Group Title: Molecular Pain 2008, 4:3
Title: Perineural resiniferatoxin selectively inhibits inflammatory hyperalgesia
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Title: Perineural resiniferatoxin selectively inhibits inflammatory hyperalgesia
Series Title: Molecular Pain 2008, 4:3
Physical Description: Archival
Creator: Neubert JK
Mannes AJ
Karai LJ
Jenkins AC
Zawatski L
Abu-Asab M
Iadarola MJ
Publication Date: 39463
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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olecular Pain ioed
Molecular Pain BlioMed Central


Research

Perineural resiniferatoxin selectively inhibits inflammatory


hyperalgesia
John K Neubert* 1,23, Andrew J Mannes4, Laszlo J Karai4, Alan C Jenkins1,
Lanel Zawatski4, Mones Abu-Asab5 and Michael J Iadarola4


Address: 'College of Dentistry Department of Orthodontics, University of Florida, Gainesville, FL, USA, 2College of Medicine Department of
Neuroscience, University of Florida, Gainesville, FL, USA, 3Evelyn F. and William L. McKnight Brain Institute, University of Florida, Gainesville,
FL, USA, 4Neurobiology and Pain Therapeutics Section, Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research,
National Institutes of Health, Bethesda, MD, USA and 5Section of Ultrastructural Pathology, Laboratory of Pathology, National Cancer Institute,
National Institutes of Health, Bethesda, MD, USA
Email: John K Neubert* jneubert@dental.ufl.edu; Andrew J Mannes amannes@mail.nih.gov; Laszlo J Karai lkarai@mail.nih.gov;
Alan C Jenkins ajenkins@dental.ufl.edu; Lanel Zawatski lzawatski@temple.edu; Mones Abu-Asab amones@mail.nih.gov;
Michael J Iadarola miadarola@dir.nidcr.nih.gov
* Corresponding author



Published: 16 January 2008 Received: 14 September 2007
Molecular Pain 2008, 4:3 doi:10.1186/1744-8069-4-3 Accepted: 16 January 2008
This article is available from: http://www.molecularpain.com/content/4/1/3
2008 Neubert et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Resiniferatoxin (RTX) is an ultrapotent capsaicin analog that binds to the transient receptor
potential channel, vanilloid subfamily member I (TRPVI). There is a large body of evidence
supporting a role for TRPVI in noxious-mediated and inflammatory hyperalgesic responses. In this
study, we evaluated low, graded, doses of perineural RTX as a method for regional pain control.
We hypothesized that this approach can provide long-term, but reversible, blockade of a portion
of nociceptive afferent fibers within peripheral nerves when given at a site remote from the
neuronal perikarya in the dorsal root ganglia. Following perineural RTX application to the sciatic
nerve, we demonstrated a significant inhibition of inflammatory nociception that was dose- and
time-dependent. At the same time, treated animals maintained normal proprioceptive sensations
and motor control, and other nociceptive responses were largely unaffected. Using a range of
mechanical and thermal algesic tests, we found that the most sensitive measure following perineural
RTX administration was inhibition of inflammatory hyperalgesia. Recovery studies showed that
physiologic sensory function could return as early as two weeks post-RTX treatment, however,
immunohistochemical examination of the DRG revealed a partial, but significant reduction in the
number of the TRPVI-positive neurons. We propose that this method could represent a beneficial
treatment for a range of chronic pain problems, including neuropathic and inflammatory pain not
responding to other therapies.



Introduction small- and medium-sized neurons in sensory ganglia [1-
Resiniferatoxin (RTX), an ultrapotent capsaicin analog, is 3]. Evidence from TRPV1 gene deletion studies supports a
a vanilloid agonist that binds to the transient receptor role for TRPV1 in noxious thermal, chemical, and inflam-
potential channel, vanilloid subfamily member 1 matory hyperalgesic responses [4,5]. Behaviorally, long
(TRPV1), a non-selective cation ion channel expressed in duration noxious heat analgesia can be induced by subcu-


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taneous RTX administration [6-9]. These data reinforce
the long-recognized idea that TRPV1 has an important
role in inflammation and pain and thus TRPV1 has
become a target for analgesic drug development [10-16].

