CV Mice Research Articles

Neuroprotective Potential of Vitamin B-12 Against Cadmium–Induced Neuroinflammation Mediated Memory Dysfunctions in Adult Albino Mice

Cadmium induces neurotoxicity in brain and results in increased enzymatic functions, accumulation of Amyloid beta plaque and Neuo Fibrillary Tangles (NFTs) causing neurodegenerative diseases. The purpose of the present work was to investigate the anti-inflammatory or anti-oxidative potential of vitaminB12 against Cd -induced memory impairment and mechanism of signaling pathway of vitamin-B12 protection in Cd-induced neuroinflammatory in mice model. Male albino mice of BALB/C trait of age 7 to 8 weeks and weighing about (30-32±3g) were used as model animal in the study. All mice were divided in three groups. Control Group (CG) treated with 0.9% saline (1mL/Kg), Cd Cl2 administered Group (CD) (1mg/kg) and Cd +Vitamin administered group (CV) B-12 (500µg/kg). CD group was treated with Cd Cl2 for three weeks on alternate days while CV group was treated with Vitamin B12 and toxin CdCl2 for the next two weeks intraperitonealy. Behavior tests like Morris Water Maze (MWM) and Y-Maze (YM) tests were conducted on the experimental mice to study the neuro protective potential of Vitamin-B12 against CdCl2 induced memory dysfunctions. After completing the above procedures, mice were sacrificed and the brains of each group were collected for protein quantification step and western blotting. The Immuno blots of Iba-1, NF-kB, TNF-α, IL-1β, BACE1 and Aβ proteins in the brain homogenates of CD and CV mice confirm the anti-neuroinflammatory potential of vitamin-B12 against Cadmium –induced Neuroinflammation. Escape latency of CV group mice receiving Methylcobalamine was less and showed better performance in MWM test. The results of Y-maze test also showed that the spontaneous percentage alternation of CD group mice was less than CV and CG group of mice. Immuno blot of p-Akt confirm the signaling pathways of vitamin-B12 protection in Cd-induced neuroinflammatory mediated memory-impairment in the model mice. Our findings suggest that Vitamin-B12 could be a potent candidate drug for treating cognitive dysfunctions due to its anti-inflammatory and anti-oxidative potential. Our study also shows that Vitamin-B12 activates p-Akt signaling pathway along with decreasing the NF-Κb, TNF-α and IL-1β to protect the brain of mice against CdCl2 neurotoxicity.

Environmental factors regulate Paneth cell phenotype and host susceptibility to intestinal inflammation in Irgm1-deficient mice.

ABSTRACTCrohn's disease (CD) represents a chronic inflammatory disorder of the intestinal tract. Several susceptibility genes have been linked to CD, though their precise role in the pathogenesis of this disorder remains unclear. Immunity-related GTPase M (IRGM) is an established risk allele in CD. We have shown previously that conventionally raised (CV) mice lacking the IRGM ortholog, Irgm1 exhibit abnormal Paneth cells (PCs) and increased susceptibility to intestinal injury. In the present study, we sought to utilize this model system to determine if environmental conditions impact these phenotypes, as is thought to be the case in human CD. To accomplish this, wild-type and Irgm1−/− mice were rederived into specific pathogen-free (SPF) and germ-free (GF) conditions. We next assessed how these differential housing environments influenced intestinal injury patterns, and epithelial cell morphology and function in wild-type and Irgm1−/− mice. Remarkably, in contrast to CV mice, SPF Irgm1−/− mice showed only a slight increase in susceptibility to dextran sodium sulfate-induced inflammation. SPF Irgm1−/− mice also displayed minimal abnormalities in PC number and morphology, and in antimicrobial peptide expression. Goblet cell numbers and epithelial proliferation were also unaffected by Irgm1 in SPF conditions. No microbial differences were observed between wild-type and Irgm1−/− mice, but gut bacterial communities differed profoundly between CV and SPF mice. Specifically, Helicobacter sequences were significantly increased in CV mice; however, inoculating SPF Irgm1−/− mice with Helicobacter hepaticus was not sufficient to transmit a pro-inflammatory phenotype. In summary, our findings suggest the impact of Irgm1-deficiency on susceptibility to intestinal inflammation and epithelial function is critically dependent on environmental influences. This work establishes the importance of Irgm1−/− mice as a model to elucidate host-environment interactions that regulate mucosal homeostasis and intestinal inflammatory responses. Defining such interactions will be essential for developing novel preventative and therapeutic strategies for human CD.

