Validated Mouse Model Research Articles

Effect of remote ischemic preconditioning on liver ischemia/reperfusion injury using a new mouse model

Ischemic preconditioning of remote organs (RIPC) reduces liver ischemia/reperfusion (IR) injury in the rabbit and rat. Mice are the only species available with a large number of transgenic strains. This study describes development and validation of a mouse model of hindlimb RIPC that attenuates liver IR injury. Mice were allocated to 4 groups: (1) Sham surgery; (2) RIPC: 6 cycles of 4 × 4 minutes ischemia/reperfusion of hindlimb; (3) IR: 40 minutes lobar (70%) hepatic ischemia and 2 hours reperfusion; (4) RIPC+IR: RIPC followed by IR group procedures. Plasma liver aminotransferases and hepatic histopathological and transmission electron microscopy studies were performed at the end of the experiment. Hepatic microcirculatory blood flow was measured throughout the experiment. Postoperative complications and animal survival were evaluated. Hindlimb RIPC using a tourniquet resulted in limb paralysis. Hindlimb RIPC using direct clamping of the femoral vessels showed no side effects. Compared to liver IR alone, RIPC+IR reduced plasma aminotransferases (P < 0.05) and histopathological and ultrastructural features of injury. Hepatic microcirculatory blood flow was preserved in the RIPC+IR compared to IR group (P < 0.05). There was no mortality in any of the groups. By demonstrating a consistent improvement in these features of liver IR injury with antecedent hindlimb RIPC and by minimizing experimental confounding variables, we validated this mouse model. In conclusion, we describe a validated mouse model of hindlimb RIPC that reduces liver IR injury. With the availability of transgenic mice strains, this model should prove useful in unraveling the mechanisms of protection of hindlimb RIPC.

Antipsychotic-induced vacuous chewing movements and extrapyramidal side effects are highly heritable in mice

Pharmacogenomics is yet to fulfill its promise of manifestly altering clinical medicine. As one example, a predictive test for tardive dyskinesia (an adverse drug reaction consequent to antipsychotic exposure) could greatly improve the clinical treatment of schizophrenia but human studies are equivocal. A complementary approach is the mouse-then-human design in which a valid mouse model is used to identify susceptibility loci which are subsequently tested in human samples. We used inbred mouse strains from the Mouse Phenome Project to estimate the heritability of haloperidol-induced activity and orofacial phenotypes. 159 mice from 27 inbred strains were chronically treated with haloperidol (3 mg/kg/day via subdermal slow-release pellets) and monitored for the development of vacuous chewing movements (VCMs, the mouse analog of TD) and other movement phenotypes derived from open field activity and the inclined screen test. The test battery was assessed at 0, 30, 60, 90, and 120 days in relation to haloperidol exposure. As expected, haloperidol caused marked changes in VCMs, activity in the open field, and EPS. Unexpectedly, factor analysis demonstrated that these measures were imprecise assessments of a latent construct rather than discrete constructs. The heritability of a composite phenotype was ~0.9 after incorporation of the longitudinal nature of the design. Murine VCMs are a face valid animal model of antipsychotic-induced TD and heritability estimates from this study support the feasibility of mapping of susceptibility loci for VCMs.

The 22q11.2 microdeletion: Fifteen years of insights into the genetic and neural complexity of psychiatric disorders

Over the last fifteen years it has become established that 22q11.2 deletion syndrome (22q11DS) is a true genetic risk factor for schizophrenia. Carriers of deletions in chromosome 22q11.2 develop schizophrenia at rate of 25–30% and such deletions account for as many as 1–2% of cases of sporadic schizophrenia in the general population. Access to a relatively homogeneous population of individuals that suffer from schizophrenia as the result of a shared etiological factor and the potential to generate etiologically valid mouse models provides an immense opportunity to better understand the pathobiology of this disease. In this review we survey the clinical literature associated with the 22q11.2 microdeletions with a focus on neuroanatomical changes. Then, we highlight results from work modeling this structural mutation in animals. The key biological pathways disrupted by the mutation are discussed and how these changes impact the structure and function of neural circuits is described.

Does the learning deficit observed under an incremental repeated acquisition schedule of reinforcement in Ts65Dn mice, a model for Down syndrome, change as they age?

