Variant Mutant55.56%

RAT

Rat Mutant

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Immunology

Veronica M. Jennings, Dirck L. Dillehay, in The Laboratory Rat (Second Edition), 2006

2. Long-Evans Cinnamon Rats

Long-Evans Cinnamon (LEC) mutant rats have a maturational arrest in the development of CD4+ T-cells, but not CD8+ T-cells, from CD4+CD8+ T-cells (Agui et al., 1990, 1991). This results in a significant decrease in the number of peripheral CD4+ T-cells; however, these cells are present in peripheral lymphoid organs and are most likely generated by an alternative pathway. The deficiency of CD4+ T-cells is owing a single recessive gene system, thid (T-helper immunodeficiency) (Yamada et al., 1991). This mutation is found in bone marrow-derived cells but not in thymic stromal cells (Agui et al., 1991). CD4+ T-cells that are present show functional abnormalities (Sakai et al., 1995). For example, there is no IL-2 production after Con A stimulation. Interestingly, the presence of the allele thid does not prevent the development of CD4+ intraepithelial lymphocytes since LEC rats possess normal numbers of both CD4+CD8+ and CD4+8− intra-epithelial lymphocytes (Sakai et al., 1994).

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Modeling the Psychopathological Dimensions of Schizophrenia

Bart A. Ellenbroek, Tim Karl, in Handbook of Behavioral Neuroscience, 2016

Transcription Activator-Like Effector Nucleases

TALENs can be used for in vivo genetic engineering of mutant rat models. TALENs are artificial restriction enzymes and can cut DNA strands at any desired sequence, which makes them an attractive tool for genetic engineering. TALENs are generated by fusing DNA binding domains of transcription activator-like (TAL) effectors to DNA cleavage domains. TAL effectors are secreted by Xanthomonas bacteria and can bind DNA sequences (via repetitive amino acid residues in the central domain) and activate gene expression. The simple relationship between amino acids in the TAL effector and the DNA bases in its target provides the possibility of engineering TAL effector proteins with an affinity for a predetermined DNA sequence (Tong et al., 2012). Several researchers have fused the FokI nuclease domain to TAL effector proteins to create TALENs (e.g., Miller et al., 2011). When TALENs are introduced into cells they can be used for genome editing in situ. For example, TALENs were used to disrupt the IgM locus in the rat and to create a heritable mutation that eliminates IgM function. For this, titrations of specifically designed TALENs were microinjected either as DNA or mRNA into one-cell rat embryos (similar to what has been described for ZFN technology), of which a proportion (DNA, 9.5%; mRNA, 58%) showed subsequent alterations to the IgM locus. IgM mutation frequency was a function of TALEN dose as was the rate of biallelically modified rats, which were generated by mRNA (but not DNA) injections only. Genetically modified rats were then bred with wild-type-like control rats and the resulting F1 generation was checked for mutant alleles using PCRs. This procedure established TALEN technology as a valid tool for the generation of in vivo gene knockouts in rats (Tesson et al., 2011).

However, the technique to generate TALEN-mediated DNA double-strand breaks described previously can be technically challenging using the regular cloning methods and is relatively expensive. Tong and coworkers recently developed TALEN-targeting vectors using Golden Gate cloning technique thereby providing a more time-efficient tool to generate gene-targeted rat ES cells (i.e., construction of a pair of TALENs targeting any sequence of interest can be completed in just 5 days) (Tong et al., 2012). Furthermore, TALEN-mediated homologous recombination has been utilized to generate a knock-in rat model using oocyte microinjections of TALENs mRNA with a linear donor (instead of a supercoiled donor, which was ineffective in producing knock-in rats) (Ponce de Leon, Merillat, Tesson, Anegon, & Hummler, 2014).

In conclusion, efficient gene targeting in rat ES cells can be achieved quickly using either TALEN-mediated DNA double-strand breaks (Tong et al., 2012) or integration of TALENs by homologous recombination (Ponce de Leon et al., 2014). Thus, TALENs are an affordable and highly efficient option for the generation of targeted and specific mutagenesis of the rat and will reduce significantly time expenditure.

To give an example, Ferguson and coworkers selected the gene for the toll-like receptor 4 (TLR4) for TALEN-mediated gene inactivation (Ferguson, McKay, Harris, & Homanics, 2013). The team developed a pair of TALEN constructs that specifically target exon 1 immediately downstream of the start of translation. TALEN mRNAs were microinjected into the cytoplasm of one-cell Wistar rat embryos and heterozygous F1 offspring were interbred to produce homozygous F2 animals. The homozygous knockout rats had a markedly attenuated increase in plasma tumor necrosis factor alpha in response to a lipopolysaccharide challenge compared to control rats. TLR4 knockout rats will also be valuable for studies of ethanol action and of inflammatory conditions including septic shock, as TLR4 appears to play a role in ethanol-induced neuroinflammation and neurodegeneration.

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Preventive Role of Renal Kallikrein–Kinin System in the Early Phase of Hypertension and Development of New Antihypertensive Drugs

Makoto Katori, Masataka Majima, in Advances in Pharmacology, 1998

1 Mutant BN-Ka Rats

Damas and Adams, at the Katholiek University of Leuven, Belgium, reported mutant rats of the BN strain (Rattus norvegicus, BN/fMai), which are devoid of plasma kallikrein-like activity and show low levels of kininogen in plasma (Damas and Adams, 1980). This was also studied by another group (Oh-ishi et al., 1982). These BN-strain rats show a prolonged kaolin-activated partial thromboplastin time (APTT) because of the lack of HMW kininogen and the low level of plasma prekallikrein (Oh-ishi et al., 1984). They were designated BN-Ka rats (Oh-ishi et al., 1982), since the original report was published by the Katholiek University of Leuven. Further studies revealed that both HMW and LMW kininogens were almost absent from the plasma (Fig. 6) (Oh-ishi et al., 1986; Majima et al., 1991) and that BN-Ka rats are practically incapable of excreting kinin in the urine (Fig. 6) (Yamasu et al., 1989; Majima et al., 1991). Normal rats of the same strain were kept at the Kitasato University animal facilities and were designated BN-Kitasato (BN-Ki) rats (Oh-ishi et al., 1982). Normal BN-Ki rats show the same levels of kininogens as rats of other strains, such as the SD strain (Majima et al., 1991). The mutant BN-Ka rats, although capable of producing kininogens in the liver, cannot release kininogens into the bloodstream because of the point mutation of Ala163 to threonine in the common heavy chain of the structures of both kininogens (Hayashi et al., 1993). The HMW and LMW kininogens and prekallikrein mRNAs that are present in the liver of BN-Ka rats are of a similar size and abundance to those in BN-Orl rats (Lattion et al., 1988).

