Acute spinal cord injury is associated with mitochondrial dysfunction in mouse urothelium.

AIM: To characterize the effects of acute spinal cord injury (SCI) on mitochondrial morphology and function in bladder urothelium and to test the therapeutic efficacy of early treatment with the mitochondrially targeted antioxidant, MitoTempo. METHODS: We used a mouse model of acute SCI by spinal cord transection between the T8-T9 vertebrae with or without MitoTempo delivery at the time of injury followed by tissue processing at 3 days after SCI. Control, SCI, and SCI-MitoTempo-treated mice were compared in all experimental conditions. Assessments included analysis of markers of mitochondrial health including accumulation of reactive oxygen species (ROS), morphological changes in the ultrastructure of mitochondria by transmission electron microscopy, and Western blot analysis to quantify protein levels of markers for autophagy and altered mitochondrial dynamics. RESULTS: SCI resulted in an increase in oxidative stress markers and ROS production, confirming mitochondrial dysfunction. Mitochondria from SCI mice developed large electron-dense inclusions and these aberrant mitochondria accumulated throughout the cytoplasm suggesting an inability to clear dysfunctional mitochondria by mitophagy. SCI mice also exhibited elevated levels of dynamin-related protein 1 (DRP1), consistent with a disruption of mitochondrial dynamics. Remarkably, treatment with MitoTempo reversed many of the SCI-induced abnormalities that we observed. CONCLUSIONS: Acute SCI negatively and severely affects mitochondrial health of bladder urothelium. Early treatment of SCI with MitoTempo may be a viable therapeutic agent to mitigate these deleterious effects.

Serological microarray for a paradoxical diagnostic of Whipple's disease.

Whipple's disease is a systemic chronic infection caused by Tropheryma whipplei. Asymptomatic people may carry T. whipplei in their digestive tract and this can be determined by PCR, making serological diagnosis useful to distinguish between carriers and patients. Putative antigenic proteins were selected by computational analysis of the T. whipplei genome, immunoproteomics studies and from literature. After expression, putative T. whipplei antigens were screened by microimmunofluorescence with sera of immunized rabbit. Selected targets were screened by microarray using sera from patients and carriers. Paradoxically, with 19 tested recombinant proteins and a glycosylated native protein of T. whipplei, a higher immune response was observed with asymptomatic carriers. In contrast, quantification of human IgA exhibited a higher reaction in patients than in carriers against 10 antigens. These results were used to design a diagnostic test with a cut-off value for each antigen. A blind test assay was performed and was able to diagnose 6/8 patients and 11/12 carriers. Among people with positive T. whipplei PCR of the stool, patients differ from carriers by having positive IgA detection and a negative IgG detection. If confirmed, this serological test will distinguish between carriers and patients in people with positive PCR of the stool.

A randomized multicenter study of remifentanil compared with halothane in neonates and infants undergoing pyloromyotomy. I. Emergence and recovery profiles.

UNLABELLED: Pyloric stenosis is sometimes associated with hemodynamic instability and postoperative apnea. In this multicenter study we examined the hemodynamic response and recovery profile of remifentanil and compared it with that of halothane in infants undergoing pyloromyotomy. After atropine, propofol, and succinylcholine administration and tracheal intubation, patients were randomized (2:1 ratio) to receive either remifentanil with nitrous oxide and oxygen or halothane with nitrous oxide and oxygen as the maintenance anesthetic. Pre- and postoperative pneumograms were done and evaluated by an observer blinded to the study. Intraoperative hemodynamic data and postanesthesia care unit (PACU) discharge times, PACU recovery scores, pain medications, and adverse events (vomiting, bradycardia, dysrhythmia, and hypoxemia) were recorded by the study's research nurse. There were no significant differences in patient age or weight between the two groups. There were no significant differences in hemodynamic values between the two groups at the various intraoperative stress points. The extubation times, PACU discharge times, pain medications, and adverse events were similar for both groups. No patient anesthetized with remifentanil who had a normal preoperative pneumogram had an abnormal postoperative pneumogram, whereas three patients with a normal preoperative pneumogram who were anesthetized with halothane had abnormal pneumograms after. IMPLICATIONS: The use of ultra-short-acting opioids may be an appropriate technique for infants less than 2 mo old when tracheal extubation after surgery is anticipated.

A randomized multicenter study of remifentanil compared with halothane in neonates and infants undergoing pyloromyotomy. II. Perioperative breathing patterns in neonates and infants with pyloric stenosis.

