A clinically relevant rodent model of the HIV antiretroviral drug stavudine induced painful peripheral neuropathy

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PAIN 154 (2013) 560–575

www.elsevier.com/locate/pain

A clinically relevant rodent model of the HIV antiretroviral drug stavudine induced painful peripheral neuropathy Wenlong Huang a,⇑,1, Margarita Calvo b,1, Kersti Karu c, Hans R. Olausen a, Gabriella Bathgate a, Kenji Okuse c, David L.H. Bennett b,d,1, Andrew S.C. Rice a,e,1 a

Department of Surgery and Cancer, Imperial College London, UK Wolfson Centre for Age Related Disease, King’s College London, UK Division of Cell and Molecular Biology, Imperial College London, UK d Nuffield Department of Clinical Neurosciences, University of Oxford, UK e Pain Medicine, Chelsea and Westminster Hospital NHS Foundation Trust, London, UK b c

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

a r t i c l e

i n f o

Article history: Received 2 November 2012 Received in revised form 10 December 2012 Accepted 20 December 2012

Keywords: Burrowing HIV Neuropathy Peripheral Stavudine Thigmotaxis

a b s t r a c t HIV-associated sensory neuropathy is the most frequent manifestation of HIV disease, afflicting 40–50% of patients whose HIV disease is otherwise controlled by antiretroviral therapy. It often presents with significant neuropathic pain and is consistently associated with previous exposure to nucleoside reverse transcriptase inhibitors including stavudine (d4T), which is widely used in resource-limited settings. Here we investigated complex pain-related behaviours associated with d4T treatment using ethologically relevant thigmotaxis and burrowing behaviours in adult rats. Detailed neuropathological response was also examined using neurochemistry, electron microscopy, and proteomics. After 2 intravenous injections of d4T (50 mg/kg, 4 days apart), rats developed hind paw mechanical hypersensitivity, which plateaued at 21 days after initial d4T injection, a time that these animals also had significant changes in thigmotaxis and burrowing behaviours when compared to the controls; reductions in hind paw intraepidermal nerve fibre density and CGRP/IB4 immunoreactivity in L5 spinal dorsal horn, suggesting injury to both the peripheral and central terminals of L5 dorsal root ganglion neurons; and increases in myelinated and unmyelinated axon diameters in the sural nerve, suggesting axonal swelling. However, no significant glial and inflammatory cell response to d4T treatment was observed. Sural nerve proteomics at 7 days after initial d4T injection revealed down-regulated proteins associated with mitochondrial function, highlighting distal axons vulnerability to d4T neurotoxicity. In summary, we have reported complex behavioural changes and a distinctive neuropathology in a clinically relevant rat model of d4T-induced sensory neuropathy that is suitable for further pathophysiological investigation and preclinical evaluation of novel analgesics. Ó 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

1. Introduction Human immunodeficiency virus (HIV)-associated sensory neuropathy (SN) is the most frequent neurological manifestation of HIV disease and is observed in 40–50% of patients whose HIV disease is otherwise well controlled by antiretroviral therapy (ART) [37,39,78]. HIV-SN is a distal symmetrical, predominantly sensory polyneuropathy. The symptoms of HIV-SN are mainly distal in nat⇑ Corresponding author. Address: Department of Surgery and Cancer, Pain Research Group, Imperial College London, Chelsea and Westminster Hospital Campus, 369 Fulham Rd., London SW10 9NH, UK. Tel.: +44 (0) 20 8746 8424; fax: +44 (0) 20 8237 5109. E-mail address: [email protected] (W. Huang). 1 The first 2 and the last 2 authors contributed equally to this work.

ure, with the feet being predominantly affected, and it is associated with significant neuropathic pain [3,25,48,71,79]. HIV-SN can result from 2 clinically indistinguishable neuropathies with distinct pathologies: a distal axonal degeneration caused by the HIV glycoprotein gp120 [8,30,38,49,58,78,81], and ART-induced toxic neuropathy associated with nucleoside reverse transcriptase inhibitors (NRTIs) [37,39,82]. Since NRTI introduction, the morbidity and mortality of HIV infection have been markedly reduced [37]. The NRTI stavudine (d4T) remains in the first-line HIV treatment in many resourcelimited countries as a result of its high effectiveness and inexpensiveness [76], despite the World Health Organization’s recommendation to gradually phase it out [88]. d4T neurotoxicity has been reported in HIV patients [11,71,76], in uninfected subjects receiving prophylaxis [85], and in animal studies [23,34,66]. A

0304-3959/$36.00 Ó 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.pain.2012.12.023

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recent survey demonstrated that the prevalence of painful HIV-SN in South African HIV patients who had d4T exposure for at least 6 months before the survey was 44% [79]. Continued d4T use as an economic reality will result in ongoing high rates of HIV-SN, particularly in resource-limited settings. HIV patients have limited peripheral nerve regenerative capacity [28], suggesting that existing HIV-SN cases caused by d4T use are likely to persist for life, because past d4T exposure is a major risk factor for HIV-SN long after exposure to the drug has ceased [25,71]. Considering that patients with access to modern ART now enjoy a near-normal life expectancy [52,77], global HIV-SN burden will remain high [24,17]. 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 [25]. In a broader context, anxiety, depression, and cognitive impairment are recognized as being associated with chronic pain [19,27,50,55,71], but these pain-associated comorbidities have not been adequately addressed in animal models. A model of painful HIV-SN with the NRTI zalcitabine (ddC) has been described previously [16,34–36,47,82,83]. However, no studies have assessed pain-associated comorbidities in animal models of d4T-induced HIV-SN to mimic the current clinical scenario of d4T use, nor have any studies detailed neuropathology after d4T treatment been described. Here we hypothesized that systemic d4T treatment in rats would produce neuropathic painlike behaviours. Initially, we validated the approach in behavioural studies, which demonstrated that d4T-treated rats developed reflex hypersensitivity and pain-related aberrations in complex, ethologically relevant behaviours [2,29,33,81–84]. We then elucidated the neuropathological responses in the peripheral nerve and spinal cord of both neurons and immune cells after d4T treatment. Finally, we supplemented with early stage proteomics analysis of the neuronal response. 2. Material and methods 2.1. Ethical statement All animal experiments conformed to the British Home Office Regulations (Animal Scientific Procedures Act 1986; Project License PPL70/7162 to Prof Andrew Rice) and International Association for the Study of Pain guidelines [90] for the care and use of animals. We followed the ARRIVE guidelines for reporting the behavioural studies in preparing this report [40].

2.2. Experimental animals Male adult Wistar rats (200–300 g; Charles River, UK) were housed in temperature-controlled standard rat individually ventilated cages (21°C, 2–3 per cage) with corncob bedding and no environment enrichment. Animals were maintained on a 12:12 h light–dark cycle, provided with normal rat chow food (RM1 pelleted form; Special Diet Services, Essex, UK) and tap water ad libitum, and allowed to acclimatize in their housing environment for at least 48 h after arrival. 2.3. Study design The numbers of experimental and control groups and timings for behavioural assays are detailed in Table 1. Necessary steps, e.g., major domains of good laboratory practice [46], were taken to minimize the impact of experimental bias (Table 2). Except the burrowing assay, which was conducted from the beginning of the dark cycle, all other behavioural experiments were conducted in the light phase. All behavioural assessments were carried out in our behavioural laboratory at Chelsea and Westminster campus of Imperial College, while procedures involving intravenous (i.v.) and intraperitoneal (i.p.) injections and small animal surgeries were carried out in our surgical laboratory. The thigmotaxis and burrowing experiments consisted of batches of subset experiments (normally 2–3 animals per group), as a result of the capacity of our laboratory, and also in order to have comparable timings between test animals. For the thigmotaxis experiment, sequences of A–B–C then C–B–A (letters assigned to mask the cage labels during testing) were used to select animals. 2.4. Procedures for d4T treatment and behavioural experiments 2.4.1. Administration of d4T For rats receiving d4T treatment, 0.5 mL of d4T (a gift from Pfizer Ltd., UK; 50 mg/kg in sterile saline) was injected i.v. via a tail vein under a brief general anaesthesia (1–2% isoflurane [Abbott, UK] in O2 and N2O at a 1:1 ratio]. A second i.v. injection with the same volume and dose was given 4 days apart. Vehicle control animals received equivalent volumes of sterile saline using the same administration protocol for d4T. Although patients are generally administered d4T orally, previous studies have demonstrated that both daily oral gavage and a single i.v. administration routes produce similar nocifensive behavioural profiles in rats [34]. Thus, we opted for the i.v. route in order to minimize the handling stress

Table 1 Details of groups, timings,a and primary outcomes for each behavioural experiment. Experiment

Description

Group

Group size

Timings for various behavioural tests (PID)

Initial

After exclusion

HPW test

Thigmotaxis

Burrowing

Rotarod

NA

NA

NA

NA

NA

NA

21

NA

NA

1

MH development

d4T/vehicle

5/5

5/5

2

Pharmacological validation for MH

Gabapentin/ vehicle Cannabinoid/ vehicle Amitriptyline/ vehicle d4T/vehicle/ naive d4T/vehicle/ naive TNT/sham

6/6

5/6

6/6

6/6

6/6

6/5

12/12/12

12/12/12

4, 7, 11, 14, 18, 21 11, 14, 15–18 11, 14, 15–18 11, 14, 15–17 18

10/10/10

7/8/6

18

NA

21

22

5/5

3/3

18

NA

21

NA

3

Thigmotaxis

4

Burrowing

MH, mechanical hypersensitivity; PID, post initial d4T injection days; HPW, hind paw withdrawal. a Timing only refers to timings of tests after d4T/saline administration or TNT/sham surgery.

