Reversal of axonal transport at a nerve crush.

Abstract— —We have compared retrograde axonal transport of ³H-labeled protein in normal rat motor and sensory axons, and axons which were injured by a distal ligation of the sciatic nerve. After injection of L-[³H]leucine into the vicinity of the neuron cell bodies, labeled protein was transported into the axons. A premature return of protein towards the cell bodies occurred in the injured axons, which we interpret as a reversal of axonal transport occurring at the site of injury. We estimate that reversal of transport occurred within 1.9–2.4 h of the arrival of labeled protein at the injury, and that the minimum velocity of the subsequent retrograde transport was 112–133 mm day^?1. The ability of the injured axons to reverse transport developed about 0.8 h after making the injury. A large fraction of the orthograde transported protein was returned towards the cell body: it is estimated that by 28 h after labeled protein in sensory axons reached the injury, 46% of the³H-labeled protein originally transported to the injury site had been returned. In intact sensory nerves at this time only 15% of the transported protein had returned. It is suggested that axonal injury produces a sudden increase in the return of newly synthesized protein to the cell body, and that this might serve as a signal for chromatolysis.     

Reversal of Axonal Transport: Similarity of Proteins Transported in Anterograde and Retrograde Directions

Reversal of axonal transport of endogenous labeled protein was studied in intact and injured nerve axons. Nerve crushes were used to collect labeled protein transported in anterograde and retrograde directions in rat sciatic nerve motoneuron axons after administration of L-[35S]methionine to the vicinity of the cell bodies. The collected proteins were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent fluorography. In injured nerves, where the nerves were ligated distally at the time of precursor injection, the polypeptide composition of proteins moving in anterograde and retrograde directions, 9-11 h after precursor injection, was identical, indicating that reversal at a ligature is a nonselective process. In intact nerves, protein moving in the anterograde direction 22-24 h after injection was different from that found 9-11 h after injection, and was also different from protein moving in the retrograde direction 22-24 h after injection. However, protein moving in the retrograde direction 22-24 h after injection was similar to protein moving in the anterograde direction 9-11 h after injection. Thus it appears that the same group of proteins originally transported into the axon are later returned toward the cell body. In intact axons, also, reversal was nonselective, except that one major labeled polypeptide was reduced in amount in the protein moving in the retrograde direction.     

Retrograde Axonal Transport of Phospholipid in Rat Sciatic Nerve

Abstract: Retrograde axonal transport of phospholipid was studied in rat sciatic motoneuron axons by placing collection crushes on the nerve at intervals after injection of [methyl-³H]choline into the lumbosacral spinal cord, and allowing labelled material undergoing anterograde or retrograde movement to accumulate adjacent to the collection crushes. Control experiments showed that the accumulations of label were not a result of local uptake of circulating precursor. The majority of the ³H label was associated with phosphatidylcholine. Accumulation of label at the distal collection crush, representing retrograde transport, was observed subsequent to the anterograde transport of phospholipid. In comparison with previous study on retrograde transport of protein, the following points were noted: (1) onset of retrograde transport occurred at approximately the same time after precursor injection (10–20 h) for both protein and phospholipid; (2) retrograde transport of lipids was more prolonged: maximum retrograde transport occurred later for phospholipid (30 h) than for protein (15–20 h), and declined to half-maximum between 49 and 99 h, compared to a corresponding value of 24–28 h for protein; (3) the proportion of total anterograde-transported activity subsequently undergoing retrograde transport was less in the case of phospholipid, at least over the time interval studied (up to 99 h after precursor injection). The similar times of onset of retrograde transport of phospholipid and protein support the concept of retrograde transport as a recycling mechanism returning to the cell body membrane fragments that were earlier transported into the axon. Coordinated retrograde transport of labelled protein and phospholipid components of the recycled membranes would be predicted. Differences between protein and phospholipid in the subsequent time course and amount of retrograde transport may reflect differences in axonal handling of protein and lipid. Both the more prolonged outflow of labelled lipids from cell body into axon and exchange with a distal pool of unlabelled phospholipid may account for the prolonged time course of retrograde transport of labelled lipid.     

