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A distal symmetrical sensory peripheral neuropathy is frequently observed in people living with Human Immunodeficiency Virus Type 1 (HIV-1). This neuropathy can be associated with viral infection alone, probably involving a role for the envelope glycoprotein gp120; or a drug-induced toxic neuropathy associated with the use of nucleoside analogue reverse transcriptase inhibitors as a component of highly active anti-retroviral therapy. In order to elucidate the mechanisms underlying drug-induced neuropathy in the context of HIV infection, we have characterized pathological events in the peripheral and central nervous system following systemic treatment with the anti-retroviral agent, ddC (Zalcitabine) with or without the concomitant delivery of HIV-gp120 to the rat sciatic nerve (gp120+ddC). Systemic ddC treatment alone is associated with a persistent mechanical hypersensitivity (33% decrease in limb withdrawal threshold) that when combined with perineural HIV-gp120 is exacerbated (48% decrease in threshold) and both treatments result in thigmotactic (anxiety-like) behaviour. Immunohistochemical studies revealed little ddC-associated alteration in DRG phenotype, as compared with known changes following perineural HIV-gp120. However, the chemokine CCL2 is significantly expressed in the DRG of rats treated with perineural HIV-gp120 and/or ddC and there is a reduction in intraepidermal nerve fibre density, comparable to that seen in herpes zoster infection. Moreover, a spinal gliosis is apparent at times of peak behavioural sensitivity that is exacerbated in gp120+ddC as compared to either treatment alone. Treatment with the microglial inhibitor, minocycline, is associated with delayed onset of hypersensitivity to mechanical stimuli in the gp120+ddC model and reversal of some measures of thigmotaxis. Finally, the hypersensitivity to mechanical stimuli was sensitive to systemic treatment with gabapentin, morphine and the cannabinoid WIN 55,212-2, but not with amitriptyline. These data suggests that both neuropathic pain models display many features of HIV- and anti-retroviral-related peripheral neuropathy. They therefore merit further investigation for the elucidation of underlying mechanisms and may prove useful for preclinical assessment of drugs for the treatment of HIV-related peripheral neuropathic pain.
Distal symmetrical polyneuropathy (DSP) afflicts 15-50% of people living with HIV (Verma et al., 2005; Marshall et al., 2005), 50-60% of whom have measurable sensory abnormalities (Martin et al., 2003) and on-going, paroxysmal or stimulus evoked pain (Dalakas and Pezeshkpour, 1988; Martin et al., 1999). There are two predominant (and clinically similar) settings in which painful HIV-DSP may occur. First, a disease-related DSP associated with HIV-infection per se; or secondly a drug-induced DSP associated with the use of nucleoside reverse transcriptase inhibitors (NRTI), particularly the dideoxynucleosides; zalcitabine (ddC), didanosine (ddI) and stavudine (d4T), as part of highly active anti-retroviral therapy (HAART) (Verma et al., 2005). The use of HAART has markedly increased patient survival (Beck et al., 1999) making, painful DSP an important source of morbidity in otherwise reasonably healthy HIV-infected individuals (Berger et al., 1993; Wulff et al., 2000; Dalakas et al., 2001). This limits viral suppression strategies and may precipitate abbreviation or discontinuation of anti-retroviral therapy. Therefore, it is important that analgesic strategies are developed to combat this pain. The exact pathogenesis of HIV-associated neuropathies is unclear. Features of peripheral neuropathy are apparent on examination of post-mortem peripheral nerve tissue taken from AIDS patients (Jones et al., 2005). Therefore, it is likely that HIV infection is commonly associated with axonal damage and that the clinical presentation of small fibre neuropathy is largely due to the effects of the virus. However, direct infection of the nervous system by HIV is thought to be unlikely (Michaels et al., 1988; Lipton, 1998; Jones et al., 2005). Instead, HIV-1 appears to interact with the nervous system via binding of the external envelope protein, gp120, to the chemokine receptors CXCR4 and/or CCR5 (depending on viral strain) expressed on neurons and glial cells (Pardo et al., 2001). Such interactions can result in neurotoxocity (Apostolski et al., 1993; Lavi et al., 1997; He et al., 1997; Oh et al., 2001; Keswani et al., 2003b) and peripheral axonal damage (Melli et al., 2006). In addition to gp120-modulated immunopathogenic factors (de la Monte et al., 1988; Rizzuto et al., 1995; Pardo et al., 2001; Verma et al., 2005) such neurotoxic mechanisms likely lead to the presentation of a prominently small-diameter axonal loss, associated with a ‘dying back’ pattern of axonal degeneration (for review see (Pardo et al., 2001). In contrast, certain anti-retroviral drugs may be neurotoxic via mechanisms involving mitochondrial dysfunction and toxicity (Kakuda, 2000; Dalakas et al., 2001; Keswani et al., 2003a; Joseph et al., 