Source Paper
Hyponatremia-induced osteoporosis
Joseph G Verbalis, Julianna Barsony, Yoshihisa Sugimura, Ying Tian, Douglas J Adams et al.
Journal of Bone and Mineral Research • 2009
Materials & Equipment Checklist
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Protocol Steps
1
Animal acquisition and baseline measurements
Obtain male albino Sprague-Dawley rats at 6 weeks old, weighing 250-300g. Measure baseline body weight.
Note: Six rats per group in experiment 2
View evidence from paper
“male albino Sprague-Dawley rats (6 weeks old, 250 to 300 g; Taconic Farms, Germantown, NY, USA)”
2
Osmotic minipump implantation
Surgically implant Alzet Model 2004 osmotic minipump subcutaneously for continuous DDAVP infusion at 5 ng/h in hyponatremic groups
Note: Minipumps replaced monthly to maintain constant desmopressin levels
View evidence from paper
“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)”
3
Diet assignment and feeding
Assign rats to experimental groups: Group 1 (hyponatremic) receives DDAVP and 70 mL/d liquid diet (F1268SP); Group 2 (control) receives solid AIN-76 diet without DDAVP; Group 3 (control) receives liquid diet without DDAVP. Administer liquid diet via feeding tubes (No. 9010, BioServ).
3 months
Note: Liquid diet supplied 30 IU/d vitamin D3 and 155 mg/d calcium at 1.0 kcal/mL caloric density
View evidence from paper
“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”
4
Vitamin D3 supplementation
Administer biweekly intramuscular injections of 50,000 U/kg vitamin D3 to hyponatremic group and solid diet control group
Biweekly for 3 months
Note: Vitamin D3 from GeroVitamins, Zalmoxian Products
View evidence from paper
“Rats in one of the hyponatremic groups and rats on the solid AIN-76 diet in experiment 2 also received biweekly intramuscular injections of 50,000 U/kg vitamin D 3”
5
Body weight monitoring
Measure body weight biweekly throughout the 3-month experimental period
Biweekly for 3 months
View evidence from paper
“Body weight was measured biweekly”
6
Osmotic minipump replacement
Replace osmotic minipumps monthly to maintain constant desmopressin levels
Monthly for 3 months
View evidence from paper
“The osmotic minipumps were replaced monthly to maintain constant desmopressin levels”
7
Serum sodium monitoring
Monitor serum or plasma sodium levels monthly to verify hyponatremia induction and stability
Monthly for 3 months
Note: Sodium levels remained stable over the duration of the 3 month experiments
View evidence from paper
“Serum or plasma sodium levels were monitored monthly and remained stable over the duration of the 3 month experiments”
8
Metabolic cage housing for urine collection
House rats intermittently in Nalgene metabolic cages (No. 650-0100) for 24-hour urine collections at the end of each month
24 hours at end of each month
Note: Urine collected both with and without preservative
View evidence from paper
“Rats were housed intermittently in metabolic cages (No. 650-0100, Nalgene) for 24 hour urine collections”
9
Survival blood collection
Collect 2 mL survival blood by tail vein venipuncture under inhalation anesthesia with isoflurane (3% in oxygen at 0.5 L/min)
Note: Performed at end of 3-month experiment
View evidence from paper
“Survival blood (2 mL) was collected by tail vein venipuncture under inhalation anesthesia with isoflurane (3% in oxygen at 0.5 L/min)”
10
Nonsurvival blood collection and euthanasia
Collect nonsurvival blood by cardiac puncture under isoflurane anesthesia. Increase isoflurane dose until lack of heart contraction and respiration indicates death.
Note: Performed at end of 3-month experiment
View evidence from paper
“Nonsurvival blood was collected by cardiac puncture, also under isoflurane anesthesia; after blood collection, isoflurane dose was increased until the lack of heart contraction and respiration indicated death”
Subjects / Specimens
- Species
- rat
- Strain
- Sprague-Dawley
- Age
- 6 weeks old
- Sex
- male
- Count
- 6
- Weight
- 250 to 300 g
albino; six rats per group in experiment 2