Adult C3H Mice Research Articles

Galectin-3 Decreases in Mice Exposed to Immobilization Stress

Using M3/38 monoclonal antibody we have analyzed effects of immobilization stress on the expression of galectin-3 in liver, spleen and peritoneal macrophages from adult RFM and C3H mice, as well as from aged C3H mice (total of 22 animals). In all analyzed tissues, immobilization stress caused a significant decrease in the expression of galectin-3, ranging from 14% to 47%. The decrease of galectin-3 was observed in both strains of mice, as well as in old animals. Moreover, the same range of decrease (approximately 50%) was observed when cells grown in vitro were exposed to subculturing, or heat-shock. Although the biological significance of this effect is not known, it is very interesting that a single episode of immobilization stress was sufficient to cause a significant decrease in galectin-3, implicating that this β-galactoside-binding lectin might be involved in the physiological response to psychological stress.

Repeated ethanol withdrawal experience selectively alters sensitivity to different chemoconvulsant drugs in mice.

Repeated ethanol withdrawal experience has been shown to result in exacerbated seizures associated with future withdrawal episodes. This sensitization of the withdrawal response has been postulated to represent a "kindling" phenomenon. The present study employed an established model of repeated ethanol withdrawals to examine the potential role of GABA(A), and NMDA and non-NMDA glutamate receptor systems in mediating enhanced seizure activity, as assessed by sensitivity to seizures induced by pentylenetetrazol (PTZ), NMDA, and kainic acid (KA) i.v. infusions, respectively. Adult C3H mice were chronically exposed to ethanol vapor in inhalation chambers. A multiple withdrawal (MW) group received four cycles of 16-h ethanol vapor exposure interrupted by 8-h periods of abstinence; a single withdrawal (SW) group was tested after a single 16-h bout of ethanol intoxication; and the third group was ethanol-naive, serving as controls (C). Results indicated that the MW group evidenced significantly lower PTZ and NMDA seizure thresholds compared to SW and C groups at 8 and 24 h post-withdrawal. In contrast, MW and SW groups exhibited reduced sensitivity (higher seizure threshold) to KA in comparison to controls, and this effect only emerged at 24 h post-withdrawal. Further, MW mice required significantly less additional PTZ or NMDA to induce more severe convulsions once initial signs of seizures were elicited. Conversely, latency and amount of KA required to transition from initial seizure signs to more severe end-stage convulsions was significantly greater for MW and SW groups compared to controls. Taken together, these results suggest that repeated ethanol withdrawal experience does not result in a global non-specific lowering of threshold to convulsive stimuli, but rather, selective changes in CNS mechanisms associated with neural excitability may underlie potentiated withdrawal responses. Thus, reduced GABA(A) receptor function and increased NMDA receptor activity may become exaggerated as a consequence of repeated withdrawal experience, while reduced sensitivity to KA induced seizures may represent a compensatory response to withdrawal-related CNS hyperexcitability.

Donor bone marrow potentiates the effect of tacrolimus on nonvascularized heart allograft survival: association with microchimerism and growth of donor dendritic cell progenitors from recipient bone marrow.

The influence of donor hematopoietic cell microchimerism on organ allograft survival has been studied largely in vascularized transplant models. Here, we examine the impact of donor bone marrow (BM) cells administered intravenously together with transient systemic tacrolimus therapy on microchimerism, the survival of nonvascularized cardiac allografts, and growth of donor antigen-presenting cells [dendritic cells (DCs)] from recipient BM. Adult male C3H (H2k) mice received heterotopic heart transplants from B10 (H2b) donors in the dorsal ear pinna. They were given no further treatment, or either a short course of tacrolimus (FK506; 2 mg/kg i.p. from day 0 to day 13), unmodified donor BM cells (50x10(6) i.v. on day 0) or both treatments. Grafts were examined daily for contractile activity. Anti-donor cytotoxic T lymphocyte responses were determined in recipients' spleens. Microchimerism (IAb+ cells) was demonstrated by immunocytochemical staining of spleens, and of cells expanded from recipient BM using cytokines and culture conditions that promote the growth of DCs. Tacrolimus alone significantly prolonged median heart graft survival time from 10 to 22 days (P<0.001). BM alone failed to prolong graft survival. By contrast, tacrolimus + donor BM resulted in a mean survival time of 42 days (P<0.01 compared with tacrolimus treatment alone). This marked increase in heart allograft survival was associated with reduced anti-donor cytotoxic T lymphocyte responses attributable to a nonspecific effect of tacrolimus. In addition, however, a link was observed between the beneficial effect of donor BM and comparatively large numbers of donor major histocompatibility complex class II (IAb+)-positive cells in recipients' spleens, and in cultures of granulocyte-macrophage colony-stimulating factor + interleukin-4-stimulated DCs from recipients' BM. No donor-derived cells were propagated from heart graft recipients given either tacrolimus or donor BM alone. This nonvascularized organ transplant model demonstrates the positive effect on allograft survival of donor BM given at the time of transplant to transiently immunosuppressed recipients. The findings also reveal links between hematopoietic cell chimerism, the presence of donor DC progenitors in recipient BM, and organ allograft survival.

