Investigating the relationship between virtual cystoscopy image quality and CT slice thickness.

OBJECTIVE: To investigate the effect of reconstruction slice thickness on image quality at CT virtual cystoscopy (VC). METHODS: Pelvic CT examinations in bladder cancer patients were reconstructed at different slice thicknesses (0.6-5 mm) and intervals, and resulting VC images assessed. Quality indicators were ridging, holes, floaters and dimpling artefacts, tumour definition, and an overall score, ranked 1 (best) to 7 (worst). CT number and standard deviation (SD) for bladder contents and bladder wall were recorded. The mean SD was used as a measure of noise, and the contrast-to-noise ratio (CNR) was calculated as the CT number difference between them divided by the average image noise. The mean CNR across the three levels was used for analysis. Each qualitative image quality measure was compared with CT number, noise and CNR measurements. RESULTS: Dimpling artefacts increased with thinner slice reconstruction and correlated with increased noise, often resulting in poor tumour definition. The best overall image quality score was seen for VC images reconstructed at 1.2 mm slice thickness, probably because of the competing effects of spatial resolution and CNR. CONCLUSION: A slice thickness reconstruction <1.2 mm does not provide for better image quality at VC owing to the presence of increased noise.

An inositol 1,4,5-triphosphate (IP3)-IP3 receptor pathway is required for insulin-stimulated glucose transporter 4 translocation and glucose uptake in cardiomyocytes.

Intracellular calcium levels ([Ca2+]i) and glucose uptake are central to cardiomyocyte physiology, yet connections between them have not been studied. We investigated whether insulin regulates [Ca2+]i in cultured cardiomyocytes, the participating mechanisms, and their influence on glucose uptake via SLC2 family of facilitative glucose transporter 4 (GLUT4). Primary neonatal rat cardiomyocytes were preloaded with the Ca2+ fluorescent dye fluo3-acetoxymethyl ester compound (AM) and visualized by confocal microscopy. Ca2+ transport pathways were selectively targeted by chemical and molecular inhibition. Glucose uptake was assessed using [3H]2-deoxyglucose, and surface GLUT4 levels were quantified in nonpermeabilized cardiomyocytes transfected with GLUT4-myc-enhanced green fluorescent protein. Insulin elicited a fast, two-component, transient increase in [Ca2+]i. Nifedipine and ryanodine prevented only the first component. The second one was reduced by inositol-1,4,5-trisphosphate (IP3)-receptor-selective inhibitors (xestospongin C, 2 amino-ethoxydiphenylborate), by type 2 IP3 receptor knockdown via small interfering RNA or by transfected Gßγ peptidic inhibitor ßARKct. Insulin-stimulated glucose uptake was prevented by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM, 2-amino-ethoxydiphenylborate, and ßARK-ct but not by nifedipine or ryanodine. Similarly, insulin-dependent exofacial exposure of GLUT4-myc-enhanced green fluorescent protein was inhibited by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM and xestospongin C but not by nifedipine. Phosphatidylinositol 3-kinase and Akt were also required for the second phase of Ca2+ release and GLUT4 translocation. Transfected dominant-negative phosphatidylinositol 3-kinase γ inhibited the latter. In conclusion, in primary neonatal cardiomyocytes, insulin induces an important component of Ca2+ release via IP3 receptor. This component signals to glucose uptake via GLUT4, revealing a so-far unrealized contribution of IP3-sensitive Ca2+ stores to insulin action. This pathway may influence cardiac metabolism in conditions yet to be explored in adult myocardium.

Isolation of viable porcine islets by selective osmotic shock without enzymatic digestion.

Islet transplantation is a potential cure for type 1 diabetes, but clinical results have been disappointing. Currently, islet isolation is by enzymatic digestion of the pancreas which has significant pitfalls: warm ischemia exposure, collagenase-induced damage to the islet mass and viability, poor reproducibility, high cost, a relatively low number of islets obtained per whole pancreas, and selection of islets for collagenase resistance rather than for glucose responsiveness. In the present study we performed a series of experiments in a porcine model to demonstrate the feasibility of a new isolation method based on selective osmotic shock (SOS) using very high glucose solutions, doubling or tripling physiological osmotic strength. The SOS method can be carried out at room temperature or in the cold eliminating warm ischemia time which damages the islets. The SOS method does not depend on the texture of the pancreas so all pancreases can be processed identically and the process can be fully automated. The SOS method isolates all the islets of the pancreas regardless of size and shape allowing a greater number of islets to be harvested. The SOS method avoids exposure to toxins in collagenase solutions, is inexpensive and selects for islets with high concentrations of Glut 2 transporters, representing the best glucose responding islets. The SOS method showed a comparable recovery of islets from young pig pancreas and the islets showed improved viability. We conclude that the selective osmotic shock (SOS) method of separating islets from the pancreatic tissue is superior to the collagenase method.