Vanilloids can trigger a Na+/Ca2+ flux in axons of primary
cultured dorsal root ganglia (DRG) neurons [17]. We have
observed that when RTX is administered in close proxim-
ity to the cell body of TRPV1-positive neurons (i.e., intra-
ganglionic), it induces a Ca2+-excitotoxicity and subse-
quent permanent neuronal cell deletion [18,19]. This
route of application has been proposed as a treatment for
pain in advanced metastatic disease [9,20,21]; however, a
reversible approach for achieving analgesia represents an
alternative route with certain advantages for pain control
in non-malignant situations. Based on prior studies using
peripheral application ofvanilloid agonists such as capsa-
icin and RTX [8,22-25], we hypothesized that perineural
application of RTX may temporarily block transmission in
TRPV1-containing axons. The percutaneous perineural
approach is proposed to broadly target a specific nerve
and its' peripheral receptive fields to produce a selective
antinociceptive action while maintaining other somatic
and proprioceptive sensations conveyed by large diameter
TRPV1-negative axons. By modifying transmission
through the peripheral nerve, this method can provide
analgesia in a wide range of chronic pain problems.
Perineural application has recently been explored using
high doses of RTX (1500 ng) to eliminate thermal and
inflammatory heat sensitivity and reduce motor reaction


threshold responses to pressure [24]. However, in the clin-
ical setting, exploration of the lower range of doses needs
to be considered in order to determine the minimum
effective dose for analgesia that reduces or eliminates
potential side effects. In the present study, we investigate
the efficacy of lower doses of RTX and evaluate the tempo-
ral recovery of function by using perineural RTX against
acute pain challenges and evaluate its effectiveness in
preemptively blocking inflammation-induced hyperalge-
sia.

Methods
All procedures complied with the guidelines of the Com-
mittee for Research and Ethical Issues of IASP and the
Institutional Animal Care and Use Committee of the
National Institute of Dental and Craniofacial Research,
National Institutes of Health.

RTX or vehicle application
Adult male Sprague Dawley rats (200-300 gm) were anes-
thetized (2.5% isoflurane, USP, inhalation), the outer
portion of rear left thigh shaved, and the area sequentially
disinfected with Betadine (10% povidone-iodine, Purdue
Frederick Co, Stamford, CT) and alcohol (70% isopropa-
nol). The femur was palpated at the superior (greater tro-
chanter) and inferior aspects (condyle) of the bone and a
sterile Stimuplex insulated needle (Stim-A25, 24 gauge x
1 inch, B. Braun Medical Inc, Bethlehem, PA) was inserted
approximately 5 mm through the skin, 1 cm ventral and 1
cm rostral to the greater trochanter (Fig. 1A). The tip of the


Figure I
Locating the sciatic nerve for electrically-stimulated percutaneous, perineural RTX injection. Note finger palpa-
tion locations and direction of the needle (see Methods). Fluorescein dye injection (50 [I, I glg/lil) using this technique demon-
strates the distribution of dye-containing fluid within the perineurium of the nerve (B).




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needle, with active electrical stimulation, was advanced
toward the left sciatic nerve until a distal dorsal flexion of
the hindpaw was elicited at 0.1 mA (1 Hz pulses). RTX (N
= 46, 25 250 ng, LC Laboratories, Woburn, MA), or vehi-
cle (N = 16, 0.25% Tween 80 in phosphate buffered saline
(PBS), 0.05% ascorbic acid) was injected in a volume of
50 |il. Note that the vehicle used was the same as the high-
est dose of RTX (250 ng). Immediate loss of paw flexion
was noted following the injection, even with continued
stimulation, as the injection fluid shunted the stimulus to
the nerve, thereby confirming a perineural distribution of
the fluid. In preliminary studies using PBS, larger currents
(0.2 0.4 mA) could produce dorsal flexion from at areas
distant from the sciatic nerve, however, the flexion
response was not lost when fluid was injected at this dis-
tant site.

While the sciatic nerve innervates the majority of the
hindpaw, a small part of the medial plantar surface of the
hindpaw is also innervated by the saphenous nerve and
the stimulus can overlap to this receptive field. Therefore
the saphenous nerve was additionally injected in a subset
of animals (N = 15) to evaluate the effects of RTX when
applied to both the sciatic and saphenous nerves. Follow-
ing percutaneous sciatic nerve injection (see above), a 0.5
cm incision was made into the medial aspect of the leg at
the mid-thigh level and RTX (250 ng, 50 |il) was injected
directly onto the saphenous nerve. A single Vicryl resorb-
able (4-0, Ethicon) suture was used to close the wound
and topical triple antibiotic cream (Neomycin, Polymyxin
B, and Bacitracin ointment, USP) was applied.

