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 from Taconic Farms. Measure baseline body weight.
Note: Nine rats per group were studied in experiment 1
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 to deliver DDAVP at 5 ng/h
Note: Minipumps were 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
Initiate liquid diet feeding
Begin administering 70 mL/d of nutritionally balanced rodent liquid formula (F1268SP, BioServ) at 1.0 kcal/mL via liquid diet feeding tubes (No. 9010, BioServ). This supplies 30 IU/d of vitamin D3 and 155 mg/d of calcium (5.16 g/kg).
Note: Hyponatremic groups received DDAVP and liquid diet
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
Assign control diet
In experiment 1, control group receives solid diet identical to liquid diet in composition (AIN-76) while receiving DDAVP infusion
Note: Control group still receives DDAVP to isolate diet effects
View evidence from paper
“In experiment 1, the control group received a solid diet identical to the liquid diet in composition (AIN-76) and were infused with DDAVP”
5
Administer vitamin D3 supplementation
Administer biweekly intramuscular injections of 50,000 U/kg vitamin D3 (GeroVitamins, Zalmoxian Products) to designated groups
Note: Rats in one of the hyponatremic groups and rats on solid AIN-76 diet in experiment 2 received this supplementation
View evidence from paper
“biweekly intramuscular injections of 50,000 U/kg vitamin D 3 (GeroVitamins, Zalmoxian Products, London, UK)”
6
Biweekly body weight measurement
Measure and record body weight of all rats biweekly throughout the 3-month experiment
3 months total
Note: Measurements taken every 2 weeks
View evidence from paper
“Body weight was measured biweekly”
7
Monthly osmotic minipump replacement
Replace osmotic minipumps monthly to maintain constant desmopressin levels
Monthly throughout 3-month experiment
Note: Critical for maintaining consistent DDAVP infusion
View evidence from paper
“The osmotic minipumps were replaced monthly to maintain constant desmopressin levels”
8
Monthly serum/plasma 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”
9
Monthly 24-hour urine collection
House rats intermittently in metabolic cages (No. 650-0100, Nalgene) for 24-hour urine collections at the end of each month, both with and without preservative
24 hours at end of each month
Note: Collections performed 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”
10
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)”
11
Nonsurvival blood collection and euthanasia
Collect nonsurvival blood by cardiac puncture under isoflurane anesthesia. After blood collection, increase isoflurane dose until lack of heart contraction and respiration indicates death.
Note: Terminal procedure performed at end of 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
- 9
- Weight
- 250 to 300 g
albino Sprague-Dawley rats obtained from Taconic Farms