Near-infrared and nuclear magnetic resonance spectroscopic assessment of tissue energetics in an isolated, perfused canine hind limb model of dysoxia.

This controlled laboratory study examined the efficacy of near-infrared spectroscopy (NIRS) and 31P-nuclear magnetic resonance (NMR) spectroscopy in measuring regional tissue oxygenation in a isolated, perfused hind limb model of tissue dysoxia. Isolated hind limb perfusion was carried out in 20 mongrel dogs and oxygen delivery was varied by manipulating either hemoglobin concentration, oxygen saturation, or flow. Hind limbs from anesthetized mongrel dogs (n = 20) were separated and isolated perfusion performed. NIRS probes for recording relative O2 saturation of tissue hemoglobin (HbO2) and cytochrome a,a3 and NMR probes for measuring 31P-high energy phosphates were placed over the limb. Measurements of physiologic parameters, blood gases, lactate, NIRS values for HbO2 and cytochrome a,a3 redox state, and 31P-phosphate levels were recorded at set intervals throughout the experiment. Measures of tissue oxygen consumption (VO2) correlated with tissue oxygenation as measured by HbO2 and cytochrome a,a3 redox state (NIRS), as well as by 31P-high energy phosphate levels (NMR) throughout the experiment. Delivery-dependent tissue oxygenation was detected at a higher DO2 by NIRS than by VO2 or NMR. Tissue oxygenation as measured by NIRS and NMR shows excellent correlation with oxygen delivery in an isolated, perfused model of shock. NIRS may allow early detection of tissue dysoxia using rapid non-invasive techniques.

Modulation of macrophage and B cell function by glycosaminoglycans.

There is increasing evidence that the behavior of antigen-presenting cells may be regulated, in part, by the surrounding microenvironment. Components of the microenvironment of solid tissues that might influence antigen-presenting cell functions include glycosaminoglycans. We previously showed that heparan sulfate glycosaminoglycans activate macrophages, leading to profound alterations in T cell responses. Here we demonstrate the functional changes that occur in murine antigen-presenting cells induced by heparan sulfate and other glycosaminoglycans, and postulate how these functional changes influence the nature of local immune responses. Heparan sulfate triggered up-regulation of ICAM-1 and I-A, caused the release by antigen-presenting cells of interleukin (IL)-1, IL-6, tumor necrosis factor, IL-12, transforming growth factor beta, and prostaglandin E2 (PGE2), and (in macrophages) induced cytotoxic capability. Heparin induced IL-12 and interferon-gamma production but did not promote the release of other cytokines. Chondroitin sulfate and dermatan sulfate, although not stimulating the production of cytokines or of PGE2, elicited the production by macrophages of nitric oxide. These findings support a model in which the glycosaminoglycan composition of a given tissue, which may be altered by inflammatory processes, helps to regulate the behavior of antigen-presenting cells, which in turn determines the characteristics of the immune response that ensues.

Enhanced cytochrome P450 IA1 activity of self-assembled rat hepatocyte spheroids.

Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes. Spheroids have been observed to exhibit enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures on dry collagen. With confocal scanning laser microscopy, CYP1A1 activity was evaluated in situ by detecting resorufin. This highly fluorescent molecule is the P450-mediated product of 7-ethoxyresorufin O-dealkylation (EROD). Significantly higher P450 activity was observed in spheroids compared to monolayers on collagen upon induction with 50 microM beta-naphthoflavone (BNF), a CYP1A inducer. This was confirmed by measuring microsomal EROD activity. The distribution of CYP1A1 activity within spheroids was heterogeneous, with higher activity localized to the hepatocytes in the interior. During the process of spheroid formation, cells were initially seen to attach and spread out as a monolayer. This stage was associated with relatively low CYP1A1 activity. As cells formed multicellular structures and aggregated into spheroids, the level of CYP1A1 activity increased over time. At least a fivefold higher fluorescence intensity was observed in spheroids compared to that of monolayers maintained on collagen. The higher P450 activity within spheroids may be associated with their ability to maintain a greater degree of differentiation compared to monolayers. These studies demonstrate the potential of hepatocyte spheroids as a model system for investigating drug metabolism, tissue engineering, and tissue self-assembly.

Development of a bioartificial liver device.

