Antiretroviral toxic neuropathy (ATN) is the commonest HIV-associated distal symmetrical polyneuropathy (DSP) , causing significant morbidity in 10–35% of HIV-positive patients [2–4] and occurring in 11–66% of patients [1;5] taking nucleoside analogue reverse transcriptase inhibitor (NRTI) drug therapy. The dideoxynucleotide analogue agents stavudine, zalcitabine and didanosine are implicated in its pathogenesis [1,4,6] and changes in prescribing pattern of these drugs, including dose reduction, have led to a reduction in the incidence of polyneuropathy . A feature of ATN is development of dysaesthetic pain , the severity of which is associated with elevated plasma levels of HIV-1 RNA ; this pain may be unresponsive to analgesia, even in combination with anticonvulsant drugs , tricyclic antidepressants , mexiletine  or peptide T . Withdrawal of certain NRTI drugs often becomes necessary [1,4] since there are no licensed effective therapies, although lamotrigine  and recombinant human nerve growth factor (NGF)  have shown some benefit. Ideally a pathogenesis-based treatment for ATN would allow patients to continue NRTI therapy, still the keystone of current highly active antiretroviral therapy regimens .
ATN is thought to result from disrupted mitochondrial oxidative metabolism [6,12] secondary to reduction in neuronal mitochondrial DNA content [6,13,14]. Consequently, neurons are unable to meet the metabolic requirements of their long peripheral axons, which undergo die-back and result in the glove and stocking distribution of ATN . In keeping with this die-back hypothesis, epidermal innervation is reduced in ATN [15,16], although the innervation of the dermis and sweat glands remains to be quantified. Development of abnormal sweating patterns in ATN suggests that an autonomic neuropathy also develops, which may be a factor in nocturnal sweating, common in HIV disease .
A proposed therapeutic agent for DSP is acetyl-l-carnitine (ALCAR), the acetyl ester of l-carnitine. ALCAR is vital for normal mitochondrial function, being a transport molecule for free fatty acids and an important acetyl-group donor in high-energy metabolism and free fatty acid β-oxidation [18,19]. Also, ALCAR potentiates NGF actions [20–22], promotes peripheral nerve regeneration [23,24] and is neuroprotective in vitro [19,25], in vivo  and in animal models of diabetic neuropathy . ALCAR has analgesic properties, possibly mediated by increasing adrenocorticotrophic hormone and β-endorphin levels , while l-carnitine also has favourable immunological benefits  in HIV infection. ALCAR treatment has been shown to improve pain scores and electrophysiological parameters in a placebo-controlled study in diabetics with DSP . Short-term ALCAR treatment  has shown symptomatic benefits in ATN, although it is unclear if this effect is long lasting because of neuronal regeneration or merely is an analgesic effect.
This study was designed to determine the effect of long-term ALCAR therapy upon symptoms and cutaneous innervation in patients with ATN, using quantitative immunohistochemistry with antisera specific for fibre types. This technique has been used in HIV-associated sensory neuropathy  and has been found to be more sensitive than clinical assessment in diabetic and leprosy neuropathy [32–34].
This study was performed in agreement with the terms of the Helsinki Declaration, after appropriate review by the local research ethics committee. The study recruited five non-neuropathic HIV-negative controls and an observational cohort of 21 patients with established ATN (grade 0–4/4 by analgesic requirement, where 0 is asymptomatic, 1 is ATN but no analgesia required, 2 is ATN requiring non-opioid analgesics, 3 is opioid analgesics required, and 4 is pain uncontrolled by opioid analgesia). Patients commenced treatment between March 1999 and October 2001. Selection criteria were age 20–60 years, either sex, documented HIV positive by a licensed HIV antibody enzyme-linked immunosorbent assay (Amplicor version 1.5 or Cobas version 1.5; Roche Molecular Systems, Branchburg, New Jersey, USA), and stable ATN diagnosis (onset within 6–12 months of commencing NRTI therapy, symptoms stable for 2 months, and no other neuropathic aetiological factors or DSP-associated therapies). The controls tested negative for HIV antibodies within the previous 3–12 months and had no neuropathic symptoms, no history of diabetes, metabolic disorders, alcohol abuse or treatment with potentially neurotoxic agents.
