Effect of cholecystokinin on human cholangiocarcinoma xenografted into nude mice.

Gastrointestinal polypeptide hormones regulate growth of various normal gastrointestinal tissues as well as certain visceral cancers. Since cholecystokinin (CCK) promotes growth of normal biliary tract, we sought to determine whether CCK affects the growth and metabolism of human cholangiocarcinoma line SLU 132. Twenty-six nude mice with s.c. xenografts of this cancer received either CCK octapeptide (50 micrograms/kg/dose) or 0.9% NaCl solution (saline) twice a day i.p. for 14 days. Tumor volume was calculated from Vernier caliper measurements. At sacrifice on Day 15, tumors were excised, weighed, and examined histologically. DNA, RNA, and protein were measured in the xenografted carcinomas. Because this cholangiocarcinoma produces carcinoembryonic antigen (CEA), we obtained serum at sacrifice for CEA radioimmunoassay and also tumor tissue for CEA immunolabeling with murine anti-CEA monoclonal antibody. Serum CEA levels were 90% higher in the CCK-treated group. Tumor tissue in the CCK-treated group also contained more CEA than did the controls. Mean tumor volume increased significantly in the saline group during the 14-day treatment period, whereas mean tumor volume did not increase significantly in the CCK group. Exogenous high-dose CCK thus appears to increase production and release of CEA from SLU-132; it also appears to retard growth of this tumor line in the nude mouse.

DNA damage in rat brain cells after in vivo exposure to 2450 MHz electromagnetic radiation and various methods of euthanasia.

The present study was done to confirm the reported observation that low-intensity acute exposure to 2450 MHz radiation causes DNA single-strand breaks (Lai and Singh, Bioelectromagnetics 16, 207-210, 1995). Male Sprague-Dawley rats weighing approximately 250 g were irradiated with 2450 MHz continuous-wave (CW) microwaves for 2 h at a specific absorption rate of 1.2 W/kg in a cylindrical waveguide system (Guy et al., Radio Sci. 14, 63-74, 1979). There was no associated rise in the core body temperature of the rats. After the irradiation or sham treatments, rats were euthanized by either CO2 asphyxia or decapitation by guillotine (eight pairs of animals per euthanasia group). After euthanasia the brains were removed and immediately immersed in cold Ames medium and the cells of the cerebral cortex and the hippocampus were dissociated separately and subjected to the alkaline comet assay. Irrespective of whether the rats were euthanized by CO2 asphyxia or decapitated by guillotine, no significant differences were observed between either the comet length or the normalized comet moment of cells from either the cerebral cortex or the hippocampus of sham-treated rats and those from the irradiated rats. However, the data for the rats asphyxiated with CO2 showed more intrinsic DNA damage and more experiment-to-experiment variation than did the data for rats euthanized by guillotine. Therefore, the guillotine method of euthanasia is the most appropriate in studies relating to DNA damage. Furthermore, we did not confirm the observation that DNA damage is produced in cells of the rat cerebral cortex or the hippocampus after a 2-h exposure to 2450 MHz CW microwaves or at 4 h after the exposure.

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.

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.

X-linked transmission of the shaker mutation in rats with hereditary Purkinje cell degeneration and ataxia.

This study reports on the mode of inheritance of the shaker mutation and the development of an inbred strain of the shaker rat mutation from Sprague Dawley outbred stock onto a Wistar Furth background. Neuroanatomical and behavioral expression of the affected phenotype, through seven generations of backcross and intercross breeding, has confirmed the mode of inheritance to be X-linked. Behaviorally, affected mutants present with a wide-based ataxic gait and whole body tremor. In affected mutants calbindin immunostaining for surviving cerebellar Purkinje cells revealed widespread degeneration in the anterior lobe and in limited areas of the posterior lobe. Fast Fourier transform analysis of the tremor revealed a frequency of 3-5 Hz. As predicted by X-linked inheritance, female descendants of an affected male are carriers for the genotype and the phenotype is expressed in one-half of her male offspring. There was spatially random and limited degeneration of Purkinje cells in carrier females, but they did not display overt clinical signs of ataxia and tremor. These data provide further support for using the shaker mutant rat as an animal model for studies of mechanisms underlying human heredodegenerative diseases.

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.

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.

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.

Measurement of DNA damage after acute exposure to pulsed-wave 2450 MHz microwaves in rat brain cells by two alkaline comet assay methods.

PURPOSE: To investigate the effect of 2450 MHz pulsed-wave microwaves on the induction of DNA damage in brain cells of exposed rats and to discover whether proteinase K is needed to detect DNA damage in the brain cells of rats exposed to 2450 MHz microwaves. MATERIALS AND METHODS: Sprague-Dawley rats were exposed to 2450 MHz pulsed-wave microwaves and sacrificed 4 h after a 2-h exposure. Rats irradiated whole-body with 1 Gy (137)Cs were included as positive controls. DNA damage was assayed by two variants of the alkaline comet assay on separate aliquots of the same cell preparation. RESULTS: Significant DNA damage was observed in the rat brain cells of rats exposed to gamma-rays using both versions of the alkaline comet assay independent of the presence or absence of proteinase K. However, neither version of the assay could detect any difference in comet length and/or normalized comet moment between sham- and 2450 MHz pulsed-wave microwave-exposed rats, regardless of the inclusion or omission of proteinase K in the comet assay. CONCLUSIONS: No DNA damage in brain cells was detected following exposure of rats to 2450 MHz microwaves pulsed-wave at a specific absorption rate of 1.2 W kg(-1) regardless of whether or not proteinase K was included in the assay. Thus, the results support the conclusion that low-level 2450 MHz pulsed-wave microwave exposures do not induce DNA damage detectable by the alkaline comet assay.

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.

Induced renal dysplasia in the young pouch opossum.

Using the North American opossum, Didelphis virginiana, we have developed a new model for studying the effects of early fetal urinary obstruction on subsequent renal development. We have successfully induced renal dysplasia in the marsupial that has a typically mammalian kidney.

The expression of transforming growth factor type beta in fetal and adult rabbit skin wounds.

Transforming growth factor, subtype beta (TGF-beta) exists in several isoforms and is known to have important roles in adult wound healing by promoting collagen and extracellular matrix component deposition. It is also believed that TGF-beta influences normal developmental processes during embryo-genesis. Immunolocalization of two isoforms, TGF-beta 1 and TGF-beta 2, in healing fetal and adult rabbit skin wounds shows distinctly different forms of expression of these molecules. TGF-beta 1 and TGF-beta 2 are both expressed within the developing fetal dermis, but no differential upregulation in the area of the healing wound is noted. In contrast, the expression of TGF-beta 1 and TGF-beta 2 is increased in adult wounds by day 7 after wounding, within macrophages that are abundant by this time. High levels of TGF-beta 1 and TGF-beta 2 within adult wounds might indicate that the relative paucity and differential distribution of these factors in fetal wounds are important in the production of scar in adults and the absence of scar in the fetus. Further, these patterns of expression suggest fundamental differences between fetal and adult tissues in accomplishing wound repair.

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.

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.

Self trauma and Staphylococcus aureus in ulcerative dermatitis of rats.

Eight cases of ulcerative dermatitis occurred in 7 groups of adult male Sprague-Dawley rats. Coagulase-positive Staphylococcus aureus was isolated from skin lesions of all affected rats; in some affected rats, coagulase-positive S aureus was also isolated from the feces and oropharynx. Amputation of toes of chronically affected rats resulted in remission of lesions. Self trauma (scratching with the hind feet) and fecal contamination of wounds with coagulase-positive S aureus were postulated as factors contributing to persistence of lesions in affected rats.