Molecular Pain
BioMed Central
Open Access
Research
Controlling neuropathic pain by adeno-associated virus driven
production of the anti-inflammatory cytokine, interleukin-10
Erin D Milligan1, Evan M Sloane1, Stephen J Langer2, Pedro E Cruz3,
Marucia Chacur1, Leah Spataro1, Julie Wieseler-Frank1,
Sayamwong E Hammack1, Steven F Maier1, Terence R Flotte3,
John R Forsayeth4, Leslie A Leinwand2, Raymond Chavez4 and
Linda R Watkins*1
Address: 1Department of Psychology & the Center for Neuroscience, University of CO at Boulder, Boulder, CO 80309 USA, 2Department of
Molecular, Cellular & Developmental Biology, University of CO at Boulder, Boulder, CO 80309 USA, 3Genetics Institute, the Powell Gene Therapy
Center & Department of Pediatrics, University of FL at Gainesville, Gainesville, FL 32610 USA and 4Avigen, Inc., Alameda, CA 94502 USA
Email: Erin D Milligan - emilligan@psych.Colorado.edu; Evan M Sloane - Sloane@psych.Colorado.edu;
Stephen J Langer - langers@Colorado.edu; Pedro E Cruz - pecruz@gtc.ufl.edu; Marucia Chacur - chacurm@icb.usp.br;
Leah Spataro - spataro@psych.Colorado.edu; Julie Wieseler-Frank - frankjw@psych.colorado.edu;
Sayamwong E Hammack - shammac@emory.edu; Steven F Maier - smaier@psych.Colorado.edu; Terence R Flotte - flottr@gtc.ufl.edu;
John R Forsayeth - johnfx@itsa.ucsf.edu; Leslie A Leinwand - leinwand@Colorado.edu; Raymond Chavez - rchavez@Avigen.com;
Linda R Watkins* - lwatkins@psych.colorado.edu
* Corresponding author
Published: 25 February 2005
Molecular Pain 2005, 1:9
doi:10.1186/1744-8069-1-9
Received: 15 January 2005
Accepted: 25 February 2005
This article is available from: http://www.molecularpain.com/content/1/1/9
© 2005 Milligan 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
Despite many decades of drug development, effective therapies for neuropathic pain remain
elusive. The recent recognition of spinal cord glia and glial pro-inflammatory cytokines as important
contributors to neuropathic pain suggests an alternative therapeutic strategy; that is, targeting glial
activation or its downstream consequences. While several glial-selective drugs have been successful
in controlling neuropathic pain in animal models, none are optimal for human use. Thus the aim of
the present studies was to explore a novel approach for controlling neuropathic pain. Here, an
adeno-associated viral (serotype II; AAV2) vector was created that encodes the anti-inflammatory
cytokine, interleukin-10 (IL-10). This anti-inflammatory cytokine is known to suppress the
production of pro-inflammatory cytokines. Upon intrathecal administration, this novel AAV2-IL-10
vector was successful in transiently preventing and reversing neuropathic pain. Intrathecal
administration of an AAV2 vector encoding beta-galactosidase revealed that AAV2 preferentially
infects meningeal cells surrounding the CSF space. Taken together, these data provide initial
support that intrathecal gene therapy to drive the production of IL-10 may prove to be an
efficacious treatment for neuropathic pain.
Background
Neuropathic pain is an especially difficult chronic pain
syndrome to treat. No compounds are yet available that
successfully resolve such pain [1-3]. To date, drug therapies developed for human neuropathic pain have targeted
neurons. However, evidence has recently accumulated
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that pathological pain, including neuropathic pain, is
dynamically and dramatically amplified as a result of spinal cord glial activation [4-6]. Spinal cord glia become
activated as a consequence of inflammation and/or
trauma to peripheral nerves [4,6]. This raises the intriguing possibility that finding ways to target glial activation,
or its downstream consequences, may provide a novel
approach for neuropathic pain control [5,7].
Several glial-selective drugs have been successful in preventing or reversing neuropathic pain in animals, but
none is optimal for clinical applications. For example,
fluorocitrate is a selective astrocyte inhibitor [8,9]. While
effective in blocking the induction of neuropathic pain in
animals [10], fluorocitrate is inappropriate for human use
due to inhibition of glial glutamate uptake and consequent seizures can occur [11]. Similarly, minocycline is a
selective microglial inhibitor [12]. It too is effective in
blocking the induction of neuropathic pain in animals
[13,14]. However, as minocycline fails to affect established neuropathic pain [13,14], this compound does not
appear to have therapeutic potential.
Other approaches have focused on the fact that glia are
"immune cell-like". Upon activation, glia and immune
cells each release pro-inflammatory substances, most
notably pro-inflammatory cytokines (interleukin [IL]-1,
tumor necrosis factor [TNF] and IL-6). The release of proinflammatory cytokines by activated spinal cord glia is key
as these cytokines enhance pain and have been implicated
in the initiation and maintenance of neuropathic pain
[10,15,16]. Immunosuppressive (methotrexate) and
immunomodulatory (propentofylline) drugs have been
tested with the aim of suppressing neuropathic pain via
suppression of glial-derived pro-inflammatory cytokines
in spinal cord. While these drugs have proven effective in
reducing enhanced pain responses [17,18], they are not
optimal for chronic use in humans, as their systemic
administration would negatively impact the peripheral
immune system. In addition, although selective proinflammatory cytokine antagonists have proven successful in resolving neuropathic pain [10,15], their lack of
CNS penetration negates systemic administration and
their relatively short duration of action poses problems
for chronic intrathecal administration in humans.
The purpose of the present series of studies was to explore
a new approach to neuropathic pain control; that is,
intrathecal gene therapy using an adeno-associated viral
(serotype II; AAV2) vector to drive the production and
release of interleukin-10 (IL-10), a powerful anti-inflammatory cytokine [19]. As IL-10 is known to suppress the
production and release of all 3 pro-inflammatory
cytokines [19], it would be predicted to be efficacious for
the treatment of neuropathic pain. Two animal models of
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neuropathic pain were employed: (a) sciatic inflammatory neuropathy (SIN) induced by localized inflammation of the sciatic nerve in the absence of frank trauma,
and (b) chronic constriction injury (CCI), a model involving both trauma to and inflammation of the sciatic nerve.
Depending upon the intensity of sciatic nerve inflammation induced, the SIN model can produce either an ipsilateral or bilateral mechanical allodynia [20]. This allows
examination of both ipsilateral and mirror-image pain.
CCI was chosen for study both because it is a classic
model of partial nerve injury [21] and because it induces
thermal hyperalgesia in addition to mechanical allodynia
[22-24]. We attempted to prevent and reverse SIN- and
CCI-induced pain changes, respectively. These studies
demonstrate that intrathecal administration of an AAV2
vector that encodes for rat IL-10 (AAV2-r-IL-10) is effective
in transiently resolving neuropathic pain in both models.
Results
AAV transgene expression in vitro and in vivo
The plasmid construct containing rat IL-10 cDNA (pTR2CB-rIL-10) was transfected into an IB3 cell line to verify
plasmid-induced rat IL-10 release (see Methods). Media
from the transfected cell cultures were collected 18, 36,
and 60 hr later and frozen at -80°C until analyzed for rat
IL-10 by ELISA. Rat IL-10 was readily detected in culture
supernatants of transfected cells versus untransfected control cultures (see Fig. 2A Inset). This construct was subsequently used to create an AAV2 vector for in vivo testing.