To further assess the efficacy of the percutaneous
approach, we examined a group of animals (N = 10) that
received RTX via a direct, open surgical procedure. Ani-
mals were anesthetized, surgically prepared as described
above, and a 1 cm incision was made through the skin at
the mid-thigh portion of the hind limb. The underlying
gluteus superficialis muscles were bluntly dissected and
the sciatic nerve exposed. The overlying fascia was care-
fully removed and RTX (250 ng, 50 Al) was bathed over 1
cm of the nerve for 5 min, after which the wound was
closed with Vicryl resorbable (4-0, Ethicon) sutures and
the animals were allowed to recover. Note that the RTX
solution was not washed off prior to suturing the area to
better simulate the exposure produced via the percutane-
ous injection method.

Fluorescein imaging
To determine the flow and uptake of fluid to and around
the sciatic nerve, Fluorescein-5-isothiocyanate (FITC, 'Iso-
mer I', 0.05 mg in 50 il PBS, Molecular Probes, Eugene,
OR) was injected into a subset of animals (N = 2) to verify
targeting of the sciatic nerve (Fig. 1B). Animals were sacri-
ficed (CO2 inhalation) and the injected sciatic nerve was


dissected free, cut in cross-section (5 rim), formalin fixed
and paraffin embedded. Sections were visualized under
an Olympus BX-60 microscope equipped with a RT-Slider
CCD camera (Diagnostic Instruments Inc. Burroughs,
MI), using the appropriate filters and the Spot Advance
software.

Behavioral Assessments
Development of heat hyperalgesia and analgesia to radi-
ant thermal stimulation was determined by placing unre-
strained animals under a small plastic box (9 x 6 x 6 in)
on an elevated clear glass platform, as described previ-
ously [26,27]. Animals were allowed to habituate for at
least 5 min prior to testing. Testing was performed prior to
injection, and at various time points (2.5 hrs, 1 day, 1
week, 2 weeks, 1 month, 3 months, 6 months) following
the nerve injection and 2.5 hrs after hindpaw carrageenan
injection. Statistical analyses (one-way and two-way
repeated measures ANOVA with Scheffe post-hoc analy-
sis) were used to compare baseline withdrawal latency
times to post-treatment times and treated versus untreated
values.

Sensitivity to mechanical stimuli was evaluated using an
electronic Von Frey anesthesiometer (Model 1601, IITC
Inc, Woodland Hills, CA). Following placement of ani-
mals under the plastic box on an elevated open-grated sur-
face and habituation (5 min), the probe tip was applied
perpendicularly to the ventral paw surface and pressure
was applied until the animal withdrew its paw. The aver-
age force (gm) to elicit paw withdrawal was calculated
from three iterations (5 min between each trial). Raw
threshold values were normalized to the initial baseline
values (base) and time and treatment effects were ana-
lyzed using a Mann-Whitney rank sum test or Kruskal-
Wallis ANOVA (P < 0.05 significance). Effects on gross
behaviors (grooming, limb guarding, and licking) follow-
ing RTX injection were also assessed.

Gross motor impairment was evaluated using a rotarod
apparatus (Model 7650, Ugo Basile, Comerio VA, Italy),
as described previously [8]. The time (sec) required for the
animal to fall off an accelerating rotating wheel drum
(4-40 r.p.m. in 5 min) was recorded and time and treat-
ment (RTX or vehicle) effects were analyzed using one-
way ANOVA and two-way repeated measures ANOVA (p
< 0.05).

Inflammation and paw thickness measurements
Carrageenan (6 mg, Sigma, St Louis, MO) was injected
(150 il, i.pl.) into the mid-plantar surface of the hindpaw
of unanesthetized rats as described previously [26].
Inflammation was initiated at the following post-injec-
tion (RTX or vehicle) time points: 1 day; 1 week; 2 weeks;
1 month; 3 months; and 6 months. Paw thickness was


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also measured at the mid-hindpaw level before and after
carrageenan inflammation for a subset of animals pre-
treated with either RTX (N = 5) or vehicle (N = 5); the dif-
ference (mm) from pre- to post-inflammation was
calculated.

Neurogenic inflammation
Animals were assessed 1 week after perineural RTX or
vehicle injection. Following anesthesia pentobarbitall, 50
mg/kg, i.p.), the hair from the right and left rear legs was
completely removed using a depilatory cream (Nair,
Carter-Wallace, Inc, Cranbury, NJ). A PE10 catheter was
placed in the jugular vein and Evans Blue (EB, Sigma, St.
Louis, MO) was infused over 10 min (30 mg/kg; 2% solu-
tion, i.v). Capsaicin cream (1%) was liberally and evenly
applied to the legs and to the dorsal and plantar surfaces
of both hindpaws and digital pictures were captured
(Sony MVC-FD 100 FD Mavica digital still camera).

