Heat shock protein inhibitors increase the efficacy of measles virotherapy.

Oncolytic measles virus strains have activity against multiple tumor types and are currently in phase I clinical testing. Induction of the heat shock protein 70 (HSP70) constitutes one of the earliest changes in cellular gene expression following infection with RNA viruses including measles virus, and HSP70 upregulation induced by heat shock has been shown to result in increased measles virus cytotoxicity. HSP90 inhibitors such as geldanamycin (GA) or 17-allylaminogeldanamycin result in pharmacologic upregulation of HSP70 and they are currently in clinical testing as cancer therapeutics. We therefore investigated the hypothesis that heat shock protein inhibitors could augment the measles virus-induced cytopathic effect. We tested the combination of a measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) and GA in MDA-MB-231 (breast), SKOV3.IP (ovarian) and TE671 (rhabdomyosarcoma) cancer cell lines. Optimal synergy was accomplished when GA treatment was initiated 6-24 h following MV infection. Western immunoblotting confirmed HSP70 upregulation in combination-treated cells. Combination treatment resulted in statistically significant increase in syncytia formation as compared to MV-CEA infection alone. Clonogenic assays demonstrated significant decrease in tumor colony formation in MV-CEA/GA combination-treated cells. In addition there was increase in apoptosis by 4,6-diamidino-2-phenylindole staining. Western immunoblotting for caspase-9, caspase-8, caspase-3 and poly(ADP-ribose) polymerase (PARP) demonstrated increase in cleaved caspase-8 and PARP. The pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected tumor cells from MV-CEA/GA-induced PARP activation, indicating that apoptosis in combination-treated cells occurs mainly via the extrinsic caspase pathway. Treatment of normal cells, such as normal human fibroblasts, however, with the MV-CEA/GA combination, did not result in cytopathic effect, indicating that GA did not alter the MV-CEA specificity for tumor cells. One-step viral growth curves, western immunoblotting for MV-N protein expression, QRT-PCR quantitation of MV-genome copy number and CEA levels showed comparable proliferation of MV-CEA in GA-treated vs -untreated tumor cells. Rho activation assays and western blot for total RhoA, a GTPase associated with the actin cytoskeleton, demonstrated decrease in RhoA activation in combination-treated cells, a change previously shown to be associated with increase in paramyxovirus-induced cell-cell fusion. The enhanced cytopathic effect resulting from measles virus/GA combination supports the translational potential of this approach in the treatment of cancer.

The glyoxylate cycle: does it function in the dormant or active bear?

The presence of or induction of an active glyoxylate cycle (GC) in the dormant black bear whose sole source of energy is body fat is an attractive concept which would allow lipid (acetate) to be directed from oxidation via the tricarboxylic acid cycle to many biosynthetic pathways. However, in spite of earlier claims, the present report establishes that isocitrate lyase and malate synthetase, GC marker enzymes, could not be detected in liver or kidney of active or dormant bears; liver peroxisome numbers were similar. The absence of brown fat (by light microscopy) and of the GC enzymes in the dormant bear raises questions about the prior report.

Alpha-lipoic acid: effect on glucose uptake, sorbitol pathway, and energy metabolism in experimental diabetic neuropathy.

