Runar Almaas

Effect of barbiturates on hydroxyl radicals, lipid peroxidation, and hypoxic cell death in human NT2-N neurons.

Background: Barbiturates have been shown to be neuroprotective in several animal models, but the underlying mechanisms are unknown. In this study, the authors investigated the effect of barbiturates on free radical scavenging and attempted to correlate this with their neuroprotective effects in a model of hypoxic cell death in human NT2-N neurons. Methods: Hydroxyl radicals were generated by ascorbic acid and iron and were measured by conversion of salicylate to 2,3-dihydroxybenzoic acid. The effect of barbiturates on lipid peroxidation measured as malondialdehyde and 4-hydroxynon-2-enal was also investigated. Hypoxia studies were then performed on human NT2-N neurons. The cells were exposed to 10 h of hypoxia or combined oxygen and glucose deprivation for 3 or 5 h in the presence of thiopental (50–600 &mgr;M), methohexital (50–400 &mgr;M), phenobarbital (10–400 &mgr;M), or pentobarbital (10–400 &mgr;M), and cell death was evaluated after 24 h by lactate dehydrogenase release. Results: Pentobarbital, phenobarbital, methohexital, and thiopental dose-dependently inhibited formation of 2,3-dihydroxybenzoic acid and iron-stimulated lipid peroxidation. There were significant but moderate differences in antioxidant action between the barbiturates. While phenobarbital (10–400 &mgr;M) and pentobarbital (10–50 &mgr;M) increased lactate dehydrogenase release after combined oxygen and glucose deprivation, thiopental and methohexital protected the neurons at all tested concentrations. At a higher concentration (400 &mgr;M), pentobarbital also significantly protected the neurons. At both 50 and 400 &mgr;M, thiopental and methohexital protected the NT2-N neurons significantly better than phenobarbital and pentobarbital. Conclusions: Barbiturates differ markedly in their neuroprotective effects against combined oxygen and glucose deprivation in human NT2-N neurons. The variation in neuroprotective effects could only partly be explained by differences in antioxidant action.

Diving seals, ischemia-reperfusion and oxygen radicals

The cardiovascular adaptations of seals that contribute to their ability to tolerate long periods of diving asphyxial hypoxia result in episodic regional ischemia during diving and abrupt reperfusion upon termination of the dive. These conditions might be expected to result in production of oxygen-derived free radicals and other forms of highly reactive oxygen species. Seal organs vary during dives with respect to the degree and persistence of ischemia. Myocardial perfusion is reduced and intermittent; kidney circulation is vigorously vasoconstricted. Heart and kidney tissues from ringed seals (Phoca hispida) and domestic pigs (Sus scrofa) were compared in reactions to experimental ischemia. Resulting production of hypoxanthine, indicative of ATP degradation, was higher in pig than in seal tissues. Activity of superoxide dismutase (SOD), an oxygen radical scavenger, was higher in seal heart. We suggest that these results indicate enhanced protective cellular mechanisms in seals against the potential hazard of highly reactive oxygen forms. SOD activity was unexpectedly higher in pig kidney.

The neuronal regulation of fracture healing: Effects of sciatic nerve resection in rat tibia

The effect of sciatic nerve resection on tibial fracture healing was studied in rats 25 days post-trauma. To prevent differences in loading between sham-operated and nerve-resected animals the fractured limbs were cast-immobilized. On radiograms 8 of 11 fractures in the sham-operated animals showed very little callus formation in contrast to only 1 of 8 fractures in the group with nerve resection. Measured by single-photon absorptiometry, animals with sciatic nerve resection had a higher bone mineral content than the sham-operated animals. However, the mechanical strength in three-point cantilever bending was not better in the nerve-resected rats, implying a defective organization of the large callus. These results suggest neural regulation plays a role in the type of fracture healing, primary or secondary, and in the amount and quality of the callus.

Bilirubin induces apoptosis and necrosis in human NT2-N neurons.