Interleukin-1-Induced Sickness Behavior Depends on Behavioral Lateralization in Mice

Neveu, P. J., R.-M. Bluthé, S. Liè GE, S. Moya, B. Michaud and R. Dantzer. Interleukin-1-induced sickness behavior depends on behavioral lateralization in mice. Physiol Behav 63(4) 587–590, 1998. Inter-individual differences in brain-immune interactions have been demonstrated previously in mice using lateralization as a behavioral trait of population heterogeneity. Lipopolysaccharide (LPS), which is known to induce neurochemical, neuroendocrine, and immune responses depending on lateralization, is also able to induce sickness behavior, via the production of interleukin-1 (IL-1). The objective of this study was to determine whether lateralization can influence the behavioral response to LPS and to IL-1. To test this hypothesis, adult female C3H mice, previously selected for paw preference in a food reaching task, were injected intraperitoneally (i.p.) with 0.75 μg LPS or 0.75 μg recombinant IL-1β. Sickness induced by these molecules was measured by depressed social behavior, increased immobility, loss of body weight, and reduced food intake during the 6 h following injection. LPS-induced sickness was similar in right- and left-pawed mice. In contrast, IL-1-induced sickness behavior was dependent on behavioral lateralization. IL-1-induced depression of social investigation was more pronounced in right-pawed mice than in left-pawed animals. Likewise, IL-1-induced immobility was more important in right-pawed mice. There was a similar trend for food intake to be lower and loss of body weight to be higher in right-pawed mice than in left-pawed animals. These results demonstrate that right-pawed mice are more sensitive to IL-1-induced sickness than left-pawed animals. They extend our previous data showing a greater susceptibility to stress of right-pawed animals. The existence of inter-individual differences in the reactivity to stress or immune activation may be useful to study the mechanisms of the various strategies used by an individual in response to environmental aggressions.

Repeated ethanol withdrawal experience increases the severity and duration of subsequent withdrawal seizures in mice

Repeated ethanol withdrawal experience has been shown to result in an exacerbation of future withdrawal episodes. This sensitization of the withdrawal response has been hypothesized to represent a “kindling” phenomenon. The present study was designed to examine whether a systematic increase in the number of previous ethanol withdrawal experiences increases both the severity and duration of a subsequent withdrawal response. An established model of repeated ethanol intoxication/withdrawal was employed in which adult C3H mice were chronically exposed to ethanol vapor in inhalation chambers. In the first experiment, multiple withdrawal (MW) groups of mice received nine (MW×9), six (MW×6), or three (MW×3) cycles of 16-h ethanol vapor separated by 8-h periods of abstinence prior to testing; a single withdrawal (SW) group was tested following a single bout of 16-h ethanol exposure; and a control (C) group did not receive any ethanol treatment throughout the experiment. In a second experiment, a group of mice (MW1–9) were repeatedly tested over nine cycles of withdrawal. A third experiment was designed to assess the effects of repeated pyrazole administration on the potentiated withdrawal seizure response. Results indicated a positive relationship between the number of previously experienced ethanol withdrawals and the severity and duration of a subsequent withdrawal episode. Blood ethanol levels were similar for all ethanol-exposed groups prior to withdrawal assessment. Further, the intensity of withdrawal seizures (handling-induced convulsions) progressively increased over nine cycles of intoxication/withdrawal and repeated testing did not significantly influence the development of this potentiated response. In addition, repeated administration of pyrazole did not appear to influence this withdrawal sensitization phenomenon. Collectively, these results provide further support for the “kindling” hypothesis of ethanol withdrawal.