UNLABELLED: Although former preterm birth infants are at risk for postoperative apnea after surgery, it is unclear whether the same is true of full-term birth infants. We evaluated the incidence of apnea in 60 full-term neonates and infants undergoing pyloromyotomy both before and after anesthesia. All subjects were randomized to a remifentanil- or halothane-based anesthetic. Apnea was defined by the presence of prolonged apnea (>15 s) or frequent brief apnea, as observed on the pneumocardiogram. Apnea occurred before surgery in 27% of subjects and after surgery in 16% of subjects, with no significant difference between subjects randomized to remifentanil or halothane anesthesia. This apnea was primarily central in origin, occurred throughout the recording epochs, and was associated with severe desaturation in some instances. Of the subjects with normal preoperative pneumocardiograms, new onset postoperative apnea occurred in 3 (23%) of 13 subjects who received halothane-based anesthetics versus 0 (0%) of 22 subjects who received remifentanil-based anesthetics (P = 0.04). Thus, postoperative apnea can follow anesthesia in otherwise healthy full-term infants after pyloromyotomy and is occasionally severe with desaturation. New-onset postoperative apnea was not seen with a remifentanil-based anesthetic. IMPLICATIONS: Abnormal breathing patterns can follow anesthesia in infants after surgical repair of pyloric stenosis. Occasionally, these patterns can be associated with desaturation. New-onset postoperative apnea was not seen with a remifentanil-based anesthetic.

Lipid induced conformation of the tachykinin peptide Kassinin.

Both the aqueous and lipid-induced structure of Kassinin, a dodecapeptide of amphibian origin, has been studied by two-dimensional proton nuclear magnetic resonance (2D 1H-NMR) spectroscopy and distance geometry calculations. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized in a distance geometry algorithm to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that, while in water Kassinin prefers to be in an extended chain conformation, in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system, helical conformation is induced in the central core and C-terminal region (K4-M12) of the peptide. N-terminus though less defined also displays some degree of order and a possible turn structure. The conformation adopted by Kassinin in the presence of DPC micelles is consistent with the structural motif typical of neurokinin-1 selective agonists and with that reported for Eledoisin in hydrophobic environment.

An automated annotation tool for genomic DNA sequences using GeneScan and BLAST.

Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated annotation of genome DNA sequences.

A randomized, double-blinded study of remifentanil versus fentanyl for tonsillectomy and adenoidectomy surgery in pediatric ambulatory surgical patients.

UNLABELLED: We compared, in a double-blinded manner, the anesthetic maintenance and recovery properties of remifentanil with a clinically comparable fentanyl-based anesthetic technique in pediatric ambulatory surgical patients. Anesthesia was induced with either halothane or sevoflurane and nitrous oxide and oxygen. Patients were randomized (computer generated) to receive either remifentanil or fentanyl in a blinded syringe with nitrous oxide and oxygen in one of four possibilities: halothane/remifentanil, halothane/fentanyl, sevoflurane/remifentanil or sevoflurane/fentanyl. In patients receiving remifentanil, a placebo bolus was administered, and a continuous infusion (0.25 microg. kg(-1). min(-1)) was begun. In patients receiving fentanyl, a bolus (2 microg/kg) was administered followed by a placebo continuous infusion. The time from discontinuation of the anesthetic to extubation, discharge from the postanesthesia care unit (PACU), and discharge to home, as well as pain scores, were assessed by a blinded nurse observer. Systolic blood pressure and heart rate were noted at selected times, and adverse events were recorded. Remifentanil provided faster extubation times and higher pain-discomfort scores. PACU and hospital discharge times were similar. There were no statistical differences among the groups for adverse events. There were statistically, but not clinically, significant differences in hemodynamic variables. We noted that continuous infusions of remifentanil were intraoperatively as effective as bolus fentanyl. Although patients could be tracheally extubated earlier with remifentanil, this did not translate to earlier PACU or hospital discharge times. In addition, remifentanil was associated with higher postoperative pain scores. The frequent incidence of postoperative pain observed in the postoperative recovery room suggests that better intraoperative prophylactic analgesic regimens for postoperative pain control are necessary to optimize remifentanil's use as an anesthetic for children. IMPLICATIONS: This is a study designed to examine the efficacy and safety of a short-acting opioid, remifentanil, when used in pediatric patients. The frequent incidence of postoperative pain observed in the postoperative recovery room suggests that better intraoperative prophylactic analgesic regimens for postoperative pain control are necessary to optimize remifentanil's use as an anesthetic for children.

