Male Fischer Rats Research Articles

Protection by GDNF and Other Trophic Factors Against the Dopamine‐Depleting Effects of Neurotoxic Doses of Methamphetamine

Repeated methamphetamine (METH) administration to animals can result in long-lasting decreases in striatal dopamine (DA) content. It has previously been shown that glial cell line-derived neurotrophic factor (GDNF) can reduce the DA-depleting effects of neurotoxic doses of METH. However, there are several other trophic factors that are protective against dopaminergic toxins. Thus, the present experiments further investigated the protective effect of GDNF as well as the protective effects of several other trophic factors. Male Fischer-344 rats were given an intracerebral injection of trophic factor (2-10 microg) 1 day before METH (5 mg/kg, s.c., 4 injections at 2-h intervals). Seven days later DA levels in the striatum were measured using high-performance liquid chromatography (HPLC). Initial experiments indicated that only intrastriatal GDNF, and not intranigral GDNF, was protective. Thereafter, all other trophic factors were administered into the striatum. Members of the GDNF family (GDNF, neurturin, and artemin) all provided significant protection against the DA-depleting effects of METH, with GDNF providing the greatest protection. Brain-derived neurotrophic factor, neurotrophin-3, acidic fibroblast growth factor, basic fibroblast growth factor, ciliary neurotrophic factor, transforming growth factor-alpha (TGF-alpha), heregulin beta1 (HRG-beta1), and amphiregulin (AR) provided no significant protection at the doses examined. These results suggest that the GDNF family of trophic factors can provide significant protection against the DA-depleting effects of neurotoxic doses of METH.

Chemopreventive Effects of Dietary Flaxseedon Colon Tumor Development

: Fatty acid composition of dietary fat plays a vital role in colon tumor development in animal models. Fats containing ω-6 fatty acids (e.g., corn oil) enhanced and ω-3 fatty acids (e.g., flaxseed oil) reduced chemically induced colon tumor development in rats. Lignans have also been shown to prevent colon tumor development in experimental animals. The objective of this investigation is to study the effects of dietary flaxseed meal, a source of both ω-3 fatty acid and lignans, on colon tumor development and compare them with the effects of dietary corn meal. Male Fischer rats, two groups of 24 each, were assigned to the AIN-93M diet supplemented with either 15% corn meal or 15% flaxseed meal, respectively. Carcinogenesis was initiated with subcutaneous injections of azoxymethane (15mg/kg) once a week for 3 consecutive wk. After 35 wk of initiation, rats were anesthetized with ether. Blood was collected by cardiac puncture, and rats were sacrificed. The gastrointestinal tract was isolated. The site, size, and number of tumors were recorded. The fatty acid analysis of the collected serum and colon samples was performed. Expression of cyclooxygenase (COX)-1 and COX-2 was performed by Western blot method. Lignan levels in serum and colon samples were assayed. Colon tumor incidence, multiplicity, and size were found to be 82.6% and 29.4%; 1.3 and 0.3; and 44.4 and 5.3 mm2 in corn and flaxseed meal groups, respectively. Colon and serum samples of the corn meal group showed higher levels of ω-6 fatty acid levels whereas the flaxseed meal group exhibited higher levels of ω-3 fatty acids. COX-1 and COX-2 expression in the flaxseed group was significantly lower (P < 0.05) as compared to the corn group. Dietary flaxseed meal containing high levels of ω-3 fatty acids and lignans is effective in preventing colon tumor development when compared with dietary corn meal possibly by increasing ω-3 fatty acid levels and decreasing COX-1 and COX-2 levels.

