EFFECT OF NITROGLYCERNE-INDUCED HYPOTENSION ON CANINE SPINAL CORD BLOOD FLOW

Twenty-four mongrel dogs were anaesthetized with pentobarbitoneand morphine sulphate. Neuromuscular blockade was achieved usingpancuronium. Spinal cord blood flow was measured using the radionuclidemicrosphere and hydrogen washout methods before, during, andfollowing nitroglycerine-induced hypotension. Heart rate, meanarterial pressure, cardiac output, pulmonary capillary wedgepressure, and acid-base balance were determined with each measurement.Mean arterial pressure was reduced by 50%. Spinal cord bloodflow, as measured by the microsphere method, increased duringthe period of hypotension, whereas values obtained using thehydrogen washout method were not significantly different fromthose at normotension. No significant change in spinal cordblood flow was detected by either method after the applicationof spinal distraction. Nitroglycerine acts predominantly onvenous capacitance vessels and it is postulated that perfusionpressure, and therefore flow, is maintained despite a reductionin arterial pressure. Presented in part at the Anual Meeting of the American Societyof Anesthesiologists, October 1985, San Francisco, California. ^*Shackleton Department of Anaesthetics, Southampton GeneralHospital, Shirley, Southampton, Hants SO9 4XY. Section of Orthopedic Surgery, Madison, Wisconsin.     

The Effects of Magnetic Resonance Imaging on Implantable Pulse Generators

The effects of magnetic resonance imaging were assessed on four dual chamber and two single chamber pulse generators. The tests were performed with a resistive, water-cooled magnet operating at 0.15 T. The 6.4-MHz radiofrequency (RF) field was operated at a maximum power of 1,000 watts with a period adjusted from 130 to 500 ms. Reed switch closure occurred in all six pulse generators tested when placed near the entrance of the magnetic resonance imaging scanner, and the generators reverted to asynchronous operation unless programmed to the "magnet off" mode. None of the pulse generators exhibited any alterations in programmed parameters or in the ability to be reprogrammed after RF pulsing. When the RF field was turned on, there was no change in the asynchronous paced cycle length in four pulse generators; however, during RF scanning there was rapid cardiac stimulation at the RF pulse period in one single chamber and one dual chamber pulse generator.     

Neurotoxicity Evaluation of N,N-Diethyl-m-toluamide (DEET) in Rats

The neurotoxic potential of N,N-diethyl-m-toluamide (DEET) wasevaluated following acute oral administration or following multigenerationplus chronic dietary administration to the rat. For the acutestudy, rats were administered undiluted DEET at dose levelsof 50, 200, or 500 mg/kg by gavage. A dose level of 500 mg/kgwas considered to be the highest practical dose that could beevaluated in this study based upon observations of overt toxicityat 500 mg/kg and mortality at 1000 mg/ kg in a dose range-findingstudy. The two measures of neurotoxicity evaluated in the acutestudy were functional observational battery (FOB) and motoractivity measurements. An apparent treatment-related effectin thermal response time (increased) was noted for both sexes1 hr after dosing at the 500 mg/kg dose level. A questionableeffect on rearing activity (decreased) also was noted at thesame dose level. For the multigeneration plus chronic dietaryadministration study, rats were administered DEET at dietaryconcentrations of 0, 500, 2000, or 5000 ppm continuously overtwo generations and then chronically for 9 months. A dietaryconcentration of 5000 ppm meets the criteria for a maximum tolerateddose (MTD) based on traditional chronic toxicology assessments.Evaluations included FOB, motor activity, discriminative acquisitionand reversal in an Mmaze, acoustic startle habituation, passiveavoidance acquisition and retention, and microscopic examinationof central and peripheral nervous tissue. The only effect thatwas considered to be possibly treatment-related was a slightincrease in exploratory locomotor activity at the 5000 ppm doselevel. Based on the results of these studies, the nervous systemdoes not appear to be a selective target when DEET is administeredto rats either as a single oral dose at high dose levels orchronically at the MTD.     

