text: Ethidium bromide (EB) is an intercalating dye extensively used to induce demyelination in the central nervous system (CNS) (1). Demyelination follows the disappearance of neuroglial cells, chiefly astrocytes, in the early hours of intoxication (2,3). The death of astrocytes makes a breach in the glial-limiting membrane through which Schwann cells invade the CNS and repair the lost myelin sheaths (4,5).\n\nNaked axons receive new myelin sheaths built by invading Schwann cells and remaining oligodendrocytes - the latter along the unaffected living tissue (2). Morphological recognition of the new sheaths is based on the thickness of the sheath and on the presence of a Schwann cell wrapping the axon and surrounded by a basal lamina associated with collagen fibers (1).\n\nThe peripheral nervous system (PNS) differs from the CNS in fundamental aspects, i.e., histological composition (6) and regenerating capacity (7,8). In addition, Schwann cells are considered extremely industrious (9) and highly resistant to a broad span of spontaneous and experimental injuries (10). The aim of the present study was to observe Schwann cell reaction to local single and multiple EB injections and to document peripheral nerve responses to multiple challenges with the drug in order to validate this intoxication as an experimental model of demyelination in the PNS.\n\n\n\n--------------------------------------------------------------------------------\n\n\nMaterial and Methods \n\nThirty-four adult Wistar rats of both sexes were maintained in individual polypropylene cages, with free access to ration and water. The rats were divided into five groups and submitted to intraneural EB injections (Table 1). Two animals from each group received an intraneural saline injection. Stance, gait and social behavior were recorded daily.\n\nDemyelination was induced by single or multiple injections of 1 µl of 0.1% EB in 0.15 M saline along 8 mm on the middle third of the left sciatic nerve. The multiple injections were made at 28-day intervals. All injections were made with a Hamilton syringe and the animals were anesthetized with fentanyl/hypnomidate (Johnson & Johnson, São José dos Campos, SP, Brazil). The localization of the lesions was facilitated by a stitch in the adjacent semitendinous muscle.\n\nThe rats were sacrificed by an ether overdose and samples of the sciatic nerves were removed. The samples were collected 7 and 15 days after injection from the rats that received a single injection (group 1), and 10 days after injection from the rats submitted to multiple injections.\n\nThe samples were cut into three transverse fragments for light and transmission electron microscopy studies. All samples were washed with PBS, fixed in 2.5% glutaraldehyde in cacodylate buffer for 48 h, post-fixed in osmium tetroxide, dehydrated in graded ethanol solutions, and embedded in Epon 812 resin. Semi-thin sections were obtained with a Reichert Super Nova ultramicrotome with glass knives, stained with methylene blue and observed under a Meiji 2000 light microscope. Thin sections were made with diamond knives, stained with uranyl and lead salts and studied under an EM10 Zeiss transmission electron microscope operated at 60-80 kV.\n\n\n\n--------------------------------------------------------------------------------\n\n\nResults \n\nThe rats injected with EB did not show any changes in either stance or gait during the experiment.\n\nThe localization of the lesions was facilitated by the stitch on the adjacent semitendinous muscle although a mild thickening of the epineurium was visible to the naked eye. This thickening was due to collagen proliferation observed in all lesions. Although some variation was detected, most of the lesions were 5-8 mm in length. All lesions showed segmental demyelination and extensive remyelination by Schwann cells.\n\nAfter intraneural injection of EB, Schwann cells showed signs of intoxication with a vacuolated cytoplasm and rejection of the mye