Role of the M3 muscarinic acetylcholine receptor in beta-cell function and glucose homeostasis.

The release of insufficient amounts of insulin in the presence of elevated blood glucose levels is one of the key features of type 2 diabetes. Various lines of evidence indicate that acetylcholine (ACh), the major neurotransmitter of the parasympathetic nervous system, can enhance glucose-stimulated insulin secretion from pancreatic beta-cells. Studies with isolated islets prepared from whole body M(3) muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose-dependent insulin secretion is exclusively mediated by the M(3) muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M(3) receptors only in pancreatic beta-cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M(3) receptors in pancreatic beta-cells showed a pronounced increase in glucose tolerance and insulin secretion and were resistant to diet-induced glucose intolerance and hyperglycaemia. These findings indicate that beta-cell M(3) muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through beta-cell M(3) muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.

Muscarinic agonists activate Ca2+ store-operated and -independent ionic currents in insulin-secreting HIT-T15 cells and mouse pancreatic beta-cells.

The neurotransmitter acetylcholine, a muscarinic receptor agonist, augments glucose-induced insulin secretion from pancreatic beta-cells by depolarizing the membrane to enhance voltage-gated Ca(2+) influx. To clarify the electrical events involved in this process, we measured ionic currents from a clonal beta-cell line (HIT-T15) and mouse pancreatic beta-cells. In whole-cell recordings, the muscarinic agonist carbachol (CCh) dose-dependently and reversibly activated a voltage-independent, nonselective current (whole-cell conductance 24 pS/pF, reversal potential of approximately -15 mV). The current, which we refer to as I(musc), was blocked by atropine, a muscarinic receptor antagonist, and SKF 96365, a nonspecific ion channel blocker. The magnitude of the current decreased by 52% when extracellular Na(+) was removed, but was not affected by changes in extracellular Ca(2+), confirming that I(musc) is a nonselective current. To determine if I(musc) activates following release of Ca(2+) from an intracellular store, we blocked intracellular IP(3) receptors with heparin. Carbachol still activated a current in the presence of heparin, demonstrating the presence of a Ca(2+) store-independent, muscarinic agonist-activated ionic current in HIT cells. However, the store-independent current was smaller and had a more positive reversal potential (approximately 0 mV) than the current activated by CCh under control conditions. This result indicates that heparin had blocked a component of I(musc), which likely activates following release of stored Ca(2+). Depleting IP(3)-sensitive calcium stores with thapsigargin also activated a non-selective, SKF 96365-blockable current in HIT cells. The properties of this putative store-operated current were similar to the component of I(musc) that was blocked by heparin, being voltage-independent and reversing near -30 mV. We conclude that I(musc) consists of store-operated and store-independent components, both of which may contribute to the depolarizing action of muscarinic agonists on pancreatic beta-cells.

Surgical reconstruction of late pelvic post-traumatic nonunion and malalignment.

We have retrospectively reviewed the clinical and radiological results in 204 consecutive adult patients who had surgical correction of 70 late post-traumatic pelvic nonunions and 134 malalignments. The deformed pelvises were subdivided into united (true), unstable, ununited, and partially stable malalignments with heterotopic bone. The principal complaints were of pain, pelvic instability, sitting imbalance, and apparent limb-length discrepancy. After surgery, 195 patients (96%) achieved a primary union and 144 (71%) had slight, intermittent or no pelvic pain, while pelvic instability was entirely eliminated. Overall, 131 patients (64.2%) were extremely satisfied, 58 (28.4%) were satisfied and 15 (7.4%) were unsatisfied. After reconstruction of the malaligned pelvises, 67 results (50%) were anatomical, 47 (35%) were satisfactory and 20 (15%) were unsatisfactory. For a pelvic nonunion with local osteopenia and malalignment, stabilisation of all three pelvic columns is recommended. True pelvic (united) malunions were the most satisfactorily realigned and had the fewest complications. Ununited and unstable malalignments, especially those with heterotopic bone, had the poorest corrections and the most neurological complications. A therapeutic alternative, by the local resection of a symptomatic bony prominence, and fixation in situ of a posterior pelvic nonunion, gives highly effective symptomatic relief with fewer complications. Despite this, many patients had persistent low back pain.

