Week-old Mice Research Articles

Age-related expression of plasma membrane Ca(2+) -ATPase isoform 2 in the cochleas of C57BL/6J mice

To investigate the location and distribution of plasma membrane Ca²⁺ -ATPase isoform 2(PMCA2) in the cochleas of C57BL/6J mice at various ages (4w, 14w, 22w, 45w), and to reveal the relationship of PMCA2 and age-related hearing loss (AHL). The distribution of PMCA2 in the cochleas of C57BL/6J mice was detected by immunohistochemistry at various ages (4w, 14w, 22w, 45w). Real-time polymerase chain reaction (Rt-PCR) was used to detect the level of PMCA2 mRNA in the cochleas of C57BL/6J mice at the ages of 4, 14, 22 and 45 weeks old respectively. Using SPSS17.0 software for statistical analysis. PMCA2 was mainly located in the hear cells, stria vascularis, and spiral ganglion cells. Faint labeling of PMCA2 was also observed in spiral ligament. Hair cells missed and the number of spiral ganglion cells reduced with age. Expression of PMCA2 in the cochleas of C57BL/6J mice also showed age-related decreasing. The results of Rt-PCR demonstrated the expression of mRNA of gene (Atp2b2) at 14 weeks age was significantly less than 4 week-old mice cochlears (P<0.05). The expression of mRNA of gene (Atp2b2) at 22 weeks age was significantly less than 14 week-old mice cochlears (P<0.05). The expression of mRNA of gene (Atp2b2) at 45 weeks age was significantly less than 14 week-old mice cochlears (P<0.01). PMCA2 is mainly located in the hear cells, stria vascularis, and spiral ganglion cells. Faint labeling of PMCA2 is also observed in spiral ligament. The expression of PMCA2 demonstrates an age-related decrease with age. The mRNA expression level of PMCA2 gene(Atp2b2) in the cochleas of C57BL/6J mice displayed an age-related decrease. PMCA2 transporters may play a critical role in maintaining the normal morphology of the inner ear and it may be related to AHL.

The Angiotensin Converting Enzyme Inhibitor Lisinopril Improves Muscle Histopathology but not Contractile Function in a Mouse Model of Duchenne Muscular Dystrophy.

Background: Angiotensin converting enzyme inhibitors (ACEi) are the current standard of care treatment for cardiac dysfunction in Duchenne muscular dystrophy patients. We previously showed treatment with an ACEi plus mineralocorticoid receptor (MR) antagonist improves limb and respiratory skeletal muscles, in addition to cardiac muscles, in a dystrophic mouse model at 20 weeks-of-age. Objective: To determine whether previously observed preclinical benefits of an ACEi plus MR antagonist on dystrophic skeletal muscles can be reproduced by increasing ACEi dosage alone. We also compared functional and histological outcome measures at 10 and 20 weeks-of-age. Methods: Dystrophin deficient utrophin haplo-insufficient (utrn +/- ; mdx) “het” mice were treated with 10, 20, or 50 mg/kg × day of the ACEi lisinopril from 4 to 10 weeks-of-age via water bottles and compared with C57BL/10 wild-type control mice and untreated hets. Data from 10 week-old het mice were also compared to data collected from an untreated het group at 20 weeks-old. In vivo cardiac and grip strength measurements, in vitro diaphragm and extensor digitorum longus muscle force measurements, and histopathological analyses were performed. One-way ANOVA followed by Dunnett post hoc comparison was used to determine significance. Results: ACEi treatment reduced skeletal muscle damage but had no significant effect on muscle force. Body weight, heart rate, grip strength and blood pressure were unaffected by treatment. Limb muscle histopathology was more informative at 10 than 20 weeks-of-age.Conclusions: These results suggest increased ACEi dosage alone cannot improve all dystrophic parameters. Further optimization of MR antagonists in 20 week-old mice is warranted.

Biochemical and histopathological ultrastructural changes caused by ZnO nanoparticles in mice

Five week-old mice were divided into a vehicle control group, and groups exposed to ZnO nanoparticles at low (0.5 g/kg), middle (1 g/kg), high (3 g/kg), and exceptionally high-dose (5 g/kg). After the first, second, third, and fourth weeks’ of exposure, blood biochemistry, histopathology, and electron microscopic ultrastructural changes in liver, kidney and spleen were investigated. Increased alkaline phosphatase activities were observed in all treated mice being statistically significant at higher dose. No changes were observed in the serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, creatinine, blood urea nitrogen, and lipid levels. During the first and second weeks of the treatment, effects on the cytoarchitecture of liver, kidney, and spleen were not perceived while during the third and fouth weeks of treatment sporadic mild effects were seen. Ultrastructural electron microscopic changes in liver, kidney, and spleen were not observed for the low-dose group on the first, second, third, and fourth weeks, suggesting that exposure to ZnO nanoparticles at low dose is safe. Long-term (i.e., more than 28 days) exposure to the exceptionally high-dose resulted in sporadic changes in nuclear chromatin condensation, irregular nuclear membrane, polymorphic mitochondria, mitochondrial swelling, and vacuolation. ZnO nanoparticles could be well tolerated and no death occurred in any group of treated mice.