Figure 6. Kininogen levels in plasma (upper panel) and urinary kinin excretion (lower panel) in normal Brown Norway Kitasato (BN-Ki) rats and mutant BN-Ka rats. Values are the means (±SEM) of four rats. BK eq, bradykinin equivalent.

From Majima et al., 1991, with permission.

In a carrageenin-induced rat pleurisy model, mutant BN-Ka rats showed less plasma exudation and lower exudate volume in the pleural cavity than did normal BN-Ki rats, indicating that the plasma kallikrein–kinin system has a definite role in inflammation (Oh-ishi et al., 1987). The roles of the kallikrein–kinin system in inflammation, using mutant BN-Ka rats, are described in a review article in Japanese (Oh-ishi, 1993). Other review articles on mutant BN-Ka rats in relation to hypertension have been published (Majima and Katori, 1995c; Katori and Majima, 1996).

BN-Ka rats, in which kininogens are congenitally deficient in the plasma, have no apparent symptoms. Changes in SBP during growth in mutant BN-Ka rats are the same as in normal BN-Ki rats, when they feed on a diet containing 0.3% NaCl and drink distilled water (see Fig. 14) (Majima et al., 1991). The dose-response curve of the increase in SBP for angiotensin II injected intravenously into anesthetized mutant BN-Ka rats is not different from that in normal BN-Ki rats, suggesting that the arteriolar smooth muscle in mutant BN-Ka rats is not more sensitive to this vasoconstrictive peptide than that of normal BN-Ki rats (Majima et al., 1994b). Breeding of mutant BN-Ka rats between sisters and brothers is difficult, since the breeding rate is low.

A congenital deficiency of kininogens in the plasma was also reported in humans (Colman et al., 1975; Lacombe et al., 1975; Wuepper et al., 1975; Donaldson et al., 1976). In the first case in Japan (Hayashi et al., 1978; Oh-ishi et al., 1981), twin sisters (Fujiwara trait), who showed prolongation of APTT, were congenitally deficient in HMW and LMW kininogens in the plasma and had reduced levels of plasma prekallikrein. HMW kininogen and plasma kallikrein are essential in the activation of coagulation factor XII (Kaplan et al., 1986). However, the sisters displayed no apparent clinical symptoms and underwent appendectomy without excessive bleeding (Hayashi et al., 1978). The susceptibility to salt and the incidence of hypertension have not been studied. A similar kininogen-deficient family was also reported in Japan (Nakamura et al., 1983). As with these patients, mutant BN-Ka rats showed no apparent disorders or symptoms when they are fed a normal or low-sodium diet. However, the following experimental results clearly indicate that mutant BN-Ka rats are very sensitive to ingested salt and respond with sodium accumulation and consequent hypertension. Further more, sodium accumulation is also readily induced by aldosterone released by a nonpressor dose of angiotensin II.

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Lymphangioleiomyomatosis

Francis X. McCormack MD, Yoshikazu Inoue MD, PhD, in Murray and Nadel's Textbook of Respiratory Medicine (Sixth Edition), 2016

Sirolimus

Sirolimus had been shown to silence S6 phosphorylation and to induce apoptosis, necrosis, and regression of renal cystadenomas in TSC mutant rats and of hepatic tumors of TSC heterozygous null mice.174,175 Goncharova and colleagues176 reported abundant S6 phosphorylation and unregulated cell proliferation in LAM cells isolated from explanted LAM lungs harvested at lung transplant. They further demonstrated that sirolimus blocked the hyperphosphorylation of S6 in cultured LAM cells and restored orderly cell growth.

The discovery of the importance of the mTOR pathway in LAM and preclinical studies in TSC animal models formed the basis for phase I/II trials of sirolimus in patients with tuberous sclerosis and LAM (Cincinnati Angiomyolipoma Sirolimus Trial [CAST]; NCT00457964).177 In this open-label study, 25 patients with AMLs, including 6 patients with S-LAM, 12 with TSC-LAM, and 7 with TSC alone, were treated for 1 year with sirolimus and followed with serial magnetic resonance imaging studies of the kidneys, chest CT scans, and pulmonary function tests. Of the 20 patients still enrolled at 1 year, AML volume shrank by an average of 47%; of the 11 patients with LAM still enrolled at 1 year, the FEV1 and forced vital capacity (FVC) increased by 118 mL and 390 mL, respectively. The residual volume fell by 440 mL, and cyst volume percent, a measurement of the fraction of the pulmonary parenchyma occupied by cysts, tended to decline on therapy.178 Parameters that did not change included total lung capacity, 6-minute walk distance, and Dlco. There were a number of adverse events while patients were taking the study agent, including mouth ulcers, cholesterol elevations, and hospitalizations for pneumonia, diarrhea, cellulitis, pyelonephritis, palpitations, and mucositis.