The Ts65Dn mouse is partly trisomic at chromosome 16 and is considered to be a valid mouse model of human Down syndrome. Prior research using an incremental repeated acquisition (IRA) schedule of reinforcement has revealed that there is a significant learning deficit in young, adult Ts65Dn mice compared to littermate controls. The purpose of this study was to examine whether this deficit changes during the life-span of these mice. In order to determine if changes in the deficit were caused by motoric or motivational deficiencies, a second group of mice was trained to respond under a performance version of the task (IRA-P). The IRA-P task required the same motor responses to produce the reinforcer, but no learning or acquisition was required. Data collected under the IRA task demonstrated that there was a significant learning impairment that persisted up to 24 months of age in the Ts65Dn mice compared to littermate controls. There was a significant decrease in the rate of responding and the number of milk presentations earned by the Ts65Dn mice after 19 months of age. However, during this time, response accuracy, which is independent of mobility and possibly motivation, did not decrease. Under the IRA-P schedule, there was no decrease observed in the number of milk presentations of either line as they aged, but the trend in the rate of responding of the Ts65Dn mice was similarly declining as the rate of responding observed in the Ts65Dn mice under the IRA task. These data indicate that the ability to learn in Ts65Dn mice does not decline with age as measured by the IRA task and suggests that perhaps Ts65Dn mice do not exhibit the same early onset Alzheimer's disease phenotype that is typically seen in human patients.

Allergic Airway Inflammation Initiates Long-Term Vascular Remodeling of the Pulmonary Circulation

Background: Asthma and allergic airway inflammation are associated with persistent structural alterations in the bronchi, i.e. airway remodeling. Previous studies have shown that during allergic airway inflammation, similar structural alterations may also be evoked in the pulmonary circulation. However, it remained unknown whether remodeling of the pulmonary circulation is as persistent as airway remodeling. The aim of this study is to investigate the reversibility and resolution of vascular remodeling, induced by allergic airway inflammation. Methods: A validated mouse model of allergic airway inflammation, utilizing ovalbumin as allergen, was employed. Animals were sacrificed 1 day, 1 week or 1 month after the last allergen exposure, and different parameters of remodeling (smooth muscle mass, proliferation of smooth muscle cells and endothelial cells as well as number of myofibroblasts and procollagen-I-producing cells) were investigated and quantified histologically. Results: Allergen exposure resulted in allergic airway inflammation characterized by a transient leukocyte infiltration and in structural alterations in both airway and vascular compartments. The increase in vascular smooth muscle mass and endothelial proliferation persisted at 1 month after the last allergen exposure. The other parameters and cellular inflammatory response returned to baseline within 1 month after the last allergen challenge. Conclusions: Based on the findings in this study, we conclude that acute allergic airway inflammation, although being initiated from the airways, is able to evoke similar long-term structural alterations in pulmonary vessels as described for bronchi.

Autoimmunity in Dry Eye Is Due to Resistance of Th17 to Treg Suppression

Dry eye disease (DED), an inflammatory autoimmune disorder affecting the ocular surface, degrades visual performance and the quality of life of >10 million people in the United States alone. The primary limitation in the effective treatment of DED is an incomplete understanding of its specific cellular and molecular pathogenic elements. Using a validated mouse model of DED, herein we functionally characterize the different T cell subsets, including regulatory T cells (Tregs) and pathogenic effector T cells, and determine their contribution to the pathogenesis of DED. Our data demonstrate the presence of dysfunctional Tregs and the resistance of pathogenic T cells, particularly Th17 cells, to Treg suppression in DED. In addition, we clearly show that in vivo blockade of IL-17 significantly reduces the severity and progression of disease, which is paralleled by a reduction in the expansion of Th17 cells and restoration of Treg function. Our findings elucidate involvement of a previously unknown pathogenic T cell subset (Th17) in DED that is associated specifically with Treg dysfunction and disease pathogenesis and suggest a new target for dry eye therapy.