Lumbosacral intrathecal injection of adenovirus has been
well characterized as infecting predominantly meningeal
cells surrounding the lower spinal cord CSF space [25]. A
comparable examination of AAV2 spread has not been
previously reported in adult rats following lumbosacral
intrathecal injection. Rats (n = 3) were injected intrathecally with AAV2 (approximately 4.1 × 10^8 infectious particles in 10 ul) engineered to express beta-galactosidase 8
days prior to tissue fixation for beta-galactosidase histochemistry. Tissues from non-injected control rats (n = 2)
were processed at the same time.
A diffuse and sporadic pattern of beta-galactosidase
expression was observed from the injection site rostral to
spinal cord C2 regions with the most robust staining
observed closest to the injection site. Close inspection of
spinal cord tissue (Fig. 1A) revealed little to no beta-galactosidase expression in control naive tissue. In contrast,
clear beta-galactosidase expression was observed in LacZ
injected rats (Fig. 1B), as indicated by stained cells in the
pial meningeal layer. Beta-galactosidase expression
decreased at more rostral regions (not shown). No betagalactosidase expression was observed in the meninges of
ventral spinal cord. The tissue distribution of AAV2-LacZ
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Figure
Photomicrographs
1
of beta-Galactosidase histochemistry; expression in spinal cord meninges
Photomicrographs of beta-Galactosidase histochemistry; expression in spinal cord meninges. Spinal cords were
removed from control, non-injected rat (A) or the AAV2-LacZ injected rat (B) 8 days after intrathecal injection. Beta-Galactosidase histochemistry was conducted with X-gal staining procedures. No sections are counter stained. Magnification in Panels A
and B are identical, scale bar, 100 microns.
induced beta-galactosidase expression was observed predominantly in the pial membrane.
Blockade of SIN-induced mechanical allodynia by
intrathecal AAV2-r-IL-10
Sciatic inflammatory neuropathy (SIN) is induced by perisciatic injection of an immune activator, such as zymosan
(yeast cell walls), around one healthy sciatic nerve at midthigh level [10,20]. This procedure creates robust mechanical allodynia, but no thermal hyperalgesia [20]. Mechanical allodynia is restricted to the injected limb when low
doses of zymosan are used. High doses of zymosan induce
allodynia in both the injected hindleg as well as the contralateral (mirror-image) hindleg [10,20]. The zymosan
injection is done in unanesthetized, unrestrained rats via
a pre-implanted peri-sciatic catheter [26]. This allows for
repeated injections of the immune activator across days so
to induce chronic neuropathic pain [10]. Prior to induction of sciatic inflammatory neuropathy (SIN), rats were
assessed for their responses to low-threshold mechanical
stimuli (0.407 – 15.136 gm) applied to the plantar surface
of their hindpaws (von Frey test). This was done prior to
(baseline; BL) and again on Day 3 after intrathecal AAV2r-IL-10 (8.5 × 10^8 infectious units in 5 ul; dose based on
pilot studies) or AAV2-GFP (Control; 8.45 × 10^8 infectious units in 5 ul; directed the intracellular production of
jellyfish green fluorescent protein; GFP). AAV2-r-IL-10
and AAV2-GFP had no effect on mechanical response
thresholds measured 3 days after virus delivery, compared
to BL (F 7,88 = 0.686, p > 0.68) (Fig. 2). Hence neither the
presence of rat IL-10 nor adeno-associated virus had
measurable effects on basal low threshold mechanical
responses.
Immediately upon completion of the Day 3 test, all rats
were peri-sciatically injected with either 4 or 160 ug
zymosan (n = 5–6/group) to induce ipsilateral or bilateral
mechanical allodynia, respectively. Zymosan was readministered to maintain allodynia across the testing
period, as previously described [10,20,27]. Behavioral
responses on the von Frey test were reassessed daily
through Day 11 and lastly on Day13 in accordance with
prior studies [10].
AAV2-r-IL-10 and AAV2-GFP had no effect on mechanical
response thresholds measured 3 days after virus delivery,
compared to BL (Fig. 2). Hence neither the presence of rat
IL-10 nor adeno-associated virus had measurable effects
on basal pain responses. As in our previous studies [10],
low dose zymosan induced a unilateral allodynia (Fig.
2A,B) while higher dose zymosan induced a bilateral allodynia (Fig. 2C,D), compared to BL measures. Both ipsilateral (Fig. 2A) and bilateral (Fig. 2C,D) allodynias were
blocked by AAV2-r-IL-10 through Day 11, as von Frey
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allodynia2
Adeno-associated
Figure
viral IL-10 blocks development of chronic sciatic inflammatory neuropathy (SIN) induced mechanical
Adeno-associated viral IL-10 blocks development of chronic sciatic inflammatory neuropathy (SIN) induced
mechanical allodynia. After baseline (BL) assessment on the von Frey test, all rats received intrathecal AAV2-GFP (Control,
encoding green fluorescent protein) or AAV2-r-IL-10. Behavior was reassessed Day 3 after intrathecal AAV, confirming that
neither AAV2-GFP (Control) nor AAV2-r-IL-10 affected behavior prior to peri-sciatic injections (F 7,88 = 0.686, p > 0.68). After
this Day 3 assessment, unilateral peri-sciatic injections of 0 (vehicle control; Panels A, B), 4 ug zymosan (to induce ipsilateral
allodynia; Panels C, D), or 160 ug zymosan (to induce bilateral allodynia; Panels E, F) were delivered, with repeated readministration across days to induce a chronic neuropathic state. Repeated measures ANOVA revealed reliable main effects of
peri-sciatic zymosan dose (F 1,40 = 12.093, p < 0.002), IL-10 (F 1,40 = 69.829, p < 0.0001), and laterality (F 1,40 = 22.315, p <
0.0001), and interactions between zymosan dose and IL-10 (F 1,40 = 6.161, p < 0.02) and between IL-10 and laterality (F 1,40 =
15.412, p < 0.001). The construct pTR2-CB-r-IL-10 employed in an AAV vector for behavioral testing induced the production
and release of rat IL-10 from transfected IB3 cells in culture. Increases in rat IL-10 protein were detected in supernatants of
transfected cells versus untransfected vehicle control cultures (Panel A Inset). Neither AAV2-GFP nor AAV2-r-IL-10
affected the behavioral responses of rats receiving chronic peri-sciatic vehicle, as illustrated by data obtained from the hindpaws ipsilateral (Panel A) or contralateral (Panel B) to the peri-sciatic injections. Allodynia was induced in the ipsilateral
hindpaw of intrathecal AAV2-GFP rats receiving 4 ug peri-sciatic zymosan (Panel C). This allodynia was largely blocked by
AAV2-r-IL-10 (p > 0.045 through Day 11 compared to BL) with allodynia reappearing on Day 13 after AAV; that is, 10 days
after initiation of chronic zymosan. Again, neither AAV2-GFP nor AAV2-r-IL-10 affected behaviors obtained from the contralateral, non-allodynic hindpaws (Panel D). Allodynia was induced in both the ipsilateral (Panel E) and contralateral (Panel F)
hindpaws of intrathecal AAV2-GFP rats receiving 160 ug peri-sciatic zymosan. These ipsilateral and contralateral allodynias
were largely blocked by AAV2-r-IL-10 (p > 0.15 through Day 11 compared to BL), until allodynia reappeared on Day 13 after
AAV; that is, 10 days after initiation of chronic zymosan.
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responses after peri-sciatic zymosan did not differ from
BL. By Day 13, both ipsilateral and bilateral allodynia
returned.