Relationship between body iron stores and diquat toxicity in male Fischer-344 rats

The effects of body iron stores on diquat (DQ)-induced toxicity were examined in male Fischer-344 rats, which are sensitive to this herbicide. The rats (5 weeks old) were fed diets containing 40 (lower iron storage [LIS] group) or 320 ppm iron (higher iron storage [HIS] group) for 5 weeks. The concentrations of nonheme iron and ferritin in the liver and kidney were significantly higher in the HIS group than in the LIS group (P<0.0001), although there was no significant differences between the HIS and LIS groups in hematological parameters, including red blood cell count, hemoglobin concentration, and mean corpuscular volume. Three hours after administration of 0.1 mmol DQ/kg, serum alanine aminotransferase and urea nitrogen were significantly higher than in controls (saline injection) for both the LIS and HIS groups (P<0.01), and, after DQ injection, these parameters were significantly higher in the HIS group than in the LIS group (P<0.01). When the rats were injected with 0.075 or 0.1 mmol DQ/kg, the survival time was significantly shorter in the HIS group than in the LIS group (P<0.05). These findings suggest that higher body iron stores result in more severe DQ toxicity in Fischer-344 rats.

Protection of Tissue Mass/Body Mass by Combination of Lifelong Voluntary Exercise and Mild (8%) Caloric Restriction in Aging Fischer-344 Rats

Advancing age is associated with reduction of skeletal muscle mass and hypertrophy of the heart. Evidence indicates that moderate (30–40%) caloric restriction (CR) conserves maintenance of skeletal muscle and heart mass and myocyte number. Exercise training in aging populations may also provide protection against sarcopenia in skeletal muscle and remodeling in the heart. PURPOSE: To investigate whether lifelong voluntary exercise or mild (8%) caloric restriction provide protection of tissue mass/body mass in rat skeletal muscle, heart, brain, kidney, and liver. METHODS: We tested the hypothesis that 8% CR or lifelong wheel running + 8% CR would conserve tissue mass/body mass ratio by dividing male Fischer-344 rats at 11 weeks into four groups: fed ad libitum until 6 mo. old (6AL, n=12); fed ad libitum until 24 mo. old (24AL, n=11); 8% caloric restriction from 11 wk to 24 mo of age (24CR, n= 12); wheel running from 11 wk to 24 mo. with 8% caloric restriction (24ExCR, n=12). RESULTS: Body mass was significantly lower in the 24ExCR group than 6AL and 24CR. Skeletal muscle mass/body mass was better conserved with age (means similar to 6AL) in 24ExCR compared with 24AL and 24CR in the gastrocnemius, tibialis anterior, extensor digitorum longus, vastus lateralis, and plantaris, and compared with 24CR in the soleus. Heart mass was higher in 24AL than 6AL, but not 24CR and 24ExCR. In addition, brain mass/body mass was significantly higher in 24ExCR than 6AL, 24AL, and 24ExCR. Kidney mass was higher in all age groups compared with 6AL. CONCLUSIONS: The combination of lifelong wheel running and mild caloric restriction conserved skeletal muscle mass/body mass and brain mass, while preventing age-induced elevation of heart mass in the Fischer-344 rat. Supported by an NIH Grant R01AG17994-01

Inhaled Sevoflurane Produces Better Delayed Myocardial Protection at 48 Versus 24 Hours After Exposure

Ischemia preconditioning produces a delayed window of cardioprotection against subsequent ischemia and reperfusion injury. Contradictory results have been reported regarding the ability of inhaled anesthetics to produce similar effects. Our investigation was designed to test whether inhaled sevoflurane is capable of producing a delayed window of anesthetic preconditioning and to compare the differences at 24 and 48 h after exposure. Male Fischer-344 rats, 2-4 mo old, were exposed to sevoflurane (2.5% for 60 min). Twenty-four or 48 h after exposure, the hearts were isolated and perfused for 30 min (equilibration) followed by 25 min of ischemia and then 60 min of reperfusion. Control hearts received no treatment before ischemia. Left ventricular (LV) function, creatine kinase (CK), and infarct size (IS) were measured. Nuclear magnetic resonance was used to measure Na+(i), [Ca2+]i, and pH(i). There was improved LV function and significant reduction in IS and CK and in both the 24- and 48-h delayed groups compared with the controls. There was also a significant recovery of LV function and reduction in IS and CK in the 48-h group when compared with the 24-h group. There was significant adenosine triphosphate preservation in both the 24- and 48-h groups, as well as a significant reduction in acidosis, [Ca2+]I, and Na+(i) in response to ischemia in both the groups versus the control. Sevoflurane is capable of producing a delayed window of preconditioning, and it takes more than 24 h to produce maximal protective effects.