The peripheral nerve of experimental diabetic neuropathy (EDN) is reported to be ischemic and hypoxic, with an increased dependence on anaerobic metabolism, requiring increased energy substrate stores. When glucose stores become reduced, fiber degeneration has been reported. We evaluated glucose uptake, nerve energy metabolism, the polyol pathway, and protein kinase C (PKC) activity in EDN induced by streptozotocin. Control and diabetic rats received lipoic acid (0, 10, 25, 50, 100 mg/kg). Duration of diabetes was 1 month, and alpha-lipoic acid was administered intraperitoneally 5 times per week for the final week of the experiment. Nerve glucose uptake was reduced to 60, s 37, and 30% of control values in the sciatic nerve, L5 dorsal root ganglion, and superior cervical ganglion (SCG), respectively, in rats with EDN. Alpha-lipoic acid supplementation had no effect on glucose uptake in normal nerves at any dose, but reversed the deficit in EDN, with a threshold between 10 and 25 mg/kg. Endoneurial glucose, fructose, sorbitol, and myo-inositol were measured in sciatic nerve. Alpha-lipoic acid had no significant effect on either energy metabolism or polyol pathway of normal nerves. In EDN, endoneurial glucose, fructose, and sorbitol were significantly increased, while myo-inositol was significantly reduced. Alpha-lipoic acid had a biphasic effect: it dose-dependently increased fructose, glucose, and sorbitol, peaking at 25 mg/kg, and then fell beyond that dose, and it dose-dependently increased myo-inositol. Sciatic nerve cytosolic PKC was increased in EDN. ATP, creatine phosphate, and lactate were measured in sciatic nerve and SCG. Alpha-lipoic acid prevented the reduction in SCG creatine phosphate. We conclude that glucose uptake is reduced in EDN and that this deficit is dose-dependently reversed by alpha-lipoic acid, a change associated with an improvement in peripheral nerve function.

Hypothermic neuroprotection of peripheral nerve of rats from ischemia-reperfusion injury: intraischemic vs. reperfusion hypothermia.

The pathophysiology of ischemic fiber degeneration (IFD) is not known, but mechanisms involved during nerve ischemia differ from those during reperfusion. We have previously demonstrated hypothermic neuroprotection of peripheral nerve from IFD. We now evaluate the efficacy of hypothermia in the intraischemic vs. the reperfusion period, using our established model of ischemia-reperfusion injury. Intraischemic hypothermia resulted in significant recovery of all indices (behavior score, electrophysiology and histology, P<0.01 or 0.05) while hypothermia during reperfusion period showed less improvement, significant only for the histological score compared to normothermia group (IFD index, P<0.05). Once hypothermia was applied in the ischemic period, the resultant neuroprotection continued into the reperfusion period, even if nerve temperature was then raised during the reperfusion period. These results indicate that hypothermic neuroprotection is more efficacious during the intraischemic period than during reperfusion, when a lesser degree of neuroprotection ensued.

Alpha-lipoic acid provides neuroprotection from ischemia-reperfusion injury of peripheral nerve.

BACKGROUND: Reperfusion aggravates nerve ischemic fiber degeneration, likely by the generation of reduced oxygen species. We therefore evaluated if racemic alpha-lipoic acid (LA), a potent antioxidant, will protect peripheral nerve from reperfusion injury, using our established model of ischemia-reperfusion injury. METHODS: We used male SD rats, 300+/-5 g. Ischemia was produced by the ligature of each of the supplying arteries to the sciatic-tibial nerve of the right hind-limb for predetermined periods of time (either 3 or 5 h), followed by the release of the ligatures, resulting in reperfusion. LA was given intraperitoneally daily for 3 days for both pre- and post-surgery. Animals received either LA, 100 mg/kg/day, or the same volume of saline intraperitoneally. Clinical behavioral score and electrophysiology of motor and sensory nerves were obtained at 1 week after ischemia-reperfusion. After electrophysiological examination, the sciatic-tibial nerve was fixed in situ and embedded in epon. We evaluated for ischemic fiber degeneration (IFD) and edema, as we described previously. RESULTS: Distal sensory conduction (amplitude of sensory action potential and sensory conduction velocity (SCV) of digital nerve) was significantly improved in the 3-h ischemia group, treated with LA (P<0.05). LA also improved IFD of the mid tibial nerve (P=0.0522). LA failed to show favorable effects if the duration of ischemia was longer (5-h ischemia). CONCLUSION: These results suggest that alpha-lipoic acid is efficacious for moderate ischemia-reperfusion, especially on distal sensory nerves.

Hypothermic neuroprotection of peripheral nerve of rats from ischaemia-reperfusion injury.

