Portal vein arterialization promotes liver regeneration after extended partial hepatectomy in a rat model

In the current study, we sought to establish a novel rat model of portal vein arterialization (PVA) and evaluate its impact on liver regeneration after extended partial hepatectomy (PH). A total of 105 Sprague-Dawley rats were randomly assigned to three groups: 68% hepatectomy (the PH group), portal arterialization after 68% hepatectomy (the PVA group), and right nephrectomy only (the control group). Liver regeneration rate (LRR), 5-bromo-2-deoxyuridine (BrdU) labeling index, and liver functions were assessed on postoperative day 2, 7, 14 and 28. The 28-day survival rates were compared among the three groups. The 28-day survival rates were similar in all groups (P  =  0.331), and the anastomotic patency was 100%. The LRR in the PVA group was significantly higher than that of the PH group within postoperative 14 days (P < 0.05). The PVA and PH group had increased serum alanine aminotransferase levels (232 ± 61 U/L and 212 ± 53 U/L, respectively) compared with the control group (101 ± 13 U/L) on postoperative day 2, whereas from postoperative day 7 to day 28 there were no differences among the three groups. Serum albumin values were higher after the PVA procedure within postoperative day 14, which gradually became comparable on postoperative day 28 among the three groups. The peaks of BrdU labeling index appeared on postoperative day 2 in all rats, and the PVA procedure was associated with increased BrdU labeling index from postoperative day 7 to 28. The 28-day survival of the PVA rats was comparable. Our findings demonstrate that the PVA procedure utilizing portal vein trunk-renal artery microvascular reconstruction promotes remnant liver regeneration and confers beneficial effects on maintaining and even optimizing liver function after extended partial hepatectomy in rats.     

Phenotypical and morphological alterations to rat sinusoidal endothelial cells in arterialized livers after portal branch ligation

The hepatic sinusoids are preferentially supplied with portal venous blood and equipped with fenestrated endothelial cells that are distinct from capillary endothelial cells. We previously observed in rats that sinusoidal capillarization proceeded concurrently with arterial blood supply during hepatocarcinogenesis. This study aimed to clarify the inducing role of arterialization in sinusoidal capillarization by investigating phenotypical, morphological and functional alterations to sinusoidal endothelial cells (SECs) in arterialized rat livers induced by portal branch ligation. At one week, after massive hepatic necrosis following ligation, the livers were restored to their normal architecture without causing post-necrotic fibrosis. At 12-21 weeks, they exhibited a normal histology except for mild pericellular fibrosis which developed along sinusoids or between adjacent hepatocytes. SECs expressed factor VIII-related antigen and showed a decrease in the number of fenestrae and porosity, still lacking any basement membrane but further retaining the functional capacity for carmine dye uptake. Stellate cells, while occasionally associated with large amounts of collagen bundles, contained many lipid droplets and expressed no alpha-smooth muscle actin, indicating a quiescent property. Kupffer cells were commonly found within the sinusoids. The present results indicate that arterialization of the liver induces a partial (but not complete) transition of SECs into capillary-type endothelial cells, suggesting that arterialization might be one of the factors which induce sinusoidal capillarization in the development of hepatocellular carcinoma.     

Effect of uterine artery ligation on ovulation in the rat.

Female rats were divided into six groups: (1) control, (2) one uterine artery (a.) ligated near the utero-tubal (U-T) junction, (3) one uterine a. ligated at the level of the cervix, (4) both uterine aa. ligated separately at the U-T junction, (5) both uterine aa. ligated separately at the cervix and (6) both uterine aa. tied with one ligature at the cervix. Segmental aa. were disrupted in all experimental groups except group 6. Animals were allowed to recover for ten days and killed the first metestrus thereafter. Number of eggs ovulated was determined by flushing the oviduct with saline solution and counting the ova. Control rats ovulated 5.0 +/- 0.4 eggs per ovary. Groups 2 and 3 had an increase in the number of eggs shed from the ovary on the non-ligated side. In contrast, a decrease in the number of ova shed occurred on the ligated side. When both aa. were ligated separately (groups 4 and 5), irrespective of location, a decrease in the number of eggs shed by both ovaries was evident. No effect was found when only one ligature was placed near the cervix (group 6). The data demonstrate that blood supply to the ovary via the uterine artery is essential for the full complement of eggs to be shed.     