Reproductive and Thyroid Effects of Low-Level Polychlorinated Biphenyl (Aroclor 1254) Exposure

As little information is available on the adverse effects ofpolychlorinated biphenyls (PCBs) on the reproductive systemof the male rat, the current study was conducted to evaluatethe effects of subchronic administration of the PCB mixtureAroclor 1254 on testicular gamete production and endocrine function.The thyroid hormone thyroxine (T4), which is critical for reproductionand development, was also measured because of the well-documentedeffects of PCBs on this hormone. Weanling (31-day-old) maleFischer rats were administered 0, 0.1, 1, 10, or 25 mg/kg/dayAroclor 1254 by gavage for 5, 10, or 15 weeks and necropsied.The hormones testosterone (T) and thyroxine were measured inthe serum, and body weight and weights of the liver, kidney,testes, seminal vesicle plus coagulating gland, cauda epididymides,and pituitary were taken. At 10 and 15 weeks, testicular interstitialfluid (IF) was collected and T concentration in the IF was measured.Sperm motility was measured from a caudal sperm sample and spermnumbers in the testis and cauda epididymis were determined.In addition, tissues were examined microscopically for histopathologicalalterations. In the high-dose group, body, seminal vesicle,cauda epididymal, and pituitary weights were depressed at 10and 15 weeks and cauda epididymal sperm numbers were reducedafter 15 weeks of dosing. In contrast, testes weights, testicularsperm numbers, sperm motility, and serum and testicular testosteronelevels were unaffected, even in the highest dose group (25 mg/kg/day).Aroclor 1254 administration produced histological alterationsin the liver and kidney at doses of 1.0 mg/kg/day and above.These results indicate that the testis of the rat is not a specifictarget organ for Aroclor 1254. In contrast, serum T4 levelswere reduced by Aroclor 1254 administration at a dose 250-foldbelow the dose that failed to alter testicular function. SerumT4 levels were depressed 25% in the 1 mg/kg dose group after5 weeks of exposure and 30% in the 0.1 mg/kg group following15 weeks of exposure. T4 levels were undetectable in the twohighest (10 and 25) dose groups at all intervals. The fact thatthe decreases in T4 were generally concurrent with increasesin liver weight suggested that Aroclor 1254 altered T4 levelsby increasing the turnover rate in the serum by enhancing themetabolism of T4 by the liver. The reduction in serum T4 reportedhere occurred at a dose 25-fold lower than the dose generallyrecognized as affecting thyroid hormone levels.     

Immunotoxicological Profile of Morphine Sulfate in B6C3F1 Female Mice

This study was undertaken to investigate a number of immuneparameters which may be compromised with exposure to morphinesulfate. Mice were implanted subcutaneously with 8-, 25-, or75-mg morphine sulfate pellets. Placebo pellets of identicalmakeup to the 75-mg morphine pellet (without morphine of course)were used as a control. Twenty-four hours after implantationof a 75-mg morphine pellet, blood levels reached a peak of 1610ng/ml. Corticosterone increased in parallel with morphine andreached a peak level of 966 ng/ml 24 hr after implantation.The dose response of morphine to increase corticosterone, however,was fiat. The weight of the lymphoid organs, spleen and thymus,and the liver were significantly reduced in the morphine-treatedgroups. Morphine treatment was associated with an increase inserum albumin, SGPT, BUN, and alkaline phosphatase indicativeof hepatic damage. In contrast to increased serum proteins,the C3 component of complement was reduced in a dose-dependentmanner. Leukocyte number in the peripheral blood was significantlyreduced, while erythro-cyte number and hematocrit were bothincreased. The number of B cells and T cells was decreased inmorphine-treated animals. However, the percentage of T cellsrelative to B cells was increased. The primary IgM antibodyresponse to the T-depen-dent antigen, sheep red blood cells,was decreased. Natural killer cell activity was reduced in responseto morphine, as was the phagocytic capacity of Kupffer cells.Host-resistance models of Listeria monocytogenes or Streptococcuspneumoniae showed an increased resistance following administrationof morphine. This increased host resistance, however, was notdue to an increase in antimicrobial action of sera obtainedfrom mice treated with morphine. The majority of morphine'seffects on the immune system exhibited a flat dose response,suggesting that these effects may be mediated secondarily throughcorticosterone.     

