Dynorphin A (1-13) neurotoxicity in vitro: opioid and non-opioid mechanisms in mouse spinal cord neurons

Dynorphin A is an endogenous opioid peptide that preferentially activates kappa-opioid receptors and is antinociceptive at physiological concentrations. Levels of dynorphin A and a major metabolite, dynorphin A (1-13), increase significantly following spinal cord trauma and reportedly contribute to neurodegeneration associated with secondary injury. Interestingly, both kappa-opioid and N-methyl-D-aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through kappa-opioid and/or NMDA receptor types. Despite these findings, few studies have systematically explored dynorphin toxicity at the cellular level in defined populations of neurons coexpressing kappa-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both kappa-opioid and NMDA receptors from embryonic mouse spinal cord and examined the effects of dynorphin A (1-13) on intracellular calcium concentration ([Ca2+]i) and neuronal survival in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. At micromolar concentrations, dynorphin A (1-13) elevated [Ca2+]i and caused a significant loss of neurons. The excitotoxic effects were prevented by MK-801 (Dizocilpine) (10 microM), 2-amino-5-phosphopentanoic acid (100 microM), or 7-chlorokynurenic acid (100 microM)--suggesting that dynorphin A (1-13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (-)-naloxone (3 microM), or the more selective kappa-opioid receptor antagonist nor-binaltorphimine (3 microM), exacerbated dynorphin A (1-13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 microM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 microM). Thus, opioid receptor blockade significantly increased toxicity, but only in the presence of excitotoxic levels of dynorphin. This provided indirect evidence that dynorphin also stimulates kappa-opioid receptors and suggests that kappa receptor activation may be moderately neuroprotective in the presence of an excitotoxic insult. Our findings suggest that dynorphin A (1-13) can have paradoxical effects on neuronal viability through both opioid and non-opioid (glutamatergic) receptor-mediated actions. Therefore, dynorphin A potentially modulates secondary neurodegeneration in the spinal cord through complex interactions involving multiple receptors and signaling pathways.     

Priming of neutrophil [Ca2+]i signaling and oxidative burst by human fracture fluids

BACKGROUND: Patients with major fracture/soft-tissue injuries are at risk for adult respiratory distress syndrome after secondary infection. Fracture fluids (FF) are rich in neutrophil (PMN) -specific chemokines such as interleukin-8. PMN respond to both interleukin-8 and bacterial stimuli with calcium ([Ca2+]i) fluxes, which can initiate respiratory burst (RB). We hypothesize that small amounts of FF entering the circulation could exaggerate PMN [Ca2+]i and RB responses, potentially increasing the risk of adult respiratory distress syndrome. METHODS: FF were obtained from 10 patients at open fixation of the femur 2 to 5 days postinjury. Volunteer PMN were isolated and loaded with fura dye. PMN were preincubated either in 30% autologous plasma (AP)/70% buffer, or in 5% FF/25% AP/70% buffer. Cells were resuspended in buffer with 1,2,3-dihydrorhodamine and stimulated with low-dose n-formyl-methionyl-leucyl-phenylalanine (fMLP). [Ca2+]i was assayed by fura fluorescence at 505 nm after excitation at 340/380 nm. RB was assessed by 1,2,3-dihydrorhodamine fluorescence at 530 nm after 488 nm excitation. RESULTS: PMN basal [Ca2+]i was higher after FF incubation than AP incubation (94+/-12 vs. 61+/-9 nmol/L, p = 0.0002). Peak [Ca2+]i response to fMLP was 475+/-47 nmol/L after FF but only 356+/-22 nmol/L after AP (p = 0.01). Two hundred seconds after fMLP, [Ca2+]i remained higher after FF (172+/-17 vs. 145+/-9 nmol/L, p = 0.04). Basal RB was slightly higher after FF than AP (13.4+/-0.3 vs. 11.3+/-0.3 units, p = 0.051) as was the maximal rate of extracellular oxidant release (1.10+/-0.17 vs. 0.76+/-0.16 units/s, p = 0.004) and total oxidant production (42.5+/-0.8 vs. 31.7+/-0.8 units, p = 0.006). CONCLUSION: Small amounts of FF in plasma can exaggerate PMN [Ca2+]i flux and RB responses to subsequent bacterial stimuli. These findings are consistent with the hypothesis that release of FF into the circulation primes PMN and, thus, may predispose to adult respiratory distress syndrome. Such PMN priming events might have important implications for both the operative and medical management of patients with major fractures.     