Liver disease continues to be a challenge clinically, with 30,000 patients dying each year from liver failure (1). Although liver transplantation can successfully treat many patients undergoing liver failure, the scarcity of donor organs severely limits this treatment's application. For this reason, many investigators are pursuing alternatives to total organ transplantation, from living donors to cell transplantation. One additional approach is the development of a hybrid, bioartificial liver as an extracorporeal device for the temporary treatment of acute liver failure. This approach has demonstrated early success, and may provide an important clinical treatment in the near future. In addition, a bioartificial liver reactor is useful for prolonged in vitro studies of hepatocyte function. This chapter will provide information on the design and use of such a reactor for in vitro applications.

Effect of pharmacokinetic sampling methods on aminoglycoside dosing in critically ill surgery patients.

We compared pharmacokinetic parameters derived from three aminoglycoside serum concentration sampling methods and evaluated their effects on recommended aminoglycoside dosing regimens in 60 critically ill surgery patients. Patients had presumed or documented gram-negative sepsis, and had at least 4 aminoglycoside serum concentrations measured. We used a one-compartment model for peak and trough, 3-point series, and 4-point series sampling methods. Dosing regimens were calculated for each patient based on values derived from each method. We found differences in regimens for nearly 50% of patients if either 4- or 3-point series sampling was used to calculate the recommended dosage rather than peak and trough sampling. However, the 3-point method required a clinically significant change in regimen in only 12% of patients compared with 4-point sampling. The variability of all values derived from 3-point sampling were well accounted for by the 4-point method (r2 > 0.80). In addition, we noted significantly greater relative precision for 3-point sampling than peak and trough sampling for estimates of clearance, elimination rate, recommended daily dosage, and recommended dosing frequency. We recommend three optimally timed samples be drawn instead of peak and trough levels in dosing aminoglycosides in critically ill surgery patients.

Brain lactate by magnetic resonance spectroscopy during fulminant hepatic failure in the dog.

A noninvasive test is needed to assess the severity of encephalopathy during fulminant hepatic failure. This feasibility study was designed to compare a noninvasive test, brain lactate measurement by magnetic resonance spectroscopy, with intracranial pressure monitoring in a large animal model of fulminant hepatic failure. Five dogs received an intraventricular catheter for intracranial pressure measurement. Liver injury was induced by intravenous bolus of D-galactosamine. Brain lactate concentrations were determined by magnetic resonance spectroscopy for up to 48 hours after D-galactosamine administration (t = 0 hour). A dose of D-galactosamine exceeding 1.5 g/kg resulted in fulminant hepatic failure. Brain lactate levels increased to > 10 mmol/L in the two dogs that developed severe intracranial hypertension of > 50 mm Hg and sustained cerebral perfusion pressures of < 40 mm Hg. Both dogs experienced brain death, 42 and 48 hours after the administration of D-galactosamine. Brain lactate concentrations determined by magnetic resonance spectroscopy were in agreement with brain tissue concentrations of lactate determined by high-performance liquid chromatography at necropsy. Plasma lactate concentrations were only mildly elevated (3.2 and 4.2 mmol/L) at the time of brain death. Elevated levels of brain lactate are associated with intracranial hypertension and poor neurological outcome during fulminant hepatic failure.

Frank B. Cerra, MD.

Though the effects of managed care can be seen all across the country, the state of Minnesota has clearly been in the forefront of change. While this has presented an opportunity to be on the leading edge of health reform, it has also had a revolutionary impact on all previously held ways of doing business. A long-time faculty member at the University of Minnesota Frank Cerra was named Dean of the medical school in May of 1995, a time which found the University of Minnesota Hospital in a precarious position. The day-to-day financial workings of the institution soon became his major focus and in 1997 he became the Senior Vice-President for Health Sciences. In this position much of the restructuring and strategic planning of the school is now under his supervision, and he has dealt with several daunting challenges both within the school and the state. Interviewed in his office in Minnesota, Cerra candidly reflected on the power of market-based health reform the frustrations involved in turning a slow moving public institution towards the future.

Characterization of the three-compartment gel-entrapment porcine hepatocyte bioartificial liver.