Although there is much information on experimental ischaemic neuropathy, there are only scant data on neuroprotection. We evaluated the effectiveness of hypothermia in protecting peripheral nerve from ischaemia-reperfusion injury using the model of experimental nerve ischaemia. Forty-eight male Sprague-Dawley rats were divided into six groups. We used a ligation-reperfusion model of nerve ischaemia where each of the supplying arteries to the sciatic-tibial nerves of the right hind limb was ligated and the ligatures were released after a predetermined period of ischaemia. The right hind limbs of one group (24 rats) were made ischaemic for 5 h and those of the other group (24 rats) for 3 h. Each group was further divided into three and the limbs were maintained at 37 degrees C (36 degrees C for 5 h of ischaemia) in one, 32 degrees C in the second and 28 degrees C in the third of these groups for the final 2 h of the ischaemic period and an additional 2 h of the reperfusion period. A behavioural score was recorded and nerve electrophysiology of motor and sensory nerves was undertaken 1 week after surgical procedures. At that time, entire sciatic-tibial nerves were harvested and fixed in situ. Four portions of each nerve were examined: proximal sciatic nerve, distal sciatic nerve, mid-tibial nerve and distal tibial nerve. To determine the degree of fibre degeneration, each section was studied by light microscopy, and we estimated an oedema index and a fibre degeneration index. The groups treated at 36-37 degrees C underwent marked fibre degeneration, associated with a reduction in action potential and impairment in behavioural score. The groups treated at 28 degrees C (for both 3 and 5 h) showed significantly less (P < 0.01; ANOVA, Bonferoni post hoc test) reperfusion injury for all indices (behavioural score, electrophysiology and neuropathology), and the groups treated at 32 degrees C had scores intermediate between the groups treated at 36-37 degrees C and 28 degrees C. Our results showed that cooling the limbs dramatically protects the peripheral nerve from ischaemia-reperfusion injury.

Blood flow and autoregulation in somatic and autonomic ganglia. Comparison with sciatic nerve.

We studied blood flow rates along the sciatic nerve and in the superior cervical and L-5 dorsal root ganglia of rats at rest and during reductions and increases in mean arterial pressure induced by partial exsanguination or blood transfusion. Blood flow was measured by the tissue distribution of [14C]iodoantipyrine and autoradiography. At rest, blood flow did not vary along the peripheral nerve, but was two to three times greater in dorsal root and superior cervical ganglia. In peripheral nerve, blood flow increased with increases in blood pressure. In contrast, blood flow in dorsal root and sympathetic ganglia did not vary with changes in pressure. Thus, peripheral nerve cell bodies have greater blood flow than their axons; ganglion blood flow is autoregulated within the range of blood pressure tested. Nerve ganglia appear to be protected against ischaemic stress by autoregulation rather than by a blood flow "safety margin', as in peripheral nerve.

Neuropathology and blood flow of nerve, spinal roots and dorsal root ganglia in longstanding diabetic rats.

Vascular perfusion and neuropathologic evaluation of the lumbar spinal roots and dorsal root ganglia (DRG) were studied in rats with longstanding (duration 12-15 months) streptozotocin-induced diabetes and age- and sex-matched control rats. We also undertook nerve conduction studies including F-wave recordings and measured blood flow in sciatic nerve, DRG, and superior cervical ganglion (SCG). Light microscopically, changes of the myelin sheath in the dorsal and ventral roots and vacuolated cells in the DRG were the major findings, being significantly higher in diabetic rats than in control rats. The effects of the diabetic state on myelin splitting were greater in the dorsal than ventral roots. Electron microscopic studies revealed a gradation of changes in myelin from mild separation to severe ballooning of myelin with relative axonal sparing. DRG cells showed vacuoles of all sizes with cristae-like residues, suggestive of mitochondria. These findings suggest that diabetes mellitus has a dual effect: it accelerates the normal age-related degenerative changes in the spinal roots and DRG, and it also has a selective effect on the sensory neuron. Nerve conduction studies showed markedly reduced conduction velocities in the distal nerve segments and prolonged F-wave latency and proximal conduction time despite the shorter conduction pathway in diabetic rats. Blood flow, which was measured using iodo[14C]antipyrine autoradiography, was significantly reduced in the sciatic nerves, DRG, and SCG of diabetic rats. We suggest that the combination of hyperglycemia and ischemia results in oxidative-stress and a predominantly sensory neuropathy.

