Salt preference of congenic strains derived from the spontaneously hypertensive rat

Previous studies using reciprocal crosses between the spontaneously hypertensive rat (SHR) and the normotensive Wistar–Kyoto (WKY) strain suggested a role for the Y chromosome in the SHR's exaggerated preference for saline solutions. We have reexamined the role of the Y chromosome in the salt preference of the SHR using a consomic strain derived from SHR and Brown Norway (BN-Lx) progenitors. We also studied congenic lines in which regions of BN-Lx chromosomes 8 and 20 had been introgressed into the SHR genome. Animals were given a choice of water and 0.9% saline to drink over a period of 7 days and their total fluid intake (TFI; water plus saline) and saline preference (proportion of the TFI taken as saline) calculated. SHR bearing the BN-Lx Y chromosome had a significantly reduced saline preference when compared to progenitor SHR. Evidence was also found for the existence of a region on chromosome 8, which influences fluid intake in the SHR. The causative genes involved in these effects however remain to be determined.     

Prepulse startle deficit in the Brown Norway rat: a potential genetic model

Prepulse inhibition (PPI), an operational measure of sensorimotor gating, is deficient in schizophrenia patients. PPI was compared among 4 strains of rats: Sprague-Dawley, Spontaneously Hypertensive, Wistar Kyoto (WKY), and Brown Norway (BN). PPI was dramatically lower in BN versus the other strains, especially WKY, for both acoustic and airpuff startle stimuli, whereas startle amplitude was similar between BN and WKY. Female BN also had lower PPI than did female WKY. Response to increasing prepulse intensities showed a right shift in the BN relative to the WKY. Visual prepulses also showed deficiencies in BN versus WKY. The absence of background noise did not negate strain differences. Auditory brainstem response to clicks and tone pips revealed no differences in auditory threshold between the 2 strains. These results are the first to demonstrate that BN have impaired sensorimotor gating compared with WKY, without impaired acoustic acuity.     

Functional genomics in rat models of hypertension: using differential expression and congenic strains to identify and evaluate candidate genes

Hypertension is a leading contributor to cardiovascular diseases such as heart attack and stroke. Genetic and environmental factors contribute to the development of hypertension. Animal models have been developed to study the genetic contributions to blood pressure (BP) regulation and to identify chromosomal regions harboring candidate genes causative of differences in BP regulation (i.e., BP quantitative trait loci [QTL]). Advances in both mammalian genome projects and global gene expression analysis present opportunities to study functional genomics in these animal models. In this article, novel approaches for designing experiments and interpreting global gene expression data using the Dahl salt-sensitive hypertension rat model are presented. We describe two-step screening protocols that can be used to identify BP QTL candidate genes. Genetically determined expression differences are identified in the target organs of inbred strains of contrasting phenotype in the first screen. Expression patterns in a panel of congenic strains or expression differences stemming from gene x environment interactions are examined in the second screen. Chromosomal locations of these genes can then be examined to determine whether they map to BP QTL-containing regions. Another approach is to study the expression of genes identified from public databases to be located within BP QTL-containing congenic regions. Several candidate genes have been identified using these strategies.     

Mechanisms of the anorexia of aging in the Brown Norway rat

Aging is associated with a loss of the ability to maintain homeostasis in response to physiologic and environmental disturbances. Age-related dysregulation of food intake and energy balance appears to be the result of impaired responsiveness of hypothalamic integrative circuitry to metabolic cues, which can lead to lack of appropriate food intake (the anorexia of aging) and thus to inappropriate weight loss in response to acute or chronic illness or other stressors. Using the Brown Norway (BN) male rat model, we have shown that old animals fail to appropriately increase food intake after the metabolic challenge of a 72 h fast, resulting in the failure to re-gain lost body weight upon refeeding. Leptin levels increase with adiposity and age, and remain elevated above levels of young animals even after a 72 h fast, suggesting that hyperleptinemia may be influencing the energy balance dysregulation. It is unclear whether this age-related response is due to a failure of the network of hypothalamic neurons to appropriately integrate hormonal and neural inputs, or due to a failure of the neurons to produce the appropriate neuropeptides. We hypothesize that sequential, age-related alterations in the expression patterns of neuropeptides that maintain melanocortinergic tone, and in the hormone mediators that inform the system of the state of energy balance, result in a diminished ability to maintain energy homeostasis with increasing age. We have undertaken a number of interventional approaches to test this hypothesis, including manipulations of the hormones ghrelin, insulin and testosterone, and direct application of neuropeptides to the central nervous system in these animals.     

Aggressive behavior in hypertensive and normotensive rat strains

Shock induced fighting, mouse killing, and jump thresholds were measured in gentically hypertensive or hypertensive-prone rat strains and in normotensive and surgically induced (renal) hypertensive rats. Differences in levels of shock induced agression and jump thresholds were observed, but were not a direct effect of hypertension and appeared to be traits genetically separate from the susceptibility to hypertension.     