Importance of TLR2 in Early Innate Immune Response to Acute Pulmonary Infection with Porphyromonas gingivalis in Mice

The periodontal pathogen Porphyromonas gingivalis is implicated in certain systemic diseases including atherosclerosis and aspiration pneumonia. This organism induces innate responses predominantly through TLR2, which also mediates its ability to induce experimental periodontitis and accelerate atherosclerosis. Using a validated mouse model of intratracheal challenge, we investigated the role of TLR2 in the control of P. gingivalis acute pulmonary infection. TLR2-deficient mice elicited reduced proinflammatory or antimicrobial responses (KC, MIP-1alpha, TNF-alpha, IL-6, IL-12p70, and NO) in the lung and exhibited impaired clearance of P. gingivalis compared with normal controls. However, the influx of polymorphonuclear leukocytes into the lung and the numbers of resident alveolar macrophages (AM) were comparable between the two groups. TLR2 signaling was important for in vitro killing of P. gingivalis by polymorphonuclear leukocytes or AM and, moreover, the AM bactericidal activity required NO production. Strikingly, AM were more potent than peritoneal or splenic macrophages in P. gingivalis killing, attributed to diminished AM expression of complement receptor-3 (CR3), which is exploited by P. gingivalis to promote its survival. The selective expression of CR3 by tissue macrophages and the requirement of TLR2 inside-out signaling for CR3 exploitation by P. gingivalis suggest that the role of TLR2 in host protection may be contextual. Thus, although TLR2 may mediate destructive effects, as seen in models of experimental periodontitis and atherosclerosis, we have now shown that the same receptor confers protection against P. gingivalis in acute lung infection.

MICRO-ULTRASOUND IMAGING FOR BLADDER CANCER IN A MURINE MODEL

4534 A validated mouse model for superficial bladder cancer is warranted to enable preclinical studies on intravesical anticancer agents. We sought to improve upon our previously described orthotopic superficial bladder cancer mouse model with validation using in vivo ultrasound imaging. Using the murine bladder tumor (MBT-2) cell line, 11 C3H/HeJ female mice were inoculated with 5 x 105 MBT-2 cells in 50 uL serum-free RPMI via transurethral catheterization without electrocautery. Using the VisualSonicsTM Vevo 770® ultrasound device, we performed transabdominal micro-ultrasound imaging 3, 6, and 15 days after tumor instillation. Imaging was also performed in all mice prior to tumor cell instillation. Tumors were measured using linear 2-dimensional measurements using sagittal and transverse imaging planes. Mouse bladders were subsequently harvested, formalin-fixed, embedded in paraffin, sectioned and stained with hematoxylin and eosin for confirmation of bladder tumors. Superficial bladder tumors were evident on micro-ultrasound imaging starting on day 3 post-MBT-2 cell instillation in all mice. Multifocal tumors were seen in 27% of mice on day 3, 91% on day 6, and 100% on day 15. Average number of tumors found on days 3, 6, and 15 was 1.3, 2.4, and 3.5. Mean tumor size (max dimension of largest tumor) on days 3, 6, and 15 was 0.7mm, 1.1mm, 1.3mm. Histology sections on all mice confirmed presence of bladder tumors in all mice. Using abdominal micro-ultrasound, we validate the presence of superficial bladder tumors in our MBT-2 mouse orthotopic bladder cancer model. Our model is reproducible and can be used to evaluate the efficacy of various intravesical anti-cancer agents. Tumors can be followed longitudinally for progression or regression with micro-ultrasound imaging.

Cognitive evaluation of disease-modifying efficacy of donepezil in the APP23 mouse model for Alzheimer’s disease

The interest for acetylcholinesterase inhibitors in the treatment of Alzheimer's disease has been greatly renewed owing to the discovery of a broad range of additional cholinergic and non-cholinergic effects, exploitable to maximize the efficacy of these drugs beyond merely improving intellectual functions at the symptomatic level. The age-dependent cognitive decline in the valid APP23 transgenic mouse model for Alzheimer's disease was employed to evaluate disease-modifying efficacy of chronic treatment with donepezil. At age 6 weeks, heterozygous APP23 mice and control littermates were subcutaneously implanted with osmotic pumps delivering saline or donepezil (0.27 or 0.58 mg/kg per day). After 2 months of treatment, a 3-week wash-out period was allowed to prevent bias from sustained symptomatic effects before cognitive evaluation in the Morris water maze commenced. Donepezil (0.27 mg/kg per day)-treated APP23 mice performed significantly better than their sham-treated counterparts during the Morris water maze acquisition phase and the subsequent probe or retention trial. Chronic donepezil (0.27 mg/kg per day) treatment improved spatial accuracy in APP23 mice as to reach the same level of performance as wild-type control animals on this complex visual-spatial learning task. This is the first study reporting disease-modifying efficacy of donepezil at the level of cognitive performance in transgenic mice modeling Alzheimer's disease.

