Neurofilament gene expression following beta,beta'-iminodipropionitrile (IDPN) intoxication

beta,beta'-Iminodipropionitrile (IDPN) is an agent that produces a disorganization of the axonal cytoskeleton with massive accumulation of neurofilaments in the proximal axon. Abnormalities in axonal transport of neurofilament proteins and in their phosphorylation occur in this model. In this study we evaluated the gene expression of neurofilament and other cytoskeletal components at an early, intermediate and late stage of intoxication to determine whether this neuropathy is directly due to or secondarily affects the expression of these components. Specific cytoskeletal mRNA expression was evaluated in the spinal cords of rats treated with IDPN for varying durations using Northern analysis and in situ hybridization. Our results show no qualitative or quantitative alteration in the mRNA expression of the neurofilament triplet, alpha-tubulin, alpha-actin or glial fibrillary acidic protein. We conclude that abnormalities at various stages of cytoskeletal processing such as the early disorganization of the cytoskeleton, the impairment of neurofilament transport, and the long-term redistribution of neurofilaments along the axon are not directly due to, nor do they affect the gene expression of cytoskeletal components in IDPN neuropathy.     

Poisoning by beta,beta'-iminodipropionitrile (IDPN) in the rat: an experimental model for elective lesions of peripheral motoneurons

Chronic or acute intoxication of rats with beta,beta'-iminodipropionitrile induces characteristic lesions of axons of anterior horn cells. Swellings of proximal axons are associated with disorganization of the cytoskeleton which includes a disorientation of neurofilaments which are segregated in the periphery of the axon, while microtubules and mitochondria are clustered centrally. Slow axonal transport, which is markedly reduced, results in accumulation of neurofilaments in the proximal part of the peripheral motoneuron and distal atrophy. In chronically intoxicated rats, proliferation of Schwann cells with onion bulb formation were observed in the anterior spinal roots after 10 months on diet. In distal sciatic nerve, axonal degeneration was associated with accumulation of neurofilaments, organelles and glycogen. Axonal regeneration occurred in spite of sustained intoxication. The intensity of the lesions induced by IDPN in the proximal part of the axon of the spinal motoneuron are reminiscent of those observed in degenerative motor neuron disease. However, the abnormalities of the myelin sheath and the proliferation of Schwann cells encountered in IDPN-intoxicated rats do not occur in degenerative motor neuron diseases in humans.     

Axonal degeneration and axonal caliber alterations following combined beta,beta'-iminodipropionitrile (IDPN) and acrylamide administration

A new model of neurofilamentous axonal abnormality is described which employs combined administration of beta,beta'-iminodipropionitrile (IDPN) and acrylamide (AC). The model was developed to test the hypothesis that IDPN-induced swelling increases the vulnerability of the distal axon to a second neurotoxic chemical insult. Rats were given a single intraperitoneal (IP) injection of IDPN (1.5 g/kg) one week before receiving a single injection of AC (75 mg/kg, IP). Axonal degeneration was observed at multiple levels along the sciatic nerve at two weeks (with reference to IDPN administration), and was not progressive up to five weeks. Quantitation of degenerating fibers demonstrated that the extent of degeneration increased distally along the sciatic nerve. Single administration of either IDPN or AC did not produce degeneration. Thus, IDPN-induced neurofilamentous swellings alter the susceptibility of the axon to AC neurotoxicity. Two variations of this model were also studied. First, rats given five daily injections of AC (30 mg/kg, IP) beginning one week following IDPN administration developed accumulations of fast axonally transported materials in IDPN-induced microtubule channels. Second, rats given chronic injections of AC (30 mg/kg, IP, five days/week, for four weeks), to reduce the delivery of neurofilaments to the proximal axon, developed less prominent axonal enlargements when challenged with IDPN. Thus, axonal atrophy can mask the development of neurofilamentous axonal swellings.     

