Chronic opiate exposure in the male rat adversely affects fertility.

We examined whether morphine administration to adult male rats adversely affected pregnancy outcome after mating with drug-naive females and at what point in the complex series of steps leading to viable offspring it exerted its actions. The results indicate that chronic paternal morphine exposure markedly influenced fertility measures in a number of important ways. There was a pronounced increase in pseudopregnancies in females mated with males treated chronically with morphine (40%) when compared to controls (<6%), indicating that vaginal penetration occurred, but successful impregnation failed; only 33% of matings between drug-naive females and morphine-treated males resulted in pregnancies, as compared to 74.5% in controls. In addition, there were fewer implantation sites in gravid females mated with morphine-treated males than in controls. Taken together, these observations suggest that morphine-exposed male rats were apparently able to copulate, but there was a failure in successful impregnation of the females. These findings suggest a primary defect in either the quality of male sexual behavior or a complete failure of the fertilization or conception processes in females mated with morphine-exposed males. This potentially important effect of paternal morphine administration on conception and/or preimplementation loss of embryos has not been previously noted and deserves more systematic study.

Micronuclei in the peripheral blood and bone marrow cells of rats exposed to 2450 MHz radiofrequency radiation.

PURPOSE: To determine the incidence of micronuclei in peripheral blood and bone marrow cells of rats exposed continuously for 24h to 2450 MHz continuous wave radiofrequency radiation (RFR) at an average whole-body specific absorption rate (SAR) of 12W/kg. MATERIALS AND METHODS: Eight adult male Sprague-Dawley rats were exposed to 2450 MHz RFR in circularly polarized waveguides. Eight sham-exposed rats were kept in similar waveguides without the transmission of RFR. Four rats were treated with mitomycin-C (MMC) and used as positive controls. All rats were necropsied 24h after the end of RFR and sham exposures, and after the 24h treatment with MMC. Peripheral blood and bone marrow smears were examined to determine the frequency of micronuclei (MN) in polychromatic erythrocytes (PCE). RESULTS: The results indicated that the incidence of MN/2000 PCE were not significantly different between RFR- and sham-exposed rats. The group mean frequencies of MN in the peripheral blood were 2.3+/-0.7 in RFR-exposed rats and 2.1+/-0.6 in sham-exposed rats. In bone marrow cells, the average MN incidence was 3.8+/-1.0 in RFR-exposed rats and 3.4+/-0.7 in sham-exposed rats. The corresponding values in positive control rats treated with MMC were 23.5+/-4.7 in the peripheral blood and 33.8+/-7.4 in bone marrow cells. CONCLUSION: There was no evidence for the induction of MN in peripheral blood and bone marrow cells of rats exposed for 24h to 2450 MHz continuous wave RFR at a whole body average SAR of 12 W/kg.

Use of intraosseous blood for repeated hematologic and biochemical analyses in healthy pigs.

OBJECTIVE: To evaluate the clinical and histologic effects of repeated intraosseous (IO) needle placement in domestic pigs and determine whether blood and serum obtained intraosseously could be used for CBC and biochemical analyses. ANIMALS: 5 healthy 10-week-old pigs. PROCEDURE: An IO needle was placed in the proximomedial region of the tibia of anesthetized pigs every other week for 2 months, and IO blood was obtained for CBC and serum biochemical analyses. Results were compared with those obtained for blood collected at the same time from the auricular vein. Two weeks after the final samples were obtained, pigs were euthanatized and tibias were processed for histologic examination. RESULTS: Clinical abnormalities, including lameness, were not detected following IO needle placement. Histologic examination revealed only mild multifocal periosteal fibrosis and slight thickening of the periosteum without evidence of osteomyelitis. Chloride, creatinine, glucose, total protein, sodium, and BUN concentrations, alanine transaminase and gamma glutamyl transpeptidase activities, RBC count, mean corpuscular volume, and Hct did not significantly differ between IO and venous samples. However, aspartate transaminase activity, potassium, hemoglobin, and mean corpuscular hemoglobin concentrations, mean corpuscular hemoglobin, and platelet and WBC counts were significantly different. CONCLUSION AND CLINICAL RELEVANCE: Repeated placement of 10 needles may be a safe and clinically useful method to obtain serial blood samples from domestic pigs, particularly when other vascular sites are not accessible. Intraosseous blood can be used for many of the tests comprising CBC and serum biochemical analyses.