In the second year of the trial, patients were observed off the study agent at 6-month intervals. At the 2-year point, the average AML volume had increased again to 86% of baseline, although in 25% of patients, AML size remained below 70% of baseline; FEV1 and FVC had declined at rates that are generally consistent with those of untreated patients with LAM,179 but remained 62 mL and 364 mL above baseline, respectively; residual volume remained 333 mL below baseline, indicative of durable reduction in gas trapping. Similar results were seen in subsequent trials.180,181

On the basis of the unexpected lung function response in the 11 patients with LAM, and a higher than expected rate of adverse events, a pivotal trial was designed to determine the risks and benefits of sirolimus in patients with LAM. The Multicenter International LAM Efficacy of Sirolimus (MILES) trial was a randomized, double-blind, controlled trial of sirolimus in 89 women with LAM and abnormal lung function (FEV1 < 70% predicted).182 During the 1-year treatment period, lung function (FEV1) stabilized on sirolimus, while declining by about 11% in the placebo group. Sirolimus also improved some measures of quality of lung and functional performance. In the observation year off therapy, lung function decline resumed in the sirolimus group and paralleled that in the placebo group. At baseline, serum VEGF-D was elevated by more than fivefold (relative to healthy volunteers73) in both groups. VEGF-D remained stable in the placebo group but, in the sirolimus group, VEGF-D decreased by more than 50%. When sirolimus was withdrawn, VEGF-D increased toward baseline again.75 The sirolimus-induced decreases in VEGF-D are intriguing in light of the strong lymphangiogenic phenotype observed in LAM and marked improvement in chylous effusions and lymphangiomyoma volume seen in patients with LAM with lymphatic involvement.183 Adverse events were common during the treatment period and were more prevalent in the sirolimus group. There was no increase in risk of infection, however, and the frequency of serious adverse events was balanced between the groups.

Collectively, the data suggest sirolimus therapy stabilizes lung function and improves some measures of quality of life in patients with LAM. The fact that decline resumes when the drug is withdrawn indicates that the therapy is suppressive and does not result in durable remission. One explanation for the beneficial but transient effects on lung function and tumor volume seen in CAST, MILES, and other trials is that the drug may shrink cells, attenuate tumor cell infiltration, or inhibit proliferation within the organs, but does not induce apoptosis of LAM cells. It is possible that mTOR inhibitors must be given continuously to maintain cellular homeostasis and avoid AML regrowth and lung function decline; indeed, there is early evidence of sustained benefit from longer-term therapy.184

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Remyelination in Multiple Sclerosis

Martin Stangel, ... Viktoria Gudi, in Neuroinflammation, 2011

Genetic Myelin Mutants

In recent years, an increasing number of genetic animal models with a disturbance of CNS myelination have been characterized. The myelin-deficient (md) rat and mouse mutants jimpy have a mutation in the gene encoding for PLP that results in central myelination deficits. Both mutants serve as animal models for the human X-chromosome-linked Pelizaeus–Merzbacher disease, which is genetically related to a defect in the PLP gene [22]. The shiverer (shi) mouse is another myelin mutant model that has a defect in the MBP gene and causes abnormal myelination [23].

The advantage of myelin mutants is the consistency in myelination defects and known area of damage, allowing transplanted cells to be identified more clearly. Furthermore, genetic mutants serve as excellent models to study human dysmyelinating diseases. The disadvantage of genetic models is that they are poorly myelinating or nonmyelinating models rather than demyelinating lesion models. Furthermore, demyelinated areas in the CNS are noninflammatory. Thus, genetic models are less suitable for studies of remyelination in MS.

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Bacterial, Mycoplasmal and Mycotic Infections

Steven H. Weisbroth, ... Ron Boot, in The Laboratory Rat (Second Edition), 2006

C. Pneumocystosis (Pneumocystis carinii)

Pneumocystis infection was originally described as a pneumonia of laboratory guinea pigs by Chagas (1909), who mistook the cysts he saw as forms of Trypanosoma cruzi. Again, in 1911. he saw the cyst forms (which he reported as trypanosomes) in lung tissue from a human case of Chagas disease (Chagas, 1911). Shortly afterwards, similar pneumonias in rats were described as caused by a new organism named P. carinii by Delanoe and Delanoe (1912). The organism was reported several years later in English laboratory mice (Porter, 1915). There are, however, few references to rodent pneumocystosis in the intervening years before the era of radiation and antiinflammatory (cortisone) research in the 1950s and 1960s (Frenkel et al., 1966). At that time, scientists began to encounter clinically overt Pneumocystis infections, principally in rats, as complications of research protocols now recognized as inducing a state of immunosuppression. Interestingly, most references to unanticipated, "spontaneous" C. piliforme and C. kutscheri infections of rats date to the same era, for the same reasons.

Animals, especially rats and mice and, to a lesser degree rabbits and ferrets, have been an indispensable element of Pneumocystis research dating from Frenkel's 1966 paper (Dei-Cas et al., 1998). The histologic features of pulmonary Pneumocystis pneumonia (PCP) in animals (see description and illustrations in Percy and Barthold, 2001) are nearly identical to those of the disease in humans. Animal models have provided the basis for most of our current concepts of the epizootiology, taxonomy, diagnosis, genetics, and therapy of pneumocystosis (Hughes, 1989; Walzer, 1991). In the past, rats and mice from conventional commercial breeding colonies of immunocompetent stocks have acted as convenient, but erratic and unreliable sources of carrier Pneumocystis infections. The term "conventional" describes a rodent population in which one or more murine viruses cycle as enzootic infections and may also be infected with a range of helminth, protozoan, arthropod, and bacterial pathogens. The pathogen load is emblematic of unshielded husbandry environments and the correspondingly high likelihood that such populations also latently (actually, inapparently) carry Pneumocystis. Investigators needing Pneumocystis- infected rodents for research programs favored procurement from such colonies in the past because it was assumed they were likely to be "latent" carriers (Russell and McGinley, 1991) although it has since been shown that Pneumocystis infection dynamics are quite independent of murine virus status (Pesanti and Shanley, 1984; Cushion and Linke, 1991). After arrival at the user institution, these animals would be deliberately subjected to an immunosuppressive protocol that would culminate, after an inductive phase of about 4 to 8 weeks, with development of severe lesions of PCP with abundant Pneumocystis. Faced with impending disappearance of conventional Pneumocystis carrier sources as commercial rodent breeders mounted intensive rederivation programs to eradicate murine viruses (Bartlett et al., 1987), investigators have turned to the more reliable method of inducing PCP in pathogen-free rodents. In this procedure, animals are first rendered susceptible to overt infection by immunosuppressive treatments and then infected by intranasal or intratracheal installation of Pneumocystis inocula (Bartlett et al., 1987, 1988, 1990, 1991; Shellito et al., 1990; Boylan and Current, 1992) or by short-term housing with infected seed rats or mice (McFadden et al., 1991). Additional models of pneumocystosis include the athymic (nude) rat, athymic (nude), and SCID mice, which offer physiologic analogs comparable to the HIV-infected human with PCP. Neonatal rabbits commonly develop transient lesions of pneumocystosis, without immunosuppressive treatment, but these infections resolve naturally as immunocompetence develops in the weeks after birth (Soulez et al., 1989).