Effects of cyclophosphamide on autoantibody synthesis in the Brown Norway rat.

The effects of cyclophosphamide on autoantibody synthesis were studied in an experimental model of glomerulonephritis due to autoantibodies to the glomerular basement membrane (GBM). Brown Norway rats develop anti-GBM antibodies, as part of a polyclonal response, when repeatedly injected with mercuric chloride (HgCl2). Anti-GBM antibody levels peak between days 11 and 14 and thereafter rapidly fall; convalescent animals show a time-dependent resistance to rechallenge with HgCl2 which remains significant for up to 3 months. The administration of cyclophosphamide, as a single intramuscular injection at day 0, has three distinct dose-dependent effects on anti-GBM antibody production. Firstly, lower doses (2.5 mg/kg) increase antibody levels at the time of peak response; secondly, higher doses (greater than or equal to 20 mg/kg) prevent antibody synthesis following HgCl2; and thirdly, the higher doses also reduce the response to rechallenge with HgCl2 3-4 months later. These effects of cyclophosphamide also apply to the polyclonal response to HgCl2, as judged by measurement of total IgG concentrations. Further investigation of the mechanisms of action of cyclophosphamide in this model should provide information relevant to the treatment of human autoimmune disease.     

Examination of oral sensitization with ovalbumin in Brown Norway rats and three strains of mice

We studied the conditions needed to sensitize animals to the oral feeding of food allergens, without induction of tolerance, in order to investigate the allergenicity of orally ingested food proteins. Brown Norway (BN) rats were sensitized by daily OVA (ovalbumin)-gavage or by drinking OVA containing water ad libitum and the ASA (active systemic anaphylaxis) response, as the immediate hypersensitivity response to antigen stimulation after oral sensitization, was examined. The oral administration of OVA by gavage produced a higher OVA-specific IgE response and an increase in serum histamine after antigen challenge, as compared to those produced by drinking water. Next, we examined the effect of murine age, the oral feeding technique and the oral feeding dose on sensitization using BALB/c, B10A and ASK mice. Twenty-week-old mice showed the strongest OVA-specific IgE and IgG1 responses and ASA-associated serum histamine contents increased with gavage in the three different age groups of BALB/c mice. Administering 0.1 mg of OVA by gavage daily for 9 weeks appeared to induce a higher response than administering 1 mg of OVA, in terms of OVA-specific IgE and IgG1 antibody responses and ASA responses. Among the three strains of mice, B10A mice exhibited the highest response in terms of OVA-specific IgE and IgG1 antibody and ASA responses. These findings suggested BN rats and B10A mice were suitable models for oral sensitization with antigen protein and that oral sensitization in mice requires low dose, intermittent antigen intakes.     

Mouse inter-subspecific consomic strains for genetic dissection of quantitative complex traits

Consomic strains, also known as chromosome substitution strains, are powerful tools for assigning polygenes that control quantitative complex traits to specific chromosomes. Here, we report generation of a full set of mouse consomic strains, in which each chromosome of the common laboratory strain C57BL/6J (B6) is replaced by its counterpart from the inbred strain MSM/Ms, which is derived from Japanese wild mouse, Mus musculus molossinus. The genome sequence of MSM/Ms is divergent from that of B6, whose genome is predominantly derived from Western European wild mouse, Mus musculus domesticus. MSM/Ms exhibits a number of quantitative complex traits markedly different from those of B6. We systematically determined phenotypes of these inter-subspecific consomic strains, focusing on complex traits related to reproduction, growth, and energy metabolism. We successfully detected more than 200 statistically significant QTLs affecting 26 traits. Furthermore, phenotyping of the consomic strains revealed that the measured values for quantitative complex traits often far exceed the range between B6 host and MSM/Ms donor strains; this may result from segregation of alleles or nonadditive interactions among multiple genes derived from the two mouse subspecies (that is, epistasis). Taken together, the results suggest that the inter-subspecific consomic strains will be very useful for identification of latent genetic components underlying quantitative complex traits.     

Circulating T-cell populations during mercuric chloride-induced nephritis in the Brown Norway rat.

Fluorescence-activated cell sorter analysis was used to study the peripheral lymphocyte populations during mercuric chloride (HgCl2)-induced autoimmune nephritis in the Brown Norway (BN) rat. Sequential studies showed a transient loss of T cells from peripheral blood attributable to decreases in the percentage of T-helper cells. In addition, there was a decrease in the percentage of T-cytotoxic/suppressor cells prior to the appearance of circulating anti-GBM antibodies, followed by elevated levels of T-suppressor cells during down-regulation of the response. This method may allow closer inspection of the events linking changes in T-cell populations and induction and termination of an autoimmune response.     

