Impaired axonal regeneration in acrylamide intoxication

Acrylamide is a neurotoxin known to impair regeneration of axons following nerve crush and to produce structurally abnormal regenerating sprouts. To investigate the mechanism of these abnormalities, protein synthesis and fast axonal transport were studied in acrylamide-intoxicated and control rats 2 weeks after sciatic nerve crush. Using an in vitro preparation of sciatic nerve-dorsal root ganglion, there was no difference in ganglion ³H-leucine incorporation between the two groups. In these preparations of sensory axons, as well as in motor axons studied in vivo, a smaller proportion of rapidly transported radioactivity was carried beyond the crush in the acrylamide-regenerating nerves compared to the control-regenerating nerves. Correlative ultrastructural studies demonstrated that this difference reflected the impaired outgrowth of the acrylamide-regenerating nerves, rather than an abnormality in fast transport. The acrylamide-treated sprouts often developed swellings filled with whorls of neurofilaments; in addition, many sprouts ended in massively enlarged growth cones containing membranous organelles. EM autoradiography showed labeled, rapidly transported organelles accumulated in the neurofilamentous whorls, and therefore suggested that these organelles might be “trapped” or impeded in passage through these regions. However, there was no evidence that the growth cones received insufficient amounts of transported protein; in fact, the distended endings were densely labeled and apparently “ballooned” by transported organelles. These results suggest that acrylamide intoxication does not impair regeneration by diminishing the delivery of rapidly transported materials to the growing tip. Rather, the marked distention of the growth cones is interpreted as the morphological consequence of continued delivery of rapidly transported organelles into sprouts unable to utilize them in outgrowth.     

Proteins of fast axonal transport in the regenerating hypoglossal nerve of the rat

The composition of proteins conveyed by fast axonal transport in growing or regenerating axons is different from that of intact, mature axons. Consistent alterations have been observed in several different types of neurons, but adult peripheral axons (rabbit hypoglossal motoneurons) seemed to be exceptions because during their regeneration there was no increased labelling of a 23 kilodalton (kD) protein associated with the growth state. We examined the composition of fast-transported proteins, labelled by application of [35S]methionine to the hypoglossal nuclei, in intact and regenerating hypoglossal nerves of the rat. Using one- and two-dimensional electrophoresis we detected both increases and decreases in the labelling of specific polypeptides during regeneration. In particular, there was increased labelling of a 23 kD polypeptide. Changes were maximal 7 days after axotomy and subsided thereafter, coincident with reinnervation of the tongue. We conclude that hypoglossal axons show the same changes in transported protein composition which are characteristic of the growth state in other axons. Thus, we have strengthened the correlation between the growth state and changes in synthesis of a set of polypeptides of unknown function.     

STUDIES ON AXOPLASMIC TRANSPORT OF INDIVIDUAL PROTEINS: 1–ACETYLCHOLINESTERASE (AChE) IN ACRYLAMIDE NEUROPATHY

Abstract— The axoplasmic transport rate and distribution of acetylcholinesterase (AChe, EC 3.1.1.7) was studied in the sciatic nerves of normal rats and those with a neuropathy due to acrylamide, by measuring the accumulation of the enzyme proximal to single and double ligatures. The single ligature experiments showed that the apparent transport rate of AChE was decreased in acrylamide neuropathy. The double ligature experiments indicated that only 8.1% of AChE was mobile in normal rat sciatic nerve. The mobility of the enzyme in acrylamide-treated rat sciatic nerves was altered to 11.8%. The absolute transport rate of AChE in normal rat sciatic nerve was 567 mm/24 h, and in acrylamide neuropathy it was decreased to 287 mm/24 h.
The amount of AChE activity transported in normal rat sciatic nerve was 2.64 μmol/24 h. The rats with acrylamide neuropathy showed a decrease in the amount of AChE activity moving in the orthograde direction (2.03 μmol/24 h).
The colchicine-binding properties of tubulin protein from sciatic nerves of normal and acrylamide-treated rats were studied. In rats with acrylamide neuropathy, a marked decrease of 75% in tubulin-colchicine binding was observed.     