Studies on primary cultures of newborn rodent neurons have suggested that neuronal death induced by unconjugated bilirubin (UCB) is mainly apoptotic in nature. We exposed a human teratocarcinoma-derived cell line, NT2-N neurons, to different concentrations of UCB and albumin at a 1.5 molar ratio and used multiple, independent measures of cell damage to evaluate neuronal injury after 6, 24, and 48 h. Low doses of UCB (0.66, 2, and 5 μM) induced a moderate loss of 3–4[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) cleavage accompanied by delayed morphologic changes consistent with apoptosis (2 and 5 μM). Moderate concentrations of UCB (10 and 25 μM) resulted in early (6 h) necrosis in a subset of neurons, while remaining neurons underwent progressive impairment of MTT cleavage and increasing lactate dehydrogenase (LDH) release accompanied by predominantly delayed apoptosis. High concentrations of UCB (100 μM) induced severe impairment of MTT cleavage, extensive LDH release, and morphologic changes consistent with necrosis within 6 h. Used as a positive control for apoptosis, 2 μM STS induced progressive impairment of MTT cleavage and morphologic changes consistent with apoptosis over the entire observation period. DNA electrophoresis at 48 h was compatible with apoptosis both after treatment with STS and UCB concentrations ≤25, but not at 100 μM. Cleavage of poly (ADP-ribose) polymerase (PARP) was only seen in neurons treated with low UCB concentrations and STS. We conclude that UCB induces early necrosis at high and moderate concentrations and predominantly delayed apoptosis at low and moderate concentrations in cultured human NT2-N neurons.

Hypoxic Cell Death in Human NT2‐N Neurons: Involvement of NMDA and Non‐NMDA Glutamate Receptors

Abstract: Human NTera2 teratocarcinoma cells were differentiated into postmitotic NT2‐N neurons and exposed to hypoxia for 6 h. The cultures were evaluated microscopically, and percent lactate dehydrogenase (LDH) release after 24 and 48 h was used as an assay for cell death. After 48 h LDH release was 24.3 ± 5.6% versus 13.8 ± 3.7% in controls (p < 0.001). Cell death was greatly diminished by MK‐801 pretreatment (15.4 ± 5.1%, p < 0.001). If glutamate was omitted from the medium, glutamate levels after 6 h of hypoxia were reduced from 101 ± 63 to 2.3 ± 0.3 µM, and cell death at 48 h was also markedly reduced (15.4 ± 4.5%, p < 0.001). The α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (18.7 ± 5.1%, p < 0.001) and mild hypothermia (33.5–34°C) during hypoxia (19.5 ± 2.75, p < 0.05) were moderately protective. Basic fibroblast growth factor (24.1 ± 3.2%), the nitric oxide synthase inhibitor NG‐nitro‐l‐arginine methyl ester (22.8 ± 8.1%), the antioxidant N‐tert‐butyl‐o‐phenylnitrone (18.9 ± 5.9%), and the 21‐aminosteroid U74389G (24.0 ± 3.4%) did not protect the cells. N‐Acetyl‐l‐cysteine even tended to increase cell death (30.1 ± 2.5%, p = 0.06). Treatment with MK‐801 at the end of hypoxia did not reduce cell death (23.3 ± 2.3%). In separate experiments, a 15‐min exposure to 1 mM glutamate without hypoxia did not result in significant cell death (14.7 ± 2.4 vs. 12.2 ± 2.1%,p = 0.07). We conclude that, although somewhat resistant to glutamate toxicity when normoxic, NT2‐N neurons die via an ionotropic glutamate receptor‐mediated mechanism when exposed to hypoxia in the presence of glutamate. As far as we know, this is the first reported analysis of the mechanism of hypoxic cell death in cultured human neuronlike cells.