Radiofrequency electromagnetic fields have no effect on the in vivo proliferation of the 9L brain tumor.

The intracranial 9L tumor model was used to determine if exposure to a radiofrequency (RF) electromagnetic field similar to those used in cellular telephone has any effects on the growth of a central nervous system tumor. Fischer 344 rats implanted with different numbers of 9L gliosarcoma cells were exposed to 835.62 MHz frequency-modulated continuous wave (FMCW) or 847.74 MHz code division multiple access (CDMA) RF field with nominal slot-average specific absorption rates in the brain of 0.75 +/- 0.25 W/kg. The animals were exposed to the RF field for 4 h a day, 5 days a week starting 4 weeks prior to and up to 150 days after the implantation of tumor cells. Among sham-exposed animals injected with 2 to 10 viable cells (group 1), the median survival was 70 days, with 27% of the animals surviving at 150 days. The median survival length and final survival fraction for animals injected with 11 to 36 viable cells (group 2) were 52 days and 14%, respectively, while the values for those injected with 37 to 100 cells (group 3) were 45 days and 0%. The animals exposed to CDMA or FMCW had similar survival parameters, and the statistical comparison of the survival curves for each of the groups 1, 2 and 3 showed no significant differences compared to sham-exposed controls.

Anomalous development of the hepatobiliary system in the Inv mouse.

Extrahepatic biliary atresia (BA) is a devastating disease of the neonate in which the hepatic and/or common bile duct is obliterated or interrupted. Infants and children with this diagnosis constitute 50% to 60% of the pediatric population that undergoes orthotopic liver transplantation. However, there is still very little known about the etiology and pathogenesis of BA. Several recent studies have demonstrated that anomalies of situs determination are more commonly associated with BA than previously recognized. In this study, we examined the pathogenesis of jaundice in the inv mouse, a transgenic mouse in which a recessive deletion of the inversin gene results in situs inversus and jaundice. The results show that these mice have cholestasis with conjugated hyperbilirubinemia, failure to excrete technetium-labeled mebrofenin from the liver into the small intestine, lack of continuity between the extrahepatic biliary tree and the small intestine as demonstrated by Trypan blue cholangiography, and a liver histological picture indicative of extrahepatic biliary obstruction with negligible inflammation/necrosis within the hepatic parenchyma. Lectin histochemical staining of biliary epithelial cells in serial sections suggests the presence of several different anomalies in the architecture of the extrahepatic biliary system. These results suggest that the inversin gene plays an essential role in the morphogenesis of the hepatobiliary system and raise the possibility that alterations in the human orthologue of inversin account for some of the cases of BA in which there are also anomalies of situs determination.

The murine tub (rd5) mutation is not associated with a primary axonemal defect.