Immunohistochemistry
Animals were deeply anesthetized pentobarbitall, 100
mg/kg, i.p.) 31 days post-RTX injection and fixed by tran-
scardial perfusion with cold phosphate buffered saline
(PBS) followed by cold Streck's Tissue Fixative (STF).
Right and left lumbar 4-5 (L4-L5) dorsal root ganglia
(DRG) were dissected and placed in STF for post-fixing.
Samples were paraffin processed and sections were cut (7
gim) and mounted on slides. Following deparaffinization
and epitope unmasking with Target Retrieval Solution
(S1700, Dako, Carpinteria, CA) at 950C for 20 min, sec-
tions were blocked with 10% normal goat serum (S-1000,
Vector Laboratories, Inc., Burlingame, CA) and incubated
overnight at 40C with the TRPV1 primary antibody
(1:10,000, Oncogene, #PC420). Antibody detection was
performed using the Vectastain Elite Rabbit IgG and Per-
oxidase Substrate Kits (SK-4700 and SK-4100, respec-
tively, Vector Laboratories, Inc., Burlingame, CA). Sites of
peroxidase activity were visualized using 3,3'-diami-
nobenzidene tetrahydrochloride (DAB). Control speci-
mens for assessment of non-specific binding were
processed in an identical way except for omission of the
primary antibody. Histological sections were visualized
with an Olympus BX 60 microscope, equipped with a RT
Slider CCD camera and processed with the Spot Advanced
software.

A total of N = 7 animals were used for the histological
analysis: N = 5 animals had RTX (250 ng, 50 gl) applied
to the left sciatic nerve and vehicle (0.25% Tween 80 in
PBS, 0.05% ascorbic acid, 50 gl) applied to the right sci-
atic nerve. A total of N = 10 DRG were examined for each
treatment from these 5 animals and a blinded observer
chose non-adjacent sections (separated by > 100 inm)
from different levels of the DRG for each treatment group
to give a final N = 15 sections/group. The remaining N =


2 untreated naive animals had 2 DRGs harvested from
each, with a total of N = 6 sections counted. The number
of TRPV1 immunoreactive and non-immunoreactive cell
bodies within a rectangular reticule were counted by vis-
ual inspection (10x magnification); the ratio ofTRPV1 to
the total number of cells was calculated and comparisons
between treatment groups (RTX, vehicle, no treatment)
were performed using a one-way ANOVA and Scheffe
post-hoc test, with significance set at a level of P < 0.05.

Results
Behavioral observations and effects on inflammatory
edema
We were interested in assessing general behavioral and
anti-inflammatory effects of perineural RTX. Following
injection (RTX or vehicle) and recovery from anesthesia (<
10 min), animals did not display nociceptive behaviors,
such as licking, guarding, which we had observed in pre-
vious studies upon injection into the hindpaw (Neubert
2003). We also never observed abnormal behaviors such
as autotomy in any of the animals, such as seen with com-
plete sciatic denervation [28]. Perineural RTX did not
affect paw edema following carrageenan inflammation
(change in hindpaw size was 6.2 0.3 mm for RTX, 6.0 +
0.2 mm for vehicle). These data indicate that the doses of
RTX used for analgesia did not inhibit the inflammatory
stimulus provided by the carrageenan injection.

RTX blocks inflammatory heat hyperalgesia when applied
directly to the sciatic nerve in a time and dose dependent
fashion
RTX produced a small but significant increase (range =
1-3 sec net change) in hind paw heat withdrawal latency
ipsilaterally when injected perineurally around the sciatic
nerve, as compared to contralateral hindpaw, which we
used as the control, or vehicle treated animals (Figs. 2 and
3). This increase was also observed when animals were
injected at both the saphenous and sciatic nerve sites, with
a significant increase noted at 1 week post-injection as
compared to the single sciatic nerve injection (Fig. 2). In
contrast to the effect on acute heat sensation, there was a
more substantial effect for perineural percutaneouss or
open surgical) RTX application on inhibiting heat hyper-
algesia following inflammation, as compared to animals
treated with vehicle and to pre-inflammation testing times
(Fig. 3). Animals treated with vehicle had a statistically
significant decrease in latency following inflammation (P
< 0.01) compared to the RTX treated group (Fig. 3). There
were no significant differences in latency times regarding
method of RTX application to the nerve, i.e. percutaneous
injection versus the open surgical application (8.4 2.1
sec versus 10.0 1.0 sec, respectively). Therefore, the dou-
ble site injection was not pursued further. These data indi-
cate that even animals that display only a modest increase
in thermal latency under non-inflammatory conditions,