2004; Fiala et al., 2004). The decision to commence anti-retroviral therapy is often based on a high viral load/low CD4 count (and/or an ‘AIDS defining’ illness which is also a manifestation of a high viral load) (Gazzard et al., 2006). Consequently, at the early stage of anti-retroviral therapy (when the peripheral neuropathy usually manifests itself) there will be a period in which contemporaneous exposure to HIV-gp120 and anti-retroviral drug of choice (>1 dideoxynucleoside drug may be used although the routine use of ddC is no longer recommended in current HIV-treatment guidelines). Therefore, it is likely that although mechanistically distinct, the combination of HIV-associated pathology and neurotoxic effects of anti-retroviral drugs is synergistic for the development of painful neuropathies (Keswani et al., 2003a; White et al., 2005). Previous studies have indicated rats to be susceptible to NRTI-induced neuropathy (Schmued et al., 1996) associated with behavioural indices of neuropathic pain (Joseph et al., 2004; Bhangoo et al., 2007). Gp120 is known to induce neuropathic changes in primary sensory neurons in vitro (Oh et al., 2001; Keswani et al., 2003a; Melli et al., 2006) and recently we characterized a rodent model in which perineural administration of HIV-gp120 leads to the development of pain-like behaviour in rats as well as neuropathic changes in axons and sensory neurones (Wallace et al., 2007). However, no studies have been performed to assess the combined effects of HIV-gp120 and NRTI’s on pain behaviour and neuropathology in rats. Therefore, in an effort to further model the predominant current clinical scenario, we have, for the first time, investigated the effect of NRTI treatment on the interaction of HIV-gp120 with the peripheral nerve in the rat. To do this, we have assessed behavioural correlates of neuropathic pain and co-morbidity behaviour in rats treated concomitantly with perineural HIV-gp120 and systemic ddC as compared to either intervention alone. We have also assessed sensory neuronal phenotype, activity of non-neuronal, particularly immune, cells in the PNS and CNS and the effects on intraepidermal nerve fibre density. Furthermore, to draw parallels between these interventions and data from human studies, we have pharmacologically validated the associated behavioural sensitivity with drugs known to possess analgesic efficacy in other rodent models of neuropathic pain and from randomized controlled trials of neuropathic pain conducted in humans, including minocycline, amitriptyline, gabapentin, morphine and the cannabinoid agonist WIN-55212-2 (Hempenstall et al., 2005; Finnerup et al., 2005).
All experiments conformed to the British Home Office Regulations and IASP guidelines (Zimmermann, 1983). Male Wistar rats (200-250 g) (B & K, Hull, UK) were housed in a temperature-controlled environment, maintained on a 14:10 h light-dark cycle (experiments conducted in light phase) and provided with feed and water ad libitum. For rats receiving ddC treatment, 0.5 ml of ddC (Roche, Basel, Switzerland; 5, 25 or 50 mg/kg in saline) was injected i.p. three times per week for 3 weeks (Mon, Wed, Fri). This was previously established as the optimal drug delivery regime as compared to a number of regimens including; a single injection (which we found to be ineffective in producing hypersensitivity) (data not shown). For perineural HIV-gp120 administration, the technique previously described (Wallace et al., 2007) was used. Briefly, under 1-2% isoflurane anaesthesia (Abbott, UK) in O2 and N20, and aseptic surgical conditions, the left sciatic nerve was exposed in the popliteal fossa without damaging the perineurium and wrapped loosely, with a 3 × 0.5 cm2 strip of oxidized regenerated cellulose (Surgicell, Ethicon); previously soaked in 200 μl of a 0.1% rat serum albumin (RSA) in saline solution (Sigma, Poole, UK) containing 200 ng gp120-MN (Immunodiagnostics, Bedford, MA.) or for sham controls; 0.1% RSA in saline. The nerve was gently manipulated back into place and incisions closed with 4/0 sutures (Ethicon). For combined perineural HIV-gp120 + systemic ddC treatment (henceforth referred to as gp120+ddC), rats were injected with 0.5 ml of ddC (50 mg/kg in saline) at the time of surgery and three times a week thereafter for a maximum of 3 weeks. Sham controls were treated with perineural RSA and i.p. saline in the same regime as ddC treatment.
As a comparison model for nerve fibre density we used a previously characterized model of varicella zoster virus (VZV)-associated hypersensitivity (Garry et al., 2005; Hasnie et al., 2007). Methods are described in full elsewhere (Hasnie et al., 2007). In brief, primary human embryonic lung (Hel) cells (gift from J. Breuer, Royal London Hospital, UK) were inoculated with VZV (Dumas) and harvested when cells exhibited ~80% cytopathic effect (cpe) on microscopic examination. Virus-infected cells were centrifuged and re-suspended in 150 μl sterile phosphate buffer solution (Invitrogen, Paisley, UK) and 50 μl injected s.c. using a 25 gauge needle into the left (ipsilateral) hind footpad of anaesthetized rats.