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healthy, appeared to gain weight normally, and exhibited
typical posture, grooming, and locomotion.
Discussion
Reversal of chronic constriction injury (CCI) induced
mechanical allodynia and thermal hyperalgesia by
intrathecal AAV2-r-IL-10
Chronic constriction injury (CCI) is a classic model of
neuropathic pain induced by partial nerve injury [21].
Like the SIN model described above, CCI is performed on
one sciatic nerve at mid-thigh level. In contrast to the SIN
model described above, CCI involves both inflammation
of, and trauma to, this nerve, To test whether AAV2-r-IL10 could reverse established thermal hyperalgesia or low
threshold mechanical allodynia induced by CCI, the
plantar surface of the rat hindpaws were first assessed for
their responses to low threshold mechanical stimuli (von
Frey test) and radiant heat stimuli (Hargreaves test) prior
to (BL) and again on Days 3 and 10 after surgery. CCI surgery produced reliable ipsilateral thermal hyperalgesia
(Fig. 3) and bilateral mechanical allodynia (Fig. 4) compared to sham surgery, in agreement with our prior
reports [22-24].
Immediately after behavioral testing on Day 10, rats (n =
5–6 /group) received intrathecal AAV2-r-IL-10 (8.5 × 10^8
infectious units in 5 ul) or AAV2-GFP (8.45 × 10^8 infectious units in 5 ul). Hargreaves and von Frey tests were
again performed on Days 3, 5, 7, 9, 11, 14, 16 and 20 after
viral administration. This corresponds to Days 13, 15, 17,
19, 21, 24, 26, and 30 after CCI or Sham surgery. The data
demonstrate that AAV2-r-IL-10 administration produced
significant reversal of both ipsilateral thermal hyperalgesia (Fig. 3) and bilateral allodynia (Fig. 4) induced by CCI,
compared to AAV2-GFP, on Days 13–26 after surgery.
Intrathecal AAV2-r-IL-10 did not permanently reverse
these ongoing pathological pain states. AAV2-r-IL-10
reversal of CCI-induced pathological pain states began
dissipating by Day 26 post-surgery. From Day 26–30,
both thermal hyperalgesia and mechanical allodynia progressively returned, with pain facilitation reaching preAAV levels by Day 30 (Figs. 3,4). At Day 30, neither behavioral response modality was significantly different from
Day 10 preinjection levels.
Lastly, 2 groups of rats (n = 10/group) were matched for
body weight prior to receiving intrathecal AAV2-r-IL-10
(8.5 × 10^8 infectious units in 5 ul) or AAV2-GFAP (8.45
× 10^8 infectious units in 5 ul). As part of an unrelated
study, these rats were weighed Day 7 post-AAV2 just prior
to sacrifice. No difference in the body weight of these 2
groups of animals was found (F 1,18 = 0.181; p > -.67). As
in all prior experiments, no adverse effects of either AAV2r-IL-10 or AAV-GFAP were noted. All rats appeared
The present experiments document the efficacy of AAV2r-IL-10 in preventing and reversing neuropathic pain. In
addition, this work provides initial evidence that
intrathecal gene therapy to express anti-inflammatory
cytokines, such as IL-10, may be an approach worth pursuing for the treatment of chronic pain. When AAV2-r-IL10 was administered intrathecally 3 days prior to chronic
inflammation of the sciatic nerve (SIN), AAV2-r-IL-10 prevented the onset of both ipsilateral and mirror-image
mechanical allodynia, as measured by the von Frey test.
Blockade of these SIN-induced allodynias lasted for 8 days
(11 days after intrathecal AAV2-r-IL-10), with allodynia
developing by day 10 (13 days after intrathecal AAV2-r-IL10). In contrast, rats receiving AAV2-GFP (Control) prior
to induction of SIN exhibited strong ipsilateral and mirror-image allodynia throughout the timecourse tested.
Such profound and prolonged allodynia with this chronic
SIN procedure is in agreement with prior studies [10].
Neither AAV2-r-IL-10 nor AAV2-GFP altered mechanical
response thresholds in sham-operated rats. AA2-r-IL-10
was also successful in reversing established CCI-induced
thermal hyperalgesia. Intrathecal AVV2-r-IL-10 administered 10 days after CCI surgery returned hindpaw
response latencies to radiant heat (Hargreaves test) to presurgery baseline values. The effect of AAV2-r-IL-10 was
again transient, with complete reversal observed for a
week, from 7 through 14 days after AAV2-r-IL-10. Thermal
hyperalgesia then progressively returned with robust
hyperalgesia recorded 20 days after AAV-r-IL-10. In contrast, rats receiving intrathecal AAV2-GFP exhibited stable
ipsilateral thermal hyperalgesia throughout the timecourse tested. Neither AA2-r-IL-10 nor AAV-GFP altered
thermal response thresholds in sham-operated rats. AAV2-r-IL-10 attenuated established ipsilateral and mirrorimage mechanical allodynia in these same animals, with
allodynia again becoming fully re-expressed by 16–20
days after AAV2-r-IL-10. Rats receiving intrathecal AAV2GFP exhibited marked bilateral mechanical allodynia
throughout the timecourse tested, in agreement with prior
studies [22-24]. Again, neither vector altered response
thresholds of sham-operated rats. AAV2 appears to predominantly infect meningeal cells surrounding the CSF
space, as indicated by beta-galactosidase staining of spinal
tissues from rats injected with AAV2-LacZ. This pattern of
meningeal staining is in accordance with prior studies of
intrathecal adenovirus administration [25].
IL-10 is only one of many endogenous anti-inflammatory
cytokines. In addition to IL-10, the anti-inflammatory
cytokine family also includes IL-4, IL-11, and IL-13
[28,29]. Leukemia inhibitory factor, interferon-alpha, IL-
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Figure 3
Adeno-associated
viral IL-10 reverses established CCI-induced thermal hyperalgesia
Adeno-associated viral IL-10 reverses established CCI-induced thermal hyperalgesia. After predrug (baseline; BL)
assessment on the Hargreaves test, sham (Panels A, B) or CCI (Panels C, D) surgery was performed (timing denoted by the
first vertical dotted line). Behavioral assessments were reassessed Days 3 and 10 after surgery to document the lack of thermal
hyperalgesia in sham operated rats and development of unilateral allodynia in CCI groups ipsilateral to sciatic surgery. ANOVA
revealed reliable main effects of CCI (F 1,40 = 140.740, p < 0.0001) and laterality (F 1,38 = 48.901, p < 0.0001), and an interaction
between CCI and laterality (F 1,40 = 104.295, p < 0.0001). After the Day 10 assessment, rats received intrathecal injections of
either AAV2-GFP (Control) or AAV2-r-IL-10 (timing denoted by the second vertical dotted line). Behavioral assessments were
again recorded on Days 13, 15, 17, 19, 21, 24, 26, and 30 after surgery; that is, Days 3, 5, 7, 9, 11, 14, 16, and 20 days after
AAV. While neither AAV2-GFP nor AAV2-r-IL-10 exerted marked effects in sham operated animals (Panels A, B) or nonallodynic hindpaws of CCI-operated animals (Panel D), AAV2-r-IL-10 transiently reversed ipsilateral CCI allodynia compared
to CCI operated AAV2-GFP treated animals (Panel C). For Days 13–26, ANOVA revealed reliable main effects of CCI (F 1,39
= 134.036, p < 0.0001), IL-10 (F 1,39 = 12.047, p < 0.01) and laterality (F 1,39 = 66.284, p < 0.0001), and interactions between
CCI and AAV2-r-IL-10 (F 1,39 = 24.486, p < 0.0001), CCI and laterality (F 1,39 = 91.956, p < 0.0001), and IL-10 and laterality (F
1,39 = 17.392, p < 0.0001). At Day 30, behavioral responses were not significantly different from Day 10 preinjection levels (F
1,39 = 7.824, p > 0.10).