Selective solubilization of high-molecular-mass neurofilament subunit during nerve regeneration

A reduction in neurofilament (NF) protein synthesis and changes in their phosphorylation state are observed during nerve regeneration. To investigate how such metabolic changes are involved in the reorganization of the axonal cytoskeleton, we studied the injury-induced changes in the solubility and axonal transport of NF proteins as well as their phosphorylation states in the rat sciatic nerve. In the control nerve, 15-25% of high-molecular-mass NF subunit (NF-H) was recovered in the 1% Triton-soluble fraction when fractionated in the presence of phosphatase inhibitors. After a complete loss of NF proteins distal to the injury site (70-75 mm from the spinal cord) 1 week after injury, NF-H detected in the regenerating sprouts at 2 weeks or later exhibited higher solubility (>50%) and lower C-terminal phosphorylation level than NF-H in the control nerve. Solubility increase was also apparent with L-[35S]methionine-labeled NF-H that was in transit in the proximal axon at the time of injury. The low-molecular-mass subunit remained in the insoluble fraction in both the normal and the regenerating nerves, indicating that selective solubilization of NF-H rather than total filament disassembly occurs during regeneration.     

AXONAL TRANSPORT OF SELECTED PARTICLE-SPECIFIC ENZYMES IN RAT SCIATIC NERVE IN VIVO AND ITS RESPONSE TO INJURY

Abstract— Orthograde and retrograde axoplasmic transport of selected axonal organelles were examined by monitoring accumulation of enzyme activities residing in various types of particles proximal and distal to a ligature placed on rat sciatic nerve as a function of time after tying. Proximal to the tie, activity of acetylcholinesterase (AChE, EC 3.1.1.7; probably in small endoplasmic reticulum-like particles) accumulated for 2 days; then, during the next 5 days, the accumulation disappeared. Activities of glutamic dehydrogenase (GDH, EC 1.4.1.3) and monoamine oxidase (MAO, EC 1.4.3.4) (both located in mitochondria) accumulated steadily for 7 days. Accumulation of monoamine oxidase activity was more rapid than that of glutamic dehydrogenase during the first day or two. Acid phosphatase (acid P'tase, EC 3.1.3.2; in lysosomes) activity also accumulated throughout the week of observation. Accumulation of all four enzyme activities proximal to the ligature was blocked by nerve crush or subepineurial vinblastine injection 1 cm or more proximal to the site of the tie. Distal to the ligature, AChE activity accumulated early (14 h), and then gradually disappeared in the course of the week. MAO activity also accumulated, with a maximum at 2 days, and no further change thereafter. GDH activity, on the other hand, showed little accumulation during the first 2 days, but did appear in modest amounts at the end of the week. Distal accumulation of acid P'tase kept pace with proximal accumulation for the first day, and continued more slowly for another day, after which there was no further change. This system has been used to study the effects of axonal crush injury upon anterograde and retrograde axoplasmic transport. A tie applied at various times after injury, proximal to the site of injury, was used to show that orthograde transport of AChE was maintained for 1 day after tying, but at 2 days had fallen 50% or more, and within a week was down to 20–25% of control. At 3 days after injury retrograde transport of AChE activity was not different from the control. Orthograde transport of acid P'tase activity was depressed 35% by injury. Retrograde transport of acid P'tase was inhibited more than 50% both at 3 and at 7 days after injury. Transport of the mitochondrial enzymes was not measurably affected.     