α-Synucleinopathy models and human neuropathology: similarities and differences

Point mutations and genomic multiplications in the alpha-synuclein (alphaSYN) gene cause autosomal-dominant Parkinson's disease. Moreover, alphaSYN fibrils are the major component of Lewy bodies, the neuropathological hallmarks of Parkinson's disease and dementia with Lewy bodies as well as of glial cytoplasmic inclusions in multiple system atrophy. These diseases are collectively referred to as alpha-synucleinopathies. Cellular mechanisms regulating alphaSYN fibril formation and toxicity are intensely studied in vitro, and in cell culture and diverse animal models. Specific neuropathology was achieved in transgenic mouse models using several promoters to express human wild-type and mutant alphaSYN in brain regions affected by the various alpha-synucleinopathies. Somatodendritic accumulation of the transgenic alphaSYN with neuritic distortions was a common finding. The nigrostriatal dopaminergic projections were surprisingly resistant to alpha-synucleinopathy in transgenic mice, although they tended to be more vulnerable to neurotoxins. In a few mouse models, alphaSYN aggregated in an age-dependent manner into genuine fibrillar amyloid. Brain region selective alphaSYN neuropathology correlated with specific behavioral impairments, such as locomotor dysfunction and cognitive decline. Thus, the alphaSYN fibrillization process is tightly linked to neuropathology. The role and thus therapeutic potential of post-translational modifications (ubiquitinylation, oxidation, phosphorylation, truncation) and modifier genes on alphaSYN neuropathology can now be assessed in valid transgenic mouse models of alpha-synucleinopathies.

A validated mouse model for orthotopic bladder cancer using transurethral tumour inoculation and bioluminescence imaging

To describe a technique for transurethral tumour inoculation, bioluminescence imaging (BLI) and validation of this approach using ex vivo magnetic resonance imaging (MRI), as a reproducible and quantifiable model of orthotopic bladder cancer is required to enable preclinical pharmacological studies of intravesically administered anticancer agents and the use of BLI provides a sensitive method to monitor tumour growth over time. Human KU-7 bladder tumour cells were transduced with a lentiviral construct to stably express the firefly luciferase gene. These cells were then inoculated in female nude mice by intravesical instillation. BLI was performed weekly and the mice were killed after 4 weeks. Ex vivo MRI and whole-mount step-sections were obtained to assess bladder tumour volume. KU-7 tumour cells were highly tumorigenic and were successfully inoculated in 96% of mice. After 4 weeks, all tumours were confined to the mucosa and submucosa (</=pT1). There was an excellent correlation between tumour volume and BLI for both ex vivo bladder MRI (R(2) = 0.929) and end-point histological measurements (R(2) = 0.836). We have established and validated a reliable model of orthotopic bladder cancer that can be used to evaluate various methods of intravesical therapy. BLI allows excellent longitudinal surveillance and quantification of tumour burden.

Mouse models of autism spectrum disorders: The challenge for behavioral genetics

Autism is a severe neurodevelopmental disorder, which typically emerges early in childhood. The core symptoms of autism include deficits in social interaction, impaired communication, and aberrant repetitive behavior, including self-injury. Despite the strong genetic component for the disease, most cases of autism have not been linked to mutations in a specific gene, and the etiology of the disorder has yet to be established. At the present time, there is no generally accepted therapeutic strategy to treat the core symptoms of autism, and there remains a critical need for appropriate animal models and relevant behavioral assays to promote the understanding and treatment of the clinical syndrome. Challenges for the development of valid mouse models include complex genetic interactions underlying the high heritability of the disease in humans, diagnosis based on deficits in social interaction and communication, and the lack of confirmatory neuropathological markers to provide validation for genetic models of the disorder. Research focusing on genes that mediate social behavior in mice may help identify neural circuitry essential for normal social interaction, and lead to novel genetic animal models of the autism behavioral phenotype.