Selective interruption of axonal transport of neurofilament proteins in the visual system by beta,beta'-iminodipropionitrile (IDPN) intoxication

Beta,beta'-iminodipropionitrile (IDPN) is an agent that produces a marked impairment in the transport of neurofilaments. Its effect on other slowly transported cytoskeletal components such as tubulin and actin is variable. Previous studies have evaluated transport of neurofilaments after IDPN intoxication in a neurofilament-rich system (sciatic motor nerves) and in a system devoid of neurofilaments (axons of the dorsal motor nucleus of the vagus). In the former, IDPN impairs the transport of tubulin and actin but to a lesser degree than it does neurofilament proteins. In the latter, tubulin and actin transport were not impaired, and neurofilament proteins were not present. In this study we evaluated the transport of the cytoskeletal components in a system with an intermediate amount of neurofilaments (the visual system). In the visual system, there is a selective and marked (50%) impairment in the transport of neurofilaments with no impairment in transport of tubulin or microtubule-associated proteins (tau group). We conclude that these different patterns of impairment in transport reflect the differences in pre-intoxication neurofilament content of the nerves examined, the effect of IDPN on the transport of the other components of slow transport being secondary to the presence of stagnated neurofilaments. This model also suggests that transport of neurofilaments can be selectively impaired without producing an effect on other major slow transport components.     

Evolution of axonal swellings in cats intoxicated with beta,beta'-iminodipropionitrile (IDPN). An electrophysiological and morphological study

beta,beta'-Iminodipropionitrile (IDPN) causes formation of axonal swellings in the proximal internodes of spinal motor axons. The swellings enlarge and demyelinate with the progression of the neuropathy. The correlation between axonal swellings and electrophysiologic function of motoneurons was examined in cats 2 to 35 days after initial administration of IDPN. Morphologic changes in intraspinal motor axons, occasionally observed 2 days after the first injection, became progressively more evident at later times, with enlargement at the first internode in some axons and appearance of fusiform or balloon-like axonal swellings. At 7 days axonal swellings were infrequently observed and the main structural feature was a reduction in myelin thickness in affected nerve fibers. Despite scant histopathologic changes, motoneuron action potential discharge at this time was significantly altered in latency to onset of spike and rate of rise. Abnormal motoneuron firing patterns were observed at this time. As the neuropathy progressed, both the frequency of occurrence and the size of axonal swellings increased markedly but at no time was there morphologic evidence of chromatolysis. At 14 and 35 days, (after two or five IDPN injections) action potential discharge became further altered in latency to spike onset, rate of rise, initial segment conduction time and somal-dendritic threshold. The incidence of repetitive firing increased and axonal conduction block was observed in several motoneuron recordings. The electrophysiologic changes closely resemble those reported in chromatolytic motoneurons after axotomy. The axonal swellings induced by IDPN may produce an axotomy-like condition which becomes more prominent as the neuropathy progresses but without morphologic evidence of chromatolysis.     

Regional effects of iminodipropionitrile (IDPN) on free radical scavengers in rat brain

Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were measured in different regions of the brains of control and IDPN-treated rats. IDPN treatment resulted in significant increase of SOD activity in the cerebellum. Other regions, except frontal cortex, exhibited a slight decrease. While there was no significant difference in GSH-Px activity in all regions compared to controls, CAT activity was slightly increased in the cerebellum. These results give credence to the notion that free radical damage to certain areas of the brain such as isodendric core of the brainstem may play a role in the development of movement disorders.     

Toxic neurofilamentous axonopathies and fast anterograde axonal transport. II. The effects of single doses of neurotoxic and non-neurotoxic diketones and beta, beta'-iminodipropionitrile (IDPN) on the rate and capacity of transport

The site and mode of action of neurotoxic chemicals producing neurofilamentous axonopathies has been speculated to be the axonal transport system. The current study determined the effects of neurotoxic and non-neurotoxic gamma-diketones as well as beta, beta'-iminodipropionitrile (IDPN) upon both the rate and quantity of protein transported in the fast anterograde component of the rat sciatic nerve. 2,5-Hexanedione (2,5-HD), given as 4, 6 and 8 mmoles/kg single ip injections reduced the rate of transport by 18.4-24.7% but more significantly reduced the quantity of protein transported 50-63%. 3,4-Dimethyl-2,5-HD (3,4-DMHD) at single doses of 0.25, 0.50 and 1.0 mmoles/kg similarly reduced the rate and capacity of protein transport. The toxicants did not alter the uptake of leucine and synthesis of protein during the three hour time frame used to measure transport. Although high doses of IDPN reduced the rate of anterograde transport, this toxicant, as well as the non-neurotoxic diketones studied, had no effect upon the quantity of protein transported. Therefore, neurotoxic gamma-diketones which produce distal nerve degeneration had a common effect in decreasing the quantity of protein delivered to the nerve after just a single exposure.     