A hybrid bioartificial liver device supporting a large mass of cells expressing differentiated hepatocyte metabolic capabilities is necessary for the successful treatment of fulminant hepatic failure. The three-compartment gel-entrapment porcine hepatocyte bioartificial liver was designed to provide "bridge" support to transplantation or until native liver recovery is achieved for patients with acute liver failure. The device is an automated mammalian cell culture system supporting 6-7 x 10(9) porcine hepatocytes entrapped in a collagen matrix and inoculated into the capillary lumen spaces of two 100 kDa molecular mass cut-off hollow fiber bioreactors. Gel contraction recreates a small lumen space within the hollow fiber which allows for the delivery of a nutrient medium. This configuration supported hepatocyte viability and differentiated phenotype as measured by albumin synthesis, ureagenesis, oxygen consumption, and vital dye staining during both cell culture and ex vivo application. The hollow fiber membrane was also shown to isolate the cells from xenogenic immunoglobulin attack. The gel-entrapment bioartificial liver maintained a large mass of functional hepatocytes by providing a three-dimensional cell culture matrix, by delivering basal nutrients through lumen media perfusion, and by preventing rejection of the xenocytes. These features make this device a favorable candidate for the treatment of clinical fulminant hepatic failure.

Comparative effects of lipopolysaccharide on newborn versus adult rat hepatocyte and nonparenchymal cell cocultures.

OBJECTIVE: To examine the effects of development on the response of hepatocytes and nonparenchymal cells to an endotoxin challenge as an in vitro model of organ system dysfunction at differing developmental ages. DESIGN: In vitro animal cell culture model. SETTING: University teaching hospital research laboratory. SUBJECTS: Adult and newborn Sprague-Dawley rats. INTERVENTIONS: The method of hepatocyte and nonparenchymal cell coculture was utilized and modified to allow evaluation of cells derived from newborns. Cells were isolated from adult rats by standard perfusion technique. Hepatocytes and nonparenchymal cells were isolated from newborn rats by use of identical enzymatic degradation after fine mincing of the organ. Isolated cells were purified by density gradient. The hepatocytes were incubated in standard cell culture plates for 24 hrs before the addition of nonparenchymal cells. Hepatocytes were incubated with similar-age nonparenchymal cells. After an additional 24 hrs, serial log dilutions of lipopolysaccharide were added as a stimulus and the system was cultured an additional 24 hrs. The response of the hepatocytes was assessed by determination of 3H-leucine incorporation in acid-precipitated protein. In addition, the production of tumor necrosis factor, interleukin (IL)-1, and IL-6 by isolated nonparenchymal cells from adult and newborn rats was determined after stimulation with serial log dilutions of lipopolysaccharide by bioassay. MEASUREMENTS AND MAIN RESULTS: Both adult and newborn hepatocytes cocultivated with nonparenchymal cells demonstrated a comparable and statistically significant dose response to lipopolysaccharide (p < .01). The newborn hepatocytes demonstrated a greater rate of protein synthesis than the adult hepatocytes at all concentrations of lipopolysaccharide. Tumor necrosis factor production by newborn and adult nonparenchymal cells was similar at all lipopolysaccharide doses. IL-1 production demonstrated a positive dose response to lipopolysaccharide in the adult and newborn nonparenchymal cells, with a trend (p = .17) toward greater IL-1 secretion by the adult cells. There were significant differences in IL-6 production by isolated nonparenchymal cells at lipopolysaccharide doses of 0.01, 0.1, and 10 micrograms/mL. While a similar trend was apparent in the cocultured cells, the significance was not apparent, except at the highest lipopolysaccharide dose. CONCLUSIONS: The dose responses of newborn and adult hepatocytes to nonparenchymal cells stimulated with lipopolysaccharide were similar, although newborn hepatocytes appeared to have an inherently higher rate of protein synthesis compared with adult hepatocytes. Cytokine production was similar in nonparenchymal cells of both ages, although IL-1 production by stimulated newborn nonparenchymal cells appeared to be less than IL-1 production by adult nonparenchymal cells.

Development of a bioartificial liver employing xenogeneic hepatocytes.