Primary outcomes

HPW threshold in response to punctate static mechanical stimulus HPW threshold in response to punctate static mechanical stimulus

Frequency of entry and duration in the inner zone Latency to burrow and gravel displaced

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Table 2 Major domains of good laboratory practice [45] to minimize the effects of experimental bias. Characteristic

Description of procedures

Sample size calculation

Group size was determined by sample size estimation for each experiment by SigmaStat software, version 3.5 (ANOVA sample size, desired power = 0.8, a = 0.05). Effect sizes for estimation were derived from previous studies in our group. Details of sample size calculation for each experiment can be found in the Supplementary materials In experiments for pharmacological validation, thigmotaxis, and burrowing, only rats that developed hind paw mechanical hypersensitivity of at least 25% change from the baseline were included. In addition, rats that did not reliably burrow more than 500 g of gravel during training were excluded in the burrowing experiment In experiments for mechanical hypersensitivity development and pharmacological studies, animals were randomized into treatmenta groups by picking numbers out of a hat. In experiments for thigmotaxis and burrowing, random cage assignment to treatments or TNT/sham surgeries were applied by picking numbers out of a hat. This was a result of our recent evidence revealing that naive rats mixed with neuropathic rats demonstrate increased thigmotaxis (Ewen Legg, personal communication) The person creating the model (i.e., injection of d4T or vehicle solution, administration of analgesic agent or vehicle solution, or performing TNT/sham surgeries) was unaware of the allocation to treatment group.b This was achieved by the blinding procedure described below, as well as masking cage labels or turning around the cages before each behavioural assessment session The TNT model was used for the purpose of internal assay assessment for burrowing. If burrowing behaviour was unsatisfactory with the TNT/sham rats, then the rest of rats within the same batch were excluded. Any rat with hunched posture, marked behavioural change, exudates around wound, or sensitivity to palpitation on handling that could be attributable to surgery, the drug, the dosing procedure, infection resulting from surgery or otherwise, was excluded. Any rat with significant surgical complications, or whose general health deteriorates, was excluded. Limited motor dysfunction or autotomy may occur after TNT injury, but very rarely. If a persistent motor impairment whereby the animal is prevented from reaching food and water was observed then the animal was excluded. Similarly, if autotomy of more than one digit was observed then the animals were excluded. The details of the number of excluded animals and the reason for exclusion are stated in the results section Codes were assigned to different treatments by an independent person and kept in a sealed envelope. The codes were not broken until the analysis had been completed. The experimenter was blinded to the treatments received and had no knowledge of the experimental group to which an animal was randomized.b In addition, burrowing videos were assigned with different names by an independent person before analysing

Inclusion and exclusion criteria

Randomization

Allocation concealment

Reporting of animals excluded from analysis

Blinded measurement, assessment, and analysis of outcome

a

Treatment here refers to drug administration, i.e., d4T vs vehicle, and each pharmacological analgesic agent vs vehicle. TNT/sham groups were difficult to apply allocation concealment and blinding procedures as rats with TNT injury demonstrate hind limb posture change compared to the sham animals. b

to animals associated with oral gavage. The dose of 50 mg/kg was chosen because previous studies have demonstrated that ddC and d4T produce a robust mechanical hypersensitivity in rats at this dose [34,82]. The treatment regimen (i.e., 2 i.v. injections) was chosen on the basis of our initial regimen exploratory studies (data not shown). 2.4.2. Behavioural experiment 1: hind paw mechanical hypersensitivity Hindpaw withdrawal to sensory stimuli was measured as previously described [82]. Briefly, the withdrawal threshold in response to punctate static mechanical stimulation was measured with an electronic von Frey device of 0.5 mm2 probe tip area (Somedic Sales AB, Sweden). Two sessions of habituation (40–50 min each) to the testing area were conducted, followed by 2 baseline measurements. Rats were allowed to acclimatize until exploratory behaviour ceased. Force was applied manually to the midplantarhind paw and then increased gradually at a rate of 8–15 g/s until the paw was withdrawn. The application was performed 5 times with a minimum interval of 1 min between applications. The hind paw reflex thresholds were determined at various time points after initial d4T injection (PID) for inclusion and exclusion purposes (Tables 1 and 2). We only measured hypersensitivity in the hind paws because HIV-SN predominantly affects the feet and is less frequent in the hands. 2.4.3. Behavioural experiment 2: pharmacological validation for mechanical hypersensitivity At PID 15–17/18, rats were provided either drug or vehicle i.p. twice daily (b.d.), and the hind paw reflex thresholds were measured once each day 1.5–2 h after the first drug administration. The following drugs were tested: gabapentin (a gift from Pfizer Ltd; 30 mg/kg in 0.5 mL sterile saline); the CB1/CB2 cannabinoid receptor agonist WIN 55,212-2 (Sigma, UK; 2.5 mg/kg in 0.75 mL sterile saline with 40% dimethyl sulphoxide); and amitriptyline

(Sigma, UK; 10 mg/kg in 0.5 mL sterile saline). The choices of drugs, doses, and administration regimens were based on our previous studies and evidence of efficacy or lack of efficacy in clinical trials [29,62,82]. 2.4.4. Behavioural experiment 3: thigmotaxis The thigmotaxis paradigm is based on natural behaviours of rodents, as follows: their instinct predator avoidance behaviour drives them to adhere to the perimeter of a novel open arena; and this avoidance behaviour conflicts with drives (e.g., exploration and foodseeking) to enter the mildly aversive central arena. The construct of this paradigm for pain studies is that although it would not be in a wild rat’s survival interest to reveal itself as easy prey to a predator by displaying overt pain-related behaviours, there is a survival benefit to the individual rat in increasing protective behaviours that would have the effect of decreasing exposure to potential predators, e.g., thigmotaxis, which have been repeatedly demonstrated in neuropathic rats [29,82,83]. At PID 21, rats were introduced to the corner of a 100  100 cm arena (inner zone 40  40 cm) to which they have not previously been exposed. Spontaneous activities were recorded for 15 min under dim lighting conditions (12 lux) with a high-sensitivity camera (VCB 3372; Sanyo, Japan). Data were analyzed by EthoVision software v.4.1 (Tracksys Ltd., UK). The number of entries into the inner zone, the time spent in the inner zone, and the total distance moved were measured and the mean calculated. In addition, the number of rearing movement was manually counted by the videos generated in this experiment. A rearing episode was defined as a rat standing on its hind limbs with forelimbs elevated. 2.4.5. Behavioural experiment 4: spontaneous burrowing behaviour Laboratory rats are fossorial animals and therefore naturally burrow (in the wild, the burrow is used for shelter and food storage), and this behaviour is highly conserved [21]. The burrowing behaviour is thought to represent tunnel maintenance behaviour,

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which would be engaged in after collapse of a burrow in the wild, and it can be measured by weighing the amount of gravel left in the burrow at the end of the test period [21]. The construct of this paradigm for pain studies is that pain will affect a rat’s motivation to burrow; this has been demonstrated in animal models of peripheral nerve injury and inflammation [2]. The burrows were made in house with hollow plastic tubes (32 cm long  10 cm in diameter; B&Q, UK), sealed at one end and open at the other, with the entrance raised by 6 cm to prevent loss of gravel (5 mm pea shingle; Fulham Palace Garden Centre, UK). All tests were carried out in separate empty individually ventilated cages in dim lighting (5 lux), and recorded with a camcorder (DVD653E; Sony, Japan). The videos were used to determine the latency to burrow, i.e., the time from the first entry into the cage to the first sign of gravel displaced by the hind paws. During 2 days’ training, rats were placed in pairs, and then a burrow containing 2500 g of gravel was placed into the cage. Animals were then allowed to burrow for 2 h. If on the first training day 1 pair of rats did not burrow at all or revealed little tendency to burrow, then 1 rat of this pair was swapped with a rat from a burrowing pair for the second training day. After training, rats that demonstrated a tendency to burrow were placed individually in the test cages and allowed to burrow for 2 h on 2 consecutive days to determine their baseline levels of burrowing, i.e., the latency to burrow and the amount of gravel displaced. At PID 21, rats were tested again for their burrowing performance to assess the effects of d4T/vehicle treatment or tibial nerve transection (TNT)/sham surgery (Supplementary materials). The TNT model was used for the purpose of internal assay assessment because we previously demonstrated that TNT rats developed significant burrowing deficits when compared to the sham controls [2]. 2.5. Motor coordination Rats from the burrowing experiment were also investigated for their motor coordination with a rotarod treadmill (Ugo Basile, Italy). Over 2 days’ training, rats were placed on a fixed-speed rotarod (5 rpm) and trained to remain on the rotarod for a total of 180 s without falling. Then baseline performance was evaluated at a higher speed (15 rpm) over an additional 2 consecutive days, with 3 tests per day and a minimum of 15 min interval between tests. The time that they remained on the rotarod before falling was recorded with a cutoff time of 180 s. At PID 22, rats were again tested on the rotarod to assess the effects of d4T/vehicle treatment. 2.6. Procedures for histological and neurochemical studies After defined survival times (7 or 21 days), animals were humanely killed with pentobarbital and transcardially perfused with 0.9% heparinized saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The lumbar spinal cord, L5 dorsal root ganglion (DRG), sciatic nerve, sural nerve, and glabrous hind paw skin were removed. Tissue for immunohistochemistry was postfixed in 4% paraformaldehyde for 6 h and cryoprotected in 30% sucrose for at least 3 days. The samples were embedded in OCT, cut with a cryostat (spinal cords at 20 lm, DRG and sciatic nerve at 10 lm, skin at 14 lm), and thaw-mounted onto glass slides. Sections were preincubated in buffer (phosphate-buffered saline, pH 7.4, containing 0.2% Triton X-100 and 0.1% sodium azide) containing 10% normal donkey serum for 30 min and then incubated with primary antibodies overnight at room temperature. Primary antibodies used are listed in Table 3. After primary antibody incubation, sections were washed and incubated for 2 h with secondary antibody solution (donkey anti-rabbit Cy3, 1:400; Stratech, UK). For detecting nonpeptidergic C-fibres, we used biotin-conjugated isolectin B4 (IB4; 0.5 mg/mL used at 1:50; Sigma, UK) and

Table 3 Primary antibodies used for immunohistochemistry.

a

Antibody

Concentration used

Company

Ionized calcium binding adapter molecule-1 antibody (rabbit antiIba1) Rabbit anti-phospho-p38MAPK

1:1000

WAKO, Japan

1:100a

Glial fibrillary acidic protein antibody (rabbit anti-GFAP) Calcitonin gene-related peptide antibody (rabbit anti-CGRP) Rabbit anti-ATF3

1:1000

Cell Signalling, USA Dako, UK

1:2000

Sigma, UK

1:500

Rabbit anti-NPY Pan neuronal marker protein gene product (rabbit PGP 9.5)

1:1000 1:1000

Santa Cruz Biotechnology, UK Sigma, UK Ultraclone, UK

Viewed by tyramide amplification (TSA Biotin System, Perkin Elmer, UK).