Over the years, until the 1980s, informed opinion had wavered on the kingdom in which to place Pneumocystis, but historically, the organism was widely thought to be a protozoan. Uncertainty still surrounds details of the life cycle of Pneumocystis. The issue is complicated, and even the terms used to describe morphologic life forms of Pneumocystis derive from protozoology. The mature form is termed a cyst. Within the cyst, visible more or less depending on the stain and stage of maturation are eight intracystic bodies or "sporozoites." When exeystation takes place, the sporozoites enter a trophic stage. The vegetative, trophic forms in turn become precysts, crescent-shaped cysts, and finally cysts (Kim et al., 1972; Yoneda et al., 1982; Cushion et al., 1988). Cyst production involves a sexually reproductive phase (Cushion et al., 1997; Cushion, 2003). An ste3-like pheromone receptor has been identified in the organism (Smulian et al., 2001). Although the morphologic stage variants described above have been recognized microscopically, the lack of a continuous in vitro culture system for propagation of Pneumocystis has hampered an understanding of the progression of the life cycle and definition of the infective form. Pneumocystis may be carried for several passages in tissue culture, with an increase of up to 10- to 20-fold in numbers, but a continuous in vitro culture method has so far proven elusive (Bartlett et al., 1979; Cushion and Walzer, 1984a,b; Cushion et al., 1985; Cushion and Ebbets, 1990; Aliouat et al., 1995; Beck et al., 1996; Atzori et al., 1998). At least one phase, the most studied, takes place in the mammalian lung, although tracing methods using PCR commonly detect Pneumocystis DNA in extrapulmonic locations, for example, bone marrow, liver, blood stream (antigenemia), and spleen (Reddy and Zammit, 1991; Schluger et al., 1991; Chary-Reddy and Graves, 1996; Rabdonirina et al., 1997). Lesions in spontaneous rodent PCP are usually confined to the lung, but there are reports of SCID mice developing extrapulmonic foci in the heart and spleen (Reddy and Zammit, 1991).

The cyst walls stain best with GMS, PAS, or toluidine blue O stains (Thompson et al., 1982; Sundberg et al., 1989). Giemsa stain does not stain the cyst wall. The best single stain for diagnostic visualization of Pneumocystis forms in concentrated smears of lung homogenates or impression smears of cut lung surface appears to be the Diff-Quik and similar stains because all stages are stained. Although several lines of evidence, for example, histologic tinctorial qualities (argyrophilia), were consistent with relatedness to fungi (Walkeden, 1990), antifungal drugs were shown to be ineffective against PCP, although Pneumocystis has since been shown, in vitro, susceptible to some antifungals (Kaneshiro et al., 2000). Conversely, drugs with confirmed activity against Pneumocystis, for example, trimethoprim-sulfamethoxazole, fansidar, and pentamidine, have an antiprotozoan spectrum. Molecular and biochemical analyses have since established that Pneumocystis is a genus of unusual eukaryotic single-celled and genetically complex fungi, most likely an ascomycete but lacking in ergosterol (Edman et al., 1988; Cushion, 1993; Stringer et al., 1992, 1993, 2002).

Genetic studies with Pneumocystis isolates derived from various mammalian hosts have shown that these organisms are quite different. The accumulating results of DNA studies directed to taxonomic analysis have established P. jiroveci and P. carinii as separate species. Based on genetic divergence studies (Stringer et al., 1993) and infective isolation for its human host, the human strains of this organism were designated P. jiroveci. (Frenkel 1999), as earlier suggested by Frenkel (1976). Although DNA sequence polymorphism has been shown with human Pneumocystis isolates (Lee et al., 1993), suggesting that numerous strains of P. jiroveci exist, this diversity has been thought insufficient to designate either special forms of P. jiroveci or additional human species (Wakefield, 1998; Stringer, 2002). Pneumocystis jiroveci has not been found in the lungs of any other mammal, including nonhuman primates (Wakefield et al., 1990; Stringer, 2002), nor may it establish productive infection even in SCID mice (Durand-Joly et al., 2002). Accordingly, it has been concluded that humans do not carry rodent Pneumocystis and, conversely, that humans do not contract pneumocystosis from animal contacts.

Experience from several lines of inquiry supports the concept that Pneumocystis strains have diverged in genetic heterogeneity, progressing to speciation according to homology with a given mammalian host species (Shah et al., 1996; Stringer, 2001). Infectivity studies have demonstrated that heterologous hosts, even if immunodeficient, are refractory to replication of Pneumocystis derived from other host species (Gigliotti et al., 1993; Aliouat et al., 1994). Similarly, the ultrastructural morphology and isoenzyme diversity among Pneumocystis isolates from different host species is host specific and supports evidence of genetic diversity demonstrated by molecular analysis (Mazars et al., 1997; Nielsen et al., 1998). For this reason, under natural circumstances, rats should not be regarded as vectors of Pneumocystis infectious for other rodent species, and conversely, other rodent species, including mice, are not carriers of Pneumocystis infectious for rats.