Liver failure is a major cause of mortality. A bioartificial liver (BAL) employing isolated hepatocytes can potentially provide temporary support for liver failure patients. We have developed a bioartificial liver by entrapping hepatocytes in collagen loaded in the luminal side of a hollow fiber bioreactor. In the first phase of development, liver-specific metabolic activities of biosynthesis, biotransformation and conjugation were demonstrated. Subsequently anhepatic rabbits were used to show that rat hepatocytes continued to function after the BAL was linked to the test animal. For scale-up studies, a canine liver failure model was developed using D-galactosamine overdose. In order to secure a sufficient number of hepatocytes for large animal treatment, a collagenase perfusion protocol was established for harvesting porcine hepatocytes at high yield and viability. An instrumented bioreactor system, which included dissolved oxygen measurement, pH control, flow rate control, an oxygenator and two hollow fiber bioreactors in series, was used for these studies. An improved survival of dogs treated with the BAL was shown over the controls. In anticipated clinical applications, it is desirable to have the liver-specific activities in the BAL as high as possible. To that end, the possibility of employing hepatocyte spheroids was explored. These self-assembled spheroids formed from monolayer culture exhibited higher liver-specific functions and remained viable longer than hepatocytes in a monolayer. To ease the surface requirement for large-scale preparation of hepatocyte spheroids, we succeeded in inducing spheroid formation in stirred tank bioreactors for both rat and porcine hepatocytes. These spheroids formed in stirred tanks were shown to be morphologically and functionally indistinguishable from those formed from a monolayer. Collagen entrapment of these spheroids resulted in sustaining their liver-specific functions at higher levels even longer than those of spheroids maintained in suspension. For use in the BAL, a mixture of spheroids and dispersed hepatocytes was used to ensure a proper degree of collagen gel contraction. This mixture of spheroids and dispersed cells entrapped in the BAL was shown to sustain the high level of liver-specific functions. The possibility of employing such a BAL for improved clinical performance warrants further investigations.

The future. Monitoring cellular energetics.

Current monitoring of critically ill patients uses measurement of global parameters such as oxygen consumption and lactate levels. With development of new monitoring technologies, it may be possible to monitor patients on an organ or tissue level, allowing manipulation of specific organ or tissue perfusion. Potentially useful techniques for monitoring tissue energetics in the future include NIR and NMR spectroscopy. However, both of these techniques are currently limited in their usefulness due to technical factors; NIR by its inability to monitor "silent" metabolically active organs and NMR by its cost, size, and interference of magnetic fields with electronic equipment. Both of these techniques may be useful for identification of dysoxia or oxygen-limited mitochondrial turnover. Experimental evidence suggests that organs in the septic state are more sensitive to dysoxia. Implications for the care of the patient with sepsis include possible decreased tolerance to factors leading to dysoxia, such as hypoxemia, hemodilution, or ischemia.

Hollow fiber bioartificial liver utilizing collagen-entrapped porcine hepatocyte spheroids.

A xenogeneic hollow fiber bioreactor utilizing collagen-entrapped dispersed hepatocytes has been developed as an extracorporeal bioartificial liver (BAL) for potential treatment of acute human fulminant hepatitis. Prolonged viability, enhanced liver-specific functions, and differentiated state have been observed in primary porcine hepatocytes cultivated as spheroids compared to dispersed hepatocytes plated on a monolayer. Entrapment of spheroids into the BAL can potentially improve performance over the existing device. Therefore, studies were conducted to evaluate the feasibility of utilizing spheroids as the functionally active component of our hybrid device. Confocal microscopy indicated high viability of spheroids entrapped into cylindrical collagen gel. Entrapment of spheroids alone into collagen gel showed reduced ability to contract collagen gel. By mixing spheroids with dispersed cells, the extent of collagen gel contraction was increased. Hepatocyte spheroids collagen-entrapped into BAL devices were maintained for over 9 days. Assessment of albumin synthesis and ureagenesis within a spheroid-entrapment BAL indicated higher or at least as high activity on a per-cell basis compared to a dispersed hepatocyte-entrapment BAL device. Clearance of 4-methylumbelliferone to its glucuronide was detected throughout the culture period as a marker of phase II conjugation activity. A spheroid-entrapment bioartificial liver warrants further studies for potential human therapy. (c) 1996 John Wiley & Sons, Inc.

Extrahepatic metabolism of 4-methylumbelliferone and lidocaine in the anhepatic rabbit.

