Cell Body Reaction Research Articles

Reparative mechanisms in the cerebellar cortex

In the adult brain, different neuronal populations display different degrees of plasticity. Here, we describe the highly different plastic properties of inferior olivary neurones and Purkinje cells. Olivary neurones show a basal expression of growth-associated proteins, such as GAP-43 and Krox24/EGR-1, and remarkable remodelling capabilities of their terminal arbour. They also regenerate their transected neurites into growth-permissive territories and may reinnervate the lost target. Sprouting and regrowing olivary axons are able to follow specific positional information cues to establish new connections according to the original projection map. In addition, they set a strong cell body reaction to injury, which in specific olivary subsets is regulated by inhibitory target-derived cues. In contrast, Purkinje cells do not have a constitutive level of growth-associated genes, and show little cell body reaction, no axonal regeneration after axotomy, and weak sprouting capabilities. Block of myelin-derived signals allows terminal arbour remodelling, but not regeneration, while selective over-expression of GAP-43 induces axonal sprouting along the axonal surface and at the level of the lesion. We suggest that the high constitutive intrinsic plasticity of the inferior olive neurones allows their terminal arbour to sustain the activity-dependent ongoing competition with the parallel fibres in order to maintain the post-synaptic territory, and possibly underlies mechanisms of learning and memory. Such a plasticity is used also as a reparative mechanism following axotomy. In contrast, in Purkinje cells, poor intrinsic regenerative capabilities and myelin-derived signals stabilise the mature connectivity and prevent axonal regeneration after lesion.

Neuro-regeneration: plasticity for repair and adaptation.

Specificity of connectivity is essential to nervous system function. It is determined by intrinsic programmes of gene expression that define neuronal phenotypes, and by activity-dependent mechanisms. Neuro-regeneration in the adult may involve re-activation of growth programmes within the constraints of neuron-type specific phenotypes. Lesion-induced re-induction of an axonal growth mode in adult neurons correlates with a vigorous cell body reaction that can also lead to apoptotic cell death. Directing the cell body reaction towards regeneration is a major goal towards improving regeneration. Extrinsic factors that prevent axonal regeneration in the adult CNS of higher vertebrates include inhibitory components on the surface of oligodendrocytes and CNS myelin, and proteoglycans associated with scar material; grafts of certain glial cells can promote regeneration. Local nerve sprouting and synaptic plasticity can produce dramatic functional adaptation to lesions in the adult and greatly enhance the impact of the partial regeneration of lesioned axons; nerve sprouting is promoted by diffusible and contact-mediated extrinsic mechanisms, and by intrinsic neuronal components. As a result of recent discoveries, significant progress in promoting axonal regeneration and recovery of function in the adult can be anticipated.

Axotomy-induced alterations in the red nucleus revealed by monoclonal antibody, Py, following a low thoracic spinal cord lesion in the adult rat.

The monoclonal antibody Py was previously developed as a tool for the identification of subpopulations of hippocampal neurons. Here, the differential distribution of Py immunoreactivity in the mid-brain is described showing that Py also serves as a useful marker for other populations of neurons. Medium to strong immunoreactivity was observed in the cell body and dendrites of neurons of the oculomotor nucleus, superior colliculus and substantia gelatinosa reticulata. However, particularly intense Py-immunoreactivity was identified in the magnocellular neurons in the caudal pole of the red nucleus. Unilateral transection of the rubrospinal tract at Th9-10 induced a marked reduction of Py immunoreactivity in the ventrolateral territory of the caudal pole of the axotomised red nucleus. A small but statistically significant reduction of Py-immunoreactivity was first seen at 7 days after surgery and a maximal loss of immunoreactivity (reduced to 66% of control levels) was observed by 21 days after surgery. Immunoreactivity in the axotomised red nucleus was reduced for the duration of the experiment but at the longer survival times studied (3 and 6 months) a small degrees of recovery of staining was observed in small-medium diameter atrophic neurons. These results indicate that monoclonal antibody Py, may be a useful novel and sensitive tool for investigating the cell body reaction of particular populations of axotomised CNS neurons following spinal cord injury.