Isolates of Pneumocystis from rats are taxonomically quite complex. A trinomial system has been adopted (Wakefield, 1998; Cushion, 1998) in which the various strains of Pneumocystis have been designated as special forms (formae speciales, or f. sp.). At least five species of rat Pneumocystis have been delineated by DNA analysis, but only two of them occur in laboratory rats, that is, P. carinii and P. wakefieldiae (formerly, P. carinii f. sp. ratti) (Cushion, 2003). The binomial P. carinii is reserved for the more common Pneumocystis species occurring in rats and the former designation P. carinii f. sp. carinii is now shortened) (Stringer et al., 2001). Three additional species have been detected in wild rats, viz., P. carinii f. sp. ratti-secundi, P. carinii f. sp. ratti-tertii, and P. carinii f. sp. ratti-quarti (Cushion et al., 1993; Palmer et al., 2000). These forms may be discriminated by PCR (Palmer et al., 1999, 2000; Schaffazin et al., 1999; Nahimana et al., 2001) or by immunoblotting (Vasquez et al., 1996), and investigators should do so because the two species vary in responses to drug therapy trials, the life cycles differ, and the use of mixed isolates from lung preparations (not uncommon) could lead to a confusing mixture of gene sequences in gene libraries. At present, P. carinii f. sp. carinii is believed to be the most prevalent strain in commercial rat producer stocks (Icenhour et al., 2001), although coinfection by more than one special form in the same rat is recognized as not uncommon (Cushion et al., 1993; Nahimana et al., 2001). The name newly given to the mouse strain is P. murina.

The common denominator of immunodeficiency that underlies PCP has been linked with the common occurrence of intercurrent or "spontaneous" PCP in immunodeficient (athymic and scid) mutant and transgenic rat and mouse stocks, with induced rodent models for human PCP, and of course, with human PCP, itself a consequence of impaired immunocompetence resulting from HIV infection. Progressive wasting (emaciation, or cachexia), cough, dyspnea, and cyanosis are clinical signs of PCP in rats. Clinically overt signs of pneumocystosis occur in rats and mice under the following circumstances:

Immunologically competent rodents that have been rendered immunodeficient as a consequence of experimental treatments in which in which PCP develops as an unplanned complication of a research protocol, as cited above during earlier eras of radiation and anti-inflammatory research. Such animals may have been infected at the source, before arrival at the user institution or first become exposed to same host species Pneumocystis shedders at the institution and infected after arrival.

Unplanned outbreaks in immunologically deficient mutants lacking cell-mediated immune capability and T cell-assisted antibody responses. Such mutants include the athymic (nude) mouse and rat, the beige triple-deficient mouse, the scid mouse, and a range of similarly affected heritably immunodeficient mutant and transgenic rodent models (Veda et al., 1977; Van Hooft et al., 1986; Weir et al., 1986; Walzer et al., 1989; Sundberg et al., 1989; Gordon et al., 1992; Deerberg et al., 1993; Furuta et al., 1993; Percy and Barta, 1993; Pohlmeyer and Deerberg, 1993). These animals need only adequate exposure to Pneumocystis to initiate development of PCP (Soulez et al., 1991). On this basis, athymic mice have been used in mouse colonies as sentinels for detection of Pneumocystis (Serikawa et al., 1991). The clinical course of PCP in immunodeficient animals may be fulminant but is more likely to be chronic. The microscopic qualities of the lesions that develop in immunodeficient mutants are the same as in immunocompetent rodents that develop PCP as a result of immunosuppressive treatments. There is no support in the literature for the view that immunodeficient mutants may carry Pneumocystis in a latent form, and further, it is established that immunosuppressive treatments of athymic mutants do not increase the incidence or severity of PCP during experimental induction (Veda et al., 1977; Walzer and Powell, 1982; Soulez et al., 1991; McFadden et al., 1991).

Immunologically competent carrier rats and mice rendered immunodeficient by chemical immunosuppressants, for example, corticosteroids or antimetabolites such as cyclophosphamide, as a planned process to develop PCP for some further experimental purpose; to propagate Pneumocystis for inoculation or antigen material; or to detect Pneumocystis carriers through the outcome of a diagnostic stress test.

Clinically overt pneumocystosis (PCP) should be viewed in all species as an unusual outcome requiring special circumstances of infection (immunodeficiency) as outlined above. More commonly, pneumocystosis occurs as a clinically inapparent infection of immunocompetent rodent stocks and strains that, because of its symptomalogic silence and difficulty of diagnostic detection (until recently), seems to have provided little incentive to commercial and institutional rodent breeders for eradication. Indeed, as recently as 2001, most rats from good commercial producers in the United States were detected as contaminated with DNA of P. carinii (Icenhour et al., 2001).

It is gradually becoming clear that the natural mode for transmission and maintenance of rodent Pneumocystis infection is by exposure via aerosol of susceptible immunocompetent hosts (Walzer et al., 1977; Hughes, 1982; Hughes et al., 1983). Even germfree rats were shown to be insusceptible to oral dosing via the diet or by ingestion of infected lung (Hughes, 1982). In enzootically infected breeding colonies, neonates are born to variably immune dams, exposed within hours of birth (Icenhour et al., 2002) but, it may assumed, are probably protected from productive infection for the first few weeks of life by colostral antibody. Thereafter, they become susceptible and cage-to-cage transmission occurs via aerosol from infected shedder animals. In enzootically infected, closed, barriered colonies, weanling (3- to 4-week-old [100 to 125 g]) rats can be envisioned as living in a cloud of Pneumocystis spores, with spore contamination (at least as denoted by PCR detection of Pneumocystis DNA) on all environmental surfaces (Icenhour et al., 2002), thus with ample opportunity for exposure. The prime infective (shedder) phase would thus appear to be the 6- to 12-week-old rat (200 to 250 g), which corresponds to the age at which most rats are procured and shipped to user institutions. Thereafter, the immunologic response to these light (and clinically inapparent) infections supervenes, and Pneumocystis populations decline until eliminated by the host (An et al., 2003). Thus, the infection in the colony would seem to be propagated by successive cohorts of the productively infected, spore-shedding 4- to 12-week age group, infectious only to the next cohort of 3- to 4-week-old rats that are in the process of losing their colostral immunity. It would appear that older rats may be immune to reinfection and younger rats protected by colostral antibody.