Developmental Neurotoxicity: Evaluation of Testing Procedures with Methylazoxymethanol and Methylmercury

Testing procedures for identification of potential developmentalneurotoxicants were evaluated using two prototypical developmentalneurotoxicants, methylazoxymethanol (MAM) and methylmercury(MeHg). Evaluation of offspring of LongEvans rats incorporatedassessments of developmental toxicity, neurochemistry, histology,and behavior, with most testing being completed near weaning.A number of endpoints in the testing strategy were sensitiveto the effects of prenatal exposure to MAM [30 mg/kg on GestationDay (GD) 15]: (1) MAM caused reduced neonatal body weights butdid not effect viability or postnatal survivorship; (2) measurementof total and regional brain weight and histological analysisshowed that a number of regions, the cortex and hippocampusin particular, were affected by MAM exposure; (3) an assay forglial fibrillary acidic protein (GFAP) showed that the concentrationof this protein was significantly increased in the cortex andhippocampus of treated offspring; (4) a T-maze delayed-alternationprocedure indicated that MAM-treated pups were slower in theacquisition phase of the task relative to control pups; (5)motor activity testing revealed hyperactivity in treated offspringthat persisted into adulthood; and (6) acoustic startle proceduresrevealed reduced startle amplitudes in preweanlings. Few endpointswere significantly affected by prenatal MeHg exposure (1, 2,or 4 mg/kg on GD 6–15). High fetal and neonatal mortalityand lower neonatal body weights were detected at the highestdose of MeHg. Although minimal effects of MeHg may reflect arelative insensitivity of the test species and/or the test methods,the combined results from both chemicals suggest that some proceduresnot currently required in the developmental neurotoxicity guidelinemay be useful in hazard identification, and further evaluationwith other chemicals, species, strains, and/or exposure paradigmsmay be warranted.     

Absorption, Metabolism, and Excretion of N,N-Diethyl-m-toluamide Following Dermal Application to Human Volunteers

The absorption, metabolism, and excretion of N,N-diethyl-m-toluamide(DEET) in male human volunteers following dermal applicationof |¹⁴C|DEET was studied. DEET was applied to two groups ofsix volunteers either as the undiluted technical grade materialor as a 15% solution in ethanol. The material was applied overa 4 x 6-cm area on the volar surface of the forearm and wasleft in contact with the skin for 8 hr, then rinsed off theskin. Application sites also were tape stripped at 1, 23, and45 hr after rinsing. Serial blood samples and all urine andfeces were collected for 5 days after application. Aliquotsof these materials were analyzed for total radioactivity inorder to define absorption and excretion patterns. Urine samplesalso were analyzed by HPLC to characterize the metabolic profileand/or to identify metabolites. Absorption of DEET as evidencedby plasma radioactivity occurred within 2 hr after dose application.Elimination of radioactivity from plasma was rapid and quantifiablelevels of radioactivity were observed in plasma for only 4 hrafter the end of the 8-hr exposure period. Urine was the principalroute of excretion of radioactivity and accounted for an averageof 5.61 and 8.33/ of the applied dose in the undiluted DEETand 15/ DEET in ethanol groups, respectively. Excretion of radioactivityin the feces was less than 0.08/ of the applied dose in bothgroups. DEET did not accumulate in the superficial layers ofthe skin as evidenced by low amounts of radioactivity in thetape strippings. The major fraction of the applied radioactivitywas recovered in the skin rinses. Absorbed DEET was completelymetabolized and six major metabolites were observed in urine.Two major urinary metabolites tenta tively were identified.Based upon the percentage of applied dose recovered in the excreta,dermal absorption of DEET ranged from 3 to 8% with a mean of5.6/ in the volunteers applied undiluted technical grade DEET.The corresponding values for the volunteers applied 15/ DEETin ethanol were 4 to 14/ and 8.4/, respectively.     