Chronic constriction model of rat sciatic nerve: nerve blood flow, morphologic and biochemical alterations.

We evaluated the nerve blood flow (NBF), light and electron microscopy, and adrenergic innervation of rat sciatic nerve at 2-45 days after the application of four loose ligatures. Ischemia developed at the lesion edge, creating an endoneurial dam. Calcitonin gene-related peptide, norepinephrine and NBF were increased within the lesion. Morphologic alterations consisted of early endoneurial edema, followed by myelinated fiber degeneration, with relative sparing of small myelinated and unmyelinated nerve fibers, and leukocyte adhesion to microvessels. Axonal degeneration predominated over demyelination. At 45 days, profuse regeneration of small myelinated fibers was seen. The mechanism of lesional sensitization is discussed.

Black bear (Ursus americanus) bile composition: seasonal changes.

Gallbladder contents from 8 active and 14 dormant black bears were analyzed for individual bile acids by high-performance liquid chromatography and for cholesterol, phospholipids, sodium, potassium, calcium, magnesium, zinc, iron, and copper. Only three bile acids occurring as taurine conjugates were detected: tauroursodeoxycholate, taurochenodeoxycholate, and taurocholate. The proportion of tauroursodeoxycholate to the sum of the three bile acids decreased. Calcium, cholesterol, phospholipids, magnesium, zinc, and copper concentrations were increased in dormancy. Standardized collection and handling procedures yielded more consistent data than previously available. The decrease in tauroursodeoxycholate and absence of deoxycholate and lithocholate are consistent with our working hypothesis that a marked reduction in metabolic activity of the gut flora is an integral part of the adaptation to metabolic stability of the dormant bear.

Ischemic reperfusion causes lipid peroxidation and fiber degeneration.

Although the neuropathology of ischemic fiber degeneration (IFD) is relatively well known, its pathogenesis is poorly understood. One putative mechanism of IFD is oxidative stress, causing a breakdown of the blood-nerve barrier (BNB) and lipid peroxidation. We evaluated the effect of ischemic reperfusion of rat sciatic-tibial nerve seeking biochemical and pathologic evidence of BNB disruption and lipid peroxidation. Ischemia, caused by the ligation of the supplying arteries to sciatic-tibial nerve, was maintained for 3 h, followed by reperfusion. Reperfusion resulted in an increase in nerve lipid hydroperoxides, greatest at 3 h, followed by a gradual decline over the next month. Nerve edema and IFD consistently became more severe with reperfusion, indicating that oxidative stress impairs the BNB (edema) and causes IFD. Reduced reperfusion was greatest over distal sciatic nerve and midtibial nerve at day 7. The most ischemic segment (midtibial), of nonreperfused ischemic nerves (duration 3 h), underwent both edema and IFD that was as pronounced as those of other segments after reperfusion, and underwent a smaller increase with reperfusion, suggesting that ischemia alone can also cause IFD and edema. The type of fiber degeneration was that of axonal degeneration.

Hypoxic effect of exogenous insulin on normal and diabetic peripheral nerve.