Some genetic syndromes causing loss of hearing and vision, such as some forms of Usher's syndrome, also cause reduced sperm cell motility, bronchiectasis, and other pathologies involving cilia- and flagella-bearing cells. In some Usher's patients, ultrastructural defects of axonemes within photoreceptor ciliary bridges, nasal cilia, and sperm cell flagella have been found, indicating a primary defect of axonemal conformation. Mice homozygous for the tub (rd5) mutation exhibit progressive retinal degeneration, sensorineural hearing loss, reduced fertility, and obesity, and presently represent the only animal model with neuroepithelial degeneration of both cochlea and retina without other neurological abnormalities. They provide a good phenotypic match to human genetic sensory syndromes, particularly human sensory/obesity syndromes, such as Alstrom's and Bardet/Biedl, although no human candidate genes have been identified. Because of their unique phenotype, tubby mice are an appropriate model in which to look for a primary axonemal defect. We studied the axonemal ultrastructure of photoreceptors and sperm cells and performed functional testing of sperm in tub/tub mice before and after the onset of obesity. Approximately 15% of photoreceptor axonemes appeared abnormal in tub/tub animals, compared to 0% in controls. Both tub homozygotes and controls exhibited approximately 10% abnormal sperm cell axonemes, and no differences in sperm cell motile function were found at any age. The modest occurrence of axonemal defects in photoreceptors of tub/tub animals is likely to be a secondary effect of retinal degeneration. We conclude that the tubby phenotype is not associated with a generalized defect of cilia- and flagella-bearing cells and that the tub mutation does not primarily affect axonemal structure.

A behavioral study of the development of hereditary cerebellar ataxia in the shaker rat mutant.

shaker Mutant rats were first identified by their abnormal motor behaviors and degeneration of cerebellar Purkinje cells and brainstem inferior olivary neurons. After 6 generations of inbreeding 77% of shaker rat mutants are mildly ataxic (identified as mild shaker mutants) and 23% are ataxic and exhibit a whole body tremor (strong shaker mutants) by 3 months of age. This study of shaker mutants from birth to 3 months of age was designed to: (1) compare the somatic and motor development of shaker mutants with age matched normal rats; (2) identify the temporal onset of motor deficits; and (3) correlate qualitative differences in Purkinje cell degeneration between 3-month-old mild and strong shaker rat mutants. Shaker mutant rats consistently weighed less than age-matched control animals. Analysis of motor-development using the hindlimb splay test demonstrated the distance between hindpaws was significantly greater in shaker mutant rats than in controls starting at 42 postnatal days (PND) of age. Hindlimb stride width was greater for shaker than control rats at 42 PNDs. However, after 42 PNDS shaker mutant average hindlimb width was narrower than controls. Forelimb stride width was consistently narrower in shaker mutants than in normal rats. Hindlimb placement was impaired in shaker rat mutants after 15 PND. Forelimb placement, cliff avoidance and surface righting were only transiently impaired in shaker mutants. Mid-air righting, performance of a geotaxic response, and climbing and jumping postural reactions were similar in shaker and normal rats. The spatial extent of Purkinje cell survival/degeneration correlated with differences in abnormal motor activity seen in 3-month-old mild and strong shaker mutants. In mild shaker rat mutants, Purkinje cells appeared to have degenerated randomly throughout the cortex. In strong shaker mutants most Purkinje cells in the anterior lobe had degenerated. In the posterior lobe Purkinje cell degeneration appeared to be numerically significant, but many surviving cells were present. Although Purkinje cell loss was not numerically quantified in this study, a strong association between the extent and type of spatial loss of Purkinje cells, and the severity of clinical signs, appears to exist.

Proximal gastric vagotomy by minimally invasive methods in an acute rat model.

In this prospective study, minimally invasive methods of proximal gastric vagotomy (PGV) were investigated in male Sprague-Dawley rats. Completeness of vagotomy by traditional operative therapy, by laser denervation of the gastric serosa, and by subserosal or transmucosal injections of chemoneurolytic agents was evaluated with postoperative Congo red testing, ulcerogenic stimulation of the gastric mucosa, and histochemical labeling of whatever vagal fibers remained in the gastric wall. Short-term results demonstrate that successful PGV can be performed with minimally invasive methods.

Preservation of fertility following doxorubicin administration in the rat.