Intranasal murine model of Bordetella pertussis infection. I. Prediction of protection in human infants by acellular vaccines.

Bicomponent, tricomponent and pertactin DTPa vaccines were tested in sublethal aerosol, and lethal and sublethal intranasal murine Bordetella pertussis respiratory challenge models. Pertactin and bicomponent vaccines induced protective immunity against lethality but with little or no bacterial clearance. Intranasal challenge discriminated in a reproducible, statistically significant manner between the efficacies of bicomponent and tricomponent DTPa, in agreement with clinical trial data. This discrimination was not observed in the aerosol challenge. Pertactin had a synergistic effect with bicomponent DTPa. Intranasal challenge may be useful as part of the preclinical evaluation of new acellular pertussis formulations or DTPa-based combinations.     

Population studies of three AMPFLPs systems in a North Polish population

Allele and phenotype frequencies for D1S80, D17S5 and ApoB were determined in a population sample of more than 200 unrelated persons from North Poland using the PCR method. For D1S80, D17S5 and ApoB 19, 13 and 21 alleles respectively were observed. No deviations from Hardy-Weinberg equilibrium were detected. All three systems have discrimination values above 92% and a cumulative discrimination index of 4.5 × 10⁴.Dedicated to Prof. Dr. J. Gerchow on the occasion of his 75th anniversary     

Cash budgeting: an underutilized resource management tool in not-for-profit health care entities

Cash budgeting is generally considered to be an important part of resource management in all businesses. However, respondents to a survey of not-for-profit health care entities revealed that some 40 percent of the participants do not currently prepare cash budgets. Where budgeting occurred, the cash forecasts covered various time frames, and distribution of the document was inconsistent. Most budgets presented cash receipts and disbursements according to operating, investing, and financing activities--a format consistent with the year-end cash flow statement. By routinely preparing monthly cash budgets, the not-for-profit health care entity can project cash inflow/outflow or position with anticipated cash insufficiencies and surpluses. The budget should be compared each month to actual results to evaluate performance. The magnitude and timing of cash flows is much too critical to be left to chance.     

Stoß- und schlagbelastung des menschlichen schienbeins im drehversuch

Zusammenfassung Die menschliche Tibia wurde bei mittlerer und schneller Drehgeschwindigkeit bis zum Bruch des Knochens belastet. Die Ergebnisse der Zehntel- und Hundertstelsekundenfraktur werden mit denen bei langsamer Drehung (Sekundenfraktur) verglichen.Beim Drehstoß ( = 236°/sec) steigen die Frakturdrehmomente durchschnittlich um 17%, beim Drehschlag ( = 597°/sec) um 25% gegenüber der langsamen Drehung ( = 6°/sec). Die Elastizitätsgrenze, die die Verletzungsgrenze der Tibia darstellt, zeigt jeweils einen noch etwas höheren Anstieg. Die menschliche Tibia ist also gegenüber Stößen und Schlägen weniger empfindlich als gegenüber langsamen Belastungen, bei Drehung noch mehr als bei Biegung. Der Durchmesser des Schienbeinkopfes korrelierte im Vergleich zu zahlreichen weiteren Parametern am besten mit den Bruchwerten. Energieaufnahme, Form, Lage und Länge der Fraktur änderten sich auch bei schneller Drehung nicht, der Frakturdrehwinkel nahm bei Schlagbeanspruchung leicht ab.Für die Verletzungsprophylaxe beim alpinen Skilauf sind bei kurzer Krafteinwirkung die Trägheitsmomente des gesamten Beines zu berücksichtigen und erhöhen die o. g. Werte der Verletzungsgrenze erheblich.Aus den Ergebnissen werden Folgerungen für den Schutz vor typischen Skiverletzungen durch die Skibindung abgeleitet und können dazu beitragen, die passive Sicherheit beim Skilauf weiter zu erhöhen.