Chronic iminodipropionitrile (IDPN) causes no changes in the rat brain phencyclidine (PCP) receptor

Chronic IDPN treatment leads to a persistent stereotypic and dyskinetic behavioral syndrome which is reminiscent of that caused by PCP in mammals. Since the neuropharmacological profile of the two syndromes are very similar, the status of the PCP binding site was studied in rats who were suffering from the IDPN-induced syndrome. The characteristics of the receptor were not altered in either the striata or the hippocampi of the animals. These results suggest that the development of chronic streotypies is not intimately linked to any perturbation of the PCP binding site in rat brain.     

Effect of changes in neurofilament content on caliber of small axons: the beta,beta'-iminodipropionitrile model

The structural role of neurofilaments in the normal axon and the consequences of altered axonal transport of neurofilaments have been extensively studied in large axons. These studies suggest that neurofilament numbers and interneurofilament spacing are major determinants of axonal cross-sectional area. In contrast, in small axons and dendrites, microtubules and membranous organelles appear to be the most closely correlated with size and shape of the cell process. In this study we have examined the effect of impairment in neurofilament transport on small axons, typical of most CNS pathways. Neurofilament transport was impaired by administration of beta,beta'-iminodipropionitrile (IDPN), resulting in proximal accumulation and distal depletion of neurofilaments. The evolution of these changes was studied in the optic nerves of guinea pigs treated with IDPN, 1-35 weeks following intoxication. The effect of this redistribution of neurofilaments on cross-sectional area of small axons was evaluated using quantitative ultrastructural methods. Our results show that with the alteration in neurofilament transport seen with IDPN intoxication, there is a wide spectrum of neurofilament densities, ranging from a 5-fold increase above normal in the proximal axon, to a 5-fold decrease below normal in the distal axon. Although the optic nerve fibers enlarge with the increase in neurofilament content, they do not atrophy significantly with the continued loss of neurofilaments. We conclude that factors other than neurofilament content are capable of maintaining size and shape of these small axons. Candidate organelles include microtubules and membranous organelles and possibly other axonal elements.     

Electrophysiological investigation of beta,beta'-iminodipropionitrile neuropathy: intracellular recordings in spinal cord

beta,beta'-Iminodipropionitrile (IDPN) was given to cats (50 mg/kg/week for 5 weeks) to induce giant axonal swellings in the proximal internodes of motor axons. Conventional intracellular recording techniques were used to investigate the influence of the axon swellings on axonal impulse conduction and generation of action potentials in unidentified lumbosacral motoneurons (MN). Action potentials recorded from axon swellings, verified by lack of orthodromically or antidromically elicited EPSPs or IPSPs, afterhyperpolarization potentials or initial segment-somaldendritic (IS-SD) inflections, were variable in shape. Some were indistinguishable from recordings in normal axons. Component or extra potentials occurred in 45% of recordings from axon swellings; their position on the action potential depended on the direction of impulse invasion into the swelling. Many action potentials were broad, with amplitudes less than 30 mV. Impulse conduction was estimated to be blocked in 19% of motor axons tested. Action potentials recorded in MN of IDPN treated cats resembled in many aspects those recorded in chromatolytic MN, with increased latencies upon antidromic stimulation and decreased IS conduction times and SD thresholds; other parameters did not differ significantly. The minimal interval between two stimuli which each evoked action potentials increased from 3.3 +/- 0.1 to 5.8 +/- 0.5 ms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased cortical serotonin-2 (5-HT-2) receptors in the iminodipropionitrile (IDPN)-model of persistent dyskinesia in the rat

Chronic treatment with IDPN causes a persistent behavioral syndrome which is reminiscent of the head shakes, backward locomotion, and hyperactivity observed after acute injection of 5-HT agonists in rodents. Evaluation of the characteristics of 5-HT receptors, using mianserin to label 5-HT-2 receptors, revealed a significant increase in the number of binding sites in the cortices of rats which demonstrated the IDPN-induced syndrome. There was an associated decrease in the affinity of the receptors. These results add more evidence to support the idea that the serotonergic system may play a significant role in the manifestation of the persistent abnormalities induced by IDPN.     