Hind-paw withdrawal to sensory stimuli was measured in conscious rats as previously described (Wallace et al., 2007). Briefly, the hind paw withdrawal threshold in response to punctate static mechanical stimulation was measured using (a) graded ‘von Frey’ nylon filaments (Alan Ainsworth, UCL, London) (Chaplan et al., 1994) and (b) an electronic ‘von Frey’ device (Moller et al., 1998) of 0.5 mm2 probe tip area (Somedic Sales AB, Sweden). The time for hind-paw withdrawal in response to noxious heat was assessed using the Plantar test (Ugo Basile, Italy) (Hargreaves et al., 1988). Hypersensitivity to a cold stimulus was assessed by acetone application (Bridges et al., 2001).
Baseline measurements were obtained for all rats over the course of a week prior to surgery. All thresholds were measured by a ‘blinded’ observer. The threshold value at each time point tested was calculated as the mean ± SEM.
On day 21 following the first ddC injection (ddC only model) or day 14 post surgery (gp120+ddC model), rats (n = 8 per group) were placed into a 100 cm × 100 cm arena (4 lux) with a defined inner zone of 40 cm × 40 cm. Locomotion of the rats within the arena was tracked for 15 min as described previously (Hasnie et al., 2007; Wallace et al., 2007) and analysed using Ethovision software v.3 (Tracksys, Notts, UK). The total distance moved, time spent in the inner zone and number of entries into the inner zone were measured and the mean calculated. Only those rats with mechanical hypersensitivity (a change of at least 30% from baseline) were included (100% inclusion rate). For minocycline experiments, activity was measured on the final day of minocycline delivery.
Rats were deeply anaesthetized with pentobarbitone and transcardially perfused with heparinized saline followed by 4% paraformaldehyde (Sigma) with 1.5% picric acid (VWR, Poole, UK) in 0.1 M phosphate buffer (PB, Sigma). The L4 and L5 DRG, sciatic nerve and lumbar spinal cord were removed, post-fixed in 4% PFA for 4 h and transferred to a 15% then 30% sucrose solution in 0.1 M PB and left overnight. Tissue was embedded in OCT mounting medium (VWR) and frozen over liquid nitrogen. Cryostat sections of DRG (12 μm), sciatic nerve (15 μm) and L5 spinal cord (20 μm; level identified under light microscope) and were thaw-mounted on poly-l-lysine-coated glass slides (VWR).
For all fluorescent immunohistochemistry, sections were pre-incubated in buffer (0.1 M PBS, pH 7.4, containing 0.2% Triton X-100) containing 10% normal donkey serum for 1 h at room temperature and incubated with primary antibodies in buffer containing 4% donkey serum overnight.
The following antibodies were used: For detection of the stress-related transcription factor ATF3; rabbit anti-ATF3 (1:400; Santa Cruz Biotechnology, Santa Cruz, CA). For the detection of neuropeptides; rabbit anti-NPY (1:1000, Peninsula Laboratories Inc., Belmont, CA) and rabbit anti-galanin (1:2000; Peninsula, Cambridgeshire, UK). For the identification of cell bodies of myelinated afferents; mouse monoclonal anti-neurofilament 200 kDa (1:2000; clone N52; Sigma). For the detection of C-fibre terminals; rabbit anti-PGP 9.5 (1:1000; Ultraclone, Isle of White, UK) which recognizes the neuronal marker protein gene product 9.5 (PGP 9.5) in axons (McCarthy et al., 1995). For the staining of the chemokine CCL2; goat anti-mouse-JE (CCL2) (1:10; R&D Systems; Oxon, UK). For the staining of peripheral macrophages; mouse anti-CD68 (ED1) (1:2500; Serotec, Oxford, UK) which recognizes the CD68/ED1 glycoprotein expressed intracellularly by phagocytic macrophages (Hu and McLachlan, 2003). For the staining of satellite cells in the DRG; rabbit anti-GFAP (1:1000; Chemicon) which recognizes the GFAP intermediate filament protein that is upregulated in activated and proliferating DRG satellite cells (Hanani et al., 2005). For the staining of microglial cells; mouse anti-OX-42 (1:800; Serotec) directed against the CD11b/c protein expressed on the cell surface of microglia (Blackbeard et al., 2007). Sections were then washed in buffer and incubated with the appropriate secondary antibodies linked to either Cy3 (donkey anti-mouse-Cy3 1:300; Jackson ImmunoResearch Laboratories, Westgrove, PA) or fluorescein isothiocyanate (FITC) (donkey anti-rabbit-FITC 1:300; Jackson ImmunoResearch) for 2 h at room temperature. Three final washes in 0.1 M PBS were conducted before cover-slipping with Vecta-Shield (Vector Laboratories, Peterborough, UK) for analysis. Control sections were processed as above omitting the primary antisera.