Reductions in Motoneuronal Neurofilament Synthesis by Successive Axotomies: A Possible Explanation for the Conditioning Lesion Effect on Axon Regeneration

Axons regenerate more rapidly after a test lesion if they received a conditioning lesion. Previous work suggests that the cell body reaction to injury is responsible for this conditioning lesion effect. Here we examined the effects of the second, test lesion on the expression of the major cytoskeletal proteins, tubulin, actin, and neurofilament proteins. Using 2D-SDS–PAGE to separate these cytoskeletal proteins synthesized in the facial nucleus, along within situhybridization and RNA blotting to measure corresponding mRNA levels, we found that previous conditioning had little effect on actin or tubulin responses to a test lesion, but resulted in a further decrease in neurofilament synthesis. Immunocytochemistry and electron microscopy revealed a greater loss of neurofilaments from the proximal conditioned axons, and axonal shrinkage. We suggest that the reduction in neurofilaments in the proximal axons of conditioned neurons reduces interference with tubulin transport. This may allow more tubulin to be transported more rapidly into the growing axon, to support the faster elongation rate of conditioned axons following a test lesion.

NGF-dependent and NGF-independent recovery of sympathetic function after chemical sympathectomy with 6-hydroxydopamine.

To help clarify the distinction between the nerve growth factor (NGF)-dependent collateral sprouting of sympathetic nerves and their NGF-independent regeneration after crush, we used 6-hydroxydopamine (OHDA) to destroy the sympathetic terminals in adult rats; this leaves the axons damage-free. Would recovery occur by regeneration and/or collateral sprouting? A single 6-OHDA injection abolished the sympathetic pilomotor field revealed by electrical stimulation of a cutaneous nerve. Recovery began within 2 days, and by 20 days the field was reestablished. If the field was "isolated" by adjacent denervations at the time of 6-OHDA treatment, the recovering pilomotor field expanded extensively into the surrounding territory. In the presence of anti-NGF, however, the pilomotor field expansion ceased at about 60% of its former size; if anti-NGF treatment was discontinued, expansion recommended and extended into the surrounding skin. We suggest that the latter, NGF-dependent, growth phase corresponds to collateral sprouting, and the initial NGF-independent one to regeneration. After simple nerve crush, however, such regeneration can triple the normal sympathetic field size. This difference between crush- and 6-OHDA-induced regeneration might relate to the "cell body reaction" (CBR); the CBR is reduced with increasing distance of the lesion and is undetectable after a 6-OHDA lesion. Since the CBR and the vigor of regeneration are both increased by repeated axonal injury, we tested the effects of multiple 6-OHDA treatments; this significantly increased the initial NGF-independent expansion. We hypothesize that regeneration is regulated largely by mechanisms associated with the CBR, and that neurotrophin-dependent collateral sprouting occurs independently of these.

Differential expression of the electrophysiological responses to axotomy in bullfrog sympathetic neurons following nerve injury at 15°C

In frogs maintained at 15°C, there is a partial failure of the cell body reaction (CBR) to axotomy. We investigated, in the sympathetic B-cells of the bullfrog, the effects of axotomy at 15°C on the changes in electrophysiological properties which we found previously to follow axotomy at 21°C. While the increase in action potential (AP) duration was delayed by about 14 days, it increased by the normal amount. In contrast, the reduction in after-hyperpolarization (AHP) duration was both delayed and attenuated. These results show that there is a differential expression of these two components of the CBR, with changes in AHP duration more sensitive to lowered temperature. In attempts to determine if the effect of reduced temperature was on the signalling of axotomy or on the cell body response to the signal, we performed experiments where the 15°C frogs were maintained at 21°C for 7 days, immediately after axotomy, or after various delays. These results suggest that at 15°C, there is an increased delay in signalling that the injury has occurred, but we were not able to show conclusively that maintained expression of the CBR requires an ambient temperature of 21°C. This study emphasizes that the CBR to axotomy is not a unitary response to a single signal, but can be dissected, by lowered ambient temperature, into differentially regulated components.

