Contribution of skin immunohistochemistry in the evaluation of nerve fibers in diabetes mellitus type 2

CONTRIBUTION OF SKIN IMMUNOHISTOCHEMISTRY IN THE EVALUATION OF NERVE FIBERS IN DIABETES MELLITUS TYPE 2 (ABSTRACT)^*.THESIS. CAMPINAS, 2001.

SOLANGE GARCIA GARIBALDI^**

Neuropathy is a common complication of diabetes mellitus type 2 (DM-2). Variability in clinical expression of diabetic neuropathy is due to the selective or concomitant involvement of unmyelinated, thin myelinated or thick myelinated nerve fibers. Myelinated fibers may be well assessed by electroneuromyography, whereas evaluation of unmyelinated nerve fibers is still a challenge.

The aims of this study were to evaluate: 1. the usefulness of skin immunohistochemistry to protein gene product 9.5 (PGP 9.5) in DM-2 neuropathy; 2. whether the number of immunoreactive epidermal nerve fibers (IRENFs) to PGP 9.5 differ significantly between subgroups of patients with DM-2, initially examined by clinic and electrophysiological methods; 3. the pattern and frequency of nerve fiber involvement in diabetic neuropathy.

Patients and controls agreed to participate in the research by written informed consent, after UNICAMP Bioethical Council approval.

Forty-nine patients (20 male and 29 female) with DM-2, according to American Diabetes Association, 1997, and 18 healthy subjects as controls (4 male and 14 female) were included. The mean age was 56.69 ± 6.44 for patients and 54.33 ± 7.48 years for controls. Patients were classified into four groups according to clinical scores for neuropathy, routine nerve conduction velocities (NCV) and sympathetic skin response (present, normal; absent after four stimuli, abnormal). Punch skin biopsies of 3 mm in diameter were obtained from the distal part of the right leg, 10 cm proximal to the external malleolus, under local anesthesia, in patients and controls. They were processed using rabbit policlonal antibody to PGP 9.5 and anti-rabbit IgG conjugated with Rhodamine. Immunofluorescence pattern and morphology of nerve fibers were analyzed in a confocal microscope. The linear density of fibers, defined as the number of IRENFs per millimeter of epidermal length, was used for measurements. Statistical analyses were performed using data of age, sex, height, weight, duration of DM-2, duration of neuropathic symptoms, score of symptoms and signs, NCV, sympathetic skin response, level of glycohemoglobin in tables of frequency, measurement of position and dispersion. Fischer, Mann-Whitney and Kruskal-Wallis tests were used with significance level of 5 %.

DM-2 patients were classified as Group 1 (n = 15), absence of neuropathy. Group 2 (n = 8), neuropathy without symptoms. Group 3 (n = 8), symptomatic neuropathy with normal NCV. Group 4 (n=18), symptomatic neuropathy with abnormal NCV.

Clinical symptoms were present in 27 (55.1%) patients, clinical signs were abnormal in 38 (77,6%), NCV were altered in 26 (53.1%), foot sympathetic skin response was negative in 14 (28.5%) DM-2 patients.

The IRENFs densities were 1.40 ± 0.97 in DM-2 patients and 7.04 ± 2.46 in controls. The densities of IRENFs in each group of DM-2 patients were: Group 1 = 2.39 ± 0.53; Group 2 = 1.51 ± 0.49; Group 3 = 1.36 ± 0.41; Group 4 = 0.54 ± 0.79. The densities of IRENFs fibers were significantly (p = 0.001) reduced in patients from Groups 3 and 4, when compared to those of Group 1.

These results suggest that measurement of IRENFs to PGP 9.5 by linear densities allows early detection of the involvement of unmyelinated (C nociceptive) and small myelinated (Ad) nerve fibers in patients with DM-2, including those patients without clinical symptoms. A significant distinction between degrees of involvement of small fibers into Groups 3 and 4 in relation to Groups 1 and 2 was possible.

Skin biopsy detected abnormalities of unmyelinated and small myelinated fibers in all DM-2 patients in Groups 1 through 4. Twenty-six (53.06%) patients had large myelinated fiber dysfunction by clinical and neurophysiologycal assessment.

This study indicates that two patterns of involvement of peripheral nerve fibers are found in patients with DM-2: dysfunction of both small and large nerve fibers or selective abnormalities of unmyelinated nerve fibers leading to small fiber neuropathy.

KEY WORDS: skin biopsy, PGP 9.5 immunohistochemistry, diabetic neuropathy, small fiber neuropathy.

** Address: Faculdade de Ciências Médicas da UNICAMP, Caixa Postal 6111, 13083-970 Campinas SP, Brasil. Fax [phone].

*

Contribuição da imuno-histoquímica cutânea na avaliação das fibras nervosas no diabete melito tipo 2 (Resumo). Tese de Doutorado, Universidade Estadual de Campinas ¾ UNICAMP (Área: Neurociências - Neurologia) Orientadora: Anamarli Nucci. Co-orientadora: Maria Júlia Marques.

Publication Dates

• Publication in this collection
  11 Apr 2002
• Date of issue
  Mar 2002