ARA290 in a Rat Model of In fl ammatory Pain - Tissue-Protective Cytokines: Methods and Protocols - page 214

4. Before applying the von Frey filaments, rats must be standing
and weight bearing, i.e., not lying down.
5. When applying the von Frey filaments, aim for the glabrous
skin on the mid-plantar surface of the hind limb (see Note 13).
Apply the monofilament perpendicular to the skin so that it
bends slightly. Hold it in this position for 5 s. Avoid the hairy
skin as well as the foot pads. If the monofilament slips or the
skin is brushed, wait 1 min before repeating the application.
A positive response is a rapid withdrawal, or a lift, followed by
a hold or lick of the paw. If the rat steps away, this should not
be considered a positive response.
6. Both hind limbs should be tested. Randomize which side to
test first and leave at least five min between sides.
7. Begin testing with the 4 g von Frey filament. Apply the
monofilament up to five times (or six if the rats' first response is
to the fifth repeat). If the rat ambulates during testing or
the response is ambiguous, dismiss the trial and apply the
monofilament up to a maximum of eight times. Leave 10 s
between repeat applications with the same monofilament.
8. If the rat responds twice in a row to the 4 g monofilament,
stop testing and repeat with the 0.4 g monofilament. If the
rat does not respond twice to the 4 g monofilament, repeat
with the 6 g monofilament. Work your way up through
monofilaments of ascending thicknesses (from either the 0.4 g
or 6 g monofilament) until a repeat response is achieved. Leave
1 min interval between different monofilaments.
9. The threshold is the lowest thickness monofilament that pro-
duces two withdrawals.
10. Test animals as pairs (i.e., Test the first hind paw of two animals
before repeating on the second paw). Allow at least 5 min
between sides.

3.5 Treatment
with ARA290

1. Administer 120 m Millipore filter)

ARA290 or vehicle (0.9% Saline) in a total volume of 200

μ g/kg sterile filtered (0.22 μ

l i.p.
using a 1 ml syringe mounted with a 25 G needle (see Note 15).
2. Animals are injected immediately after behavioral testing at 1,
2, 3, and 4 days following surgery (see Note 16).

μ

A study was carried out in 18 adult male Sprague-Dawley rats
(250-300 g) to assess the effects of ARA290 on neuritis-induced
mechanical allodynia and heat hyperalgesia. Prior to induction of
the neuritis, baseline behavioral values were obtained on three con-
secutive days. Behavioral testing was further performed on days 1,
2, 3, 4, and 7 post-neuritis. Animals were assigned into two groups
( n = 9 per group). One group was administered 120

3.6 Typical Results

g/kg
ARA290, whereas the other group received only the vehicle. All
testing was performed blind with respect to the drug.

μ