Extrahepatic drug metabolism was studied in an anhepatic rabbit model. Plasma concentrations of 4-methylumbelliferone (4-MU) and its major metabolites, 4-methylumbelliferyl-O-glucuronide and 4-methyumbelliferyl sulfate, along with lidocaine and its major metabolites, monoethylglycinexylidide and 3-hydroxylidocaine, were measured in sham rabbits (n = 4) and anhepatic rabbits (n = 4) following bolus intravenous administration of each drug. Along with concentration profiles of the drugs and metabolites, pharmacokinetic analyses of 4-MU metabolism and lidocaine metabolism were used to assess the extrahepatic metabolism of these classical substrates. Total body clearance of 4-MU in the anhepatic rabbits was about 50% that of the sham animals. Extensive extrahepatic glucuronidation of 4-MU was revealed by comparing the AUC ratios of 4-methylumbelliferyl-O-glucuronide and 4-MU in anhepatic and sham rabbit groups. Sulfation of 4-MU was reduced significantly in the anhepatic group, although some extrahepatic sulfation was observed. Total body clearance of lidocaine was reduced 3-fold in anhepatic animals. 3-Hydroxylidocaine was only detected in plasma samples from sham animals. These results emphasize the importance of extrahepatic sites in drug metabolism, especially glucuronidation of phenolic compounds such as 4-MU.

Efficient assembly of rat hepatocyte spheroids for tissue engineering applications.

Freshly harvested primary rat hepatocytes cultivated as multicellular aggregates, or spheroids, have been observed to exhibit enhanced liver-specific function and differentiated morphology compared to cells cultured as monolayers. An efficient method of forming spheroids in spinner vessels is described. Within 24 h after inoculation, greater than 80% of inoculated cells formed spheroids. This efficiency was significantly greater than that reported previously for formation in stationary petri dishes. With a high specific oxygen uptake rate of 2.0 x 10(-9) mmol O(2)/cell/h, the oxygen supply is critical and should be monitored for successful formation. Throughout a 6-day culture period, spheroids assembled in spinner cultures maintained a high viability and produced albumin and urea at constant rates. Transmission electron microscopy indicated extensive cell-cell contacts and tight junctions between cells within spheroids. Microvilli-lined bile canaliculus-like channels were observed in the interior of spheroids and appeared to access the exterior through pores at the outer surface. Spheroids from spinner cultures exhibited at least the level of liver-specific activity as well as similar morphology and ultrastructure compared to spheroids formed in stationary petri dishes. Hepatocytes cultured as spheroids are potentially useful three-dimensional cell systems for application in a bioartificial liver device and for studying xenobiotic drug metabolism. (c) 1996 John Wiley & Sons, Inc.

Mechanistics of formation and ultrastructural evaluation of hepatocyte spheroids.

Freshly harvested rat hepatocytes form spheroids on uncoated positively charged polystyrene surfaces. Time lapse microscopy revealed that cell movement and reorganization were involved in spheroid formation. Ultrastructural evaluation using scanning and transmission electron microscopy indicated polarized cellular morphology and extensive cell-cell communication within spheroids. Bile canalicular structures were observed to surround each individual hepatocyte, forming an intricate three-dimensional continuous network of channels that appeared to end as pores/holes on the surface of the spheroid. The maintenance of differentiated cellular morphology coincided with preservation of hepatocyte viability and enhanced levels of tissue specific functions in spheroids.

Evidence based critical care medicine; what is it and what can it do for us? Evidence Based Medicine in Critical Care Group.

OBJECTIVE: To describe the philosophy and approach to patient care called evidence based medicine, and to highlight how it can enhance the practice of intensive care. DATA SOURCES: We searched MEDLINE, reference lists, and our personal files to identify relevant literature. STUDY SELECTION: Articles on intensive care practice, critical appraisal, clinical research, and healthcare delivery were selected for discussion. DATA SYNTHESIS: We summarize the rationale for evidence based medicine, its applications and future developments, and suggest several methods for intensivists to use evidence based medicine in their practice and teaching. CONCLUSIONS: Evidence based medicine can complement other foundation disciplines in intensive care. This is the first article in a series entitled "Evidence Based Critical Care Medicine" which will demonstrate how this approach can be used at the bedside.