Axonal degeneration within the tympanal nerve of Schistocerca gregaria

This study describes time course and ultrastructural changes during axonal degeneration of different neurones within the tympanal nerve of the locust Schistocerca gregaria. The tympanal nerve innervates the tergit and pleurit of the first abdominal segment and contains the axons of both sensory and motor neurones. The majority of axons (approx. 97%) belong to several types of sensory neurones: mechano- and chemosensitive hair sensilla, multipolar neurones, campaniform sensilla and sensory cells of a scolopidial organ, the auditory organ. Axons of campaniform sensilla, of auditory sensory cells and of motor neurones are wrapped by glial cell processes. In contrast, the very small and numerous axons (diameter <1 microm) of multipolar neurones and hair sensilla are not separated individually by glia sheets. Distal parts of sensory and motor axons show different reactions to axotomy: 1 week after separation from their somata, distal parts of motor axons are invaded by glial cell processes. This results in fascicles of small axon bundles. In contrast, distal parts of most sensory axons degenerate rapidly after being lesioned. The time to onset of degeneration depends on distance from the lesion site and on the type of sensory neurone. In axons of auditory sensory neurones, ultrastructural signs of degeneration can be found as soon as 2 days after lesion. After complete lysis of distal parts of axons, glial cell processes invade the space formerly occupied by sensory axons. The rapid degeneration of distal auditory axon parts allows it to be excluded that they provide a structure that leads regenerating axons to their targets. Proximal parts of severed axons do not degenerate.     

RAPID AXOPLASMIC TRANSPORT OF PROTEINS IN REGENERATING SENSORY NERVE FIBERS

Abstract— Utilizing an in vitro labeling procedure, the proteins carried by rapid axoplasmic transport in normal and regenerating sensory fibers of the rat sciatic nerve were compared. No statistically significant differences were found when the total amount of transported protein was compared in control and sectioned nerves at times from 2 to 76 days following axotomy. Fractionation of labeled proteins on polyacrylamide slab gels enabled the identification of some 25 individual transported proteins. By this criterion, no differences were detectable in the composition of proteins synthesized in the dorsal root ganglia from which sectioned vs control sciatic nerves project. When the electrophoretic distributions of transported proteins from control and sectioned nerves were compared, significant' differences were observed. The appearance and disappearance of two proteins were temporally related to chromatolytic changes in the nerve cell body. In addition, the composition of transported proteins in undamaged control nerves contralateral to the sectioned nerves exhibited changes which were not observed in either normal control nerves or sectioned nerves. Changes in the composition of transported proteins as a function of time following the onset of chromatolysis may be involved in controlling nerve regeneration in sensory nerve fibers.     

Deletion of p75NTR impairs regeneration of peripheral nerves in mice

AimsAfter peripheral nerve injury, p75NTR was upregulated in Schwann cells of the Wallerian degenerative nerves and in motor neurons but down-regulated in the injured sensory neurons. As p75NTR in neurons mediates signals of both neurotrophins and inhibitory factors, it is regarded as a therapeutic target for the treatment of neurodegeneration. However, its physiological function in the nerve regeneration is not fully understood. In the present study, we aimed to examine the role of p75NTR in the regeneration of peripheral nerves.Main methodsIn p75NTR knockout mice (exon III deletion), the sciatic nerves and facial nerves on one side were crushed and regenerating neurons in the facial nuclei and in the dorsal root ganglia were labelled by Fast Blue. The regenerating fibres in the sciatic nerve were also labelled by an anterograde tracer and by immunohistochemistry.Key findingsThe results showed that the axonal growth of injured axons in the sciatic nerve of p75NTR mutant mice was significantly retarded. The number of regenerated neurons in the dorsal root ganglia and in the facial nuclei in p75NTR mutant mice was significantly reduced. Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR mutant mice.SignificanceOur data suggest that p75NTR plays an important role in the regeneration of injured peripheral nerves.     

Fast axonal transport in motor nerve regeneration.