Secretion from islets and single islet cells following cryopreservation

The ability to cryopreserve pancreatic islets has allowed the development of low-temperature banks that permit pooling of islets from multiple donors and allows time for sterility and viability testing. However, previous studies have shown that during cryopreservation and thawing there is a loss of islet mass and a reduction in islet function. The aim of this study was to measure and compare insulin secretion from cultured nonfrozen and frozen-thawed canine islets and beta-cells. Canine islets were isolated from mongrel dogs using intraductal collagenase distention, mechanical dissociation, and EuroFicoll purification. One group of purified islets was cultured overnight before dissociation into single cells and subsequent analysis. Remaining islets were cultured overnight (22 degrees C) and then cryopreserved in 2 M dimethyl sulfoxide (DMSO) solution using a slow stepwise addition protocol with slow cooling to -40 degrees C before storage in liquid nitrogen (-196 degrees C). Frozen islets were rapidly thawed (200 degrees C/min) and the DMSO removed using a sucrose dilution. From a series of seven consecutive canine islet isolations, islet recovery following postcryopreservation tissue culture was 81.5 +/- 4.8% compared to precryopreservation counts. In vitro islet function was equivalent between cultured nonfrozen and frozen-thawed islets with a calculated stimulation index of 10.4 +/- 1.5 (mean +/- SEM) for the frozen-thawed islets, compared with 12.4 +/- 1.2 for the cultured nonfrozen controls (p = ns, n = 7 paired experiments). Amperometric detection of secretion from single beta-cells in vitro has the sensitivity and temporal resolution to detect single exocytotic events and allows secretion to be monitored from single beta-cells in real time. Secretion from single beta-cells elicited by chemical stimulation was detected using a carbon fiber microelectrode. The frequency of exocytosis events was equivalent between the cultured nonfrozen and frozen-thawed beta-cells with an average of 7.0 +/- 1.32 events per stimulation for the cultured nonfrozen group compared with 6.0 +/- 1.45 events from the frozen then thawed preparations (minimum of 10 cells per run per paired experiment, p = ns) following stimulation with tolbutamide. The average amount of insulin released per individual exocytosis event was equivalent for the cultured nonfrozen and frozen-thawed islets. In addition, beta-cells responded to both tolbutamide and muscarinic stimulation following cryopreservation. It was determined that beta-cells recovered following cryopreservation are capable of secreting insulin at levels and frequencies comparable to those of cultured nonfrozen islet preparations.     

The effects of microencapsulation on pancreatic islet osmotically induced volumetric response