Orthograde, Retrograde, and Turnaround Axonal Transport of Dopamine-β-Hydroxylase: Response to Axonal Injury

Reversal of the direction (turnaround) of orthograde axonal transport of dopamine-beta-hydroxylase (DBH) activity was studied at a ligature placed on rat sciatic nerve. DBH was allowed to accumulate at a ligature in vivo for selected intervals, at which time a second ligature was placed proximal to the first and turnaround transport measured just distal to the second tie after incubation in vivo or in vitro. Orthograde accumulation of DBH activity proximal to a ligature peaked at 2 days, and then rapidly decreased as a result of turnaround transport and injury-induced reduction of orthograde transport. Destruction of postganglionic sympathetic axon terminals in vivo with 6 hydroxydopamine resulted in a decrease in orthograde transport similar to that seen after axotomy and turnaround at or proximal to the site of chemical injury. Turnaround transport of DBH in vitro was blocked by incubation in the cold and in the presence of NaCN and vinblastine. Orthograde transport of DBH appeared to reverse direction within a few millimeters of a ligature.     

Changes in lectin binding of lumbar dorsal root ganglia neurons and peripheral axons after sciatic and spinal nerve injury in the rat

Summary The effects of chronic lesions of rat lumbar spinal or sciatic nerves on the binding of Glycine max (soybean) agglutinin to galacto-conjugates, in small-and medium-size primary sensory neurons of the L₄ and L₅ dorsal root ganglia, were examined over a 580-day period. Spinal nerve section resulted in a marked decrease in the population of stained neurons within 7 days. However, despite some retrograde morphological changes triggered by axonal injury, the proportion of stained nerve cells was normalized 180 days postoperatively. This temporary decrease in perikaryal lectin reactivity was initially associated with a marked accumulation of stained material in the nerve, proximal and distal to the site of section, with similar accumulations also being noticeable at each level of injury in sciatic nerves subjected to double ligature. This may reflect the presence of glycocompounds linked to the autolysis of nerve fibers during the phase of retrograde dying-back and Wallerian degeneration. At later stages, stained deposits could be seen scattered along central and peripheral axonal processes of the dorsal root ganglion neurons in the vicinity of the cell body. They may indicate a disturbance in the peripheral turnover of glycoproteins in chronically-transected nerves, with piling up of neuronal products. Sciatic nerve injury caused similar but less severe effects which, except for the L₄ ganglion cells, were rapidly reversible.     

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.     

β,β''-Iminodipropionitrile Impairs Retrograde Axonal Transport

beta,beta'-Iminodipropionitrile (IDPN), a neurotoxin, causes redistribution of neurofilaments in axons followed by the development of proximal axonal swellings and, in chronic intoxication, a distal decrease in axonal caliber. The latter changes are caused by a selective impairment in the slow anterograde axonal transport of neurofilament proteins. To assess the role of retrograde axonal transport in IDPN toxicity, we used [3H]N-succinimidyl propionate ([3H]NSP) to label covalently endogenous axonal proteins in sciatic nerve of the rat and measured the accumulation of radioactively labeled proteins in the cell bodies of motor and sensory neurons over time. IDPN was injected intraneurally 6 h or intraperitoneally 1 day before subepineurial injection of [3H]NSP into the sciatic nerve, and the animals were killed 1, 2, and 7 days after [3H]NSP injection. Neurotoxicity was assessed by electron microscopic observation of the nerves of similarly treated animals. Both intraneural and intraperitoneal injection of IDPN caused an acute reduction in the amount of labeled proteins transported back to the cell bodies. The early appearance of these changes suggests that alterations in retrograde transport may play a role in the production of the neuropathic changes.     

Changes in Rapid Transport of Phospholipids in the Rat Sciatic Nerve During Axonal Regeneration

