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Final Survey. Questionnaire administered at the end of the study to compare the three treatment modalities. (PDF 30 kb)

VERGE Survey. Questionnaire administered following the VERGE therapy addressing system specific details. (PDF 28 kb)

Weekly Survey. Questionnaire administered weekly following each therapy modality (including VERGE). (PDF 311 kb)

Home Exercise Program (HEP) handout. The HEP consisted of pre-defined sets of seated, self-paced arm-hand exercises derived from the standard of care. These 16 exercises were presented to the participant in the form of a printed handout. (PDF 261 kb)

The physiological role of nitric oxide (NO) in portal venous and hepatic arterial haemodynamics in the rat in vivo during healthy and diseased conditions remains unclear. The present study determined the physiological role of nitric oxide in hepatic haemodynamics in the rat in vivo during healthy conditions as a basis for future pharmacological work. Male Wistar rats (300-350 g) were anaesthetised with fentany/fluanisone (0.3 mg/kg s.c.) and midazolam (0.3 mg/kg s.c.) and heparinised (30 U/100 g i.v.) via a cannulated left carotid artery for measurement of heart rate, mean arterial pressure, and the difference between systolic and diastolic blood pressures (P(S-D)). Following laparotomy, two distal ileocolic veins were cannulated, one catheter introduced to a distance of 1 cm and used for intraportal drug injections and the other to the main trunk of the portal vein for continuous measurement of portal venous pressure. The portal venous trunk and hepatic artery were carefully isolated and electromagnetic probes placed around each of them for measurement of portal venous flow and hepatic arterial flow. Augmentation of NO production was achieved by intraportal injection of 0.2, 0.4, 0.6 and 0.8 g/kg L-arginine and the NO donor, 3-morpholinosydnonimine (SIN-1), was injected intraportally at 0.2, 0.4, 0.6 and 0.8 mg/kg. L-NAME, the non-selective NOS inhibitor, was injected intraportally in increasing doses of 5, 10, 15 and 20 mg/kg in the absence or presence of L-arginine in doses of 0.2 and 0.5 g/kg. L-arginine increased portal blood flow by 25% without significant changes in systemic haemodynamics. SIN-1 decreased mean arterial pressure by 33% with no effect on portal blood flow. Both L-arginine and SIN-1 reduced portal venous pressure by 25% in a dose-dependent manner. L-NAME had no effect on portal haemodynamics despite a significant increase in systemic arterial pressure of 60% that was reduced dose-dependently by L-arginine. Hepatic arterial flow increased by 88% and 49% at the second and third doses of L-arginine and by 68% and 27% at the first two doses of L-NAME. No significant changes in hepatic arterial flow were found when L-NAME and L-arginine were given together. It is concluded that augmented endogenous NO production increased portal flow. Inhibition of endogenous NO had no effect on portal haemodynamics. Endogenous NO may not play a major role in regulation of portal haemodynamics in the rat in vivo.

Background/Aims : Hepatotoxin-induced rat models of liver cirrhosis are limited by the wide heterogeneity of cirrhosis produced. The present study developed a modified, reliable, and reproducible technique by which hepatic and systemic responses to thioacetamide during induction of cirrhosis were monitored by weekly weight changes. Methods : Male Wistar rats (200–230 g) were divided into three groups. Group 1 ( n =20) received continuous administration of 0.03% (w/v) thioacetamide in the drinking water for 12 weeks. Group 2 ( n =20) received the same concentration of 0.03% thioacetamide as an initial concentration that was modified according to weekly weight changes in response to thioacetamide during the induction of cirrhosis. Group 3 ( n =6) received normal water and served as controls. Results : Mortality of Group 1 was 30% and the production of cirrhosis was only 45%. In contrast, there were no deaths in Group 2 and well-developed macronodular cirrhosis was found in 90% of the rats which was associated with significant portal hypertension, as indicated by increased portal venous pressure (13.6±0.4 vs. 9.1±0.3 mmHg, cirrhotic vs. control, respectively, P t -test). Conclusions : Variations in responses to thioacetamide can be easily monitored by weekly weight changes to reduce mortality to zero and simultaneously increase the production and quality of cirrhosis induced in rats.