Microencapsulation of pancreatic islets has been proposed as a means to prevent allograft rejection and to protect islets during cryopreservation. The aim of this study was to investigate: 1) the effects of the cryoprotectants (CPAs) dimethyl sulfoxide (DMSO) and ethylene glycol (EG) on the volume of Ca2+ alginate microcapsules, and 2) the effects of microencapsulation on the volumetric response of human and canine pancreatic islets during CPA equilibration. Stock sodium alginate with a high mannuronic acid content (HM) or a high guluronic acid content (HG) was used to generate empty capsules (mean diameter 200 microm) with an electrostatic generator. The capsules were held in place by a holding pipette system and videotaped during the addition of 2 or 3 M CPA at 22 degrees C. Islets (isolated from human cadaveric donors and mongrel dogs and then cultured overnight at 37 degrees C) were encapsulated in alginate (HM), loaded into a microperfusion chamber, and the change in islet volume was videotaped after exposure to the same CPAs and concentrations. These were compared to the volume responses of nonencapsulated islets. Images were analyzed using a computerized image analysis system and the data were analyzed using ANOVA. HG microcapsules showed a significant (p < 0.05) increase in volume following exposure to EG but not to DMSO. HM microcapsule volume did not change significantly following exposure to either EG or DMSO and was therefore chosen as the substrate for islet encapsulation. Free, nonencapsulated canine and human islets responded to the osmotic challenge of the 2 M DMSO by shrinking to 70.00 +/- 1.04% (mean +/- SEM) and 70.11 +/- 1.05%, and in 2 M EG to 72.89 +/- 1.93% and 69.33 +/- 1.38%, respectively, of the isotonic volume before returning to the original cell volume. Exposure to 3 M DMSO or EG resulted in a further dehydration to 65.89 +/- 0.91% and 67.67 +/- 1.91% for canine and 62.22 +/- 0.66.% or 65.89 +/- 1.30% for human islets. Minimum volumes were reached within 30-40 s after exposure to the cryoprotectant. Encapsulated human islets reached 86.88 +/- 1.47% of their original volume in 2 M and 80.33 +/- 0.89% in 3 M DMSO, and 87.33 +/- 1.86% in 2 M and 82.80 +/- 1.57% in 3 M EG. This volume change was significantly less (p < 0.01) than that observed in corresponding free islets. Encapsulated canine islets reached 83.67 +/- 2.13% of their original volume in 2 M and 78.22 +/- 0.95% in 3 M DMSO, and 85.44 +/- 1.92% in 2 M and 78.11 +/- 2.01% in 3 M EG. As with human islets, this was significantly different than free islets (p < 0.01). These minimal volumes were reached within 30-50 s. These results demonstrate that there are cryoprotectant and alginate-specific interactions and that microencapsulation modulates the degree of osmotically induced shrinkage of islets. The development or modification of existing cryopreservation protocols to improve postcryopreservation recovery or function must account for these factors.     

Subchronic Effects of Dieldrin and Phenobarbital on Hepatic DNA Synthesis in Mice and Rats

Dieldrin, an organochlorine pesticide, has been shown to behepatocarcinogenic in mice but not rats. Phenobarbital, in contrast,induces hepatic tumors in both mice and rats. Previous studieshave shown that acute dietary exposure of rats or mice to eitherdieldrin or phenobarbital produces several liver changes, includingcentrilobular hypertrophy, induction of hepatic cytochrome P450,and increased liver weight. The present study examined the subchroniceffect of dieldrin (0.1, 1.0, 3.0, 10.0 mg dieldrin/kg diet)and phenobarbital (10, 50, 100, 500 mg phenobarbital/kg diet)on the induction of hepatic DNA synthesis and hepatocyte lethalityin male B6C3F1 mice and male F344 rats. Eight-week-old animalswere treated as above and evaluated for hepatic DNA synthesisafter 7, 14, 21, 28, and 90 days of continual treatment to dieldrinor phenobarbital. Maximal induction of hepatic DNA synthesisin mice was seen at the 14-, 21-, and 28-day sampling times.In rats, no significant increase in hepatic DNA synthesis orhepatocyte lethality was observed at any dose of dieldrin investigated.Phenobarbital produced a significant increase in hepatic DNAsynthesis in both rat and mouse liver following 7 days of treatment.The induction of DNA synthesis in rat liver was transient, withthe labeling index returning to control levels by 14 days oftreatment. In contrast, mice treated with phenobarbital showeda significant increase in hepatic DNA synthesis throughout thetreatment. In both mice and rats, dieldrin and phenobarbitalinduced hepatic DNA synthesis selectively in the centrilobularregion of the hepatic lobule. The lack of an increase in serumenzymes indicative of hepatic damage and the absence of liverhistopathology in mice or rats fed dieldrin or phenobarbitalindicate that the induction of DNA synthesis was not mediatedby a cytolethal, compensatory hyperplastic response, suggestinga mitogenic mechanism. Therefore, the species-specific inductionof hepatic DNA synthesis by either dieldrin or phenobarbitalcorrelated with the previously observed species-specific inductionof hepatic cancer by these two compounds.