Ventilatory response to CO2 in children with obstructive sleep apnea from adenotonsillar hypertrophy.

UNLABELLED: We measured the ventilatory response to CO2 as an indicator of respiratory control dysfunction in children with obstructive sleep apnea (OSA) scheduled for adenotonsillectomy. Measurements were performed in unpremedicated children via an endotracheal tube under 0.4%-0.5% end-tidal halothane anesthesia. Mean ventilatory CO2 response slopes for 11 children with OSA requiring adenotonsillectomy (Group I) were compared with those for 14 children without OSA requiring adenotonsillectomy (Group II) and 15 children without OSA requiring nonairway surgery (Group III). The mean ventilatory slope corrected for body surface area for Groups I, II, and III were 539 +/- 338, 828 +/- 234, and 850 +/- 380 mL.min-1.mm Hg ETCO2(-1).m-2, respectively (P < 0.05, Group I versus Groups II and III). Historical data--including snoring, apneic episodes > 10 s, daytime hypersomnolence, and nocturnal enuresis--defined those with OSA. Obesity occurred more frequently in patients with OSA and with depressed ventilatory responses (P < 0.001). Children with OSA from adenotonsillar hypertrophy have a diminished ventilatory response to CO2 stimulation, compared with those without OSA symptoms. The depressed response may account, in part, for the reported increased risk of perioperative respiratory complications in this population. IMPLICATIONS: Children with obstructive sleep apnea undergoing adenotonsillar surgery are at risk of postoperative respiratory compromise. We found that patients with a clinical history suggesting obstructive sleep apnea have a diminished ventilatory response to CO2 rebreathing, compared with controls.

Regulation of Gi by the CB1 cannabinoid receptor C-terminal juxtamembrane region: structural requirements determined by peptide analysis.

A CB1 cannabinoid receptor peptide fragment from the C-terminal juxtamembrane region autonomously inhibits adenylyl cyclase activity in a neuroblastoma membrane preparation. The cannabinoid receptor antagonist, SR141716A, failed to block the response. The peptide was able to evoke the response in membranes from Chinese hamster ovary (CHO) cells that do not express the CB1 receptor. These studies are consistent with a direct activation of Gi by the peptide. To test the importance of a BXBXXB sequence, Lys403 was acetylated, resulting in a peptide having similar affinity but reduced efficacy. N-Terminal truncation of Arg401 resulted in a 6-fold loss of affinity, which was not further reduced by sequential truncation of up to the first seven amino acids, four of which are charged. N-Terminal-truncated peptides exhibited maximal activity, suggesting that Gi activation can be conferred by the remaining amino acids. Truncation of the C-terminal Glu417 or substitution of Glu417 by a Leu or of Arg401 by a Norleucine reduced activity at 100 microM. The C-terminal juxtamembrane peptide was constrained to a loop peptide by placement of Cys residues at both terminals and disulfide coupling. This modification reduced the affinity 3-fold but yielded near-maximal efficacy. Blocking the Cys termini resulted in a loss of efficacy. Circular dichroism spectropolarimetry revealed that all C-terminal juxtamembrane peptide analogues exist in a random coil conformation in an aqueous environment. A hydrophobic environment (trifluoroethanol) failed to induce alpha-helix formation in the C-terminal juxtamembrane peptide but did so in less active peptides. The anionic detergent sodium dodecyl sulfate induced alpha-helix formation in all analogues except the loop peptide, where it induces a left-handed PII conformation. It is concluded that alpha-helix formation is not required for Gi activation.

Use of remifentanil in infants.

We describe the use of remifentanil in three infants with complex medical issues (hepatic failure, cyanotic heart disease and renal compromise). The short duration of opioid effect even after a long period of drug infusion (18 h) suggests this drug may be useful in some infants. Continued study is warranted.

Pharmacokinetics and side effects of milrinone in infants and children after open heart surgery.