Insulin administration can cause or worsen experimental and human diabetic neuropathy ("insulin neuritis"). In this study, we tested the hypothesis that insulin administration impairs tissue oxygenation. We infused insulin under nonhypoglycemic conditions and evaluated its effect on endoneurial oxygen tension, nerve blood flow, and the oxyhemoglobin dissociation curve of peripheral nerve in normal and diabetic rats. Intravenous insulin infusion resulted in a dose-dependent reduction in endoneurial oxygen tension in normal nerves (from 26% at 0.04 U/kg insulin to 55% at 32 U/kg). The nerves of rats with streptozotocin-induced diabetes were resistant, but with control of hyperglycemia this susceptibility to the endoneurial hypoxic effect of insulin returned. The reduction in endoneurial oxygen tension regressed with glycosylated hemoglobin (Y = 53.8-2.7X, where Y = %reduction in endoneurial oxygen tension and X = HbA1; r = 0.87; P = < 0.001). Diabetes or insulin administration resulted in only minimal and physiologically insignificant alterations in the oxygen dissociation curve and 2,3-diphosphoglycerate of sciatic nerve. Instead, insulin administration resulted in a reduction in nerve nutritive blood flow and an increase in arteriovenous shunt flow. When the latter was eliminated by the closure of arteriovenous shunts (infusion of 5-hydroxytryptamine), endoneurial oxygen reverted to normal. These findings indicate a deleterious vasoactive effect of insulin and may explain the development of insulin neuritis.

Different reinnervation patterns in the celiac/mesenteric and superior cervical ganglia following guanethidine sympathectomy in adult rats.

We sought to determine whether chronic guanethidine (Gu) treatment in adult rats produces depletion of sympathetic neurons and hyperinnervation by sensory neuropeptides in the celiac/superior mesenteric (C/SMG) ganglion. Rats received Gu 40 mg/kg per day i.p or saline for 5 weeks. Upon completion of treatment, the C/SMG and the superior cervical ganglion (SCG) were examined for neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP), both by immunocytochemistry (ICC) and radioimmunoassay (RIA). Gu produced marked depletion of NPY-containing neurons and NPY content in the C/SMG, similar to that in the SCG (-89 +/- 2 vs. -92 +/- 4%, respectively). SP and CGRP immunoreactivities were significantly higher in control C/SMG as compared with SCG; after Gu treatment, there was no significant increase in either SP or CGRP in the C/SMG, however, both increased in the SCG. In contrast, VIP levels were similar in the SCG and C/SMG in controls and increased in the C/SMG but not in the SCG after Gu treatment. Thus, in adult rats, the C/SMG is as susceptible as the SCG to Gu treatment; the different pattern of hyperinnervation by SP, CGRP and VIP of the C/SMG as compared with the SCG may reflect the different sources for these neuropeptides in prevertebral as compared with paravertebral ganglia.

The influence of dose of microspheres on nerve blood flow, electrophysiology, and fiber degeneration of rat peripheral nerve.

Microsphere embolization of peripheral nerve results in a variable degree of ischemic fiber degeneration. To enhance the utility of the model, we evaluated the relationship between dose of microspheres to the supplying arteries of the sciatic-tibial nerve to nerve blood flow (NBF), electrophysiology, morphology, and behavioral changes. There was considerable variability in the effect of embolization on nerve pathology in individual nerves. However, the dose of microspheres regressed with the degree of hindlimb paresis, reduction in NBF, degree of fiber pathology, and ischemic conduction failure of the tibial nerve, evaluated at day 7. All nerves with severe (grade 4) fiber degeneration had flows of < 3 mL x 100 g-1 x min-1. We conclude that it is possible to predict with a high degree of accuracy the severity of fiber degeneration by the degree of NBF reduction and by the electrophysiologic abnormalities.

Guanethidine sympathectomy increases substance P concentration in the superior sympathetic ganglion of adult rats.

Adult rats received intraperitoneal injections of guanethidine or saline for 5 weeks. Six to 8 weeks following completion of treatment, concentrations of substance P and neuropeptide Y (NPY) were measured by radioimmunoassay in the superior cervical ganglion (SCG) and thoracic spinal cord. The SCG was also immunostained for NPY and substance P. No differences were observed in thoracic spinal cord content of either NPY or substance P. We observed depletion of NPY immunoreactive neurons and NPY levels in the SCG, and pharmacologic evidence of postganglionic denervation in guanethidine-treated rats. In guanethidine-treated rats, there was a marked increase of substance P levels in the SCG, where substance P was localized in fibers, but not cell bodies. Thus, sprouting of substance P-containing sensory fibers in the sympathetic ganglia occurs following postganglionic sympathectomy in adult rats.

