Determinants of axonal regeneration.

Axons often regrow to their targets and lost functions may be restored after an injury in the peripheral nervous system. In contrast, axonal regeneration is generally very limited after injuries in the central nervous system, and functional impairment is usually permanent. The regenerative capacity depends on intrinsic neuronal factors as well as the interaction of neurons with other cells. Glial cells may, in different situations, either support or inhibit axonal growth. This review discusses the molecular mechanisms that are involved in promoting and inhibiting axonal regeneration in the nervous system after injuries.

End-to-side neurorrhaphy resulting in limited sensory axonal regeneration in a rat model.

This study evaluated reinnervation of an end-to-side neurorrhaphy and the resultant functional recovery in a rat model. The cut distal posterior tibial nerve was repaired to the side of an intact peroneal nerve. In one group, the epineurium of the peroneal nerve was left intact; in another group, the epineurium was stripped; in the third experimental group, a perineurial slit was created. Evaluations included walking track analysis, nerve conduction studies, muscle mass measurements, retrograde nerve tracing, and histologic evaluation. Walking tracks indicated poor functional recovery. No significant difference in nerve conduction between the experimental and control groups was seen. Gastrocnemius muscle mass measurements revealed no functional recovery in the end-to-side groups. Retrograde nerve tracing revealed minimal staining of motor neurons. However, sensory neuronal staining of the dorsal root ganglia occurred in all groups. Histology revealed minimal myelinated axonal regeneration. These results suggest that predominantly sensory neural regeneration occurs in an end-to-side neurorrhaphy at an end point of 16 weeks.

Anterior chiasmal optic nerve avulsion.

In a case of traumatic avulsion of the optic nerve at the anterior chiasm, MR imaging provided highly specific images of the injury site, including the absence of the optic nerve within the optic canal and the point of transection at the anterior portion of the chiasm. This was confirmed clinically and histopathologically. MR imaging should be considered in cases of suspected chiasmal injury.

Retrograde abolition of conditional fear after excitotoxic lesions in the basolateral amygdala of rats: absence of a temporal gradient.

The role of the basolateral amygdala (LA) in the acquisition and expression of Pavlovian fear conditioning was examined in 80 rats. Excitotoxic lesions were made in the BLA using N-methyl-D-aspartate 7 days before or 1, 14, or 28 days after Pavlovian fear conditioning. Conditioning consisted of three pairings of a tone with an aversive footshock in a novel chamber, and freezing behavior served as an index of conditional fear. BLA lesions abolished conditional freezing to both the contextual and acoustic conditional stimuli at all training-to-lesion intervals, and the magnitude of the impairment did not vary as a function of the training-to-lesion interval. Reacquisition training elevated levels of freezing in rats with BLA lesions but did not reduce the magnitude of their deficit in relation to that of controls. These results reveal that neurons in the BLA have an enduring role in the expression of conditional fear.

Response properties of long-range axon-bearing amacrine cells in the dark-adapted rabbit retina.

Axon-bearing amacrine cells in mammalian retinae are encountered relatively infrequently during electrophysiological investigations, and thus very little is known about their physiological properties. Patch-clamp electrodes were used to record light responses from two axon-bearing amacrine cells in flat-mounted, dark-adapted rabbit retina. The recorded cells were stained, and the morphology visualized. Both cells were capable of generating action potentials. In one case, a linear relationship between mean depolarization and action-potential frequency was demonstrated. The cells had a proximal dendritic arbor and a morphologically distinct, much larger axon terminal system. The receptive field of the center response was coextensive with the dendritic arbor, and thus also much smaller than the axon terminal system. The center response was suppressed by activation of an inhibitory surround. Both cells responded to center illumination with an inward current which became more transient as the size of the illuminating spot was increased. It is suggested that axon-bearing amacrine cells receive input over a receptive field defined by the dendritic arbor, and distribute their output over a much more extensive axon terminal system, most probably via action potentials.

Delayed posttraumatic brain hyperthermia worsens outcome after fluid percussion brain injury: a light and electron microscopic study in rats.

The morphological consequences of delayed posttraumatic brain hyperthermia (39 degrees C) after fluid percussion brain injury were assessed in rats. Sprague-Dawley rats anesthetized with 4% halothane and maintained on a 70:30 mixture of nitrous oxide:oxygen and 0.5% halothane underwent moderate (1.5-2.0 atm) traumatic brain injury with the injury screw positioned parasagittally over the right parieto-occipital cortex. At 24 hours after traumatic brain injury, the rats were reanesthetized and randomized into two groups in which either a 3-hour period of brain normothermia (36.5 degrees C, n = 18) or hyperthermia (39 degrees C, n = 18) was maintained. Sham-operated controls (n = 10) underwent all surgical and temperature-monitoring procedures. After the 3-hour monitoring period, the rats were allowed to survive for 3 days for light microscopic analysis or were injected with the protein tracer horseradish peroxidase and were perfusion-fixed 15 minutes later for light and electron microscopic analysis. At 4 days after traumatic brain injury, delayed posttraumatic hyperthermia (n = 12) significantly increased mortality (47%) and contusion volume (1.7 +/- 0.69 mm3, mean +/- standard error of the mean), compared to normothermia (n = 12) (18% mortality and 0.13 +/- 0.21 mm3 contusion volume) (P < 0.01, analysis of variance). At 15 minutes after the 3-hour hyperthermic period, the area of hemorrhage and horseradish peroxidase extravasation overlying the lateral external capsule was significantly increased (2.52 +/- 0.71 mm2, mean +/- standard error of the mean, versus 0.43 +/- 0.16 mm2) (P < 0.01), compared to normothermic rats. Examination of toluidine blue-stained plastic sections demonstrated a higher frequency of abnormally swollen myelinated axons per high microscopic field with hyperthermia. For example, numbers of swollen axons within the sixth layer of the right somatosensory cortex, corpus callosum, and internal capsule were 7.3 +/- 1.3, 4.2 +/- 1.4, and 3.0 +/- 1.2 axons (mean +/- standard error of the mean) with normothermia, respectively, compared with 24.7 +/- 12.1, 33.1 +/- 4.2, and 27.3 +/- 3.1 axons with hyperthermia, respectively (P < 0.01). An ultrastructural examination of the swollen axons demonstrated a severely thinned myelin sheath containing axoplasm devoid of cytoskeletal components. These experimental results indicate that posttraumatic brain hyperthermia might increase morbidity and mortality in patients with head injury by aggravating axonal and microvascular damage.