Primary total hip arthroplasty after acetabular fracture.

In most cases, when THA is performed after an acetabular fracture, it is done to manage secondary degenerative change or, possibly, osteonecrosis of the femoral head. Secondary complicating factors may be encountered during the THA. After initial nonsurgical treatment of an acetabular fracture, an occult or frank acetabular nonunion and malunion are not uncommon and may extend to the residual pelvic ring. After surgical treatment, intrusive hardware, heterotopic bone, dense scar tissue, ischemic muscle or bone, and occult infection are additional hazards that may be encountered. When acute sciatic nerve palsy, whether induced traumatically or iatrogenically, accompanies the initial acetabular injury, the palsy is likely to be exacerbated during a subsequent THA. A careful clinical and radiographic evaluation is needed, along with the formulation of a detailed surgical strategy. The need for specialized arthroplasty instruments, fixation devices, and autograft or, occasionally, allograft has to be identified. When heterotopic bone is evident, an extensile approach may be needed to allow adequate exposure for its complete removal. After a bone defect and/or a nonunion with displacement has been characterized, one or more strategies for obliteration of the defect are considered; these include the use of impaction grafting, a structural graft, a cup inserted with multiple screws, mesh, or a suitable ring or other fixation device. Evaluation of results has shown that, overall, the late outcome of THA after acetabular fracture is inferior to that of arthroplasty performed because of degenerative arthritis. Although open reduction and internal fixation of an acute acetabular fracture was previously hypothesized as an effective way to improve the anticipated late outcome of THA by the elimination of a large fracture gap or the prevention of a potential nonunion, current observations do not support that hypothesis. An initial open reduction may compromise the outcome of a subsequent THA by compromising the blood supply of the acetabulum and by initiating the formation of scar tissue, heterotopic bone, or an occult or frank infection. For a highly selected group of especially severe acetabular fractures, particularly those in elderly patients, THA appears to be a promising therapeutic alternative.

Partial resection of the scapula and a release of the long head of triceps for the management of Sprengel's deformity.

Previous surgical methods to address Sprengel's deformity by an attempted relocation of the scapula have achieved a limited functional improvement. A novel method was devised that includes a partial scapular resection, a removal of any omovertebral communication, and a release of the long head of triceps from the scapula. The results of eight cases are presented in which this method was used on 5 males and 3 females patients (age range, 19 months to 9 years). Early postoperative, active-assisted motion exercises for the patients were encouraged. On average, flexion improved from 100 degrees to 175 degrees and abduction improved from 90 degrees to 150 degrees. In one patient, a second operation was performed to remove an exostosis that followed the primary procedure. Initially, two keloid scars followed the use of a curvilinear incision. However, subsequently, this problem was eliminated by the use of a transverse incision. The new method seems to provide highly favorable functional and cosmetic results with a low morbidity.

Factors affecting hepatocyte viability and CYPIA1 activity during encapsulation.

Hepatocytes encapsulated in alginate-poly-1-lysine-alginate (APA) are used in transplantation studies and in bioartificial liver support systems. Loss of cell viability in the process of APA encapsulation is usually 20-30% while the effect on cytochrome CYP450 activity is rarely reported. This work investigates the negative influences on hepatocyte viability and CYPIA1 activity during APA encapsulation, and reports methods to alleviate these influences by incorporating certain reagents into the encapsulation solution. The results show that loss of hepatocyte viability and CYPIA1 activity was caused almost entirely by extracellular calcium toxicity rather than by mechanical damage (p < 0.05). Use of 10 mM instead of 100 mM calcium chloride (CaCl2) in the encapsulation process improved CYPIA1 activity (p < 0.05), but did not improve hepatocyte viability (p > 0.05) or result in satisfactory microcapsules. Hepatocyte viability was 25% higher (p < 0.05) in CaCl2 than in calcium lactate (CaLa) when the cells were gelled by contact with these calcium solutions at room temperature (RT). Hepatocyte viability showed little improvement by processing at 4 degrees C than at RT in CaCl2 (p > 0.05) but was 23% higher at 4 degrees C than at RT in CaLa (p < 0.05). Calcium used in the process of encapsulation caused cell necrosis rather than apoptosis. Addition of Dulbecco's modified Eagle's medium (containing 10% foetal bovine serum) or 20 mM fructose to the calcium solution did not improve cell survival. However, nifedipine at a final concentration of 25 mM modestly improved hepatocyte survival in solution containing 100 mM CaCl2 (p = 0.003). Glutathione and taurine in certain concentrations showed protective effects against loss of CYPIA1 activity (p < 0.05 and <0.01 respectively). In conclusion, to optimise the use of calcium during the process of encapsulation, CaCl2 is preferred to CaLa and inclusion of nifedipine, glutathione or taurine in 100 mM CaCl2 solution is recommended.