Endothelium-dependent and -independent vasorelaxation in ring segments of rabbit thoracic aorta is reduced and noradrenaline-induced vasoconstriction unaltered after prolonged storage in University of Wisconsin solution (UW) compared to arteries stored in extracellular-type solutions such as Krebs-Bülbring buffer (KBB). The aims of the present study were to determine whether angiotensin-II-induced vasoconstriction, alterations in myosin light chains, protein synthetic capacity, and subcellular structures are altered after 8 days of UW storage at 4 degrees C. The present study showed reduced contractility to angiotensin II, following 8 days of cold storage in UW, that was not reversed in the presence of a nitric oxide synthase inhibitor, N(G)-nitro-l-arginine methyl ester (100 microM). Measurements of contractile protein ratios in the same tissues after cold storage in UW or KBB did not show any significant alterations in smooth muscle myosin light chains or protein synthetic capacity (reflected by total RNA). It is concluded that reductions in vasoconstriction in UW-stored tissue are unlikely to be due to increased release of nitric oxide nor reduced availability of myosin light chains for phosphorylation and vasoconstriction.

Inclusion of albumin in the perfusate has been previously shown to be detrimental to liver function, but its effect on hepatic vascular reactivity remains unknown. The aim of this study was to determine the effects of albumin on hepatic arterial vascular reactivity and liver viability in the isolated dual-perfused rat liver. A total of 12 rat livers were perfused with Krebs–Bulbring buffer without (Group 1) and with (Group 2) addition of 1% bovine serum albumin (BSA) through the hepatic artery and portal vein for up to 5 h. Hepatic arterial responses to acetylcholine and sodium nitroprusside were studied at 30-min intervals. Liver viability was assessed by bile volume production, release of aspartate serine aminotransferase (AST) and lactic acid dehydrogenase (LDH), and histological examination. Hepatic arterial responses to acetylcholine were significantly attenuated in Group 2. No significant differences in sodium nitroprusside responses were noted. However, bile volume production in Group 2 was significantly decreased compared to Group 1. Effluent AST and LDH release increased significantly in Group 1 but not in Group 2. Histological results showed that sinusoidal endothelial cells and hepatocytes were well preserved without significant deterioration in either group, although there was a marked decrease in vasodilatation to acetylcholine in Group 2. This data suggested that the presence of albumin in the perfusate did not improve retention of smooth muscle reactivity and reduced endothelium-dependent vasodilatation and bile volume production during perfusion. However, improved liver parenchymal cell function was observed.

The pathogenesis of portal-systemic encephalopathy (PSE) and hepatic encephalopathy (HE), disorders of the brain attributed to abnormal liver function, are poorly understood. This study was conducted to examine if a fundamental, and possibly exclusive, homeostatic interrelationship exists between the liver and brain that might deteriorate during liver failure to result in the syndrome of PSE and HE. An isolated organ perfusion circuit was devised to accommodate an isolated rat brain and an isolated rat liver preparation perfused concomitantly. The survival time of the brain preparation was measured by the maintenance of the spontaneous electroencephalocorticogram and was extended from a median survival time of 35 min (range 22 to 53 min), when perfused alone, to 210 min (range 172 to 480 min), when perfused simultaneously with a liver. Also, concomitant perfusion with an isolated rat liver reduced perfusate glucose concentrations from 200 mg% to a range between 45 to 60 mg%. These data support our hypothesis that a homeostatic interrelationship exists between the liver and brain that is independent of other metabolic influences; disturbance of this relationship may contribute towards the syndrome of PSE and HE.

Endothelial dysfunction has been reported in donor blood vessels destined for organ transplantation following cold-storage preservation with University of Wisconsin solution (UW). This was investigated in the present work. Segments of rabbit thoracic aorta were mounted on isometric fine-wire myographs at 37°C and gassed with 95% O2/5% CO2. Concentration-dependent vasodilatations to acetylcholine and adenosine-5′-triphosphate (ATP) were obtained in freshly-harvested rabbit aortic rings, with and without the endothelium, and after 8 days of cold-storage, at 4°C, in either UW, Krebs-Bülbring buffer (KBB) or saline. The action of the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (100 μM) was evaluated upon the concentration-response curves to determine whether nitric oxide (NO) exerted any modulatory actions. Endothelium-dependent, NO-mediated responses to acetylcholine were unaltered after eight days of storage in UW, reduced after storage in KBB and absent after removal of the vascular endothelium, saline storage or after testing in the presence of L-NAME, suggesting improved NO-mediated endothelial function with the use of UW. Structural preservation was also confirmed using scanning electron microscopy. In contrast, endothelium-dependent responses to ATP were unchanged after eight days of storage in KBB but were reduced after storage in UW and saline, suggesting purinergic (ATP) endothelial dysfunction after storage in UW. L-NAME markedly reduced vasodilatation to ATP in freshly harvested rings and after eight days of storage in KBB. This reduction was statistically significant (P < 0·05, Student's two tailed, unpaired t-test) at —log (M) ATP concentrations of 5·5, 5·0, 4·5, 4·0 and 3·5. NO-dependent vasodilatation to ATP was not attenuated by L-NAME in UW-stored rings. Eight days' UW-storage of rabbit thoracic aortic rings appeared to have differential and paradoxical effects upon NO-dependent vasodilatation to acetylcholine and ATP. Morphological observations using electron microscopy suggested that UW preserved the vascular endothelium and this was verified by retained vascular reactivity of endothelium-dependent vasodilatations to acetylcholine. UW-storage however, significantly reduced endothelium-dependent relaxation to ATP thereby suggesting that P2Y-purinoceptors, which are located on the vascular endothelium, may be more susceptible to biodegradation than cholinergic receptors and may be responsible for endothelial dysfunction following transplantation.