All analysis was performed in a ‘blind’ manner Fluorescent images were visualized using a Leica DMR fluorescence microscope (Leica, Milton Keynes, UK) and analysed using Leica QWin software (Leica) as described previously (Wallace et al., 2007). For DRG immunohistochemistry, analysis was performed at ×20 on 6-8 randomly selected sections of DRG (separation of 180 μm) from each of four rats per group; only neurons with clear nuclei were counted. In each case, the analysis was performed in an automated manner using constant detection threshold levels. The total number of neurons was determined by counting both NF-200 labelled and non-labelled neuronal cells bodies and the number of DRG neurons expressing immunoreactivity (IR) for the protein of interest and reported as percent of total number of neurons/section. To measure alterations in DRG satellite cell populations the number of neuronal cell bodies associated with GFAP-IR satellite cells were counted and expressed as a percentage of total DRG neurons per section. To quantify the activated or infiltrating macrophages in DRG, CD68-IR cellular profiles were counted in a defined area (450 × 450 μm) and expressed as the total number/area. For CD68-IR in the sciatic nerve, six sections from each of four rats per group were captured at the site of HIV-gp120 or RSA application and the number of CD68-IR cellular profiles expressed as total number per unit area (450 × 250 μm). To quantify epidermal nerve fibre density (McArthur et al., 1998), PGP 9.5-IR nerves were counted in the epidermis of four footpad sections from each of four rats per group at ×20 using previously described methods (Hsieh et al., 2000; Lin et al., 2001; Keswani et al., 2006; Wallace et al., 2007). The total length of the epidermis along the upper margin of the stratum corneum in each footpad was measured and epidermal nerve density was expressed as the number of fibres per millimetre of epidermal length.
Analysis of spinal cord immunoreactivity was performed on a minimum of five randomly selected L5 coronal spinal cord sections from each of four rats per group. The percentage area of immunoreactivity (determined by the number of positive pixels) for GFAP or OX-42 staining in the ipsilateral and contralateral dorsal horn (laminae I-IV) was determined in a defined grid area (860 um × 580 um) using an automated grey-scale detection system using Leica QWin software version 3.0 (Leica) and expressed as mean% area ± SEM (Blackbeard et al., 2007).
For all experiments, animals were randomized into treatment group and the experimenter ‘blinded’ to drug treatments received. Hind-paw withdrawal thresholds to punctuate mechanical stimuli were measured as described previously using an electronic von Frey device (Moller et al., 1998; Hasnie et al., 2007; Wallace et al., 2007). Baseline measurements were obtained at least twice prior to surgery. For investigations into the effect of minocycline (Sigma, 40 mg/kg in saline), ddC rats received a single dose 1 h prior to the first dose of ddC and once daily thereafter for 21 days. gp120+ddC rats received a single dose of minocyline 1 h prior to perineural HIV-gp120 surgery and once daily thereafter for 14 days. Hind-paw withdrawal thresholds were tested at regular intervals during minocycline treatment. For all other pharmacological investigations, reflex thresholds were determined on days 11 and 14, only rats that developed mechanical hypersensitivity of at least 30% change were included. On days 15-18, animals were administered the drug or vehicle twice daily (b.d.), and behavioural tests performed once each day 1.5-2 h following the first injection. The following drugs were tested: morphine sulphate (Sigma; 2.5 mg/kg in 0.5 ml saline i.p. b.d.); the CB1/CB2 cannabinoid receptor agonist WIN 55,212-2 [(mesylate (R)-(+)-(2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo(1,2,3-de)-1,4-benzoxazin-6-yl)-1-naphthalenylmethanone) Sigma, 2.5 mg/kg in 0.75 ml saline with 40% dimethylsulfoxide (DMSO) i.p. b.d.]; gabapentin (Pfizer Ltd 30 mg/kg in 0.5 ml saline i.p. b.d.); and amitriptyline (Sigma 10 mg/kg in 0.5 ml saline i.p. b.d.). Doses were based on previous studies (Hasnie et al., 2007; Wallace et al., 2007).
Sigmastat version 2.03 (SPSS Inc., Surrey, UK) was used to calculate statistical significance (P < 0.05) throughout the study. For behavioural measures all groups were compared using a one-way Analysis of Variance (ANOVA) with Dunn’s all pairwise multiple comparisons post hoc analysis. For immunohistochemical and pharmacological studies, experimental groups were compared using a Kruskal-Wallis one-way ANOVA on ranks with an all pairwise multiple comparisons procedure (Dunn’s method).