Electrophysiological investigation of beta, beta'-iminodipropionitrile neurotoxicity: monosynaptic reflexes and recurrent inhibition

beta-beta'-iminodipropionitrile (IDPN) neurotoxicity is morphologically characterized by the presence of giant axon swellings in the first proximal internodes of motor axons. The electrophysiological consequences of these proximal giant axon swellings on monosynaptic reflexes and recurrent inhibition were investigated along dorsal root-ventral root, medial gastrocnemius-ventral root and soleus-ventral root pathways of IDPN-intoxicated cats 35, 50 and 100 days following initial administration of the toxin (50 mg/kg/wk for 5 weeks). Monosynaptic reflex action potentials, normally relatively synchronous spike potentials, frequently appeared as doublet potentials which did not represent temporal fractionation of the spike potential. Latencies to the monosynaptic reflex action potentials were prolonged. Amplitudes of unconditioned monosynaptic reflex action potentials were significantly decreased at all time points, although there was a tendency for the amplitudes to recover with time. Post-tetanic potentiation was variously altered. Recurrent inhibition was reduced by 42-49% along monosynaptic reflex pathways. These results demonstrate that electrophysiological function in the spinal cord of IDPN-intoxicated cats is profoundly altered and the dysfunction partly results from the presence of the giant axon swellings.     

Regulation of aberrant neurofilament phosphorylation in neuronal perikarya. III. Alterations following single and continuous beta, beta'-iminodipropionitrile administrations.

beta,beta'-Iminodipropionitrile (IDPN) administration produces giant neurofilament-filled axonal swellings in the first proximal internodes of large myelinated sensory and motor fibers without any accompanying axonal degeneration. In the present study, we asked whether proximal giant axonal swellings are sufficient to elicit aberrant neurofilament (NF) phosphorylation in neuronal perikarya. Rats were given a single intraperitoneal (i.p.) injection of IDPN (2 g/kg) followed by IDPN (0.1%) in the drinking water (continuous IDPN exposure) or tap water (single IDPN exposure) for two days to 7 weeks. Immunoreactivity to phosphorylated NF (pNF) epitopes (using monoclonal antibodies 6-17 and 7-05) was observed in L4 and L5 dorsal root ganglia (DRG) neurons beginning between one and 5 days, corresponding to the development of proximal giant axonal swellings. Quantitation of DRG neurons demonstrated maximal numbers of immunoreactive cell bodies to pNF epitopes (46-51%) by one week. The number of immunostained DRG cells was maintained in animals given continuous IDPN exposure, but declined significantly (P less than 0.001) in rats given a single injection of IDPN to 26 +/- 0.80% and 6 +/- 0.04% at 3 and 5 weeks, respectively. Ventral and dorsal root fibers, which undergo axonal atrophy distal to axonal swellings, showed intense immunoreactivity to pNF epitopes and a marked reduction or a complete lack of immunostaining to antibody 2-135 (directed against non-phosphorylated NF epitopes); pretreatment with alkaline phosphatase reversed this staining pattern. In a separate study, a similar alkaline phosphatase-sensitive lack of staining to antibody 2-135 was also observed in atrophic motor fibers in the DRG 4 weeks following nerve crush. It is suggested that aberrant NF phosphorylation in DRG neuronal cell bodies from IDPN-treated rats arises secondarily to an alteration in a retrogradely transported 'trophic' signal(s) to the neuron due to the presence of giant axonal swellings. Furthermore, pNFs in atrophic axons may correspond to stationary or slowly moving NFs in the axoplasm.     

