Methylprednisolone treatment of an organophosphorus-induced delayed neuropathy

Diisopropylfluorophosphate (DFP) injected into the femoral artery of cats causes a localized organophosphorus-induced delayed neuropathy (OPIDN). Gait disturbances develop in the treated leg 14 days after DFP exposure and reaches a maximum at 21 to 28 days after DFP. In vivo high-frequency conditioning of soleus motor nerve endings evokes stimulus-bound repetitive neural discharges (SBR) and an obligatory potentiation of the muscle contractile response (PTP). In this OPIDN model, SBR and PTP are maximally suppressed at 21 to 28 days after DFP. A high-dose regimen of methylprednisolone started 30 to 40 min after DFP exposure and lasting for 20 days prevented the development of OPIDN. In the methylprednisolone-DFP treated cats, SBR and PTP functions were not suppressed and not different from those in untreated normal cats.     

An electrophysiologic and ultrastructural study of the phenylmethanesulfonyl fluoride protection against a delayed organophosphorus neuropathy

The delayed organophosphorus neuropathy caused by diisopropylfluorophosphate (DFP) can be prevented by pretreatment with phenylmethanesulfonyl fluoride (PMSF). A single injection of DFP (2 mg/kg) into a cat femoral artery produced a delayed neuropathy in the injected leg. Clinical neurotoxic signs in the DFP treated leg were most prominent at 21 to 28 days after DFP administration: a high-step gait with some tip-toe walking. During that time the capacity of the cat soleus alpha-motor nerve terminals to generate a stimulus-evoked repetitive discharge, known as SBR, was greatly attenuated. At that time, the ultrastructure of the motor nerve terminals demonstrated prominent alterations that correlated well with the motor nerve terminal SBR deficit. These alterations included the presence of extensive whorls in nerve terminals and axoplasms, the retraction and disruption of nerve terminals from the synaptic cleft, and a widening of secondary junctional folds. From the sampled population, the incidence of normal terminals in soleus muscles of the DFP-treated leg was only 2%. Cats which received PMSF (30 mg/kg ip) 24 hr before DFP administration did not develop any neurotoxic signs. Motor movements were normal. The SBR function of the soleus alpha-motor nerve terminals was not lost and its incidence approached normal values. Moreover, the ultrastructure was normal in 86% of examined neuromuscular junctions in the PMSF pretreated DFP cats. Thus, in this model, pretreatment with PMSF protected cats against the delayed neurotoxic effects of organophosphorus poisoning.     

Motor nerve dysfunction in delayed DFP neuropathy

A localized neuropathy was produced in cats by an intra-arterial injection of diisopropyl fluorophosphate (DFP). The soleus nerve-muscle system, and especially the motor nerve endings, were functionally evaluated during the following 1–56 days. Nerve conduction velocities remained unchanged. At 24–72 hr after DFP, the indirectly evoked but not directly evoked contractile strength of muscle fell to about 20% of normal; recovery had commenced by the 7th day. Motor nerve ending function was assessed by measurement of the neurogenic post-tetanic potentiation (PTP) of soleus muscle. PTP was initially unaffected, even though indirectly evoked contractile strength was severely attenuated. However, at day 7, a PTP reduction was evident and reached a loss of 62% on day 21. The underlying post-tetanic repetition (PTR) was lost at this time. Recovery of PTP and PTR occured over the next 5 weeks. These findings indicate that there is an initial functional damage of nerve endings in delayed organophosphate neuropathy. The pattern of nerve ending impairment suggests a trophic deprivation.     

Relative refractory period as a measure of peripheral nerve neurotoxicity

A method is presented for converting the relative refractory period to a linear form which can be used for quantifying changes in peripheral nerve conduction and membrane excitability. Since the relative refractory period is very consistent in nerves taken from animals of a given age, strain, and size, small alterations due to exposure by neurotoxic agents can readily be detected. In the present study rat sciatic nerves exposed to phenol developed a peripheral neuropathy which was detected by an increased relative refractory period. Diisopropylfluorophosphate (DFP), another neuropathic agent, produced a shift in the relative refractory period which was also consistent with the time course of its known neurotoxicity. On the other hand, acrylamide did not change the relative refractory period although the rats exhibited deficits when measured behaviorally (roto-rod and inclined screen tests). This observation is consistent with the view that the acrylamide toxicity initially affects nerve endings and spares the nerve axon. Erythrosin B, an agent which enhances nerve excitability, produced a shift in the relative refractory period which indicated that the nerve was less refractory. These examples point to the value of the relative refractory period as an index of the extent and type of neurotoxicity induced by agents which affect peripheral nerve axons, and suggest that this method may be useful in determining the mechanism of action of neurotoxic agents.     