Chromosome Condensation 1-Like (Chc1L) Is a Novel Tumor Suppressor Involved in Development of Histiocyte-Rich Neoplasms.

Human chromosomal region 13q14 is a deletion hotspot in prostate cancer, multiple myeloma, and chronic lymphocytic leukemia. This region is believed to host multiple tumor suppressors. Chromosome Condensation 1-like (CHC1L) is located at 13q14, and found within the smallest common region of loss of heterozygosity in prostate cancer. Decreased expression of CHC1L is linked to pathogenesis and progression of both prostate cancer and multiple myeloma. However, there is no direct evidence for CHC1L’s putative tumor suppressing role in current literature. Presently, we describe the generation and characterization of Chc1L knockout mice. Chc1L -/- mice do not develop cancer at a young age, but bone marrow and spleen cells from 8–12 week-old mice display an exaggerated proliferative response. By approximately two years of age, knockout and heterozygote mice have a markedly increased incidence of tumorigenesis compared to wild-type controls, with tumors occurring mainly in the spleen, mesenteric lymph nodes, liver and intestinal tract. Histopathological analysis found that most heterozygote and knockout mice succumb to either Histiocytic Sarcoma or Histiocyte-Associated Lymphoma. Our study suggests that Chc1L is involved in suppression of these two histiocyte-rich neoplasms in mice and supports clinical data suggesting that CHC1L loss of function is an important step in the pathogenesis of cancers containing 13q14 deletion.

Abstract LB-140: Investigating cancer stem cells in glioblastoma initiation and recurrence

Abstract In this study, I examine the unique features of quiescent cancer stem cells (CSCs) in glioblastoma initiation and recurrence in genetically engineered mouse models. A nestin-TK-GFP transgene was used to label CSCs with GFP and to render them susceptible to ganciclovir (GCV) treatment in a fully penetrant mouse model of GBM (M7: hGFAP-Cre; Nf1fl/+; p53fl/fl; Ptenfl). Using food-mediated GCV delivery (GCV chow) to optimize the drug delivery, Doublecortin-positive and NG2-positive cells derived from the nestin-TK-GFP transgene labeled neural stem cells were significantly reduced after six weeks. In addition, on GCV chow the tumor-bearing mice containing the nestin-TK-GFP transgene live significantly longer than the ones without the transgene. Isolation and transplantation of the GFP+/GFP- tumor cells demonstrated that the GFP+ cells have enhanced tumorigenic potential over the GFP- cells. These results support the hypothesis that the nestin-TK-GFP transgene targets the CSCs that are responsible for the sustained GBM growth. In the M7 model, the hGFAP transgene has a broader expression, and thus broader Cre targeting, than the nestin transgene. To eradicate the problem of differing scopes of Cre expression versus targeting of CSCs, I generated a new transgene (CGD: nestin-CreERT2-H2BeGFP-hDTR) that enables Cre-mediated recombination, specific cell labeling and targeted cell ablation. The CGD is designed to express a fusion protein that consists of CreERT2, H2B-eGFP, and hDTR (human diphtheria toxin receptor). Containing P2A linkers between the three gene cassettes, the fusion protein can be efficiently cleaved into three active. I have screened for a transgenic line that can induce GBM formation upon tamoxifen induction. Six to eight week-old mice with the following genetic configuration: CGD; Nf1fl/+; p53fl/+; Ptenfl/+, were given one dose of tamoxifen and all mice died of high grade gliomas three to six months later. The analysis of the CGD-induced tumors demonstrated that the CGD transgene labels tumor cells that are relatively quiescent, compared to the highly proliferative Ki67+ cells. Thus our CGD transgene labels putative CSCs within the tumor. In this model, the same cells initiating tumor formation (Cre-expressing) are also expressing H2B-eGFP and hDTR. Thus, all the tumor cells are derived from the CGD transgene and all the CSCs should be labeled with the H2B-eGFP protein. This dramatically facilitates the sorting of the tumor-initiating cells. Transplantation of the GFP+/GFP- cells in these tumors revealed that the quiescent GFP+ cells are more tumorigenic than the proliferating GFP- cells. Furthermore, diphtheria toxin-mediated ablation of the hDTR-expressing cells significantly elongates the lives of tumor-bearing mice. To mimic the endogenous environment for GBM growth, wild-type mice were also used to prove the differential tumorigenic ability of the GFP+/- cells. These results validate the essential roles of CSCs in GBM initiation and recurrence. Citation Format: xuanhua xie, Xiuping Zhou, German B. Sánchez, Luis F. Parada. Investigating cancer stem cells in glioblastoma initiation and recurrence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-140. doi:10.1158/1538-7445.AM2015-LB-140