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Figure 4
Adeno-associated
viral IL-10 attenuates established CCI-induced mechanical allodynia
Adeno-associated viral IL-10 attenuates established CCI-induced mechanical allodynia. After predrug (baseline;
BL) assessment on the von Frey test, sham (Panels A, B) or CCI (Panels C, D) surgery was performed (timing denoted by
the first vertical dotted line). Behavioral assessments were reassessed Days 3 and 10 after surgery to document the lack of
allodynia in sham operated rats and development of bilateral allodynia in CCI groups. ANOVA revealed reliable main effects of
CCI (F 1,40 = 197.446, p < 0.0001) and laterality (F 1,40 = 6.356, p < 0.05). After the Day 10 assessment, rats received intrathecal
(i.t.) injections of either AAV2-GFP (Control) or AAV2-r-IL-10 (timing denoted by the second vertical dotted line). Behavioral
assessments were again recorded on Days 13, 15, 17, 19, 21, 24, 26, and 30 after surgery; that is, Days 3, 5, 7, 9, 11, 14, 16, and
20 days after AAV. While neither AAV2-GFP nor AAV2-r-IL-10 exerted marked effects in sham operated animals (Panels A,
B), AAV2-r-IL-10 transiently attenuated bilateral CCI allodynia compared to CCI operated AAV2-GFP treated animals (Panels C, D). For Days 13–26, ANOVA revealed reliable main effects of CCI (F 1,40 = 496.336, p < 0.0001), IL-10 (F 1,40 = 59.636,
p < 0.0001), and laterality (F 1,40 = 28.565, p < 0.0001), and interactions between CCI and IL-10 (F 1,40 = 72.988, p < 0.0001) and
CCI and laterality (F 1,40 = 9.325, p < 0.01). At Day 30, behavioral responses were not significantly different from Day 10 preinjection levels (F 1,40 = 0.696, p > 0.40).
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6, and transforming growth factor-beta are categorized as
either anti-inflammatory or pro-inflammatory cytokines,
under various circumstances [29-32]. Anti-inflammatory
effects are also exerted by a variety of endogenous agents
as well, such as IL-1 receptor antagonist, soluble and
membrane-bound IL-1 decoy receptors, and soluble TNF
decoy receptors [29]. Thus a number of anti-inflammatory
substances exist which may potentially exhibit therapeutic
effects for enhanced pain states.
IL-10 was chosen for the present study for several reasons.
First, it is considered to be the most powerful anti-inflammatory cytokine, potently downregulating TNF, IL-1 and
IL-6 production and release [19,29,33]. In addition, IL-10
can upregulate endogenous anti-cytokines and downregulate pro-inflammatory cytokine receptors [19,29,33].
Thus, it can counter-regulate production and function of
pro-inflammatory cytokines at multiple levels. Second,
simultaneous suppression of multiple pro-inflammatory
cytokines, rather than targeting a single cytokine, has
advantages for 2 reasons: (a) pro-inflammatory cytokines
are redundant, such that blockade of a single pro-inflammatory cytokine results in its functions being taken over
by other pro-inflammatory cytokines [34], and (b) TNF,
IL-1 and IL-6 can vary greatly in their relative magnitude
of production, dependent both upon the inciting stimulus
and time. This has been observed in spinal cord under
conditions of pain facilitation as well [35]. Third, acute
administration of IL-10 protein has been documented in
previous studies to suppress the development of spinallymediated pain facilitation in diverse animal models,
including intrathecal dynorphin, peri-sciatic snake venom
phospholipase A2, and spinal cord excitotoxic injury [3640]. As IL-10 has a very short half-life (~2 hr) in rat cerebrospinal fluid (L. He, R. Chavez and K. Johnson, unpublished observations), IL-10 gene therapy may provide an
efficient means of attaining neuropathic pain control
across days. Lastly, evidence to date indicates that spinal
cord neurons do not express receptors for IL-10 under
either basal or inflammatory conditions [41]. Therefore,
in spinal cord, IL-10 may selectively target glia, without
disrupting neuronal function. Indeed, the major reported
effect of IL-10 on neurons is enhancement of neuronal
survival, an effect thought to be indirect via the inhibition
of glially-derived neuroexcitotoxic products, including
pro-inflammatory cytokines [42-45].
Replication-defective adeno-associated viral vectors offer
numerous advantages for use in human gene therapy. This
vector rarely inserts into host DNA, thus avoiding insertional effects common to other gene therapy approaches
[46]. In addition, AAV is less inflammatory than other
gene delivery vectors, such as adenovirus [47]. For such
reasons, adeno-associated viruses have recently attracted
attention as vectors for human gene therapy [48].
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However, the present report found the efficacy of AAV2 to
be short-lived upon intrathecal administration. This was
surprising, given the generally accepted long-term persistence of AAV transgene expression. When AAV is administered directly into spinal cord tissue or dorsal root ganglia,
AAV-directed gene expression persists for at least 4–8
months [49,50]. Since AAV injection into spinal cord or
dorsal root ganglia requires traumatic surgery to expose
the site, such approaches are not optimal for human
application. Our intent is to explore therapies with potential use in humans, and therefore an acute intrathecal
injection via percutaneous lumbar puncture route of
administration was used. It is possible that AAVs of different serotypes and promoters may produce far more longlasting effects following intrathecal administration, as
serotype and promoter tropism of AAV can greatly affect
what cell type(s) are infected and/or express the transgene
[50-53]. Indeed, our ongoing studies suggest that far
longer IL-10 protein expression can be attained by altering
the AAV serotype used for intrathecal gene therapy (T. Liu,
R. Chavez, and K. Johnson, unpublished data). Thus,
while the results with AAV2 reported in the present studies
were transient, they do indicate the potential for intrathecal IL-10 gene therapy for pain.
Conclusion
The conclusions from the present experiments are clear.
First, intrathecal IL-10 gene therapy shows promise for
control of neuropathic pain. This is exciting as it demonstrates that targeting products of spinal cord glial activation can produce prolonged suppression of both
mechanical allodynia and thermal hyperalgesia. Second,
neuropathic pain was not only prevented but also
reversed by intrathecal IL-10 gene therapy. This is important, as it implies that spinal cord glial activation contributes to both the initiation and maintenance of
neuropathic pain. Third, intrathecally administered AAV2
targets primarily meningeal cells. This may be advantageous, as infection of meningeal cells avoids retrograde
transport of AAV to distant sites, as has been observed in
brainstem neurons following intra-spinal cord injections
[25]. Lastly, it is anticipated that the duration and potency
of AAV-IL-10 gene therapy can be improved by careful
selection of the optimal AAV serotype.
Methods
Subjects
Pathogen-free adult male Sprague-Dawley rats (300–425
g; Harlan Labs) were used in all experiments. Rats were
housed in temperature (23+/-3°C) and light (12:12 light:
dark; lights on at 0700 hr) controlled rooms with standard rodent chow and water available ad libitum. Behavioral testing was performed during the light cycle. The
Institutional Animal Care and Use Committee of the University of Colorado at Boulder approved all procedures.