ExAvidin–fluorescein isothiocyanate (1:400; Sigma, UK). Slides were washed with phosphate-buffered saline, coverslipped with Vectashield mounting medium (Vector Laboratories, UK), and visualized under a Zeiss Axioplan 2 fluorescent microscope (Zeiss, UK). 2.7. Procedures for electron microscopy Sural nerves were dissected from perfused animals, and a 5 mm length of nerve was extracted from the point at which it traverses the gastrocnemius muscle. The nerves were postfixed in 3% glutaraldehyde at 4°C overnight, washed in 0.1 M phosphate buffer, osmicated, dehydrated, and embedded in epoxy resin (TAAB Embedding Materials, UK). Transverse semithin sections with 1 lm thickness were cut on a microtome and stained with toluidine blue before being examined under a light microscope. Transverse ultrathin sections were cut on an ultramicrotome and stained with lead uranyl acetate by the Centre for Ultrastructural Imaging (King’s College London, UK). Ultrathin sections were collected on uncoated 100 lm mesh grids and visualized on a Hitachi H7600 transmission electron microscope (Hitachi, Japan). 2.8. Quantitative analysis for immunohistochemistry and electron microscopy In all cases, the observer was blinded to the experimental group when performing analysis. 2.8.1. Immunohistochemistry For immunohistochemical analysis of macrophages or microglia, quantitative assessment was carried out by determining the numbers of Iba1 immunoreactive cells within 4 areas of 10,000 lm2 in the superficial dorsal horn of the L5 spinal cord as previously described [15], as well as in the sciatic nerve or L5 DRG on 5–7 randomly selected sections from each animal. Microglia in which process length was less than double the soma diameter were classified as presenting an ‘effector’ morphology, whereas microglia in which the process length was double the soma diameter were classified as surveying (previously called resting) cells [73]. For IB4, Calcitonin gene related peptide (CGRP), Activating transcription factor 3 (ATF3), or Neuropeptide Y (NPY) expression in the DRG, analysis was performed from 6–8 sections from each animal; the total number of DRG cell profiles and the number of profiles expressing immunoreactivity for the protein of interest was counted and reported as a percentage over the total. Cell profiles were sampled only if the nucleus was visible within the plane of section and if the cell profiles exhibited distinctly delineated borders. Glial fibrillary acid protein (GFAP) in the spinal cord was measured in 6–8 randomly selected L5 spinal cord sections

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from each animal. The intensity of immunoreactivity was measured in the dorsal horn and expressed in arbitrary units. IB4 and CGRP in the dorsal horn were also analysed measuring the intensity of immunoreactivity in laminae I and II. Epidermal fibres were counted live on the microscope at a 40 objective magnification using a previously described protocol [43]. Only single fibres crossing the dermal–epidermal junction were counted, excluding secondary branching from quantification. The length of the section was measured by the open source Image J software 1.45 (National Institutes of Health, USA) and used to calculate the epidermal innervation density (IENFD) per millimeter. 2.8.2. Electron microscopy For analysis of semithin sections, mosaic images of the entire section of the left sural nerve from each animal were taken at 63 magnification. The diameter of the nerve and the number of myelinated axons were measured by Image J software, version 1.45. For ultrathin sections, analysis was performed from photographs taken at 8000 magnification. The number of unmyelinated axons was counted in 1 grid, and the density was calculated by dividing by the area of the grid. The diameter of myelinated and unmyelinated axons, the g ratios, and the number of C-fibres per Remak bundle were analysed in 1 grid per nerve. In total, 5225 unmyelinated axons and 2111 myelinated axons were analysed. 2.9. Sural nerve proteomics During the investigation of behavioural and neuropathological changes after d4T treatment, we observed hind paw mechanical hypersensitivity at PID 7, which was in contrast to a lack of injury response in the DRG and a lack of changes in hind paw skin IENFD and central terminals of C-fibres in the spinal dorsal horn at this time point. To address this discrepancy, we collected fresh sural nerves at PID 7 and investigated protein changes in the sural nerve using proteomics, an approach that may be able to detect an earlier response than the histology. Detailed methods for sural nerve proteomics and subsequent Western blot verification are described in the Supplementary materials. Briefly, sural nerves (rightsides) from d4T-treated (n = 3) and vehicle control rats (n = 3) were dissected and tissue lysed. Sural nerve proteins (20 lg of protein, determined by bicinchoninic acid assay) were separated by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1D-SDS–PAGE) into bands. Gel bands were excised. The mixtures of proteins within each band were digested with trypsin. Resulting peptides mixtures were analysed online by nano–liquid chromatography–tandem mass spectrometry (LC–MS/MS) on a linear ion trap Orbitrap (ThermoScientific, UK) in CID mode. Each protein measurement, consisting of 125 min gradients, was repeated in triplicate. We used label-free quantitation of proteins from LC–MS/MS data to identify differences between d4T-treated and vehicle control samples utilising a spectral index quantitation (SINQ) software developed by Dr. C. Trudgian. SINQ software is freely available to obtain and use as part of the central proteomics facilities pipeline, which is released under an opensource license at Oxford University, UK. 2.10. Statistical analysis The primary outcomes for each behavioural experiment are listed in Table 1. For the thigmotaxis experiment, the total distance travelled and rearing behaviour in the whole arena were included as secondary outcomes. For the burrowing experiment, motor coordination was included as a secondary outcome. Statistical analysis was performed by SPSS statistical software, version 19 (IBM, USA). Paired Student’s t test was used to determine whether mechanical hypersensitivity or other parameters within each

group was significantly different from baseline at postinjection/injury time points. One-way ANOVA was used to evaluate whether there was any difference between groups (d4T, vehicle, and naive) at baseline or at a time after the first d4T injection or if the percentage change from the baseline was different between groups. The Tukey–Kramer multicomparison adjustment was used as the post hoc test to calculate the significance levels. All measurements are expressed as mean values ± standard error of the mean. P < .05 was considered as statistically significant. Unpaired Student’s t test was used to compare the TNT and sham groups. For histological analysis, data sets were tested for normality by the Kolmogorov–Smirnov test and for homogeneity of variance by Levene’s test. Parametric or nonparametric tests were used accordingly. Differences between vehicle- and d4T-treated animals were determined by Student’s t test or the Mann–Whitney test. Frequency distribution of axon diameter and of number of fibres per Remak bundle were compared statistically by the Kolmogorov– Smirnov test. Data are reported as mean values ± standard error of the mean. P < .05 was considered as statistically significant. 3. Results 3.1. Withdrawals, exclusions, and sample sizes Two d4T-treated rats in experiment 2 and one d4T-treated rat in experiment 4 were withdrawn from the study because they developed less than 25% change from baseline in hind paw withdrawal threshold to the mechanical stimulus (Table 1). Two naive rats in experiment 4 were withdrawn from the study because they burrowed less than 500 g of gravel during the baseline test (Table 1). In addition, the second batch of experiment 4 animals was excluded (2 naive, 2 d4T treated, and 2 vehicle treated) as a result of inconsistent burrowing behaviour in the naive animals and no difference in burrowing behaviour between TNT and sham-treated animals in the same batch (Tables 1 and 2; Supplementary materials). The group sizes after withdrawals and exclusions were double-checked with post hoc sample size calculation by SigmaStat 3.5 with a desired power of 0.8 and alpha of 0.05, and were found to be sufficient. 3.2. Behavioural experiment 1: d4T treatment results in hind paw hypersensitivity to mechanical stimuli Rats treated with d4T developed bilateral hind paw mechanical hypersensitivity as measured by application of a punctate mechanical stimulus. This change developed as early as PID 4 and reached a plateau between PID 14 and PID 21 (Fig. 1). Mean percentage changes of withdrawal thresholds from the baseline were 42%, 48%, and 39% for PID 14, 18, and 21, respectively. Because there was no difference in mechanical hypersensitivity between the left and right hind paws at each time point examined (data not shown), the withdrawal thresholds from both hind paws were pooled. No such response was observed in vehicle-treated rats. In a subsequent pharmacological validation study, we demonstrated that hind paw mechanical hypersensitivity after d4T treatment persisted until PID 30 (Fig. 2). Moreover, 4 d4T-treated rats from experiment 3 were maintained until PID43, when their hind paw withdrawal thresholds demonstrated a trend to return to the baseline (17% mean change from the baseline). 3.3. Behavioural experiment 2: hind paw hypersensitivity in d4Ttreated rats: response to analgesic drugs The pharmacological validity of the animal model was tested by examining the response to analgesic drug categories that are effective or ineffective for treatment of HIV-SN [62]. We found that

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565

Fig. 1. Development of hind paw reflex sensitivity to punctate mechanical stimuli in rats treated with d4T. Hindpaw withdrawal thresholds were measured with an electronic von Frey device at baseline (BL) and after treatment with d4T (50 mg/kg, twice at 4 days apart) or vehicle. Statistical significance of differences (P < .05) between the 2 groups was determined by a 1-way ANOVA with Tukey–Kramer post hoc multiple comparisons. ⁄Significant difference from the vehicle group; àSignificant difference from baseline. Each value is the mean ± SEM.