Abstract 898: Exercise inhibits the growth of breast cancer and reduces fat mass in postmenopausal obese mice associated with a circulating angiostatic phenotype and the inhibition of angiogenesis in those tissues

Abstract Obesity and physical inactivity are important risk factors for cancers, especially for postmenopausal breast cancer. However, potential mechanisms of exercise in reducing obesity and preventing postmenopausal breast cancer are poorly understood. The animal models in this area are very limited. In the present study, we used a postmenopausal obese mouse model inoculated with mouse breast cancer (E0771) cells to examine the potential effects of long-term exercise in reducing fat mass and breast cancer progression. Ovariectomy (OVX) was performed in 12 sixty week-old female mice (C57BL/6J), then followed by a high-fat (60%) diet for 12 weeks. In the eighth week of the dietary program, the body weight (BW) was significantly increased from 32.1±1.8 to 42.5±2.3 g; P&amp;lt;0.01), then 10^6 E0771 (mouse breast cancer) cells were injected in the left fourth mammary gland. The mice were randomized into two groups, exercise and sedentary (n = 6). The exercise was conducted in a voluntary running cage system, i.e., 19.7±2.8 m/min, 30 min in the morning and 30 min in the afternoon, 7days/week, for 4 weeks. The lean mass (LM) or fat mass (FM) composition was monitored by EchoMRI weekly. In the end of experiment, the mice were anesthetized by isoflurane 2 hours after the last exercise, and blood samples, visceral fat and tumors were collected for measuring the expression of endostatin, sFlt-1 and free-VEGF using ELISA and blood vessel density (BVD) using CD31 immunochemistry. There was a significant decrease in BW (40.5±1.9 vs. 45.7±2.3 g; P&amp;lt;0.01) and tumor weight (1.95±0.41 vs. 2.63±0.11 g; P&amp;lt;0.01) in exercised-group compared to sedentary group, respectively. Exercise significantly decreased FM (17.8±0.9 vs. 22.7±1.1 g; P&amp;lt;0.01), tumor VEGF levels (44.1±2.1 vs. 65.4±3.3 pg/mg protein; P&amp;lt;0.01), and BVD (147.2±6.3 vs. 168.3±5.1 BV#/mm^2; P&amp;lt;0.01) in comparison with the sedentary, respectively. Exercise significantly decreased a ratio of capillaries to adipocytes of visceral fat in comparison with sedentary group (0.63±0.02 vs. 0.89±0.05; P&amp;lt;0.01). Exercise significantly increased endostatin (33.3±1.6 vs. 24.1±1.2 ng/ml; P&amp;lt;0.01) and sFlt-1 (59.5±3.1 vs. 48.3±2.5 pg/ml; P&amp;lt;0.05), and decreased free-VEGF (42.5±2.1 vs. 61.3±3.1 pg/ml; P&amp;lt;0.01) in the circulation in comparison with sedentary group, respectively. In postmenopausal obese mice, a long term exercise significantly 1) inhibited breast tumor angiogenesis and growth, 2) limited fat mass accumulation by reducing a ratio of capillaries to adipocytes, and 3) induced a circulating angiostatic phenotype characterized by increased endostatin and sFlt-1 and decreased free VEGF. These findings support the hypothesis that exercise-induced circulating angiostatic phenotype may contribute to the prevention of breast cancer progression and obesity in postmenopausal women. Citation Format: Shelby A. Cole, Jian-Wei Gu. Exercise inhibits the growth of breast cancer and reduces fat mass in postmenopausal obese mice associated with a circulating angiostatic phenotype and the inhibition of angiogenesis in those tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 898. doi:10.1158/1538-7445.AM2015-898

In vivo micronucleus test of n-butyl acetate to classify a chemical’s mutagenicity according to GHS

To clarify the chemical hazard and classify the category of germ cell mutagenicity, a micronucleus (MN) assay of n-butyl acetate was conducted in mouse to investigate its mutagenicity using male Institute for Cancer Research (ICR) mice bone marrow cells. Also this test has not been conducted yet. The seven week-old male ICR mice were tested at three dosages for this chemical. After 24 hr of oral administration with n-butyl acetate, the mice were sacrificed and their bone marrow cells were prepared for smearing slides. As a result of counting the micronucleated polychromatic erythrocyte (MNPCE) of 2,000 polychromatic erythrocytes, the n-butyl acetate did not inhibit the bone marrow cell proliferation in all treated groups, and did make the MN induction. Based on the results of this test, it is concluded that this chemical did not increase the incidence of micronuclei under the present test conditions, and did not classified as a mutagen in the globally harmonized system of classification and labelling of chemicals (GHS).