As more has been learned about the pathogenesis of pneumocystosis in immunocompetent rat and mouse (and human) hosts, doubt has been cast as to whether the term "latent" ever actually applies to Pneumocystis infections. Even after PCP induced by immunsuppression, most rat hosts allowed to immunologically recover eliminate Pneumocystis from the lungs (Vargas et al., 1995), as do infected SCID mice immunologically reconstituted with normative spleen cells (Chen et al., 1993). The basis for inadvertent introduction of asymptomatic and lightly infected shedder rodents into user institutions has been outlined above as reflecting the enzootic infective cycle in breeding colonies in which it can be envisioned that essentially all rodents in the 4- to 6-week to 12-week adolescent age group become infected for a brief period of time, after which they become immune noncarriers (An et al., 2003). During this period (the shedder phase), they are infective hazards for other Pneumocystis free rodents of the same species and have been a frequent cause of clinically overt infective "breaks" in immunodeficient stocks at user institutions (Dumoulin et al., 2000). This same infectivity model is believed to hold with humans as well, who become almost universally exposed and immune at a very young age (Stringer et al., 2002). In the same way, it is now understood that human PCP derives not from activation of endogenous "latent" infections but more probably from chance or nosocomial aerosol exposure to spores during a receptive phase of immunodeficiency (Chen et al., 1993; Vargas et al., 1995). Although the infective form of Pneumocystis in aerosols has not been defined, there is ample evidence of Pneumocystis DNA detected by PCR of spore trap air samples (Wakefield, 1994, 1996; Olsson et al., 1995). Immunosuppression, as by HIV infection or preparation for organ transplantation in humans or by immunosuppressive treatment of immunocompetent rodents, can effectively override naturally acquired or experimental immunity and render even immune hosts susceptible to PCP (Shellito et al., 1990; Harmsen et al., 1995). Antibodies, whether derived actively after infection or passively by injection of immune sera, adequately protect against experimental homologous Pneumocystis infection (Gigliotti and Harmsen, 1997; Bartlett et al., 1998).

Definitive diagnosis of PCP in rats continues to be based on morphologic criteria of consistent gross and microscopic lesions of lobar pneumonia, with microscopic correlates of paravascular cuffing and alveolar distension with foamy, pinkish material in hematoxylin and eosin–stained material Fig. 11-14). With use of special stains (e.g., GMS), multiple discoid blackish staining Pneumocystis cysts may be demonstrated in the alveoli Fig. 11-15). Smears of lung tissue prepared at necropsy may be stained with toluidine blue O or GMS to detect cyst forms of Pneumocystis in postmortem material. Morphologic diagnosis of PCP can be confirmed by PCR tests (Kitada et al., 1991; Schluger et al., 1992; O'Leary et al., 1995), and for this purpose, individual or pooled lung tissue, bronchioalveolar lavage, or oral swab samples may be used.

Fig. 11-14. Pneumocystis carimi: rat lung with patchy foci of histioalveolitis. Note foamy alveolar contents (arrow). Hematoxylin and eosin, 40x.

(Courtesy of Dr. Shari R. Hamilton.)

Fig. 11-15. Pneumocystis carinii: lung section demonstrating silver staining (black) cyst forms in alveolar foam. Gomori's methenamine silver, 1000×.

(Courtesy of Dr. Shari R. Hamilton.)

In considering the use of serology as a diagnostic screening tool, the many practical hurdles and potential sources of false-negative results impose important limitations to adoption of this method. It has been shown that the host species origin of Pneumocystis antigen determines the serologic specificity of anti-Pneumocystis antibodies (Walzer and Rutledge, 1980; Kovacs et al., 1989; Bauer et al., 1991, 1993). Although many investigators use serology to detect prior exposures to Pneumocystis (Peglow et al., 1990), rats are quite variable in the age when detectable levels of antibody appear (Walzer et al., 1987). Antibody levels in sera after homologous experimental infections, that is, rat origin Pneumocystis used to infect rats, have a much higher titer against homologous Pneumocystis antigen than do the same sera tested against Pneumocystis antigens derived from other host species. Scientists recognized as early as 1966 (Frenkel) that Pneumocystis antigens derived from either rat or human would fix complement only with serum from homologous hosts, although when tested by immunoblotting, sufficient cross-reactivity of human sera against rat trophozoite antigens has been shown as to be diagnostically useful (Chatterton et al., 1999). A further potential cause of false negative could accrue to the point that, at least in rats, there are multiple special forms of P. carinii with an unestablished degree of cross-reactivity. These problems combine to make unlikely the development of a universal antigen that would detect antibodies to Pneumocystis in all laboratory species, or even in just rats and mice. Rather, it would seem that homologous antigen/antibody systems would need to be used. Because of the practical issues related to the present inability for large-scale propagation of Pneumocystis, rat origin antigens are not commercially available and serodiagnosis is not commonly used for diagnostic screening.

Diagnosis of light and clinically inapparent rat infections, as is the case in immunocompetent rat stocks, in the past depended on use of a diagnostic stress test (for a review of procedural parameters used in conducting rodent stress tests, see Weisbroth, 1995). The traditional stress test required histologic demonstration of Pneumocystis in stained lung sections after use of chemical immunosuppressants and a low-protein diet during a 2- to 4-week induction period (Frenkel et al., 1966; Walzer et al., 1979, 1980; Milder et al., 1980). The test was also used, when negative, to provide evidence that the institutional or commercial source of the animals in the test were free of contamination by Pneumocystis. Stress tests are lengthy, cumbersome, and expensive and have been shown to be no more effective in detection of inapparent infection than the are same rats tested by PCR at the start of the procedure before induction (Weisbroth et al., 1999b). For this reason, the stress test as a screening method for diagnosis of Pneumocystis infection has largely been discarded in favor of PCR. As mentioned above, for this purpose individual or pooled lung tissue, bronchioalveolar lavage, or oral swab samples can be used for screening purposes (Feldman et al., 1996; Icenhour et al., 2001). Because of the sensitivity of PCR, immunocompetent (but Pneumocystis free) rats may be used as sentinels to monitor Pneumocystis status of given rat environments (Icenhour et al., 2001).