amitriptyline was not associated with any reversal of the heightened sensitivity to punctuate mechanical stimuli in d4T-treated rats (Fig. 2C). In contrast, systemic administration of gabapentin or the mixed CB1/CB2 cannabinoid receptor agonist WIN 55,212-2 (Fig. 2A and B) was associated with a complete attenuation of hind paw mechanical hypersensitivity observed with d4T treatment. In all cases, vehicle administration had no significant effect. For all drugs tested, hind paw mechanical hypersensitivity was reestablished by PID 30, indicating reversal of the drug effect and reestablishment of the neuropathic state. 3.4. Behavioural experiment 3: d4T treatment results in increased thigmotaxis We used an open field apparatus to assess the effect of d4T treatment on thigmotaxis behaviour, which we have previously demonstrated to be a correlate of pain behaviour in traumatic nerve injury-, varicella zoster virus-, HIV viral protein gp120-, and antiretroviral drug ddC-induced models [29,81–84]. First we demonstrated that d4T-treated rats developed hind paw mechanical hypersensitivity at PID 18 (Fig. 3A); then analysis of track pattern in the open field revealed a significant d4T treatment effect on the explorations of the inner zone at PID 21, as calculated by the number of entries and the time spent in the inner zone over the 15 min observation period. Thus, d4T-treated rats had significantly lower entry number (4.8 ± 1.1) and time spent (3.3 ± 0.9 s) in the inner zone compared to those of the naive (15.6 ± 2.8 and 8.9 ± 1.5 s; P < .05 ANOVA/Tukey–Kramer post hoc test) and vehicle-treated (17.6 ± 3.3 and 11.5 ± 1.8 s; P < .05 ANOVA/Tukey–Kramer post hoc test) animals (Fig. 3B and C). We also examined the rearing behaviour of the animals in the arena and found no significant difference between the naive, vehicle-treated, and d4T-treated rats (the numbers of rearing episodes were 85.2 ± 5.5, 82.2 ± 6.3, and 76.8 ± 6.5, respectively; P < .05, Tukey–Kramer post hoc test). 3.5. Behavioural experiment 4: d4T treatment results in a burrowing behavioural deficit We used a burrowing paradigm, which we have previously demonstrated to be a correlate of pain behaviour in traumatic nerve injury and inflammatory pain models [2], to assess the effect

Fig. 2. Effect of systemic administration of analgesic compounds on hind paw mechanical hypersensitivity in d4T-treated rats. (A–C) Effect of drugs. (A) Gabapentin 30 mg/kg, i.p. b.d. (B) Cannabinoid receptor agonist WIN 55,212-2, 2.5 mg/kg, i.p. b.d. (C) Amitriptyline 10 mg/kg, i.p. b.d.). Each was delivered at the time of peak behavioural change on hind paw withdrawal thresholds in response to punctate mechanical stimuli in rats treated with d4T (50 mg/kg, i.v., twice, 4 days apart). The values are displayed before, during, and after i.p. administration of each drug (open circle) vs vehicle (solid triangle) as paw withdrawal threshold (g). Drug administration period is represented by a shaded area, with arrows and arrowheads indicating the start and end of drug treatment, respectively. For all measurements, each value is the mean ± SEM, and statistical significance (⁄P < .05) of any difference between drug and vehicle threshold values was determined by a 1-way ANOVA with Tukey–Kramer post hoc multiple comparisons.

of d4T treatment on the general well-being of the rat. During baseline assessment, the naive, vehicle-treated, and d4T-treated rats displaced comparable amount of gravel (Fig. 4B). At PID 18,

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d4T-treated rats but not vehicle-treated rats had hind paw mechanical hypersensitivity (Fig. 4A). There was a significant effect of d4T treatment on the amount of gravel displaced at PID 21. d4Ttreated rats displaced significantly less gravel (1580 ± 248.2 g) than those of naive and vehicle-treated rats (1932 ± 120.8 and 1762 ± 130.4 g respectively; P < .05 ANOVA/Tukey–Kramer post hoc test), the equivalent of a 4% reduction from the baseline in d4T-treated rats, in comparison to 61% and 45% increases from the baseline in the naive and vehicle-treated rats, respectively (Fig. 4C). We also examined latency to burrow and found no significant difference at baseline between the naive, vehicle-treated, and d4T-treated rats (202.3 ± 33.2 s, 186.9 ± 20.9 s, and 163.4 ± 36.4 s respectively). At PID 21, d4T-treated rats demonstrated a trend towards an increase of burrowing latency (810.9 ± 423.6 s) compared to the naive and vehicle-treated rats (273.3 ± 27.3 s and 230.6 ± 47.3 s; Fig. 4D). In a parallel experiment that used the TNT model (batch 5), we confirmed our previous finding [2] that rats with TNT injury developed a significant burrowing deficit when compared to sham-operated rats at PID 21 (Supplementary Fig. 1), confirming the sensitivity of the assay.

addition, we did not find any significant difference in motor coordination as assessed by rotarod test between the naive, vehicletreated, and d4T-treated animals at PID 22 (same animals from experiment 4; Fig. 5B). These results suggest a lack of overt motor deficit in the d4T model, which is compatible with the clinical presentation of HIV-SN. 3.7. Treatment with d4T produces a ‘dying back’ peripheral SN Immunostaining with PGP 9.5 was used to visualize the terminations of small-diameter axons within the epidermis of glabrous hind paw skin, which are the sensory terminals arbors of both unmyelinated and myelinated primary afferents [7]. After d4T treatment at PID 21, but not PID 7, there was a significant reduction in IENFD, signifying a withdrawal of the terminations of small diameter axons from the epidermis (PID 7: 56 ± 2.5 vs 58 ± 3.6, d4T and vehicle, respectively, P = .68; PID 21: 39 ± 5.7 vs 64 ± 4.76, d4T and vehicle, respectively, P < .01; Fig. 6).

3.6. Treatment with d4T does not affect motor function

3.8. Systemic d4T treatment was associated with a reduction in expression of IB4 and CGRP in the central innervation territories of lumbar afferents in the spinal dorsal horn

As an indicator of normal locomotion, there was no significant difference in the total distance travelled in the open field by d4Ttreated rats vs the naive or vehicle-treated rats at PID 21 (same animals from experiment 3; Fig. 5A). This is consistent with our previous studies that demonstrated that rats move in the region of 6–8000 cm in the 15 min observation period [81–84]. In

At 21 days after the first d4T injection, a time when nociceptive responses are well established, we assessed changes in markers for small fibres in the dorsal horn of the L5 segment of the spinal cord. The IB4 binding nonpeptidergic subpopulation of C-fibres terminates in lamina II–inner (IIi) of the dorsal horn [72]. Traumatic peripheral nerve injury results in a disappearance of this labelling

Fig. 3. Alterations in thigmotaxis behaviour over 15 min as measured in an openfield arena in d4T-treated rats at PID 21. (A) Development of hind paw hypersensitivity to punctate mechanical stimuli at PID 18 in d4T-treated (50 mg/kg, i.v., twice, 4 days apart) rats. (B) Number of entries into the inner zone (dotted square in D, 40  40 cm2). (C) Time spent in the inner zone of the open field assessed in naive, vehicle-treated, and d4T-treated rats. (D) Example traces of (i) naive, (ii) vehicle-treated, and (iii) d4T-treated rats over 15 min in the open field arena. The total distance moved is shown in Fig. 5. The statistical significance of differences between the d4T group and its relevant control (#P < .05, ⁄P < .05) was determined by a 1-way ANOVA with Tukey–Kramer post hoc multiple comparisons. Value in A–C are the mean ± SEM.

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Fig. 4. Alterations in spontaneous burrowing behaviour in d4T-treated rats at PID 21. (A) Development of hind paw hypersensitivity to punctate mechanical stimuli at PID 18 in d4T-treated (50 mg/kg, i.v., twice, 4 days apart) rats. (B) Baseline gravel displaced measured at 1 day before d4T treatment. (C) Percentage change of gravel displaced from the baseline at PID 21. (D) Percentage change of burrowing latency from the baseline (no differences between the groups, data not shown) at PID21. The statistical significance of differences between the d4T group and its relevant control (#P < .05 and ⁄P < .05) was determined by 1-way ANOVA with Tukey–Kramer post hoc multiple comparisons. Each value is the mean ± SEM.

Fig. 5. Lack of effect of d4T treatment on motor function. (A) Total distance moved in the open arena during the thigmotaxis experiment at PID 21. (B) Percentage change of time remaining on the rotarod from the baseline during the burrowing experiment at PID 22. No significant differences were found by1-way ANOVA with Tukey–Kramer post hoc multiple comparisons. Each value is the mean ± SEM.

[53]. Treatment with d4T significantly reduced IB4 binding in the medial extent of the superficial dorsal horn, which corresponds to the terminal region of sciatic nerve afferents (n = 5, vehicle 100 ± 11.07, d4T 47.26 ± 10.8, t test, P = .009; Fig. 7). CGRP is normally expressed in primary afferent terminations within lamina I and lamina II outer (IIo), as well as less profuse arborisation in deeper laminae. Treatment with d4T also significantly reduced CGRP immunoreactivity in the medial extent of the superficial dorsal horn (n = 5, vehicle 100 ± 5.7, d4T 30.05 ± 7.2, t test, P < .001, Fig. 7). The effects of d4T on the central terminals of primary afferents appeared to be length dependent in that they were less marked at the thoracic level where sensory axons are shorter than at the lumbar and sacral levels (Supplementary Fig. 4).

3.9. Treatment with d4T results in increased axon diameters in the sural nerve Review of semithin sections of the sural nerve trunk at PID 21 indicated that there was no evidence of Wallerian degeneration, demyelination, or an inflammatory cellular infiltrate in nerves from d4T-treated animals. Detailed morphometric analysis revealed that the total number of unmyelinated axons did not differ between nerves collected from d4T- and vehicle-treated animals (1522 ± 122 vs 1839 ± 394 fibres per nerve d4T and vehicle, respectively, P = .47), nor did the number of unmyelinated axons per Remak bundle (4 ± 0.5 vs 3.5 ± 0.2, d4T and vehicle, respectively; Kolmogorov–Smirnov test P = .965; Fig. 8). Interestingly, the

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Fig. 6. Decreased intraepidermal nerve fibre density (IENFD) after d4T treatment. Representative sections of the glabrous hind paw skin of a vehicle (a) and of a d4T-treated animal (b) at day 21 after the first injection are shown. The sections were immunostained with PGP9.5, a pan-neuronal marker, to quantify fibres crossing the dermal– epidermal junction. There is a frank reduction in the number of intraepidermal fibres in the d4T-treated animals. This effect is quantified in (c), where the results obtained at 7 and 21 days after the first injection are shown. At day 7, there was no significant difference between the 2 groups (t test, n = 5, P = .68). At day 21, there was a significant reduction in IENFD in the d4T-treated animals (t test, n = 5, P = .01). Error bars denote SEM. Scale bar = 50 lm.