Acceleration of axonal outgrowth in rat sciatic nerve at one week after axotomy

Following injury of sciatic motor axons in the rat, the rate of axonal outgrowth is faster if there has been a prior "conditioning" axotomy. The acceleration of outgrowth is due to an acceleration of SCb, the rate [slow (SC)] component of axonal transport that carries cytomatrix proteins; this occurs throughout the axon by 7 days after the conditioning axotomy (Jacob and McQuarrie, 1991a, J. Neurobiol. 22:570-583). To further characterize the conditioning lesion effect (CLE), it is important to know (1) the minimum effective conditioning interval (time between conditioning and testing lesions), (2) whether the cell body reaction is required, and (3) whether outgrowth accelerates after a single axotomy. Outgrowth distances were measured by radiolabeling all newly synthesized neuronal proteins and detecting those carried to growth cones by fast axonal transport. When the conditioning and testing lesions were made simultaneously (0 day conditioning interval), there was no CLE. With a conditioning interval of 3 days, there was a shortening of the initial delay (before the onset of outgrowth) without a change in outgrowth rate. With conditioning intervals of 7, 14, and 21 days, the rates of outgrowth were increased by 8%, 22%, and 11%, respectively. To determine whether the cell body reaction to axotomy is necessary for the CLE, a nonaxotomizing stimulus to axonal growth (partial denervation) was used in place of a conditioning axotomy. This had no effect on the rate of outgrowth from a testing lesion made 14 days later. Finally, we examined the possibility that outgrowth accelerates after a single lesion. Outgrowth was faster at 6-9 days after axotomy than at 3-6 days (p < 0.001), and accelerated further at 9-12 days (p < 0.001). We conclude that (1) the shortest effective conditioning interval is 3 days; (2) the cell body reaction is necessary for the CLE; (3) axonal outgrowth from a single axotomy accelerates in concert with the anabolic phase of the cell body reaction. The SCb motor is, in turn, upregulated by this reaction. This suggests that the SCb motor responds to a fast-transported signal that is a product of the cell body reaction.

Regeneration of an adult peripheral nerve preparation in culture.

The methods used to maintain the vagus nerve from the adult rat in culture and how regeneration is studied in this preparation are described. A hypothesis is presented on the triggering of the cell body reaction. It is suggested that this reaction is initiated by proteins synthesized in nonneuronal cells at the site of a nerve lesion. These proteins, referred to as regenerins, reach the nerve cell body by retrograde axonal transport, where they initiate the regeneration process.

Does insulin-like growth factor I (IGF-1) trigger the cell body reaction in the rat sciatic nerve?

Regeneration was measured after the infliction of a crush lesion on rat sciatic nerves which 4 days earlier had been subjected to a distal conditioning transaction. Such nerves exhibited an increased outgrowth of nerve fibers as compared to nerves subjected to a single crush lesion. This increased outgrowth could be prevented, if the nerve was locally perfused around the site of the transection during the 4 days conditioning interval, with cycloheximide, actinomycin D and vinblastine, inhibitors of protein-, RNA-synthesis and retrograde axonal transport, respectively. The inhibitory effect of cycloheximide could be overcome by simultaneous perfusion with insulin-like growth factor I (IGF-1). The results suggest that proteins including IGF-1 which are synthesised locally around a nerve lesion and then transported retrogradely could trigger regenerative events in the neuronal cell body.

Response of facial and rubrospinal neurons to axotomy: changes in mRNA expression for cytoskeletal proteins and GAP-43