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Drugs and adeno-associated viral vectors
Zymosan (yeast cell walls; Sigma Chemical Co., St. Louis,
MO) was made fresh daily by suspension in a vehicle of
incomplete Freund's adjuvant (Sigma Chemical Co., St.
Louis, MO). The final concentrations were 0 (vehicle control), 0.08, or 3.2 ug/ul delivered peri-sciatically in 50 ul
as described previously [10].
A replication-defective adeno-associated virus (AAV)
expression vector (serotype II) containing the cDNA
encoding for rat IL-10 (r-IL-10) was created, packaged and
purified at the Powell Gene Therapy Center, University of
Florida at Gainesville, as previously described [54].
Briefly, co-transfection was conducted with the proviral
cassette with plasmid (pDG) that provides the AAV rep
and cap genes in trans as well as adenoviral genes E2a, E4
and VA. The E1a and E1b genes were in the complimentary cell line, HEK 293. The vector cassette containing the
cDNA encoding rat IL-10 (AAV2-r-IL-10) was driven by
the hybrid cytomegalovirus (CMV) enhancer/chicken
beta actin promoter/hybrid intron (pTR2-CB-rIL-10). The
control AAV (AAV2-GFP) was an analogous AAV expression vector in which the CMV enhancer/chicken beta actin
promoter directs the expression of the reporter gene
encoding jellyfish green fluorescent protein (GFP). Viral
titers were determined by infectious center assay as previously described [54]. Here, viral titers were approximately
1.7 × 10^11 infectious particles/ml (total dose given was
approximately 8.5 × 10^8 infectious particles in 5 ul) and
1.69 × 10^11 infectious particles/ml (total dose given was
8.45 × 10^8 infectious particles in 5 ul) for AAV2-r-IL-10
and AAV2-GFP, respectively.
A replication-defective AAV expression vector (serotype II)
containing the cDNA encoding LacZ, driven by the CMV
promoter that directs the expression of beta-galactosidase
was obtained from Avigen (Alameda, CA. USA). Briefly,
HEK 293 cells were co-transfected by the calcium phosphate method with pAAV4.6CMV-lacZ (a transgene vector
encoding beta-galactosidase), an adenovirus helper plasmid and an AAV plasmid encoding the AAV2 rep and cap
genes as described [55]. For each transfection, 10 ug of
each plasmid was used. The cells were harvested after 48
hrs, centrifuged, and resuspended in Tris-buffered saline
(TBS). Cell lysates were collected after three freeze-thaw
cycles (alternating between dry ice-ethanol and 37°C
baths). The lysates were made free of debris by centrifugation. This supernatant was precipitated with PEG (8000)
and the AAV pellet was fractionated on a CsCl gradient
overnight. The AAV band containing functional viral particles was removed and precipitated again. The final material was resuspended in a buffer containing TBS and
Pluronic F-68 (0.01%). Viral titers were approximately 4.1
× 10^10 vector genomes/ml (total dose given was approximately 4.1 × 10^8 vector genomes in 10 ul)
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Behavioral Measures
von Frey Test
The von Frey test [56] was performed within the sciatic or
saphenous innervation area of the hindpaws as previously
described [20,27,57,58]. Briefly, a logarithmic series of 10
calibrated Semmes-Weinstein monofilaments (von Frey
hairs; Stoelting, Wood Dale, IL) was applied randomly to
the left and right hind paws to determine the stimulus
intensity threshold stiffness required to elicit a paw withdrawal response. Log stiffness of the hairs is determined
by log10 (milligrams × 10). The 10 stimuli had the following log-stiffness values (values in milligrams are given in
parenthesis): 3.61 (407 mg), 3.84 (692 mg), 4.08 (1,202
mg), 4.17 (1,479 mg), 4.31 (2,041 mg), 4.56 (3,630 mg),
4.74 (5,495 mg), 4.93 (8,511 mg), 5.07 (11,749 mg), and
5.18 (15,136 mg). The range of monofilaments used in
these experiments (0.407–15.136 gm) produces a logarithmically graded slope. Interpolated 50% response
threshold data is expressed as stimulus intensity in log10
(milligrams × 10). Assessments were made prior to (baseline) and at specific times after peri-sciatic and intrathecal
drug administration, as detailed below for each experiment. Behavioral testing was performed blind with
respect to drug administration. The behavioral responses
were used to calculate the 50% paw withdrawal threshold
(absolute threshold), by fitting a Gaussian integral psychometric function using a maximum-likelihood fitting
method [59,60], as described in detail previously [57,58].
This fitting method allows parametric statistical analyses,
as discussed previously [57,58].
Hargreaves Test
Thresholds for behavioral response to heat stimuli
applied to each hindpaw were assessed using the Hargreaves test [61], as previously described [58]. Briefly,
baseline (BL) paw withdrawal values were calculated from
an average of 3–6 consecutive withdrawal latencies of
both the left and right hindpaws measured during a 1 hr
period. Voltage to the heat source was adjusted to yield BL
latencies ranging 8–12 sec and a cut off time of 20 sec was
imposed to avoid tissue damage. This procedure was followed by intrathecal injections and a timecourse of postdrug behavioral assessments, as described below. Behavioral testing was performed blind with respect to drug
administration. The order of paw testing varied randomly.
Surgery and microinjections
Acute lumbar punctures
An injection catheter was temporarily inserted under brief
isoflurane anesthesia (1–2% in oxygen). Here, a 25 cm
PE-10 catheter (attached by a 30-gauge sterile needle to a
sterile, 50 ul glass Hamilton syringe) was marked 7.7–7.8
cm from the open end and placed in a sterile, dry container until the time of injection. Under light anesthesia,
the dorsal pelvic area was shaved and swabbed with 70%
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Molecular Pain 2005, 1:9
alcohol. An 18-gauge sterile needle with the plastic hub
removed was inserted between lumbar vertebrae L5 and
L6. The open end of the PE-10 catheter was inserted via
the 18-gauge needle and threaded to the 7.7 cm mark
allowing for intrathecal PE-10 catheter-tip placement at
the level of the lumbosacral enlargement. Drugs were
injected over 1 min with a 1 ul pre- and post 0.9% sterile,
isotonic saline flush. The PE-10 catheter was immediately
withdrawn and the 18-gauge needle was removed from
the L5-L6 inter-vertebral space. This acute injection
method took 2–3 min to complete, and rats showed full
recovery from anesthesia within 10 min. No abnormal
motor behavior was observed after any injection. Lumbar
puncture injections were only performed by investigators
proven to have a 100% accuracy rate with the identical
procedure using Evans' blue dye for injection confirmation in >12 rats.
Chronic peri-sciatic catheters
Peri-sciatic catheters were constructed and implanted at
mid-thigh level of the left hind leg as previously described
[10,20,27]. This method allowed multi-day recovery from
isoflurane anesthesia prior to unilateral microinjection of
an immune activator or vehicle around the sciatic nerve.
This avoids the deleterious effects of anesthetics on the
function of both immune [62-64] and glial cells [65-68].
In addition, this indwelling catheter method allowed perisciatic immune activation to be either acute (single injection of an immune activator) or chronic (repeated injections across weeks) [10]. Both methods were used in the
present experiments in awake, unrestrained rats. These
acute and chronic peri-sciatic microinjections over the left
sciatic nerve were performed as previously described
[10,20]. Catheters were verified at sacrifice by visual
inspection. Data were only analyzed from confirmed sites.