MECHANISM OF ANGIOTENSIN II VASOREACTIVITY IN RAT SOLEUS MUSCLE ARTERIOLES: EFFECTS OF AGING AND EXERCISE TRAINING

Aging appears to attenuate muscle blood flow in humans and rats, whereas exercise training ameliorates this aging effect. Because angiotensin II (Ang II) exerts a vasoconstrictor effect on the microcirculation, one possible mechanism for the aging or training associated alteration in muscle perfusion is through altered Ang II-mediated vasoconstriction. Therefore, we tested the hypothesis that, 1) aging increases Ang II vasoconstriction in rat soleus muscle arterioles, 2) the increased vasoconstriction in aged soleus arterioles is related to an endothelial nitric oxide synthase (NOS) signaling pathway, and 3) exercise training can attenuate the increased Ang II vasoconstriction in aged soleus arterioles. First-order arterioles were isolated from soleus muscles of young (4 months) and aged (24 months) male Fischer-344 rats. Ang II vasoconstrictor responses (3×10−11−3×10−5 M) were determined in arterioles with and without an intact endothelium. Ang II dose- responses were also examined in the presence of L-NAME (10−5 M), a NOS inhibitor. Ang II-mediated vasoconstriction was greater (P<0.05) in aged vs. young soleus muscle arterioles. Both the removal of the endothelium and NOS inhibition abolished this age-associated difference in Ang II vasoconstriction. Exercise training also eliminated the effects of aging on arteriolar vasoconstriction to Ang II. In conclusion, aging enhances Ang II vasoconstrictor responses through an endothelial NOS signaling mechanism, and exercise training can ameliorate the age-associated increase in Ang II vasoconstriction through an as yet unknown mechanism. Supported by NASA grants NAG2-1340 and NCC2-1166

Lifelong Exercise and Mild (8%) Caloric Restriction Conserve Cell Morphology of the Plantaris in the Aging Fischer‐344 Rat

Sarcopenia is a reduction of skeletal muscle mass as a result of reduced muscle fiber cross-sectional area and fiber number, resulting in impaired contractile function. Exercise training, either resistive or endurance ameliorates sarcopenia in aging muscle. Recent data indicate that moderate caloric restriction (30–40%) conserves skeletal muscle fiber cross-sectional area, and fiber number, particularly in muscle with a high % of fast-twitch fibers. We tested the hypothesis that 8% CR or lifelong wheel running + 8% CR would conserve fiber morphology in the plantaris muscle. We divided male Fischer-344 rats at 11 weeks into four groups: fed ad libitum until 6 mo. old (6AL, n=12); fed ad libitum until 24 mo. old (24AL, n=11); 8% caloric restriction from 11 wk to 24 mo of age (24CR, n=12); wheel running from 11 wk to 24 mo. with 8% caloric restriction (24ExCR, n=12). Aging increased the number of plantaris fibers/μm2 in 24AL; however, fiber number/μm2 was similar in both 24CR and 24ExCR to 6AL. Mean plantaris fiber cross-sectional area decreased markedly in 24 AL compared with 6 AL; however, fiber area in 24 CR and 24 ExCR significantly greater than 24AL. In addition, 24CR and 24ExCR protected against the large increase (+373%) in connective tissue found in 24 AL plantaris when compared with 6AL. These results support the hypothesis that lifelong voluntary exercise and mild caloric restriction preserve fast-twitch muscle morphology in the aging plantaris. Supported by an NIH Grant R01AG17994-01

NOS expression and activity in cerebral resistance arteries: Effects of age and exercise training