This report describes the fast transport of [3H]-leucine-labeled proteins in regenerating rat sciatic motor nerves. A normal rate of fast transport (383 +/- 33 mm/day) was present in the regenerating sprouts, as well as in the central stumps. The rapidly transported proteins passed the level of axotomy without impediment, and accumulated in the endings of the regenerating sprouts, as shown by electron microscope autoradiography. In addition, transported proteins accumulated in terminal neuromas. The relative amount of protein-incorporated radioactivity in the crest of transport in the regenerating nerves was increased compared to control nerves. These results are interpreted to suggest that the mechanism of fast transport is the same in regenerating nerves was increased compared to control nerves. These results are interpreted to suggest that the mechanism of fast transport is the same in regenerating sprouts as in normal axons; during regeneration fast transport appears to add newly synthesized materials to the growing tip.     

The effect of age on motor neurone death following axotomy in the mouse.

The ability of mouse motor neurones to survive axotomy during the first month of life was studied. The motor neurones that lie in the dorsolateral columns of spinal segments C7 and C8 and supply the flexor muscles of the forepaw were axotomized by cutting and removing part of the median and ulnar nerves above the elbow. The number and position of cell bodies with axons in these nerves were confirmed by retrograde labelling of the cut axons with horseradish peroxidase. The ability of these neurones to survive axotomy varies with the age of the animal at the time of axotomy. When the axons are sectioned within the first four postnatal days, 80-90% of the cell bodies will die, more than half of this death occurring in less than one week after axotomy. If the animals are one week old at the time the nerves are cut, a significantly smaller number (50%) die (P = 0.013), and the time-course of death is different, with eight to ten days elapsing before half the death has occurred. 40% of the neurones will die if sectioned at two weeks of age, and it is not until four weeks of age that more than 90% of the cells can survive axotomy. We conclude, therefore, that the kinetics of motor neurone death, as well as the final extent of neuronal loss, are affected by the age at which the animal is axotomized.     

Fast axonal transport in motor nerve regeneration

This report describes the fast axonal transport of [³H]-leucine-labeled proteins in regenerating rat sciatic motor nerves. A normal rate of fast transport (383 ± 33 mm/day) was present in the regenerating sprouts, as well as in the central stumps. The rapidly transported proteins passed the level of axotomy without impediment, and accumulated in the endings of the regenerating sprouts, as shown by electron microscope autoradiography. In addition, transported proteins accumulated in terminal neuromas. The relative amount of protein-incorporated radioactivity in the crest of fast transport in the regenerating nerves was increased compared to control nerves. These results are interpreted to suggest that the mechanism of fast transport is the same in regenerating sprouts as in normal axons; during regeneration fast transport appears to add newly synthesized materials to the growing tip.     

A Comparison of the Effects of Acrylamide and Experimental Diabetes on the Retrograde Axonal Transport of Proteins in the Rat Sciatic Nerve: Analysis by Two-Dimensional Polyacrylamide Gel Electrophoresis

Retrogradely transported proteins that accumulated for 18 h distal to a ligature on the sciatic nerve of rats were separated by two-dimensional polyacrylamide gel electrophoresis and visualised with silver stain. A total of 14 proteins were detectable in this way as being retrogradely transported. In rats rendered diabetic 14 days previously by a single intravenous dose of streptozotocin, the accumulation of four of those proteins was noticeably decreased. Administration of acrylamide to a cumulative dose of 150 mg.kg-1 or 350 mg.kg-1 decreased the accumulation of four and eight proteins, respectively. Three of the four protein changes were common to the diabetic and acrylamide-treated animals, and were present before signs of neuropathy could be detected. The results suggest that those alterations may play a causal role in the development of neuropathy.     