frequency distribution of the unmyelinated axon diameter of the d4T-treated group is shifted to the right, indicating an increase in axon diameter. This effect was small but statistically significant (Kolmogorov–Smirnov test P < .001, n > 2000 axons per group; Fig. 8). The total number of myelinated axons within the sural nerve did not differ between d4T- and vehicle-treated animals (1157 ± 49 vs 1005 ± 82, d4T and vehicle treatment, respectively; Fig. 8), and the g ratio (axonal diameter/fiber diameter) was also unaltered (0.677 ± 0.01 vs 0.673 ± 0.01, d4T and vehicle treatment, respectively; Fig. 8). Quantification of axon diameter revealed a small shift to the right in the frequency distribution in d4Ttreated,animals indicating an increase in axon diameter, which was statistically significant (Kolmogorov–Smirnov test P < .001, n > 1000 axons per group; Fig. 8). There was no clear ultrastructural abnormality associated with this, such as abnormalities of neurofilaments or microtubules. 3.10. Systemic d4T induces a minimal glial or inflammatory cell response We investigated whether systemic d4T treatment could induce a response in glial cells within the dorsal horn of the spinal cord. Lumbar spinal cord sections were immunostained with Iba1 to label microglia, and the number of these cells within the superficial dorsal horn with an ‘effector’ morphology (hypertrophy of the cell body and process retraction) was counted. Animals treated with d4T demonstrated a slight but significant increase in the number of microglia presenting an effector morphology (d4T: 0.44 ± 0.15 vs vehicle: 0.06 ± 0.04 cells with effector morphology per 10,000 lm2, n = 5 per group, P = .008, Mann–Whitney rank sum test; Fig. 9). The magnitude of this change is much less than we had previously observed after spinal nerve ligation (Fig. 9). Expression of the active (phosphorylated) form of p38 MAP kinase is increased in effector microglia. Phospho-p38 immunostaining revealed no difference in the percentage of microglia that were

pp38 positive between the d4T-treated and control groups at day 21 (d4T: 40 ± 7.5% and vehicle: 35 ± 8%; n = 5, t test P = .6; Supplementary Fig. 3). In addition, GFAP immunostaining and intensity analysis revealed a transient small but significant increase in GFAP immunoreactivity in the dorsal horn at PID 7 (a 35% increase in GFAP immunoreactivity vehicle 1 ± 0.08, d4T 1.3 ± 0.05, n = 5 per group, P < .05, t test; Fig. 9), but this was not maintained at PID 21 (n = 5 per group, vehicle 1 ± 0.1, d4T 0.77 ± 0.06, P = .114, t test; Fig. 9). GFAP is also expressed in DRG satellite cells and is up-regulated after nerve injury [86]. GFAP expression in the DRG was not increased by d4T treatment (Fig. 10). Nerve injury and treatment with HIV GP120 induces the recruitment of immune cells such as macrophages into the DRG and the sciatic nerve [1,82]. We investigated whether d4T-induced painful peripheral neuropathy was accompanied by such an infiltration. Immunolabeling macrophages with Iba1 demonstrated that the number of these cells do not increase after treatment with d4T in either DRG or sciatic nerves (n = 5, DRG vehicle 2.04 ± 0.12, d4T 1.76 ± 0.14, P = .17, sciatic nerve: vehicle 2.88 ± 0.28, d4T 2.5 ± 0.39, P = .45, t test; Supplementary Fig. 2). 3.11. Systemic d4T does not alter the histochemistry of lumbar DRG We investigated the expression of histochemical markers for different subsets of DRG cells after d4T treatment. The IB4 antibody binds to the nonpeptidergic population of small-diameter DRG cells, and CGRP is expressed in the majority of peptidergic smalldiameter DRG cells [5]; both of these markers are down-regulated by traumatic nerve injury [6]. The percentage of DRG cell profiles binding the lectin IB4 or expressing the neuropeptide CGRP did not change after d4T treatment (n = 5, IB4: vehicle 34.6% ± 2.5 d4T 35.7% ± 2.2, P = .76, CGRP: vehicle 22.6% ± 1, d4T 23.1% ± 1.4, P = .79, t test in both cases; Fig. 10). Traumatic forms of nerve injury and treatment with the HIV glycoprotein GP120 also induce an up-regulation of the stress-related transcription factor ATF3 [75,82] and the neuropeptide NPY [80] in sensory neurons.

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Fig. 7. Reduced expression of IB4 and CGRP in the central innervation territories of lumbar afferents in the dorsal horn after d4T treatment. (a) IB4 binding in vehicle-treated animals (green) labels nonpeptidergic primary afferent terminals within lamina IIi. (b) CGRP (red) is expressed in the terminals of peptidergic afferents within laminae I and IIo. (c) Merged image. At day 21 after the first d4T injection, there was reduced IB4 binding (d and f) and CGRP expression (e and f) in medial dorsal horn. A high-power view is shown in (f), and arrows denote the margins within which there is very little IB4 or CGRP expression. Quantification reveals a significant reduction of CGRP and IB4 immunoreactivity at day 21. ⁄⁄P < .01 and ⁄⁄⁄P < .001, t test, d4T vs vehicle, n = 5 per group. Error bars denote SEM. Scale bar = 500 lm.

Therefore, we assessed the presence of such changes after systemic d4T treatment. We found no significant change in the normal low level of expression of ATF3or NPY in the DRG (n = 5, ATF3 P = .84, NPY P = .31, t test in both cases; Fig. 10). 3.12. Down- and up-regulated proteins in the sural nerve after systemic d4T treatment We chose the sural nerve for proteomics because our electron microscopic study of the sural nerve at PID 21 had suggested axonal swelling in the sural nerve after d4T treatment. Detailed results of sural nerve proteomics are described in the Supplementary materials. Briefly, a total of 600 proteins were identified by mass spectrometry (Fig. 11A). Among 10 down-regulated proteins with at least 10-fold changes at PID 7 after d4T treatment when compared to the vehicle control are 3 enzymes directly related to mitochondrial function (Fig. 11B; Supplementary Table 1). Among 19 up-regulated proteins with at least 10-fold changes at PID 7 after d4T treatment when compared to the vehicle control is the protein kininogen-1, the precursor of peptide bradykinin (Fig. 11C; Supplementary Table 2). Western blot verification revealed a 50% reduction of mitochondrial long-chain-specific acyl-CoA dehydrogenase and an approximate 1.8-fold increase of kininogen-1 in the sural nerve after d4T treatment at PID 7 (Fig. 11D).

4. Discussion We have demonstrated that rats treated with the antiretroviral drug d4Tdeveloped 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. We have demonstrated that this neuropathy is characterized by an axonal retraction from the skin and also a novel finding of reduced CGRP and IB4 expression in the central innervation territories of lumbar afferents in the spinal dorsal horn, which has important implications for the pathology of this condition. We have also demonstrated 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 [9,89]. We have also examined axonal proteomic response to d4T treatment. Importantly, we have demonstrated that this neurotoxicity occurs independently of HIV infection, a confounding that is difficult to explain in humans because the 2 conditions coexist, as the interaction between HIV and sensory neurons is also the cause of a painful neuropathy [82]. One major challenge in current pain research is improving the predictive validity of animal models of conditions associated with

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Fig. 8. Systemic d4T treatment induced an increase in axonal diameter in myelinated and unmyelinated fibres in the sural nerve. Transverse ultrathin sections obtained from sural nerve were photographed in an electron microscope at a magnification of 8000. Representative images of (a) vehicle-treated and (b) d4T-treated animals at day 21 after the first injection. (c) Frequency distributions of the axon diameter of unmyelinated fibres. The frequency distribution of the axon diameter of the d4T-treated group is shifted to the right, indicating an increase in axon diameter. This effect was statistically significant (Kolmogorov–Smirnov test P < .001, n > 2000 axons per group). The number of unmyelinated fibres per Remak bundle was also quantified (d), but no effect of group was found (Kolmogorov–Smirnov test P = .965). The axon diameter of myelinated fibres was also analysed. (e and f) Representative images of the sural nerve of a (e) vehicle-treated and (f) d4T-treated animal at 21 days after the first injection are shown. (g) Quantification of axon diameter demonstrated a shift to the right in the frequency distribution in the d4T-treated animals, which was statistically significant (Kolmogorov–Smirnov test P < .001, n > 1000 axons per group). Analysis of relation between the g ratio (axon diameter/fibre diameter) and the axon diameter demonstrated no difference between the groups (h). Error bars denote SEM. Scale bars = 1 lm (a and b) and 5 lm (e and f).

Fig. 9. Systemic d4T induced minimal gliosis in the lumbar spinal cord. GFAP immunostaining was used to demonstrate astrocyte processes. Treatment with d4T resulted in increased GFAP immunoreactivity at day 7 after the first injection (b) vs vehicle (a); this was not maintained at day 21. (c) It is less marked than previous data we have generated using the same quantification after spinal nerve ligation. IBA1 immunoreactivity reveals microglia within the spinal dorsal horn. Treatment with d4T resulted in a small increase in the number of microglia with effector morphology (d and f) compared to vehicle treatment (e). The magnitude of this change is less than that evoked by spinal nerve ligation quantified in the same manner (f). ⁄P < .05, t test, n = 5 per group; ⁄⁄P < .01, Mann–Whitney rank sum test, n = 5 per group. Error bars denote SEM. Scale bar = 50 lm (a and b) and 100 lm (d and e).

neuropathic pain. The widely used outcome measures that exploit hind paw reflex withdrawal to sensory stimuli are relevant only to a subgroup of neuropathic pain patients who display sensory gain phenomena. This is germane to HIV-SN because only a subgroup of patients with punctuate mechanical hyperalgesia benefit from pregabalin [70]. Nevertheless, to assess neuropathic pain in rodents, it is vital to move beyond simple reflex measures to better reflect

measures by which effectiveness is evaluated in clinical trials [67,68]. To this end, we have used 2 ethologically relevant measures of complex pain-related behaviours, which we have extensively validated for their relevance to pain [2,9,29,81–84]. We have previously used a regimen involving repeated i.p. injections of the NRTI ddC [82]. Because thigmotaxis is also used to study anxiety, such an invasive regimen could confound our

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Fig. 10. Systemic d4T treatment did not induce an injury response in the lumbar DRG cells. Expression of the neuropeptide CGRP (red) and the binding of the lectin IB4 (green) by L5 DRG cells was not altered by treatment with d4T (b) vs vehicle (a), quantified in (c). In vehicle-treated animals, there was a very low level of ATF3 expression in L5 DRG cell profiles (d), and this was not altered by d4T treatment (e), quantified in (f). Similarly, NPY expression was expressed by very few L5 DRG cell profiles in animals treated vehicle (g) or d4T (h), quantified in (i). Occasional GFAP immunoreactive satellite cells were observed in animals treated with vehicle (j) and d4T (k), with no significant difference between the groups, quantified in (l). In all cases, the time points at which images were taken were day 7 and day 21 after the first injection. Error bars denote SEM. Scale bars = 100 lm (a and b) and 50 lm (d, e, g, h, j, and k).