Patterns of expression of class I antigens in the tissues of congenic strains of rat

A comparative study of expression of class I major histocompatibility (MHC) antigens among lung, spleen, kidney, heart, liver, and brain tissues of the rat was performed. Monoclonal antibodies (MAbs) against RT1Aa determinants were conjugated to 125I and applied to frozen sections. Resulting autoradiograms showed antigen reactivity in lymphoid tissue, bronchial and alveolar epithelium, and endothelium of the lung. The lymphoid tissue of the lung and spleen demonstrated the antigen after shorter autoradiographic exposures than was required for the epithelial components of these organs. The kidneys were heavily labeled over the glomeruli, but less intensely over the tubular epithelium. RT1A antigen content of capillary endothelium of the heart was demonstrable before that of the muscle bundles. In the liver, autoradiographic sections revealed high determinant density in sinusoidal regions. Brain sections show reproducibly low levels of labeling, with the exception of vascular structures. All of these tissues from PVG-RT1c and PVG-RT1u animals show only background labeling.     

Strain restricted typing sera (SRTS) for use in monitoring the genetic integrity of congenic strains

A relatively simple procedure for serologic monitoring of the genetic integrity of congenic strains housed in a conventional colony is presented. Using a combination of 3 or 4 F₁ immunizing cells, sera can be produced in each strain housed in the colony which will react in a complement-mediated cytotoxicity assay with peripheral lymphocytes from most if not all other strains in the colony. Routine screening of breeding stock with these strain restricted typing sera (SRTS) permits the sensitive detection of genetic contamination between the stocks maintained. These sera detect H-2, minor histocompatibility differences, and other cell surface differentiation antigens, and can also be used to identify the nature of a contaminant when isolated. In addition, when used within appropriate strain combinations, the sera can be useful in detecting antigenic determinants otherwise difficult to identify.     

Genetic analysis of complex cardiovascular traits in the spontaneously hypertensive rat

Identification of the genetic determinants of common diseases is a major challenge for current biomedical research. Combining linkage analyses of essentially monogenic cis-regulated expression phenotypes with oligogenic intermediate physiological phenotypes represents a promising approach for identification of quantitative trait loci at the molecular level. In the present review, a genetic analysis of cardiovascular phenotypes studied at several levels of complexity in rat recombinant inbred strains is described.     

Early vasculitis in the mercuric chloride induced Brown Norway rat model is neutrophil independent

In the Brown Norway rat, mercuric chloride (HgCl2) induces an autoimmune syndrome characterized by necrotizing vasculitis, predominantly affecting the caecum, and a polyclonal B-cell response. The time course of vasculitis is biphasic, with an alphabeta T-cell independent phase occurring within 24 h, and a T-cell and neutrophil dependent phase, maximal at two weeks. The pathogenesis of the early phase of vasculitis is unclear, and this study aims to examine the role of neutrophils. Rat neutrophils were depleted using cyclophosphamide. RP3, an antirat neutrophil monoclonal antibody, inhibited neutrophil leucocytosis but did not deplete neutrophils. Vasculitis was induced by subcutaneous HgCl2 injection. Serial measurements of peripheral blood leucocyte count were made. Rats were killed after 24 or 72 h. The macroscopic appearance of the caecum was scored by an experienced observer, and samples taken for histological examination. Caecums were excised and myeloperoxidase, a marker enzyme for neutrophil infiltration, assayed. Cyclophosphamide induced marked neutropaenia whereas RP3 inhibited the neutrophilia observed after HgCl2 injection. Vasculitis was present in both treated and control animals, with no significant differences in macroscopic or microscopic scores between the groups. Tissue myeloperoxidase activity was low in all animals and did not differ significantly between groups. The data do not support a role for neutrophils in the initial pathogenesis of vasculitis in this model.     

Nitric oxide contributes to resistance of the Brown Norway rat to experimental autoimmune encephalomyelitis

The Brown Norway (BN) rat is reported to be resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and a number of mechanisms have been suggested to explain this resistance. In work reported here we provide evidence that such resistance in the BN rat can be accounted for, at least in part, by their ability to produce higher levels of nitric oxide (NO) than susceptible strains of rats. Spleen cells from the BN rat make significantly more NO following in vitro stimulation than do cells from the Lewis or PVG rat and following in vivo immunization using complete Freund's adjuvant (CFA) the BN rat makes substantially more NO than either susceptible strain. If carbonyl iron is used as adjuvant in vivo there is no increase in NO levels in the BN rat and they are rendered highly susceptible to EAE. Immunizing with CFA simultaneously with neuroantigen and carbonyl iron drives up NO levels and the resistance is restored. EAE produced using carbonyl iron is characterized by extensive macrophage/microglia presence in the central nervous system lesions of the BN rat yet the cytokine profile in the lymph nodes does not differ from that in the EAE Lewis rats.     