Immunohistological,microscopical and neurochemical studies on encephalitides

Summary LEC virus derived from a patient with SSPE was inoculated intracerebrally into beagle puppies. All three animals that received cell-associated virus and two of three animals inoculated with cell-free virus developed encephalitis. None of the other animals inoculated with cell-free and cell-associated wild measles or attenuated measles virus developed any disease. Whereas the animals that received inoculation of measles virus developed an antibody response, the animals with encephalitis due to the SSPE virus remained free of antibodies up to the point of death. These results are consistent with those previously reported by us for calves and lambs.This investigation was supported in part by: Hartford Foundation Grant. Deutsche Forschungsgemeinschaft (AZ: Me 270/8), and grants from Sonderforschungsbereich (33), the Stiftung Volkswagenwerk, the Forschungsmittel des Landes Niedersachsen, and Freudenberg Stiftung.These studies have been carried out in collaboration with Drs. S. L. Notermans and W. F. Tijl of the Department of Neurology, Katholieke Universiteit, Nijmegen, Holland. The results of their neurophysiologic studies will be published elsewhere.     

Immunohistological,microscopical and neurochemical studies on encephalitides

Summary A cytophotometric analysis was made of 238 nerve cells isolated from histological sections obtained from the trigeminal nucleus of a SSPE brain and they were compared with 236 control cells of the same region. The measurements were carried out in the ultraviolet spectrum on fixed, unstained cells and in the visible light spectrum after staining the sections with methylene blue, and according to the Feulgen, ninhydrin Schiff, and Millon methods. The SSPE cells showed an increase in proteins by 40%, an increase in RNA by 34% and a decrease in DNA 29% in comparison with control cells.Computer analysis of the data following the ninhydrin Schiff staining revealed protein increase not only within the cytoplasm, but also within the nucleus and nucleolus.This demonstration of RNA increase in SSPE nerve cells may perhaps be explained by a possible infection with an RNA virus.This paper is dedicated to Prof. Dr. Dr. h. c. W. Scholz for his 80th birthday.     

Immunohistological,microscopical and neurochemical studies on encephalitides

Summary Histochemical and immunohistological studies were carried out with brain cell cultures derived from two patients with subacute sclerosing panencephalitis (SSPE). Morphologically the cultures showed polygonal, probably histiocytic, cell formations and spindle intranuclear inclusion bodies. An increase in intracellular proteins and ribonucleoprotein was demonstrated by histochemical techniques. Fluorescent microscopy revealed an antigen that reacted with measles-specific immunoglobulin in the spindle cells, in the multinucleated giant cells, and in some of the polygonal cells. The findings described resemble the histochemical and immunohistological cell changes in brains of SSPE patients.This investigation was supported by the Deutsche Forschungsgemeinschaft (AZ: Me 270/4), the Stiftung Volkswagenwerk and the Forschungsmitteln des Landes Niedersachen, and also, in part, by the U.S. Public Health Service Research Grant No. R01-06859 to the Wistar Institute from the National Institute of Neurological Diseases and Stroke.     

Immunhistological,microscopical and neurochemical studies on encephalitides

Summary Studies on a case of subacute progressive panencephalitis are presented. The etiology of this disease has been investigated with the aid of virological and immunohistological techniques. Since myxoviruses have been implicated as causative agents, brain tissue has been examined for the presence of antigens, and serum, cerebro spinal fluid, and brain homogenates for antibodies.Measles virus is considered to be the causative agent in this case, because extremely high antibody titers to this virus were found in serum, C.S.F., and brain homogenates. Brain tissue did not reveal infectious virus, hemagglutinating or complement fixing antigens. In sections of brain tissue G globulin, complement and measles antigen were demonstrated by immunofluorescence in nerve and glia cells. In addition, G globulin was present in lymphocytes and plasmocytes of the perivascular infiltrations. These cells are regarded as source of part of the C.S.F. G globulin. The significance of these results is discussed in relation to the etiology and pathogenesis of subacute progressive panencephalitis.This paper is dedicated to Prof. Dr.Dr.h.c.Hugo Spatz for his 80^th birthday.