Review of support systems used in the management of fulminant hepatic failure.

Fulminant hepatic failure has an exceedingly high mortality. Liver transplantation is the treatment option of choice. Unfortunately, one-third of patients with fulminant hepatic failure die awaiting a donor liver. For over 35 years attempts to remove or dilute putative toxins in the blood have been unsuccessful in improving survival rates. The use of biocompatible interfaces with blood or plasma and current hepatocyte culture techniques have led to the development of new support systems. This generation of bioartificial livers will hopefully provide the necessary hepatic functions and prevent many of the complications associated with fulminant hepatic failure. This paper will review the support systems tried and currently under investigation, with an emphasis on bioartificial livers.

Effects of hepatocyte growth factor on viability and biotransformation functions of hepatocytes in gel entrapped and monolayer culture.

OBJECTIVES: An extracorporeal bioartificial liver device must maintain viability and differentiated function of hepatocytes cultivated at high cell density. Growth factors, such as hepatocyte growth factor, found in high concentrations in the plasma of patients with fulminant hepatic failure, have the potential to promote hepatocyte dedifferentiation and thus, decrease function. We tested the hypothesis that hepatocyte growth factor would improve viable cell density and decrease biotransformation functions of liver cells in monolayer culture and in hepatocytes entrapped in collagen cylindrical gel "noodles" as found in the extracorporeal bioartificial liver. DESIGN: In vitro, controlled study. SETTING: University research laboratory. SUBJECTS: Adult Sprague Dawley Rats. INTERVENTIONS: Hepatocytes were harvested by a two-step collagenase technique. Harvested hepatocytes were plated onto type 1 collagen coated plates or entrapped in type 1 collagen cylindrical gels and cultured in different concentrations of hepatocyte growth factor. Interval measurements of 3H-thymidine incorporation, albumin synthesis, biotransformation functions, and viability were made. MEASUREMENTS AND MAIN RESULTS: In monolayer culture, the addition of hepatocyte growth factor caused a dramatic increase in 3H-thymidine incorporation. This increase was accompanied by a decrease in the appearance of the lidocaine metabolite, monoethyglycinexylidide. Albumin production was unchanged. In cylindrical gel entrapment cultures, hepatocyte growth factor caused a significant increase in 2-day viability but had no effect on the metabolite appearance of lidocaine or 4-methyl umbelliferone or albumin production. CONCLUSIONS: Hepatocyte growth factor induces dedifferentiation of hepatocytes in monolayer culture. Collagen matrix entrapment appears to abrogate this effect and improve liver cell viability. There may be reciprocal regulation of hepatocyte reproductive and differentiated functions, such as biotransformation, which can be influenced by the entrapment of hepatocytes in an extracellular type 1 collagen matrix.

Gel-entrapment bioartificial liver therapy in galactosamine hepatitis.

A need exists for an effective, safe bioartificial liver to support patients in fulminant hepatic failure (FHF). The purpose of this study was to determine the treatment efficacy of the novel gel-entrapment porcine hepatocyte bioartificial liver (BAL) in a fatal model of canine hepatic failure. FHF was produced in 27- to 30-kg halothane-anesthetized dogs by bolus infusion of the hepatotoxin D-galactosamine (D-Gal). Three groups were studied during the 48-hr experiment: Group D-Gal (n = 5) received galactosamine, 1.0 g/kg, iv at Time O, Group HepBAL (n = 5) received D-Gal followed by continuous hemoperfusion with the BAL device loaded with approximately 6 billion viable pig hepatocytes starting at Time 24 hr, and three dogs served as healthy controls (Group Control) and received no galactosamine. The primary endpoints were survival and coma development. Group D-Gal demonstrated 100% mortality from liver failure by 42 hr, characterized by a progressive rise in liver enzymes, total bilirubin, ammonia, and lactate and associated with coagulopathy, hypoglycemia, coma, and brain death. BAL therapy significantly delayed the onset of coma and improved survival (median 47 hr vs D-Gal median 36 hr). A significant delay in the rise of lactate and ammonia was also noted. BAL therapy prolonged survival and improved both laboratory and clinical markers of fatal liver failure. These data indicate that this BAL may have clinical utility in supporting human liver failure.