The locations of the vascular resistance sites which regulate vascular tone in the hepatic arterial and portal venous vasculatures of the rat liver were identified using a new, in vitro, dual-perfused liver preparation. Twelve livers of male Wistar rats were perfused via the hepatic artery and portal vein at fixed flow and at physiological pressure. Dose-related vasoconstriction to injections or infusions of noradrenaline was measured as transient or sustained increases in perfusion pressure, respectively, in the hepatic arterial and portal venous vasculatures. Direct injections/infusions of noradrenaline refer to those administered into the vasculature from which pressure was recorded, e.g., the effects of hepatic arterial (direct) injections/infusions of noradrenaline upon hepatic arterial perfusion pressure. Indirect injections/infusions of noradrenaline were those administered to the adjacent afferent vasculature, e.g., the effects of portal venous (indirect) injections of noradrenaline upon hepatic arterial perfusion pressure. The converse applies for recordings of portal venous perfusion pressure. The -log(M) ED50 values to direct (hepatic arterial) and indirect (portal venous) injections in the hepatic artery were 4.25+/-0.20 and 3.40+/-0.10, respectively, and were significantly different (P0.01, Student's unpaired t-test); the -log(M) ED50 values to direct (portal venous) and indirect (hepatic arterial) injections in the portal vein were 3.91+/-0.08 and 3.85+/-0.11, respectively, and were not significantly different (P0.05, Student's unpaired t-test). Similarly, the -log(M) ED50 values to direct (hepatic arterial) and indirect (portal venous) infusions in the hepatic artery were 5.28+/-0.11 and 3.75+/-0.12, respectively, and were significantly different (P0.01, Student's unpaired t-test); the -log(M) ED50 values to direct (portal venous) and indirect (hepatic arterial) infusions in the portal vein were 5.31+/-0.19 and 5.70+/-0.16, respectively, and were not significantly different (P0.05, Student's unpaired t-test). These results demonstrated that there is little transfer of noradrenaline from the portal venous to the hepatic arterial resistance sites, but significant transfer from the hepatic artery to the portal venous suggesting that; (a) the portal venous resistance sites are located at the sinusoidal or post-sinusoidal level; and (b) the hepatic arterial resistance sites are located at the pre-sinusoidal level.

1. Mechanisms underlying the haemodynamic activity of diastereoisomers of S-adenosyl-L-methionine (SAM) were investigated using inhibitors of purinoceptors and nitric oxide (NO) synthase in perfused rat livers damaged by sequential 24 h cold and 20 min rewarming ischaemia + reperfusion. 2. Stored livers were flushed with 10 ml saline alone (control) or with added (R,S) or (S,S) SAM (100 microM) and reperfused in the absence (control) or presence of 10 microM 8-phenyltheophylline (8-PT) or 100 microM L-N-monomethylarginine (L-NMMA). 3. Both SAM diastereoisomers rapidly increased blood flow and bile production versus controls (P