Validation of transgenic models of breast cancer: ductal carcinoma in situ (DCIS) and Brca1-mutation-related breast cancer

Available mouse models of ductal carcinoma in situ (DCIS) and BRCA1-mutation-related breast cancer are reviewed. The best validated mouse models of human DCIS are the conditional estrogen receptor α in mammary tissue (CERM) model initiated by deregulated estrogen receptor α and the serial explant mouse model initiated by p53 deficiency. At present the most useful and best validated mouse model of BRCA1-mutation-related breast cancer uses the cre-lox system to make a conditional Brca1 deletion targeted to mammary epithelial cells. The major shortcoming of the non-conditional Brca1 models is the high incidence of non-mammary tumor development. The use of mammary gland transplants or explants from these mice into nude hosts is one approach that could be used to circumvent this deficiency. Development and validation of a Brca1-mutation-related mouse model of basal cell breast cancer is an important next step.

Oral administration of recombinant Lactococcus lactis expressing bovine β‐lactoglobulin partially prevents mice from sensitization

The use of probiotics such as Lactococcus lactis and other lactic acid bacteria (LAB) has been proposed for the management of food allergy. However, no experimental study has clearly demonstrated any preventive or therapeutic inhibition of an allergen-specific IgE response. We aimed to study the immunomodulatory effect of recombinant L. lactis expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, in a validated mouse model of allergy. Six-week-old female Balb/c mice received five repeated doses of BLG, of L. lactis plus BLG, or of recombinant L. lactis by gavage. Different recombinant strains were inoculated, which corresponded to BLG doses ranging from 4 to 70 microg/mice. Mice were then sensitized by intra-peritoneal injection of BLG emulsified in incomplete Freund's adjuvant to induce high IgE concentrations. Pre-treatment with natural L. lactis plus BLG allowed induction of BLG-specific T-helper type 1 (Th1) response, and abrogated the oral tolerance induced by BLG alone, demonstrating the adjuvant effect of this non-colonizing LAB. Moreover, pre-treatment with some of the recombinant strains favoured the development of a Th1 response inhibiting the Th2 one: it induced a significant decrease of specific IgE response, and an intense increase of specific IgG2a and IFN-gamma productions. The most efficient strains that inhibited the IgE response were those producing the highest amounts of the BLG protein. Oral administration of some recombinant L. lactis was demonstrated to induce a specific Th1 response down-regulating a further Th2 one. Prophylaxis protocols will thus be evaluated using the most efficient strains.

Introducing a mouse model for pre-eclampsia: adoptive transfer of activated Th1 cells leads to pre-eclampsia-like symptoms exclusively in pregnant mice.

Pre-eclampsia (PE) is the most severe pregnancy-related disease, leading to high maternal and fetal morbidity/mortality. Immunological imbalances associated with endothelial cell dysfunction have been hypothesized as a cause for the onset and perpetuation of PE. Valid and reliable animal models are urgently required to test this hypothesis and to better understand the mechanisms underlying PE. We developed a novel PE-model by adoptively transferring activated BALB/c Th1-like splenocytes into allogeneically pregnant BALB/c female mice during late gestation; the model mimicked the symptoms of PE, i.e. increased blood pressure and glomerulonephritis accompanied by proteinuria. Interestingly, these PE-like symptoms were not detectable in non-pregnant recipients of activated Th1-like cells. Adoptive cell transfer adversely affected the outcome of pregnancy by increasing fetal rejection, with uterine immune cells showing an inflammatory profile. In conclusion, we have established a valid and reliable PE mouse model, which opens vast opportunities for therapeutic interventions.

Transgenic overexpression of caveolin-3 in the heart induces a cardiomyopathic phenotype.