Abstract: Axonal transport of phospholipids in normal and regenerating sciatic nerve of the rat was studied. At various intervals after axotomy of the right sciatic nerve in the midthigh region and subsequent perineurial sutures of the transected fascicles, a mixture of 60 μCi [Me-^HC]choline and 15 μCi [2-³H]glycerol in the region of the spinal motor neurons of the L5 and L6 segments was injected bilaterally. The amount of radioactive lipid (and in certain cases its distribution in various lipid classes) along the nerve was determined as a function of time. Three days after fascicular suture and 6 h after spinal cord injection of precursors, there was an accumulation of labeled phospholipids and sphingolipids in the transected sciatic nerve in the region immediately proximal to the site of suture. Nine days after, there was a marked increase in the accumulation of radioactivity in the distal segments of the injured nerve, which increased up to 14 days after cutting and disappeared as regeneration proceeded (21–45 days). In all segments of both normal and regenerating nerve fibers, as well as in L5 and L6 spinal cord segments, only phosphatidylcholine and sphingomyelin were labeled with [¹⁴C]choline. These results suggest that the regeneration process in a distal segment of a peripheral neuron, following cutting and fascicular repairing by surgical sutures, is sustained in the first 3 weeks by changes in the amount of phospholipids rapidly transported along the axon towards the site of nerve fiber outgrowth.     

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.     

Tropic 1808基因在大鼠损伤神经组织中的表达

目的观察Tropic 1808基因在大鼠正常和损伤坐骨神经组织中的表达,探讨Tropic 1808基因在周围神经损伤与再生过程中的作用.方法采用地高辛标记的Tropic 1808 cDNA探针、抗大鼠S-100蛋白抗体,以原位杂交和免疫组织化学双重染色法,观察Tropic 1808基因在正常和损伤大鼠坐骨神经组织中的表达.结果免疫组化结果显示,大鼠正常坐骨神经可表达S-100蛋白,但表达量较低;神经损伤后,其远侧端S-100蛋白的表达量明显增加.原位杂交结果显示,大鼠正常坐骨神经组织未见Tropic 1808 mRNA杂交信号;损伤神经的远侧端呈现较强的阳性信号,而且在部分S-100强阳性反应区可见Tropic 1808 mRNA杂交信号.结论 Tropic 1808基因在正常坐骨神经组织中未见表达;坐骨神经损伤后,其远侧端增殖的雪旺氏细胞可表达Tropic 1808 mRNA.提示,Tropic 1808是一种周围神经损伤后特异表达的基因.     

肌肉注射人神经生长因子基因逆轴突传递修复神经损伤的研究进展

神经生长因子(NGF)促进中枢及外周神经系统神经元细胞存活、分化、轴突再生等重要作用已得到临床的广泛证实。目前临床上主要以局部或肌肉注射NGF蛋白的方式对神经系统的损伤进行治疗。但NGF半衰期短、局部应用副作用大、费用昂贵、难以透过血脑屏障等缺点而限制临床应用。长期以来,科研工作者致力于寻求一种理想的途径或方法以克服这一缺陷。随着基因工程技术的飞速发展,研究人员发现通过骨骼肌肌肉注射途径,以非病毒载体介导外源的NGF基因体内表达并逆轴突传递到神经损伤部位,有望解决这一难题。本文将就NGF及受体的基本结构和特性、逆轴突传递的机制、非病毒载体结合骨骼肌肌肉注射的基因治疗等方面进行总结和阐述。     

Changes in neurofilament transport coincide temporally with alterations in the caliber of axons in regenerating motor fibers

The delivery of neurofilaments via axonal transport has been proposed as an important mechanism for regulating axonal caliber. If this hypothesis is correct, alterations in axonal caliber should appear coincident with changes in the delivery of neurofilaments to the axon. The purpose of this study was to determine whether alterations in the caliber of axons in the proximal stumps of transected motor fibers precede, coincide with, or occur substantially later than changes in the delivery of neurofilaments via axonal transport. Between 3 d and 12 wk after crushing the sciatic nerves of 7-wk-old rats, lumbar motor neurons were labeled by the intraspinal injection of [35S]methionine. In neurons labeled between 3 d and 6 wk after axotomy, the relative amount of neurofilament protein in the slow component, as reflected by the ratio of the radioactivities of the 145-kD neurofilament protein to tubulin, was reduced to 30-40% of the control value. Moreover, as determined by immunoreactivity on blots, the amounts of neurofilament protein and tubulin in these nerve fibers were reduced fourfold and twofold, respectively. Thus, changes in the ratio of labeled neurofilament protein to tubulin correlated with comparable changes in the quantities of these proteins in nerve fibers. This decrease in the quantity of neurofilament proteins delivered to axons coincided temporally with reductions in axonal caliber. After regeneration occurred, the delivery of neurofilament proteins returned to pre-axotomy levels (i.e., 8 wk after axotomy), and caliber was restored with resumption of normal age-related radial growth of these axons. Thus, changes in axonal caliber coincided temporally with alterations in the delivery of neurofilament proteins. These results suggest that the majority of neurofilaments in these motor fibers continuously move in the anterograde direction as part of the slow component of axonal transport and that the transport of neurofilaments plays an important role in regulating the caliber of these axons.     