Some immunological studies on a new vaccine derived frombacteriophage lysate of Pasteurella multocida in mice

The study was undertaken to evaluate the humoral immune response in mice induced by a novel phage lysate vaccine against Hemorrhagic Septicemia developed from Pasteurella multocida grown under iron-restricted conditions and lysed by a bacteriophage. Two groups of mice were immunized with lysate vaccine (LV) and conventional killed HS vaccine (CV) and blood was collected at various days post – immunization (DPI). The serum protein concentration in lysate vaccinated mice (4.675±0.223) was significantly higher (p less than 0.05) than the conventional vaccinated mice (4.100±0.282) at 150 DPI. The serum globulin levels at 90 DPI and 180 DPI in LV mice (1.330±0.071 and 0.650±0.100) were significantly (p less than 0.01) higher than the CV mice (0.850±0.084 and 0.366±0.098). The serum IgG levels at 150 DPI and 180 DPI in LV (0.564±0.188 and 0.485±0121) mice were significantly higher (p less than 0.01) than the CV mice (0.178±0.039 and 0.121±0.026). Although our results in mice are promising, further studies involving bovines are needed.

Regulation of Hepatic Drug-Metabolizing Enzymes in Germ-Free Mice by Conventionalization and Probiotics.

Little is known regarding the effect of intestinal microbiota modifiers, such as probiotics and conventionalization with exogenous bacteria, on host hepatic drug metabolism. Therefore, the goal of this study was to determine the effect of these modifiers on the expression of various drug-metabolizing enzymes of the host liver. VSL3 is a probiotic that contains eight live strains of bacteria. Five groups of mice were used: 1) conventional mice (CV), 2) conventional mice treated with VSL3 in drinking water, 3) germ-free (GF) mice, 4) GF mice treated with VSL3, and 5) GF mice exposed to the conventional environment for 2 months. All mice were 3 months old at tissue collection. GF conditions markedly downregulated the cytochrome P450 (P450) 3a gene cluster, but upregulated the Cyp4a cluster, whereas conventionalization normalized their expression to conventional levels [reverse-transcription quantitative polymerase chain reaction (qPCR) and western blot]. Changes in the Cyp3a and 4a gene expression correlated with alterations in the pregnane X receptor and peroxisome proliferator-activated receptor α-DNA binding, respectively (chromatin immunoprecipitation-qPCR). VSL3 increased each bacterial component in the large intestinal content of the CV mice, and increased these bacteria even more in GF mice, likely due to less competition for growth in the GF environment. VSL3 given to conventional mice increased the mRNAs of Cyp4v3, alcohol dehydrogenase 1, and carboxyesterase 2a, but decreased the mRNAs of multiple phase II glutathione-S-transferases. VSL3 given to germ-free mice decreased the mRNAs of UDP-glucuronosyltransferases 1a9 and 2a3. In conclusion, conventionalization and VSL3 alter the expression of many drug-metabolizing enzyme s in the liver, suggesting the importance of considering "bacteria-drug" interactions for various adverse drug reactions in patients.

Up-regulation of intestinal type 1 taste receptor 3 and sodium glucose luminal transporter-1 expression and increased sucrose intake in mice lacking gut microbiota

The chemosensory components shared by both lingual and intestinal epithelium play a critical role in food consumption and the regulation of intestinal functions. In addition to nutrient signals, other luminal contents, including micro-organisms, are important in signalling across the gastrointestinal mucosa and initiating changes in digestive functions. A potential role of gut microbiota in influencing food intake, energy homeostasis and weight gain has been suggested. However, whether gut microbiota modulates the expression of nutrient-responsive receptors and transporters, leading to altered food consumption, is unknown. Thus, we examined the preference for nutritive (sucrose) and non-nutritive (saccharin) sweet solutions in germ-free (GF, C57BL/6J) mice compared with conventional (CV, C57BL/6J) control mice using a two-bottle preference test. Then, we quantified mRNA and protein expression of the sweet signalling protein type 1 taste receptor 3 (T1R3) and α-gustducin and Na glucose luminal transporter-1 (SGLT-1) of the intestinal epithelium of both CV and GF mice. Additionally, we measured gene expression of T1R2, T1R3 and α-gustducin in the lingual epithelium. We found that, while the preference for sucrose was similar between the groups, GF mice consumed more of the high concentration (8%) of sucrose solution than CV mice. There was no difference in either the intake of or the preference for saccharin. GF mice expressed significantly more T1R3 and SGLT-1 mRNA and protein in the intestinal epithelium compared with CV mice; however, lingual taste receptor mRNA expression was similar between the groups. We conclude that the absence of intestinal microbiota alters the expression of sweet taste receptors and GLUT in the proximal small intestine, which is associated with increased consumption of nutritive sweet solutions.

