Intra-epidermal Nerve Fibers Density and Nociception in EPO-Treated Type 1 Diabetic Rats with Peripheral Neuropathy - Tissue-Protective Cytokines: Methods and Protocols - page 193

ganglia, nerve axons and Schwann cells ( 32 ). They are over-expressed
after nerve injury, and there appears to be an endogenous neuropro-
tective pathway, this sets the basis for therapeutic use of exogenous
EPO ( 26, 33 ). Exogenous EPO can be beneficial when locally or
virus-mediated delivered by promoting the regeneration of periph-
eral nervous system axons and preventing the progression of diabetic
neuropathy ( 34, 35 ). In vivo experimental models have shown that
EPO and its carbamylated non-hematopoietic derivative CEPO can
both prevent and treat diabetic peripheral neuropathy in rats and
mice ( 21 ).
The procedure to investigate the innervation of the skin by
bright-field immunohistochemistry in rats with STZ-induced DPN
is reported. The technique of IENF density count is described with
the aim of evaluate the number of IENF that clearly cross the epi-
dermal-dermal junction (extending off dermal fibers) and are
located in the epidermis and the length (in mm) of the epidermis
in order to determine the mean number of IENF/mm/skin biopsy
(length density).

2 Materials

2.1 Solutions

Prepare all solutions using ultrapure water (prepared by purifying
deionized water to attain a sensitivity of 18 M Ω cm at 25°C) and

analytical grade reagents.

1. 0.4 M Sorenson's phosphate buffer (PB; use at 0.1 M): Dissolve
7.176 g sodium phosphate monobasic monohydrate in 100 mL
water. Dissolve 49.4 g sodium phosphate dibasic anhydrate in
750 mL water and mix solutions the two solutions in a 1-L
graduated cylinder adding water up to 900 mL. Adjust the pH
to 7.6; with base or acid. Make up to 1 L with water. Store at
4°C up to 1 month.
2. 0.1 M Phosphate saline buffer (PBS). Dissolve 2.759 g sodium
phosphate monobasic monohydrate in 200 ml water (MONO).
Dissolve 8.898 g sodium phosphate dibasic dihydrate in 500 mL
water (DIBA). Dissolve 4.5 g NaCl in 100 mL DIBA; add to
this solution DIBA and MONO to obtain 500 mL PBS 0.1 M
pH 7.4 (see Note 1). Store at 4°C up to 1 month.
3. 8% paraformaldehyde stock (see Note 2): Add 40 g of “Prill
Grade” Paraformaldehyde to 500 mL PB and stir continuously
heating up to 55°C (see Note 3) on stir plate until diluted. Do
not allow powder to settle on bottom of beaker. After 2-3 h
the solution will be transparent. Stir until solution is clear. A
small amount of flocculent material will remain. Vacuum filter
(paper filter) through a 3 mm Whatman filter in a Buchner
funnel on a filter flask. Store at 4°C up to 1 month.