Several hundred thousand men receive chemotherapy each year; many are sterilized by this treatment. Testicular circulatory isolation (TCI), a regional drug exclusion approach to circumvent chemotherapy-related infertility, lessens doxorubicin-induced testicular injury in the rat. We evaluated the effect of TCI on doxorubicin-induced infertility in this study. Thirty-two eight-week-old male Sprague-Dawley rats were used. Eight rats received TCI for 45 min. Eight received doxorubicin (i.v. bolus) plus sham surgery. Eight received i.v. doxorubicin given immediately after institution of TCI. Eight controls received sham surgery alone. Mating studies began 2 months later. Six of the 8 males receiving TCI alone were fertile. In the doxorubicin-treated, sham-operated group, 0 of 7 animals were fertile. In the doxorubicin-treated group which also received TCI, 2 of 7 males were fertile. In the sham-operated group, all 8 rats were fertile. This is the first evidence that regional drug exclusion technique can improve fertility in this model.

Long-term outcome following testicular ischemia in the rat.

Male germ cells are quite sensitive to interruption in blood flow. Eight weeks after subjection to 45 minutes of testicular ischemia, the spermatogenic epithelium of the rat remains significantly damaged, though other cell types are well preserved. The authors evaluated the testicular recovery of the rats at 8 and 72 weeks after the 45-minute period of warm ischemia. Twenty-eight rats were studied: 14 underwent 45 minutes of total left testicular ischemia; 14 received no treatment. Four rats from each group were necropsied at 8 weeks to document the ischemic injury. At 72 weeks, the 18 surviving rats were necropsied to evaluate the long-term outcome of the treatment. At 8 weeks, significant left testicular injury was documented. However, at 72 weeks there was no difference in testicular weight or sperm head count between the groups: in all 36 testicles, the repopulation index was 1.00, the epididymal index was 3+, the modified Johnsen index was 14, and the spermatic cord score was 7 (all are maximum obtainable scores). Neither contralateral orchiopathy nor injury to spermatic cord structures was observed. Our work shows that ischemia-induced testicular injury is fully reversible with time in this model.

Response to exogenous cholecystokinin of six human gastrointestinal cancers xenografted in nude mice.

Gastrointestinal hormones regulate growth of cancers as well as normal tissues. We investigated whether long-term cholecystokinin (CCK) administration might affect growth or metabolism of human tumors xenografted in nude mice. In each experiment, approximately 20 nude mice bearing subcutaneous xenografts of the particular cancer line being studied were used. Half received CCK and half received saline solution intraperitoneally twice daily for 14 days. Tumor volume and body weight were measured every 3 days. If the tumors produced marker substances, these were measured in nude mouse serum and also in the xenografts. Tumor growth was significantly retarded by CCK in two of the six cancers studied. In each case, DNA, RNA, and protein reflected tumor volumes. In one of these tumors (SLU 077), serum carcinoembryonic antigen (CEA) levels paralleled the tumor volumes. In another tumor (SLU 132), serum CEA levels and tumor immunolabeling for CEA and pancreatic oncofetal antigen increased in response to CCK administration, whereas tumor volumes did not. These findings suggest that exogenous highdose CCK altered the growth and metabolism in two of six human cancers studied.

Effect of chronically administered cholecystokinin on the nude mouse.

The nude mouse has been used to evaluate the effect of cholecystokinin (CCK) on xenografted tissues, but little is known about long-term actions of cholecystokinin on native organs in this animal. We investigated the impact of chronically administered synthetic cholecystokinin octapeptide on the nude mouse. Six groups of eight animals each received intraperitoneal injections twice daily for 14 days with diluent or a 4-log range of cholecystokinin. Overall health, behavior, and body weight were unaffected by this treatment. Among the seven organs examined at necropsy, pancreas alone showed a dose-related increase in weight. Pancreatic DNA content decreased with increasing dosages of CCK-8, while RNA content exhibited a biphasic response to CCK-8. The only histological abnormality occurred in the pancreas and was confined to the higher doses. These data indicate for the first time the action of CCK on the non-tumor-bearing nude mouse. Unlike other animal models, the nude mouse responds to cholecystokinin administration with pancreatic hypoplasia and hypertrophy, which is accompanied by pancreatitis at higher doses.