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O Control
* Sciatic nerve
O Sciatic and saphenous nerves


Base 2.5 hrs 1 day

Pre-nflammation
Pre-inflammation


1 week
Post-nflammation
Post-Inflammation


Testing Time
Figure 2
Percutaneous application of RTX to the sciatic nerve
and sciatic plus saphenous nerves minimally effects
response to heat stimuli. RTX (250 ng, 50 il) application
to only the sciatic nerve produced a transient change in heat
withdrawal latency with respect to testing time (*P < 0.05, I
way ANOVA, Scheffe post-hoc analysis), as compared to the
baseline. Additional application of RTX to the saphenous
nerve produced a modest increase in heat latency, but this
effect was not significantly different, as compared to the sci-
atic group. The "sciatic nerve" group refers to animals
treated only with percutaneous RTX (N = 24) and the "sci-
atic and saphenous nerves" group refers to animals that were
injected percutaneously near the sciatic nerve and had an
open field injection around the saphenous nerve (N = 15).
The "control group" represents the uninjected right hindpaw.
There was no difference between contralateral controls for
the sciatic nerve group (N = 24) and the sciatic/saphenous
group (N = 15), therefore these groups were combined (N =
39) for subsequent comparisons.


exhibit a strong anti-hyperalgesic effect during carra-
geenan inflammation. Since this endpoint was the most
sensitive parameter, we used it to follow the time course
of recovery from the RTX analgesic effect.

Perineural application of RTX (250 ng, 50 pil) to the sciatic
nerve produced a significant inhibition of heat hyperalge-
sia following carrageenan-induced inflammation as soon
as 1 day post-injection and lasting for 2 weeks (Fig. 4, P <
0.05, 1 way RM ANOVA). At later times, using a repeated
measure ANOVA, no statistically significant block of
hyperalgesia was seen at 1, 3, and 6 months post-injec-
tion. These data show that recovery of inflammatory heat
hyperalgesia occurs between 2 weeks and 6 months. This
is consistent with the idea that the behavioral analgesic
effects ofperineural RTX are reversible (Fig. 4A). In vehicle


Testing Time
Figure 3
RTX blocks inflammatory heat hyperalgesia when
applied directly to the sciatic nerve. Both percutaneous
and open field injection of RTX (250 ng, 50 il) inhibited
inflammatory heat hyperalgesia following carrageenan injec-
tion (6 mg, 150 l). There was no significant difference in
latency time regarding RTX application method (i.e. percuta-
neously vs. direct open field application); therefore data were
pooled (N = 15). However, for this set of animals, there was
a significant increase in normal heat latency as compared to
vehicle treated animals (*P < 0.05, I way ANOVA, Scheffe
post-hoc analysis). Animals pretreated with vehicle (0.25%
Tween-80, PBS 50 [il, N = 10) had a significant decrease in
their heat withdrawal latency (+P < 0.05, I way RM-ANOVA)
following induction of inflammation.



treated rats there was a significant reduction in the heat
withdrawal latency compared to pre-inflammation
responses and to the 1 day, 1 week, and 2 weeks RTX
treated groups. We tested for an effect of vehicle as well at
48 hrs and 1 week following perineural vehicle injection,
but prior to carrageenan inflammation, and there was no
change in comparison to the contralateral side, therefore
the groups (N = 16) are combined in Fig. 4A.

RTX inhibited inflammatory heat hyperalgesia in a dose-
dependent fashion, with the effective dose being 2 125 ng
(Fig. 4B). The lower doses did not significantly block
inflammatory hyperalgesia, as compared to vehicle. For
the 250 ng group following inflammation, there was no
significant difference compared to pre-inflammation val-
ues, and these groups had significantly higher values as
compared to doses of 62.5 ng and lower. We chose to use
the 250 ng dose throughout the rest of the study to mini-
mize variability in responses at the lower effective dose
(125 ng). There were no significant differences between


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0 Vehicle
* RTX


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B*
B _ _


_T_


O Vehicle
O RTX


*



I.I


14
,S12
10
a.1
J8
S6
4


0


1
000
E E E
T- CO)