Abstract 626: Vascular Endothelial HSP60 and Von Willebrand Factor Induction Are Both Involved in Promoting Fatty-Streak Formation in ApoE Null Mice

Objectives: This study was designed to determine the murine atherosclerosis in ApoE null mice with inducible expression of human heat shock protein 60 (hHSP60) in vascular endothelium. Background: Autoimmunity to HSP60 may be involved in eliciting early atherosclerotic lesions. Most classical risk factors of atherosclerosis were previously shown in cell culture models to induce HSP60 expression in vascular ECs and in vivo particularly at regions predilected to lesions. However, it has not been demonstrated directly in an animal model whether HSP60 induction in ECs are capable of influencing lesion formation. Methods: We developed ApoE -/- ::Cdh5-CreER T2 ::G-Lox-HSP60 triple Tg mouse model with tamoxifen-induced hHSP60 expression in ECs. Eight week-old triple TG mice were fed by tamoxifen citrate-contained chow for 2 weeks, followed by high fat chow (HF) for additional 4 and 8 weeks before sacrificed for oil red staining for fatty streaks and IHC staining. Results: In tamoxifen-fed triple Tg mouse, vascular EC expression of hHSP60 increased fatty-streak formation and increased the severity of lesions in comparison with uninduced triple Tg or wildtype ApoE mouse. In triple Tg mouse, we found tamoxifen strongly induced HSP60 expression in ECs of the lesions and to a lesser degree in ECs of veins and other arterial regions not predilected to lesions. Concomitant VWF induction at ECs and subendothelial regions was observed at areas with increased HSP60 expression. Conclusions: hHSP60 induction at vascular ECs accelerates fatty-steak formation in ApoE null mice that may involve increasing local VWF expression.

714. Gene Therapy Rescues Cone Function and Viability in an Rp2 Knockout Mouse Model for X-Linked Retinitis Pigmentosa Over a Wide Dose Range and a Broad Therapeutic Time Window

Retinitis pigmentosa (RP) refers to a diverse group of inherited retinal dystrophies characterized by a progressive loss of photoreceptors, resulting in vision impairment or blindness. The prevalence of the disease is 1 in 3,500 to 4,000 worldwide. X-linked RP (XLRP) accounts for ~15% of all RP cases and is one of the most severe forms of retinal degeneration. About 10% of the total XLRP cases are caused by mutations in the RP2 gene. A recently developed Rp2 knockout (Rp2-KO) mouse model exhibits early onset, progressive cone photoreceptor dysfunction and degeneration, mimicking early cone involvement in patients with RP2 mutations. To develop gene therapy for the disease, we generated and characterized a self-complementary AAV8 vector carrying the human RP2 expression cassette (AAV8-scRK-RP2), and examined its efficacy in the Rp2-KO mice. Our initial results demonstrated successful cone function rescue following subretinal administration of the vector to the mice (ASGCT 2014, poster#122). In the present study, we continued to examine the long term safety and efficacy of the vector using a broader dose range. Four to six week-old Rp2-KO mice received unilateral subretinal injections of the vector with doses ranging from 5×107 to 1×109 vector genomes (vg)/eye. The fellow eyes were injected with vehicle as controls. Retinal function was assessed by electroretinography (ERG) and optomotor test up to 18 months of age. Our results revealed significantly higher amplitude and faster recovery of cone ERG in vector-treated eyes than control eyes in almost all dose groups through the duration of monitoring starting from 4 months of age. In addition, remarkably better visual acuity was observed in the vector-treated eyes at 18 months of age. The treatment also reversed M-opsin mis-localization, restored cone PDE expression and maintained cone viability. Interestingly, this rescue was achieved in the Rp2-KO mice even when treated at 10-month of age, suggesting the potential benefit of the treatment to older patients. Retinal toxicity was only observed in mice receiving the highest vector dose (1×109 vg/eye), as reflected by reduced rod ERG amplitude and thinner photoreceptor layer. We conclude that our RP2 AAV vector can rescue cone function and viability over a wide dose range and a broad therapeutic time window, which paves the way for future clinical studies.

