Search

The optimal site for implantation of isolated hepatocytes has not been established. We have developed a novel technique which allows repeated infusion of hepatocytes into the portal system via an indwelling catheter. Seven Nagase Analbuminemic rats (NAR) underwent single intra-portal infusion of 2 × 107 isolated normal albumin-producing rat hepatocytes. Another seven NAR rats underwent placement of indwelling catheters into the portal venous system via the gastroduodenal vein. Each of them received six batches of 5 × 106 normal albumin producing hepatocytes. Seven control NAR rats were infused repeatedly (intraportally) with saline only. Plasma albumin (ELISA) showed significant increase in experimental animals and was more pronounced (p < 0.05) in rats transplanted repeatedly than in those given a single dose of cells. Immunohistochemical staining of the liver sections confirmed the presence of transplanted albumin producing hepatocytes. Rats transplanted with a single large batch of isolated hepatocytes showed liver tissue damage, whereas those subjected to repeated cell infusions had normal liver histology. We have developed a novel intraportal transplantation method which allows successful engraftment of a large number of isolated hepatocytes.

Renewed interest in the transplantation of isolated hepatocytes into the liver as a potential therapy for liver disease has stimulated the development of methods for the identification of donor cells within the recipient organ. We describe a method for cellular tagging and in vivo identification of intraportally transplanted hepatocytes using an intracellular fluorescent dye, 5(6)-carboxyfluorescein diacetate, succinimidyl-ester (CFSE). Rat and porcine hepatocytes were isolated and labelled with CFSE. The optimal conditions for labelling consisted of a buffered saline suspension of hepatocytes (5 × 106 cells/mL) in 20.0 μM CFSE incubated for 15 min at 37°C. In vitro, labelled hepatocytes were cultured either on fibronectin-coated chamber slides or in culture flasks. Cultures were evaluated in situ by fluorescence photomicrography or by fluorescence-activated cell sorting (FACS) after cell detachment. Cell viability was assessed serially and cultured, labelled hepatocytes retained the dye for up to 3 wk (last day of study). CFSE did not effect hepatocyte viability and there was no evidence of intercellular diffusion of the dye. In vivo, syngeneic Lewis rats underwent selective portal vein infusion of freshly isolated, labelled hepatocytes (2.0 × 107 cells/2.0 mL saline/animal) into the posterior liver lobes. All recipients were sacrificed 48 h and 96 h later and their livers examined. Transplanted hepatocytes were identified by fluorescence microscopy in tissue sections and by FACS following collagenase digestion of the liver tissue. CFSE persisted in a population of viable, engrafted hepatocytes. FACS analysis demonstrated that 9 ± 3% of the hepatocytes in the posterior liver lobes were labelled 48 and 96 h after transplantation. At 96 h following transplantation, multiple engrafted hepatocytes could be observed by fluorescence microscopy around the central veins. CFSE labelling allows for both in vitro identification and in vivo localization of donor hepatocytes. Furthermore, it appears to be more stable and specific for labelling hepatocytes than other tested dyes (especially DiI).

OBJECTIVE: The authors developed an extracorporeal liver support system and tested its efficacy in experimental animals with liver failure. The first clinical use of this system to treat a patient with liver failure is reported. SUMMARY BACKGROUND DATA: Multiple attempts have been made, ranging from plasma exchange to use of charcoal columns, to develop liver support systems for treating patients with acute severe liver failure. None of these systems has achieved wide clinical use. There is a need for providing liver support as a "bridge" to transplantation and for treating patients with potentially reversible liver dysfunction. METHODS: A hybrid liver support system has been developed consisting of plasma perfusion through a charcoal column and a porous hollow fiber module inoculated with 5 x 10(9) matrix-attached hepatocytes. The system was tested in dogs with ischemic liver failure (n = 7) who underwent plasmapheresis; a control group (n = 6) underwent charcoal perfusion alone. A patient with liver failure was treated with this hybrid system. RESULTS: After 6 hours of hybrid liver support treatment, animals had significantly decreased serum ammonia and lactate levels, increased glucose level, normal prothrombin time, and increased systolic blood pressure compared with controls treated with charcoal perfusion alone. Use of the system to treat a patient was well tolerated with evidence of clinical improvement. CONCLUSIONS: Plasma perfusion through a system consisting of a charcoal column and matrix-attached porcine hepatocytes had significant beneficial effects in animals with liver failure and was well tolerated by a patient with liver failure.