Patients’ clinical grade of DSP was monitored while receiving ALCAR 1500 mg twice daily (500 mg tablets; Sigma-Tau, Italia), and a record of changes in other medications was kept.
Skin biopsies (1 cm × 0.5 cm) were excised from a standardized point in the lower third of the more symptomatic leg as an outpatient procedure under local anaesthesia. Wounds were closed with 4/0 Surgipro sutures (Surgipro, Chicago, Illinois, USA), which were removed after 14 days. A maximum of four biopsies were taken from each patient, the first prior to commencing ALCAR therapy (pretreatment), and then subsequently from the same leg at 6–12 month intervals while on treatment. A single biopsy was taken from an equivalent site in each control.
Immunostaining and quantification
Biopsies were fixed in Zamboni's solution (16 h at 4°C) prior to equilibration in phosphate-buffered saline, 0.1% sodium azide, 15% sucrose, followed by rapid freezing in OCT compound (Tissuetec, Elkhart, Indiana, USA). Both pre- and post-treatment biopsies were processed together to eliminate variabilities between staining runs and all staining and quantification were blinded. Systematic random samples of 15 μm cryosections from each biopsy  were collected onto glass slides coated with Vectabond (Vector, Peterborough, UK). Sections were permeabilized in 0.2% Triton-X (BDH Laboratory Supplies, Bristol, UK), and stained by indirect immunohistochemistry using primary antisera against polyclonal rabbit antisera for protein gene product 9.5 (PGP; Affiniti, Chesham, UK), calcitonin gene-related peptide (CGRP; Affiniti) and vasoactive intestinal polypeptide (VIP; Affiniti). PGP antibodies identify all fibre types as it is a constitutive component. CGRP antibodies are specific for C and Aδ sensory fibres and VIP antibodies are specific for cholinergic sympathetic efferent fibres. Staining was visualized using a fluorescein isothiocyanate-conjugated secondary antibody (conjugated goat anti-rabbit serum, Vector).
After staining, each section was examined by fluorescence microscopy using a 20× objective. For each antibody, a random sample of six visual fields, including both epidermis and dermis (PGP and CGRP) or subcutaneous sweat glands alone (PGP and VIP), was captured for analysis using a high-resolution digital camera (Model 1-3-0; Diagnostic Instruments, Sterling Heights, Michigan, USA). The immunostaining area in each captured image was quantified by image analysis using Image-Pro-Plus (version 4.0; MediaCybernetics, Silver Spring, Maryland, USA) software. First, the colour immunofluorescent image was converted into greyscale; then the area of interest (epidermis, dermis or sweat gland) was outlined by tracing its margins. An intensity threshold was applied to differentiate bright immunostaining from any low-level background autofluorescence, and the immunostaining area was calculated automatically. The immunostaining area approximates closely the actual area of each nerve fibre type, thus permitting quantification of cutaneous innervation by each fibre type.
To facilitate comparisons, the immunostaining area was expressed as a fraction of the total frame area of epidermis, dermis or sweat glands. From six figures for each patient at each time point, a mean value (fractional area of immunostaining) was calculated and used for statistical comparisons.