Caveolins are structural protein components of caveolar membrane domains. Caveolin-3, a muscle-specific member of the caveolin family, is expressed in skeletal muscle tissue and in the heart. The multiple roles that caveolin-3 plays in cellular physiology are becoming more apparent. We have shown that lack of caveolin-3 expression in skeletal muscle resembles limb-girdle muscular dystrophy-1C. In contrast, we have demonstrated that overexpression of caveolin-3 in skeletal muscle tissue promotes defects similar to those seen in Duchenne muscular dystrophy (DMD). Thus, a tight regulation of caveolin-3 expression is fundamental for normal muscle functions. Since caveolin-3 is also endogenously expressed in cardiac myocytes, and cardiomyopathies are observed in DMD patients, we looked at the effects of overexpression of caveolin-3 on cardiac structure and function by characterizing caveolin-3 transgenic mice. Our results indicate that overexpression of caveolin-3 causes severe cardiac tissue degeneration, fibrosis and a reduction in cardiac functions. We also show that dystrophin and its associated glycoproteins are down-regulated in caveolin-3 transgenic heart. In addition, we demonstrate that the activity of nitric oxide synthase (NOS) is down-regulated by high levels of caveolin-3 in the heart. Taken together, these results indicate that overexpression of caveolin-3 is sufficient to induce severe cardiomyopathy. In addition, these findings suggest that caveolin-3 transgenic mice may represent a valid mouse model for studying the molecular mechanisms underlying cardiomyopathies associated with Duchenne muscular dystrophy.

Modulation of myoblast fusion by caveolin-3 in dystrophic skeletal muscle cells: implications for Duchenne muscular dystrophy and limb-girdle muscular dystrophy-1C.

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-3 is the principal structural component of caveolae in skeletal muscle cells in vivo. We have recently generated caveolin-3 transgenic mice and demonstrated that overexpression of wild-type caveolin-3 in skeletal muscle fibers is sufficient to induce a Duchenne-like muscular dystrophy phenotype. In addition, we have shown that caveolin-3 null mice display mild muscle fiber degeneration and T-tubule system abnormalities. These data are consistent with the mild phenotype observed in Limb-girdle muscular dystrophy-1C (LGMD-1C) in humans, characterized by a approximately 95% reduction of caveolin-3 expression. Thus, caveolin-3 transgenic and null mice represent valid mouse models to study Duchenne muscular dystrophy (DMD) and LGMD-1C, respectively, in humans. Here, we derived conditionally immortalized precursor skeletal muscle cells from caveolin-3 transgenic and null mice. We show that overexpression of caveolin-3 inhibits myoblast fusion to multinucleated myotubes and lack of caveolin-3 enhances the fusion process. M-cadherin and microtubules have been proposed to mediate the fusion of myoblasts to myotubes. Interestingly, we show that M-cadherin is downregulated in caveolin-3 transgenic cells and upregulated in caveolin-3 null cells. For the first time, variations of M-cadherin expression have been linked to a muscular dystrophy phenotype. In addition, we demonstrate that microtubules are disorganized in caveolin-3 null myotubes, indicating the importance of the cytoskeleton network in mediating the phenotype observed in these cells. Taken together, these results propose caveolin-3 as a key player in myoblast fusion and suggest that defects of the fusion process may represent additional molecular mechanisms underlying the pathogenesis of DMD and LGMD-1C in humans.

Progress toward valid transgenic mouse models for Alzheimer’s disease

A transgenic mouse model for Alzheimer’s disease (AD) should mimic the age-dependent accumulation of β-amyloid plaques, neurofibrillary tangles, neuronal cell death as well as display memory loss and behavioral deficits. Age-dependent accumulation of Aβ deposits in mouse brain has been achieved in mice overexpressing mutant alleles of the amyloid precursor protein (APP). In contrast, mice bearing mutant alleles of the presenilin genes show increased production of the Aβ42 peptide, but do not form amyloid deposits unless mutant alleles of APP are also overproduced. Furthermore, the onset of Aβ deposition is greatly accelerated, paralleling the involvement of presenilins in early onset AD. Studies of APP and presenilin transgenic mice have shown 1) the absence of a requirement for a maturation step in dense core plaque formation, 2) evidence that β-amyloid deposition is directed by regional factors, and 3) behavioral deficits are observed before Aβ deposition. Crosses of APP transgenic mice with mice modified for known AD risk factors and “humanizing” the mouse may be necessary for complete replication of AD.