Characterization of neuronal regeneration in the abdominal ganglion of Aplysia californica

The ability of neurons in the abdominal ganglion of Aplysia to regenerate their axons following branchial nerve crush was studied using retrograde staining and intracellular dye injection. The duration of the gill withdrawal reflex (GWR) was measured prior to and following nerve crush. Three days after crushing the nerve, the duration of the gill withdrawal reflex was reduced to 20% of control levels. There was rapid recovery 19 days after crushing the branchial nerve. The GWR duration returned to control levels by postlesion days 25–27. Some of the behavioral recovery can be attributed to axonal regeneration. Regeneration, as evidenced by retrograde staining, was first observed by postlesion day 15. The number of stained neurons in ganglia with crushes increased until postlesion day 33. The number of stained neurons in experimental animals was always less than that of controls (67 ± 9% at postlesion day 56). More axonal regeneration was seen in the hemiganglion ipsilateral to the branchial nerve. Regeneration after 32 days postlesion was 60 ± 5% of controls in the ipsilateral hemiganglion, as opposed to 29 ± 6% in the contralateral hemiganglion. Regeneration of individual neurons was also demonstrated. Identified neuron R2 was shown by intracellular dye injection and electrical stimulation of antidromic action potentials to have an axon in the branchial nerve in all ganglia allowed to regenerate for longer than 32 days. These results indicate that in Aplysia, despite behavioral recovery, complete axonal regeneration does not occur in a large segment of the neurons in the adult central nervous system. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 160–172, 1998     

Axonal transport of synaptic vesicle proteins in the rat optic nerve

The optic nerve, as a part of the central nervous system (CNS), has been used to study axonal transport for decades. The present study has concentrated on the axonal transport of synaptic vesicle proteins in the optic nerve, using the “stop-flow/nerve crush” method. After blocking fast axonal transport, distinct accumulations of synaptic vesicle proteins developed during the first hour after crush-operation and marked increases were observed up to 8 h postoperative. Semiquantitative analysis, using cytofluorimetric scanning (CFS) of immunoincubated sections, revealed that the ratio between distal accumulations (organelles in retrograde transport) and proximal accumulations (organelles in anterograde transport) was much higher (up to 80–90%) for the transmembrane proteins than that for surface adsorbed proteins (only 10–20%). The pattern of axonal transport in the optic nerve was comparable to that in the sciatic nerve. However, clathrin and Rab3a immunoreactivities were accumulated in much lower amounts than that in the sciatic nerve. Most synaptic vesicle proteins were colocalized in the axons proximal to the crush. A differential distribution of synaptobrevin I and II, however, was observed in the optic nerve axons; synaptobrevin I was present in large-sized axons, while synaptobrevin II immunoreactivity was present in most axons, including the large ones. The two isoforms were, thus, partially colocalized. The results demonstrate that (1) cytofluorimetric scanning techniques could be successfully used to study axonal transport not only in peripheral nerves, but also in the CNS; (2) synaptic vesicles are transported with fast axonal transport in this nerve; and (3) some differences were noted compared with the sciatic nerve, especially for Rab3a and clathrin. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 237–250, 1997.     