Genome-wide association study identifies nox3 as a critical gene for susceptibility to noise-induced hearing loss.

In the United States, roughly 10% of the population is exposed daily to hazardous levels of noise in the workplace. Twin studies estimate heritability for noise-induced hearing loss (NIHL) of approximately 36%, and strain specific variation in sensitivity has been demonstrated in mice. Based upon the difficulties inherent to the study of NIHL in humans, we have turned to the study of this complex trait in mice. We exposed 5 week-old mice from the Hybrid Mouse Diversity Panel (HMDP) to a 10 kHz octave band noise at 108 dB for 2 hours and assessed the permanent threshold shift 2 weeks post exposure using frequency specific stimuli. These data were then used in a genome-wide association study (GWAS) using the Efficient Mixed Model Analysis (EMMA) to control for population structure. In this manuscript we describe our GWAS, with an emphasis on a significant peak for susceptibility to NIHL on chromosome 17 within a haplotype block containing NADPH oxidase-3 (Nox3). Our peak was detected after an 8 kHz tone burst stimulus. Nox3 mutants and heterozygotes were then tested to validate our GWAS. The mutants and heterozygotes demonstrated a greater susceptibility to NIHL specifically at 8 kHz both on measures of distortion product otoacoustic emissions (DPOAE) and on auditory brainstem response (ABR). We demonstrate that this sensitivity resides within the synaptic ribbons of the cochlea in the mutant animals specifically at 8 kHz. Our work is the first GWAS for NIHL in mice and elucidates the power of our approach to identify tonotopic genetic susceptibility to NIHL.

Metabolic dysfunction and altered mitochondrial dynamics in the utrophin-dystrophin deficient mouse model of duchenne muscular dystrophy.

The utrophin-dystrophin deficient (DKO) mouse model has been widely used to understand the progression of Duchenne muscular dystrophy (DMD). However, it is unclear as to what extent muscle pathology affects metabolism. Therefore, the present study was focused on understanding energy expenditure in the whole animal and in isolated extensor digitorum longus (EDL) muscle and to determine changes in metabolic enzymes. Our results show that the 8 week-old DKO mice consume higher oxygen relative to activity levels. Interestingly the EDL muscle from DKO mouse consumes higher oxygen per unit integral force, generates less force and performs better in the presence of pyruvate thus mimicking a slow twitch muscle. We also found that the expression of hexokinase 1 and pyruvate kinase M2 was upregulated several fold suggesting increased glycolytic flux. Additionally, there is a dramatic increase in dynamin-related protein 1 (Drp 1) and mitofusin 2 protein levels suggesting increased mitochondrial fission and fusion, a feature associated with increased energy demand and altered mitochondrial dynamics. Collectively our studies point out that the dystrophic disease has caused significant changes in muscle metabolism. To meet the increased energetic demand, upregulation of metabolic enzymes and regulators of mitochondrial fusion and fission is observed in the dystrophic muscle. A better understanding of the metabolic demands and the accompanied alterations in the dystrophic muscle can help us design improved intervention therapies along with existing drug treatments for the DMD patients.

Short and Long‐term Pro‐Inflammatory Mediator Responses in Prostate Adenocarcinoma Progression in TRAMP Mice after Goniothalamin Therapy

The role of pro‐inflammatory mediators has been widely investigated due to their relevance in prostate cancer (CaP). The aim of the study was to evaluate the short and long term responses of pro‐inflammatory mediators correlated with different grades of CaP in TRAMP mice after Goniothalamin treatment. The control groups were TC8, TC12 and TC22 (8, 12 and 22 week‐old mice). The treated groups received Goniothalamin (150mg/Kg, orally) for 4 weeks (from 8 to 12 week‐old mice) and were sacrificed at different ages, (T1GTN group/12 week‐old mice and T2GTN group/22 week‐old mice). The ventral lobe was collected for light microscopy, ELISA and Western Blotting analyses. Low‐grade and high‐grade PIN in TC8 and TC12 groups were observed. Well‐developed and undifferentiated adenocarcinoma characterized the prostate in the TC22 group. The Goniothalamin treatment led to a decrease of lesion frequency in the treated groups. Decreased NF‐κB and p‐STAT3 levels were seen in TRAMP mice immediately after the treatment. Despite having higher levels of these molecules in the T2GTN group, these levels still showed a decrease in relation to the TC22 group. TNF‐α, IL‐17 and IL1‐β serum levels showed a tendency to decrease with the treatment. The Goniothalamin reduced pro‐tumorigenic effects related to inflammation in the prostate in early and late neoplastic development. Also, the reduction of NF‐kB and p‐STAT3 transcription factor levels led to decreased proliferative process frequency. Thus, the Goniothalamin significantly lessened the pro‐inflammatory signaling, retarding lesion progression and highlighting the relevance of the inflammatory process in early CaP development. Fapesp 2013/01294‐8, 23049‐5

Chronic Sweetener Intake and its Effects on IGF‐II and IGF‐IIR Expression in the Mouse Brain.