Reevaluation of a genetic model for the development of exostosis in hereditary multiple exostosis

EXT1 and EXT2 are genes that have been shown to cause hereditary multiple exostosis (HME), a syndrome marked by the formation of bony growths juxtaposed to the growth plate. These genes are members of a growing family of proteins with glycosyltransferase activity required for the synthesis of heparan sulfate chains. This protein activity is predicted to play a role in the expression of proteoglycans on the cell surface and in the extracellular matrix. We and others have previously suggested that a two-hit mutational model applies to the development of an exostosis where a germline mutation coupled with a somatic mutation results in the loss of EXT1 or EXT2 function and subsequent tumor formation. We report the direct sequencing and loss of heterozygosity (LOH) analysis of 12 exostoses from 10 HME families, 4 solitary exostoses, and their corresponding constitutional DNA. Of the 16 exostoses screened, we find only one solitary case in which two somatic mutations, a deletion and an LOH, are present. This provides limited support for the two-hit hypothesis involving the EXT1 and EXT2 genes for the development of an exostosis. Alternative models are developed based on the functional significance of EXT proteins in heparan sulfate biosynthesis.     

Transplant arteriosclerosis in a rat aortic model.

Transplant arteriosclerosis (TA) has emerged as an obstacle to the long-term survival of transplanted organs, especially cardiac transplants. The animal models that have been used to study TA have not been fully characterized with regard to features such as the time course of cell proliferation and the sequence of cell types arriving in the developing intimal lesion. We present a model of TA based on a transplanted segment of abdominal aorta that helps address these questions. Two strains of rats (PVG x DA) underwent orthotopic aortic transplantation without immunosuppression and were killed at 14, 20, 40, and 60 days after transplantation. The within-strain control group displayed minimal evidence of cellular rejection with minimal to absent intimal lesions. In contrast, the allograft group showed a linearly increasing intimal lesion, up through 60 days after transplantation. The mechanism of intimal thickening was by an increase in cell number at the earlier time points with the later deposition of extracellular matrix. The early intimal lesion consisted mostly of mononuclear inflammatory cells (45%) with gradually increasing presence of smooth muscle cells (SMC) in the intima between 20 and 60 days. Conversely, the media showed gradual infiltration by macrophage-type cells with virtual loss of all SMC from the media by 40 days. The proliferative index showed a peak of 6% and 8% at 20 days in both the intima and media, respectively, and was preceded by the presence of macrophages. In fact, most of the proliferating cells at the earlier time points were either monocytes/macrophages, or were immediately adjacent to monocyte-/macrophage-rich regions. This straight artery segment model of transplant arteriosclerosis provides an easily quantifiable system in which the effects of different interventions (e.g., immunosuppressive regimens) can be tested.     

Reevaluation of the yeast killer phenomenon.

The killer effect of 36 Hansenula, Pichia, Saccharomyces, and Candida species on 26 hyphomycetes isolates, 1 isolate of the achlorophyllous microorganism Prototheca, 4 isolates of the lipophilic yeast Malassezia, 1 isolate of the aerobic actinomycete Nocardia, and 19 isolates of bacteria was studied. The killer phenomenon, which was previously considered to be restricted to yeasts, was found to occur among unrelated microorganisms.     

Reevaluation of bacteriocinogeny in Neisseria gonorrhoeae.

Bacteriocin typing has been described previously and proposed for typing gonococci. A survey has been made of 150 strains of N. gonorrhoeae from various places to determine the feasibility of a gonocin typing system. All strains were found to produce an inhibitory substance which inhibited all strains of gonococci tested, one strain of Neisseria flavescens, two strains of Neisseria meningitidis, as well as the producing strain itself. The inhibitory activity was enhanced by supplementary glucose, reduced by supplementary serum, and unaffected by the addition of HEPES buffer, by the temperature of incubation, or by the exposure of potential producer strains to sublethal concentrations of mitomycin C. This nonspecific inhibitory activity differed from that of a putative bacteriocin produced by a strain of N. meningitidis, in that the latter inhibited most other meningococci but not the producer strain itself. Bacteriocinogeny has not yet been convincingly demonstrated in N. gonorrhoeae, and gonocin typing has not yet been shown to be feasible. Production of the nonspecific inhibitor may have obscured past attempts to demonstrate type-specific gonococcal bacteriocin.     

Effect of uterine artery ligation on ovulation in the rat

Female rats were divided into six groups: (1) control, (2) one uterine artery (a.) ligated near the utero-tubal (U-T) junction, (3) one uterine a. ligated at the level of the cervix, (4) both uterine aa. ligated separately at the U-T junction, (5) both uterine aa. ligated separately at the cervix and (6) both uterine aa. tied with one ligature at the cervix. Segmental aa. were disrupted in all experimental groups except group 6. Animals were allowed to recover for ten days and killed the first metestrus thereafter. Number of eggs ovulated was determined by flushing the oviduct with saline solution and counting the ova. Control rats ovulated 5.0 ± 0.4 eggs per ovary. Groups 2 and 3 had an increase in the number of eggs shed from the ovary on the non-ligated side. In contrast, a decrease in the number of ova shed occurred on the ligated side. When both aa. were ligated separately (groups 4 and 5), irrespective of location, a decrease in the number of eggs shed by both ovaries was evident. No effect was found when only one ligature was placed near the cervix (group 6). The data demonstrate that blood supply to the ovary via the uterine artery is essential for the full complement of eggs to be shed.     