Effect of ischemia and reperfusion in vivo on energy metabolism of rat sciatic-tibial and caudal nerves.

Our model of severe nerve ischemia consistently results in extinction of the compound nerve and muscle action potentials (NAP; CMAP) within 30 min. Since impulse transmission may depend on nerve energy metabolism (NEM), we studied the effects of ischemia with reperfusion on sciatic-tibial nerve NEM in vivo and compared these results with NEM of this nerve in deoxygenated Ringer's solution in vitro and postmortem. Ischemia for 30 min postmortem or in deoxygenated Ringer's solution resulted in marked depletion of adenosine triphosphate (ATP) and creatine phosphate (CP) and an increase in lactate (LAC) of sciatic-tibial nerve of adult male Sprague-Dawley rats. In vivo ischemia for up to 3 h to sciatic-tibial nerve was sufficient to extinguish CMAP but not NAP and did not deplete ATP, CP, or GLU nor did it increase LAC. Ischemia sufficient to extinguish NAP resulted in reduction of energy substrates to about 50% of resting. Muscle fails to conduct impulses before nerve and in vivo reductions of energy substrates are milder than in vitro changes. These changes are explainable in terms of energy requirements and supply. These findings support an energetic basis of ischemic conduction failure.

Characterization of muscarinic receptor subtype of rat eccrine sweat gland by autoradiography.

The muscarinic cholinergic receptor of rat eccrine sweat gland was characterized using quantitative autoradiography and [3H]QNB as radioligand. The distribution of radioligand was maximal in the secretory coil. Autoradiographic competition binding studies were performed using selective antagonists to M1 (pirenzepine), M2 (AF-DX 116), and M3 (4-DAMP) and the classical nonselective antagonist atropine. pKi for pirenzepine, AF-DX 116, 4-DAMP, and atropine was 6.58, 5.47, 8.50, and 8.66 respectively indicating that the eccrine sweat gland muscarinic receptor was predominantly M3.

Augmentation cystoplasty utilizing de-epithelialized sigmoid colon: a preliminary study.

The use of bowel segments for bladder replacement or augmentation has been associated with metabolic complications and obstruction due to mucus production. Establishment of a transitional epithelium over the de-epithelialized surface of a segment of intestine might alleviate these complications. Twenty Holstein bull calves underwent sigmoidocystoplasty. Fourteen experimental animals had the epithelium of the sigmoid removed before augmentation. Six calves with intact mucosa served as controls. Fifteen calves survived the study: 11 experimentals and four controls. Cystectomies were performed at four, six, eight, or 12 weeks. Ninety-one percent (10/11) of the experimental calves had almost complete epithelialization of the de-epithelialized graft. All experimental animals had residual colonic mucosa or mucoceles. Nine of 11 experimental calves (82%) had greater than 25% contracture of the sigmoid graft. Two animals had less than 25% graft contracture (1) or formed a wide-mouthed true diverticulum (1) in the grafted segment. All control animals formed a wide-mouthed true diverticulum and had no graft contracture.

Cholinergic urticaria, passive transfer experiments from human to monkey.

Passive transfer experiments in cholinergic urticaria were carried out from 16 patients to a Macaca cymnologous monkey. Intravenous Evans blue dye was used to demonstrate vascular permeability. The animal was challenged after 24 h first by heating the serum-injected dorsal skin to 45 degrees C and secondly by superinjection of acetylcholine into serum-injected sites, and a control site. Local heat proved insufficient to evoke a response. Seven of 16 serum-injected sites showed positive reaction to acetylcholine, control injection of acetylcholine did not. These experiments suggest the presence of a serum factor in cholinergic urticaria which, with acetylcholine, causes increased vascular permeability.