A biodialysis system for liver support tested in a porcine hepatic failure model.

BACKGROUND: A practical liver support system for patients in fulminant hepatic failure (FHF) remains a needed therapeutic modality. A new method of bioartificial liver support, the liver biodialysis system (LBDS), is described. METHODS: Porcine hepatocytes, removed from direct contact with the treated subject's circulation, are in culture in a bioreactor which is combined in a dialysis circuit for patient treatment. The LBDS was tested in a porcine ischaemic hepatic failure model. RESULTS: The viable hepatocyte content of the bioreactor was 2.49 +/- 0.72 x 10(10). Cells remained viable in culture throughout the experiments (30 +/- 3 h) without evidence of immunological damage. A decrease in the degree of accumulation in the blood of ammonia (P < 0.02) and of 14 amino acids (P < 0.001) was achieved by the LBDS. Cerebral perfusion pressure was maintained at significantly higher levels in LBDS-treated animals (P < 0.05). CONCLUSIONS: In the LBDS, hepatocytes in large numbers and satisfactory culture conditions in a bioreactor have sustained viability and function. When combined in a dialysis circuit for the treatment of FHF pigs, immune reactions between the blood and hepatocytes were prevented and beneficial metabolic effects were observed.

Defects in inositol 1,4,5-trisphosphate receptor expression, Ca(2+) signaling, and insulin secretion in the anx7(+/-) knockout mouse.

The mammalian anx7 gene codes for a Ca(2+)-activated GTPase, which supports Ca(2+)/GTP-dependent secretion events and Ca(2+) channel activities in vitro and in vivo. To test whether anx7 might be involved in Ca(2+) signaling in secreting pancreatic beta cells, we knocked out the anx7 gene in the mouse and tested the insulin-secretory properties of the beta cells. The nullizygous anx7 (-/-) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (+/-) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (+/-) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8- to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent Ca(2+) channel functions are normal. However, electrooptical recordings indicate that the (+/-) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate (IP(3))-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in IP(3) receptor expression and function in pancreatic islets. The profound increase in islets, beta cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic beta cells. This is a direct demonstration of a connection between glucose-activated insulin secretion and Ca(2+) signaling through IP(3)-sensitive Ca(2+) stores.

Surgical treatment of acetabular fractures in elderly patients with osteoporotic bone.

The incidence of acetabular fractures in the elderly has recently shown a marked increase due to the combination of greater longevity for the population as a whole and a relative decrease in the incidence of alcohol-related trauma in younger adults. The compromised physiologic reserve and the diminished healing capacity of the typical elderly patient have an adverse effect on the potential for a favorable clinical outcome. The presence of osteopenic bone or degenerative arthritis and the effects of previous radiation therapy to the hip and pelvis hamper diagnostic imaging and the utility of some treatment alternatives that were designed primarily for younger patients. The diverse clinical presentations include major polytrauma, minor trauma, and insufficiency fractures. An assessment of the prior health and functional status of the patient is crucial in determining the optimal therapeutic protocol. Treatment options vary according to the clinical presentation and include conservative methods, percutaneous fixation in situ, open reduction, and acute total hip arthroplasty. The feasibility of acute total hip arthroplasty rests on the use of newly developed techniques for minimally invasive stabilization of the acetabular fracture with cables and the application of morselized or structural autograft harvested from the femoral head. Whichever surgical method is chosen, the objective is rapid mobilization of the patient on a walker or crutches. Late complications that may occur after nonoperative or operative treatment include posttraumatic arthritis, nonunion, wound infection, and heterotopic bone formation.

