ± 0.13, 1.45 ± 0.25, and 4.90 ± 0.48 µm/min at the three test temperatures. The resulting activation energy was 14.60 kcal/mol. The corresponding L p values were 0.071 ± 0.007, 0.068 ± 0.006, and 0.219 ± 0.012 µm/min/atm with an activation energy of 10.96 kcal/mol. The solute permeabilities in the presence of 2 M EG for newborn pig islet cells were 1.03 ± 0.15, 1.42 ± 0.23, and 5.56 ± 0.22 µm/min; the activation energy was 15.70. The L p values for cells in the presence of 2 M EG were 0.068 ± 0.008, 0.071 ± 0.006, and 0.225 ± 0.010 µm/min/atm; the activation energy for these values was 11.49 kcal/mol. These key cryobiological parameters permit the mathematical modeling of osmotic responses of intact islets during the cryopreservation process, which may lead to further improvements ...

Many lower vertebrates (reptilian and amphibian species) are capable of surviving natural episodes of hypoxia and hypothermia. It is by specific metabolic adaptations that anurans are able to tolerate prolonged exposure to harsh environmental stresses. In this study, it was hypothesized that livers from an aquatic frog would possess an inherent metabolic ability to sustain high levels of ATP in an isolated organ system, providing insight into a metabolic system that is well-adapted for low temperature in vitro organ storage. Frogs of the species, R. pipiens were acclimated at 20 degrees C and at 5 degrees C. Livers were preserved using a clinical preservation solution after flushing. Livers from 20 degrees C-acclimated frogs were stored at 20 degrees C and 5 degrees C and livers from 5 degrees C-acclimated frogs were stored at 5 degrees C. The results indicated that hepatic adenylate status was maintained for 96 h during 5 degrees C storage, but not longer than 4-10 h during 20 degrees C storage. In livers from 5 degrees C-acclimated animals subjected to 5 degrees C storage, ATP was maintained at 100% throughout the 96-h period. Warm acclimation (20 degrees C) and 20 degrees C storage resulted in poorer maintenance of ATP; energy charge values dropped to 0.50 within 2 h and by 24 h, only 24% of control ATP remained. Lactate levels remained less than 25 mumol/g dry weight in all 5 degrees C-stored livers; 20 degrees C-stored livers exhibited greater accumulation of this anaerobic endproduct (lactate reached 45-50 mumol/g by 10 h). The data imply that hepatic adenylate status is largely dependent on exposure to hypothermic hypoxia and although small amounts of ATP were accounted for by anaerobic glycolysis, there must have been either a substantial reduction in cellular energy-utilization or an efficient use of low oxygen tensions.