Acrylamide axonopathy revisited

Distal swelling and eventual degeneration of axons in the CNS and PNS have been considered to be the characteristic neuropathological features of acrylamide (ACR) neuropathy. These axonopathic changes have been the basis for classifying ACR neuropathy as a central-peripheral distal axonopathy and, accordingly, research over the past 30 years has focused on the primacy of axon damage and on deciphering underlying mechanisms. However, based on accumulating evidence, we have hypothesized that nerve terminals, and not axons, are the primary site of ACR action and that compromise of corresponding function is responsible for the autonomic, sensory, and motor defects that accompany ACR intoxication (NeuroToxicology 23 (2002) 43). In this paper, we provide a review of data from a recently completed comprehensive, longitudinal silver stain study of brain and spinal cord from rats intoxicated with ACR at two different daily dosing rates, i.e., 50 mg/kg/day, ip or 21 mg/kg/day, po. Results show that, regardless of dose-rate, ACR intoxication was associated with early, progressive nerve terminal degeneration in all CNS regions and with Purkinje cell injury in cerebellum. At the lower dose-rate, initial nerve terminal argyrophilia was followed by abundant retrograde axon degeneration in white matter tracts of spinal cord, brain stem, and cerebellum. The results support and extend our nerve terminal hypothesis and suggest that Purkinje cell damage also plays a role in ACR neurotoxicity. Substantial evidence now indicates that axon degeneration is a secondary effect and is, therefore, not pathophysiologically significant. These findings have important implications for future mechanistic research, classification schemes, and assessment of neurotoxicity risk.     

Neurotoxicology of vincristine in the cat

Cats were given vincristine sulfate (50 g/kg i.v. every 4 days) and studied after 7–29 injections when neurological deficits became evident. The conduction velocity spectrum of individual afferent nerves from soleus muscles was shifted toward slower velocities. Relatively few functional muscle spindles or other proprioceptors which responded to muscle stretch were encountered. Those primary endings of soleus muscle spindles which did respond were significantly reduced in dynamic but not length sensitivity. Length sensitivity of secondary endings was unchanged. Thresholds of secondary but not primary endings were elevated. The average conduction velocity of soleus motor axons was reduced 30% but no deficit was detected in motor nerve terminal function. Indirectly-elicited contractile tension of the soleus muscles of the neuropathic cats was not significantly lower than that in untreated animals. Amplitudes of spinal monosynaptic reflexes were unaffected. These data indicate, that in the cat, neurological impairment results partly from dysfunction in muscle spindles and peripheral nerves.     

Dantrolene effects on neuromuscular function in cat soleus muscle.

Dantrolene sodium (DS) was investigated for its effects on cat soleus muscle contractile properties and motor nerve terminal activity in particular. DS, 0.1-1.5 mg/kg i.v., caused a dose-dependent depression of indirectly elicited contractile strength which was more pronounced at lower frequencies of stimulation. Maximum tetanic strength at frequencies of 10-400 Hz was depressed to a lesser degree than contractile responses evoked by lower frequencies of stimulation; the twitch/tetanus contraction ratios were reduced with increasing dose, primarily because of diminished twitch. DS was without effect on motor nerve terminals as evidenced by normal post-tetanic repetition in the nerves following DS administration. Post-tetanic potentiation became relatively larger in amplitude as contractile strength was diminished. These data suggest that DS depresses neuromuscular function at a site other than the neural apparatus at the neuromuscular junction.     

Spontaneous age-related peripheral neuropathy in B6C3F1 mice

Peripheral neuropathy, which accompanies aging, occurs during the long-term rearing of laboratory animals. The present study set out to delineate the clinical and functional features of this neuropathy. A total of 200 B6C3F1 female mice, in groups of 5 to 20 mice, were sacrificed and autopsied each week beginning at 5 weeks and continuing to 130 weeks of age. Examination for histopathologic changes was conducted on the dorsal nerve roots, sciatic nerves, peroneal nerves, tibial nerves, plantar nerves and brachial nerve plexuses. At 90 weeks of age or later, peripheral neuropathy, characterized by axonal degeneration and Schwann cell proliferation, were observed mainly in the sciatic nerves, brachial nerve plexus and peroneal nerves. These spontaneous age-related nerve lesions appeared in all animals by 100 weeks of age in all nerves, and increased with increasing age. The nerve lesions were most prominent in the distal sciatic nerve. The rectal and hind-limb surface temperatures, motor nerve conduction velocity, blood glucose and HbA1C decreased with increasing age. Elevation of sorbitol contents in sciatic nerves and reduction of myo-inositol levels were also detected in 120-week-old mice. However, except for blood glycemic parameters, no correlation with peripheral nerve lesions could be demonstrated. Spontaneous hypoglycemia (< 40 mg/dL) persisted throughout the day in a small percentage (< 5%) of animals aged 80 weeks or more; these animals had extensive lesions in the peripheral nerves and showed decreased plasma levels of HbA1C and frucutosamines and increased plasma levels of ketones. These results suggest that spontaneous peripheral nerve disorders which accompany aging might worsen if spontaneous age-related hypoglycemia is also present. Such age-related changes must be taken into consideration in experimental studies performed on mice of this age.     