Commensal bacteria promote migration of mast cells into the intestine

Mast cells differentiate from hematopoietic stem cells in the bone marrow and migrate via the circulation to peripheral tissues, where they play a pivotal role in induction of both innate and adaptive immune responses. In this study, the effect of intestinal commensal bacteria on the migration of mast cells into the intestine was investigated. Histochemical analyses showed that germ-free (GF) mice had lower mast cell densities in the small intestine than normal mice. It was also shown that GF mice had lower mast cell proportion out of lamina propria leukocytes in the small intestine and higher mast cell percentages in the blood than normal mice by flow cytometry. These results indicate that migration of mast cells from the blood to the intestine is promoted by intestinal commensal bacteria. In addition, MyD88 −/− mice had lower densities of intestinal mast cells than CV mice, suggesting that the promotive effect of commensals is, at least in part, TLR-dependent. The ligands of CXC chemokine receptor 2 (CXCR2), which is critical for homing of mast cells to the intestine, were expressed higher in intestinal tissues and in intestinal epithelial cells (IECs) of normal mice than in those of GF or MyD88 −/− mice. Collectively, it is suggested that commensals promote migration of mast cells into the intestine through the induction of CXCR2 ligands from IECs in a TLR-dependent manner.

Intestinal commensal bacteria promote T cell hyporesponsiveness and down-regulate the serum antibody responses induced by dietary antigen

Colonization of the gut by commensal bacteria modulates the induction of oral tolerance and allergy. However, how these intestinal bacteria modulate antigen-specific T cell responses induced by oral antigens remains unclear. In order to investigate this, we used germ-free (GF) ovalbumin (OVA)-specific T cell receptor transgenic (OVA23-3) mice.Conventional (CV) or GF mice were administered an OVA-containing diet. Cytokine production by CD4+ cells from spleen (SP), mesenteric lymph nodes (MLN) and Peyer's patches (PP) was evaluated by ELISA, as was the peripheral antibody titer. T cell phenotype was assessed by flow cytometry.CD4+ cells from the SP and MLN of CV and GF mice fed an OVA diet for 3 weeks produced significantly less IL-2 than the corresponding cells from mice receiving a control diet, suggesting that oral tolerance could be induced at the T cell level in the systemic and intestinal immune systems of both bacterial condition of mice. However, we also observed that the T cell hyporesponsiveness induced by dietary antigen was delayed in the systemic immune tissues and was weaker in the intestinal immune tissues of the GF mice. Intestinal MLN and PP CD4+ T cells from these animals also produced lower levels of IL-10, had less activated/memory type CD45RBlow cells, and expressed lower levels of CTLA-4 but not Foxp3 compared to their CV counterparts. Furthermore, GF mice produced higher serum levels of OVA-specific antibodies than CV animals. CD40L expression by SP CD4+ cells from GF mice fed OVA was higher than that of CV mice. These results suggest that intestinal commensal bacteria promote T cell hyporesponsiveness and down-regulate serum antibody responses induced by dietary antigens through modulation of the intestinal and systemic T cell phenotype.