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Hit and blow endurance of the human tibia in torsion testing

Summary Whole human tibiae were fractured with rapid loading rates of tenths and hundreds of a second for torsional fracture. Results were compared with data of experiments in slow loading during seconds.Loading time of 100 msec ( = 236°/s) increased the mean maximum torque of 17%, loading time of 10–40 msec ( = 597°/s) showed increase of 25%. In dynamic twisting the applied torque at the elasticity threshold, which represents the injury threshold of the tibia, has relatively higher values than the data at failure. The best correlation to the maximum torque exists at the frontal diameter of the tibia head.In dynamic twisting energies, site, figuration and length of fracture did not differ from those in quasistatic twisting procedures. With loading time of hundreds of a second the angle of fracture diminished.To prevent injuries in alpine skiing it is important to consider inertial effects of the whole leg in dynamic twisting. These effects raise the values of injury threshold distinctly. Practical conclusions are drawn from the results to get better passive safety in alpine skiing by perfecting release bindings.

     

Inhaled environmental combustion particles cause myocardial injury in the Wistar Kyoto rat.

Epidemiologists have associated particulate matter (PM) air pollution with cardiovascular morbidity and premature mortality worldwide. However, experimental evidence demonstrating causality and pathogenesis of particulate matter (PM)-induced cardiovascular damage has been insufficient. We hypothesized that protracted, repeated inhalation by rats of oil combustion-derived, fugitive emission PM (EPM), similar in metal composition to selected sources of urban air PM, causes exposure duration- and dose-dependent myocardial injury in susceptible rat strains. Zinc was the only primary water-leachable/bioavailable element of this EPM. Male Sprague-Dawley (SD), Wistar Kyoto (WKY), and spontaneously hypertensive (SH) rats were exposed nose-only to EPM (2, 5, or 10 mg/m(3), 6 h/day for 4 consecutive days or 10 mg/m(3), 6 h/day, 1 day/week for 4 or 16 consecutive weeks). Two days following the last EPM exposure, cardiac and pulmonary tissues were examined histologically. The results showed that particle-laden alveolar macrophages were the only pulmonary lesions observed in all three rat strains. However, WKY rats exposed to EPM (10 mg/m(3) 6 h/day, 1 day/week for 16 weeks) demonstrated cardiac lesions with inflammation and degeneration. To further characterize the nature of EPM-associated lesions, more rigorous histopathological and histochemical techniques were employed for WKY and SD rats. We examined the hearts for myocardial degeneration, inflammation, fibrosis, calcium deposits, apoptosis, and the presence of mast cells. Decreased numbers of granulated mast cells, and multifocal myocardial degeneration, chronic-active inflammation, and fibrosis were present in 5 of 6 WKY rats exposed to EPM for 16 weeks. None of these lesions were present in WKY exposed to clean air. EPM-related cardiac lesions were indistinguishable from air-exposed controls in SD and SH rats. This study demonstrates that long-term inhalation exposures to environmentally relevant PM containing bioavailable zinc can cause myocardial injury in sensitive rats. These findings provide supportive evidence for the epidemiological associations of cardiovascular morbidity and ambient PM.     

Noninvasive Tracking of Patient's Head Movements During Computer-Assisted Intranasal Microscopic Surgery

A noninvasive system designed for patient tracking during image-guided intranasal sinus surgery is described. It is based on optical digitizing with a custom-made registration and reference system, locatable surgical instruments, and a self-localizing operating microscope. Experimental and clinical results reveal a high degree of accuracy for the system. A mean spatial error of 0.82 ± 0.31 mm was determined for repositioning of the reference system in a plastic model of the skull. For the positioning of the microscope, a mean error of 2.3 ± 0.83 mm was calculated. Measurements of repositioning accuracy in 24 patients who received surgery for various sinus diseases had a mean spatial error of 1.56 ± 0.76 mm. The 95% error interval for locating intranasal structures using the surgical instrument was 2.05 mm, and it was 4.92 mm using the microscope. These results suggest that the use of our noninvasive registration and reference system may be effective, accurate, and useful for noninvasive tracking of patient movements in computer-assisted intranasal surgery.