It is unclear how the omission of a perfusate oxygen carrier may influence data derived from prolonged in vitro liver perfusions. The effects of reduced perfusate oxygen content upon liver function tests conducted in livers from male Sprague-Dawley rats perfused for 6 h, were studied using diluted rat blood (PCV = 10%) (group 1) and compared to lines perfused with Krebs' buffer (group 2). Perfusate and bile samples were withdrawn at the start and at hourly intervals following the commencement of perfusion for biochemical analysis of aspartate-serine transaminase, alkaline phosphatase, bilirubin, urea and potassium concentrations, and also for perfusate gas measurements for calculations of hepatic oxygen uptake. There were no significant differences between the initial perfusion pressures of 11.4+/-1.6 and 15.7+/-2.4 mm Hg at the start of perfusion (group 1 versus group 2, respectively). Maintenance of comparable perfusion pressures at the start of perfusion, however, resulted in a significant difference in the fixed flow rate between the two groups of 1.9+/-0.1 and 2.6+/-0.2* ml/min/g liver and resulted in significantly higher perfusion pressures in group 2 than in group 1 after 6 h of perfusion (18.2+/-2.0 versus 8.7+/-1.7 mmHg, respectively). Bile volume production was significantly greater in group 1 than in group 2, at 784+/-84 vs 458+/-75 microl/h** respectively. Hepatic oxygen uptake (HOU) was significantly greater in group 1 than in group 2, and maximal at 0.93+/-0.13 vs 0.32+/-0.08** micromol/min/g, respectively. Bile concentrations of bilirubin and potassium were significantly greater in group 1 than in group 2, 23.0+/-0.6 vs 4.9+/-0.4 micromol/l*** and 4.7+/-0.5 vs 1.1+/-0.2 mmol/l**, respectively. Perfusate concentrations of urea, bilirubin, and glucose were significantly higher in group 1 than group 2, 5.7+/-0.5 versus 1.4+/-0.2 micromol/l**; 2.4+/-0.5 versus 0.7+/-0.1 micromol/l** and 22.1+/-1.4 versus 17.8+/-0.9 micromol/l***. It is concluded that in vitro perfusion of the rat liver with Krebs'-Henseleit buffer (KHB) via the portal vein alone may be insufficient to maintain optimum liver function as assessed by the tests used. Inclusion of a perfusate oxygen carrier is optimal if livers are to be perfused at physiological pressures. *p < 0.05; **p < 0.01; ***p < 0.001; group 1 versus group 2, Student's unpaired t test.

Nitric oxide (NO) may regulate hepatic metabolism directly by causing alterations in hepatocellular (hepatocyte and Kupffer cell) metabolism and function or indirectly as a result of its vasodilator properties. Its release from the endothelium can be elicited by numerous autacoids such as histamine, vasoactive intestinal peptide, adenosine, ATP, 5-HT, substance P, bradykinin, and calcitonin gene-related peptide. In addition, NO may be released from the hepatic vascular endothelium, platelets, nerve endings, mast cells, and Kupffer cells as a response to various stimuli such as endotoxemia, ischemia-reperfusion injury, and circulatory shock. It is synthesized by nitric oxide synthase (NOS), which has three distinguishable isoforms: NOS-1 (ncNOS), a constitutive isoform originally isolated from neuronal sources; NOS-2 (iNOS), an inducible isoform that may generate large quantities of NO and may be induced in a variety of cell types throughout the body by the action of inflammatory stimuli such as tumor necrosis factor and interleukin (IL)-1 and -6; and NOS-3 (ecNOS), a constitutive isoform originally located in endothelial cells. Another basis for differentiation between the constitutive and inducible enzymes is the requirement for calcium binding to calmodulin in the former. NO is vulnerable to a plethora of biologic reactions, the most important being those involving higher nitrogen oxides (NO2-), nitrosothiol, and nitrosyl iron-cysteine complexes, the products of which (for example, peroxynitrite), are believed to be highly cytotoxic. The ability of NO to react with iron complexes renders the cytochrome P450 series of microsomal enzymes natural targets for inhibition by NO. It is believed that this mechanism provides negative feedback control of NO synthesis. In addition, NO may regulate prostaglandin synthesis because the cyclooxygenases are other hem-containing enzymes. It may also be possible that NO-induced release of IL-1 inhibits cytochrome P450 production, which ultimately renders the liver less resistant to trauma. It is believed that Kupffer cells are the main source of NO during endotoxemic shock and that selective inhibition of this stimulation may have future beneficial therapeutic implications. NO release in small quantities may be beneficial because it has been shown to decrease tumor cell growth and levels of prostaglandin E2 and F2 alpha (proinflammatory products) and to increase protein synthesis and DNA-repair enzymes in isolated hepatocytes. NO may possess both cytoprotective and cytotoxic properties depending on the amount and the isoform of NOS by which it is produced. The mechanisms by which these properties are regulated are important in the maintenance of whole body homeostasis and remain to be elucidated.