Laminin A, B1, and B2 Chain Gene Expression in Transected and Regenerating Nerves: Regulation by Axonal Signals

Abstract: Laminin A, B1, and B2 chain mRNA levels in degenerating and regenerating mouse sciatic nerves were examined using northern blot analysis. In normal intact nerves, B1 and B2 mRNA steady-state levels were high, but when the nerves were crushed, the steady-state levels of B1 and B2 mRNA per milligram wet tissue weight of the distal segments of the nerves increased five- to eightfold over that of control levels as the total RNA and β-actin mRNA levels increased, suggesting that these increases were the consequence of Schwann cell proliferation after axotomy. When the steady-state levels of B1 and B2 mRNA were normalized as the ratio to total RNA or β-actin mRNA levels, however, they drastically decreased to about 20% of the normal nerve levels in the nerve segments distal to both the crush and transaction sites 1 day after injury. In the crushed nerves, B1 and B2 mRNA levels gradually increased as the regenerating nerves arrived at the distal segments and reestablished normal axon–Schwann cell contact, and then returned to normal levels on the 21 st day. In the transected nerves, where Schwann cells continued to be disconnected from axons, both B1 and B2 mRNA levels remained low. Cultured Schwann cells expressed detectable levels of B1 and B2 chain mRNA which significantly increased when the cells were cocultured with sensory neurons. However, mRNA for A chain was not detectable in the normal, axotomized nerves or in cultured Schwann cells. These data indicate that Schwann cells express laminin B1 and B2 chain mRNA that are up-regulated by axonal or neuronal contact, but they do not express A chain mRNA.     

Functional Recovery of Regenerating Motor Axons is Delayed in Mice Heterozygously Deficient for the Myelin Protein P0 Gene

Mice with a heterozygous knock-out of the myelin protein P₀ gene (P₀+/?) develop a neuropathy similar to human Charcot–Marie–Tooth disease. They are indistinguishable from wild-types (WT) at birth and develop a slowly progressing demyelinating neuropathy. The aim of this study was to investigate whether the regeneration capacity of early symptomatic P₀+/? is impaired as compared to age matched WT. Right sciatic nerves were lesioned at the thigh in 7–8 months old mice. Tibial motor axons at ankle were investigated by conventional motor conduction studies and axon excitability studies using threshold tracking. To evaluate regeneration we monitored the recovery of motor function after crush, and then compared the fiber distribution by histology. The overall motor performance was investigated using Rotor-Rod. P₀+/? had reduced compound motor action potential amplitudes and thinner myelinated axons with only a borderline impairment in conduction and Rotor-Rod. Plantar muscle reinnervation occurred within 21 days in all mice. Shortly after reinnervation the conduction of P₀+/? regenerated axons was markedly slower than WT, however, this difference decayed with time. Nevertheless, after 1 month, regenerated P₀+/? axons had longer strength-duration time constant, larger threshold changes during hyperpolarizing electrotonus and longer relative refractory period. Their performance at Rotor-Rod remained also markedly impaired. In contrast, the number and diameter distribution of regenerating myelinated fibers became similar to regenerated WT. Our data suggest that in the presence of heterozygously P₀ deficient Schwann cells, regenerating motor axons retain their ability to reinnervate their targets and remyelinate, though their functional recovery is delayed.     

Increased regeneration rate in peripheral nerve axons following double lesions: Enhancement of the conditioning lesion phenomenon

The rate of regeneration of rat sciatic nerve sensory axons was measured using the pinch-reflex test method, and confirmed by studying the transport of labelled protein into the regenerating axons. For nerves receiving a single test crush lesion the rate was 4.02 ± 0.03 (SE) mm/day. For nerves with a conditioning lesion made at the knee seven days prior to the test lesion at the hip the rate was 5.73 ± 0.06 mm/day, and for nerves where both conditioning and test lesions were made at the same site (hip or knee) but separated by seven days, the rate was 6.76 ± 0.04 mm/day, a 68% increase over the normal rate, showing that pre-degeneration of the nerve distal to the site of the test lesion increases the rate of regeneration. It is concluded that the rate of axon regeneration can be influenced by the environment through which the regenerating axons grow.     