We have previously shown that endothelium-dependent vasodilation to acetylcholine (ACh) is preserved in cerebral resistance arteries from aged rats; however, the role of specific isozymes of nitric oxide synthase (NOS) in mediating dilation to ACh changes with age and exercise training. In the present study, we investigated the effects of age and exercise training on expression and activity of NOS isoforms. Young (4 months) and old (24 months) male Fischer-344 rats underwent exercise training or remained sedentary for 10 weeks. Posterior communicating arteries (PCoA) were isolated for assessment of ACh-stimulated NO production and protein levels of NOS isozymes. ACh-stimulated NO production was similar in PCoA from young and old rats. Treatment with aminoguanidine, an inhibitor of iNOS significantly reduced NO production in PCoA from young but not old rats; however, iNOS protein was not detectable in PCoA from rats in any of the groups. Although protein levels for endothelial NOS (eNOS) and neuronal NOS (nNOS) were not altered by age or exercise training; calcium-free production of NO was significantly lower in PCoA from old rats. These data indicate that NOS activity differs in PCoA from young and old rats; however, alterations of NO production that occur with age and training are likely related to changes in NOS regulatory mechanisms rather than changes in enzyme expression.

Individual differences in glucose homeostasis: Do our early life interactions with bacteria matter?

Exposure to endotoxin during the neonatal period in the rat has been shown to alter the development of the hypothalamic–pituitary–adrenal axis, inducing hyper-responsivity and increased glucocorticoid production in later-life. Glucocorticoids are known to have major metabolic effects, therefore, early life endotoxin exposure may have potentially serious consequences for metabolic homeostasis in the exposed animal. The aims of this study were therefore to assess the effect of neonatal bacterial endotoxin exposure on subsequent glucose homeostasis, insulin action and corticosterone production from puberty through to senescence. Male Fischer-344 rat pups were treated with bacterial endotoxin (0.05 mg/kg Salmonella enteritidis i.p.) or vehicle (sterile pyrogen free saline) on days 3 and 5 postnatally. Insulin and glucose levels were assessed before and during and intraperitoneal glucose tolerance test (IPGTT) together with body mass on postnatal days 40, 80, and 400. In addition, circulating levels of corticosterone were measured at 0, 30, and 90 min following a 30-min restraint challenge at these ages. Neonatal endotoxin challenge did not alter fasting plasma glucose or insulin, but impaired glucose tolerance at puberty ( p < .05), improved glucose tolerance in adulthood ( p < .05) and had no effect at senescence. During the IPGTT insulin was reduced at all ages ( p < .05) following neonatal endotoxin challenge, but insulin sensitivity was unaltered, except for an increase in adulthood ( p < .05), which is consistent with the observed improvement in glucose tolerance at this age. Neonatal endotoxin challenge reduced body mass during puberty and senescence ( p < .05) but did not alter basal or stressed plasma corticosterone levels at any of the three developmental time points examined. These findings suggest that variations in an individual’s early life bacterial environment may contribute to differences in glucose homeostasis, insulin action and disease susceptibility later in life.

Identification of Valid Housekeeping Genes and Antioxidant Enzyme Gene Expression Change in the Aging Rat Liver

Valid housekeeping genes (HKG) are a prerequisite for accurate gene quantification. We performed real-time reverse transcription-polymerase chain reaction to investigate the gene expression of five commonly used HKGs (beta-actin, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ubiquitin C [UBC], hypoxanthine phosphoribosyl-transferase [HPRT], and cyclophilin A [CYPa]) and antioxidant enzymes in the liver of young and old male Fischer rats. A wide variation in HKG expression existed during the aging process, and HPRT was identified as the most stable HKG in rat liver aging. When Cu/Zn-superoxide dismutase gene expression was normalized to HPRT, there was no detectable difference between young and old rats; however, a significant difference was seen when it was normalized to UBC. The variation of UBC caused the misinterpretation of Cu/Zn-superoxide dismutase expression. Catalase expression was significantly decreased, whereas glutathione peroxidase expression was not altered with age. We demonstrated that HPRT was an appropriate HKG, validation of HKGs was vital for accurate quantification, and decreased catalase expression might be involved in the decline of antioxidant defenses during rat liver aging.