Changes in brain 5-HT turnover which have been associated with portal-systemic encephalopathy (PSE) in man were studied in rats with experimental PSE for intervals up to 15 weeks following the surgical construction of end-to-side portacaval shunts (PCS). These were compared to changes measured in portacaval transposed rats (PCT) which, show little hepatic dysfunction or cerebral abnormalities but, in common with the PCS rat, sustain total portal-systemic diversion. Thus any differences between these two groups were indicative of hepatic dysfunction and not the systemic diversion of portal blood. After 15 weeks, sustained increases were measured in brainstem and cerebral concentrations of the catabolite of 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA), from 0.25±0.01 to 0.68±0.01*** μg g−1 brain and from 0.18±0.01 to 0.31±0.03*** μg g−1 brain respectively in PCS rats and were statistically greater to those measured in the brainstem and cerebrum of PCT and control rats. Sustained increases in cerebral concentrations alone of 5-hydroxytryptophan (5-HTP), the precursor of 5-HT, from 0.17±0.01 to 0.23±0.02 μg g−1 brain were measured in PCS rats and were significantly*** greater than in PCT control rats after 15 weeks. Some early increases in 5-HTP were measured in PCS above control rats but these were not significant after 15 weeks. No sustained significant differences between the 3 groups were measured in 5-HT after 15 weeks. These data confirm previous evidence that the elevations in 5-HTP and 5-HIAA concentrations observed in experimental chronic liver failure and PSE are due to liver dysfunction and not portal-systemic diversion and may contribute additional information regarding the role of derangements in central 5-HT turnover as one of the causes of PSE. *** p

Background Hepatocellular maturation is characterised by the progessive transition from an architecture in which hepatocyte plates are at least two cells thick to the familiar adult pattern in which liver cell plates are predominantly single-cell in thickness. A similar process also has been noted during compensatory hyperplasia following damage, or destruction to the hepatic parenchyma. The pathological events that underlie these processes remain inadequately explained. A new morphological approach has been developed to study the maturity of rat neonatal livers in order to identify the factors which govern the structured morphogenesis of the liver in greater detail. Methods Sections of hepatic tissue obtained from the left lateral and right posterior lobes from neonatal rats were studied at 8, 10, 11, 13, 14, 18, 23, and 28 days postpartum. These were mounted, fixed, and stained with haematoxylin and eosin and analysed using a modified point-counting technique. Following validation of the technique, the proportion of single-cell plates to double-cell plates was then calculated at each time point. Results Liver sections from 8-day neonatal rats had the lowest percentage of single-cell thick plates of 16.9 ± 4.6% and the lowest standard deviation (mean ± SD). The hepatic architecture had fully matured by 28 days and was characterised by predominantly single-cell plates (84.6 ± 4.6%) lining the hepatic sinusoids. During the intervening time, the standard deviations increased significantly, peaking between 18–23 days, and reflected the rapidly changing morphology of the liver during this maturation process of the conversion of double-cell plates to single-cell plates. Conclusions It is concluded that the process of hepatocellular maturity in the neonatal Sprague-Dawley rat is reproducibly complete by 28 days and that further studies may now be conducted to determine the anatomical and pathophysiological changes that govern this important transition. Anat. Rec. 248:104–109, 1997. © 1997 Wiley-Liss, Inc.

The cerebral vasculature of five anaesthetised rabbits was perfused with a perfluorocarbon emulsion via the internal carotid arteries, and the effluent from the jugular veins analysed for ATP, substance P (SP), endothelin (ET) and arginine vasopressin (AVP). Viability of the preparation was monitored periodically by the electrocorticogram, oxygen uptake, carbon dioxide release and perfusion pressure. The basal rate of infusion of 7.8±1.26ml·min−1 resulted in an infusion pressure of 114.0±22.1 mmHg and when increased first to 10.5±1.53ml·min−1 and then to 15.0±1.87ml·min−1, rose to 163.0±33.1mmHg and to 170.0±33.2mmHg, respectively. Between each 3-min period of increased flow the rate was returned to the basal rate for 6 min. of the four vasoactive substances, ET was released at the largest rate during the initial period of basal flow, 65.3±10.7pmol·min−1. This increased further when the infusion rate rose to 10.5 ml·min−1, but was significant only when the infusion rate was increased to 15.0 ml·min−1. ATP was released at 41.5±11.5pmol·min−1 during the initial period of basal flow. Its release significantly increased with flow and peaked at 15.0 ml·min −1. SP was released at a rate of 13.3±8.2pmol·min−1 during the initial period of basal flow. Its rate of release was increased significantly the second time the flow was increased to 10.5 ml·min−1 and increased even further when the flow was increased to 15.0 ml·min−1. AVP was detected in one third of the samples collected during the initial period of basal flow, being released at a rate of 0.65±047pmol·min−1. Only in the period following the 15.0 ml·min−1 rate of flow was there a sustained increased release of AVP. These results are consistent with the view that ATP, SP, ET and AVP are released from vascular endothelial cells during changes in flow, and may contribute to the adjustments in local cerebrovascular tone.