Intestinal anaphylaxis in the rat as a model of food allergy.

An animal model of food allergy has been developed in which some aspects of the allergic response could be quantified and the effects of various drugs evaluated. The change in permeability of the intestinal tract of actively sensitized rats, after oral challenge with the sensitizing antigen, was the parameter measured. Rats were sensitized by injection of egg albumin and B. pertussis vaccine to induce reaginic antibody to egg albumin. Two weeks after sensitization, 125I-labelled bovine serum albumin (125I-labelled BSA) was injected intravenously, followed by oral challenge with egg albumin. Pieces of intestinal tissue were obtained and the amount of 125I-labelled BSA determined in a gamma counter. The amount of 125I-labelled BSA in the intestinal tissue of sensitized and challenged rats regularly showed an increase of greater than 100% above values for control rats.     

Spontaneous nephrotic syndrome in a genetic rat model.

Advances in our understanding of the mechanisms of proteinuria in humans have depended on a variety of animal models. Most of these have been partially satisfactory because they require pretreatment of the animal with chemicals or toxins or they depend on an aging-related glomerular protein leakiness. The strain in this study was obtained by Koletsky after selective inbreeding of the offspring from a hypertensive Kyoto-Wistar and a normotensive Sprague-Dawley rat. The affected animals appear in 25% of the litters, indicating an autosomal recessive gene, and present with a spontaneous and progressive nephrotic syndrome detected as early as 3-5 weeks and associated with obesity, hypertension, hypoalbuminemia, hypercholesterolemia, and hyperlipidemia. Preliminary morphologic and immunofluorescence studies of their kidneys show progressive glomerular segmental sclerotic lesions and prominent mesangial deposition of IgM, a picture which resembles a steroid-resistant form of idiopathic nephrotic syndrome in humans, namely, focal glomerular sclerosis.     

Discharge patterns of preganglionic neurones with axons in a cardiac vagal branch in the rat

The fibre types that run in a vagal branch projecting to the rat heart are described in this study. In order to obtain spontaneous discharge in this vagal branch and optimal recording conditions, we compared the decerebrate state to urethane, urethane-chloralose and pentobarbital-chloralose anaesthesia with regard to level of chronotropic cardiac vagal tone. Administration of atropine (2 mg kg(-1), I.V.) significantly decreased baseline cardiac interval only in the decerebrate and urethane-anaesthetised rat (by 0.018 +/- 0.001 s and 0.019 +/- 0.002 s, respectively). As a result of these experiments, urethane was chosen as the anaesthetic for all subsequent studies. Using a heart rate signal-averaging method we demonstrated that rat cardiac vagal preganglionic neurones innervating the sinoatrial node should have an expiratory discharge pattern, as reported in other species. However, only 5 % of chronotropic vagal tone was found to be subject to respiratory sinus arrhythmia. A suction microelectrode method, combined with spike-triggered averaging, was employed to record activity from a total of 58 vagal afferents that had axons in this branch. Approximately 75 % of these latter sensory fibres displayed cardiac rhythm. In a separate study we also recorded 318 preganglionic neurones with axons in the right cardiac vagal branch of the rat. Respiratory-modulated preganglionic units were statistically less common than tonically firing units. Six preganglionic subtypes were categorised according to conduction velocity and respiratory discharge pattern. Myelinated B-fibre and unmyelinated C-fibre types were found to be equally prevalent and equally likely to be reflexly excited during the pulmonary chemoreflex and the peripheral arterial chemoreflexes. The electrophysiological analysis has shown how diverse the discharge patterns of the preganglionic neurones or interneurones are whose axons course in the right cardiac vagal branch of the rat. The results of these experiments demonstrate the usefulness of combining spike discrimination with multiple spike-triggered averaging to simultaneously record B and C centrifugal vagal efferents.     