Exacerbation of Ethanol Withdrawal Seizures in Mice With a History of Multiple Withdrawal Experience

Repeated ethanol withdrawal experience has been shown to result in an exacerbation of future withdrawal episodes. This sensitization of the withdrawal response has been hypothesized to represent a “kindling” phenomenon. We previously demonstrated that mice exposed to ethanol vapor for a total of 48 h exhibited more severe withdrawal seizures if the exposure was divided into three 16 h intoxication/8 h abstinence cycles than if the 48 h of exposure occurred in a single bout. The present study was designed to further characterize this model of ethanol withdrawal “kindling” and determine whether such a “kindled” response may be evident when withdrawal testing is conducted after an additional bout of intoxication that is the same for all groups. Adult C3H mice were chronically exposed to ethanol vapor in inhalation chambers for 40 h prior to withdrawal testing. Prior to this 40 h intoxication period, one group (Multiple Withdrawal; MW) received three cycles of 16 h ethanol vapor separated by 8 h abstinence; a second group (Single Withdrawal; SW) did not receive any ethanol exposure prior to the 40 h test cycle; a third group (Continuous Exposure; CE) received the same total ethanol exposure as the MW group (48 hr), but without interruption; and a control group (C) did not receive any ethanol treatment throughout the experiment. Blood ethanol levels following the 40 h bout of ethanol intoxication were 100–140 mg/dl for all ethanol-exposed groups. The severity of handling-induced convulsions during withdrawal was significantly greater in the MW group compared to CE and SW groups. These results suggest that differences in the severity of ethanol withdrawal seizures due to differences in prior withdrawal experience can be demonstrated even when later ethanol exposure patterns are equated. As such, the results provide further support for the “kindling” hypothesis of ethanol withdrawal.

Repeated episodes of ethanol withdrawal potentiate the severity of subsequent withdrawal seizures: an animal model of alcohol withdrawal "kindling".

Prior experience with ethanol (EtOH) withdrawal may sensitize an individual to subsequent withdrawal episodes. It has been hypothesized that the progressive intensification of the EtOH withdrawal syndrome following repeated episodes of EtOH intoxication and withdrawal may represent the manifestations of a "kindling" mechanism. The purpose of this study was to develop an animal model of EtOH withdrawal that is sensitive to the effects of prior withdrawal experience. Adult male C3H mice were chronically exposed to EtOH vapor in inhalation chambers prior to withdrawal testing. A multiple withdrawal (MW) group received 3 cycles of 16 hr EtOH vapor separated by 8-hr periods of abstinence; a single withdrawal (SW) group received a single bout of EtOH exposure (16 hr); a third group (SW-CONT) experienced a single withdrawal episode after receiving the equivalent amount of EtOH intoxication as the MW group (16 x 3 = 48 hr), but in a continuous (uninterrupted) fashion; and a fourth group (C) served as controls, not receiving any EtOH exposure throughout the study. Severity of the withdrawal response was assessed by scoring handling-induced convulsions hourly for the first 10 hr and then at 24 hr postwithdrawal. The results indicated that the severity of EtOH withdrawal seizures was significantly greater in animals that had a prior history of withdrawal episodes (MW group) in comparison to a separate group of animals that were tested following a single withdrawal from the same 16-hr intoxication period (SW group).(ABSTRACT TRUNCATED AT 250 WORDS)

Murine Inter-Sex Difference in Methylcholanthrene Induced Tumor and its Correlation with NK Activity