Rats treated with 50 mg/kg ddC developed hind-paw mechanical hypersensitivity as measured by (a) graded von Frey filaments (gvF) (Fig. 1A) and (b) the electronic von Frey device (evF) (Fig. 1B). No such behaviour was observed in vehicle-treated control rats. This change peaked between day 14 and day 32 post initial injection and persisted until day 43. At 5 mg/kg, ddC produced no development of sensitivity, whereas at 25 mg/kg, ddC was associated with a small but significant mechanical sensitivity that peaked between days 14 and 21 (Fig. 1A and B) and recovered quickly. Therefore, we used 50 mg/kg as for subsequent experiments. At all time points, there was no evidence of hypersensitivity to thermal (Fig. 1C) or cold stimuli (Fig. 1D). We observed no overt motor deficits during or after ddC treatment which was further confirmed by measuring spontaneous locomotor activity in the open field area (see later).
Perineural administration of HIV-gp120 results in hypersensitivity to punctuate static mechanical stimuli (Wallace et al., 2007). We therefore investigated the effect of systemic ddC treatment on this behaviour. gp120+ddC-treated rats developed an increased mechanical hypersensitivity as compared to either treatment alone (Fig. 2A and B) without sensitivity to either thermal (Fig. 2C) or cold stimuli (Fig. 2D). Again we observed no overt motor deficit. In all cases, sham controls, showed no such effect.
We have shown repeatedly that the presence of mechanical hypersensitivity is detectable using either graded von Frey filaments or the electronic von Frey device (Bridges et al., 2001; Wallace et al., 2007; Hasnie et al., 2007), therefore for the remainder of experiments, we used only the electronic von Frey device to measure mechanical hypersensitivity.
We have previously shown spontaneous exploratory activity to be pathologically altered in rats following perineural delivery of gp120 (Wallace et al., 2007). Therefore, we assessed whether this behaviour was apparent in ddC and gp120+ddC-treated rats. As an indicator of normal locomotion, there was no significant difference in the total distance moved by ddC-treated or gp120+ddC-treated rats versus sham controls (Fig. 3A), suggesting a lack of overt motor deficit in these models. However, at the time of peak mechanical hypersensitivity (day 21), exploration of the centre of the arena (as calculated by the number of entries into a defined inner zone and the time spent in the inner zone) was significantly decreased in ddC-treated rats as compared to vehicle controls [Fig. 3B and D(ii)] indicative of thigmotaxis (anxiety-like behaviour). This abnormal pattern of behaviour was also a feature of gp120+ddC rats (Fig. 3Diii) when tested within the period of peak mechanical hypersensitivity (14-18 days post surgery), as compared to sham controls [Fig. 3C and D(i)]. These findings concur with those previously published for rats treated with perineural HIV-gp120 alone (Wallace et al., 2007) as well as models of traumatic neuropathy and VZV infection (Hasnie et al., 2007), indicating that thigmotaxis is a consistent feature of rat models of neuropathic pain in rats.
We have previously demonstrated that following perineural HIV-gp120 there is an up-regulation of the stress-related transcription factor ATF3 and the neuropeptides, galanin and NPY in sensory neurons, and of the intermediate filament, GFAP, in satellite cells (Wallace et al., 2007). Therefore, we assessed the presence of such changes following systemic ddC treatment. We found no significant expression of ATF3, galanin or NPY in the DRG of ddC-treated rats at day 21. There was, however, a small but significant increase in GFAP-IR satellite cells (Fig. 4A). In contrast, at day 14, DRG ipsilateral to perineural HIV-gp120 from gp120+ddC rats, expressed ATF3, galanin and, NPY in a significant number of neurones (Table 1) as well as GFAP in satellite cells (Fig. 4A). This expression was apparent in a similar proportion of cells as has been previously reported for perineural HIV-gp120 alone (Wallace et al., 2007) (Table 1). This suggests that there is no additive effect of ddC on such mechanisms and that ddC-induced neuropathic pain involves processes distinct from other models of neuropathy.
As a novel mechanistic correlate of perineural HIV-gp120 and systemic ddC treatment we assessed the induction of CCL2 expression in the DRG. CCL2 was significantly expressed in DRG from ddC rats and DRG ipsilateral to perineural HIV-gp120 treatment with no expression in sham-treated DRGs (Table 1 and Fig. 4B). CCL2 expression was greater in ipsilateral DRG from gp120+ddC rats as compared to either treatment alone (Table 1 and Fig. 4B). In gp120-treated rats (+/- ddC); a small proportion of CCL2 positive neurons were ATF-3 positive. Whereas, no ATF3-IR neurons were observed in DRG from ddC-treated rats. This highlights CCL2 as a novel mechanistic target for all three models that is not necessarily associated with cell damage/stress.
Perineural HIV-gp120 is associated with macrophage infiltration into the peripheral nerve and the DRG (Wallace et al., 2007). No increased macrophage population was observed in the peripheral nerve of ddC-treated rats (data not shown). However there was a small increase in the average number of CD-68-IR macrophages present in the DRG from ddC-treated rats at day 21 as compared to control DRG. This expression was of a similar magnitude to that seen in rats treated with perineural HIV-gp120 alone and gp120+ddC-treated rats again suggesting an overlap of possible mechanisms (Table 1).