Anticarcinogenic Effects of Isoflavones May Be Mediated by Genistein in Mouse Mammary Tumor Virus-Induced Breast Cancer

Isoflavones are known to exert anticancer effects. These effects were examined using two isoflavones, biochanin A and daidzein, in a mouse mammary tumor virus (MMTV)-induced spontaneous breast cancer model. Emphasis was placed on isoflavone metabolism by the intestinal microflora and changes in estrogen levels. Germ-free (Gf) mice and their conventionalized (Cv) counterparts were assigned to three diet groups: (1) control diet, (2) biochanin A and (3) daidzein. In all groups, urine was collected from virgin female mice to analyze isoflavone metabolism by high performance liquid chromatography. These studies revealed changes of biochanin A into genistein, and of daidzein into equol, which were accelerated in the Cv animals. However, the Gf mice could not transform biochanin A into genistein, or daidzein into equol. Estrogen levels in the control and daidzein diet groups were lower in the Gf mice than in the Cv mice. The biochanin A group showed no differences in estrogen levels between the Cv and Gf animals. Four-week-old male and female animals were paired in the Gf and Cv groups. The female animals delivered and lactated repeatedly and were observed for the development of mammary cancer by palpation, twice weekly, until 15 months of age. The Cv mice showed a significantly lower incidence of breast cancer in the biochanin A diet group than in the control or daidzein groups (p < 0.05). These results suggest that the anticarcinogenic effects in this system might be produced not by daidzein or equol, but by biochanin A and/or genistein. In the Gf animals, the incidence of breast cancer was significantly higher in the biochanin A group than in the control group (p < 0.05), probably due to the increased level of estradiol in the former group. The biochanin A group tended to have a higher incidence of breast cancer than the daidzein group in the Gf group, although no significant differences were noted. Thus, no anticarcinogenic effect was produced by biochanin A alone in the Gf mice. In view of the results presented, genistein derived from biochanin A following metabolic processes in the intestinal microflora most likely acts as an inhibitor in breast carcinogenesis; biochanin A is most likely a precursor of genistein.

Spontaneous polyposis in the small intestine of germ-free and conventionalized BALB/c mice

The spontaneous polyposis in the small intestine of germfree (Gf) and conventionalized (Cv) BALB/c mice was studied. Gf mice were bred in our laboratory and maintained Gf in vinyl isolators. The first generation offspring of the Cv mice derived from the Gf mice was used as Cv animals. When they were 12 months old, the animals were killed under CO2 inhalation and autopsied carefully for the number and size of polyps with the aid of a dissecting microscope. The incidence of polyposis was higher in the Gf mice (68% in female and 89% in male) than in the Cv mice (37% in female and 51% in male). The number of polyps/mouse was also higher in the Gf mice (2.3 in female and 5.7 in male) than in the Cv mice (0.8 in female and 1.3 in male). All of the polyps were histopathologically adenomatous and developed only in the upper part (mainly duodenum) of the small intestine. The present study demonstrated that development of polyposis in the small intestine of BALB/c mice was suppressed by the presence of intestinal microflora.

Controlled genetic variation in a subchronic toxicity assay: susceptibility to induction of bladder hyperplasia in mice by 2-acetylaminofluorene.

Five different dose levels of 2-acetylaminofluorene (2-AAF) were fed to weanling mice of 4 different genotypes from three unrelated F1 hybrids for 13 wk to determine differences in susceptibility to induction of bladder hyperplasia. Differences in the prevalence of hyperplasia per se and in the average grade of hyperplasia were interpreted as indicating greater susceptibility. On this basis, males of all genotypes were more susceptible than females. Among the genotypes, (AEX YS)F1 mice (AY) were most susceptible, followed closely by yellow A vy/A(BALB/cXVY)F1 mice (CV). Agouti A/a(BALB/cXVY)F1 mice were less susceptible than their yellow siblings and similar to the (C57BL/6XC3H)F1 mice. Neither body weight gain nor any of the biochemical parameters measured appeared to be affected at any dose level of 2-AAF. However, quantitative differences in several biochemical characteristics were detected among the genotypes. Serum gamma-glutamyl transpeptidase activity was higher in the AY mice than in the other hybrids. Among the CV mice, the yellow animals had lower glutathione S-transferase (GST) activity than their agouti siblings. Hepatic GST activity was lower in CV mice than in either of the other hybrids. Hepatic cytochrome P-450 and bs activities were similar in all hybrids.