Susceptibility to 3-methylcholanthrene (MC) induced tumor development between adult male and female C3H mice at 3–4 mo. old was examined. Animals that were injected (s.c.) with a tumorigenic dose (150 μg/mouse) of MC demonstrated a significant difference in the latent period of tumor development between both sexes. Female mice were more responsive and developed tumors at 3 wks after treatment, whereas, male mice were less responsive as indicated by the longer latent period (10 wks). In order to correlate these data with activity of natural killer (NK) cells, splenic NK activity was measured by 4 hr 51Cr-release assay. The results showed male mice having higher activity: 27 lytic units (LU) in comparison to female ones (3 LU). This was attributed to a decrease in the lytic effect of female NK cells (11%) compared to male mice (20%), since the binding capacity of effector cells to their targets was similar in both sexes (15%), as indicated by single cell assay. The possible role of an immunosuppressive factor (IF) in the sera of female mice was investigated. NK cells from young females demonstrated 33% suppression in activity post culture with sera from 3–4 mo. old female mice. In conclusion, this study demonstrates a sex-linked tumor development by MC treatment, and correlates this phenomenon with differences of NK activity between young male and female mice as well as in the age-dependent decline of reactivity. A possible immunosuppressive mechanism may be operative in explaining as to why MC is more effective in tumorigenesis in female mice as opposed to males.

PROLONGATION OF ADULT SKIN ALLOGRAFT SURVIVAL BY COTRANSPLANTATION OF NEONATAL SKIN IN ANTILYMPHOCYTE SERUM AND DONOR BONE MARROW CELL-TREATED MICE

Adult male B6AF1 (H-2KIb/kDb/d) mice were given skin allografts from adult male C3H (H-2k) mice, with and without contralateral cotransplants. The cotransplants were of either adult or neonatal (less than 24 hr) C3H skin. In recipient mice treated peritransplantation with antilymphocyte serum and posttransplantation with an i.v. injection of donor-strain bone marrow cells, the presence of a neonatal cotransplant resulted in significantly longer survival of the adult grafts. Median survival time (MST) for adult grafts, for the group that received a neonatal cotransplant was greater than 100 days, in comparison to MSTs of 59 days and 55 days for the groups that received only single adult grafts or the adult graft with an adult cotransplant. These results suggest an active immunomodulatory contribution of neonatal tissue, and we term this phenomenon the "cotransplant effect." This prolongation of survival of the adult grafts by the neonatal cotransplants was statistically significant in animals treated with ALS and BMC, but not in recipients that were treated with ALS only (MST = 34 days, in comparison with 29 days for groups that received either a single adult graft or an adult graft with an adult cotransplant). The graft-prolonging effects of an infusion of donor BMC thus appear to potentiate the expression of the cotransplant effect. An understanding of this effect may lead to improved methods of controlling the allograft response to adult tissues in the clinical setting.

Effects of 5-azacytidine and 5-aza-2-deoxycytidine on alphafetoprotein levels in mice

1. Production of alphafetoprotein in adult C3H mice was monitored by radial immunodiffusion both in controls, and in animals treated with carbon tetrachloride, 5-azacytidine, or 5-aza-2-deoxycytidine, either alone or in combination. 2. Carbon tetrachloride routinely induced alphafetoprotein synthesis in our experiments, but neither of the cytidine analogues showed any effects on the serum levels of this protein when administered alone. 3. Treatment of mice with either cytidine analogue prior to carbon tetrachloride injection markedly reduced the consequent production of alphafetoprotein, whereas if carbon tetrachloride injection was followed by a subsequent injection with either cytidine analogue, a markedly enhanced level of serum alphafetoprotein was detected. 4. It is suggested that carbon tetrachloride induces alphafetoprotein production in adult mice by inducing liver damage, followed by synthesis of the protein in the dividing and differentiating cells during recovery. We also propose that the cytidine analogues ablate this response by a cytotoxic effect on the liver cells when they are administered prior to the CCl 4, but enhance the alphafetoprotein levels when administered after the CCl 4 because they inhibit the methylation of cytidine residues in the recovering cell population in the liver and thus prevent early cessation of synthesis of the protein.

Structural changes in the ribosomal genes of immortalized and transformed mouse embryo C3H/10T1/2 cell lines

Comparative studies of DNA isolated from adult C3H mouse liver, immortalized mouse embryo cells (C3H/10T1/2 Cl 8) and the tumorigenic methylcholanthrene transformed C3H/10T1/2 Cl 16 cell line have been carried out in order to analyze possible structural changes in the ribosomal genes associated with the immortalization and tumorigenic transformation of mouse cells. Southern blot hybridization experiments revealed a mutation hotspot within repetitive sequences 13 kb upstream from the 18S rRNA genes in the non-transcribed spacer (NTS). Other DNA changes were localized near the initiation and termination regions of rRNA transcription. The differences found in the restriction maps of the 5'-region resided 5 to 6 kd upstream from the 18S 5'-end and the changes located in the 3'-end mapped approximately 5 kb downstream from the 28S 3'-end. Thus, oncogenic transformation of the C3H/10T1/2 Cl 8 cells by methylcholanthrene treatment was associated with stable genetic changes in the 18S rRNA gene. There was no evidence for rRNA gene amplification.