UNLABELLED: We investigated the pharmacokinetics and side effects of milrinone in infants and children (< or = 13 yr) after open heart surgery in this prospective, open-label study. Milrinone binding to cardiopulmonary bypass (CPB) circuitry was also examined in out two groups. Children in the small dose group (n = 11) received two 25-microg/kg boluses with a final infusion rate of 0.5 microg kg(-1) x min(-1); those in the large dose group (n = 8) received a 50-microg/kg bolus and a 25-microg/kg bolus with a final infusion rate of 0.75 microg x kg(-1) x min(-1). Blood samples for milrinone concentration were drawn 30 min after each bolus, at steady state, and after discontinuing the milrinone infusion. Pharmacokinetics were evaluated using traditional and nonlinear mixed effects modeling analysis. Milrinone kinetics best fit a two-compartment model. Steady-state plasma levels in the small and large dose groups were within the adult therapeutic range (113 +/- 39 and 206 +/- 74 ng/mL, respectively). The volumes of distribution (Vbeta) in infants (0.9 L/kg) and children (0.7 L/kg) were not different, but infants had significantly lower milrinone clearance (3.8 vs 5.9 mL x kg(-1) x min(-1)). Thrombocytopenia (defined as platelet count < or = 100,000 mm(-3)) occurred in 58%, and the risk increased significantly with duration of infusion. Tachyarrythmias were noted in two patients. Milrinone did not bind to CPB circuitry. We conclude that milrinone is cleared more rapidly in children than in adults. The major complication was thrombocytopenia. IMPLICATIONS: Most pediatric dosing is based on data published for adults. Infants and children have kinetics that differ from adults. We studied the distribution of I.V. milrinone in infants and children after open heart surgery. Milrinone had a larger volume of distribution and a faster clearance in infants and children than in adults, and dosing should be adjusted accordingly.

Thyroid function in infants following cardiac surgery: comparative effects of iodinated and noniodinated topical antiseptics.

OBJECTIVES: Cardiopulmonary bypass has profound effects on thyroid hormone metabolism. These effects may be exacerbated in infants because they are able to absorb large quantities of iodine transcutaneously. The purpose of this study was to test the hypothesis that preoperative povidone-iodine contributes to postoperative thyroid depression in infants who undergo cardiac surgery. DESIGN: Prospective, randomized, controlled trial. SETTING: Children's Hospital and Medical Center, Seattle, WA. PATIENTS: Thirty-seven infants undergoing repair of congenital cardiac defects. INTERVENTIONS: Infants requiring cardiopulmonary bypass were divided into two groups: Group 1 received povidone-iodine; group 2 received chlorhexidine as a topical preoperative antiseptic. Group 3 did not require cardiopulmonary bypass for repair of cardiac defects and received povidone-iodine as a preoperative antiseptic. MEASUREMENTS AND MAIN RESULTS: Thyrotropin (TSH), total triiodothyronine (T3), and thyroxine (T4) were measured at four intervals: a) before preparation for surgery; b) immediately after surgery; c) at 2 days after surgery; and d) at 5 to 8 days after surgery. There was a significant decrease in TSH concentrations immediately after surgery in the two bypass groups. This change was significantly greater than in the change in TSH concentration in the thoracotomy group. Total T3 and T4 concentrations decreased by postoperative day 2 in both groups 1 and 2, and the changes were significant compared with group 3. Total T3 and T4 concentrations increased significantly in all groups after postoperative day 2, with no significant difference between the three groups. CONCLUSION: Cardiopulmonary bypass has a more significant effect on thyroid hormone metabolism than does the preoperative antiseptic.

Morphine pharmacokinetics during continuous infusion of morphine sulfate for infants receiving extracorporeal membrane oxygenation.

OBJECTIVES: To determine a) if serum morphine concentration changes during the first 3 hrs of extracorporeal membrane oxygenation (ECMO); and b) if absorption of morphine onto the membrane oxygenator is responsible for these changes. Also, morphine clearance during the first 5 days of ECMO was studied. DESIGN: Prospective, open-label study with consecutive patient enrollment. SETTING: Neonatal intensive care unit at a university-affiliated, children's hospital. SUBJECTS: Eleven neonates with severe persistent pulmonary hypertension of the newborn receiving continuous intravenous infusions of morphine sulfate and requiring ECMO. INTERVENTIONS: Blood samples were obtained from the subjects and ECMO circuits at predetermined time intervals. MEASUREMENTS AND MAIN RESULTS: Serum morphine concentration was determined using high-performance liquid chromatography. Morphine concentrations were no different from baseline at 5 mins, 1 hr, or 3 hrs after beginning ECMO. There was no significant difference in morphine concentration from samples taken immediately proximal and distal to the membrane oxygenator at 5 mins, 1 hr, and 3 hrs after the start of ECMO. Morphine clearance was calculated on days 1, 3, and 5 of ECMO. The mean value for morphine clearance was 11.7 +/- 9.3 (SD) ml/min/kg (range 2.6 to 34.5). CONCLUSIONS: The initiation of ECMO does not lead to a significant decrease in serum morphine concentration and there is no uptake of morphine onto the membrane oxygenator of the ECMO circuit. Morphine clearance for infants receiving ECMO is variable.