Tetracaine stimulates insulin secretion from the pancreatic beta-cell by release of intracellular calcium.

The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+]i) in cultured rat and mouse beta-cells. The tetracaine-specific [Ca2+]i rise also occurs in the absence of glucose, or in beta-cells depolarized by exposure to a Ca(2+)-deficient medium (< 1 microM) or elevated [K+]o. Furthermore, tetracaine (> or = 300 microM) depolarized the beta-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+]i rise in cultured rat beta-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 microM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic beta-cell, and thapsigargin (3 microM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect beta-cell insulin secretion.

Developments in blood management: the potential therapeutic role for epoetin alfa in orthopedic trauma.

Orthopedic trauma is a major source of morbidity and mortality in the United States and other countries. Major orthopedic trauma often results in significant blood loss, which is the most common cause of shock in the trauma setting. Transfusion of allogeneic blood and blood products may be used to maintain blood pressure but may not be the most effective therapy for the acute anemia that results from trauma-induced hemorrhage. Because acute anemia can interfere with successful and timely rehabilitation of these patients, it is important to be aggressive in treating anemia. One approach is to administer Epoetin alfa to stimulate erythropoiesis. A pilot study is currently in progress to test the efficacy of this approach in major trauma patients.

Glucagon induces suppression of ATP-sensitive K+ channel activity through a Ca2+/calmodulin-dependent pathway in mouse pancreatic beta-cells.

Glucagon is known to increase intracellular cAMP levels and enhance glucose-induced electrical activity and insulin secretion in pancreatic beta-cell perfused with Krebs-Ringer bicarbonate solution. The present experiments were aimed at evaluation of the hypothesis that changes in beta-cells ATP-sensitive K+ (K(ATP)) channel activity are involved in the glucagon-induced enhancement of electrical activity. Channel activity was recorded using the cell-attached configuration of the patch-clamp technique. Addition of glucagon (2.9 x 10(-7) m) in the presence of 11.1 mm glucose caused closure of K(ATP) channels followed by an increase in the frequency of biphasic current transients (action currents) due to action potential generation in the cell. Three calmodulin-antagonists (W-7, chlorpromazine, and trifluoperazine) restored with similar efficacy K(ATP) channel activity in cells being exposed to glucagon. At 2.8 mm glucose, glucagon did not affect K(ATP) channel activity until Ca2+ was released from Nitr-5 by flash photolysis, at which point channel activity was transiently suppressed. Similar effects were seen when db-cAMP was used instead of glucagon. These results support the view that glucagon and other cAMP-generating agonists enhance glucose-induced beta-cell electrical activity through a Ca2+/calmodulin dependent-closure of K(ATP) channels.

Comparison of porcine hepatocytes with human hepatoma (C3A) cells for use in a bioartificial liver support system.

Cells from primary porcine hepatocytes (PPH) and the immortalized human hepatoma cell line C3A are both used in bioartificial liver support systems (BALSS). In this work the viability and metabolic capacity of PPH and C3A cells cultured in different media were compared. Also, because the cells come into direct or indirect contact with human blood components in BALSS, the effects of human complement on survival and functions of the cells was evaluated. For short-term culture, maintenance of PPH viability was essential for retention of P450IA1 activity (r = 0.882, p < 0.01) and effective ammonia clearance (r = -0.791, p < 0.01). When cell viability was below 60% P450IA1 activity could not be recorded and nitrogen elimination activity significantly diminished. In contrast to PPH, ammonia levels were markedly increased for C3A cells in all culture media tested (p < 0.01). Ammonia increase correlated with C3A viability (r = 0.896, p < 0.05). PPH metabolic function was superior to that of the C3A cell line when evaluated by P450IA1 activity, ammonia removal, and amino acid metabolism. When PPH were incubated in human plasma (HP) or human serum (HS) there was rapid and irreversible deterioration of viability occurring within 9 h. This toxic effect could be prevented by the inactivation of complement. When sodium citrate dissolved in dextrose was added to medium, there was considerable damage to both PPH and the C3A cell line. However, there was no demonstrable toxic effect when hepatic cells of either type were exposed to heparin. We conclude that PPH cultivated in complement-inactivated HP or HS are to be preferred to C3A for clinical application of BALSS, and that heparin should be preferred for anticoagulation in BALSS.