Early handling enhances mitogen responses of splenic cells in adult C3H mice

Experiments were done to investigate the effects of early postnatal handling in mice on humoral and cellular immune response at 21 and 60 days of age. In Experiment 1, C3H mice were either handled or left undisturbed from birth to weaning and their mitogen-induced, splenic B- and T-cell proliferative responses were measured at Day 21. No differences were found. In Experiment 2, mice were handled as in Experiment 1, except that at weaning the two groups were further subdivided into postweaning handled or nonhandled groups and their splenic B- and T-cell proliferative responses were measured at 60 days of age. These data showed significantly enhanced B- and T-cell proliferative responses due to preweaning handling and no effect of postweaning handling.

Effect of cyclosporin A on MHV3-induced chronic CNS infection in mice.

Intraperitoneal injection of MHV3 into adult C3H mice results either in early death due to hepatitis or in the development of a chronic disease with neurologic manifestations and virus persistence in surviving animals. The chronic neurologic disease which is described in another report in this volume, leads to meningitis, ependymitis, encephalitis and hydrocephalus followed by a chronic thrombotic vasculitis. In view of the potential therapeutic interest of Cyclosporin A (CsA), we decided to test its effects on the CNS lesions induced by MHV3.

Influence of maternal immunity on the outcome of murine coronavirus JHM infection in suckling mice.

Adult C3H mice are resistant to intraperitoneal infection with murine coronavirus JHM, whereas suckling offspring of non-immune females are susceptible. Resistance can be conferred on suckling C3H mice by postnatal transmission of maternal immunity, if transfer preceeds infection. Suckling mice succumb to infection even when they receive maternal antibodies within 1 day after infection. Prenatal transmission alone without subsequent postnatal transmission of maternal immunity is not sufficient to provide resistance. Persistence of virus without clinical consequences was observed when the supply of breast milk anti-JHMV antibodies was terminated 5 days before infection. Immune reactions restricted by histocompatibility antigens do not play a crucial role in bestowing resistance. As neutralizing anti-JHM serum antibody titers of adult mice only rise sharply 5 to 7 days after infection, these results indicate that infection of adults can be arrested by immunological means but that, in addition, the rate of virus dissemination must be limited by other non-immunological mechanisms.

A comparison of two commercial diets on growth of young adult C3H mice

The effect of two different commercial chow-type diets on body weight and adipose tissue growth of young adult male C3H mice was investigated. Mice, 6 weeks old, were fed Purina Rodent Chow (#5001) (5% fat by weight) for 10 weeks. At 16 weeks of age half the mice were switched to Purina Mouse Chow (#5015) (11% fat by weight) while the other half were maintained on the original diet. After an additional 10 weeks, the mice were killed. Body weight and epididymal and retroperitoneal fat pad weights were significantly increased in the mice fed the diet with the higher fat content. The increased epididymal pad weight in the 11% fat group was due primarily to an increase in fat cell number. Lipoprotein lipase activity in the retroperitoneal pad was significantly increased by the 11% fat diet. These results indicate that a moderate increase in dietary fat in young adult mice can lead to an increase in fat cell number.

Lethal Role of Interferon in Lymphocytic Choriomeningitis Virus-induced Encephalitis

After intracerebral inoculation of adult C3H mice, the 'docile' strain of lymphocytic choriomeningitis (LCM) virus multiplied to high titre in several visceral organs. Although the virus content of lung, liver, spleen and brain was high, these mice did not die but became long-term carriers of the virus. Injection of mice with the same dose of the 'aggressive' strain of LCM virus resulted in much lower virus titres in these organs; nevertheless, 100% of the mice died within 7 to 9 days. The results presented here show that mice infected with the 'aggressive' virus do not die if treated with anti-interferon globulin. Under these conditions the titres of 'aggressive' virus were as high in the different organs as in mice injected with the 'docile' virus. These results are consistent with the hypothesis that inhibition of LCM virus multiplication in various organs by interferon results in a lethal disease. The possible mechanisms underlying this seemingly paradoxical phenomenon are discussed.

