Hyponatremia-Induced Osteoporosis methods
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Materials and Methods
All experimental procedures were accomplished according to the guidelines in the "Care and Use of Animals," available at http://www.nap.edu/openbook.php?isbn=0309053773. The Institutional Animal Care and Use Committee of Georgetown University approved all animal experimentation according to federal and institutional policies and regulations. To produce hyponatremia, male albino Sprague-Dawley rats (6 weeks old, 250 to 300 g; Taconic Farms, Germantown, NY, USA) were infused with desmopressin (DDAVP, Aventis, Bridgewater, NJ, USA) at a rate of 5 ng/h via a subcutaneously implanted osmotic minipump (Alzet Model 2004; Durect Co., Cupertino, CA, USA) and were fed 70 mL/d of a nutritionally balanced rodent liquid formula (F1268SP, BioServ, Frenchtown, NJ, USA) at a caloric density of 1.0 kcal/mL.( ) At typical daily intakes, this supplied 30 IU/d of vitamin D 3 and 155 mg/d of...
Evaluation of excised bones
Bone histology and histomorphometry were done on femora and tibiae in experiment 1 and on femora, tibiae, and lumbar spine in experiment 2. To obtain dynamic histomorphometric parameters, rats were injected intraperitoneally at the end of experiment 2 with calcein (15 mg/kg) 14 days before euthanasia and with xylenol orange (90 mg/kg) 3 days before euthanasia. Excised femora and tibiae from experiment 1 were fixed in 70% ethanol, dehydrated in 95% ethanol, and stored in 100% ethanol. Sample preparation and staining were carried out at the Armed Forces Institute of Pathology, Department of Scientific Laboratory (Washington, DC). The distal parts of femora were embedded in methyl methacrylate resin without prior decalcification. Consecutive 5 µm thick longitudinal sections were stained according to Von Kossa and Goldner's Masson-trichrome methods. In addition, the proximal halves o...
Evaluation of excised bones
From all sections, histomorphometric measurements were taken from the secondary spongiosa and restricted to a 4 mm 2 area 400 µm distal to the growth plate-metaphyseal junction, according to standard procedures.( ) Histomorphometric measurements were made in a blinded, nonbiased manner using the BioQuant computerized image analysis system (BioQuant, R&M Biometrics, Nashville, TN, USA) interfaced with a Zeiss 410 inverted microscope. Static histomorphometric parameters were obtained from both experiments. Multinucleated TRAP-positive cells in close proximity to trabecular surfaces were counted as osteoclasts. Dynamic histomphometric parameters were obtained from 5 µm undecalcified sections using fluorescence microscopy and a dual excitation filter from Chroma Technology (Bellows Falls, VT, USA).
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All experimental procedures were accomplished according to the guidelines in the "Care and Use of Animals," available at http://www.nap.edu/openbook.php?isbn=0309053773. The Institutional Animal Care and Use Committee of Georgetown University approved all animal experimentation according to federal and institutional policies and regulations. To produce hyponatremia, male albino Sprague-Dawley rats (6 weeks old, 250 to 300 g; Taconic Farms, Germantown, NY, USA) were infused with desmopressin (DDAVP, Aventis, Bridgewater, NJ, USA) at a rate of 5 ng/h via a subcutaneously implanted osmotic minipump (Alzet Model 2004; Durect Co., Cupertino, CA, USA) and were fed 70 mL/d of a nutritionally balanced rodent liquid formula (F1268SP, BioServ, Frenchtown, NJ, USA) at a caloric density of 1.0 kcal/mL.( ) At typical daily intakes, this supplied 30 IU/d of vitamin D 3 and 155 mg/d of calcium (5.16 g/kg). Liquid diet was administered via liquid diet feeding tubes (No. 9010, BioServ). Rats were housed intermittently in metabolic cages (No. 650-0100, Nalgene) for 24 hour urine collections. In experiment 1, the control group received a solid diet identical to the liquid diet in comp...
Bone histology and histomorphometry were done on femora and tibiae in experiment 1 and on femora, tibiae, and lumbar spine in experiment 2. To obtain dynamic histomorphometric parameters, rats were injected intraperitoneally at the end of experiment 2 with calcein (15 mg/kg) 14 days before euthanasia and with xylenol orange (90 mg/kg) 3 days before euthanasia. Excised femora and tibiae from experiment 1 were fixed in 70% ethanol, dehydrated in 95% ethanol, and stored in 100% ethanol. Sample preparation and staining were carried out at the Armed Forces Institute of Pathology, Department of Scientific Laboratory (Washington, DC). The distal parts of femora were embedded in methyl methacrylate resin without prior decalcification. Consecutive 5 µm thick longitudinal sections were stained according to Von Kossa and Goldner's Masson-trichrome methods. In addition, the proximal halves of the tibiae were preserved in 10% neutral buffered formalin, decalcified using formic acid solution, embedded in paraffin, and stained for tartrate-resistant acid phosphatase (TRAP) using the acid phosphatase reagents and the recommended procedure from Sigma, St. Louis, MO, USA. Similarly, excised...
From all sections, histomorphometric measurements were taken from the secondary spongiosa and restricted to a 4 mm 2 area 400 µm distal to the growth plate-metaphyseal junction, according to standard procedures.( ) Histomorphometric measurements were made in a blinded, nonbiased manner using the BioQuant computerized image analysis system (BioQuant, R&M Biometrics, Nashville, TN, USA) interfaced with a Zeiss 410 inverted microscope. Static histomorphometric parameters were obtained from both experiments. Multinucleated TRAP-positive cells in close proximity to trabecular surfaces were counted as osteoclasts. Dynamic histomphometric parameters were obtained from 5 µm undecalcified sections using fluorescence microscopy and a dual excitation filter from Chroma Technology (Bellows Falls, VT, USA).
Machine-readable layer
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