There are very few options for control and eradication of Pneumocystis from rat populations. Owing almost entirely to the role of rats as prime models for exploration of treatment modalities for human PCP, there is an extensive body of experience in treatment of clinically apparent PCP in rats. The generality is that although a fair number of treatments—including trimethoprim-sulfamethoxazole, dapsone, or pentamidine, as well as an impressive array of newer therapeutics (beyond the scope of this text for review)— have been shown effective in amelioration of clinical signs (and lung counts), none have been shown effective in eradication of Pneumocystis (Hughes, 1979, 1988). For that reason, treatment options would appear limited to those few instances in which the life of a small population of immunodeficient animals needed to be prolonged for some purpose.

Gnotobiotic rederivation into isolators followed by introduction to a rodent barrier stringently managed for exclusion of rodent pathogens is recommended as effective for eradication and restart of rat populations free of Pneumocystis. There is no convincing evidence that P. carinii can traverse the rodent placenta to effect vertical transmission (Icenhour et al., 2000), rather, there is a good deal of experience demonstrating efficacy of gnotobiotic rederivation (Wagner, 1985; Ito et al., 1991).

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Crigler-Najjar Syndrome

C.E. CORNELIUS, in Spontaneous Animal Models of Human Disease, 1979

II ANIMAL MODEL

Hereditary nonhemolytic unconjugated hyperbilirubinemia also occurs in Gunn rats due to an absence of UDP-glucuronyltransferase activity identical to that observed in the type I deficiency in man. The mutant rats, first observed at the Connaught Laboratory in Toronto in 1939, are jaundiced and represent an apparent spontaneous mutation occurring in a Wistar rat colony. This acholuric jaundice is transmitted as an autosomal recessive characteristic. Gunn rats have hepatic UDPGA dehydrogenase activities comparable to that in normal rat liver. Heterozygous rats appear normal, are not jaundiced, and their livers exhibit glucuronide formation in vivo and in vitro that is intermediate between that observed in genetically normal and homozygous jaundiced littermates. Maximal rates for biliary excretion of injected sulfobromophthalein (BSP) or conjugated bilirubin are similar in Gunn mutants and normal Wistar rats. Bile obtained from Gunn rats is nearly colorless, lacks bilirubin glucoronide, and contains only trace amounts of unconjugated bilirubin. The deficiency in glucuronyltransferase activity is also present in the intestines and kidney as well as the liver. These observations suggest that pigment in mutants is disposed of through alternate pathways, as in Crigler-Najjar syndrome in man. The major portion of bilirubin is catabolized to diazo-negative polar bilirubin derivatives which are excreted in the bile and urine. A smaller amount of unconjugated bilirubin is transferred from blood across the mucosa into the intestine.

A large number of studies have been performed using this animal model and have provided unusual opportunities to study bilirubin transport, the mechanisms of jaundice, and the prevention of kernicterus in man.

Colonies of Gunn rats are maintained by Dr. Irwin M. Arias, Albert Einstein College of Medicine of Yeshiva University; Dr. Roger Lester, Boston University School of Medicine; Dr. Gerald Lucey, College of Medicine, University of Vermont; Dr. Donald Ostrow, University of Pennsylvania; and Dr. Rudi Schmid, University of California Medical Center, San Francisco.

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Models with Spontaneous Seizures and Developmental Disruption of Genetic Etiology

RADDY L. RAMOS, JOSEPH J. LOTURCO, in Models of Seizures and Epilepsy, 2006

Flathead Mutant Rat

In the first postnatal week, flathead pups display severe generalized seizures and motor deficits. During ambulation, astatic episodes can be observed and are characterized by the splaying of limbs. Mutant rats also often fall to one side during walking but are able to right themselves quickly. The falling and astatic episodes in flathead mutants are likely related to general muscle weakness, since flathead mutants cannot grip a small metal bar and are unable to rear onto their hind limbs. Seizures begin reliably toward the end of the first postnatal week and increase in duration and frequency until the third postnatal week (Figure 1). Seizures subsequently become more rare, but more severe, as identified by behavioral observations and electrophysiologic recordings.

FIGURE 1. Flathead seizures are generalized and increase in duration with age. A: Representative six-channel, electrographic recordings of a P16 flathead rat. Electrodes were placed across the rostrocaudal extent of the cortical surface (top to bottom: anterior left hemisphere; anterior right hemisphere; middle left hemisphere; middle right hemisphere; posterior left hemisphere; posterior right hemisphere). B: The average duration of seizures increases with age in flathead. C: There were no significant differences in average interval between seizures across age. D: Representative in vitro extracellular recording from a flathead hippocampal slice (electrode positioned in CA1 pyramidal layer). Ictal and interictal periods are identifiable in the upper trace (10-minute recording). Burst-type spiking can be seen in the expanded (lower) trace. Scale bars: A, 1000 ms/200 µV; D, upper trace, 2 min/70 µV; lower trace, 8 sec/70 µV.

As early as postnatal day 8 (P8), flathead mutants display intermittent episodes of tail flexion and extension (strub tail) and tremor of the limbs and head. These observations are the earliest signs of spontaneous generalized seizures of any of the models described here. Alternating forelimb clonus becomes more prevalent between P15 and P19. During clonic episodes, animals frequently ambulate around the home cage, propelling themselves by their forelimbs. Bursts of forelimb movements often result in forward lunges. Clonus of the neck musculature produces rhythmic head movements. Flathead rats display episodes of tonus that can involve one or more limbs or the entire body; tonus persists for approximately 1 minute and is sometimes preceded by loud vocalizations. Behavioral seizures are rare after P21, although single episodes of loud vocalization, jumping, and tonus have been observed in animals as late as P25—near the time of premature death, which is thought to be caused by a lethal seizure.