experiments by provoking stress-related behaviours. Hence, we refined the administration regimen to 2 i.v. injections of d4T at 4 days apart under brief general anaesthesia. We demonstrated that when hind paw hypersensitivity plateaued, d4T-treated rats displayed increased thigmotaxis when compared to vehicle-treated rats. Thigmotaxis is an innate rodent behaviour that involves conflicting drivers (predator avoidance vs exploration/foodseeking) and is probably also associated with motivation and the decision-making process. Increased thigmotaxis is evident in other rodent models associated with pain [29,54,59,81–84], and it is appropriately sensitive to pharmacological perturbation [29,83]. We demonstrated that d4T-treated rats developed burrowing deficits when hind paw hypersensitivity plateaued. A previous mouse study has demonstrated a learning effect after burrowing training and baseline testing [22], which we also observed in the naive and vehicle-treated rats with increased burrowing at PID 21 (61% and 45% increases from baseline respectively). In contrast, d4T-treated rats had reduced burrowing at PID 21 (4% decrease from baseline), suggesting a loss of this learning. Burrowing is an evolutionarily conserved rodent behaviour, and alterations in such

activity reflect illness-associated effects on motivation and general well-being [20,21]. Reduced burrowing is also evident after nerve injury and inflammation in rats, and it is appropriately sensitive to analgesics [2]. By using the rotarod assay and the total distance moved in the open field, we demonstrated no overt motor dysfunction after d4T treatment, which discounts any theoretical confound in the thigmotaxis and burrowing behaviours, which could have been caused by a motor deficit. We did not measure forepaw mechanical hypersensitivity after d4T treatment, which might also contribute to the observed burrowing deficits. An important consequence of d4T treatment was hind paw skin IENFD reduction, which is also evident in other painful neuropathies [42], including ddC-induced SN [82], direct HIV-mediated neurotoxicity [81], and paclitaxel-induced SN [7]. IENFD is an important clinical diagnostic tool, and reduced IENFD correlates inversely with the development of neuropathic pain [63]. Here, IENFD was reduced at PID 21, but not at PID 7, when hind paw hypersensitivity had already established, suggesting the consequential process of ‘‘dying back’’ may not be sufficiently developed at PID 7 to be detected by IENFD measurement. However, it does

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Fig. 11. Sural nerve proteomics at PID 7. (A) Relative quantification of proteins in the sural nerve from rats treated with d4T (n = 3) or vehicle (n = 3) by the SINQ method. Results from 3 repeated nano-LC–MS/MS experiments are shown as the log2 ratio of the d4T divided by the vehicle control of proteins identified and quantified in rat sural nerves. Twenty micrograms of protein lysate from the d4T and vehicle control groups were separated by 1D-SDS–PAGE. Both lanes were excised and cut into bands, digested in gel. Label-free SINQ normalized spectral index quantitation was achieved by nano-LC–MS/MS analysis. The dashed lines represent thresholds for up- and down-regulated proteins (Supplementary Tables 1 and 2). (B) Functional categorization of down-regulated sural nerve proteins at 7 days after the first d4T injection. The protein expression was at least 10-fold down-regulated compared to those of vehicle-treated controls. Functional categories were created according to Supplementary Table 1. (c) Functional categorization of the up-regulated sural nerve proteins at 7 days after the first d4T injection. The protein expression was at least 10-fold up-regulated as compared to those of the vehicle controls. Functional categories were created according to Supplementary Table 2. (D) Validation of label-free quantitative proteomics data from the d4T and vehicle control sural nerve samples by Western blot test. The anti-acyl-CoA dehydrogenase (ACADL) primary antibody was used to detect mitochondrial long-chain-specific ACADL, and Western blot analysis revealed a 50% reduction (based on 2 technical repeats, 41% and 58%) of the protein level of mitochondrial ACADL after d4T treatment compared to the vehicle control. The anti-kininogen HC primary antibody was used to detect kininogen-1, and Western blot analysis revealed about a 1.8-fold increase (based on 10 technical repeats, 1.78 ± 0.23) of the protein level of kininogen-1 after d4T treatment when compared to the vehicle control. Total protein lysates of the sural nerves from either d4T or vehicle control rats were used. b-Actin was used as a loading control.

appear that there are ongoing biochemical events in the sural nerves of d4T-treated rats at PID 7: our proteomic analysis demonstrated a 11-fold up-regulation of kininogen-1, the precursor of bradykinin, which can activate nociceptive terminals, sensitize other fibres to become nociceptors, and stimulate the release of substance P and cytokines involved in nociception [44]. Importantly, at PID 21, we observed clear changes in primary afferents’ central projections, i.e., reductions in CGRP expression in lamina I and IIo and IB4 binding in lamina IIi within the medial portion of the lumbar spinal dorsal horn, where afferents innervating hind paws terminate. Given the normal expression of CGRP and IB4 in the DRG, these reductions are likely to represent specific changes in the sensory terminals. The reduced CGRP expression may be indicative of active CGRP release from the terminals as presynaptic CaV2.2 channel complex increases CGRP release in the dorsal horn after d4T treatment [69]. Whether these reductions within the dorsal horn represent altered transport or targeting of CGRP and IB4, or physical withdrawal/injury to sensory terminals is unclear. We also demonstrated that the changes in CGRP and IB4 in the dorsal horn were length dependent, as similar changes were observed at sacral levels where afferents from the tail terminate, but not at thoracic levels, where sensory axons are shorter. These findings therefore mirror those in the peripheral projections in which there was a clear decrement in cutaneous innervation; however, axon counts in the nerve trunk were normal. The length-dependent observation fits well with HIV-SN clinical presentation, which is a typical length-dependent dying back axonopathy. Furthermore,

sural nerve proteomics at PID 7 revealed 3 down-regulated proteins related to mitochondrial functions and adenosine triphosphate (ATP) production, supporting the notion that mitochondrial dysfunction in the distal axons results in axonal degeneration in d4T-induced SN. Microtubule-associated protein 1B, which controls branching and axonal growth in degenerating DRG neurons [10], was also down-regulated and may contribute to the observed axonopathy. Reduced IENFD, together with a possible injury to the central terminals, is likely to up-regulate brain-derived neurotrophic factor (BDNF) expression in the dorsal horn, which can modulate nociceptive processing [51,57], increase neuronal excitability [45], and contribute to the development of central sensitization [87]. BDNF up-regulation in the dorsal horn is observed after nerve injury [56,61] and injury to DRG central terminals [65]. A mouse study has demonstrated that a single i.v. injection of d4T at 50 mg/kg significantly increases BDNF expression in the dorsal horn [66]. At PID 21, we revealed a small but significant increase in the diameters of both unmyelinated and myelinated axons in the sural nerve. Other toxic neuropathies including those produced by b,b0 iminodiproprionitrile, acrylamide, and vincristine can produce axon swelling [18,64,74], which is likely to be caused by impaired axon transport and neurofilament accumulation. The potassium chloride cotransporter KCC3 can regulate axon diameter; mice lacking this gene have axon swelling [12]. However, we did not observe periaxonal fluid accumulation. One possibility, therefore, is that d4T impacts on ion transport homeostasis required to

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maintain normal axon diameter. ATP is required for axon transport and maintenance of ionic gradients [41]. Therefore, it is possible that mitochondrial dysfunction and resultant ATP reduction could lead to increased axon diameters observed here. We did not detect DRG abnormalities after d4T treatment, which is in contrast to nerve injury models. ATF3 was not increased, nor was there any change in CGRP, IB4, and neuropeptide Y in small- and large-diameter DRG neurons. No increase in GFAP expression in satellite cells was observed, nor any macrophage recruitment to the DRG—events that have been observed in chemotherapy- and nerve injury-induced neuropathy models [26,31,60]. In contrast to an early microgliosis followed by an astrocytosis in the dorsal horn after nerve injury [4,13], we only found a small increase (15%) in microglia with an effector morphology at PID 21, contrasting to an approximately 200% increase after spinal nerve ligation. Phosphorylated p38, which is increased after nerve injury [32], demonstrated no change after d4T treatment. GFAP expression within the dorsal horn was increased at PID 7 but was not maintained at PID 21. Our findings of a minimal glial and immune response in the peripheral and central nervous systems are similar to those found in other models of painful neuropathy, such as those induced by chemotherapy or metabolic agents [9,14,89], which is distinct from those evoked by nerve injury models, emphasizing the need to develop animal models relevant to specific clinical scenarios. In conclusion, we have developed a robust rodent model of d4Tmediated painful SN that shares a number of features of the clinical condition. With more people, in particular those from developing countries, gaining access to ART every day, the burden of HIV-SN pain is a problem of enormous global importance. Our model provides a valuable tool to better understand its pathogenesis, to develop strategies to prevent new cases, and to find effective treatments for HIV-SN.

[3]

[4]

[5] [6]

[7]

[8]

[9]

[10]

[11]

[12] [13] [14] [15]

[16]

Conflict of interest statement [17]

The authors report no conflict of interest. [18]

Acknowledgements The research leading to these results is part of the Europain Collaboration, which has received support from the Innovative Medicines Initiative Joint Undertaking, under Grant agreement 115007, resources of which are composed of financial contribution from the European Union’s Seventh Framework Program (FP7/2007-2013) and EFPIA companies’ in-kind contribution. We thank Pfizer Ltd for providing d4T and gabapentin. We acknowledge Dr. Benjamin Thomas and Dr. Svenja Hester for their help with mass spectrometry analysis at the Central Proteomics Facility; the Sir William Dunn School of Pathology, University of Oxford, Oxford, UK; and Dr. Benjamin Thomas for his continued advice in all areas of proteomics mass spectrometry analysis.

[19]

[20] [21] [22] [23]

[24]

[25]

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pain.2012.12.023.