Immunostaining of skin biopsies for the neuronal marker protein gene product 9.5 (PGP 9.5), (McCarthy et al., 1995; Kennedy et al., 1996) is used to quantify intraepidermal nerve fibre (IENF) density (Smith et al., 2005) in humans and rodents (Lauria et al., 2007; McCarthy et al., 1995; Herrmann et al., 1999; Lin et al., 2001; Keswani et al., 2006). We quantified IENF densities in the lateral plantar surface of the rat hind paw [i.e. the innervation of the sciatic nerve (Decosterd and Woolf, 2000)] treated with systemic ddC or with gp120+ddC. In ddC-treated rats, there were significantly fewer nerve fibres as compared to controls (Fig. 5). Similarly, in gp120+ddC rats, there were significantly fewer nerve fibres ipsilateral to perineural HIV-gp120 as compared to sham controls (Fig. 5). As a comparator to another non-nerve injury associated model that is characterized by mechanical hypersensitivity, we assessed the IENFD in rats 21 days following injection of VZV into the hind paw (Hasnie et al., 2007). Similarly, this model was associated with a significantly decreased IENFD as compared to controls (Fig. 5).
Spinal gliosis occurs following perineural HIV-gp120 administration (Wallace et al., 2007). We therefore assessed microgliosis and astrocytosis (by measurement of OX-42-IR and GFAP-IR, respectively) in ddC and gp120+ddC-treated rats. In ddC rats at day 21, there was a small increase in OX-42-IR in the spinal dorsal horn as compared to vehicle controls (Fig. 6A). In gp120+ddC rats at day 14 there was a greater increase in OX-42-IR ipsilateral to perineural gp120 treatment, as compared to either treatment alone, or to contralateral values or ipsilateral to sham (Fig. 6A). In ddC rats at day 21, there was a small increase in GFAP-IR as compared to vehicle control rats (Fig. 6B). GFAP-IR was also increased ipsilateral to perineural HIV-gp120 in both the gp120 and gp120+ddC models as compared to controls (Fig. 6B). However this expression was not different between the two models suggesting that unlike the effect on microgliosis, ddC did not exacerbate astrocytosis.
To assess the contribution of microglial-related processes to mechanical hypersensitivity and thigmotactic behaviour, we assessed the effect of the microglial activation inhibitor, minocycline, on behavioural responses in our models. When administered daily, commencing 1 h prior to the first ddC administration, minocycline (40 mg/kg i.p.) was not associated with any effect on the development of the mechanical hypersensitivity associated with ddC treatment (Fig. 7A). Nor did it affect measures of thigmotaxis in the open field. In contrast, when administered to gp120+ddC-treated rats, minocycline (40 mg/kg i.p.) significantly delayed the onset of mechanical sensitivity ipsilateral to perineural gp120 treatment as compared to vehicle controls (Fig. 7B). However, by day 14 post surgery, the minocycline-treated rats had developed mechanical hypersensitivity of a similar magnitude to that of vehicle-treated controls. Minocycline also decreased thigmotaxis in gp120+ddC rats as measured by time spent in the central zone of the open field arena, at day 14 (Fig. 7C).
Analysis of spinal OX-42-IR at the end of minocycline treatment confirmed that minocycline attenuated microgliosis in the dorsal horn of ddC-treated rats at day 21 (Fig. 7D) and of gp120+ddC rats at day 14 (Fig. 7D).
Amitriptyline (10 mg/kg i.p. b.d.) was not associated with any reversal of the heightened sensitivity to punctuate mechanical stimuli in ddC-treated rats [Fig. 8A(i)] or gp120+ddC rats [Fig. 8A(ii)]. In contrast, gabapentin (30 mg/kg, i.p. b.d. on d15-d18), was associated with a complete attenuation of the mechanical hypersensitivity observed with ddC treatment [Fig. 8B(i)] and a partial reversal of mechanical hypersensitivity in gp120+ddC rats [Fig. 8B(ii)]. Morphine (2.5 mg/kg i.p b.d.) reversed mechanical hypersensitivity displayed by both ddC and gp120+ddC-treated rats (Fig. 8C). In all cases, vehicle administration had no significant effect. Finally, systemic administration of the mixed CB1/CB2 cannabinoid receptor agonist WIN 55,212-2 (2 mg/kg, i.p. b.d.) completely reversed the mechanical hypersensitivity associated with ddC treatment [Fig. 8D(i)] and partially reversed mechanical hypersensitivity in gp120+ddC rats [Fig. 8D(ii)]. In both models, the effect of WIN 55,212-2 was significantly greater than the effect of the vehicle (40% DMSO). For all drugs tested, mechanical hypersensitivity was re-established by d28, indicating reversal of the drug effect and re-establishment of the neuropathic state.