Acrylamide-Induced Increases in Deposition of Axonally Transported Glycoproteins in Rat Sciatic Nerve

The axonal transport of proteins, glycoproteins, and gangliosides in sensory neurons of the sciatic nerve was examined in adult rats exposed to acrylamide via intraperitoneal injection (40 mg/kg of body weight/day for nine consecutive days). The L5 dorsal root ganglion was injected with either [35S]methionine to label proteins or [3H]glucosamine to label, more specifically, glycoproteins and gangliosides. At times ranging from 2 to 6 h later, the sciatic nerve and injected ganglion were excised and radioactivity in consecutive 5-mm segments determined. In both control and acrylamide-treated animals, outflow profiles of [35S]methionine-labeled proteins showed a well defined crest which moved down the nerve at a rate of approximately 340 mm/day. Similar outflow profiles and transport rates were seen for [3H]glucosamine-labeled glycoproteins in control animals. However, in animals treated with acrylamide, the crest of transported labeled glycoprotein was severely attenuated as it moved down the nerve. This finding suggests that in acrylamide-treated animals, axonally transported glycoproteins were preferentially transferred (unloaded or exchanged against unlabeled molecules) from the transport vector to stationary axonal structures. We also examined the clearance of axonally transported glycoproteins distal to a ligature on the nerve. The observed impairment of clearance in acrylamide-treated animals relative to controls is supportive of the above hypothesis. Acrylamide may directly affect the mechanism by which axonally transported material is unloaded from the transport vector. Alternatively, the increased rate of unloading might reflect an acrylamide-induced increase in the demand for axonally transported material.     

Consequences of slow Wallerian degeneration for regenerating motor and sensory axons.

The time course of Wallerian degeneration in the tibial and saphenous nerves was compared in Balb/c mice and mice of the C57BL/Ola strain (Lunn et al., 1989). Axons, particularly myelinated ones, in nerves of C57BL/Ola mice are very slow to degenerate, many still being present 3 weeks after axotomy. Nuclear numbers in the distal stump peak much later and do not reach the levels found in Balb/c mice; debris removal is very slow, and Schwann cell numbers only rise slightly above normal levels in the long term. Regeneration was investigated electrophysiologically and by electron microscopy (EM). Myelinated sensory axons regenerated slowly and incompletely compared with motor ones which were only slightly slowed after nerve crush (although they were significantly hindered after nerve section). Total myelinated axon numbers were still some 20% less than normal even after 200 days in sensory nerves. Even after all axons had degenerated in C57BL/Ola mice, regeneration rates of neither myelinated nor unmyelinated sensory axons reached those achieved in Balb/c mice. It is concluded that while regeneration can eventually proceed slowly when Wallerian degeneration is much delayed, the usual rapid time course of Wallerian degeneration is necessary if axons, particularly sensory ones, are to regenerate at optimal rates and to maximum extent. While local obstruction to axon growth probably impedes the early phase of regeneration in C57BL/Ola mice, it seems possible that a lack of adequate early signals affects regeneration permanently by minimizing the cell body reaction to injury.     

DLK: the "preconditioning" signal for axon regeneration?

In this issue of Neuron, Shin et?al. (2012) show that the dual leucine zipper kinase (DLK) is responsible for the retrograde injury signal in spinal sensory and motor neurons. DLK is required for the accelerated regeneration seen after axotomy and for the improved regeneration seen after a conditioning injury. DLK KO axons have severely reduced axon regeneration in?vivo.     

Org.2766 improves functional and electrophysiological aspects of regenerating sciatic nerve in the rat

The beneficial effect of short-term (8 days) melanocortin therapy on regenerating peripheral nerves is demonstrated using functional and electrophysiological tests. Following a crush lesion of the rat sciatic nerve, recovery of sensory function is monitored by assessing the responsiveness of the rat to a small electric current applied to the footsole. Recovery of motor function is assessed by means of an analysis of walking patterns. Normalization of the walking pattern reflects reinnervation of different muscle groups. The motor and H-reflex related sensory nerve conduction velocity of the regenerated nerves are longitudinally investigated in the same rats in which the recovery of motor and sensory function had been assessed previously. Functional tests show an enhanced recovery under melanocortin therapy, but in the end both saline- and melanocortin-treated rats show 100% recovery. However, when compared to the contralateral sciatic nerve, in the peptide-treated animals motor nerve conduction in the regenerated nerves has fully recovered after about 90 days following the crush lesion and the sensory conduction after about 120 days, whereas in the saline-treated rats a deficit of 20-40% in both motor and sensory conduction remains. This difference is observed even 214 days following crush.     

