Hyponatremia-Induced Osteoporosis methods
Aim. Evidence-backed execution summary for Hyponatremia-Induced Osteoporosis methods from Hyponatremia-Induced Osteoporosis.
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This experiment, in seven questions
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rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Materials and Methods
reagent used in the protocol.
- Use
- 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...
Evaluation of excised bones
reagent used in the protocol.
- Use
- 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 xyleno...
Statistical analysis
reagent used in the protocol.
- Use
- Rat study results are expressed as the mean ± SEM. Statistical significance was determined by analysis using Student's t test for two-group comparison. Results from experiment 2 with four groups were evaluated using analysis of variance (ANOVA) followed by the Holme-Sidak test for multiple pairwise group compar...
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 xyleno...
- Use
- 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 xyleno...
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...
- Use
- 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...
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First confirmation
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Procurement checkpoint
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Open quote workflowStep-by-step procedure
What do I do, in order?
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).
Measurement outputs
What raw and processed outputs should exist?
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 p...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
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-metaphysea...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Rat study results are expressed as the mean ± SEM. Statistical significance was determined by analysis using Student's t test for two-group comparison. Results from experim...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
p <.05 using Kruskal Wallis one-way ANOVA on Ranks and Dunn's method for multiple comparisons against the solid diet + vitamin D group.
from paperScoring or quantification
Quantify the primary readouts for this experiment: 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 p...; 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-metaphysea...; Rat study results are expressed as the mean ± SEM. Statistical significance was determined by analysis using Student's t test for two-group comparison. Results from experim....
from paperStatistical comparison
p <.05 using Kruskal Wallis one-way ANOVA on Ranks and Dunn's method for multiple comparisons against the solid diet + vitamin D group.; p <.05 using one-way ANOVA and Holm-Sidak method for multiple comparisons against the liquid diet group.; Rat study results are expressed as the mean ± SEM. Statistical significance was determined by analysis using Student's t test for two-group comparison. Results from experim...
from paperReporting output
Report representative outputs alongside summary comparisons for 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 p..., 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-metaphysea..., Rat study results are expressed as the mean ± SEM. Statistical significance was determined by analysis using Student's t test for two-group comparison. Results from experim....
inferred from protocolStructured statistical methods
p <.05 using Kruskal Wallis one-way ANOVA on Ranks and Dunn's method for multiple comparisons against the solid diet + vitamin D group.; p <.05 using one-way ANOVA and Holm-Sidak method for multiple comparisons against the liquid diet group.; Rat study results are expressed as the mean ± SEM. Statistical significance was determined by analysis using Student's t test for two-group comparison. Results from experim...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (3)
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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