Regeneration restores some of the altered electrical properties of axotomized bullfrog B-cells

In bullfrog B-type sympathetic neurones axon injury produces substantial changes in somal membrane properties. These include a shortening of action potential afterhyperpolarization (AHP) and an increase in action potential (AP) duration. In the present experiments we compared two injury situations: nerve crush, which was followed by regeneration, and nerve cut, after which regeneration to the original target was prevented, to investigate whether these electrophysiological changes were related to axon regeneration. Both crush and cut injuries produced a similar maximum decrease in AHP duration (to 33 and 30%) by 14 days after axotomy. After nerve crush, AHP duration recovered to within control values by 42 days, while after cut it remained depressed. AHP amplitude decreased to the same extent after nerve crush or cut (to 62 and 58%), but the rate of decrease was slower following crush when compared with cut, and following both types of injury it still remained depressed at 42 and 49 days. Changes in AP duration also took longer to occur following nerve crush, reaching maximal values at 35-42 days, at which time AHP duration had returned to within the normal range. The early reduction in AHP duration and its rapid recovery in regenerating neurones suggests that the current underlying this membrane property is regulated by events associated with axon outgrowth and peripheral reconnection. In contrast, changes in AHP amplitude and AP repolarization appeared to be independent of the occurrence of axon regeneration and remained abnormal at 49 days despite the recovery of AHP duration. These results imply that the electrophysiological changes seen in B-cells following injury are differentially regulated during subsequent regeneration.     

RETROGRADE AXONAL TRANSPORT OF RAPIDLY MIGRATING LABELLED PROTEINS AND GLYCOPROTEINS IN REGENERATING PERIPHERAL NERVES

Abstract— The redistribution of rapidly migrating [³H]leucine-labelled proteins and [³H]fucose-labelled glycoproteins was studied in ligated regenerating hypoglossal and vagus nerves of the rabbit. When regenerating and contralateral hypoglossal nerves were ligated 16 h after labelling of the nerve cell bodies, rapidly migrating proteins and glycoproteins accumulated distal to the ligatures indicating a rapid retrograde transport from the peripheral parts of the nerves within 6 h. The retrograde accumulation of both proteins and glycoproteins was greater on the regenerating side than on the contralateral side at both 1 and 5 weeks after a nerve crush. Labelled proteins and glycoproteins also accumulated proximal to the ligatures, indicating a delayed rapid anterograde phase of axonal transport. The accumulation of this phase was also greater on the regenerating side 1 week after a nerve crush for both labelled proteins and glycoproteins. One week after a crush of the cervical vagus nerve, rapidly migrating proteins and glycoproteins redistributed between he crush zone and a proximal ligature applied 16 h after labelling of the nerve cell bodies. A retrograde accumulation occurred distal to the ligature within 6 h, indicating a rapid retrograde transport from the crush zone.     

The intra-axonal transport of acetylcholine and cholinergic enzymes in rat sciatic nerve during regeneration after various types of axonal trauma

The proximo-distal intra-axonal transport of acetylcholine (ACh) and cholinergic enzymes (choline acetyltransferase, CAT, and ACh-esterase, AChE) in rat regenerating sciatic nerve was studied by accumulation technique. Four types of axonal trauma were performed: freezing with solid CO₂, crushing, ligating the nerve with remaining tight silk ligature, and cutting the nerve. Normal and sham-operated rats were used as controls. One to twenty-nine days later, the nerves were crushed about 15 mm proximal to the trauma. The nerve segment proximal to this crush was dissected out 12 hr later and assayed for ACh-content and enzyme activities. The increase in this segment 12 hr after crushing was taken as an indication of proximo-distal transport in the regenerating nerves. ACh transport did not seem to vary during regeneration as compared to controls. In contrast, the transport of both CAT and AChE was initially markedly depressed. Towards the end of the observation period (29 days), a recovery of CAT-transport occurred in all groups. Recovery of AChE-transport was marked in the freeze and crush groups. In the cut group no recovery was seen and in the ligated group only a small recovery occurred. Thus, in the nerves where regeneration was facilitated by the presence of intact connective tissue sheaths (freezing and crushing) recovery of transport occurred earlier than in cut or ligated nerves.