The outcomes were analysed using generalized linear models. The model fit was best when the immunostaining fractional areas were fitted on a log scale, so that parameter estimates given are multiplicative increases. Each observation (i.e., each individual biopsy at each time point) was included individually in the model, which assumed normal covariance structure, with the correlation between individual patient's values accounted for using generalized estimating equations. Time-on-therapy covariate was dichotomized into a categorical variable because of evidence of a non-linear trend in the data. Biopsies taken between 3 and 9 months after starting treatment were taken to be the value at 6 months. Similarly, values at 12, 18 and 24 months were that time point ±3 months. Each variable was fitted into the model compared with the baseline immunostaining fractional area variables to gain an estimate of the average increase rate from baseline to that time point. Parameter estimates of increase in immunostaining fractional area on therapy were adjusted for potential confounders. These were baseline CD4 cell count (< 250 or > 250 × 106 cells/l), baseline age (< 40 or > 40 years), clinical grade of peripheral neuropathy (grade 1, 2 and 3) and antiretroviral treatment history (exposed to more or less than six different drugs). Models were examined for goodness of fit using residual plots and log-likelihood tests.
The data were analysed using an intent-to-treat analysis. Analysis was performed using SAS version 8.0 (SAS Institute Inc, Cary, North Carolina, USA). All P values are two sided.
Clinical and immunological parameters
Baseline data and patient demographics are summarized in Table 1. Median age at recruitment was 40 years (range, 29–60) for neuropathic patients, and 32 years (range, 29–53) for controls. In patients with established ATN, the median neuropathy duration prior to recruitment was 11 months (range, 2–41). None had a history of diabetes, excessive alcohol consumption or metabolic disorders. The median follow-up length was 14 months (range, 5–33). After the baseline biopsy, 11 patients had one further biopsy, 10 had two and 1 had three. During follow-up, two patients died. The causes of death were Mycobacterium avium intracellularae and cytomegaloviral infection for one patient, and liver failure for the second.
Clinical grade of neuropathic pain improved in 15 of 21 patients overall; seven of eight patients whose baseline grade was 1 and four of ten whose baseline was grade 2 became asymptomatic (grade 0); two of ten patients with baseline grade 2 improved to grade 1; one of three patients improved from grade 3 to 1 and one of three to grade 0. The neuropathic grade of five patients was unaffected by treatment, and one patient progressed from grade 1 to 2.
Although not formally documented, those who stopped ALCAR treatment suffered rapid symptom worsening, including return of dysaesthesiae. ALCAR treatment was well tolerated with no side effects, no adverse events or wound complications.
Three patients changed antiretroviral medication during the study period (one from didanosine to lamivudine at month 12 because of raised amylase, two from stavudine to nevirapine because of persistent symptoms of ATN).
Baseline median CD4 cell count was 286 × 106 cells/l [interquartile range (IQR), 155–423] and CD8 cell count was 1162 × 106 cells/l (IQR, 794–1918). These values did not change significantly at 6 months (CD4 cell count, P = 0.9; CD8 cell count, P = 0.9) and remained stable throughout the study. CD4/CD8 ratios also remained stable, with baseline median 0.19 (IQR, 0.14–0.39) not altered significantly at 6 months (P = 0.7) or thereafter. Median viral load values showed a similar pattern, with baseline median < 400 copies/ml (IQR, < 50 to 26 900) and P = 0.4 when compared with values at 6 months.
Morphology of cutaneous innervation
A normal cutaneous innervation pattern was seen in control biopsies, which derived from the reticular dermal plexus branching into plexi enveloping sweat glands or into the finer subepidermal plexus running parallel to the skin surface within the papillary dermis (Figs 1a and 2a). These fibres terminated within dermo-epidermal junction or entered the epidermis as free-end terminals. Within the epidermis, fibres ran perpendicular to the skin surface or ended in fine branches lying parallel to the stratum corneum. PGP-immunoreactive fibres predominated in all skin regions (Fig. 1a,d), while CGRP-positive fibres lay principally in the subepidermal plexus and deeper epidermal layers (Fig. 2a). VIP-positive fibres enveloped the sweat glands (Fig. 2d).
In patients with established DSP, there was an almost complete absence of fibres within the epidermis, the subepidermal plexus (Figs 1b and 2b) and around the sweat glands (Figs 1e and 2). ALCAR treatment was associated with normalization of morphology (Figs 1c,f and 2c,f), although epidermal fibres remained less evident than in control skin.