The weights of genital organs in germfree and conventional ICR mice administered with hCG, estradiol benzoate, progesterone and testosterone propionate (author's transl)

Female germfree (Gf) and conventional (Cv) ICR mice aged 21-25 days were administered with hCG (50IU/head/day for 2 days). Six hours after the last administration, uterine weights of Gf and Cv mice were 175.8 mg% and 238.7 mg% respectively, the difference between Gf and Cv animals was statistically significant (p less than 0.05). Ovariectomized Gf and Cv mice aged 80-150 days were successively administered with estradiol benzoate (0.02 mg/head/day for 5 days). Six hours after the last administration, uterine weights of Gf and Cv mice were 358.8 mg% and 462.7 mg% respectively, being statistically significant between Gf and Cv animals (p less than 0.01). After administration of estradiol benzoate (0.02 mg/head/day for 2 days), 1.0 mg/head/day of progesterone for 3 days were given ovariectomized Gf and Cv mice aged 80-150 days. Six hours after the last administration, uterus of Gf and Cv mice were weight 337.6 mg% and 423.5 mg% respectively, the difference was statistically significant (p less than 0.01). Castrated Gf and Cv mice aged 80-150 days were administered with 1.0 mg of testosterone propionate (head/day for 5 days). Six hours after the last administration, weights of seminal vesicle and coagulating gland of Gf and Cv mice were 376.0 mg% and 611.8 mg% respectively, the difference was statistically significant (p less than 0.01).

Suppression of the incidence of death with spontaneous tumours in DBA/2 mice after Corynebacterium parvum-mediated rejection of syngeneic tumours.

KILLED Corynebacterium parvum has been effective when administered into tumours or when included as a subcutaneous inoculum containing tumour cells (the subcutaneous site, unlike the intradermal site1, facilitates early tumour cell dissemination)2–4. After tumour rejection, the augmented host resistance prevented progressive growth of potentially lethal metastases present at the time of therapy5. In addition, animals rejecting tumours formed from primary grafts of tumour cells admixed with killed C. parvum (compared with those rejecting tumours after intratumour therapy with C. parvum) exhibited an augmented tumour-specific protection against inoculation of large challenge cell doses6. Whitmire and Huebner7 reported a significant decrease in incidence of 3-methyl-cholanthrene-induced malignancies in C57BL/Cum and BALB/c Cv mice after preimmunisation with formalin-killed wild-type virus and Rauscher leukaemia virus vaccines administered with Freund's complete adjuvant. We have now found a considerable decrease in the incidence of spontaneous tumours in the high-incidence strain of DBA/2 mice after rejection of tumours formed from subcutaneous transplants of syngeneic mammary adenocarcinoma cells admixed with killed C. parvum.

Plasmodium berghei and Eperythrozoon coccoides: Antibody and immunoglobulin synthesis in germfree and and conventional mice simultaneously infected

The immune response to Eperythrozoon coccoides and the malaria parasite Plasmodium berghei was evaluated in germfree (GF) and conventionally reared (CV) mice infected with both parasites. Following infection, the mice showed significant changes in the levels of the immunoglobulins IgM, 7Sγin1, 7Sγ2a, and 7Sγ2b, but no detectable changes in IgA. Increases in immunoglobulin levels were first observed in GF mice, but by the twelth day both GF and CV mice had comparable levels. 7Sγ2a globulin had a bimodal distribution in both groups of mice which probably was due to heterogeneity in the allotype of this immunoglobulin. IgM levels closely paralleled the antibody responses to P. berghei suggesting that most of the antibody to this parasite was IgM. Relatively low levels of antibody to both parasites, in comparison to the large immunoglobulin response, were detected in GF and CV mice. The possible causes for the low titers are discussed.

The effects of heterologous anti-thymocyte sera in mice. III. High susceptibility of germfree mice to the suppressive effects of IgG from rabbit anti-mouse thymocyte serum