Lymphocytic choriomeningitis virus killer T cells are lethal only in weakly disseminated murine infections.

Two types of lymphocytic choriomeningitis (LCM) viruses were studied which, upon intracerebral injection into adult C3H mice, provoked either (a) acute fatal central nervous system (CNS) disease or (b) life-long persistent infection. Both virus types, (a) aggressive and (b) docile, had been found to induce LCM-specific lymphocytes with comparable in vitro lytic activity (11). Because the requirement for T cells in the development of adult LCM disease has been extensively documented, we sought other reasons for the lack of acute disease in mice infected with docile virus. A striking correlation was found between the outcome of the infection and spread of virus to visceral organs. Adoptive transfer experiments showed that a 300-plaque forming unit inoculum of docile virus induced a population of T cells in donor mice fully capable of causing CNS disease in identically infected recipients. This disease causing ability was lost if the interaction was delayed beyond 3 d after infection of the recipients, but could be preserved by lowering the size of the viral inoculum in the recipients. Furthermore, without adoptive transfer, very low intracerebral doses of docile virus (which mimicked the normally slow spread of aggressive virus) were lethal. On the other hand, very high doses of aggressive virus, which mimicked the normally rapid spread of docile virus, did not induce fatal CNS disease. The results suggest that rapid dissemination of the LCM infection creates multiple target organs which divert the focused lethal T cell attack on the brain.

Development of two cloned epithelial cell lines from normal adult mouse and rat ventral prostates.

Two epithelial cell lines were established, one from adult C3H mouse and one from adult Fischer rat ventral prostate. These cell lines were obtained from explant cultures, using Ham's F12 medium supplemented with HEPES, insulin, testosterone, hydrocortisone, epidermal growth factor, and 7.5% fetal bovine serum. A low concentration of trypsin and EDTA in Ca++- and Mg++-free phosphate buffer was used for passaging the cells. The rat cell line was established following implantation of prostate tissue in nude mice. These cell lines stained positively for acid phosphatase and were dependent upon epidermal growth factor for growth. Morphological studies, including electron microscopy, revealed a highly characteristic epithelial morphology of both cell lines. These cell lines have hypotetraploid chromosome number and are capable of metabolizing benzo(a)pyrene. We propose the application of these cells as models for the study of prostate carcinogenesis.

Age-related changes of cAMP and prostaglandin content in the brain and lymphocytes of spontaneously tumor-producing C3H mice

Age caused concurrent changes in the activation of the adenylate-cyclase activity by dopamine in the diencephalon and the stimulation of the basal level of cAMP by isoproterenol in the lymphocytes were demonstrated in the spontaneously tumor producing C3H mice. These changes may be related to the occurrence of tumors in these animals. The above-mentioned changes in the cAMP system were not found in AB mice. By treating the adult C3H mice with substances which increase the cAMP level, it was possible to achieve a renewed stimulation of the cAMP system as seen in young mice. With this treatment the spontaneous tumor-induction rate was also reduced. In the adenocarcinomas of the C3H mice a decrease in the level of prostaglandin E and an increase in the level of prostaglandin F2 alpha was observed.

Toxicité de l'acide polyinosinique-polycytidylique pour les souris infectées par le virus de la chorioméningite lymphocytaire

While injection of polyinosinic-polycytidylic acid (poly-IC) does not protect adult C3H mice against fatal brain infection with lymphocytic choriomeningitis (LCM) virus, it can have a toxic effect and bring on death four to eight hours after administration. Toxicity depends on the stage of infection of mice and on the dose of poly-IC used, and can be reduced or eliminated by administration of water and electrolytes. Administration of poly-IC gives rise to an increased induction or ⪡ priming ⪢ of interferon in relation to the level of circulating interferon induced by LCM virus. Toxicity is not, however, related to viremia. Poly-IC injected into congenitally infected mice is not toxic but does elicit a priming effect. Several hypotheses are proposed to explain this toxicity of poly-IC in mice infected with LCM virus.