Tolerance of rat testis to graded periods of total circulatory isolation.

Infertility because of testicular damage is a distressing problem for survivors of cancer chemotherapy. We have previously described a cytoprotective effect of temporary testicular circulatory isolation (TCI) during doxorubicin administration in the rat. In this report we define the relationship between duration of TCI and testicular ischemic injury, using paired groups of animals with normothermic TCI and hypothermic TCI. Sixty rats were used and received TCI for varying lengths of time up to 180 minutes, with or without hypothermia. On day 56 +/- 4, all rats were killed and necropsied. Testicular injury was evaluated qualitatively by histology and quantitatively by testicular weight, sperm head count, repopulation index, and epididymal index. Warm TCI for up to 60 minutes induced modest damage to germ cells, while injury was greater with cold TCI. Leydig cells and vascular tissues were essentially unaffected by TCI, although Sertoli cell abnormalities were noted in rats receiving a long duration of TCI.

Effect of chronically administered pentagastrin on the nude mouse.

The nude mouse has been used to evaluate the effect of gastrin on xenografted tissues, but little is known about long-term actions of gastrin on native organs in this species. We investigated the impact of chronically administered synthetic pentagastrin on the nude mouse. Six groups of mice (eight animals each) received intraperitoneal injections twice daily for 14 days with saline or pentagastrin at 0.5, 5, 50, 500, or 5,000 micrograms/kg. Behavior, overall health, and body weight were unaffected by this treatment. Of the seven organs examined at necropsy, only the pancreas showed a weight gain in response to pentagastrin treatment, and this occurred only at the highest dose. Total DNA content of the pancreas decreased in a dose-related manner, indicating hypoplasia, whereas pancreatic RNA content increased, indicating hypertrophy. No effect on the stomach was observed. This work indicates that the nude mouse is less sensitive than other species to visceral growth regulation by pentagastrin, and that toxicity is low.

Effect of chronically-administered secretin on the nude mouse.

Secretin plays an important role in the growth regulation of certain cancers in vitro. The nude mouse is a suitable model for evaluation of the effects of this hormone on tumor xenografts in vivo, but little is known about long-term actions of secretin in this species. We investigated the impact of chronically administered synthetic porcine secretin in the nude mouse. Six groups of mice (eight animals each) received twice-daily intraperitoneal injections of saline or secretin at 0.5, 5, 50, 500, or 5,000 micrograms/kg for 14 days. Body weight and general health were unaffected by exogenous secretin, and no apparent behavioral effects were observed. Seven abdominal organs were examined at necropsy and all were histologically normal. The only organ that showed a weight change was the pancreas (13% decrease at the highest secretin dose). This was accompanied by decreases in DNA and RNA content, indicating pancreatic hypoplasia. Secretin administration caused changes in DNA and/or RNA content (but not protein content or weight) in liver, small bowel, cecum, and large bowel. No effect of secretin on stomach or kidney was observed. Our work demonstrates the safety of frequent injections of pharmacologic doses of secretin in this frail animal and suggests that the nude mouse is an appropriate model for the in vivo study of tumor growth regulation by secretin.

Characterization of Erysipelothrix rhusiopathiae isolated from an opossum (Didelphis virginiana) with septicemia.

Erysipelothrix rhusiopathiae was isolated from a wild-caught opossum (Didelphis virginiana). The opossum was quarantined in isolation and removed from contact with other animals. After a 2-mo period it was found dead in its cage, and presented for postmortem examination. Pure cultures of Erysipelothrix rhusiopathiae (SLU isolate) were recovered from heart blood, liver, spleen and lungs. To compare pathogenicity, an experimental infection was attempted in CF1 mice with a single dose of 1.5 x 10(7) organisms of both an ATCC standard strain and SLU isolate of E. rhusiopathiae. Similar signs, lesions and results of culture were found for both strains. The findings suggest that opossums can be infected with E. rhusiopathiae.