Ascorbic acid enhances hydroxyl radical formation in iron-fortified infant cereals and infant formulas

Abstract Infant cereals and formulas are usually fortified with iron to prevent iron deficiency. To enhance iron bioavailability, supplemental ascorbic acid is recommended. Ascorbic acid is considered to be an antioxidant in vivo, but has pro-oxidant effects when exposed to non-protein-bound iron. We measured formation of free radicals in cereals and infant formulas after addition of ascorbic acid. The production of hydroxyl radicals was assessed by hydroxylation of salicylic acid to 2,5-dihydroxybenzoic acid (2,5-DHBA). Production of 2,5-DHBA increased with increasing ascorbic acid doses added. Addition of 0.8 mM ascorbic acid to breast milk produced less radicals (0.03 ± 0.05 μM) than addition of ascorbic acid to low-iron formula (0.13 ± 0.08 μM, P = 0.019), medium-iron formula (0.34 ± 0.12 μM, P < 0.0001) or high-iron formula (0.44 ± 0.08 μM, P < 0.0001). Even when iron content in breast milk was adjusted to a level comparable with that of formulas, production of 2,5-DHBA was lower. Breast milk seems to contain substances that reduce hydroxyl radical formation. Conclusion Supplemental ascorbic acid causes hydro-xyl radical formation in iron-fortified infant nutrients in vitro.

Acidosis has opposite effects on neuronal survival during hypoxia and reoxygenation

To study the effect of extracellular acidosis on apoptosis and necrosis during ischemia and reoxygenation, we exposed human post‐mitotic NT2‐N neurones to oxygen and glucose deprivation (OGD) followed by reoxygenation. In some experiments, pH of the cell medium was lowered to 5.9 during either OGD or reoxygenation or both. Staurosporine, used as a positive control for apoptosis, caused Poly(ADP‐ribose)‐polymerase (PARP) cleavage and nuclear fragmentation, but no PARP cleavage and little fragmentation were seen after OGD. Low molecular weight DNA fragments were found after staurosporine treatment, but not after OGD. No protective effect of caspase inhibitors was seen after 3 h of OGD and 21 h of reoxygenation, but after 45 h of reoxygenation caspase inhibition induced a modest improvement in 3‐(4,5‐dimethylthiazol‐2‐yl)2,5‐diphenyltetrazolium bromide (MTT) cleavage. While acidosis during OGD accompanied by neutral medium during reoxygenation protected the neurones (MTT: 228 ± 117% of neutral medium, p < 0.001), acidosis during reoxygenation only was detrimental (MTT: 38 ± 25%, p < 0.01). We conclude that apoptotic mechanisms play a minor role after OGD in NT2‐N neurones. The effect of acidosis on neuronal survival depends on the timing of acidosis, as acidosis was protective during OGD and detrimental during reoxygenation.

Evaluation of risk factors for bleeding after liver biopsy in children.

Objectives: Acetylsalicylic acid is used in liver-transplanted children to prevent thrombosis of the hepatic artery. We evaluated whether acetylsalicylic acid and other risk factors were associated with bleeding after percutaneous liver biopsy. Methods: Medical charts, laboratory results, imaging studies, and anesthesia charts of 275 ultrasound-guided liver biopsy procedures in 190 children were reviewed. A total of 178 biopsies were performed on native livers and 97 on transplanted livers. Results: Three major and 28 minor bleeding incidents were found. The mortality rate was 0%. Acetylsalicylic acid had been given the last 5 days before 55 of the biopsy procedures and no increased risk of bleeding was found (odds ratio 0.96 [0.37–2.26]; P = 1.00). Low-molecular-weight heparin and biopsies from focal lesions were risk factors for bleeding complications. Acute liver failure was associated with increased risk for major complications (odds ratio 26.1 [3.3–205]; P = 0.01) and was a risk factor for major bleeding. Postbiopsy ultrasound the day after the procedure (n = 266 [96% of 275 biopsies]) revealed minor bleeding after 7.1% of the biopsies and after 2.6% of the ultrasounds revealed unsuspected bleeding, but none of these required intervention. Conclusions: Ultrasound-guided liver biopsy in children is a procedure with a low rate of major complications and a high rate of minor bleeding not requiring intervention. Treatment with low-dose acetylsalicylic acid did not increase bleeding incidence or total complication rate. Low-molecular-weight heparin and biopsies from focal lesions were risk factors for bleeding complications. Routine ultrasound the day after the procedure did not change handling of the patients.