Chronic constriction injury (CCI)
CCI was created at mid-thigh level of the left hindleg as
previously described [21]. Four sterile, absorbable surgical
chromic gut sutures (cuticular 4-0, chromic gut, 27", cutting FS-2; Ethicon, Somerville, NJ) were loosely tied
around the gently isolated sciatic nerve under isoflurane
anesthesia (Phoenix Pharm., St. Joseph, MO). The sciatic
nerves of sham-operated rats were identically exposed but
not ligated. Suture placements were verified at sacrifice by
visual inspection. Data were only analyzed from confirmed sites.
Histochemistry for the expression of AAV2 driven betagalactosidase
Histochemistry for spinal cord beta-galactosidase was
conducted as previously described [25] with following
changes described here. Briefly, rats were deeply anaesthetized with sodium pentobarbital and transcardially perfused with 0.9% saline (5 min) followed by chilled, fresh
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2% paraformaldehyde in 0.1% PBS (5 min). Whole spinal
cords 3 cm rostral and 3.5 cm caudal to the injection site
were collected and post-fixed in 2% paraformaldehyde for
15 min at room temperature. As a first examination of the
spread of AAV2 driven beta-galactosidase, spinal cord was
sectioned into 0.5–1 cm segments, collected into 12-well
plates and washed (30 min each at room temperature
with gentle agitation) 3 times with LacZ wash solution
(0.1 M PBS, 0.1% deoxycholic acid [Sigma-Aldrich, St.
Louis, MO], 0.2% IGEPAL CA-630 [Sigma-Aldrich, St.
Louis, MO] and 2 mM MgCl2 in distilled water). Tissues
were transferred to clean 12-well plates containing LacZ
stain solution (5 mM K-ferri/ferrocyanide [Sigma-Aldrich,
St. Louis, MO], 10% X-gal [Sigma-Aldrich, St. Louis, MO]
in LacZ wash solution) and incubated in the dark for 3 hr
and 45 min at 37°C followed by overnight incubation at
4°C, post-fixed for 24 hr in 4 % paraformaldehyde at 4°C
and stored in 70% ethanol at 4°C. After inspection of
beta-galactosidase staining in rostro-caudally re-constructed spinal cords of AAV2 injected and control rats,
spinal segments (control and LacZ treated) were sliced at
45-micron sections and photomicrographed according to
methods previously described [57]. Briefly, large segments
were cryoprotected (30% sucrose in phosphate buffer
overnight), frozen embedded in OCT compound (Tek,
Ted Pelli Inc, Redding, CA), cryostat-sectioned at 25
microns and thaw- mounted, (0.5% gelatin-treated glass
slides; Fisherbrand Superfrost Plus Slides; Fisher Scientific, Pittsburgh, PA). Spinal cord segments from the injection site (L3–L6) to 1.5 cm rostral to the injection site, and
at the most caudal area of spinal cord (C2–C4) were collected because these areas best represent the degree of
spread of beta-galactosidase expression from the injection
site. The thaw mounted, cryostat sections (3–4 per slide)
were viewed for beta-galactosidase expression with an
Olympus bright-field microscope (model BX61). Images
were collected with an Olympus Magnafire camera coupled to a Dell computer equipped with Olympus MagnaFire SP for windows software.
Data Analysis
All statistical comparisons were computed using Statview
5.0.1 for the Macintosh. Data from the von Frey test were
analyzed as the interpolated 50% threshold (absolute
threshold) in log base 10 of stimulus intensity (monofilament stiffness in milligrams × 10). Baseline measures for
both the von Frey and Hargreaves tests, and dose response
effects of adenovirus, were analyzed by one-way ANOVA.
Timecourse measures for each behavioral test were analyzed by repeated measures ANOVAs followed by Fisher's
protected least significant difference posthoc comparisons, where appropriate.
List of Abbreviations
AAV2 – adeno-associated virus, serotype II
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Molecular Pain 2005, 1:9
AAV2-r-IL-10 – adeno-associated virus (serotype II)
encoding for rat interleukin-10
ANOVA – analysis of variance
BL – baseline
CCI – chronic constriction injury
CMV – cytomegalovirus
CsCl – cesium chloride
CSF – cerebrospinal fluid
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surgeries and tissue collections; SH performed surgeries
and animal perfusions; PEC designed, created and tested
the AAV2-rat IL-10 vector in a cell expression system, JRF
contributed to data interpretation and manuscript preparation; TRF oversaw the design, creation and testing of the
AAV2-rat IL-10 vector and provided comments on manuscript drafts, SFM assisted in manuscript preparation and
consulted on experimental design, statistics, and interpretation, LAL oversaw the production of AAV and assisted in
manuscript preparation and experiment interpretation,
RC participated in data interpretation and manuscript
preparation as well as advising on AAV2-LacZ anatomy
methodology and producing the AAV2-LacZ vector, LRW
consulted on experimental design and interpretation and
was responsible for manuscript and figure preparation.
GFP – green fluorescent protein
Note added in proof
IL – interleukin
PE – polyethylene
SIN – sciatic inflammatory neuropathy
A previously undetected point mutation, resulting in an
amino acid change (F129S), has been identified in the rat
IL-10 gene expressed in this study. This mutation lies outside of identified receptor binding regions. Its effect on IL10 bioactivity in vitro, if any, is currently under investigation
TBS – Tris-buffered saline
Acknowledgements
TNF – tumor necrosis factor
Competing interests
Avigen is a designated collaborator on NIH grant
DA018156 (Cutting Edge Biomedical Research Award,
Phase 2) awarded to ED Milligan (Principal Investigator).
Avigen has provided additional research support for this
project.
This work was supported by NIH grants DA015642, NS40696, DA015656,
DA015591, DA018156, and HL56510, and grants from Avigen and JDRF.
References
1.
2.
Avigen authors hold stocks or shares in Avigen.
3.
University of Colorado and University of Florida authors
do not hold stocks or shares in Avigen.
4.
5.
The University of Colorado has applied for a patent relating to the content of the manuscript, for which the Watkins laboratory has received licensing fees.
No other financial competing interests exist.
6.
7.
8.
Authors' contributions
EDM performed surgeries, designed and performed the
behavioral and anatomical studies, oversaw data entry,
performed statistical analyses, and contributed to manuscript and graphics preparation; EMS performed surgeries
and behavioral studies as well as participated in data
entry, SJL grew and purified vectors, MC performed surgeries and behavioral studies as well as participated in
data entry, LS: performed surgeries and behavioral studies
as well as participated in data entry, JWF performed
9.
10.
11.
12.
Collins SL, Moore A, McQuay HJ, Wiffen P: Antidepressants and
anticonvulsants for diabetic neuropathy and postherpetic
neuralgia: a quantitative systematic review. J Pain Symptom
Manage 2000, 20:339-457.
McQuay H, Tramer M, Nye BA, Carroll D, Wiffen P, Moore RA: A
systematic review of antidepressants in neuropathic pain.
Pain 1996, 68:217-227.
McQuay H, Carroll D, Jadad AR, Wiffen P, Moore A: Anticonvulsant drugs for management of pain: a systematic review. Brit
Med J 1995, 311:1047-1052.
DeLeo JA, Tanga FY, Tawfik V: Neuroimmune activation and
neuroinflammation in chronic pain and opioid tolerance/
hyperalgesia. The Neuroscientist 2004, 10:40-52.
Watkins LR, Maier SF: Glia: a novel drug discovery target for
clinical pain. Nature Reviews Drug Discovery 2003, 2:973-985.
Watkins LR, Milligan ED, Maier SF: Glial activation: a driving force
for pathological pain. Trends Neurosci 2001, 24:450-455.