Whole organ transplantation remains the only clinically effective method of treating acute and chronic liver failure due to specific genetic and other defects of liver function. However, there are several factors which limit the application of liver transplantation in the treatment of liver insufficiency: high cost, relatively high morbidity and limited organ donor availability. As a result, investigators have attempted to develop alternative methods to treat liver insufficiency due to genetic defects of liver function by using either isolated normal hepatocyte transplantation and/or in vitro and in vivo gene therapy techniques.

Because of the complexity and the vast number of metabolic and other physiologic functions provided by the liver, it was felt that in constructing an extracorporeal liver support system, viable, isolated, intact hepatocytes should be utilized rather than specific cell components or enzymes. Previously, Reid and Rojkind [1] demonstrated that isolated hepatocyte viability and expression of differentiated function in vitro were enhanced when the hepatocytes were maintained as a monolayer on a collagen biomatrix; several types of biomatrix have been described for maintaining primary hepatocyte cultures for long periods of time. We hypothesized that “anchored” hepatocytes attached to a collagen matrix would be better able to express differentiated liver functions in an extracorporeal liver support system because of the more physiologic environment which would allow cell-cell and cell-matrix interactions.

Introduction: The effects of cold ischemic preservation and degree of donor-recipient histoincompatibility on graft coronary arteriosclerosis(GCA)were examined using a rat heterotopic cardiac transplant model. Methods: Three donor-recipient rat strain combinations were utilized. Lewis-F344 rats which vary slightly in class I Major Histocompatibility (MHC) Loci genes, ACI-Lewis which vary significantly in MHC class I and II genes, and Lewis-Lewis (control), donor-recipient strain combinations were subjected to varying lengths of cold ischemic preservation prior to transplantation(0, 4, and 24h in University-of-Wisconsin solution at 4’C).There were 9 rats per group.Grafts were harvested at 90 days post-op. and sections of the epicardial arteries were examined (H&E and van Gieson elastin stains).Intimal area ratio (IAR = intimal area/area within internal elastic lamina), intimal thickness score (ITS-based on % luminal compromise, perimeter of involvement), and rejection score (RS) were determined for each specimen using computerized image morphometry. Results: IAR, ITS and RS were significantly higher at 24hrs vs. 0hrs (p ∗ . IAR (hr) LEW-LEW (%) LEW-F344 ∗ (%) LEW-ACI ∗ (%) 0 0.38 ± 0.15 0.33 ± 0.16 0.57 ± 0.16 4 0.34 ± 0.11 0.42 ± 0.21 0.72 ± 0.29 24 0.41 ± 0.18 0.63 ± 0.14 ITS (hr) 0 0.36 ± 1.00 0.67 ± 0.84 2.00 ± 1.40 4 0.14 ± 0.29 0.82 ± 0.94 2.80 ± 2.10 24 0.91 ± 0.91 1.90 ± 0.87 RS (hr) 0 0.11 ± .33 3.30 ± 0.89 3.10 ± 1.4 4 0.56 ± .88 3.80 ± 0.71 5.00 ± 0.0 24 1.10 ± 1.4 4.50 ± 0.53 4.40 ± .71 ∗ p Conclusions: GCA is significantly increased by greater MHC disparity and longer cold ischemic times. GCA may be caused by an Immune-mediated antigen dependent vasculopathy secondary to histoincompatability. In the control group, longer cold ischemic times did not have a significant effect on GCA, suggesting that better histocompatibility may mitigate the adverse effects of longer ischemic times.