Counteraction on experimentally induced diabetic neuropathy by levocarnitine acetyl.

The effect of levocarnitine acetyl on diabetic peripheral neuropathy induced by a single injection of streptozotocin or alloxan was studied. Levocarnitine acetyl was administered intraperitoneally one week after induction of diabetes at the dose of 50 mg/kg/day for five and ten weeks. At the end of treatment, neuromuscular conduction velocity (m/sec) was evaluated by stimulating the sciatic nerve and recording the soleus muscle potentials evoked, and the muscle contraction force (mm) by measuring the isometric muscular tension. Motor coordination was evaluated on the Rota-rod apparatus. Treatment with levocarnitine acetyl fully prevented the reduction (20%) in the neuromuscular conduction velocity observed in both experimental models of diabetes. The decrease (30-33%) in muscle contraction force was prevented partially in streptozotocin-induced diabetes and fully in alloxan-induced diabetes. Levocarnitine acetyl also improved the concomitantly reduced motor performance. The results of the present study suggest a beneficial effect of levocarnitine acetyl on peripheral neuropathy and muscle performance.     

The effect of ions and second messengers on long-term potentiation of chemical transmission in avian ciliary ganglia.

1. The effects of tetanic stimulation of the oculomotor nerve on transmission through the avian ciliary ganglion have been determined by use of the amplitude of the compound action potential recorded in the ciliary nerve, in the presence of hexamethonium (300 microM), as a measure of synaptic efficacy. 2. Tetanic stimulation for 20 s at 30 Hz potentiated the chemical phase of the compound action potential by at least 100% of its control level. This potentiation, reflecting an increase in synaptic efficacy, decayed over two distinct time courses: firstly, a rapid decay with a time constant in the order of minutes, and secondly, a slower decay, representing a smaller potentiation, with a time constant in the order of an hour. The large increase in synaptic efficacy is attributed to post-tetanic potentiation (PTP) whereas the smaller but longer lasting increase is attributed to long-term potentiation (LTP). 3. Higher frequencies of tetanic stimulation gave increased PTP and LTP. 4. In order to test whether the influx of calcium ions into the nerve terminal during the tetanus is likely to be involved in potentiation, facilitation was measured during PTP and LTP. Facilitation was reduced to approximately zero during PTP but recovered to normal values about 15 min into LTP. A requirement for the induction of LTP was shown to be the presence of calcium in the bathing solution. However, blocking synaptic transmission with a high concentration of hexamethonium (3 mM) during the tetanic stimulation did not block the induction of LTP. 5. Application of the muscarinic inhibitor, atropine (2 microM), did not affect the magnitude of PTP or LTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of phenylmethanesulfonyl fluoride on delayed organophosphorus neuropathy

A delayed localized neuropathy of peripheral nerves in a single hind leg of the cat develops after a single intraarterial 2 mg/kg injection of diisopropylfluorophosphate (DFP). This neuropathy is manifested by a maximum loss of the capacity of soleus -motor nerve terminals to generate stimulus-bound repetition 21 days after DFP exposure. Phenylmethanesulfonyl fluoride (PMSF) is a protective inhibitor of the neurotoxic esterase which is associated with the development of the delayed organophosphorus neuropathy. Pretreatment of cats with PMSF (30 mg/kg i.p.) 24 h before the DFP injection protected the cats from the delayed neuropathy. No clinical neurotoxic signs were observed at 21 days after DFP. The stimulus-bound repetitive capacity of soleus -motor nerve terminals was not lost at this time and its incidence was much greater than that which occurred in cats not pretreated with PMSF.This work was supported by U.S. Public Health Service National Institute for Neurological and Communicative Diseases and Stroke Grants NS-01447 and NS-11948     

Synaptic terminal degeneration and remodeling at the rat neuromuscular junction resulting from a single exposure to acrylamide

