PROJECT PERIODIC REPORT
APPENDIX I
Report of deliverable D2.1b.i
Grant Agreement number:
115007
Project acronym:
EUROPAIN
Appendix number to be filled in by coordinator, when all
Appendix number:
WP 2
reports has been collected
New animal models and new outcome
measures of chronic pain
Report of deliverable D 2.1b.i
Validation of new rat models of
antiretroviral drug (stavudine, d4T and
Indinavir)-induced neuropathic pain
Month of delivery:
M 36
Author(s):
Wenlong Huang
Report Release date:
Date
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Contents
Summary………………………………………………………………………………. 2
Background and objective…………………………………………………………….. 2
Study design…………………………………………………………………………… 2
Results…………………………………………………………………………………. 2
Objectives achieved…………………………………………………………………… 3
Deviations………………………………………………………………………………3
Publications……………………………………………………………………………. 3
Summary
We have shown that rats treated with the key antiretroviral drug stavudine (d4T), developed a
persistent painful peripheral sensory, but not motor, neuropathy, mirroring a major clinical
problem in HIV disease management. In keeping with the clinical presentation, we have
demonstrated simple reflex pain behaviour that is appropriately sensitive to pharmacological
perturbation as well as complex pain behaviours. Furthermore, we have shown, using
histology, that this neuropathy is characterized by an axonal retraction from the skin and also
a novel finding of central spinal axonal retraction, which has important implications for the
pathology of this condition. We have also shown that glial and immune cell responses,
characteristic of nerve injury, are minor features of d4T neurotoxicity, thus calling into
question the direct generic relationship of such phenomena with pain-related behaviours. We
have also examined the axonal proteomic response to d4T treatment. Importantly, we have
shown that this neurotoxicity occurs independently of HIV infection, a confound that is
difficult to dissect in humans since the two conditions co-exist as the interaction between HIV
and sensory neurons is also the cause of a painful neuropathy.
We have shown that rats treated with the protease inhibitor Indinavir developed hindpaw
mechanical and cold hypersensitivity, but not heat hypersensitivity. There appeared to be an
increased thigmotaxis behaviour following Indinavir treatment. Initial histological
examination revealed a loss of hindpaw intra-epidermal nerve fibres and an infiltration of
macrophages in the L4 DRG following Indinavir treatment. Further experiments are needed to
characterise the thigmotaxis behavioural and histological changes.
Background and objective
HIV-associated sensory neuropathy (HIV-SN) is the most frequent neurological manifestation
of HIV disease and is seen in 40-50% of patients whose HIV disease is otherwise well
controlled by antiretroviral therapy (ART). It is a distal symmetrical, predominantly sensory,
polyneuropathy and is associated with significant neuropathic pain. HIV-SN can result from:
the HIV glycoprotein gp120 and/or ART such as the nucleoside reverse transcriptase
inhibitors (NRTIs). The NRTI d4T remains in the first-line HIV treatment in many resourcelimited countries due to its high effectiveness and inexpensiveness. Its neurotoxicity has been
reported in HIV patients, uninfected subjects receiving prophylaxis, and animal studies.
Continued d4T use as an economic reality will result in on-going high rates of HIV-SN,
particularly in resource-limited settings. Furthermore, a very large proportion of people living
with HIV in well-resourced settings have been exposed to d4t. HIV patients have limited
peripheral nerve regenerative capacity, suggesting that existing HIV-SN cases caused by d4T
use are likely to persist for life as past d4T exposure is a major risk factor for HIV-SN long
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after exposure to the drug has ceased. Considering that patients with access to modern ART
now enjoy a near-normal life expectancy, global HIV-SN burden will remain high. HIV-SN
has a negative impact on patients’ quality of life. The CHARTER study has revealed strong
associations of disability in daily activities and depression with painful HIV-SN. In a broader
context, anxiety, depression, and cognitive impairment are recognised as being associated
with chronic pain, but these pain-associated co-morbidities have not been adequately
addressed in animal models. A model of painful HIV-SN with the NRTI zalcitabine has been
described previously. However, no studies have assessed pain-associated co-morbidities in
animal models of d4T-induced HIV-SN to mimic the current clinical scenario of d4T use, nor
have detailed neuropathology following d4T treatment been described.
Recent studies have shown that the prevalence of HIV-SN does not decline even in resource
rich countries where neurotoxic NRTI drugs including d4T are no longer being used,
suggesting there are other important factors that may contribute to the development of HIVSN in NRTI drug naïve patients. One possibility is the toxicity of other drugs being used in
ART, e.g. the protease inhibitors. Evidence has suggested that exposure to indinavir, a
protease inhibitor commonly used as a part of the ART, is associated with high prevalence of
HIV-SN. In vitro studies have shown neurotoxicity in cultured adult rat dorsal root ganglion
cells when exposed to indinavir.
The objective of the deliverables was to validate rat models of HIV antiretroviral-induced
neuropathies, i.e. the NRTI d4T and the protease inhibitor Indinavir induced neuropathies, for
the purposes of mechanism and target identification and preclinical evaluation of novel
agents. The post doc for these experiments (Dr Wenlong Huang) took up his post in mid July
2010, so far we are 2 years and 2.5 months into 3-year the project (Dr Huang’s contract is for
2 year and 10 months), but this has been further extended by additional funding as a
consequence of Astellas joining EUROPAIN.