Pathfinding, target recognition, and synapse formation of single regenerating fibers in the adult grasshopper Schistocerca gregaria

After lesion of the peripheral tympanal nerve of the adult locust (Schistocerca gregaria), sensory axons regenerate into their original target areas. We examined the individual behavior of single regenerating auditory afferents during pathway and target selection by intracellularly recording and labeling them at different times postlesion. During axotomy, spontaneous activity is not increased in either the distal or proximal part of the cells. Stimulus response properties of lesioned cells with or without regenerating axons are not influenced. Surprisingly, only 55% of sensory neurons regenerate through the lesion site and often give rise to more than one axonal fiber. Within the central nervous system, 70% of regenerated axons consistently follow an incorrect pathway to reach the correct target region. Often, one of two processes formed by a cell chooses the correct pathway, and the other the incorrect one. In the target region, regenerated axons reconstitute somatotopically ordered projections and form synapses that resemble those of intact fibers in number and structure. The regeneration process does not induce a detectable expression of antigens that are known to be expressed during neural development in these neurons. Our study clearly demonstrates that precise synaptic regeneration is possible in adult animals within a completely differentiated central nervous system, although pathfinding and formation of arborizations are disturbed in a particular and probably system-related manner. The results strongly suggest that accurate pathfinding is unlikely to be a decisive factor in target area recognition and synaptogenesis.     

Acrylamide impairs fast and slow axonal transport in rat optic system

Effects of single and repeated doses of acrylamide on fast and slow axonal transport of radio labeled proteins following the injection of L-[4,5-³H] leucine have been studied in the optic system of male Sprague-Dawley rats. A single dose of acrylamide (100 mg/kg) had no effect, but higher concentrations (200–300 mg/kg) altered the distribution of fast axonally transported materials in optic nerves and optic tracts. Repeated doses of acrylamide (30 mg/kg/day, 5 days per week for 4 weeks) produced degeneration of tibial nerves but spared optic nerves and optic tracts. Fast axonal transport rate in optic axons was reduced by 50% (reduced to 4 mm/h from 8 mm/h) in acrylamide treated animals. Acrylamide also slowed the velocity of slow axonal transport of labeled proteins in optic axons to 1.0 mm per day from 1.3 mm per day. Since acrylamide impaired the rate of both fast and slow axonal transport in the absence of overt morphological damage, it can be concluded that deficit in axonal transport is an important factor in the pathogenesis of axonal degeneration in acrylamide neuropathy.     

Diverse mechanisms for assembly of branchiomeric nerves

The formation of branchiomeric nerves (cranial nerves V, VII, IX and X) from their sensory, motor and glial components is poorly understood. The current model for cranial nerve formation is based on the Vth nerve, in which sensory afferents are formed first and must enter the hindbrain in order for the motor efferents to exit. Using transgenic zebrafish lines to discriminate between motor neurons, sensory neurons and peripheral glia, we show that this model does not apply to the remaining three branchiomeric nerves. For these nerves, the motor efferents form prior to the sensory afferents, and their pathfinding show no dependence on sensory axons, as ablation of cranial sensory neurons by ngn1 knockdown had no effect. In contrast, the sensory limbs of the IXth and Xth nerves (but not the Vth or VIIth) were misrouted in gli1 mutants, which lack hindbrain bmn, suggesting that the motor efferents are crucial for appropriate sensory axon projection in some branchiomeric nerves. For all four nerves, peripheral glia were the intermediate component added and had a critical role in nerve integrity but not in axon guidance, as foxd3 null mutants lacking peripheral glia exhibited defasciculation of gVII, gIX, and gX axons. The bmn efferents were unaffected in these mutants. These data demonstrate that multiple mechanisms underlie formation of the four branchiomeric nerves. For the Vth, sensory axons initiate nerve formation, for the VIIth the sensory and motor limbs are independent, and for the IXth/Xth the motor axons initiate formation. In all cases the glia are patterned by the initiating set of axons and are needed to maintain axon fasciculation. These results reveal that coordinated interactions between the three neural cell types in branchiomeric nerves differ according to their axial position.