One generation of n-3 polyunsaturated fatty acid deprivation increases depression and aggression test scores in rats

Male rat pups at weaning (21 days of age) were subjected to a diet deficient or adequate in n-3 polyunsaturated fatty acids (n-3 PUFAs) for 15 weeks. Performance on tests of locomotor activity, depression, and aggression was measured in that order during the ensuing 3 weeks, after which brain lipid composition was determined. In the n-3 PUFA-deprived rats, compared with n-3 PUFA-adequate rats, docosahexaenoic acid (22:6n-3) in brain phospholipid was reduced by 36% and docosapentaenoic acid (22:5n-6) was elevated by 90%, whereas brain phospholipid concentrations were unchanged. N-3 PUFA-deprived rats had a significantly increased (P = 0.03) score on the Porsolt forced-swim test for depression, and increased blocking time (P = 0.03) and blocking number (P = 0.04) scores (uncorrected for multiple comparisons) on the isolation-induced resident-intruder test for aggression. Large effect sizes (d > 0.8) were found on the depression score and on the blocking time score of the aggression test. Scores on the open-field test for locomotor activity did not differ significantly between groups, and had only small to medium effect sizes. This single-generational n-3 PUFA-deprived rat model, which demonstrated significant changes in brain lipid composition and in test scores for depression and aggression, may be useful for elucidating the contribution of disturbed brain PUFA metabolism to human depression, aggression, and bipolar disorder.

543. Monitoring Transgene Expression by Radio-Peptide Positron Emission Tomography

Background Progress towards Clinical application of gene therapy is hindered by the lack of possibility to image and trace transgene expression. Optical methods using fluorescent gene products or chemiluminesce inducing enzymes are not fully quantitative because of tissue absorption and fluorescence quenching. By using radioactive probes such as positron emitter labelled radiopeptides, a quantitative method for measuring transgene expression would be available. Aim The feasibility of electroporation mediated gene transfer of human somatostatin receptor 2 (hsstr2) in rats and subsequent PET/CT imaging was evaluated. Methods100 |[mu]|g of Pcik-hSSTR2 plasmid was injected in the forelimb muscle of anesthetised adult male Fischer rats (n = 12) followed by in vivo electroporation with 4, 20ms pulses of 200v/cm. Assessment was performed at 24 hrs after gene transfer and repeated at different time points till day 6. 2-5MBq of Ga-68-DOTATOC (0.5nmol DOTATOC peptide) was injected via the penile vein.30 minutes after injection, PET/CT Siemens/CTI,Erlangen/Knoxville;LSO Biograph16-pico3D High res,4.0mm intrinsic resolution)scans were performed (10min emission). After reconstruction, segmentation, attenuation and scatter correction of the PET/CT, activity accumulations were measured in the right and left fore limb of the rats. Results All the animals showed moderate to high accumulation of radioactivity in the right fore limb. No significant activity accumulation was found in the contralateral muscles. Correct CT anatomical co registration into the forelimb muscle was found for all animals. Relative expression was found to be increased by a factor of 3-8 as correlated to the contralateral activity. Induction of expression increased by a factor of 2-4 at 24hrs and 48hrs after gene transfer. No activity accumulation could be detected in the forelimbs 6 days after gene transfer. Conclusion Expression of hsstr2 as reporter gene may be detected and quantified by PET/CT.

Early life host–bacteria relations and development: Long-term individual differences in neuroimmune function following neonatal endotoxin challenge