Whilst the exact pathogenesis of HIV-DSP is unclear, it is likely that in those receiving HAART therapy, a combination of HIV-associated pathology and anti-retroviral neurotoxicity occurs (Pardo et al., 2001). Therefore, to investigate the mechanisms of HIV-DSP, we have pharmacologically and mechanistically characterized two rodent models: the first models the effects of systemic treatment with the NRTI, ddC, and the second investigates, for the first time, the combined effect of systemic ddC and perineural exposure to HIV-gp120 in the rat. In both models behavioural and mechanistic correlates of neuropathic pain are evident, a number of which are enhanced by the combination of anti-retroviral treatment and perineural HIV-gp120.
As with other drug-induced neuropathy models (Aley et al., 1996; Authier et al., 2000; Polomano et al., 2001; Authier et al., 2003a, b; Joseph et al., 2004), systemic ddC treatment caused a dose-dependant mechanical hypersensitivity. Furthermore, when combined with perineural HIV-gp120, this mechanical hypersensitivity was increased suggesting that an additive effect of gp120 and ddC-related mechanisms occurs. Additionally, in both models, and in contrast to many neuropathic pain models, no hypersensitivity to heat or cold stimuli developed. This is consistent with clinical data showing that patients with HIV-related painful neuropathy do not usually present with thermal hypersensitivity (Martin et al., 2003). At the time of peak mechanical hypersensitivity, both models display decreased spontaneous exploratory behaviour and thigmotaxis in the open field, a behaviour thought to reflect anxiety (Cryan and Holmes, 2005). This suggests the presence of pain-driven alterations in affect, which may be representative of ongoing pain and/or pain-related affective co-morbidities which are known to be a feature of neuropathic pain in humans (Meyer-Rosberg et al., 2001; Nicholson and Verma, 2004; Gore et al., 2005). This supports the use of this paradigm in the future to test the efficacy of novel drugs. A lack of overt motor deficit in both models was indicated by this test which may be further confirmed by additional tests of motor function.
Perineural HIV-gp120 is associated with signs of neuronal insult, as indicated by expression of ATF3 in the DRG (Wallace et al., 2007) and the presence of macrophages in the nerve, a change which correlates well with the clinical scenario (de la Monte et al., 1988; Yoshioka et al., 1994; Shapshak et al., 1995; Nagano et al., 1996; Pardo et al., 2001). This implies that HIV-gp120 may interact directly with axons, or indirectly via stimulation of macrophages to precipitate damage and that these factors may underlie the axonal ‘dying back’ observed in the skin of HIV patients. In line with a model of diabetic neuropathy (Wright et al., 2004), gp120+ddC-associated ATF3 expression indicates the presence of axonal stress in models of disease states, a phenomenon that merits further investigation. In contrast, we found no nerve macrophage infiltration or DRG expression of ATF3 in ddC-treated rats suggesting that such mechanisms may be associated with viral effects on the nerve alone. However, we did find a reduced numbers of C-fibre axon terminals in the plantar hind paw of ddC and gp120+ddC rats at times of peak mechanical hypersensitivity. IENF density, representing the number of terminals of C (nociceptive) fibres, is commonly used for diagnosis of sensory neuropathy (McCarthy et al., 1995; Kennedy et al., 1996; Lin et al., 2001; Lauria et al., 2007). Intraepidermal nerve fibres, are known to degenerate in patients with HIV-DSP and their density is inversely correlated with the presence of neuropathic pain in HIV patients (Polydefkis et al., 2002). Therefore, our results reflect the clinical scenario of a small fibre neuropathy affecting the distal nerve (Martin et al., 2003; Verma et al., 2005). The cause for IENF density decrease is uncertain. The lack of ATF3 in the ddC model indicates that neuronal injury or death is not necessarily required for reduced peripheral innervation density suggesting this may be due to a local effect on axons. Nevertheless, these findings suggests that even though ddC is associated with neuropathic changes distinct to those of gp120, the presentation of axonal ‘dying back’ in patients may be associated with both the presence of the virus and of the use of anti-retroviral drugs. This supports the possibility that NRTI drugs are exacerbating a virally mediated neuropathy and support the use of these models in the investigation of such mechanisms. These data are in contrast to a recent study in which the effects of gp120+ didanosine were assessed in mice (Keswani et al., 2006). However, this study investigated a gp120 expressing transgenic mouse treated with an alternative anti-retroviral drug, didanosine. Therefore, the differences between the models in question suggest that little comparison can be made between these studies.
