Cutaneous Denervation in Alcoholic Neuropathy

Alcoholic patients and control subjects. Alcoholic patients will be recruited from neurologic clinics and ward at the Far Eastern Memorial Hospital, Taipei, Taiwan. A detailed clinical history that includes daily alcohol consumption, daily dietary intake, lifestyle, and occupation will be obtained from patients as well as their families. The inclusion criteria include daily uptake of at least 100 g ethanol for more than 3 years prior to the onset of neuropathic symptoms (Behse and Buchthal, 1977). All patients will undergo clinical and neurologic assessment, cranial MRI or CT, and neuropsychological evaluation to rule out CNS disorders, which interfere the evaluation of neuropathic symptoms and signs and psychophysic test. Laboratory investigations will include complete blood count, fasting plasma glucose, hemoglobin A1C, liver and renal function tests, ethanol level, tumor markers, antinuclear antibodies, complement factors, serum protein electrophoresis, thyroid function, human immunodeficiency virus, hepatitis virus, and vitamin B12 level. Thiamine status will be assessed at the time of the first referral to the hospital by measuring total thiamine concentration in whole blood by high performance liquid chromatography, as described elsewhere (Koike et al., 2001 and 2003). None of the patients will administrate thiamine at the time of determination. Age- and gender-matched control subjects will be retrieved from our database, who will be evaluated by detailed questionnaires and neurological examinations to exclude any neurological disorder or clinical neuropathy (McCarthy et al., 1995).

Skin biopsy. Skin biopsy will be performed following established procedures after informed consent has been obtained (McCarthy et al., 1995; Pan et al., 2003; Shun et al., 2004). Under local anesthesia with 2% lidocaine, punches 3mm in diameter will be taken from the following locations: (1) the extensor side of the distal forearm, 5 cm above the middle point of a line connecting the radial styloid process and the ulnar styloid process; and (2) the lateral side of the distal leg, 10 cm above the lateral malleolus. No suturing will be required, and the wounds will be covered with a piece of gauze. Wound healing takes 7-10 days, the same as would a typical abrasion wound. The protocol is under review by the Ethics Committee of Far Eastern Memorial Hospital.

Immunohistochemistry. For immunohistochemistry on freezing microtome sections, the skin samples will be fixed with 4% paraformaldehyde in 0.1M phosphate-buffered saline (PBS), pH 7.4, for 48 h (McCarthy et al., 1995; Hsieh et al., 2000). Sections of 50 mm perpendicular to the dermis will be cut on a sliding microtome (model 440E; Microm, Walldorf, Germany). They will be quenched with 1% H2O2, blocked with 5% normal goat serum, and incubated with rabbit antiserum to PGP 9.5 (UltraClone, UK, diluted 1: 1000 in 1% normal serum/Tris) at 4C for 16-24 h. After rinsing in Tris, sections will be incubated with biotinylated goat anti-rabbit IgG at room temperature for 1 h, followed by incubation with avidin-biotin complex (Vector, Burlingame, CA) for another hour. The reaction product will be demonstrated with chromogen SG (Vector, Burlingame, CA), and counterstained with eosin (Sigma, St. Louis, MO).

Quantitation of epidermal innervation. Epidermal innervation will be quantified following established protocols, and slides will be coded to ensure that measurements are blinded (Hsieh et al., 2000; Pan et al., 2003; Shun et al., 2004). PGP 9.5-immunoreactive nerve fibers in the epidermis of each section will be counted at a magnification of  40 with an Olympus BX40 microscope (Tokyo, Japan) through the depth of the entire section. Each individual nerve with branching points inside the epidermis will be counted as one. For epidermal nerves with branching points in the dermis, each individual nerve will be counted separately. The length of the epidermis along the upper margin of the stratum corneum in each section will be measured using Image-Pro PLUS (Media Cybernetics, Silver Spring, MD). END will therefore be derived and expressed as fibers/mm. For each tissue, there will be 48-50 sections, and all sections will be sequentially labeled. Every fifth section will be immunostained and quantified. The mean of values from these sections will be considered the END of the tissue specimen. In the distal leg, normative values from our laboratory (mean  SD, 5th percentile) of END are 11.16  3.70, 5.88 fibres/mm for subjects aged < 60 years and 7.64  3.08, 2.50 fibres/mm for subjects aged  60 years. The cut-off point of END is 5.88 and 2.50 fibres/mm in the two age groups, respectively.

Quantitative sensory testing. We will perform quantitative sensory testing (QST) with a Thermal Sensory Analyzer and Vibratory Sensory Analyzer (Medoc Advanced Medical System, Minneapolis, MN) to measure thresholds of warm, cold, and vibratory sensations following the established protocol (Pan et al., 2001; Pan et al., 2003; Yarnitsky and Ochoa, 1991). Briefly, the machine will deliver a stimulus of constant intensity which had been determined by the test algorithm. The intensity of the next stimulus will be either increased or decreased by a fixed ratio according to the response of the subject, i.e., whether or not the subject perceives the stimulus. Such procedures will be repeated until a pre-determined difference of intensity is reached. The mean intensity of the last two stimuli will be the threshold for the level method. Thermal thresholds recorded on the toe will be expressed as warm threshold temperature and cold threshold temperature, respectively. These temperatures will be compared with normative values (Pan et al., 2003). Vibratory thresholds recorded on the malleolus will be measured with similar algorithms. Normative values are documented previously and are similar to those of previous reports (Pan et al., 2001; Pan et al., 2003). Threshold values greater than the 95th percentile value for each age group will be considered abnormal.