[26]

[27]

References [1] Abbadie C, Lindia JA, Cumiskey AM, Peterson LB, Mudgett JS, Bayne EK, DeMartino JA, MacIntyre DE, Forrest MJ. Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2. Proc Natl Acad Sci USA 2003;100:7947–52. [2] Andrews N, Legg E, Lisak D, Issop Y, Richardson D, Harper S, Pheby T, Huang W, Burgess G, Machin I, Rice ASC. Spontaneous burrowing behaviour in the rat is

[28]

[29]

573

reduced by peripheral nerve injury or inflammation associated pain. Eur J Pain 2012;16:485–95. Banerjee S, McCutchan JA, Ances BM, Deutsch R, Riggs PK, Way L, Ellis RJ. Hypertriglyceridemia in combination antiretroviral-treated HIV-positive individuals: potential impact on HIV sensory polyneuropathy. AIDS 2011;25: F1–6. Beggs S, Salter MW. Stereological and somatotopic analysis of the spinal microglial response to peripheral nerve injury. Brain Behav Immun 2007;21: 624–33. Bennett DL. Neurotrophic factors: important regulators of nociceptive function. Neuroscientist 2001;7:13–7. Bennett DL, Michael GJ, Ramachandran N, Munson JB, Averill S, Yan Q, McMahon SB, Priestley JV. A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurons after nerve injury. J Neurosci 1998;18:3059–72. Bennett GJ, Liu GK, Xiao WH, Jin HW, Siau C. Terminal arbor degeneration—a novel lesion produced by the antineoplastic agent paclitaxel. Eur J Neurosci 2011;33:1667–76. Bhangoo S, Ripsch M, Buchanan D, Miller R, White F. Increased chemokine signaling in a model of HIV1-associated peripheral neuropathy. Mol Pain 2009;5:48. Blackbeard J, Wallace VC, O’Dea KP, Hasnie F, Segerdahl A, Pheby T, Field MJ, Takata M, Rice AS. The correlation between pain-related behaviour and spinal microgliosis in four distinct models of peripheral neuropathy. Eur J Pain 2012;16:1357–67. Bouquet C, Soares S, von Boxberg Y, Ravaille-Veron M, Propst F, Nothias F. Microtubule-associated protein 1B controls directionality of growth cone migration and axonal branching in regeneration of adult dorsal root ganglia neurons. J Neurosci 2004;24:7204–13. Browne MJ, Mayer KH, Chafee SBD, Dudley MN, Posner MR, Steinberg SM, Graham KK, Geletko SM, Zinner SH, Denman SL, Dunkle LM, Kaul S, McLaren C, Skowron G, Kouttab NM, Kennedy TA, Weitberg AB, Curt GA. 20 ,30 -Didehydro30 -deoxythymidine (d4T) in patients with AIDS or AIDS-related complex: a phase I trial. J Infect Dis 1993;167:21–9. Byun N, Delpire E. Axonal and periaxonal swelling precede peripheral neurodegeneration in KCC3 knockout mice. Neurobiol Dis 2007;28:39–51. Calvo M, Bennett DL. The mechanisms of microgliosis and pain following peripheral nerve injury. Exp Neurol 2012;234:271–82. Calvo M, Dawes JM, Bennett DL. The role of the immune system in the generation of neuropathic pain. Lancet Neurol 2012;11:629–42. Calvo M, Zhu N, Tsantoulas C, Ma Z, Grist J, Loeb JA, Bennett DL. NeuregulinErbB signaling promotes microglial proliferation and chemotaxis contributing to microgliosis and pain after peripheral nerve injury. J Neurosci 2010;30: 5437–50. Chen X, Levine J. Mechanically-evoked C-fiber activity in painful alcohol and AIDS therapy neuropathy in the rat. Mol Pain 2007;3:5. Cherry CL, Kamerman PR, Bennett DLH, Rice ASC. HIV-associated sensory neuropathy: still a problem in the post-stavudine era? Future Virol 2012;7: 840–54. Clark AW, Griffin JW, Price DL. The axonal pathology in chronic IDPN intoxication. J Neuropathol Exp Neurol 1980;39:42–55. Daniel HC, Narewska J, Serpell M, Hoggart B, Johnson R, Rice ASC. Comparison of psychological and physical function in neuropathic pain and nociceptive pain: implications for cognitive behavioral pain management programs. Eur J Pain 2008;12:731–41. Deacon R. Assessing burrowing, nest construction, and hoarding in mice. J Vis Exp 2012;59:e2607. Deacon RM. Burrowing in rodents: a sensitive method for detecting behavioral dysfunction. Nat Protoc 2006;1:118–21. Deacon RM, Raley JM, Perry VH, Rawlins JN. Burrowing into prion disease. Neuroreport 2001;12:2053–7. Dorsey SG, Leitch CC, Renn CL, Lessans S, Smith BA, Wang XM, Dionne RA. Genome-wide screen identifies drug-induced regulation of the gene giant axonal neuropathy (Gan) in a mouse model of antiretroviral-induced painful peripheral neuropathy. Biol Res Nurs 2009;11:7–16. Doth AH, Hansson PT, Jensen MP, Taylor RS. The burden of neuropathic pain: a systematic review and meta-analysis of health utilities. PAINÒ 2010;149: 338–44. Ellis RJ, Rosario D, Clifford DB, McArthur JC, Simpson D, Alexander T, Gelman BB, Vaida F, Collier A, Marra CM, Ances B, Atkinson JH, Dworkin RH, Morgello S. Grant I; CHARTER study group. Continued high prevalence and adverse clinical impact of human immunodeficiency virus-associated sensory neuropathy in the era of combination antiretroviral therapy: the CHARTER study. Arch Neurol 2010;67:552–8. Gehrmann J, Monaco S, Kreutzberg GW. Spinal cord microglial cells and DRG satellite cells rapidly respond to transection of the rat sciatic nerve. Restor Neurol Neurosci 1991;2:181–98. Gore M, Brandenburg NA, Dukes E, Hoffman DL, Tai KS, Stacey B. Pain severity in diabetic peripheral neuropathy is associated with patient functioning, symptom levels of anxiety and depression, and sleep. J Pain Symptom Manage 2005;30:374–85. Hahn K, Triolo A, Hauer P, McArthur JC, Polydefkis M. Impaired reinnervation in HIV infection following experimental denervation. Neurology 2007;68: 1251–6. Hasnie FS, Breuer J, Parker S, Wallace V, Blackbeard J, Lever I, Kinchington PR, Dickenson AH, Pheby T, Rice ASC. Further characterization of a rat model of

574

[30]

[31]

[32]

[33]

[34]

[35] [36] [37] [38]

[39] [40]

[41] [42] [43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55] [56]

Ò

W. Huang et al. / PAIN 154 (2013) 560–575 varicella zoster virus-associated pain: relationship between mechanical hypersensitivity and anxiety-related behavior, and the influence of analgesic drugs. Neuroscience 2007;144:1495–508. Herzberg U, Sagen J. Peripheral nerve exposure to HIV viral envelope protein gp120 induces neuropathic pain and spinal gliosis. J Neuroimmunol 2001;116:29–39. Hu P, McLachlan EM. Macrophage and lymphocyte invasion of dorsal root ganglia after peripheral nerve lesions in the rat. Neuroscience 2002;112:23–38. Jin SX, Zhuang ZY, Woolf CJ, Ji RR. P38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J Neurosci 2003;23:4017–22. Jirkof P, Cesarovic N, Rettich A, Nicholls F, Seifert B, Arras M. Burrowing behavior as an indicator of post-laparotomy pain in mice. Front Behav Neurosci 2010;4:165. Joseph EK, Chen X, Khasar SG, Levine JD. Novel mechanism of enhanced nociception in a model of AIDS therapy – induced painful peripheral neuropathy in the rat. PAINÒ 2004;107:147–58. Joseph EK, Levine JD. Caspase signalling in neuropathic and inflammatory pain in the rat. Eur J Neurosci 2004;20:2896–902. Joseph EK, Levine JD. Mitochondrial electron transport in models of neuropathic and inflammatory pain. PAINÒ 2006;121:105–14. Kamerman PR, Wadley AL, Cherry CL. HIV-associated sensory neuropathy: risk factors and genetics. Curr Pain Headache Rep 2012;16:226–36. Keswani SC, Chander B, Hasan C, Griffin JW, McArthur JC, Hoke A. FK506 is neuroprotective in a model of antiretroviral toxic neuropathy. Ann Neurol 2003;53:57–64. Keswani SC, Pardo CA, Cherry CL, Hoke A, McArthur JC. HIV-associated sensory neuropathies. AIDS 2002;16:2105–17. Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG. Animal research: reporting in vivo experiments—the ARRIVE guidelines. J Cereb Blood Flow Metab 2011;31:991–3. Kwong JQ, Beal MF, Manfredi G. The role of mitochondria in inherited neurodegenerative diseases. J Neurochem 2006;97:1659–75. Lauria G, Lombardi R, Camozzi F, Devigili G. Skin biopsy for the diagnosis of peripheral neuropathy. Histopathology 2009;54:273–85. Lauria G, Majorana A, Borgna M, Lombardi R, Penza P, Padovani A, Sapelli P. Trigeminal small-fiber sensory neuropathy causes burning mouth syndrome. PAINÒ 2005;115:332–7. Liang YF, Haake B, Reeh PW. Sustained sensitization and recruitment of rat cutaneous nociceptors by bradykinin and a novel theory of its excitatory action. J Physiol 2001;532:229–39. Lu VB, Biggs JE, Stebbing MJ, Balasubramanyan S, Todd KG, Lai AY, Colmers WF, Dawbarn D, Ballanyi K, Smith PA. Brain-derived neurotrophic factor drives the changes in excitatory synaptic transmission in the rat superficial dorsal horn that follow sciatic nerve, injury. J Physiol 2009;587:1013–32. Macleod MR, Fisher M, O’Collins V, Sena ES, Dirnagl U, Bath PM, Buchan A, van der Worp HB, Traystman R, Minematsu K, Donnan GA, Howells DW. Good laboratory practice. Preventing introduction of bias at the bench. Stroke 2009;40:e50–2. Maratou K, Wallace VCJ, Hasnie FS, Okuse K, Hosseini R, Jina N, Blackbeard J, Pheby T, Orengo C, Dickenson AH, McMahon SB, Rice ASC. Comparison of dorsal root ganglion gene expression in rat models of traumatic and HIVassociated neuropathic pain. Eur J Pain 2009;13:387–98. Maritz J, Benatar M, Dave JA, Harrison TB, Badri M, Levitt NS, Heckmann JM. HIV neuropathy in South Africans: frequency, characteristics, and risk factors. Muscle Nerve 2010;41:599–606. Marshall S, Cox S, Rice ASC. Pain in human immunodeficiency virus and acquired immunodeficiency syndrome. In: Ballentyne JC, editor. Massachusetts general hospital handbook of pain management. Philadelphia: Lippincott Williams & Wilkins; 2005. p. 446–60. Meyer-Rosberg K, Kvarnstrom A, Kinnman E, Gordh T, Nordfors LO, Kristofferson A. Peripheral neuropathic pain—a multidimensional burden for patients. Eur J Pain 2001;5:379–89. Miletic G, Miletic V. Increases in the concentration of brain derived neurotrophic factor in the lumbar spinal dorsal horn are associated with pain behavior following chronic constriction injury in rats. Neurosci Lett 2002;319:137–40. Mills EJ, Bakanda C, Birungi J, Chan K, Ford N, Cooper CL, Nachega JB, Dybul M, Hogg RS. Life expectancy of persons receiving combination antiretroviral therapy in low-income countries: a cohort analysis from Uganda. Ann Intern Med 2011;155:209–16. Molander C, Wang HF, Rivero-Melian C, Grant G. Early decline and late restoration of spinal cord binding and transganglionic transport of isolectin B4 from Griffonia simplicifolia I after peripheral nerve transection or crush. Restor Neurol Neurosci 1996;10:123–33. Morland RH, Novejarque A, Pheby T, Huang W, Rice ASC. Comorbidities of visceral pain: study of the affective changes associated with acute and extended models of bladder inflammation. Program No. PH419. 2012. Program of the fourteenth World Congress on Pain. Milan, Italy: International Association for the Study of Pain. Nicholson B, Verma S. Comorbidities in chronic neuropathic pain. Pain Med 2004;5:S9–S27. Obata K, Noguchi K. BDNF in sensory neurons and chronic pain. Neurosci Res 2006;55:1–10.