Repetitive exposure to low doses of acrylamide results in extensive pathological changes at the neuromuscular junction (NMJ), but it remains undetermined if a single exposure to a larger dose will produce a similar neuropathological outcome. In the present study, morphometric and ultrastructural analyses of rat soleus NMJ were performed to determine early pathological effects of an intraperitoneal injection of 100 mg/kg acrylamide. Widespread nerve terminal degeneration, terminal sprouting, and endplate lengthening were evident as early as 4 days after injection. Degenerating terminal branches were swollen and exhibited enhanced argyrophilia. Ultrastructurally, the majority of terminals exhibited axolemmal abnormalities, neurofilament accumulations, and a paucity of synaptic vesicles; occasional swollen terminals lacked neurofilaments but contained increased numbers of tubulovesicular profiles. This early morphological pattern of nerve terminal changes suggests that acrylamide may disrupt both synaptic vesicle recycling and neurofilament degradation. These findings indicate that a single high dose of acrylamide triggers pathological lesions and remodeling in motor nerve terminals virtually identical to those resulting from multiple low doses.     

Altered retrograde axonal transport of nerve growth factor after single and repeated doses of acrylamide in the rat

The retrograde axoplasmic transport of iodinated nerve growth factor (125I-NGF) was markedly altered by systemic acrylamide treatment. Single doses of acrylamide (25 to 100 mg/kg, ip) inhibited the retrograde transport of 125I-NGF in a dose-dependent manner within 12 hr after administration. A dose of acrylamide (15 mg/kg, ip) which did not alter retrograde transport when given as a single dose, markedly inhibited retrograde transport when given repeatedly. Alterations in retrograde transport appeared prior to detectable alterations in peripheral nerve function and correlated with cumulative dose of acrylamide. These data suggest that single doses of acrylamide produce a biochemical lesion which accumulates to produce neuropathy. Altered retrograde axoplasmic transport may reflect the primary biochemical event responsible for the induction of neuropathy by acrylamide.     

神经传导速度对亚临床型糖尿病周围神经病的诊断价值

目的通过检测无周围神经病症状的糖尿病患者的周围神经传导速度（NCV）,以探讨NCV在亚临床型DPN早期诊断中的价值。并探讨早期药物干预,对亚临床型DPN预后的影响。方法糖尿病组：60例,按1999年WTO糖尿病诊断标准确诊为糖尿病但无DPN临床症状的患者。对照组：30例,为无糖尿病及可引起周围神经病的其他疾患的健康人,糖尿病组与对照组在性别、年龄上无显著差异。两组患者均检测正中神经、尺神经、胫神经、腓总神经的运动神经传导速度,正中神经、尺神经、腓肠神经的感觉神经传导速度。结果①糖尿病组异常率为48．33％。对照组异常率为3．33％。P〈0．01．两者相比差异有极显著统计学意义;②下肢NCV异常率高于上肢;③感觉神经传导速度（SCV）异常率高于运动神经传导速度（MCV）;④早期给予干预治疗,明显改善率为75．86％。结论①神经传导速度检查可早期诊断亚临床型糖尿病周围神经病;②早期给予干预治疗可能逆转周围神经病损害。     

Effect of alcohol intake on some disturbances induced by chronic exposure to carbon disulphide in rats. II. Biochemical and ultrastructural alterations in the peripheral nerves

The effect of chronic ethanol (EtOH) administration on CS2-induced peripheral neuropathy in rats was studied. Rats were exposed to 0.8 mg/l CS2 for 12-15 months and to 10% EtOH as only drink during the last 4 months of the experiment. Some biochemical correlates of Wallerian degeneration of the peripheral nerve were estimated and ultrastructural examinations of the peripheral nerves were performed. It was shown that EtOH augmented the alterations in lipid content provoked by CS2 in the peripheral nerves, i.e. an increase in cholesterol esters and in the ratio of cholesterol esters to free cholesterol (E/F ratio) and a decrease in phospholipid content. The magnitude of ultrastructural changes induced by CS2 in the nerves was increased by EtOH.     

Influence of ganglioside treatment on acrylamide neuropathy in mice. A morphometric study

Female CBA/J mice were given oral doses of 40 mg/kg acrylamide 3 times weekly for 7 or 11 weeks in order to induce peripheral neuropathy. Half the animals were concurrently treated with 20 mg/kg, subcutaneously, of a ganglioside mixture (Cronassial). The other half received physiological saline and served as control. At the end of 7 or 11 weeks, the tibial nerves were removed and examined histomorphometrically. Every animal had developed a neuropathy. However, the mice treated with the ganglioside mixture showed on both the examination dates a substantially larger proportion of small sprouting nerve fibres than the control. It was concluded from this that treatment with gangliosides stimulated the regenerative potential of the damaged nerves.     