14
-12
l10
8,

S6
S4


0-


Pre-
Inflammation
Treatment


Figure 4
Percutaneous RTX produces long-lasting, reversible inflammatory heat hyperalgesic inhibition (A) in a dose-
dependent fashion (B). Inflammatory heat hyperalgesia is significantly inhibited I day (N = 8), I week (N = 6), and 2 weeks
(N = 5) following application of RTX (250 ng, 50 Il) to the sciatic nerve (*P < 0.05, I way ANOVA). An intermediate response
was seen at I month (N = 8), and at 3 months (N = 8). Complete recovery with a normal heat withdrawal response following
inflammation was observed 6 months (N = I I) following treatment, indicating that the effect of RTX had reversed. The dose-
response results (B) indicate that 2 125 ng is necessary for a significant anti-inflammatory effect, as compared to Pre-Inflamma-
tion values and to vehicle (P < 0.05). Note that the Pre-inflammation testing point represents the same testing day that the ani-
mal was to be inflamed. There was no difference between groups treated with RTX (N = 46); therefore data were pooled for
the baseline (Baseline) and Pre-inflammation testing sessions. Animals treated with vehicle (N = 16) were also pooled in the
Baseline, Pre-inflammation, and Post-inflammation groups for comparison.


any of the doses and vehicle on pre-inflammatory heat
withdrawal latency for these sets of animals (Fig. 4B).

Mechanical sensitivity and rotarod responsiveness
There were no differences in response to von Frey
mechanical stimuli following RTX or vehicle injection
(Fig. 5A). At the 1-week time point, carrageenan was given
and testing was performed 2.5 h later (when the inflam-
mation was well developed). In both vehicle and RTX
treated groups, we observed a pronounced mechanical
hyperalgesia, in comparison to baseline or the other test-
ing sessions. The route of RTX administration (percutane-
ous or open surgical procedure) made no difference in the
results, therefore these groups were pooled. After inflam-
mation, there was a small but significant analgesic effect
of RTX as compared to vehicle, but this did not result in a
substantial reversal of inflammatory mechanical hyperal-
gesia as both the vehicle and RTX treated groups had sig-
nificantly (P < 0.05) lower thresholds for withdrawal
following inflammation compared to the prior, pre-
inflammation testing sessions.

Rotarod results demonstrate that there were no significant
differences between animals that received vehicle or RTX
perineurally next to the sciatic nerve when comparing the


duration (sec) the animals remained on the accelerating
rotating rod (Fig. 5B). Moreover, the increase in rotarod
performance over the testing times was similar for both
groups, suggesting that comprehensive or delayed motor
problems did not develop. Note that data derived from
the accelerating rod test provides a sensitive measure for
minor coordination problems since it forces both control
and treated rats to perform to the point of failure. Deficits
that the rat can compensate for in a slowly moving rod are
revealed using the accelerating paradigm.

Perineural RTX partially depletes TRPVI- immunopositive
cells in sensory ganglia
RTX is known to kill cell bodies of TRPV1-positive neu-
rons when applied a directly into sensory ganglia, produc-
ing a permanent analgesic state [9,20]. Therefore we
explored whether the axonally directed, spatially remote
application of RTX used here would preserve the integrity
of the neuronal cell bodies in the DRG. To test this,
TRPV1-positive immunoreactive (TRPV1-IR) cells were
counted in the dorsal root ganglia corresponding to the
sciatic nerve innervation from the RTX-treated side, vehi-
cle-treated side, and non-treated control rats. There was a
significant decrease (F(2,35) = 17.95, P < 0.05) in the lum-
bar (L4-5) DRG for the TRPV1-IR/total cells ratio 1 month


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Base- Pre- Post-
line Inflammation Inflammation
Testing Time


*
r


Post-
Inflammation


Molecular Pain 2008, 4:3


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0 Vehicle
* RTX


250

200

150

100

50


Pre-Inflammation
Testing Time


1 week

Post-nflammation
Post-Inflammation


o Vehicle
ARTX


Base


1 day


Testing Time


Figure 5
RTX does not affect normal mechanical sensitivity or rotarod performance. Withdrawal force latencies were simi-
lar for vehicle and RTX treated animals except after inflammation, where there was a significant difference (*P < 0.05, Mann-
Whitney rank sum test) between the two groups (A). Data presented as a median normalized threshold (bars: interquartile
range). Pre-inflammation values for both vehicle and RTX-treated groups were all significant versus the post-inflammation value
(Krukal-Wallis ANOVA, P < 0.05). Values are represented by a median normalized threshold and the interquartile range is
denoted by the bars. Rotarod performance was identical for both groups (B).


after perineural RTX application (250 ng), as compared to
perineural vehicle application or naive animals (Fig. 6).