Neurons confined within the mammalian CNS usually do not regenerate after axonal injury, while axonal regeneration is the rule in the PNS. It has been hypothesized that this may be related to differences in the microenvironment of the PNS versus CNS and to differences in the neuronal response to injury. In order to test the latter hypothesis, we compared changes in gene expression after axotomy in two populations of neurons: rat facial motoneurons and rat rubrospinal neurons. In situ hybridization with cDNA probes for the medium and light neurofilament protein revealed a reduced mRNA content in both facial and rubrospinal neurons at all times investigated (i.e., 1, 2, and 3 weeks after axotomy). On the other hand, mRNAs for actin and tubulin were increased in both neuronal populations during the first week after axotomy. While this increase was sustained in facial motoneurons for several weeks, total tubulin mRNA and actin mRNA were decreased in rubrospinal neurons at 2 and 3 weeks after axotomy, coincident with their atrophy. The developmentally regulated T alpha 1 tubulin mRNA, which was previously shown to be reexpressed in facial motoneurons after axotomy, was elevated severalfold in axotomized rubrospinal neurons, and increased levels persisted in some rubrospinal neurons as late as 7 weeks after axotomy. Similarly, the developmentally regulated GAP-43 mRNA increased in both axotomized facial and rubrospinal neurons, and increased levels were sustained in some axotomized rubrospinal neurons for at least 7 weeks. The response of rubrospinal neurons to axotomy in the cervical spinal cord is, in the first week, qualitatively similar to the response of facial motoneurons. However, by 2 weeks after axotomy there is a generalized reduction in mRNA levels for all three cytoskeletal proteins that is associated with neuronal atrophy. During this period, mRNA levels for the two specific markers of the growth state, T alpha 1 tubulin and GAP-43, remain elevated. Thus, axotomy of rubrospinal neurons appears to set in motion two independent events. First, an axotomy signal initiates a cell-body reaction similar to that of PNS neurons, including increased mRNA levels for T alpha 1 tubulin and GAP-43. Later, a generalized cellular atrophy and decrease in mRNA levels occur without reversing the specific responses of T alpha 1 and GAP-43 to axotomy. We conclude that the failure of rubrospinal neurons to regenerate is not due to a failure to initiate gene-expression changes characteristic of regenerating peripheral neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

The initial period of peripheral nerve regeneration and the importance of the local environment for the conditioning lesion effect

The aim of this study was to investigate the early period of neurite outgrowth in the regenerating rat sciatic nerve and to determine if the non-neuronal cells were important for the conditioning lesion effect. Regeneration distance was evaluated with the pinch-reflex test 6 h to 5 days after a test crush lesion. The regeneration velocity accelerated during approximately 3 days, whereupon outgrowth continued with a constant velocity. In unconditioned nerves the initial delay was 2.8 h and the constant rate of regeneration was 3.2 mm/day. In nerves with a distal conditioning lesion the initial delay was 2.4 h and the rate of regeneration increased by 52%. When the test crush was applied at the same place as the conditioning crush the initial delay was 1.9 h and the rate of regeneration increased by 61%. The conditioning lesion effect was not influenced by the distance between the cell body and the conditioning crush lesion. Furthermore, the conditioning lesion effect could not be expressed if conditioned axons grew into a freeze injured nerve section. Incorporation of [ 3H]thymidine increased in the regenerating nerve segment. The increase occurred earlier if this segment had been subjected to a conditioning crush lesion. The results of these experiments showed that peripheral neurites start to regenerate within a few hours after an injury, suggesting that growth cone formation is independent of the cell body reaction. A conditioning crush lesion increases the regeneration velocity and its acceleration, and the conditioning lesion effect cannot be expressed in the absence of living Schwann and other non-neuronal cells.

Effect of conditioning lesions on regeneration of goldfish optic axons: time course of the cell body reaction to axotomy

The time course of the cell body reaction to axotomy was determined in goldfish retinal ganglion cells by measuring cell body size and the amount of labelled protein conveyed by fast axonal transport to the optic tectum, both of which increase during regeneration of the optic axons. Following a single testing lesion of the optic nerve, the regenerating axons began to innervate the tectum at about 14 days after the lesion and the cell body reaction began to decline 2–3 weeks thereafter. If the testing lesion had been preceded by a conditioning lesion 2 weeks earlier, the time for the regenerating axons to arrive in the tectum was reduced by a week, because of the faster rate of axonal outgrowth, but the interval between their arrival and the beginning of the decline of the cell body reaction was unchanged. Electrophysiological measurements showed that synaptic transmission was initiated earlier when the axons reached the tectum faster. These results indicate that the mechanisms initiating the recovery of cell body metabolism are independent of those governing the rate of axonal outgrowth. The recovery of the cell body may begin shortly after synapses are established, regardless of whether they are correctly or incorrectly targetted. The correctness of the target may be a separate factor in determining how rapidly and completely the cell body recovers.

The influence of regeneration and nerve growth factor on the neuronal cell body reaction to injury.