Quantification: all fibre types
Initial analysis compared the baseline PGP immunostaining fractional area to that found after 6 months of ALCAR treatment (Table 2). Subsequently, the median and range of patients’ immunostaining fractional area at baseline, 6, 12 and 18 months were calculated and plotted against comparable values obtained from HIV-negative controls (Fig. 3).
The effect of ALCAR treatment upon cutaneous innervation as determined by quantitative immunohistochemistry, with biopsy intervals from 6 to 24 months, is summarized in Table 3. Each skin area showed significant overall immunostaining increases during the course of ALCAR treatment. When compared with controls, patients with established DSP exhibited reduced total innervation of epidermis (79% of control), dermis (54%) and, most markedly, sweat glands (43%) (Table 2; Fig. 3). On average, after 6 months of ALCAR treatment, the PGP immunostaining percentage in epidermis increased 1.34 times from baseline [95% confidence interval (CI), 1.06–1.70], equivalent to a 34% increase. After 12 months of ALCAR treatment, the patients had, on average, a 2.01 times increase in epidermal PGP-immunoreactive fibres (equivalent to 101% increase) compared with baseline (95% CI, 1.45–2.77; Table 3).
After 6 months of ALCAR treatment, PGP-immunoreactive fibre increase in the dermis was 1.65 times than at baseline, equivalent to a 65% increase (Table 3). Compared with HIV-negative controls, total epidermal and dermal innervation reached normal range within 6 months of commencing ALCAR treatment and thereafter remained supranormal (Fig. 3), with an overall statistically significant increase over 24 months (Table 3). After 6 months of ALCAR treatment, PGP-immunoreactive fibres around sweat glands showed a 1.75 times relative increase over baseline, corresponding to a 75% increase (Table 3). This increase remained within normal control range for the remaining treatment period (Fig. 3) and persisted up to the last analysis point at 24 months, with the overall increase being very highly significant (P < 0.0001; Table 3).
Quantification: small sensory (C, Aδ) fibres
Dermal CGRP-positive fibres were significantly reduced (75% of control) in established DSP, while epidermal innervation was almost absent (20% of control). Highly significant increases occurred in both epidermal (mean 100%; P = 0.006) and dermal (mean 133%; P < 0.0001) innervation after 6 months of ALCAR treatment, such that mean immunostaining fractional area reached 40% of control in epidermis, and 175% of control in dermis (Table 2).
Six months of ALCAR treatment increased the proportion of all nerves (PGP positive), which displayed CGRP immunoreactivity in both epidermis (4% pre-treatment; 8% at the 6 month biopsy; 36% in controls), and dermis (22% pretreatment; 44% at the 6 month biopsy; 19% in controls) (Table 2). These CGRP staining increases in both areas were sustained at all time points up to 24 months, with an overall statistically significant immunostaining increase over the study duration (P ≤ 0.0001; Table 3).
Quantification: sympathetic sudomotor fibres
In patients with established ATN, the reduction in sympathetic innervation of the sweat glands (VIP-immunoreactive fibres) (41% of control) was similar to the reduction in their total innervation (43% of control for PGP-immunoreactive fibres; Table 2). Sympathetic innervation increased by 41% (P = 0.0003) after 6 months of ALCAR treatment (Table 2). The proportion of total innervation (PGP positive) that was VIP immunoreactive remained similar to controls (pretreatment 84%; post-treatment 85%; control 76%). The increase observed at 6 months was sustained up to 24 months of treatment, with an overall statistically significant increase (P < 0.0001; Table 3).
In this patient cohort, NRTI-induced DSP (ATN) was diagnosed by a neurologist. All patients had stable ATN prior to recruitment, making spontaneous resolution unlikely to have accounted for the changes in symptoms and innervation reported here.