[57] Obata K, Yamanaka H, Dai Y, Tachibana T, Fukuoka T, Tokunaga A, Yoshikawa H, Noguchi K. Differential activation of extracellular signal-regulated protein kinase in primary afferent neurons regulates brain-derived neurotrophic factor expression after peripheral inflammation and nerve injury. J Neurosci 2003;23:4117–26. [58] Oh SB, Tran PB, Gillard SE, Hurley RW, Hammond DL, Miller RJ. Chemokines and glycoprotein120 produce pain hypersensitivity by directly exciting primary nociceptive neurons. J Neurosci 2001;21:5027–35. [59] Parent AJ, Beaudet N, Beaudry H, Bergeron J, Berube P, Drolet G, Sarret P, Gendron L. Increased anxiety-like behaviors in rats experiencing chronic inflammatory pain. Behav Brain Res 2012;229:160–7. [60] Peters CM, Jimenez-Andrade JM, Jonas BM, Sevcik MA, Koewler NJ, Ghilardi JR, Wong GY, Mantyh PW. Intravenous paclitaxel administration in the rat induces a peripheral sensory neuropathy characterized by macrophage infiltration and injury to sensory neurons and their supporting cells. Exp Neurol 2007;203:42–54. [61] Pezet S, McMahon SB. Neurotrophins: mediators and modulators of pain. Annu Rev Neurosci 2006;29:507–38. [62] Phillips TJ, Cherry CL, Cox S, Marshall SJ, Rice ASC. Pharmacological treatment of painful HIV-associated sensory neuropathy: a systematic review and metaanalysis of randomised controlled trials. PLoS One 2010;5:e14433. [63] Polydefkis M, Yiannoutsos CT, Cohen BA, Hollander H, Schifitto G, Clifford DB, Simpson DM, Katzenstein D, Shriver S, Hauer P, Brown A, Haidich AB, Moo L, McArthur JC. Reduced intraepidermal nerve fiber density in HIV-associated sensory neuropathy. Neurology 2002;58:115–9. [64] Prineas J. The pathogenesis of dying-back polyneuropathies. II. An ultrastructural study of experimental acrylamide intoxication in the cat. J Neuropathol Exp Neurol 1969;28:598–621. [65] Ramer MS. Endogenous neurotrophins and plasticity following spinal deafferentation. Exp Neurol 2012;235:70–7. [66] Renn CL, Leitch CC, Lessans S, Rhee P, McGuire WC, Smith BA, Traub RJ, Dorsey SG. Brain-derived neurotrophic factor modulates antiretroviral-induced mechanical allodynia in the mouse. J Neurosci Res 2011;89:1551–65. [67] Rice AS, Cimino-Brown D, Eisenach JC, Kontinen VK, Lacroix-Fralish ML, Machin I, Mogil JS, Stohr T. Animal models and the prediction of efficacy in clinical trials of analgesic drugs: a critical appraisal and call for uniform reporting standards. PAINÒ 2008;139:243–7. [68] Rice ASC. Predicting Analgesic efficacy from animal models of peripheral neuropathy and nerve injury: a critical review from the clinic. In: Mogil JS, editor. Pain 2010—an updated review: refresher course syllabus. Seattle: IASP Press; 2010. [69] Ripsch MS, Ballard CJ, Khanna M, Hurley JH, White FA, Khanna R. A peptide uncoupling CRMP-2 from the presynaptic Ca(2+) channel complex demonstrates efficacy in animal models of migraine and AIDS therapy – induced neuropathy. Trans Neurosci 2012;3:1–8. [70] Simpson DM, Schifitto G, Clifford DB, Murphy TK, Durso-De Cruz E, Glue P, Whalen E, Emir B, Scott GN, Freeman R. Pregabalin for painful HIV neuropathy: a randomized, double-blind, placebo-controlled trial. Neurology 2010;74:413–20. [71] Smyth K, Affandi J, McArthur J, Bowtell-Harris C, Mijch A, Watson K, Costello K, Woolley I, Price P, Wesselingh S, Cherry C. Prevalence and risk factors for HIVassociated neuropathy in Melbourne, Australia 1993–2006. HIV Med 2007;8:367–73. [72] Snider WD, McMahon SB. Tackling pain at the source. New ideas about nociceptors. Neuron 1998;20:629–32. [73] Stence N, Waite M, Dailey ME. Dynamics of microglial activation: a confocal time-lapse analysis in hippocampal slices. Glia 2001;33:256–66. [74] Topp KS, Tanner KD, Levine JD. Damage to the cytoskeleton of large diameter sensory neurons and myelinated axons in vincristine-induced painful peripheral neuropathy in the rat. J Comp Neurol 2000;424:563–76. [75] Tsujino H, Kondo E, Fukuoka T, Dai Y, Tokunaga A, Miki K, Yonenobu K, Ochi T, Noguchi K. Activating transcription factor 3 (ATF3) induction by axotomy in sensory and motoneurons: a novel neuronal marker of nerve injury. Mol Cell Neurosci 2000;15:170–82. [76] UNAIDS JUNPOH. AIDS epidemic update, ; 2009 [accessed 17.10.12]. [77] van Sighem AI, Gras LA, Reiss P, Brinkman K, de Wolf F. Life expectancy of recently diagnosed asymptomatic HIV-infected patients approaches that of uninfected individuals. AIDS 2010;24:1527–35. [78] Verma S, Estanislao L, Simpson D. HIV-associated neuropathic pain: epidemiology, pathophysiology and management. CNS Drugs 2005;19:325–34. [79] Wadley AL, Cherry CL, Price P, Kamerman PR. HIV neuropathy risk factors and symptom characterization in stavudine-exposed South Africans. J Pain Symptom Manage 2011;41:700–6. [80] Wakisaka S, Kajander KC, Bennett GJ. Increased neuropeptide Y (NPY)-like immunoreactivity in rat sensory neurons following peripheral axotomy. Neurosci Lett 1991;124:200–3. [81] Wallace VCJ, Blackbeard J, Pheby T, Segerdahl AR, Davies M, Hasnie F, Hall S, McMahon SB, Rice ASC. Pharmacological, behavioural and mechanistic analysis of HIV-1 gp120 induced painful neuropathy. PAINÒ 2007;133: 47–63. [82] Wallace VCJ, Blackbeard J, Segerdahl AR, Hasnie F, Pheby T, McMahon SB, Rice ASC. Characterization of rodent models of HIV-gp120 and anti-retroviralassociated neuropathic pain. Brain 2007;130:2688–702.

Ò

W. Huang et al. / PAIN 154 (2013) 560–575 [83] Wallace VCJ, Segerdahl AR, Blackbeard J, Pheby T, Rice ASC. Anxiety-like behaviour is attenuated by gabapentin, morphine and diazepam in a rodent model of HIV anti-retroviral-associated neuropathic pain. Neurosci Lett 2008;448:153–6. [84] Wallace VCJ, Segerdahl AR, Lambert DM, Vandevoorde S, Blackbeard J, Pheby T, Hasnie F, Rice ASC. The effect of the palmitoylethanolamide analogue, palmitoylallylamide (L-29) on pain behaviour in rodent models of neuropathy. Br J Pharmacol 2007;151:1117–28. [85] Winston A, McAllister J, Amin J, Cooper D, Carr A. The use of a triple nucleoside–nucleotide regimen for nonoccupational HIV post-exposure prophylaxis. HIV Med 2005;6:191–7. [86] Woodham P, Anderson PN, Nadim W, Turmaine M. Satellite cells surrounding axotomised rat dorsal root ganglion cells increase expression of a GFAP-like protein. Neurosci Lett 1989;98:8–12.

575

[87] Woolf CJ. Evidence for a central component of post-injury pain hypersensitivity. Nature 1983;306:686–8. [88] World Health Organization. Antiretroviral therapy for HIV infection in adults and adolescents: recommendations for a public health approach, ; 2010 revision [17.10.12]. [89] Zheng FY, Xiao WH, Bennett GJ. The response of spinal microglia to chemotherapy-evoked painful peripheral neuropathies is distinct from that evoked by traumatic nerve injuries. Neuroscience 2011;176: 447–54. [90] Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. PAINÒ 1983;16:109–10.