RTX blocks neurogenic inflammation (Fig. 7)
RTX eliminated the plasma extravasation following neu-
rogenic inflammation induced by topical capsaicin cream
(Fig. 7). In all animals, a clear demarcation demonstrating
an effect on the peroneal division of the sciatic nerve is
noted on the lateral aspect of the hind leg (A) and on the
dorsum of the lateral two toes of the hindpaw in animals
treated with RTX (C). The medial blue extravasation indi-
cates that the saphenous nerve was spared during the RTX
treatment, therefore this region was not protected from
the capsaicin-induced neurogenic inflammation. The ani-
mal illustrated is typical of seven rats tested for plasma
extravasation in which only the sciatic nerve was treated.

Discussion
The selective analgesic approach to pain control without
the loss of other functions has been the focus of many lab-
oratories. For example, targeting of sodium channels such
as NaV 1.8 has led to development of antagonists in the
quest for novel analgesics (Veneroni et al, Pain 2003).
TRPV1 antagonists have been shown to be effective for
reducing chemical, thermal, and inflammatory pain with-
out significant motor effects (Garcia-Martiez et al PNAS


2002, Gavva et al, JPET 2005). In this study, we demon-
strated that perineural application of RTX produces a
dose- and time-dependent inhibition of inflammatory
nociceptive processes, while maintaining normal proprio-
ceptive sensations and motor control. Remarkably, most
other pain sensations were preserved except for the
change in inflammatory heat hyperalgesia. There was a
small but statistically significant effect on the response to
mechanical stimuli when comparing RTX-treated animals
with vehicle-treated animals following inflammation
(Fig. 5). However, in reference to pre-inflammatory test-
ing sessions, the difference between RTX- and vehicle-
treated animals does not appear to be substantial (Fig. 5).
Additional application of RTX to the saphenous nerve did
not significantly affect the anti-inflammatory hyperalgesia
response as compared to application to the sciatic nerve
alone, although application to both the saphenous and
sciatic nerves did increase the normal thermal latency
(Fig. 2). However, sciatic perineural application may be
sufficient for producing regional effects while maintain-
ing normal thermal, mechanical, and proprioceptive sen-
sations. We demonstrate a blockade of neurogenic
inflammation mediated by the sciatic nerve with this per-
cutaneous approach (Fig. 7) based on plasma extravasa-
tion in peripheral receptive fields. We observed a



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S100
o
e-
| 75
50
250

S25
"i


1 week


f t





Molecular Pain 2008, 4:3







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RTX


Control


Vehicle


Treatment
RTX
Vehicle
None


N
(sections)
15
15
6


% TRPV1+
Cells/Total
Cell Count
*19.2
35.0
33.4


Figure 6
Dorsal root gangliaTRPVI immunopositive cells are
eliminated following perineural RTX treatment. Lum-
bar dorsal root ganglia sections (7 [im, paraffin-embedded)
were stained for TRPVI following RTX, vehicle or no treat-
ment of the sciatic nerve. There was a significant difference in
the number of TRPVI positively stained cells in the RTX-
treated animals, as compared to vehicle and untreated ani-
mals (Table inset).


significant reduction of inflammatory hyperalgesia which
was the most sensitive nociceptive endpoint.

Jancso and Lawson demonstrated that capsaicin applied
to the saphenous nerve produced a loss of approximately
a third of the unmyelinated fibers compared to control
nerves, while the proportion of myelinated fibers
remained unchanged [29]. Additionally, this group found
that perineural capsaicin application produced a reduc-
tion in the proportion of small-sized neurons in the ipsi-
lateral DRG. Similarly, in this study, we found a
significant loss of TRPV1-positive cells following
perineural RTX application in the corresponding DRG one
month following treatment, but based on the behavioral
outcomes, larger myelinated nerve fibers appeared to be
unaffected by RTX. Furthermore, even with this perma-
nent cell loss we observed a recovery of inflammatory
hyperalgesia beginning approximately 2 weeks after injec-
tion. This is consistent with several possibilities: (a) func-
tional repair of the RTX-affected axons occurs after that
time, (b) there could be a return of function due to collat-
eral sprouting of un-injured axons into the denervated
paw, (3) sensitivity of the existing axons could be