Quantification of cutaneous innervation has previously been found to correlate with clinical tests of sensory function in leprosy  and diabetic neuropathy , where immunohistochemical changes preceded those of sensory testing during disease progression . The morphology of cutaneous innervation in DSP has previously been described qualitatively, and the density of epidermal fibres shown to be reduced along a proximal–distal gradient in the limb in DSP  and ATN . Epidermal nerve fibre density has recently been described as a therapeutic outcome measure in HIV ; however, fibre counts do not assess the health of surviving fibres, and atrophy or regeneration of fibres needs to be determined by immunostaining area quantification, as used in this study.
Since PGP is a constitutive component of peripheral nerve fibres, any decrease in immunoreactivity will reflect actual loss or atrophy of fibres rather than reduction in function of surviving fibres, corresponding to decreased immunoreactivity for specific fibre-type neuropeptide transmitters (e.g., CGRP, VIP). In established ATN, the symptoms reflect a cutaneous innervation reduction of the lower leg, with epidermal innervation being most affected, consistent with the die-back hypothesis of sensory neuropathy . There is also marked atrophy of dermal and sweat gland plexi. This cutaneous denervation may explain why neuropathic symptoms frequently do not begin to resolve for some weeks after starting ALCAR treatment and may then continue to improve for many months, since this time frame matches the slow rate at which peripheral nerves regenerate.
As expected from the ATN dysaesthetic algesic symptomatology, small sensory (C, Aδ) fibres are most affected, and we have demonstrated that these fibres show the greatest reduction in immunostaining area when compared with controls. Furthermore, the proportion of CGRP:PGP-positive fibres was markedly reduced in epidermis from neuropathic patients (ATN 4%; control 36%), implying loss of epidermal fibres and reduced function of the surviving sensory fibres. In contrast, fibre loss or atrophy may be the predominant feature in sweat glands, since the VIP:PGP ratio (ATN 84%; control 76%) is relatively preserved.
Six months of oral ALCAR treatment resulted in significant increases in the innervation in epidermis, dermis and sweat glands, an improvement maintained throughout the treatment for all patients. The results demonstrate that dermal PGP-immunostaining nerve fibres regenerated sufficiently to reach the range found in normal skin. Intraepidermal fibres also regenerated, although more gradually. This difference in regrowth rate in separate skin areas is unsurprising as reinnervation occurs more slowly in epidermis than in other tissues .
The increased ratio of CGRP and VIP to PGP immunostaining found after ALCAR treatment suggests that regenerating fibres may be functional, although electrophysiological and sensory testing will be required for confirmation. The increase in dermal CGRP immunoreactivity to supranormal levels after long-term treatment with ALCAR probably reflects the incomplete epidermal regeneration, hence the anterogradely transported CGRP accumulated in the dermal fibres through a distal ‘damming’ effect, as described in early diabetic neuropathy .
ALCAR treatment resulted in significant reduction in neuropathic pain grade experienced by the patients after long-term treatment. However, a randomized controlled trial utilizing a validated visual analogue pain scale is required to demonstrate conclusive symptomatic benefits. Such a trial is now underway to determine the functional effect of ALCAR treatment in preventing ATN. While there was no placebo arm to this study, the natural history of untreated DSP suggests that the continuous and long-term beneficial effects found in this patient cohort resulted from ALCAR treatment.
The mechanism underlying the effects of ALCAR is unclear. Although ATN pathogenesis has not been clarified, the DNA polymerase-γ hypothesis remains the most likely explanation . In eukaryote cells, DNA formed from individual deoxynucleotide trisphosphates by DNA polymerase, and the γ- and β-isoforms are significantly inhibited by the dideoxynucleotide trisphosphate NRTI . Unlike nuclear DNA replication, mitochondrial DNA replication is dependent upon the γ-isoform, which is most inhibited in non-mitogenic cells where only mitochondrial DNA is replicated [6,14]. Because of its proximity to free radical species generated by the respiratory chain complexes, mitochondrial DNA is particularly liable to damage. Since no mitochondrial DNA repair mechanisms exist, overall oxidative metabolic function must be maintained by replication . Hence by impairing mitochondrial DNA replication, a NRTI reduces the cell capacity for oxidative phosphorylation, the critical energy pathway in neurons. Consequently, neurons may apoptose  or fail to meet the metabolic requirements for maintaining long peripheral axons, resulting in die-back glove and stocking neuropathy.