Introduction: Previously our laboratory identified a population of cells within the bone marrow, known as Beta2m−/Thy1+ bone marrow derived stem cells (BMSC) that are able to differentiate into hepatocytes. The effect of liver injury on the genes expressed by Beta2m−/Thy1+ BMSC is not known. The objective of this study was to determine the gene expression profile of Beta2m−/Thy1+ BMSC in common bile duct-ligated (CBDL) rats using DNA microarray analysis. Methods: Male Lewis rats underwent CBDL (n = 3)or a sham operation (n = 3). After 10 days, their bone marrow was harvested and Beta2m−/Thy1+ BMSC isolated using a two-step magnetic bead cell sorting technique. The mRNA expression profile of primary hepatocytes harvested from sham-operated male Lewis rats (n = 3) was used for comparison. The gene expression profile of Beta2m−/Thy1+ BMSC was determined using Affymetrix rat genemone U34a array. Results: In sham-operated rats, 24 common genes were found between Beta2m-/Thy1+ BMSC and normal hepatocytes. In the CBDL rats, 189 commonly expressed genes were found between the Beta2m-/Thy1+ BMSC and normal hepatocytes. Several liver-specific genes were identified including HNF1, Complement component C3, cholesterol esterase, Cytochrome P50 3A2, D site albumin promoter binding protein and mitochondrial import stimulation factor. Conclusions: Our results demonstrate that liver injury by CBDL alters the gene expression profile of Beta2m-/Thy1+ BMSCs. Cluster analysis of hepatocyte genes revealed Beta2m-/Thy1+ BMSCs from CBDL rats shared more genes in common with primary hepatocytes than Beta2m-/Thy1+ BMSCs from sham-operated rats. Download full-size image

A prospective study was carried out over a 10-month period to compare nutritional status, gastric colonization, and rates of nosocomial pneumonia in intensive care unit (ICU) patients randomized to either gastric tube feeding or feeding by an endoscopically placed jejunal tube. Sixty-nine patients were considered eligible for the study. Thirty-eight patients were enrolled from medical and surgical ICUs at two hospitals and were equally divided into two groups. The groups were similar in age, sex, race, underlying disease, type of surgical procedure, number of days fed, duration of ICU stay, duration of mechanical ventilation, days with fever, and days of antibiotic therapy. The jejunal tube (JT) group had more patients who were in circulatory shock on admission (79%) than the gastric feeding (GT) group (68.4%). Additionally, the JT group had higher Acute Physiology Scores (24.0 vs 21.7) and fewer patients with pneumonia at the time of entry into the study (26.3% us 31.6%). The JT patients received a signi...

Several investigators, including the authors, have demonstrated that patients receiving long-term total parenteral nutrition (TPN) have low plasma choline levels.1-3 In the present study, 15 patients who were receiving home TPN were randomly chosen from a group of long-term TPN patients found to have low levels of plasma free choline on initial screening.3The patients received TPN for 7.0 ± 1.1 years (range, 1.5 to 12.5 years). Their TPN consisted of 2 to 3 liters of 15% to 25% dextrose solution with 3.5% to 4.25% amino acids, electrolytes, trace elements, and vitamins. Patients received 20.1 ± 2.2 kcal/kg per day actual body weight. Parenteral lipid supplied was 22 ± 5% of daily intravenous caloric intake (Intralipid 20%, Kabi-Vitrum, Alameda, CA).Patients received either lecithin supplementation (20 g/12 hours in 2.5 tablespoons of soybean oil-based liquid) or placebo (2.5 tablespoons of oil) for 6 weeks in a double-blind fashion. Patients were evaluated at baseline and at 2 and 6 weeks. Laboratory meas...

The authors examined the effects of hydrolytic products of carbohydrate, protein, and lipid digestion on jejunal injury and restitution in anesthetized rats. Mucosal epithelial integrity was assessed by measuring blood-to-lumen clearance of 51Cr-labeled EDTA administered as a bolus into the femoral vein. When the intestinal lumen was perfused with 3% hydrolyzed casein or 150 mmol/L glucose there was no effect on 51Cr-EDTA clearance when compared with saline controls. However, perfusion with emulsified lipids (20 mmol/L sodium taurocholate and 10 to 40 mmol/L oleic acid) resulted in a statistically significant (p < .05) increase in 51Cr-EDTA clearance in a dose-dependent fashion. When lipid infusion was terminated and saline perfusion resumed, 51Cr-EDTA clearance returned to control levels.Histologic examination of the jejunal mucosa revealed damage of the epithelial lining of the villous tips in animals treated with lipid infusion. The injury was characterized by gaps in the epithelial lining of the villo...