Chronic excessive intake of refined sugars contributes to the incidence of pathologies such as type 2 diabetes, metabolic syndrome and cardiovascular disease. Sweeteners are an alternative used to replace sugar in food. Although they are considered innocuous, recent studies have shown they may have unwanted effects in humans. The objective of this study is to determine the expression of the growth factor IGF‐II and its receptor in the central nervous system of mice supplemented with sweeteners for prolonged periods of time. Four groups of 8 week‐old mice were used: A control group (without supplementation), and three groups supplemented with either sucrose, stevia or sucralose in their drinking water. The animals were supplemented for 6 weeks. After supplementation, the brain and spinal cord were dissected and their proteins purified to determine the expression of IGF‐II or IGF‐IIR by western blot. Our results show decreased expression of IGF‐II only on brain tissue from male individuals in the stevia‐supplemented group, compared to all other groups, whereas the expression of IGF‐IIR decreased for both males and females also in the stevia group, but not in the other groups. Our results suggest that chronic intake of stevia may alter the expression of IGF‐II and IGF‐IIR in cells from the central nervous system, which may have detrimental effects on essential physiologic mechanisms in nervous tissue.

Spatially Localized Disruptions of Voltage Activated Calcium Release in Mtm1-Deficient Muscle Fibers

Mutations in the gene encoding the phosphoinositide phosphatase myotubularin (Mtm1) are responsible for myotubular myopathy. We previously showed that muscle fibers from Mtm1-deficient mouse suffer from defective excitation-contraction coupling. Here we measured voltage-clamp activated Ca2+ signals in fibers from the flexor digitorum brevis muscles of 4 week-old wild type (WT) and Mtm1-ko mice under line-scan confocal microscopy using the dye rhod-2. Ca2+ release in the diseased fibers was deficient over the full range of activation: fitting the voltage dependence of the peak rate of rise of the line-averaged rhod-2 F/F0 signals with a Boltzmann function gave mean values for maximum rate, midpoint voltage and slope of 0.29 ± 0.02 and 0.14 ± 0.02 F/F0.ms−1, −8.8 ± 1.5 and 0.95 ± 2.3 mV, 7.0 ± 0.4 and 9.2 ± 0.7 mV in WT (n=22) and Mtm1-ko fibers (n=15), respectively. Furthermore, the mean time to peak rate was significantly delayed by 5-10 ms in Mtm1-ko as compared to WT fibers. These global alterations were associated with severe spatial inhomogeneity of Ca2+ release in the diseased fibers with rhod-2 transients yielding localized disruptions along the scanned line including reduced peak amplitude but also delayed or slower rate of onset, suggestive of specific alteration of the early peak component of the rate of Ca2+ release. In fibers treated with wortmannin and LY294002, the properties of the line-averaged rhod-2 F/F0 signals were unchanged in WT fibers but the mean maximum rate of rise of the rhod-2 signal was enhanced by 60% in Mtm1-deficient fibers. Results show that Mtm1-deficiency provokes EC coupling failure through accumulation of spatially localized disruptions of Ca2+ release. They also suggest that pharmacological inhibition of PtdIns-3-kinase activity has the potency to alleviate defective EC coupling in the diseased fibers.

Therapeutic potential of human adipose tissue-derived multi-lineage progenitor cells in liver fibrosis

IntroductionLiver fibrosis is characterized by excessive accumulation of extracellular matrix. In a mouse model of liver fibrosis, systemic injection of bone marrow mesenchymal stem cells (BM-MSCs) was considered to rescue the diseased phenotype. The aim of this study was to assess the effectiveness of human adipose tissue-derived multi-lineage progenitor cells (hADMPCs) in improving liver fibrosis. Methods and resultshADMPCs were isolated from subcutaneous adipose tissues of healthy volunteers and expanded. Six week-old male nude mice were treated with carbon tetra-chloride (CCl4) by intraperitoneal injection twice a week for 6weeks, followed by a tail vein injection of hADMPCs or placebo control. After 6 more weeks of CCl4 injection (12weeks in all), nude mice with hADMPCs transplants exhibited a significant reduction in liver fibrosis, as evidenced by Sirius Red staining, compared with nude mice treated with CCl4 for 12weeks without hADMPCs transplants. Moreover, serum glutamic pyruvate transaminase and total bilirubin levels in hADMPCs-treated nude mice were lower levels than those in placebo controls. Production of fibrinolytic enzyme MMPs from hADMPCs were examined by ELISA and compared to that from BM-MSCs. MMP-2 levels in the culture media were not significantly different, whereas those of MMP-3 and -9 of hADMPCs were higher than those by BM-MSCs. ConclusionThese results showed the mode of action and proof of concept of systemic injection of hADMPCs, which is a promising therapeutic intervention for the treatment of patients with liver fibrosis.