A number of changes in DRG cell phenotypic occur following perineural HIV-gp120 (Wallace et al., 2007) which were not associated with systemic ddC treatment, again suggesting a limited effect of the drugs on the primary sensory neurons themselves. There was, however, evidence for changes in satellite cells and for the presence of macrophages in the DRG, both of which are changes associated with perineural HIV-gp120 and are features of models of nerve-lesion-associated pain (Ma and Bisby, 1998; Wynick et al., 2001). Furthermore, as a novel target for HIV-related neuropathies, there was significant up-regulation of the chemokine CCL2 (MCP-1) in the DRG of ddC and/or perineural HIV-gp120-treated rats. CCL2 is expressed in the DRG following nerve injury (Tanaka et al., 2004) and has been linked to the pathogenesis of pain both in the PNS and CNS (Zhang and Koninck, 2006; Sun et al., 2006). We found that the majority of CCL2 expressing neurons were not positive for ATF3 highlighting CCL2 as a novel mechanistic target for all three models that is not necessarily associated with cell damage/stress. Furthermore, the apparent synergism of ddC and gp120 in the up-regulation of CCL2, as well as the presence of macrophages and activated satellite cells in the DRG, suggests that such inflammatory-linked pathways may provide drug targets for treatment of HIV and drug-related neuropathies. Importantly, this study has highlighted changes that may represent mechanisms by which the anti-retroviral drugs can indirectly influence the function of the neurons and promote the generation of pain.
Spinal astrocytes and microglia can produce and/or respond to a number of pronociceptive substances (Tsuda et al., 2005). Although a direct causal relationship is controversial (Colburn et al., 1997), their activation has been demonstrated in various models of neuropathic pain (Colburn et al., 1997; Milligan et al., 2003; Tanga et al., 2004) making them potential targets for therapeutic intervention. Here we have shown for the first time that rats treated with ddC displayed a modest level of microgliosis whereas; gp120+ddC rats (which display a greater magnitude of mechanical hypersensitivity) display significantly greater levels of microglial activation. This suggests that microgliosis may be important, but not necessary, for the development of behavioural indices of pain in these different rodent models. In support of this hypothesis, inhibition of microglial activation had no effect on the development of ddC-associated hypersensitivity or thigmotaxis. In contrast, the onset of gp120+ddC-associated hypersensitivity was significantly affected by microglial inhibition which also attenuated thigmotaxis. These data suggest that microglial activation may not be necessary for the eventual development and maintenance of a persistent pain state but plays an important role in the onset of mechanical hypersensitivity as well as non-reflex-based measures of pain-associated behaviour and therefore merits further investigation for possible mechanistic targets. Of course, minocycline may also be targeting the immune system systemically (Zanjani et al., 2006) which may also play a role in the observed effects. Nevertheless, these data suggest a differential role for microglia in neuropathic pain states.
To assess the suitability of these models for the investigation of neuropathic pain mechanisms, we have tested the effects of compounds known to be analgesic in rodent models of pain and in humans. Amitriptyline has known analgesic efficacy in neuropathic pain in experimental models (Abdi et al., 1998; De Vry et al., 2004) and in the clinic (Hempenstall et al., 2005; Finnerup et al., 2005). However, clinical trial data, which are largely based on patients receiving HAART and therefore who are exposed to both HIV and anti-retrovirals, suggests that amitriptyline is not effective for the treatment of HIV-related neuropathic pain (Kieburtz et al., 1998; Shlay et al., 1998). In concordance with these clinical data amitriptyline had no effect on mechanical hypersensitivity in either of our models. In contrast, gabapentin and morphine were both effective. Gabapentin is analgesic in rodent (Abdi et al., 1998; De Vry et al., 2004; Donovan-Rodriguez et al., 2005) and human neuropathic pain conditions (Hempenstall et al., 2005; Finnerup et al., 2005), including HIV neuropathy (Hahn et al., 2004). Morphine attenuates hypersensitivity in rodent neuropathy models (Lee et al., 1994; Backonja et al., 1995) and evidence is accumulating to suggest that opioids relieve symptoms of human neuropathic pain of peripheral origin (Hempenstall et al., 2005; Finnerup et al., 2005). The effects of these analgesics in our models correlate well with their clinical efficacy further supporting the use of this model for the assessment of novel therapeutic agents. For example, evidence indicates that cannabinoids may exert beneficial effects including analgesia in human neuropathic pain (Rice, 2005; Finnerup et al., 2005; Rice et al., in press) including HIV-DSP (Abrams et al., 2007). Here we have shown that in line with previous rodent neuropathic pain studies (Bridges et al., 2001; Wallace et al., 2003; Hasnie et al., 2007), the CB1/CB2 receptor agonist, WIN 55,212-2 effectively attenuated mechanical hypersensitivity in both models further supporting the potential therapeutic benefit of cannabinoids.
In conclusion, we have characterized two neuropathic pain models that mimic many features of HIV-DSP and anti-retroviral-induced neuropathy. The associated characteristics, especially the robust but specific behavioural changes, highlight the utility of these models for many further mechanistic studies and potentially the discovery of new therapeutic targets for the treatment of neuropathic pain in general.
Wellcome Trust (London Pain Consortium), Pfizer for donation of gabapentin and Roche for donation of ddC. Funding to pay the Open Access publication charges for this article was provided by The Wellcome Trust.