This is a prospective study that compares the effects of tube feeding to those of a regular diet in patients with alcohol-induced liver disease. Study inclusion criteria were a clinical diagnosis of acute liver disease (ALD), serum bilirubin greater than 51 μmol/L, and one of the following: albumin less than 30 g/L, prothrombin time 4 seconds or greater over control, presence of ascites on physical examination. Fifty-two patients met the inclusion criteria; however, 21 patients were excluded from the study because of the following reasons: objection to the length of the study, refusal of nasoduodenal tube placement, continuing gastrointestinal bleeding, elevated serum creatinine (>221 μmol/L), and inability to give informed consent.Thirty-one patients were randomly assigned to either a regular diet control group (n = 15) or a tubefed treatment group (n = 16). Patients in the treatment group received, in addition to a regular diet, Isocal HCN (Mead Johnson, Evansville, Ind) tube feeding formula that provid...

This study compares the effect of drug treatment for Crohn's disease with that of enteral nutrition alone as a therapeutic modality for this disease. Patients were randomly divided into two groups: One group (n = 52) was treated with 6-methylprednisolone (48 mg/day, subsequently tapered) and sulfasalazine (3 g/day). The second group (n = 55) was treated with enteral nutrition consisting of an oligopeptide diet. The two groups were matched for severity of illness and demographic characteristics. Remission was defined as a decrease in Crohn's Disease Activity Index by 40% (or at least 100 points). Significantly more (p < .01, χ 2) patients in the drug treatment group (41 of 52) achieved remission, compared with the dietary treatment group (29 of 55). The median time to remission for the drug treatment group was 8.2 days compared with 30.7 days for the dietary treatment group (p < .01; Mantel Cox). Further data analysis was carried out to determine whether certain subgroups within the dietary group might hav...

This study examines the effects of diet and cimetidine on theophylline metabolism in 12 men. Subjects, all nonsmokers within 15% of their ideal body weight, were 23 to 26 years of age and had no unusual dietary habits. Energy needs were calculated using the Harris-Benedict equation. Subjects were placed either on a high-protein, low-carbohydrate diet (44% of total calories as protein, 35% as carbohydrate, and 21% as fat) or a low-protein, high-carbohydrate diet (10% of total calories as protein, 70% as carbohydrate, and 20% as fat) for 15 days on each diet. The order of the dietary periods was reversed in one half of the subjects, and there was a 24-day washout period between the two dietary periods. Cimetidine (800 mg/d at bedtime) was administered on days 10 to 15 of each dietary period. Aminophylline (3 mg of theophylline base/kg body weight) was administered intravenously as a bolus over 20 to 30 minutes on days 8 and 15 of each dietary period. Blood was withdrawn before and at 1, 2, 4, 6, 8, 12, and ...

The authors undertook this prospective study to determine whether the presence of a nasogastric tube results in a change in the bacterial flora in the oropharynx. Patients scheduled to undergo an elective surgical procedure under general endotracheal anesthesia were studied. Sixteen patients had a nasogastric tube placed and 14 patients did not. The two patient groups were comparable in terms of age, gender, severity of underlying illness, and use of perioperative antibiotics. A commercially available, presterilized, clear plastic nasogastric tube was used in all patients. The tube was placed either for gastric decompression or feeding. All patients had a high oropharyngeal swab taken just before surgery. A similar swab was obtained 48 and 72 hours later. Patients were examined for evidence of otitis, sinusitis, and pharyngitis at the beginning of the study and 48 hours later. The authors found a significant increase in the frequency of oropharyngeal colonization by pathogenic Gram-negative bacteria after...

We have shown previously that supplemental vitamin A (Vit. A) increases the early inflammatory response to wounding and enhances the collagen content of the intestine of normal and injured rats. We now report the effect of dietary supplementation with Vit. A on the prevention of duodenal ulcer (DU) in rats caused by intragastric administration of cysteamine-HCl. A major way cysteamine-HCl induces DU formation is by enhancing gastric acid secretion. Adult male rats were divided into two groups: (1) rats fed a standard rat Chow (Purina) (15 IU Vit. A/g diet) containing two to three times the National Research Council recommended daily allowance for Vit. A for normal rats; (2) rats fed the same supplemented with 150 IU of Vit. A palmitate per/g Chow. One week later, all rats were given 1 ml of cysteamine-HCl (135 mg) intragastrically. The rats were maintained on their respective diets. Two days later, all rats were killed with ether, the stomach and duodenum excised, and examined for the presence of ulcers. No gastric ulcers were found in either group. There was a statistically significant decrease in the incidence of DUs in the Vit. A-supplemented group when compared to the control group (p less than 0.01) 48 hr following cysteamine-HCl administration; 32% of the Vit. A-supplemented rats developed a DU whereas 74% of rats fed standard Chow had DUs. Most rats had a single DU in the first part of the duodenum, occasionally a second ulcer was noted in the same area. Dietary supplementation with Vit. A had no effect on gastric acid production. In conclusion, our data show that Vit. A dietary supplementation is effective in preventing formation of DUs caused by cysteamine-HCl administration to rats. This effect does not appear to be due to reduction of gastric acid output.

