The day after surgery, the diet was changed from Purina Prolab?RMH 3000 to a casein- and lactalbumin-based atherogenic diet prepared by the Wake Forest University or college Diet Laboratory [4.45% total fat (w/w) and 0.001% cholesterol (w/w)]. Sle/LDLr?/? mice experienced no significant difference in serum cholesterol concentration, lipoprotein distribution, anti-dsDNA autoantibody concentration, antibody isotype concentration and renal histopathology score compared to placebo. However, they had significantly lower mean urine protein to urine creatinine percentage (UP:UC). There was no correlation between atherosclerosis lesion size and either the renal histology score or UP:UC percentage in Sle/LDLr?/? mice. Summary These results show that 17-estradiol is definitely atheroprotective within the context of murine SLE self-employed of changes in serum cholesterol concentration, autoantibody concentration, or renal pathology. The SLE phenotype in Sle/LDLr?/? mice is not exacerbated by exogenous 17-estradiol administration, Chlorpropamide and the reduced UP:UC percentage suggests a protecting effect against lupus nephritis. mice is definitely transplanted into irradiated C57BL/6 mice that lack a functional low denseness lipoprotein receptor (LDLr?/?) [14, 17]. The B6.strain is congenic for three lupus susceptibility loci ((Sle/LDLr?/?) bone marrow cells as previously explained [14, 17]. All mice were housed in ventilated racks and dealt with in accord with the Guidebook for the Care and Use of Laboratory Animals and with the authorization of the Wake Forest University or college Institutional Animal Care and Use Committee. 2.2 Estradiol treatment and necropsy Six weeks post-bone marrow transplant, the B6/LDLr?/? and Sle/LDLr?/? mice were ovariectomized and implanted subcutaneously with either a 90-day time continuous launch 5.6 Chlorpropamide g/day time 17-estradiol (E) pellet (0.5 mg total dose) or a placebo (P) pellet (Innovative Research of America, Sarasota, FL) (Fig. 1A). This 2 2 experimental design resulted in the following organizations: Group 1 = B6/LDLr?/? + P (n=16), Group 2 = B6/LDLr?/? + E (n=15), Group 3 = Sle/LDLr?/? + P (n=18), and Group 4 = Sle/LDLr?/? + E (n=34). Due to the higher mortality rate in Sle/LDLr?/? mice , more of these mice were produced to ensure statistical power for our endpoints. The day after surgery, the diet was changed from Purina Prolab?RMH 3000 to a casein- and lactalbumin-based atherogenic diet prepared by the Wake Forest University or college Diet Laboratory [4.45% total fat (w/w) and 0.001% cholesterol (w/w)]. This diet was formulated to accomplish moderate (~500 mg/dL) plasma cholesterol concentrations within the LDLr?/? background. Saturated, monounsaturated, and polyunsaturated extra fat made up 48.9%, 36.1%, and 15.0% of total fat in the diet, respectively. At the end of the 10-week treatment period the mice were anesthetized, blood was collected via cardiac puncture, and the arterial system was flushed with normal saline at 100 mmHg for 10 minutes. In the absence of reliable serum estradiol assays for mice , estradiol bioavailability was confirmed by uterine excess weight as previously explained . Open in a separate window Open in a separate window Open in a separate window Open in a separate window Open in a separate window Open in a separate window Open in a separate windowpane Fig 1 Study design and development of SLE disease. A) Six week older female mice were irradiated and received bone marrow transplants of either wild-type C57BL/6 or B6.b1 marrow. Six weeks post-transplant (day time zero) the mice were ovariectomized and randomized into estrogen (5.6 g 17-estradiol/day time pellet) or placebo (5.6 g placebo/day time pellet) treatment organizations and transferred to an atherogenic Chlorpropamide diet (0.001% cholesterol and 4.45% fat) for 10 weeks. B) Sle/LDLr?/? mice have improved renal disease compared to B6/LDLr?/? mice (n = 10C15 mice/group). Estrogen treatment did not impact the renal histopathology score when compared to placebo. C) Representative bHLHb24 H&E sections of renal glomeruli (63X for those 4 frames). Glomeruli of the Sle/LDLr?/? mice are enlarged and hypercellular with thickened capillary basement membranes and hypertrophic Bowmans epithelium. D) Serum anti-dsDNA antibody concentrations are improved in Sle/LDLr?/? mice compared to B6/LDLr?/? mice. Estrogen treatment did not alter anti-dsDNA antibody concentration when compared to placebo. E) Placebo treated Sle/LDLr?/? mice have an increased UP:UC ratio compared to placebo treated B6/LDLr?/? mice and estrogen treated Sle/LDLr?/? mice. F) Sle/LDLr?/? mice have splenomegaly when compared to B6/LDLr?/? mice (n = 10C15 mice/group). Estrogen treatment did not affect spleen excess weight when compared to placebo. G) Representative images of post-treatment spleens (mm). Data indicated as mean SEM; * p 0.05. 2.3 Artery histomorphometry Serial 5 m sections of frozen Tissue-Tek? O.C.T.-embedded aortic root were mounted about glass slides. Section 1 was defined as probably the most proximal section in which all three mitral valve leaflets were present. Sections 8C12 were stained.
- CHY drafted the manuscript
- (NIH/NIDCD, R01 DC005805