Levocarnitine acetyl stimulates peripheral nerve regeneration and neuromuscular junction remodelling following sciatic nerve injury.

The effects of levocarnitine acetyl were investigated on both peripheral nerve regeneration and neuromuscular remodelling in male Sprague-Dawley rats, three months of age, following crush of their left sciatic nerve. Levocarnitine acetyl, 150 mg/kg/day in drinking water, was given from one week before to 5, 15, 20, and 60 days after nerve crush. The sciatic nerve was examined morphologically at all given times and morphometrically at 15, 20, and 60 days after the lesion. Morphology, at 5, 15, and 60 days, and morphometry, at 60 days after the nerve crush, were also performed on the neuromuscular junction in the soleus and extensor digitorum longus muscles. Five days after nerve crush, complete axonal degeneration was observed in both control and treated rats. At 15 and 20 days, recovery from injury in treated animals was better than in controls, as shown by a significantly higher increase in the number of regenerating axons. At the same times, denervated endplates were present in both groups. At 60 days, axonal regeneration restored the number of axons to normal values in all injured animals, while their size maturation was greater in treated rats than in controls. A markedly lower number of degenerating elements was found in treated animals. In the neuromuscular junctions of the soleus and extensor digitorum longus muscles, nerve terminal branch points were reduced in the lesioned rats in comparison with uninjured ones. However, morphometric analysis revealed a greater endplate complexity in treated animals in which, at 60 days after nerve crush, nerve terminal branching and sprouting index values were significantly higher than in controls. It is concluded that levocarnitine acetyl exerts a beneficial effect on nerve regeneration processes and synaptic remodelling in crush-induced neuropathies.     

Selective promotion by phenylmethanesulfonyl fluoride of peripheral and spinal cord neuropathies initiated by diisopropyl phosphorofluoridate in the hen

This paper reports studies in hens showing that diisopropyl phosphorofluoridate (DFP) neuropathy is promoted by PMSF when initiated either in central (spinal cord) or peripheral nervous system. Moreover, the critical site for promotion is in peripheral nerve axons rather than in their cell bodies. Selective promotion in peripheral nerves was achieved by giving PMSF into sciatic artery monolaterally (7 mg/kg) to birds where neuropathy was initiated by DFP, either systemically (0.3 mg/kg s.c.) or intra-arterially (0.04 mg/kg in the same artery). Birds developed monolateral neuropathy in the leg where PMSF was delivered. Promotion of spinal cord neuropathy was achieved by giving PMSF (120 mg/kg s.c.) to birds where neuropathy was initiated selectively in spinal cord. This was obtained by protecting peripheral axons with intra-arterial bilateral injections of PMSF (0.55 × 2 mg/kg) followed by DFP (0.3, 0.4 or 0.7 mg/kg s.c.). The resulting syndrome was characterized by spastic ataxia.     

Motor nerve terminal response to edrophonium in delayed DFP neuropathy

A localized peripheral neuropathy was induced in cats with di-isopropyl fluorophosphate (DFP). Soleus nerve-muscle preparations, and the motor nerve terminals in particular, were evaluated for responsiveness to edrophonium (200 μg/kg i.v.). Potentiation of contractile strength was absent 24 hr after DFP, and showed a trend toward recovery 7–14 days post-DFP; it then fell to about 25% of normal 3 weeks following DFP administration. During the ensuing 5 weeks this aspect of edrophonium responsiveness was largely regained. The underlying post-drug repetition which gives rise to the potentiated responses was not demonstrable in either the nerve or muscle 3 weeks after DFP, but was again observed 8 weeks after poisoning. These findings suggest a delayed peripheral neuropathy indicative of a trophic deprivation and further illustrate a motor nerve terminal deficit as the initial function alteration in DFP neuropathy.     

睫状神经营养因子对周围神经损伤后功能恢复的影响

目的：研究睫状神经营养因子（ＣＮＴＦ）对受损周围神经功能恢复的影响。方法：将８０只大鼠左侧坐骨神经切断后行神经外膜吻合,随机分为两组。实验组腹腔内注入基因重组人ＣＮＴＦ,对照组注入等量生理盐水（ＮＳ）。术后行坐骨神经功能指数（ＳＦＩ）测定、电生理检测、轴突图像分析及霍乱毒素－辣根过氧化物酶（ＣＢ－ＨＲＰ）逆行追踪。结果：ＣＮＴＦ组ＳＦＩ恢复率、各项电生理及轴突图像分析指标、ＣＢ－ＨＲＰ标记的脊髓前