Changes in apnea and autoresuscitation in piglets after intravenous and intrathecal interleukin-1β injection

UNLABELLED Piglets were given IL-1 beta intravenously (Index I, n = 8) or intrathecally (Index II, n = 9) prior to apnea to test the hypothesis that IL-1 beta may prolong periods of apnea during sleep and infection. Apnea variables and the quality of autoresuscitation were recorded and compared to an apnea control group (Ctr. I, n = 7, apnea without injection of IL-1 beta) and a procedure control group (Ctr. II, n = 6, apnea after intratecally injected sterile water). Hypoxanthine (Hx) and xanthine (X) were analyzed in plasma, CSF and vitreous humor. The duration of apnea was significantly longer in the Index I - mean: 38, intraquartile range: 27-52 sec and Index II-group 26 (24-36) sec than in the Ctr. 1-12 (10-13) sec (p < 0.01) and the Ctr. II-group 14 (6-18) sec (p < 0.01). The number of respirations per 2 min following apnea in the Index I-group mean: 21, intraquartile range: 7-40 and was significantly less than in the Ctr. I-group 109 (39-150) (p < 0.01), while a similar tendency was found in the Index II-group 42 (27-55) (p = 0.06). IN CONCLUSION Intravenous and intrathecal injection of IL-1 beta prolong the duration of apnea and modifies autoresuscitation.

Early bedside detection of ischemia and rejection in liver transplants by microdialysis

This study was performed to explore whether lactate, pyruvate, glucose, and glycerol levels sampled via microdialysis catheters in the transplanted liver could be used to detect ischemia and/or rejection. The metabolites were measured at the bedside every 1 to 2 hours after the operation for a median of 10 days. Twelve grafts with biopsy‐proven rejection and 9 grafts with ischemia were compared to a reference group of 39 grafts with uneventful courses. The median lactate level was significantly higher in both the ischemia group [5.8 mM (interquartile range = 4.0‐11.1 mM)] and the rejection group [2.1 mM (interquartile range = 1.9‐2.4 mM)] versus the reference group [1.5 mM (interquartile range = 1.1‐1.9 mM), P < 0.001 for both]. The median pyruvate level was significantly increased only in the rejection group [185 μM (interquartile range = 155‐206 μM)] versus the reference group [124 μM (interquartile range = 102‐150 μM), P < 0.001], whereas the median lactate/pyruvate ratio and the median glycerol level were increased only in the ischemia group [66.1 (interquartile range = 23.9‐156.7) and 138 μM (interquartile range = 26‐260 μM)] versus the reference group [11.8 (interquartile range = 10.6‐13.6), P < 0.001, and 9 μM (interquartile range = 9‐24 μM), P = 0.002]. Ischemia was detected with 100% sensitivity and greater than 90% specificity when a positive test was repeated after 1 hour. In 3 cases of hepatic artery thrombosis, ischemia was detected despite normal blood lactate levels. Consecutive pathological measurements for 6 hours were used to diagnose rejection with greater than 80% sensitivity and specificity at a median of 4 days before the activity of alanine aminotransferase, the concentration of bilirubin in serum, or both increased. In conclusion, bedside measurements of intrahepatic lactate and pyruvate levels were used to detect ischemia and rejection earlier than current standard methods could. Discrimination from an uneventful patient course was achieved. Consequently, intrahepatic graft monitoring with microdialysis may lead to the earlier initiation of graft‐saving treatment. Liver Transpl, 2012.