Molecular mechanisms of apoptosis in the liver of rats after portal branch ligation with and without retrorsine

The mechanisms accounting for the atrophy of the portal blood-deprived liver lobes after portal branch ligation (PBL) are still unclear. The first aim of this study was to confirm the role of apoptosis in this process and to determine which apoptotic pathways are involved. The second aim of the study was to evaluate the effect of blocking compensatory hyperplasia of the nonligated lobes with retrorsine on the mechanisms of apoptosis in the ligated lobes. Mitochondrial Bax, Bcl-2 and Bcl-X(L), cytosolic cytochrome c, caspase-3, -8 and -9 activities and TNF-alpha levels were assessed in the liver of rats before and at various time points, ranging from 30 min to 7 days, after PBL. Caspase activities were also measured in rats pretreated with retrorsine. Both the mitochondrial and the death receptor-mediated pathways are activated in the ligated liver lobes after portal branch ligation. Caspase activation is inhibited by retrorsine pretreatment, resulting in fewer apoptotic bodies. Apoptosis accounts for the atrophy of the ligated lobes after PBL. It is inhibited by retrorsine, suggesting an attempt to reduce the loss of liver mass when hyperplasia of the nonligated lobes is impaired     

Renal blood flow after bilateral ureteral ligation in the rat

Renal blood flow (RBF) in rat was measured by using a noncannulating electromagnetic flowmeter. In the sham control rats, anesthetized with Inactin, RBF averaged 7 ml/min/g KW when arterial blood pressure was above 110 mm Hg. Auroregulation of RBF was observed when the arterial blood pressure was in the range of 110–150 mm Hg. Glomerular filtration rate (GFR), measured by polyfructosan clearance, averaged 1.08 ml/min/g KW. In experimental rats with 24 h of bilateral ureteral ligation (BUL), RBF averaged 38% of control value. During 1/2–3 h following release of the left ureteral occlusion, RBF increased to 60% of control value. The autoregulatory ability of the damaged kidney was reduced during BUL and did not improve after releasing occlusion. During the post-obstructive period arterial blood pressure remained stable. Thus, a high total renal vascular resistance was responsible for the depressed RBF. GFR in these rats averaged only 9% of control value. The reduction in RBF alone does not explain the drastic reduction in GFR in this model of renal failure.     

Morphologic changes of the junctional complex of the hepatocytes in rat liver after bile duct ligation.

Using freeze-fracture techniques, we have examined the changes in the morphology of the tight junctional network around the canalicular lumen of the hepatocytes in rat liver after experimental bile-duct ligation. The more or less regular belt of parallel strands formed by the tight junctions around the canalicular lumen of normal hepatocytes is changed after extrahepatic obstruction. A more irregular network is formed withe a reduced number of strands which also extend more in an abluminal direction with formation of irregular loops. Striking changes are seen at the gap junctions: the small patches normally situated within the tight junctional network become less numerous; at some canaliculi they are even absent; also the larger gap junctional areas normally present in deeer abluminal extensions of the lateral cell membrane become hard to find or are even absent. This altered tight junctional pattern suggests an increased permeability so that the pathway of intercellular escape of biliary constituents towards the blood stream in cholestasis becomes as plausible as transhepatocytic regurgitation. The disappearance of the gap junctions would result in a lack of intercellular communication and uncoupling of liver cells, which may lead to a more individual behaviour of adjecent hepatocytes, explaining the heterogeneity in canalicular changes in cholestasis.     

Modification of pancreatic carcinogenesis in the hamster model. 6. The effect of ductal ligation and excision

The effect of ligation and excision of the pancreatic duct in pancreatic carcinogenesis was examined in the hamster model. Animals were treated with a single dose (20 mg/kg body weight) of N-nitrosobis(2-oxopropyl)amine (BOP) either immediately (Group 1) or on Days 1 (Group 2), 3 (Group 3) or 7 (Group 4) after ligation and excision of the duct of the splenic lobe. Group 5 received BOP shortly after laparoscopy, and Group 6 consisted of BOP-treated controls. All hamsters were killed 46 weeks after BOP treatment. The results showed that despite advanced atrophy of the splenic lobe distal to the excised duct in Groups 1-4, some hamsters in Groups 2, 3, and 4 showed hyperplasia, dysplasia, and increased mitotic activities of ductal and ductular cells. However, carcinomas in the duct-excised atrophic lobe were found only in Groups 1-3. These data indicate that BOP carcinogenesis is mediated through blood circulation, and that cancer development is not inhibited in the duct-excised lobe for up to 3 days after surgery. However, in the entire pancreas, a significant reduction in tumor incidence was seen when the carcinogen was given immediately, or to a lesser extent, 1 day after surgery, regardless of whether or not excision was made. On the contrary, BOP, when given 3 and 7 days after duct excision, enhanced tumor development in the nonexcised (intact) pancreas, compared with other test groups and with BOP controls. Both inhibition and enhancement seemed due to a proportional decrease and increase, respectively, of BOP-responsive cells throughout the intact pancreas.