Figure 7
RTX blocks capsaicin-induced efferent neurogenic
inflammation. Following Evans blue injection (i.v., 30 mg/
kg, 2% solution), a 1% capsaicin cream was liberally applied to
the shaved hind legs and paws. Areas of neurogenic-induced
plasma extravasation are seen as blue. Note the delineation
between the blue area (positive for plasma extravasation)
versus the white skin (negative for plasma extravasation) for
the RTX treated side (A, C) and extravasation on only the
three medial toes for the RTX-treated side. Perineural vehi-
cle application did not effect plasma extravasation (B). These
data indicate selective targeting of the sciatic nerve while
preserving the saphenous division via this percutaneous injec-
tion.



enhanced. The cell loss may impact other general func-
tions but we did track a set of animals for 6 months and
these animals displayed normal behaviors throughout.
This is consistent with the Karai et al study that demon-
strated no adverse events for up to a year following intra-
trigeminal ganglia injection [9]. The mechanism for func-
tional recovery and long-term effects will be the subject of
a follow-up study.





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Targeting of primary afferent nociceptive transmission at
the peripheral axons provides an approach for producing
regionally specific therapeutic effects [9,24,25,30]. Earlier
studies using capsaicin demonstrated that direct applica-
tion to peripheral nerves produced transient nociceptive
activity, followed by a prolonged inhibition of responses
to noxious stimuli, especially heat and inhibition of neu-
rogenic inflammation [23,31,32]. However in this con-
text, capsaicin produced permanent impairment of a
proportion of C-fibers in sensory nerves as these capsaicin
treated rats continued to exhibit a deficit when tested
between 3-12 months [23,33]. In contrast, we demon-
strate recovery of sensitivity to inflammatory hyperalgesia
with RTX between 2 weeks and 6 months, suggesting the
more potent agent has fewer non-specific toxic side
effects. The specificity of RTX is further supported by pres-
ervation of motor and mechanical sensitivity, as demon-
strated in this study (Fig. 5) and previously [9]. This is not
a feature of studies performed with higher doses of RTX
which reported deficits in mechanical endpoints [24].

Based on our study with intradermal application of RTX,
we selected a dose range for RTX from 25 250 ng [8] and
evaluated these doses for analgesia and potential side
effects. We found that for perineural application of RTX
there was a steep dose response relationship occurring
between 62.5 and 125 ng, with doses 2 125 ng able to sig-
nificantly block the hyperalgesic response to inflamma-
tion (Fig. 3). We used the 250 ng dose of RTX throughout
the remainder of the study to reduce variability in
responses at the lower doses. In the absence of concurrent
inflammation, we observed that animals were able to
respond normally to noxious thermal stimuli (Fig. 2). We
also noted that there were no obvious effects on the
edema produced following carrageenan inflammation
when pretreated with RTX, as compared to vehicle. While
the neurogenic component of inflammation was elimi-
nated, there are other signs of inflammation such as
edema that suggest other inflammatory pathways are not
suppressed by RTX. While this animal model suggests that
it may not show efficacy in reducing mechanical allody-
nia, this should not exclude the potential therapeutic
action in human pathological pain states, since some of
these pain states can be maintained by peripheral inputs.
Others have shown that higher doses of RTX given in a
variety of routes are effective for reducing nociception
[7,24]. Kissin et al described a more broad-spectrum effect
of RTX administered percutaneously to the sciatic and
saphenous nerves and showed decreased sensitivity to
normal and inflammatory mechanical and heat hyperal-
gesia [24]. However at the doses used in that study (1,500
ng), systemic or non-specific effects of RTX cannot be
ruled out and may confound the utility for translation
into clinical pain control. Recently this group demon-
strated reduction of incisional post-operative pain with


perineural treatment of the sciatic and saphenous nerve
using a lower dose of RTX (450 ng)[25], supporting our
contention that smaller amounts of RTX are sufficient for
pain control.

Thermal sensing channels, in particular TRPV1, provide
targets for discovering new pain therapeutics [34,35]. The
use ofTRPV1 agonists such as RTX represents an exciting
approach for management of pain clinically especially
using site-directed application methods [8,9]. In the cur-
rent study, we found that an anatomically directed
perineural application of RTX, blocked inflammatory
hyperalgesia, while sparing normal somatosensory input,
including thermal and mechanical modalities. As such,
localized application of RTX has the potential for a range
of uses in pain management, from acute post-operative
care to treatment of regional pain disorders.

Competing interests
The authors) declare that they have no competing inter-
ests.

Acknowledgements
Support from this research was provided by the Division of Intramural
Research, and from grant #1 K22DE014865-0 IAI, National Institute of
Dental and Craniofacial Research, National Institutes of Health, Depart-
ment of Health and Human Services, Bethesda, MD, USA, and NIDCR.

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