Neonatal animals have a proportionately greater risk, relative to the adult animal, of developing a bacterial infection. Research has revealed that such infections can influence biological processes long after the actual infection has been resolved. Indeed, studies examining the long-term alterations induced by early-life infection, simulated using endotoxin, have indicated that some aspects of the systemic inflammatory response in the adult animal are susceptible to modification. Available evidence suggests that altered inflammatory activities observed in the neonatally endotoxin challenged adult may be the result of potentiated hypothalamic pituitary adrenal (HPA) activity, specifically increased corticosterone production. Few studies, however, have examined whether altered corticosterone production is actually associated with changes in systemic inflammation in the neonatally endotoxin challenged adult animal. The aim, therefore, of the current study was to simultaneously examine the relationship between altered inflammatory activities and corticosterone production in the neonatally endotoxin challenged adult rat. Our findings demonstrate no significant differences exist between adults neonatally treated with saline or endotoxin in terms of their production of corticosterone following endotoxin challenge. While not appearing to influence the production of corticosterone neonatal endotoxin challenge did result in a marked attenuation in the adult's febrile response following endotoxin challenge. Interestingly, circulating levels of IL-1β and TNF-α were found to be equivalent in neonatal treatment groups following endotoxin administration in adulthood, indicating that the reduction in fever was unlikely to be the result of altered pro-inflammatory cytokine production. Further, no differences were found between neonatal treatment groups in net food consumption, water consumption or weight loss following endotoxin challenge in adulthood. Collectively, these findings demonstrate that neonatal endotoxin challenge does not affect a blanket down regulation of inflammatory processes but rather appears to induce highly specific alterations in the adult male Fischer-344 rat.

A Toxicogenomics Approach to Identify New Plausible Epigenetic Mechanisms of Ochratoxin A Carcinogenicity in Rat

Ochratoxin A (OTA) is a mycotoxin occurring naturally in a wide range of food commodities. In animals, it has been shown to cause a variety of adverse effects, nephrocarcinogenicity being the most prominent. Because of its high toxic potency and the continuous exposure of the human population, OTA has raised public health concerns. There is significant debate on how to use the rat carcinogenicity data to assess the potential risk to humans. In this context, the question of the mechanism of action of OTA appears of key importance and was studied through the application of a toxicogenomics approach. Male Fischer rats were fed OTA for up to 2 years. Renal tumors were discovered during the last 6 months of the study. The total tumor incidence reached 25% at the end of the study. Gene expression profile was analyzed in groups of animals taken in intervals from 7 days to 12 months. Tissue-specific responses were observed in kidney versus liver. For selected genes, microarray data were confirmed at both mRNA and protein levels. In kidney, several genes known as markers of kidney injury and cell regeneration were significantly modulated by OTA. The expression of genes known to be involved in DNA synthesis and repair, or genes induced as a result of DNA damage, was only marginally modulated. Very little or no effect was found amongst genes associated with apoptosis. Alterations of gene expression indicating effects on calcium homeostasis and a disruption of pathways regulated by the transcription factors hepatocyte nuclear factor 4 alpha (HNF4alpha) and nuclear factor-erythroid 2-related factor 2 (Nrf2) were observed in the kidney but not in the liver. Previous data have suggested that a reduction in HNF4alpha may be associated with nephrocarcinogenicity. Many Nrf2-regulated genes are involved in chemical detoxication and antioxidant defense. The depletion of these genes is likely to impair the defense potential of the cells, resulting in chronic elevation of oxidative stress in the kidney. The inhibition of defense mechanism appears as a highly plausible new mechanism, which could contribute to OTA carcinogenicity.

Sympathetic neuroaxonal dystrophy in the aged rat pineal gland

Dysfunction of circadian melatonin production by the pineal gland in aged humans and rats is thought to reflect the functional loss of its sympathetic innervation. Our ultrastructural neuropathologic studies of the sympathetic innervation of the pineal gland of aged (24 months old) Fischer-344 and Sprague–Dawley rats showed loss of nerve terminals as well as the development of neuroaxonal dystrophy (NAD), an ultrastructurally distinctive distal axonopathy, far in excess of that in young control rats. Immunolocalization of tyrosine hydroxylase confirmed the age-related loss of normal noradrenergic innervation and development of NAD. NAD was more frequent in aged female rats compared to males and was particularly severe in aged female Sprague–Dawley rats compared to Fischer-344 rats. Pineal NGF content was significantly increased or unchanged in female and male aged Fischer-344 rats, respectively, compared to young controls. The rat pineal is a sensitive experimental model for the quantitative ultrastructural examination of age-related neuropathological changes in nerve terminals of postganglionic noradrenergic sympathetic axons, changes which may reflect similar changes in the diffusely distributed sympathetic innervation of other targeted endorgans.

