Pyridoxine-induced toxicity in rats: a stereological quantification of the sensory neuropathy

Abstract

Excess ingestion of pyridoxine (vitamin B6) causes a severe sensory neuropathy in humans. The mechanism of action has not been fully elucidated, and studies of pyridoxine neuropathy in experimental animals have yielded disparate results. Pyridoxine intoxication appears to produce a neuropathy characterized by necrosis of dorsal root ganglion (DRG) sensory neurons and degeneration of peripheral and central sensory projections, with large diameter neurons being particularly affected. The major determinants affecting the severity of the pyridoxine neuropathy appear to be duration and dose of pyridoxine administration, differential neuronal vulnerability, and species susceptibility. The present study used design-based stereological techniques in conjunction with electrophysiological measures to quantify the morphological and physiological changes that occur in the DRG and the distal myelinated axons of the sciatic nerve following pyridoxine intoxication. This combined stereological and electrophysiological method demonstrates a general approach that could be used for assessing the correlation between pathophysiological and functional parameters in animal models of toxic neuropathy.

Introduction

Vitamin B₆ is a dietary requirement of nonruminant animals and is a coenzyme in many important biological reactions. The vitamin has three natural occurring forms: pyridoxol (pyridoxine), pyridoxal, and pyridoxamine. It is a relatively simple compound with three substituted pyridine derivatives that differ only in the functional group in the four position: alcohol (pyridoxine or pyridoxol), aldehyde (pyridoxal), or amine (pyridoxamine). Of these, pyridoxine is most commonly used as a dietary supplement and therapeutic agent. High doses of pyridoxine have been used to treat conditions such as premenstrual or carpal tunnel syndromes, and as therapy for intoxication secondary to the false morel mushroom Gyromitra esculenta (Hanrahan and Gordon, 1984). The rationale supporting this is that the active toxin monomethylhydrazine competitively inhibits a pyridoxine-dependent step in the synthesis of the neurotransmitter gamma-aminobutyric acid.

While pyridoxine deficiency is manifested by distal, predominantly sensory neuropathy, pyridoxine has also been identified as a neurotoxicant. During the 1980s, attention was drawn to a neurologic disease, which presented in individuals consuming large quantities of vitamin B₆ for prolonged periods of time. Schaumburg et al. (1983) has described a severe sensory neuropathy of insidious onset and course associated with chronic abuse of oral pyridoxine supplements. The recommended oral daily dose is 2–4 mg in human adults. Daily oral doses of up to 6 g for 12–40 months lead to a progressive sensory neuropathy manifested by sensory ataxia, diminished distal limb proprioception, paresthesia, and hyperesthesia (Dalton and Dalton, 1987, Foca, 1985, Schaumburg and Spencer, 1979, Schaumburg et al., 1983). Parry and Bredesen (1985) subsequently reported that as little as 200 mg of pyridoxine per day could induce this syndrome. It has been successfully reproduced in both acutely and chronically treated dogs (oral dosages of 50–300 mg kg⁻¹ day⁻¹ for up to 112 days) (Hoover et al., 1981, Krinke et al., 1980, Montpetit et al., 1988, Schaeppi and Krinke, 1982) as well as rats (Bowe and Veale, 1988, Krinke and Fitzgerald, 1988, Krinke et al., 1985, Nisar et al., 1990, Windebank et al., 1985, Xu et al., 1989), and appears to be secondary to a reversible sensory nerve axonopathy at low and intermediate doses and an irreversible sensory ganglion neuropathy at high doses (Krinke et al., 1980, Krinke et al., 1985, Phillips et al., 1978, Schaeppi and Krinke, 1982, Windebank et al., 1985). In rat studies, three intraperitoneal dosing regimens were generally employed, as follows: short term/high dose 1200 mg kg⁻¹ day⁻¹ for 1–15 days (Krinke and Fitzgerald, 1988, Xu et al., 1989); intermediate dosing, 600 mg kg⁻¹ day for 1–15 days (Krinke et al., 1985, Xu et al., 1989); long term/low dose, 100–300 mg kg⁻¹ day⁻¹ for up to 12 weeks (Krinke and Fitzgerald, 1988, Windebank et al., 1985, Xu et al., 1989). Such experimental studies confirmed the morphologic pattern of peripheral nervous system lesions of pyridoxine neurotoxicity, reflecting primary injury to the cytons of neurons residing in peripheral sensory (dorsal root, trigeminal) ganglia, with secondary degeneration of axons of affected cells. These are some of the largest neurons with long processes, and hence represent cells with the greatest metabolic susceptibility. The cell body changes are manifested in cytoplasmic alterations, such as vacuolization, neurofilament aggregates, and chromatolysis. More advanced neurotoxic changes lead to neuronal death with accompanying phagocytosis by satellite cells.

Compelling evidence exists in dogs (Schaeppi and Krinke, 1982), rodents (Xu et al., 1989), and in humans (Albin et al., 1987) relating to the functional and physiological parameters induced by pyridoxine neurotoxicity. However, the anatomical and morphological anomalies within the spinal cord and peripheral nerves resulting from excessive doses of pyridoxine have to date been solely subjective. Quantitative studies, involving information about number and size of axons within a particular nerve, or the size and distribution of cell bodies within specific dorsal root ganglia (DRG), are invaluable for studying developmental, experimental, and pathological changes. Design-based stereological techniques (Mouton, 2002) allow for precise and unbiased estimates of both number and sizes of axons from a small population. Indeed, a rapidly emerging literature exists that uses these techniques in peripheral nerves and nerve roots (Larsen, 1998, Larsen et al., 1998, Schionning and Larsen, 1997, Schionning et al., 1998a, Schionning et al., 1998b). Using design-based stereological techniques, this study has quantified the morphological changes that occur in the dorsal root ganglia and the sciatic nerve as a result of pyridoxine toxicity. This approach involving morphology correlates of sensory and motor nerve conduction velocity provides a general methodology for assessment of animal models of toxic neuropathy.