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MRP2, THE APICAL EXPORT PUMP FOR ANIONIC CONJUGATES

JÖRG KÖNIG, ... DIETRICH KEPPLER, in ABC Proteins, 2003

MOLECULAR CHARACTERIZATION OF THE APICAL CONJUGATE EXPORT PUMP MRP2

The first cDNA fragment of Mrp2 (Abcc2; formerly described as cMrp and cMoat) was identified in 1995 in a comparative analysis of normal and transport-deficient GY/TR− mutant rat liver (Mayer et al., 1995). Using degenerate oligonucleotides complementary to human MRP1 mRNA, an MRP1-related 347 bp cDNA fragment was amplified from normal rat liver but not from RNA from transport-deficient liver (Mayer et al., 1995). Subsequently, the full-length cDNA encoding an MRP1-related protein now known as Mrp2 was cloned and further analyzed (Büchler et al., 1996; Paulusma et al., 1996). At present, the full-length MRP2 cDNA sequences and deduced amino acid sequences from five mammalian species are known, including the orthologues from human, rat, rabbit, mouse and dog (Büchler et al., 1996; Conrad et al., 2001; Fritz et al., 2000; Paulusma et al., 1996; Taniguchi et al., 1996; van Aubel et al., 1998). These five mammalian MRP2 proteins are highly homologous, with amino acid identities ranging from 77% for the identity between the MRP2 proteins from rat and dog, to 87% identity for the proteins from rat and mouse. Furthermore, MRP2-related sequences from other organisms including Caenorhabditis elegans (Broeks et al., 1996) and the plant Arabidopsis thaliana (Rea et al., 1998) (Chapter 17) have been described and, in part, functionally characterized. Within the human MRP (ABCC) subfamily, MRP2 shows the highest degree of similarity to MRP1 with 48% identity (Cole et al., 1992), followed by MRP3 with 47% identity (Kiuchi et al., 1998), and MRP6 with 38% identity (Kool et al., 1999). The lowest degree of amino acid identity was found between MRP2 and MRP8 (GenBank accession XM_040766) and CFTR (Riordan et al., 1989) with 29% and 26% identity, respectively.

The identity of human MRP2 with respect to MDR1 (Ambudkar et al., 1999), a member of the P-glycoprotein (ABCB) subfamily, is only 18%, underlining a major difference between the ABCB and the ABCC transporter subfamilies. Differences between the proteins belonging to the two subfamilies are also apparent based on studies of the membrane topology of these transporters. In contrast to the typical organization described for members of the ABCB subfamily with two transmembrane domains and two ATP-binding domains, MRP2, as well as MRP1, MRP3, MRP6 and MRP7, contains an additional NH2-proximal membrane-spanning domain (Figure 20.1) (Borst et al., 1999; Büchler et al., 1996; Hipfner et al., 1997; König et al., 1999a). This additional domain is represented by an extension of approximately 200 amino acids when compared with the length of the ABCB subfamily members. Another striking feature of MRP2 was found in studies on the localization of the NH2-terminus. Owing to an odd number of predicted transmembrane helices, the NH2-terminus was predicted to be extracytosolic on the basis of computational analysis by the TMAP program (Büchler et al., 1996) (see section on mutations in the MRP2 gene). This was recently directly established by immunofluorescence microscopy studies using an antibody directed against the NH2-terminus of MRP2 (Cui et al., 1999). The extracellular localization of the NH2-terminus of MRP1 was also demonstrated by glycosylation site mutational studies and epitope insertion experiments (Hipfner et al., 1997; Kast and Gros, 1998) and, based on sequence similarities, it is expected that MRP3 and MRP6 will be the same.

Figure 20.1. A predicted membrane topology model for human MRP2. Amino acids in the nucleotide-(ATP)-binding domains are indicated, with the Walker A and B motifs in red and the ABC transporter family signature in blue. Mutated amino acids in patients with Dubin–Johnson syndrome are indicated as white stars on the polypeptide chain, whereas splice site mutations are indicated as pentagonal stars near the polypeptide chain. (See Chapter 3 for detailed discussion on topologies).

The human MRP2 gene has been localized to chromosome 10q23–q24 (Taniguchi et al., 1996). It spans approximately 65 kbp and contains 32 exons with a high proportion of class 0 introns (Tsujii et al., 1999). The size of coding exons ranges from 56 bp (exon 6) to 255 bp (exon 10), and each nucleotide-binding domain is encoded by three exons (Toh et al., 1999; Tsujii et al., 1999). Comparison of the genomic organization of the human MRP2 and MRP1 genes shows that they display remarkable similarities as indicated by size and number of exons (Grant et al., 1997). Furthermore, human MRP1, MRP2 and MRP3 (GenBank accession AC004590) have 21 identical splice sites based on an amino acid alignment of the three cognate proteins (Tsujii et al., 1999). Despite the fact that these three human MRP family members share a relatively moderate degree of amino acid identity, their similar genomic organization suggests a close evolutionary relationship, possibly originating from gene duplication (Keppler et al., 2000).

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Rodent Behavior: Approaches

L.H. Tecott, E.J. Nestler, in Encyclopedia of Neuroscience, 2009

Standard behavioral analysis of rats and mice is typically time-consuming and labor-intensive. Several factors have driven the field to consider more high-throughput methods to assess rodent behavior. The still accelerating creation of mutant mice and rats by mutagenesis and viral-mediated gene transfer has swamped traditional behavioral assays. The difficulty in developing psychotherapeutic medications with fundamentally novel mechanisms of action has caused some to place behavioral screens of potential drugs earlier in the drug discovery process, which is not as feasible with traditional approaches. Finally, existing assays are incomplete in terms of the range of rodent behavior they can assess. Consequently, new strategies combining automated behavioral monitoring and information technologies are under development in both academic and industrial settings. These hold promise both for improving the throughput of mouse behavioral assessment and for providing new insights into the neurobiology of mammalian complex behavior.

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