A quantitative graft-vs.-host (GVH) assay was used to compare the reactivity of spleen cells from germfree (GF) and conventionally reared (CV) mice against allogeneic tissue before and after treatment with rabbit anti-mouse thymocyte serum (ATS) and its IgG fraction (AT-IgG). AT-IgG produced a far greater and longer lasting suppression of this reactivity in GF than in CV mice. Moreover, CV mice recovered from suppression twice as rapidly as did GF mice. In both groups, the rate of recovery was exponential. These results suggest that recovery from the suppressive effects of ATS or AT-IgG was the result of generation of new cells. Transfer of mice born and initially reared in a conventional animal room to germfree isolators, with subsequent maintenance on the same diet that the germfree mice received, did not change the reactivity of their spleen cells in the assay used nor their susceptibility to AT-IgG. Removal of GF mice to a conventional animal room resulted in a prompt reduction in susceptibility to AT-IgG. The possibility that this might be related to the elaboration of a plasma factor affecting lymphocyte stability was discussed. Spleen cells taken from GF mice at various times after such conventionalization showed a transient but marked hyperreactivity to tissues of the allogeneic recipients. The amount of reduction in reactivity of spleen cells from such mice treated with AT-IgG was always proportional to the activity of spleen cells from comparable untreated mice. It was suggested that the increased reactions evoked should be ascribed to an adjuvant effect rather than to specific immunologic sensitization. Blood lymphocyte counts correlated very poorly with the state of suppression, confirming previous observations. It was also shown that while AT-IgG had little or no effect on blood granulocyte counts in both GF and CV mice, marked reductions in circulating granulocytes followed administration of AT-IgG during the period of increased granulocytopoiesis that resulted from conventionalization. This demonstrated that AT-IgG can produce functional impairment of target cells other than lymphocytes.

THE EFFECTS OF HETEROLOGOUS ANTI-THYMOCYTE SERA IN MICE

A quantitative graft-vs.-host (GVH) assay was used to compare the reactivity of spleen cells from germfree (GF) and conventionally reared (CV) mice against allogeneic tissue before and after treatment with rabbit anti-mouse thymocyte serum (ATS) and its IgG fraction (AT-IgG). AT-IgG produced a far greater and longer lasting suppression of this reactivity in GF than in CV mice. Moreover, CV mice recovered from suppression twice as rapidly as did GF mice. In both groups, the rate of recovery was exponential. These results suggest that recovery from the suppressive effects of ATS or AT-IgG was the result of generation of new cells. Transfer of mice born and initially reared in a conventional animal room to germfree isolators, with subsequent maintenance on the same diet that the germfree mice received, did not change the reactivity of their spleen cells in the assay used nor their susceptibility to AT-IgG. Removal of GF mice to a conventional animal room resulted in a prompt reduction in susceptibility to AT-IgG. The possibility that this might be related to the elaboration of a plasma factor affecting lymphocyte stability was discussed. Spleen cells taken from GF mice at various times after such "conventionalization" showed a transient but marked hyperreactivity to tissues of the allogeneic recipients. The amount of reduction in reactivity of spleen cells from such mice treated with AT-IgG was always proportional to the activity of spleen cells from comparable untreated mice. It was suggested that the increased reactions evoked should be ascribed to an adjuvant effect rather than to specific immunologic sensitization. Blood lymphocyte counts correlated very poorly with the state of suppression, confirming previous observations. It was also shown that while AT-IgG had little or no effect on blood granulocyte counts in both GF and CV mice, marked reductions in circulating granulocytes followed administration of AT-IgG during the period of increased granulocytopoiesis that resulted from conventionalization. This demonstrated that AT-IgG can produce functional impairment of target cells other than lymphocytes.

Modifications of behaviour associated with inherited convulsions or whirling in three strains of peromyscus

Measures of behaviour of three strains of deermice ( Peromyscus maniculatus) which exhibit inherited types of convulsions or whirling are compared with similar measures made of a normal strain. Differences between affected and normal strains occur in the means of time taken to enter and to traverse several kinds of experimental apparatus, response time to bright light, maze learning, persistence in front of a glass barrier, and amount of spontaneous activity per unit of time. The mice of the EP strain exhibit sound-induced convulsions and also become deaf early in life. It is suggested that the inability of the adults of this strain to hear warning sounds may in part explain why they made better records than normal Peromyscus in a number of the measures. It is tentatively suggested also that a deficiency in the sense of smell leading to failure to detect warning odours may in part explain why the CV mice, which are osmogenic convulsives, made better records than normal animals for some of the measures. The tendency of the WZ mice to whirl evidently handicaps them in traversing the maze and other experimental pathways. This perhaps explains in part their poor records in some of these measures of behaviour. Their tendency to whirl may also in part explain why they respond more quickly than normal animals to the stimulus of bright light. Not all the differences between the affected and normal Peromyscus in these measures of behaviour, however, have an obvious relationship to the major defects of convulsions, deafness, defective smell, or whirling. Some of these modifications of behaviour of the abnormal strains may be due to pleiotropic effects of their major defective genes or to differences between the strains in their other genes.