Epidural and intravenous bolus morphine for postoperative analgesia in infants.

PURPOSE: To compare two doses of bolus epidural morphine with bolus iv morphine for postoperative pain after abdominal or genitourinary surgery in infants. METHODS: Eighteen infants were randomly assigned to bolus epidural morphine (0.025 mg.kg-1 or 0.050 mg.kg-1) or bolus iv morphine (0.050-0.150 mg.kg-1). Postoperative pain was assessed and analgesia provided, using a modified infant pain scale. Monitoring included continuous ECG, pulse oximetry, impedance and nasal thermistor pneumography. The CO2 response curves and serum morphine concentrations were measured postoperatively. RESULTS: Postoperative analgesia was provided within five minutes by all treatment methods. Epidural groups required fewer morphine doses (3.8 +/- 0.8 for low dose [LE], 3.5 +/- 0.8 for high dose epidural [HE] vs. 6.7 +/- 1.6 for iv, P < 0.05) and less total morphine (0.11 +/- 0.04 mg.kg-1 for LE, 0.16 +/- 0.04 for HE vs 0.67 +/- 0.34 for iv, P < 0.05) on POD1. Dose changes were necessary in all groups for satisfactory pain scores. Pruritus, apnoea, and haemoglobin desaturation occurred in all groups. CO2 response curve slopes, similar preoperatively (range 36-41 ml.min-1.mmHg ETCO2-1.kg-1) were generally depressed (range, 16-27 ml.min-1.mmHg ETCO2-1.kg-1) on POD1. Serum morphine concentrations, negligible in LE (< 2 ng.ml-1), were similar in the HE and iv groups (peak 8.5 +/- 12.5 and 8.6 +/- 2.4 ng.ml-1, respectively). CONCLUSION: Epidural and iv morphine provide infants effective postoperative analgesia, although side effects are common. Epidural morphine gives satisfactory analgesia with fewer doses (less total morphine); epidural morphine 0.025 mg.kg-1 is appropriate initially. Infants receiving epidural or iv morphine analgesia postoperatively need close observation in hospital with continuous pulse oximetry.

Comparison of hetastarch with albumin for postoperative volume expansion in children after cardiopulmonary bypass.

OBJECTIVE: Hetastarch has been studied as a volume expander in adults after cardiopulmonary bypass (CPB) and in recommended dosages has not altered coagulation studies or increased clinical bleeding. Hetastarch was compared with albumin in children after CPB to determine whether hetastarch use was associated with increased clinical bleeding or alteration of coagulation studies. DESIGN: Randomized double-blinded study. SETTING: University-affiliated children's hospital. PARTICIPANTS: Forty-seven children age 1 year or greater (mean 72.8 months; range 12 months to 15.5 years) scheduled for repair of congenital heart disease with moderate hypothermia were randomized to receive hetastarch or albumin as a postoperative volume expander during the first 24 hours after surgery. INTERVENTIONS: Thirty-eight children required colloid replacement therapy. Blood pressure, central venous pressure, urine output, and chest tube drainage were used to determine colloid requirement. MEASUREMENTS AND MAIN RESULTS: Clinical bleeding and laboratory studies of coagulation were evaluated as were requirements for colloid, crystalloid, and blood products. Twenty children received 6% hetastarch, and 18 received 5% albumin. No differences were found in the amount of replacement fluids required, or in coagulation parameters in children receiving 20 mL/kg or less of either colloid replacement therapy. An increase in prothrombin time was demonstrated in children who received greater than 20 mL/kg of 6% hetastarch (p = 0.006); however, no difference in clinical bleeding or blood product requirement was demonstrated between the hetastarch or albumin groups receiving more than 20 mL/kg. CONCLUSION: This study demonstrated that 6% hetastarch is safe and an effective plasma volume expander in the postoperative management of children, using volumes up to 20 mL/kg. Close laboratory monitoring and careful evaluation of clinical bleeding are suggested when larger doses of hetastarch are administered because of prolongation of the prothrombin time with more than 20 mL/kg of hetastarch.