ALCAR treatment may counteract the dideoxynucleotide trisphosphate NRTI toxicity (ATN) by several mechanisms. First, it may reduce mitochondrial DNA damage by a direct antioxidant effect [38,39]. Second, ALCAR facilitates mitochondrial homeostasis of acetyl-coenzyme A, promotes long-chain free fatty acid transport across mitochondrial membranes  and, via regulation of malonyl-coenzyme A, promotes cytosolic free fatty acid oxidation . By promoting glucose utilization and high-energy substrate oxidative metabolism, ALCAR improves neuronal metabolic capacity . In addition, ALCAR may facilitate distal neurotrophic support, particularly of myelinated (Aδ) and unmyelinated (C) fibres, since it increases NGF binding capacity , enhances the response to NGF  and promotes neurite extension in response to NGF . Furthermore, ALCAR promotes peripheral nerve regeneration [23,30] and function [42–45] independently of its role in preventing NRTI toxicity. Also, patients with NRTI-associated peripheral neuropathy have reduced serum ALCAR levels, which is not seen in asymptomatic HIV-positive controls . ALCAR penetrates the blood–brain barrier , and serum levels can be increased by its oral or parenteral administration [48,49], which also reverses the depletion occurring in peripheral nerves of diabetic rats .
ALCAR may offer additional benefits in HIV infection management since the mitochondrial DSP pathogenesis shares similarities with that of the lipodystrophy syndrome , associated with highly active antiretroviral therapy . Lipodystrophy might be responsive to ALCAR treatment, although an open small uncontrolled study of ALCAR in HIV-infected individuals with facial wasting showed no clear benefit . Furthermore, the related compound l-carnitine (with which ALCAR equilibrates in vivo) improves CD4 T cell counts by limiting ceramide production and apoptosis [53–56], and may also improve general health in HIV disease by promoting mononuclear cell proliferation , serum lipid profiles , and exercise capacity . In this study, an oral dose of ALCAR 1500 mg twice daily was safe, well tolerated and, unlike with recombinant human NGF treatment , no significant side effects were noted. ALCAR treatment was not associated with any progression of HIV infection, since viral load, CD4/CD8 ratio, CD4 cell count and CD8 cell count remained stable throughout.
Although NRTI agents have been partly superseded by other agents such as protease inhibitors, they remain fundamental for combination therapy [4,11] and are likely to remain important components in HIV management for the foreseeable future . Peripheral neuropathy has been the principal complication limiting the use of these agents [1,4] and ALCAR may now offer an effective, pathogenesis-based management approach, allowing patients to remain on NRTI therapy. Data from a full randomized controlled trial is now awaited to validate the results of this study and to determine the required duration and dose of ALCAR treatment.
The authors are grateful for the help given by the nursing staff of the Ian Charleson Day Centre, by the research nurses Zoe Cuthbertson, Pat Byrne and Tony Drinkwater, by the Research Fellows of the Blond McIndoe Centre, by Ben Wills, Paul Slade and Andrea Price of Bristol-Myers Squibb Pharmaceuticals and Menotti Calvani of Sigma-tau Farmaceutiche Italia.
Sponsorship: Unrestricted medical grant for laboratory materials from Bristol-Myers-Squibb Pharmaceuticals, Hounslow, UK.
Note: Andrew M. Hart and Andrew D. H. Wilson contributed equally to this paper.
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Keywords:© 2004 Lippincott Williams & Wilkins, Inc.
acetyl-l-carnitine; neurons; AIDS; HIV; nerve regeneration; antiretroviral toxic neuropathy; reverse transcriptase inhibitors; neuropathy