The consequence of neuronal regeneration on the affected cell body has not been well documented previously. The long-term effects of either successful peripheral nerve (sciatic) regeneration or exogenously administered nerve growth factor (NGF) on dorsal root ganglion (DRG) neurons were determined. The degree of neuronal death and changes in neuronal size were measured after various injuries and treatments. The regenerative influence of the transected, distal sciatic-nerve segment on the neuronal cell body was examined under various standardized conditions (e.g. crush, transection followed by immediate epineurial anastomosis or transection with capping of the proximal nerve stump). Neuronal death was greatest in smaller neurons with diameters between 16 and 28 microns. The data showed no difference in the degree of neuronal death between the crush injury and the anastomosis (both able to regenerate). However, the capped, proximal nerve (regeneration prevented) had a significantly higher incidence of neuronal death and less complete recovery from the early neuronal atrophy, which was initially observed in all three groups. The long-term effect on neuronal survival of transient NGF administration (three weeks) at the site of injury demonstrated partial protection by a decrease (55%) in the neuronal loss nine weeks after injury compared to controls. Either the distal nerve segment during regeneration or exogenously applied NGF is capable of mitigating the long-term effects of axotomy in the DRG neuronal cell body.

Expression of growth-associated proteins (GAPs) in injured sensory axons of frogs acclimatized to 15 °C and 25 °C

Labelled proteins conveyed by fast axonal transport into the sensory axons of frog sciatic nerve following axotomy have been studied by 2D gel electrophoresis. Previous work showed that in frogs acclimatized to 25 °C a cell body reaction occurs, along with regeneration of axons to their targets. In contrast, frogs acclimatized to 15 °C showed no cell body reaction and though regeneration began, it stalled after approximately 35 days. We found that axotomy at 25 °C was followed by an increase in transport of specific labelled proteins corresponding to growth-associated proteins (GAPs) identified in other regenerating systems. Surprisingly, axotomy at 15 °C also induced a similar increase, though with a slower onset, so that the highest levels of expression of GAPs occurred during the time when the axons had stalled. We conclude that sensory neurons in 15 °C frogs do detect that their axons have been injured, as shown by their ability to increase GAP synthesis. Slow and limited axonal growth is possible during a period when GAP synthesis is low compared to levels in rapidly regenerating nerves, but even when the ability to produce GAPs increases, this alone is not sufficient to sustain regeneration in the absence of other components of the cell body reaction to injury.

Proteins of fast axonal transport in regenerating rat sciatic sensory axons: A conditioning lesion does not amplify the characteristic response to axotomy

The regeneration rate of axons following injury can be influenced by the past history of the neuron: if an axon received a “conditioning” lesion its regeneration rate from a subsequent “test” lesion is increased. Because regeneration is associated with the increased synthesis and fast axonal transport of growth-associated proteins of unknown functions, we investigated whether the increased regeneration rate (to 81%) seen in conditioned rat sciatic sensory axons following a test lesion was associated with an amplification of the increase in growth-associated protein expression. The test lesion did not produce any amplification of the increase in growth-associated protein expression resulting from the preceding conditioning lesion. We conclude that this aspect of the cell body reaction to axotomy is not involved in the conditioning lesion effect on regeneration rate, and, as a corollary, it appears that fast-transported proteins whose expression alters after axotomy are not involved in the regulation of the regeneration rate.

Neural activity in the regenerating optic nerve of the goldfish.

1. Retinal ganglion cells of one eye were axotomized in goldfish either by sectioning the contralateral optic tract or by ablating the contralateral lobe of the optic tectum. Between 2 and 40 days later, multiunit activity in response to diffuse light flashes was recorded from the axotomized and normal optic nerves, and from the optic tectum. 2. Two days after tract section, the amplitude of the integrated multiunit response of the axotomized nerve was normal. By 16 days it had fallen to 15% of control values, at which time visual responses carried by the regenerating tract were first recorded in tectum. Activity in the axotomized nerve then recovered gradually. 3. After ablation of one tectal lobe, multiunit responses in the axotomized nerve had not recovered by 40 days. 4. Integrated spontaneous activity in the axotomized nerve was depressed with a similar time course to the depression of light-evoked activity, both after tract section and tectal ablation. 5. Retinal ganglion cell nuclear size, a morphological indicator of the cell body reaction, varied inversely with evoked activity, whether axotomy was by tract section or by tectal ablation. 6. Electrically evoked compound action potentials of normal amplitude could be recorded from an axotomized nerve despite depressed responses to light flashes. 7. It is concluded that optic nerve axotomy in goldfish reduces the number of optic fibres carrying impulses and/or the frequency of their discharge. The effect is closely linked to morphological changes occurring in the retinal ganglion cell bodies. Recovery of impulse activity and morphology depends upon the regenerating optic fibres innervating an appropriate target.