Comparative activities of glutathione- S-transferase and dialdehyde reductase toward aflatoxin B1 in livers of experimental and farm animals

In order to gain a better understanding of the relative activities of glutathione- S-transferase (GST) and aldehyde reductase toward aflatoxin B1 (AFB1) in relation to the variation of species susceptibilities, we studied the in vitro cytosolic GST and reductase activities in liver tissues from male Fischer rats, ICR mice and golden hamsters, adult male rainbow trouts and female piglets. The GST activity was determined by incubating the liver cytosol with glutathione (GSH) and AFB1 in the presence of the hamster liver microsomes to metabolize AFB1 to AFB1-8, 9-epoxide. The reaction product, AFB1 and GSH conjugate (AFB1-GSH), was quantified with HPLC. The reductase activity was determined by incubating liver cytosol with AFB1-dialdehyde, followed by the quantification of the metabolic product, AFB1-dialcohol, with HPLC. All the animal species possessed the GST activities, and AFB1–GSH formed increasingly with the increase of the AFB1 concentration according to the model of first-order enzyme reaction kinetics. The V max and K m values of the GST activities in rodent species were higher and lower, respectively, than those in the trout and pig, being consistent with the relative susceptibilities to AFB1 of these animal species. However, no relationship was noted between the reductase activity and species susceptibility. Thus, the result of this study shows that GST toward AFB1, but not aldehyde reductase, is a determinant of the variation of species susceptibilities.

Persistent upregulation of μ‐opioid, but not adenosine, receptors in brains of long‐term withdrawn escalating dose “binge” cocaine‐treated rats

There is evidence showing that the opioid and adenosine systems play an important role in cocaine addiction; fewer studies have examined their roles in cocaine withdrawal. To determine whether cocaine and/or chronic withdrawal from cocaine alters the specific components of the opioid and adenosine systems, we carried out quantitative autoradiographic mapping of mu-opioid, A1 and A2A adenosine receptors in the brains of rats treated with an escalating dose "binge" cocaine administration paradigm and of rats chronically withdrawn from cocaine. Male Fischer rats were injected with saline or cocaine (15 x 3 mg/kg/day for 4 days, 20 x 3 mg/kg/day for 4 days, 25 x 3 mg/kg/day for 4 days and 30 x 3 mg/kg/day for 2 days) at 1-h intervals for 14 days. Similarly treated rats were withdrawn from that paradigm for 14 days. A significant increase in [(3)H]DAMGO binding to mu-receptors was detected in the frontal and cingulate cortex, as well as in the caudate putamen, of long-term withdrawn rats after an escalating dose "binge" cocaine administration paradigm and in chronic cocaine-treated rats. No significant cocaine-induced change was found in A1 or A2A receptor binding in any region analyzed. These results reconfirm that mu-opioid (MOP) receptors undergo upregulation in response to chronic escalating dose "binge" cocaine administration. This upregulation was shown for the first time to persist at least 14 days into withdrawal after escalating "binge" cocaine.

Rapid Uptake, Metabolism, and Elimination of Inhaled Sulfuryl Fluoride Fumigant by Rats

Sulfuryl fluoride (SO(2)F(2)) is a structural fumigant gas used to control drywood termites and wood-boring beetles. The pharmacokinetics and metabolism of inhaled SO(2)F(2) were evaluated in male Fischer-344 rats exposed to 30 or 300 ppm (35)S-labeled SO(2)F(2) for 4 h. Blood, urine and feces were collected during and after the exposures and analyzed for radioactivity, (35)S-labeled fluorosulfate and sulfate, and fluoride (urine and feces only). Selected tissues were collected 7 days post-exposure and analyzed for radioactivity. During and after unlabeled SO(2)F(2) exposures, blood, brain, and kidney were collected and analyzed for fluoride ion. SO(2)F(2) was rapidly absorbed, achieving maximum concentrations of radioactivity in both plasma and red blood cells (RBC) near the end of the 4-h exposure period. Radioactivity was rapidly excreted, mostly via the urine. Seven days post-exposure, small amounts of radioactivity were distributed among several tissues, with the highest concentration detected in respiratory tissues. Radioactivity associated with the RBC remained elevated 7 days post-exposure, and highly perfused tissues had higher levels of radioactivity than other non-respiratory tissues. Radioactivity cleared from plasma and RBC with initial half-lives of 2.5 h after 30 ppm and 1-2.5 h after 300 ppm exposures. The terminal half-life of radioactivity was 2.5-fold longer in RBC than plasma. Based on the radiochemical profiles, there was no evidence of parent (35)SO(2)F(2) in blood. Identification of fluorosulfate and sulfate in blood and urine suggests that SO(2)F(2) is hydrolyzed to fluorosulfate, with release of fluoride, followed by further hydrolysis to sulfate and release of the remaining fluoride.