Albumin has been implicated as both an inhibitor and a stimulator of liver regeneration. We examined the rate of liver regeneration following partial hepatectomy in rats which are genetically analbuminemic. Adult male analbuminemic and Sprague-Dawley control rats underwent either a 70% partial hepatectomy or a sham operation. Twenty-four hours postoperatively, rats were injected iv with tritiated thymidine and the liver remnants were resected 1 hr later. Liver weight, protein and DNA contents, and the rate of tritiated thymidine incorporation into DNA were determined. The rate of DNA synthesis in control sham-operated rats was greater (P less than 0.05) than that of analbuminemic rats. Following partial hepatectomy, there was a statistically significant (P less than 0.01) increase in DNA synthesis in control rats; no significant increase was noted in the analbuminemic rats. No significant differences in liver remnant weight or protein and DNA contents were noted between control and analbuminemic sham-operated or partially hepatectomized rats. Our data suggest that albumin plays a role in the regulation of DNA synthesis in both the resting and the regenerating liver and it may be essential in maintaining a normal rate of liver regeneration.

After a 30 minute period of superior mesenteric artery occlusion in adult rats, there was a significant decrease in peritoneal inflammatory reaction, ileus, peritoneal adhesion formation, and histologically proved bowel wall necrosis in animals given intravenous dimethyl sulfoxide at the end of the ischemic period. In contrast, control rats given normal saline solution intravenously demonstrated severe inflammatory reaction, ileus, hemorrhagic peritoneal fluid, extensive adhesion formation, and areas of bowel wall necrosis. Intravenous glycerol did not have the beneficial effect seen with dimethyl sulfoxide. Neither compound was effective when given intraperitoneally. We conclude that intravenous dimethyl sulfoxide has a significant protective effect in rats with acute intestinal ischemia due to the superior mesenteric artery occlusion in the rat.

The purpose of this study was to determine whether enterocyte transplantation could be used to correct a specific genetic metabolic defect. Bilirubin uridine diphosphoglucuronyl transferase (UDPGT) activity has been demonstrated in normal rat intestinal mucosa. We hypothesized that normal rat enterocyte transplantation may restore the ability of Gunn rats, which lack bilirubin UDPGT, to conjugate bilirubin. Small intestine was resected from normal Wistar rat donors. Enterocytes were harvested and suspended in Dulbecco's modified Eagle's medium with 10 percent fetal calf serum. Isolated enterocytes were attached to collagen-coated dextran microcarriers and transplanted into congeneic Gunn rat recipients. Recipient rats underwent bile duct cannulation after transplantation, and bile was analyzed for bilirubin glucuronides by high-pressure liquid chromatography. Fifty percent of the transplanted rats showed a significant increase in the concentration of bilirubin monoglucuronide and diglucuronide in their bile 4 and 7 days posttransplantation. Recipient rats had well vascularized microcarrier-enterocyte aggregates in the peritoneal cavity. Our method for intraperitoneal transplantation of isolated enterocytes in rats could potentially be used to correct specific enzymatic and metabolic defects.