Acrylamide Neuropathy: Changes in the Composition of Proteins of Fast Axonal Transport Resemble Those Observed in Regenerating Axons

Proteins conveyed by fast axonal transport along sensory and motor axons of rat sciatic nerve were labelled with L-[35S]methionine and characterized by one- and two-dimensional electrophoresis on polyacrylamide gels, followed by fluorography. Nerves from normal or bis-acrylamide-treated animals were compared with nerves from acrylamide-treated animals and nerves regenerating after a crush axotomy. In both sensory and motor axons significant changes in the pattern of labelled bands on one-dimensional gels occurred after 10 days of acrylamide treatment (50 mg/kg daily, i.p.). These changes resembled those seen in regenerating axons, but were less pronounced. No changes were detectable after shorter periods of treatment, even though the onset of the neuropathy, assessed by a behavioral test, occurred on days 4-6 of treatment. Two-dimensional separations of the labelled proteins revealed increased labelling of growth-associated protein 43 in acrylamide-treated animals, but again this was less pronounced than in regenerating nerves. Acrylamide treatment induces changes in composition of fast-transported protein that are qualitatively similar to those seen after axotomy. Since these changes are not detectable until the neuropathy is advanced, it is unlikely that they are causative factors. Instead, they are most likely a result of the cell body reaction previously observed in acrylamide intoxication, a reaction that resembles that produced by axotomy.

Nucleolar changes in goldfish retinal ganglion cells in response to an optic nerve lesion are not perturbed by a second lesion

The reaction of the goldfish retinal ganglion cells to optic nerve crush, as indicated by an increase in nucleolar incidence 4 to 5 days after this lesion, was not significantly affected if a second nerve crush was made within 3 days after the first. This appears to rule out axon sprouting as the initiating event for the cell body reaction to axotomy.

Comparison of the effects of sciatic nerve crush or resection on the proteins of fast axonal transport in rat dorsal root ganglion cell axons

Proteins of fast axonal transport in rat sciatic nerve axons were separated and characterized by SDS-polyacrylamide gel electrophoresis and fluorography, after injection of l-[ 35S]methionine into the dorsal root ganglion. The effects of crushing or resecting the sciatic nerve on the relative labeling of specific polypeptide bands were compared. Initially, both types of axon injury produced the same response, but after 3 weeks there was a partial return to normal composition in crushed nerves. In resected nerves, the changes characteristic of axon injury persisted beyond 7 weeks. Behavioral testing showed that crushed nerves reinnervated foot skin, whereas no reinnervation was detected after resection. We conclude that in sensory neurons, as in several other neuronal types, the restoration in normal composition of fast-transported protein after axon injury depends on reinnervation of target tissues. This aspect of the cell body reaction to injury seems to be regulated by a retrograde trophic interaction with the target.

Removal of optic tectum prolongs the cell body reaction to axotomy in goldfish retinal ganglion cells

Injury to the optic axons of goldfish elicits dramatic changes in the cell bodies of the neurons from which these axons arise, the retinal ganglion cells. The changes include a large increase in cell size and in synthesis and axonal transport of protein. The cells begin to return to normal about 3 weeks after the injury, when the axons invade the contralateral (homotopic) lobe of the optic tectum, and recovery is essentially complete by 8–10 weeks after the lesion. However, if the homotopic lobe of the tectum was removed at the time of nerve crush, we found that the cell body reaction was greatly prolonged. The cells remained enlarged, and [ 3H]proline incorporation and fast axonal transport of protein remained elevated, until at least 10–12 weeks after nerve crush, although by this time most of the regenerating axons had probably regained their normal length and many had entered the remaining ipsilateral (heterotopic) lobe of the tectum. The cells showed partial recovery by the latest time tested, 26 weeks after nerve crush, when the projections from the two eyes had segregated into separate bands in the heterotopic tectal lobe.