Abstract 13407: A Mouse Model of Heart Failure with Preserved Ejection Fraction (HFpEF) due to Chronic Infusion of a Low Subpressor Dose of Angiotensin II

Introduction: Heart failure with preserved ejection fracture (HFpEF) is a clinical syndrome of HF symptoms associated with impaired diastolic function. Although it represents approximately 50% of all patients with HF, the mechanisms of disease are poorly understood, animal models of HFpEF not due pressure overload are lacking, and therapies for HFpEF are generally ineffective. Hypothesis: A continuous infusion of low dose of angiotensin II (ATII) may be sufficient to induce changes in left ventricular (LV) diastolic function without increasing blood pressure nor induce LV hypertrophy. Methods: Osmotic pumps were implanted subcutaneously in 8 week-old CD1 male mice randomly assigned to the ATII (200 ng/kg/day) or vehicle (N=8/group). Transthoracic echocardiography was performed at baseline and 4 weeks to measure LV dimensions, systolic and diastolic function. Aortic systolic and diastolic pressures (AoP), LV peak systolic and end-diastolic pressures (LVPSP, LVEDP) were measured at LV catheterization. Fibrosis was measured using Masson’s trichrome stain. The expression of Interleukin (IL)-18 mRNA levels, a cytokine associated with impaired cardiomyocyte relaxation, was measured at 4 weeks. Results: When compared to the baseline values or vehicle group, ATII infusion had no effects on AoP, LVPSP and HR, and had no effects on LV dimensions or mass, nor on LVEF (all P&gt;0.2). ATII induced a significant impairment in LV diastolic function as measured by an increase (worsening) of the myocardial performance index (MPI), the LV isovolumetric relaxation time (IVRT) and of LVEDP (Figure). Cardiac expression of IL-18 mRNA was significantly increased 7-fold after ATII infusion (P&lt;0.001), suggesting a potential mechanistic role of IL-18. Conclusion: Chronic infusion of low dose ATII recapitulates the HFpEF phenotype in the mouse, without increasing blood pressure. The use of this mouse model may help understanding the mechanisms leading to HFpEF syndrome in patients.

Mesenchymal stem cells alleviate atherosclerosis by elevating number and function of CD4(+)CD25 (+)FOXP3 (+) regulatory T-cells and inhibiting macrophage foam cell formation.

Atherosclerosis is a chronic inflammatory disease characterized by the formation of plaques inside arteries, leading to narrowing and blockage. Potential therapeutic strategies include expanding the population of regulatory T-cells (Tregs) to enhance atheroprotective immunity, and inhibiting the formation of macrophage foam cells. Here, we studied the effect of bone marrow-derived mesenchymal stem cells (BM-MSCs) on atherosclerotic plaque formation in Apolipoprotein E(-/-) (ApoE-KO) mice, and elucidated the underlying mechanism. BM-MSCs isolated from 4 week-old ApoE-KO mice were evaluated by flow cytometry for expression of MSC-specific markers. Thirty eight week-old ApoE-KO mice were randomly divided into three experimental groups (n = 10 per group): 1. MSC group-received BM-MSCs intravenously; 2. Vehicle group-received DMEM; 3. Control group-did not receive any treatment. Administration of MSCs resulted in a marked decrease in the size of atherosclerotic plaques 3 months after treatment. In addition, the number and function of CD4(+)CD25(+)FOXP3(+) regulatory T-cells (Tregs) in cultured splenocytes, and the expression of FOXP3 at both mRNA and protein levels, was significantly increased in the MSC group. In vitro experiments further indicated that the formation of macrophage foam cells was inhibited by treatment with MSCs, accompanied by a significant downregulation in CD36 and scavenger receptor A (SRA). Our findings suggest that MSCs play an atheroprotective role by enhancing the number and function of Tregs and inhibiting the formation of macrophage foam cells. Hence, administration of MSCs to atherosclerotic patients might have significant clinical benefits.

Humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice sustain the complex vertebrate life cycle of Plasmodium falciparum malaria.

BackgroundMalaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries. Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria. Since humans are the only natural hosts for P. falciparum, the lack of convenient animal models has hindered the understanding of disease pathogenesis and prompted the need of testing anti-malarial drugs and vaccines directly in human trials. Humanized mice hosting human cells represent new pre-clinical models for infectious diseases that affect only humans. In this study, the ability of human-immune-system humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice to sustain infection with P. falciparum was explored.MethodsFour week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes. Upon challenge with infectious P. falciparum sporozoites (NF54 strain) humanized DRAG mice were examined for liver stage infection, blood stage infection, and transmission to Anopheles stephensi mosquitoes.ResultsHumanized DRAG mice reconstituted human hepatocytes, Kupffer cells, liver endothelial cells, and erythrocytes. Upon intravenous challenge with P. falciparum sporozoites, DRAG mice sustained liver to blood stage infection (average 3–5 parasites/microlitre blood) and allowed transmission to An. stephensi mosquitoes. Infected DRAG mice elicited antibody and cellular responses to the blood stage parasites and self-cured the infection by day 45 post-challenge.ConclusionsDRAG mice represent the first human-immune-system humanized mouse model that sustains the complex vertebrate life cycle of P. falciparum without the need of exogenous injection of human hepatocytes/erythrocytes or P. falciparum parasite adaptation. The ability of DRAG mice to elicit specific human immune responses to P. falciparum parasites may help deciphering immune correlates of protection and to identify protective malaria antigens.

Establishment of nude mice xenograft model of papillary thyroid carcinoma with the ability of 99mTc-intake

Objective To establish the nude mice xenograft model of papillary thyroid carcinoma with the ability to accumulate 99mTc,and to provide a tool for further study in both pathogenesis and radioactive iodine treatment of papillary thyroid carcinoma.Methods 1 × 107 cells in 200 μl suspension liquids of the human papillary thyroid carcinoma cell lines IHH-4 were inoculated into nine nude mice (five week-old female nude mice,BALB/c-nu/ nu).Tumor formation was recorded,and the size and morphology of tumor were observed by PET-CT.Emission computed tomography (ECT) was used to observe the function of 99mTc intake by xenografts.Results The percentage of the formation of xenografts in nude mice was 100.0％ (9/9).One week after the cells were injected into nude mice,visible tumors were formed.PET-CT performed by the fourth week demonstrated that tumor size was about 1.8 cm × 2.4 cm.Xenograft' image was displayed by ECT after 15 minutes of intraperitoneal injection of 99mTc.Conclusion The nude mice xenograft model of papillary thyroid carcinoma was successfully established by using the human papillary thyroid carcinoma cell lines IHH-4.The xenografts have the ability to accumulate 99mTc,thus providing a good animal model for the study of pathogenesis and radioactive iodine with treatment of papillary thyroid carcinoma. Key words: Papillary thyroid carcinoma; Tumors in nude mice model; 99mTc; Emission computed tomography

Differential organ phenotypes after postnatal Igf1r gene conditional deletion induced by tamoxifen in UBC-CreERT2; Igf1r fl/fl double transgenic mice.

Insulin-like growth factor type 1 receptor (IGF1R) is a ubiquitously expressed tyrosine kinase that regulates cell proliferation, differentiation and survival. It controls body growth and organ homeostasis, but with specific functions depending on developmental time and cell type. Human deficiency in IGF1R is involved in growth failure, microcephaly, mental retardation and deafness, and its overactivation is implicated in oncogenesis. Igf1r-deficient mice die at birth due to growth retardation and respiratory failure. Although multiple Igf1r tissue-specific mutant lines have been analyzed postnatally, using Igf1r-floxed (Igf1r (fl/fl) ) mice mated with diverse cell-type recombinase Cre-expressing transgenics, no mouse models for the study of generalized Igf1r deficiency in adults have been reported. To this end we generated UBC-CreERT2; Igf1r (fl/fl) transgenic mice with an inducible deletion of Igf1r activated by tamoxifen. Tamoxifen administration to 4 week-old prepuberal male mice delayed their growth, producing a distinct impact on organ size 4weeks later. Whereas testes were smaller, spleen and heart showed an increased organ to body weight ratio. Mosaic Igf1r genomic deletions caused a significant reduction in Igf1r mRNA in all organs analyzed, resulting in diverse phenotypes. While kidneys, spleen and cochlea had unaltered gross morphology, testes revealed halted spermatogenesis, and liver and alveolar lung parenchyma showed increased cell proliferation rates without affecting apoptosis. We demonstrate that UBC-CreERT2 transgenic mice efficiently delete Igf1r upon postnatal tamoxifen treatment in multiple mouse organs, and corroborate that IGF1R function is highly dependent on cell, tissue and organ type.