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3. Day 3. Twenty-micron-thick sections perpendicular to the
epidermis were cut serially with a cryostat, sequentially
labeled and stored in antifreez solution at −20°C.
4. Day 4. Three sections from a single footpad were randomly cho-
sen, rinsed in 0.1 M TBS at room temperature on shaker table,
placed in 0.25% potassium permanganate for 15 min at room
temperature on shaker table, rinsed in TBS at room temperature
on shaker table, placed in 5% oxalic acid for 2 min, rinsed twice
in TBS, placed in block solution (4% normal goat serum, 0.1%
Triton X-100, in TBS) for 1 h on shaker table at room tempera-
ture and then incubated overnight with rabbit polyclonal anti-
protein gene product 9.5 (PGP 9.5, Biogenesis Ltd.) antibody
AbD serotec (1:1,000 diluted in 2% normal goat serum, 0.05%
Triton X-100, TBS), using modified free-floating protocol (
38
).
5. Day 5: After rinsing in TBS at room temperature, sections were
incubated with biotinylated goat anti-rabbit IgG for 1 h (1:100
diluted in TBS; Vector), quenched in 30% methanol/1%
hydrogen peroxide (in PBS) for 30 min, rinced in PBS and
then placed in avidin-biotin complex (Vector) for 1 h. The
reaction product was demonstrated by the blue chromogen/
peroxidase substrate (Vector SG substrate kit) according to
manufacturer indications.
Two expert measurers, blinded to the healthy or neuropathic
status of the rats, independently count the total number of PGP
9.5-positive IENF in each section under a light microscope (40×),
with the assistance of a microscope-mounted video camera.
Individual fibers were counted as they crossed the dermal-epidermal
junction, while secondary branching within the epidermis was
excluded from the quantification. The length of the epidermis was
measured using a computerized system (Microscience Inc.), and
the linear density of IENF (IENF/mm) was obtained. Difference
between groups was assessed by two-tailed Student's
t
test.
Pearson's test was used to assess the interobserver agreement in
IENF counts (see Note 8).
IENFs were intensely immunostained by PGP 9.5 (see Note 9)
as shown in a representative microphotograph of in the footpad of
control (Fig.
6a
), diabetic (Fig.
6b
), and EPO-treated diabetic
rats (Fig.
6c
). It is clearly seen that diabetic rats have a marked
decrease in cutaneous innervation density; this is completely
reversed by EPO administration (Fig.
6
).The density of footpad
IENF was quantified in healthy and diabetic Sprague Dawley rats
and in rats treated with EPO (Fig.
6
). In diabetic rats, the IENF
density is only slightly changed at 5 weeks of diabetes (data not
shown), but it was significantly reduced in diabetic rats at week 11
at the end of therapeutic schedule: A quantification of IENF den-
sity, indicating a statistically significant protective effect of EPO, is
shown in Fig.
6d
.
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