Selective breeding,congenic strains,and other classical genetic approaches to the analysis of alcohol-related polygenic pleiotropisms

Dimensions of behavioral sensitivities to alcohol in mice are under control of polygenic systems of relatively small size. The mode of inheritance of these phenotypes is frequently additive, with no evidence of dominance, epistasis, or sex linkage. The utility of classical breeding methodologies, such as selection, for assessment of genetic correlations is reviewed. A distinction is drawn between pleiotropisms in these polygenic systems, and the statistical concept of a genetic correlation. Development of congenic strains is argued to be a powerful alternative methodology, heretofore unused in alcohol pharmacogenetics. Using the phenotype of behavioral activation produced by a low dose of ethanol, we describe the production of an activated congenic strain on the non-activated background of the C57BL/6 mouse strain. Through five generations of repeated backcrossing, from a genetically heterogenous stock, activational alleles are being successfully transferred to the C57BL/6 background. Theoretical issues in the creation of congenic strains in potentially polygenic systems are covered, including number of effective loci and heritability.     

Extrarenal immune complex type deposits induced by mercuric chloride in the Brown Norway rat.

It has been reported previously that HgCl2 chronically injected in the BN rat induced a biphasic renal disease. During the first stage, anti-glomerular basement membrane antibodies appeared and during the second stage, an immune-complex type glomerulonephritis was observed. In the present study, a systemic immune disease is described. During the first stage, antibasement membrane antibodies were observed in various extrarenal structures. Their localization has been found to depend mainly on the characteristics of the endothelium. During the second stage, immune-complex type deposits containing IgG and C3 were found in most vascular structures. Their localization did not apparently depend on the endothelial characteristics. Among the organs tested the lung was most often spared. The occurrence of immune complex deposits was found to depend on the dose of HgCl2 injected: deposits were absent in some high dose HgCl2-injected rats but they were very numerous in low dose HgCl2-injected rats. These deposits probably have a pthogenic role although no major histological lesion could be found. This model may help to explain immune complex type deposits in systemic diseases.     

Spontaneous hypertension and hypertensive renal disease in the fawn-hooded rat

The fawn-hooded (FH) rat, a strain characterized by a platelet storage-pool disease, developed focal and segmental glomerulosclerosis at the age of 2-3 months (males) and approximately 6 months (females). Male animals died spontaneously at 11-13 months, and females at 15 months of age, both with overt malignant nephrosclerosis. During the first half year of life focal glomeruli showed depositions of IgG, IgA, IgM, C3 and fibrinogen in a segmental pattern and mainly in mesangial areas. Mesangial IgG and IgA were already demonstrable at the age of 5 weeks. On electron microscopy no electron-dense deposits suggestive of immune complexes were found. Mean arterial blood pressure in 5.5-month-old male FH rats was increased compared with that of matched Wistar rats. One-year-old FH rats had severe hypertension. The presumed relationship between the hypertension, the renal lesions and the blood platelet defect is discussed.     

Association between susceptibility to experimental allergic encephalomyelitis and the major histocompatibility system in congenic rat strains.

The genetics of the inducibility of experimental allergic encephalomyelitis (EAE) was studied in ten inbred rat strains by injection of guinea pig spinal cord in complete Freund's adjuvant. LEW rats were highly susceptible and AS2, AVN, BDV and BN rats were poorly susceptible. Congenic strains which carried the major histocompatibility haplotype of the poorly susceptible strains on the genetic background of the susceptible LEW strain were only moderately susceptible. It is concluded that major histocompatibility-linked genes exert a strong influence on the susceptibility to EAE, but that genetic background genes are also involved.     

Effects of ketotifen on airway responses to allergen challenge in the actively sensitized Brown Norway rat

The effects of the phorbol ester, phorbol-12-myristate-13-acetate (PMA), and of the calcium ionophore A23187 on DNA synthesis in murine quiescent and mitogen-stimulated lymphocytes have been examined. Neither PMA nor A23187 had any mitogen effect on their own on quiescent lymphocytes. However, stimulation of cells sequentially with A23187 and then PMA resulted in a proliferative response in proportion to the duration of the exposure to A23187, and the sustained simultaneous presence of both agents was necessary for maximum proliferation. On the other hand, while short incubations with A23187 potentiated mitogen-induced DNA synthesis, prolonged exposure inhibited it. Furthermore, on lymphocytes stimulated with two T cell mitogens, the effects of A23187 and PMA depended on the proliferation-inducing mitogens and the responsiveness level induced by them. Therefore, while PMA and short pretreatments with A23187 had no effect on high intensity mitogenic responses, low intensity responses were significantly enhanced. These results demonstrate differential effects of A23187 and PMA on DNA synthesis that should be useful in studies on the mechanisms of activation of lymphocyte proliferation.