Isolated cryopreserved human liver cells, attached to collagen-coated microcarriers, were injected intraperitoneally into mutant rat recipients genetically deficient in either bilirubin uridine diphosphoglucuronosyltransferase activity (Gunn rats) or albumin synthesis (Nagase analbuminemic rats). One group of the recipient Gunn and analbuminemic rats were made genetically immunodeficient by interbreeding with athymic rats with inherited T-cell deficiency. Injected microcarriers and cells formed aggregates on the surface of the pancreas. There was no morphologic evidence of rejection in athymic recipients, whereas immunocompetent recipients demonstrated rejection within 5 days of transplantation. Athymic-Gunn rat recipients demonstrated excretion of bilirubin glucuronides in bile for 30 days and reduction in their serum bilirubin levels. In recipient athymic-analbuminemic rats, plasma albumin levels increased from pretransplantation levels of 0.025–0.05 mg/ml to 3.9–4.8 mg/ml 8 days posttransplantation and remained nearly at that level throughout the 30 days of the study. A method of harvesting, attaching to microcarriers, cryopreserving, and in vivo testing of human hepatocytes with prolonged survival and function in athymic-Gunn and athymic-analbuminemic hybrid rats is reported. These rats are excellent animal models for testing xenograft function.

We have previously shown that supplemental vitamin E has a cytoprotective effect in the liver of rats with chronic CCL4-induced liver cirrhosis. In this study, we hypothesized that vitamin E would have a protective effect in acute liver injury induced by D-galactosamine. D-Galactosamine-induced injury has been thought to be due to a synergistic direct toxic effect and presence of intestinal bacteria and/or endotoxins. D-Galactosamine was used to induce acute "hepatitis" (1.5-2.0 g/Kg body weight, ip). Rats were placed on either standard chow or the same chow supplemented with vitamin E (300 mg DL-alpha-tocopherol/Kg diet) and 6 days later were given D-galactosamine. There was significantly improved early (5-day) survival and late (14-day) survival in the vitamin E-supplemented group. The vitamin E beneficial effect was manifested also by decreased liver fat and collagen content and decreased SGPT level. Because bacterial endotoxins have been implicated as playing a role in the pathogenesis of D-galactosamine hepatitis, the same experiment was carried out using germ-free and conventional rats. There was significantly improved survival in both the germ-free and conventional vitamin E-supplemented groups both at 5 and 14 days. There was no significant difference between conventional and germ-free rats with or without vitamin E supplementation. In summary (a) vitamin E improves the early fat and collagen accumulation in the liver, decreases SGPT level, and improves survival in the D-galactosamine experimental model of acute liver injury in both conventional and germ-free rats; and (b) D-galactosamine toxicity is probably not mediated through intestinal bacteria and/or endotoxins.

The enzyme ornithine decarboxylase, one of the key enzymes involved in polyamine biosynthesis, catalyzes the decarboxylation of ornithine to give putrescine. The activity of this enzyme in an in vitro hepatocyte culture assay system was measured because it is known that ornithine decarboxylase levels increase in instances where active protein synthesis, DNA synthesis, and cell growth is initiated. A good correlation was found between ornithine decarboxylase activity and the rate of tritiated thymidine incorporation into hepatocyte DNA. The increase in enzyme activity precedes the incorporation of tritiated thymidine into DNA (enzyme activity increases 2-3 hr following stimulation of cell growth; whereas the tritiated thymidine uptake increases at about 14-18 hr). Experimental results obtained with this assay system, suggest that hepatocytes from the regenerating liver remnant, grown in vitro, secrete a factor(s) into the culture medium which stimulates DNA synthesis of normal hepatocytes. Use of the increase in ornithine decarboxylase activity in this hepatocyte monolayer culture system confirmed the observation made by several investigators: that the serum of rats which underwent partial hepatectomy contains a factor(s) which stimulates hepatocyte DNA synthesis in vitro. In conclusion, these results suggest that ornithine decarboxylase activity can be used as a sensitive, early indicator of the degree of stimulation of hepatocyte DNA synthesis and thus be of use in determining the effect of various trophic factors on hepatocyte DNA synthesis in vitro.