Regional doxorubicin delivery reduces testicular toxicity.

Many anti-cancer drugs cause infertility. Regional delivery of these agents is a potential method to avoid this problem. We investigated the protective effect of normothermic testicular circulatory arrest on gonadal toxicity during doxorubicin administration in the Sprague-Dawley rat. Four groups of eight rats each were used. Animals in group 1 received no treatment. Rats in group 2 were anesthetized and received a bolus of intravenous doxorubicin (6 mg/kg). In groups 3 and 4, normothermic circulatory isolation of the left testis was induced by cross-clamping of the spermatic cord and gubernaculum immediately before doxorubicin administration. This was maintained for 15 min after doxorubicin administration in group 3 and for 45 min in group 4. Cessation and return of testicular blood flow were confirmed by Doppler. On Day 56, all rats were killed and necropsied. Testicular toxicity was evaluated qualitatively by histology and quantitatively by measurement of testicular weight, sperm count, repopulation index, and epididymal index. The results indicated that 15 min of testicular circulatory isolation mitigated testicular toxicity to a small extent and that 45 min of circulatory isolation provided moderate protection against doxorubicin-induced testicular toxicity.

Exogenous cholecystokinin (CCK) reduces neonatal rat brain opioid receptor density and CCK levels.

Newborn rats were given saline or cholecystokinin8 (CCK8) (5 micrograms/kg, twice daily) i.p. for 3 weeks. On day 21, effects on brain development were assessed. CCK-like immunoreactivity was measured in 7 brain regions; a small (12-18%) but significant decrease in endogenous levels of this peptide was detected in cerebral cortex, medulla and pons of the CCK-treated rats. Morphometric measurements revealed a slight reduction in thickness of most cerebral cortical sections within the CCK-treated group. The area of a midsagittal section of the cerebellum was unchanged except for the Purkinje/granule cell layer, which was smaller in CCK-treated animals. Levels of mu-, delta- and kappa-opioid receptors were estimated by homologous displacement binding assays using selective radioligands. The CCK treatment resulted in a significant decrease in levels of mu- (11%) and delta- (13%)-sites in the cerebral cortex. Neither binding affinities nor kappa-receptor densities were altered. Other animals received the same treatment regimens for 21 days and were maintained for an additional 29 days without treatment; these rats had reductions only in cortical mu-sites (15%). Chronic intraventricular administration of CCK (0.1 microgram/h) to adult rats did not elicit a similar down-regulation of cortical mu or delta receptors, suggesting that the effects observed in neonates reflected developmental processes.

Testicular cytotoxicity of intravenous doxorubicin in rats.

Although the testicular cytotoxicity of many chemotherapeutic drugs has been evaluated in mice, their small size can pose technical problems. In this report, we describe doxorubicin-induced testicular toxicity in a larger animal model, the Sprague-Dawley rat. Fifty-three rats were used for this study. On day 0, rats in the treatment groups were anesthetized and given different single intravenous doses of doxorubicin (0.1 to 30 mg./kg.). On day 56 +/- 2, all surviving rats were killed and necropsied. Testicular toxicity was evaluated qualitatively by histology and quantitatively by testicular weight, sperm head count, repopulation index and epididymal index. The histologic effects of doxorubicin on the heart, liver and kidney were qualitatively evaluated. Progressive dose-dependent testicular atrophy and oligospermia occurred at low and intermediate dosages of doxorubicin (0.1 to 5 mg./kg.). Marked testicular atrophy, oligospermia and germinal aplasia were observed at high dosage of doxorubicin (10 mg./kg.). LD50 for animal mortality at day 56 +/- 2 for doxorubicin appears to be 10 mg./kg. These findings are similar to those reported in mice. The rat is a suitable model for the study of techniques to avoid drug-induced testicular damage.