Watkins LR, Maier SF: Targeting glia to control clinical pain: An
idea whose time has come. Drug Discovery Today: Therapeutic
Strategies 2004.
Berg-Johnsen J, Paulsen RE, Fonnum F, Langmoen IA: Changes in
evoked potentials and amino acid content during fluorocitrate action studied in rat hippocampal cortex. Exp Brain Res
1993, 96:241-246.
Hassel B, Paulsen RE, Johnson A, Fonnum F: Selective inhibition of
glial cell metabolism by fluorocitrate. Brain Res 1992,
249:120-124.
Milligan ED, Twining C, Chacur M, Biedenkapp J, O'Connor KA, Poole
S, Tracey KJ, Martin D, Maier SF, Watkins LR: Spinal glia and proinflammatory cytokines mediate mirror-image neuropathic
pain. J Neurosci 2003, 23:1026-1040.
Willoughby JO, Mackenzie L, Broberg M, Thoren AE, Medvedev A,
Sims NR, Nilsson M: Fluorocitrate-mediated astroglial dysfunction causes seizures. J Neurosci Res 2003, 74:160-166.
Tikka T, Fiebich BL, Godsteins G, Keinanen R, Koistinaho J: Minocycline, a tetracycline derivative, is neuroprotective against
Page 11 of 13
(page number not for citation purposes)
Molecular Pain 2005, 1:9
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
excitotoxicity by inhibiting activation and proliferation of
microglia. J Neurosci 2001, 21:2580-2588.
Raghavendra V, Tanga F, DeLeo JA: Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy. J Pharmacol Exp Ther
2003, 306:624-630.
Ledeboer A, Sloane E, Chacur M, Milligan ED, Maier SF, Watkins LR:
Selective inhibition of spinal cord microglial activation attenuates mechanical allodynia in rat models of pathological
pain. Proc Soc Neurosci 2003, 29:.
Sweitzer SM, Martin D, DeLeo JA: Intrathecal interleukin-1
receptor antagonist in combination with soluble tumor
necrosis factor receptor exhibits an anti-allodynic action in a
rat model of neuropathic pain. Neurosci 2001, 103:529-539.
DeLeo JA, Colburn RW, Nichols M, Malhotra A: Interleukin (IL)-6
mediated hyperalgesia/allodynia and increased spinal IL-6 in
a rat mononeuropathy model. J Interferon Cytokine Res 1996,
16:695-700.
Sweitzer SM, Schubert P, DeLeo JA: Propentofylline, a glial modulating agent, exhibits anti-allodynic properties in a rat
model of neuropathic pain. J Pharmacol Exp Ther 2001,
297:1210-1217.
Hashizume H, Rutkowski MD, Weinstein JN, DeLeo JA: Central
administration of methotrexate reduces mechanical allodynia in an animal model of radiculopathy/sciatica. Pain 2000,
87:159-169.
Moore KW, Ho ASY, Xu-Amano J: Molecular biology of interleukin-10 and its receptor. In Interleukin-10 Edited by: DeVries JE
and de Waal Malefyt R. , R.G. Landes Company; 1995:1-9.
Chacur M, Milligan ED, Gazda LS, Armstrong C, Wang H, Tracey KJ,
Maier SF, Watkins LR: A new model of sciatic inflammatory
neuritis (SIN): induction of unilateral and bilateral mechanical allodynia following acute unilateral peri-sciatic immune
activation in rats. Pain 2001, 94:231-244.
Bennett GJ, Xie YK: A peripheral mononeuropathy in rat that
produces disorders of pain sensation like those seen in man.
Pain 1988, 33:87-107.
Spataro LE, Sloane EM, Milligan ED, Wieseler-Frank J, Schoeniger D,
Jekich BM, Barrientos RM, Maier SF, Watkins LR: Spinal gap junctions: Potential involvement in pain facilitation. J Pain 2004,
5:392-405.
Twining CM, Sloane EM, Schoeniger DK, Milligan ED, Martin D, Marsh
H, Maier SF, Watkins LR: Activation of the spinal cord complement cascade may contribute to mechanical allodynia
induced by three animal models. Journal of Pain 2004 in press.
Milligan ED, Zapata V, Chacur M, Schoeniger D, Biedenkapp J, O’Connor K, Verge GM, Chapman G, Green P, Foster AC, Naeve GS, Maier
SF, Watkins LR: Evidence that exogenous and endogenous
fractalkine can induce spinal nociceptive facilitation. Eur J
Neurosci 2004, 20:2294-2302.
Mannes AJ, Caudle RM, O'Connell BC, Iadarola MJ: Adenoviral
gene transfer to spinal cord neurons: intrathecal vs. intraparenchymal administration. Brain Res 1998, 793:1-6.
Milligan ED, Maier SF, Watkins LR: Sciatic Inflammatory Neuropathy (SIN) in the rat: Surgical procedures, induction of
inflammation and behavioral testing. In Pain Research: Methods
and Protocols Volume 99. Edited by: Luo ZD. Totowa, Humana Press;
2004:67-89.
Gazda LS, Milligan ED, Hansen MK, Twining CM, Paulos N, Chacur M,
O’Connor KA, Armstrong C, Maier SF: Sciatic inflammatory neuritis (SIN): behavioral allodynia is paralleled by peri-sciatic
proinflammatory cytokine and superoxide production. J
Peripheral Nerv Sys 2001, 6:111-129.
Yoshimura A, Mori H, Ohishi M, Aki D, Hanada T: Negative regulation of cytokine signaling influences inflammation. Curr
Opion Immunol 2003, 15:704-708.
Opal SM, DePalo VA: Anti-inflammatory cytokines. Chest 2000,
117:1162-1172.
Knight D: Leukaemia inhibitory factor (LIF): a cytokine of
emerging importance in chronic airway inflammation. Pulm
Pharmacol Ther 2001, 14:169-176.
Jones SA, Horiuchi S, Topley N, Yamamoto N, Fuller GM: The soluble interleukin 6 receptor: mechanisms of production and
implications in disease. FASEB J 2001, 15:43-58.
http://www.molecularpain.com/content/1/1/9
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
Tilg H, Peschel C: Interferon-alpha and its effects on the
cytokine cascade: a pro- and anti-inflammatory cytokine.
Leuk Lymphoma 1996, 23:55-60.
Strle K, Zhou JH, Broussard SR, Johnson RW, Freund GG, Dantzer R,
Kelley KW: Interleukin-10 in the brain. Crit Rev Immunology 2001,
21:427-449.
Bluthe RM, Laye S, Michaud B, Combe C, Dantzer R, Parnet P: Role
of interleukin-1beta and tumour necrosis factor-alpha in
lipolysaccharide-induced sickness behaviour: a study with
interleukin-1 type I receptor-deficient mice. Eur J Neurosci
2000, 12:4447-4456.
Raghavendra V, Tanga FY, DeLeo JA: Complete Freunds adjuvant-induced peripheral inflammation evokes glial activation
and proinflammatory cytokine expression in the CNS. Eur J
Neurosci 2004, 20:467-473.
Laughlin TM, Bethea JR, Yezierski RP, Wilcox GL: Cytokine
involvement in dynorphin-induced allodynia. Pain 2000,
84:159-167.
Chacur M, Milligan ED, Sloane EM, Wieseler-Frank J, Barrientos RM,
Martin D, Poole S, Lomonte B, Gutierrez JM, Maier SF, Cury Y, Watkins LR: Snake venom phospholipase A2s (Asp49 and Lys49)
induce mechanical allodynia upon peri-sciatic administration: involvement of spinal cord glia, proinflammatory
cytokines and nitric oxide. Pain 2004, 108:180-191.