The effect of dietary supplementation with vitamin A on the healing of colon anastomoses was studied. Fifty adult male Sprague-Dawley rats were divided into two groups: (1) rats fed a standard chow which contains the equivalent of about 15 IU vitamin A/g diet; (2) rats fed the chow supplemented with an additional 150 IU vitamin A/g diet. Rats were prefed for 5 days; on Day 6 under ether anesthesia the colon was divided 1-in. distal to the ileocecal junction and then reanastomosed. The rats were maintained on the above diets for 5 days and killed on the sixth postoperative day with ether and the segment of colon containing the anastomosis was resected. In 15 rats of each group, the breaking strength of the anastomosis was measured. In the remaining 10 rats of each group, the bursting strength of the anastomotic site and a segment of normal distal colon was measured. Samples of colon from the anastomotic site and the normal segment were analyzed for hydroxyproline. There was a significant decrease in hydroxyproline content at the anastomotic site when compared to the normal distal colon segment in each group of rats (P less than 0.01). The hydroxyproline content of both normal colon and the anastomotic site was significantly higher in the vitamin A-supplemented rats than in the control diet rats (P less than 0.01). There was also a significant increase in bursting strength in the vitamin A-supplemented rats both of the anastomotic site (P less than 0.01) and of the normal colon segment (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Six week-old male CBA/J mice fed a commercial powdered laboratory chow or the same chow supplemented with vitamin A palmitate (150,000 U/kg) were inoculated with either the Moloney strain of murine sarcoma virus (M-MuSV) or poxvirus. Central body temperature was measured daily. Both viruses elicited fevers, but the fevers were less pronounced and of shorter duration in the mice ingesting the vitamin A-supplemented diet. Palpable M-MuSV-induced tumors appeared later, were less frequent, grew more slowly, and were resorbed sooner in the mice fed the vitamin A supplement. Similarly, in these mice the appearance of pox lesions was delayed, their numbers reduced, and their disappearance hastened.

Vitamin A palmitate was incorporated into a laboratory chow (150,000 IU/kg diet) and fed ad libitum to C3H/HeJ female mice inoculated with 1 times 10-6 C3HBA tumor cells, beginning the day of inoculation. Control female mice of the same strain similarly inoculated were fed the laboratory chow alone. Vitamin A did not affect rate for the first 19 days, after which growth rates were independent of treatment. Vitamin A-treated mice survived for significantly longer times than did control mice.

Isolated hepatocytes, harvested from normal rat livers by portal vein collagenase perfusion, can be attached to collagen-coated dextran microcarriers and transplanted by intraperitoneal injection into rats. Survival and function of the transplanted hepatocytes have been demonstrated in mutant rats lacking bilirubin-uridine diphosphate glucuronosyltransferase activity (Gunn strain) and rats with inherited lack of plasma albumin (Nagase analbuminemia rat strain). This simple technique promises to be useful in the treatment of acute liver failure in humans.

It has been suggested by several investigators that hypoalbuminemia results in impaired wound healing. In most studies, however, hypoalbuminemia is a manifestation of malnutrition or underlying liver disease. In this study, we examined the effect of isolated hypoalbuminemia on wound healing. Analbuminemic (NAR) rats which are Sprague-Dawley mutants with trace levels of plasma albumin due to a defect in albumin synthesis were studied. Adult NAR and Sprague-Dawley rats (n = 10 each) underwent a 7 cm dorsal skin incision and implantation of a polyvinyl alcohol sponge subcutaneously under pentobarbital anesthesia. Seven days postoperatively all rats were killed with ether, the wounds were excised, and breaking strength was measured. Sponge hydroxyproline content was determined colorimetrically. There were no significant differences in wound breaking strength (fresh or formalin fixed) or sponge collagen content between the Sprague-Dawley and analbuminemic rats. We conclude that isolated hypoalbuminemia has no detrimental effect on wound healing in rats.

Male CBA mice received graded doses (450-750 rad) of total-body gamma-radiation (TBR) from a dual-beam 137Cs irradiator. Commencing directly after TBR, 2 days later, or 6 days later, groups of mice received supplemental vitamin A (Vit A) or beta-carotene (beta-Car), compounds previously found to reduce radiation disease in mice subjected to partial-body X-irradiation. Given directly after TBR, supplemental Vit A decreased mortality, evidenced by increases in the radiation dose required to kill 50% of the mice within 30 days (LD50/30). In one experiment, Vit A increased the LD50/30 from 555 to 620 rad; in another experiment, Vit A increased the dose from 505 to 630 rad. Similarly, in a third experiment, supplemental beta-Car increased the LD50/30 from 510 to 645 rad. Additionally, each compound increased the survival times, even of those mice that died within 30 days. In addition to reduction of mortality and prolongation of survival time, supplemental Vit A moderated weight loss, adrenal gland hyperemia, thymus involution, and lymphopenia--all signs of radiation toxicity. Delaying the supplementation for 2 days after irradiation did not greatly reduce the efficacy of Vit A; however, delaying supplementation for 6 days decreased its effect almost completely.