Study design
For the d4T study, first, we validated the approach in behavioural studies in adult rats, using
simple reflex hypersensitivity and pain-related aberrations in complex, ethologically relevant
behaviours. We then elucidated the neuropathological responses in the peripheral nerve and
spinal cord, of both neurons and immune cells, following d4T treatment. Finally, we
supplement with early stage proteomics analysis of the neuronal response.
For the Indinavir study, we will validate simple reflex pain behaviours as well as complex
ethologically relevant behaviours as used for the d4T study. We will examine in detail the
neuropathological responses in the peripheral nerve and spinal cord following Indinavir
treatment.
Results
For the d4T study, adult Wistar male rats were given two i.v. injections of d4T (50 mg/kg, 4
days apart). Following d4T treatment, rats developed hindpaw mechanical hypersensitivity
that was appropriately sensitive to pharmacological perturbation. We then used the open field
paradigm to assess thigmotaxis behaviour. On day 21 post the first d4T injection, increased
thigmotaxis behaviour was observed. The number of entries and the time spent in the inner
zone were significantly decreased as compared to those of the naïve and sham animals.
However, the total distance moved in the arena showed no difference between the groups
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indicating a lack of overt motor deficit in d4T-treated animals. We also used the burrowing
assay to examine the general well-being following d4T treatment. At day 21, d4T-treated rats
developed a significant deficit in burrowing when compared to the naïve and sham rats.
During the burrowing experiment, we also examined the locomotor function by using the rota
rod and we found no significant difference between the groups again indicating a lack of overt
motor deficit following d4T treatment. We also generated d4T tissues at days 7 and 21 post
first d4T injection and provided them to Dr David Bennett and Dr Margarita Calvo at King’s
College for neurochemical and electron microscopic studies. We found significant losses of
intraepidermal nerve fibres in the hindpaw skins and IB4+ and CGRP+ afferents in the L4/5
dorsal horns in d4T-treated animals. There was a minimal glial and immune cell responses
following d4T treatment. In collaboration with Dr Kenji Okuse and Dr Karu Kersti from
WP1, investigation of protein changes in the sural nerve at 7 days following d4T treatment
using proteomics approach showed down-regulated proteins related to mitochondrial
functions. Sural nerve proteomics also showed up-regulated proteins including Kininogen-1,
the precursor of bradykinin, known for its role in pain processing. We also provided fresh
spinal cord and DRG tissues from 7- and 21-day d4T animals for the next generation
sequencing study, in collaboration with Prof Steve McMahon and Dr Bennett from WP1.
For the Indinavir study, adult Wistar male rats were given two i.v. injections of Indinavir (50
mg/kg, 4 days apart). Following Indinavir treatment, rats developed hindpaw mechanical
hypersensitivity that peaked around day 14 and then returned to the baseline around day 42
following the first Indinavir injection. We also observed the development of hindpaw cold
hypersensitivity that persisted up to day 45 following the first Indinavir injection. However,
we did not observe any hindpaw heat hypersensitivity following Indinavir treatment. No
differences in body weights and blood glucose levels between indinavir- and vehicle-treated
animals were found, suggesting that our treatment regime will not cause diabetics as reported
in patients treated with Indinavir. Initial immunohistochemical assessments revealed loss of
intra-epidermal nerve fibres in the hindpaw skin and increased recruitment of macrophages in
the L4 DRG following indinavir treatment, however, further quantitative analysis is needed to
confirm these initial observations. In an initial thigmotaxis experiment, we have shown a
trend of reduced entries and time spent in the inner zone following Indinavir treatment (n=4),
when hindpaw mechanical hypersensitivity peaked, in comparison to those of the naïve (n=4)
and vehicle-treated (n=4) animals. Further experiments are needed to confirm the initial
thigmotaxis finding.
Objectives achieved
The characterisation of the d4T model has been completed. A manuscript has been prepared
and is currently in submission for publication. We have completed demonstration of
thigmotaxis as an ethologically relevant outcome measure. We have completed demonstration
of burrowing as an ethologically relevant outcome measure. The characterisation of the
indinavir model has just been started and is in a good progress. We have initially replicated
the novelty discrimination paradigm in the TNT model.
Deviations
No deviations.
Publications
Huang W et al 2012
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Manuscript in submission
W. Huang, M. Calvo, K. Karu, H. Olausen, G. Bathgate, K. Okuse, D.L.H. Bennett, A.S.C.
Rice. A clinically relevant rodent model of HIV antiretroviral drug stavudine induced painful
peripheral neuropathy.
Huang W et al 2012
Abstract and Poster at SfN 2011
W. Huang, T. Pheby, A.S.C. Rice. Assessment of anxiety-like behaviour as a pain comorbidity in a refined rat model of HIV antiretroviral drug (stavudine d4T)-induced sensory
neuropathy. Program No.583.11/II11. 2011 Neuroscience Meeting Planner. Washington, DC:
Society for Neuroscience, 2011. Online.
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