Osteogenic Potential of Estriol-Treated Cultured Bone - In Vitro and In Vivo -

Osseous tissue can be formed by culturing marrow cells with compounds such as dexamethasone and that a bone matrix cultured in this manner possesses BMP activity. We have reported that artificial bones with a high level of osteogenic potential can be prepared by culturing artificial bone materials with cultured osseous tissue. Here, in an attempt to develop activated cultured bone constructs with even greater osteogenic potential, the effects of the female hormone estriol on osteogenesis were investigated. Bone marrow cells were collected from the femur shafts of 7-week-old male Fischer rats, and subjected to primary and secondary cultures. During secondary culture with or without dexamethasone (Dx), 10-5, 10-6, 10-7, 10-8 or 10-9 M of estriol was added to a standard culture medium containing ascorbic acid and β-glycerophosphosphate. The alkaline phosphatase(ALP) activity and Ca levels were measured and statistically analyzed. There was a significant difference in ALP activity between the control group and the estriol groups, and ALP activity was the highest in the 10-7 and 10-8 M groups, being about 2.5 times higher than in the control group. Similar results were seen for Ca levels. Furthermore, in vivo study showed10-7M-estriol-treated-cultured bone/ceramic construct has significant high osteogenic potential when it is grafted into in vivo. Estriol has been reported to increase bone mass, and the results of the present study suggest that the osteogenic potential of cultured bone constructs can be more than doubled by adjusting the concentration of estriol in bone marrow cell culture. Therefore, the use of estriol may be able to facilitate osteogenesis in bone regeneration therapy.

Decrease in the AP-2 DNA-Binding Activity and in the Protein Expression of AP-2 α and AP-2 β in Frontal Cortex of Rats Treated with Lithium for 6 Weeks

Lithium chloride (LiCl), when fed to rats for 6 weeks, has been reported to decrease brain mRNA, protein, and activity levels of arachidonic acid (AA)-selective cytosolic phospholipase A2 (cPLA2), without affecting secretory sPLA2 or Ca2+-independent iPLA2. We investigated whether transcription factors known to regulate cPLA2 gene expression are modulated by chronic lithium treatment. Male Fischer-344 rats were fed a LiCl-containing diet for 6 weeks to produce a therapeutically relevant brain lithium concentration. Control animals were fed a LiCl-free diet. Using a gelshift assay, we found that LiCl significantly decreased activating protein 2 (AP-2)-binding activity, and protein levels of the AP-2 alpha and AP-2 beta but not of the AP-2 gamma subunits in the frontal cortex. Activating protein 1 (AP-1)-binding activity was increased, whereas glucocorticoid response element, polyoma enhancer activator 3, and nuclear factor kappa B DNA-binding activities were not changed significantly. Since both cPLA2 and AP-2 can be activated by protein kinase C (PKC), we examined the frontal cortex protein levels of PKC alpha and PKC epsilon, as well as AA-dependent PKC activity. The protein levels of PKC alpha and PKC epsilon were decreased significantly, as was AA-dependent PKC activity, in the lithium-treated compared to control rats. Our results suggest that the reported decrease in brain gene expression of cPLA2 by chronic lithium may be mediated by reduced AP-2 transcriptional activity, and that decreased expression of PKC alpha and PKC epsilon contributes to lowering the AP-2 activity.