Basic fibroblast growth factor (bFGF) stimulates extracellular matrix metabolism, growth, and movement of mesodermally derived cells. We have previously shown that collagen content in polyvinyl alcohol sponges increased after bFGF treatment. We hypothesized that bFGF-treated incisional wounds would heal more rapidly. After intraperitoneal pentobarbital anesthesia, male, 200- to 250-g, Sprague-Dawley rats (n = 27) each underwent two sets of paired, transverse, dorsal incisions closed with steel sutures. On Day 3 postwounding, 0.4 ml of bFGF (recombinant, 400 ng. Synergen) or normal saline was injected into one of each paired incisions. Animals were killed with ether on postwounding Days 5, 6, and 7 and their dorsal pelts were excised. Fresh or formalin-fixed wound strips were subjected to tensile strength measurements using a tensiometer. Breaking energy was calculated. Wound collagen content (hydroxyproline) was measured in wound-edge samples following hydrolysis using high-performance liquid chromatography. There was an overall significant increase in fresh wound tensile strength (13.7 +/- 1.06 vs 19.1 +/- 1.99 g/mm, P less than 0.01) and wound breaking energy (476 +/- 47 vs 747 +/- 76 mm2, P less than 0.001) in bFGF-treated incisions. There was an increase in wound collagen content which was not statistically significant and there was no difference in fixed incisional tensile strength. Histologic examination showed better organization and maturation in bFGF wounds. Recombinant bFGF accelerates normal rat wound healing. This may be due to earlier accumulation of collagen and fibroblasts and/or to greater collagen crosslinking in bFGF-treated wounds.

The effect of MCCH on wound healing was studied in animals and patients. A full thickness dorsal skin defect was made in mice and standard amounts of MCCH applied immediately to the wound in half the mice. There were no significant differences in wound closure rates between control and MCCH treated mice. A standard dorsal skin incision was made in rats. In half the rats, MCCH was put into the incision just before suturing with fine stainless steel sutures. There were no statistically significant differences in breaking strengths between control and MCCH treated rats when tested on the 8th, 20th and 40th days postoperatively. Histologic examination of the wounds showed mild inflammatory reaction surrounding the MCCH-fiber fragments, but no giant cells. Small amounts of MCCH were demonstrable at 40 days. Full thickness skin burns in pigs were excised one day after burning. MCCH was applied immediately in some pigs; excess MCCH was removed by saline irrigation. Split thickness skin autografts were applied. MCCH was applied to donor sites. The "takes" of the grafts were excellent and not affected by the use of MCCH and the donor sites healed uneventfully. MCCH was used in four patients with burns, three of whom underwent early excision of full thickness skin burns and immediate autografting. The fourth underwent skin grafting to the granulating areas 3 months after injury. The MCCH was applied to some donor sites and to some areas of excision. In one patient with severe burns, wound sepsis developed equally in areas with and without MCCH and the grafts were lost. In the other three patients, there were excellent takes of the grafts at all sites. All donor sites, treated and untreated, healed normally in all patients.

A technique has been developed by the authors that allows hepatocyte attachment on collagen-coated microcarriers resulting in prolonged hepatocyte viability and function both in vivo and in vitro. Rat hepatocytes were obtained by portal vein collagenase perfusion. Intraperitoneally transplanted microcarrier-attached normal hepatocytes into congeneic Gunn rats were functioning 3-4 weeks later, as shown by the presence and persistence of conjugated bilirubin in recipient bile, sustained decrease in serum bilirubin, uptake of Tc99m-DESIDA, and morphologic criteria. Intraperitoneal transplantation of normal microcarrier-attached hepatocytes into genetically albumin deficient rats (NAR) resulted in marked increase in plasma albumin levels (6 days without and 21 days with Cyclosporin A immunosuppression). Microcarrier-attached hepatocytes transplanted after 2 weeks of storage at -80 C into congeneic Gunn rats were viable and functional as assessed by criteria outlined above. An extracorporeal liver perfusion system was developed using the microcarrier-attached hepatocytes that was capable of synthesizing and conjugating bilirubin and synthesizing liver-specific proteins.