Biology Reference
In-Depth Information
18. ATP solution: in 20 mL tube, to 10 mL water add 0.4 g MgCl
2
and 1.1 g adenosine 5¢-triphosphate (ATP).
19. Xylazine-ketamine solution. Solution A: Dissolve 1 g of Xylar
in 10 mL of saline (0.9% NaCl). Solution B: dissolve 1 g of
ketamine in 10 mL of saline as above. Take 1.0 mL of solution
A and 1.5 mL of solution B and adjust to 10 mL with saline.
Administer 10 mL/kg body weight to animals.
The experimental examples shown were done according to national
legislation. Statement of Compliance (Assurance) with Standards
for Humane Care and Use of Laboratory Animals has been recently
reviewed (10/28/2008) and will expire on 10/31/2013 and has
been accepted (id. #A5023-01) by the NIH-Office for Protection
from Research Risks.
Animals are housed in groups of two rats (male Sprague Dawley,
180-200 g). Animal room temperature and relative humidity are
set at 22 ± 2°C and 55 ± 10%. Artificial lighting provides a 12 h
light-12 h dark cycle (7 a.m. to 7 p.m.). The animals have free
access to diet and water.
2.2
Animals
3
Methods
The methods described below outline (1) the use of animals, (2)
the induction of diabetes, (3) the experimental design and drug
treatments, (4) the characterization of the model in terms of basic
changes and EPO effects, (5) and skin biopsy and immunohis-
tochemistry technologies and determination of IENF density.
1. Diabetes is induced in rats fasted overnight by a single intrap-
eritoneal (i.p.) injection of 60 mg/kg of STZ dissolved in
sodium citrate buffer (pH 4.5). The other rats are injected
with vehicle and serve as nondiabetic controls. Hyperglycemia
is confirmed by measuring glycosuria 72 h after STZ injection,
using commercially-available strips. Only animals with glyco-
suria >5% are classified as diabetic and included in the study.
2. Two studies were performed with rats in which were tested
the efficacy of EPO in protecting (preventive schedule) or
reversing (therapeutic schedule) nerve dysfunction in STZ-
induced diabetes and their correlation with IENF density.
The flowchart of the studies is reported in Fig.
1
. Study 1
investigated the efficacy of EPO in protecting the develop-
ment of DPN in the STZ-induced diabetes rat model. Study
2 examined the in vivo efficacy of EPO chronically admin-
istered in treating established DPN in STZ-diabetic rats.
For study 1, diabetic and control rats were treated with
rhEPO (Dragon Pharmaceuticals, Vancouver) 40
g/kg of
b.w., i.p., three times per week for 5 weeks starting on week 2.
μ
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