Plunkett JA, Yu CG, Easton J, Bethea JR, Yezierski RP: Effects of
interleukin-10 (IL-10) on pain behavior and gene expression
following excitotoxic spinal cord injury in the rat. Exper Neurol
2001, 168:144-154.
Yu CG, Fairbanks CA, Wilcox GL, Yezierski RP: Effects of agmatine, interleukin-10 and cyclosporin on spontaneous pain
behavior following excitotoxic spinal cord injury in rats. J Pain
2003, 4:129-140.
Abraham KE, McMillen D, Brewer KL: The effects of endogenous
interleukin-10 on gray matter damage and pain behaviors
following excitotoxic spinal cord injury in the mouse. Neurosci
2004, 124:945-922.
Ledeboer A, Wierinckx A, Bol JGJM, Floris S, Renardel de Lavaletter
C, DeVries HE, van den Berg T, Dijkstra CD, Tilders FJH, Van Dam
AM: Regional and temporal expression patterns of interleukin-10, interleukin-10 receptor and adhesion molecules in
the rat spinal cord during chronic relapsing EAE. J
Neuroimmunol 2003, 136:94-103.
Koeberle PD, Gauldie J, Ball AK: Effects of adenoviral-mediated
gene transfer of interleukin-10, interleukin-4, and transforming growth factor-beta on the survival of axotomized retinal
ganglion cells. Neurosci 2004, 125:903-920.
Lynch AM, Walsh C, Delaney A, Nolan Y, Campbell VA, Lynch MA:
Lipopolysaccharide-induced increase in signalling in hippocampus is abrogated by IL-10 -- a role for IL-1 beta? J Neurosci
2004, 88:635-646.
Grilli M, Barbieri I, Basudev H, Brusa R, Casati C, Lozza G, Ongini E:
Interleukin-10 modulates neuronal threshold of vulnerability
to ischaemic damage. Eur J Neurosci 2000, 12:2265-2272.
Brewer KL, Bethea JR, Yezierski RP: Neuroprotective effects of
interleukin-10 folowing spinal cord injury. Exp Neurol 1999,
159:484-493.
Buchschacher GLJ, Wong-Staal F: Development of lentiviral vectors for gene therapy for human diseases. Blood 2000,
95:2499-2504.
Gudmundsson G, Bosch A, Davidson BL, Hunninghake GW: Interleukin-10 modulates the severity of hypersensitivity
pneumonitis. Amer J Resp Cell & Molec Biol 1998, 19:812-818.
Daly TM: Overview of adeno-associated viral vectors. Methods
Mol Biol 2004, 246:157-165.
Blits B, Carlstedt TP, Ruitenberg MJ, DeWinter F, Hermens WT,
Dijkhuizen PA, Claasens JW, Eggers R, Van der Sluis R, Tenenbaum L,
Boer GJ, Verhaagen J: Rescue and sprouting of motoneurons
following ventral root avulsion and reimplantation combined
with intraspinal adeno-associated viral vector-mediated
expression of glial cell line-derived neurotrophic factor or
brain-derived neurotrophic factor. Exp Neurol 2004,
189:303-316.
Xu Y, Gu Y, Wu PC, Li GW, Huang LYM: Efficiencies of transgene
expression in nociceptive neurons through different routes
of delivery of adeno-associated viral vectors. Human Gene Ther
2003, 14:897-906.
Page 12 of 13
(page number not for citation purposes)
Molecular Pain 2005, 1:9
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
Bartlett JS, Samulski RJ, McCown TJ: Selective and rapid uptake
of adeno-associated virus type 2 in brain. Hum Gene Ther 1998,
9:1181-1186.
Kugler S, Lingor P, Scholl U, Zolotukhin S, Bahr M: Differential
transgene expression in brain cells in vivo and in vitro from
AAV-2 vectors with small transcriptional control units. Virology 2003, 311:89-95.
Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin S, Reier PJ, Mandel RJ, Muzyczka N: Recombinant AAV
viral vectors pseudotyped with viral capsids from serotypes
1, 2, and 5 display differential efficiency and cell tropism after
delivery to different regions of the central nervous system.
Mol Ther 2004, 10:302-317.
Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, Chestnut K,
Summerford C, Samulski RJ, Muzyczka N: Recombinant adenoassociated virus purification using novel methods improves
infectious titer and yield. Gene Ther 1999, 6:973-985.
Matsushita T, Elliger S, Elliger C, Podsakoff G, Villarreal L, Kurtzman
GJ, Iwaki Y, Colosi P: Adeno-associated virus vectors can be
efficiently produced without helper virus. Gene Ther 1998,
5:938-945.
Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL: Quantitative assessment of tactile allodynia in the rat paw. J Neurosci
Meth 1994, 53:55-63.
Milligan ED, O'Connor KA, Nguyen KT, Armstrong CB, Twining C,
Gaykema R, Holguin A, Martin D, Maier SF, Watkins LR: Intrathecal
HIV-1 envelope glycoprotein gp120 enhanced pain states
mediated by spinal cord proinflammatory cytokines. J
Neurosci 2001, 21:2808-2819.
Milligan ED, Mehmert KK, Hinde JL, Harvey LOJ, Martin D, Tracey KJ,
Maier SF, Watkins LR: Thermal hyperalgesia and mechanical
allodynia produced by intrathecal administration of the
Human Immunodeficiency Virus-1 (HIV-1) envelope glycoprotein, gp120. Brain Res 2000, 861:105-116.
Treutwein B, Strasburger H: Fitting the psychometric function.
Percept Psychophys 1999, 61:87-106.
Harvey LOJ: Efficient estimation of sensory thresholds. Behav
Res Meth Instrum Comput 1986, 18:623-632.
Hargreaves K, Dubner R, Brown F, Flores C, Joris J: A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1998, 32:77-88.
Lockwood LL, Silbert LH, Laudenslager ML, Watkins LR, Maier SF:
Anesthesia-induced modulation of in vivo antibody levels: a
study of pentobarbital, chloral hydrate, methoxyflurane,
halothane, and ketamine/xylazine. Anesthes Analg 1993,
77:769-774.
Sato W, Enzan K, Masaki Y, Kayaba M, Suzuki M: The effect of isoflurane on the secretion of TNF-alpha and IL-1 beta from
LPS-stimulated human peripheral blood monocytes. Masui
1995, 44:971-975.
Miller LS, Morita Y, Rangan U, Kondo S, Clemens MG, Bulkley GB:
Suppression of cytokine-induced neutrophil accumulation in
rat mesenteric venules in vivo by general anesthesia. Int J
Microcirc Clin Exp 1996, 16:147-154.
Tas PW, Kress HG, Koschel K: General anesthetics can competitively interfere with sensitive membrane proteins. Proc Natl
Acad Sci U S A 1987, 84:5972-5975.
Feinstein DL, Murphy P, Sharp A, Galea E, Gavrilyuk V, Weinberg G:
Local anesthetics potentiate nitric oxide synthase type 2
expression in rat glial cells. J Neurosurg Anesthesiol 13:99-105.
Mantz J, Cordier J, Giaume C: Effects of general anesthetics on
intercellular communications mediated by gap junctions
between astrocytes in primary culture. Anesthesiology 1993,
78:892-901.
Miyazaki H, Nakamura Y, Arai T, Kataoka K: Increase of glutamate
uptake in astrocytes: a possible mechanism of action of volatile anesthetics. Anesthesiology 1997, 86:1359-1366.
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