Nimodipine accelerates axonal sprouting after surgical repair of rat facial nerve.

Facial-facial anastomosis (FFA), i.e., suture of transected facial nerve, was performed in adult Wistar rats. For 10-112 d post-operation (DPO), half of the animals received standard food (placebo) and half received food pellets containing 1000 ppm nimodipine, a Ca2+ channel blocker. The time course of mimetic reinnervation between these two groups was compared by counting all retrogradely labeled motoneurons after injection of horseradish peroxidase (HRP) into the whiskerpad. In unoperated animals, injection of HRP labeled 1280 +/- 113 motoneurons. After FFA, this number dropped to zero, and the first HRP-labeled facial motoneurons reappeared in both placebo- and nimodipine-treated animals at 14 DPO. The treatment with nimodipine yielded two beneficial effects. (1) It accelerated axonal sprouting until 28 DPO. Whereas the number of HRP-labeled cells in the placebo group was 171 +/- 9 (mean +/- SD) at 16 DPO, 372 +/- 43 at 21 DPO, and 636 +/- 187 at 28 DPO, the number of sprouted motoneurons in nimodipine-treated rats was twice as high: 386 +/- 34 at 16 DPO, 620 +/- 28 at 21 DPO, and 756 +/- 257 at 28 DPO. (2) Nimodipine reduced the polyneuronal innervation of the target muscles. Whereas the number of HRP-labeled cells in the placebo group increased to 1430 +/- 36 at 56 DPO and 1600 +/- 31 at 112 DPO, the number of labeled motoneurons in nimodipine-treated rats remained almost within the normal range: 1315 +/- 31 at 56 DPO and 1354 +/- 33 at 112 DPO.

Traumatically induced axonal damage: evidence for enduring changes in axolemmal permeability with associated cytoskeletal change.

Recent studies have demonstrated that delayed or secondary axotomy is a consistent feature of traumatic brain injury in both animals and man. Moreover, these studies have shown that the pathogenesis of this secondary axotomy involves various forms of initiating pathology, with the suggestion that, in some cases, only the axonal cytoskeleton is perturbed, while, in other cases, both the axonal cytoskeleton and related axolemma manifest traumatically induced perturbations. In the current communication, we continue in our investigation of the significance of these traumatically induced alterations in axolemmal permeability and their relation to any related intra-axonal cytoskeletal change. This was accomplished in cats which received intrathecal infusions of peroxidase, an agent normally excluded by the intact axolemma. These animals were subjected to traumatic brain injury, and sites showing altered axolemmal permeability to the peroxidase were assessed at the light and electron microscopic level. Through this approach, we recognized that a traumatic episode of moderate severity evoked changes in axolemmal permeability which surprising endured for up to 5 hrs postinjury. At such focal sites of altered permeability, the related cytoskeleton showed a statistically significantly neurofilament compaction, with the strong suggestion of concomitant neurofilament sidearm loss, microtubular dispersion, and mitochondrial abnormality. Over time, these events led to further disorganization of the axonal cytoskeleton which translated into impaired axoplasmic transport and secondary axotomy. Most likely, these alterations in axolemmal permeability result in either the direct or indirect effects upon the axonal cytoskeleton that precipitate the damaging sequences resulting in delayed axotomy.

Expression of beta-amyloid precursor protein in axons of periventricular leukomalacia brains.

Human beta-amyloid precursor protein immunoreactivity was demonstrated in axonal swellings (spheroids) around periventricular leukomalacia (PVL) of neonates. Immunoreactive axons were found at the early, but not late stage of PVL. beta-Amyloid precursor protein immunoreactivity was homogeneous in damaged axons at the early stage of PVL manifesting microglial activation, concentrated at the center of axonal swellings at the subsequent stage manifesting astrogliosis, and undetectable at the terminal stage of cavitation or neovasculation. Immunostaining for beta-amyloid precursor protein was useful in localizing PVL lesions at their early stages.

Influence of age on the late retrograde effects of sciatic nerve section in the rat.

The influence of age on the late retrograde effects of unilateral sciatic nerve section was investigated in rats. Operations were performed on young rats aged 3 months and older rats aged 15 and 18 months, with survival times ranging from 6 to 15 months depending upon age at the time of operation. As in previous studies, axonal atrophy was found in myelinated fibres proximal to nerve transection. This was observed to be greater in animals operated upon at 3 months of age than in those in which the sciatic nerve was transected at 15 and 18 months. In the sciatic nerve, focal intramyelinic oedema was present at a low frequency on the operated side just proximal to the section at all survival times but not on the unoperated side except in 1 old animal. Its frequency increased with age both in the dorsal and ventral roots on both sides but it was not more common on the operated side. Retrograde axonal atrophy is therefore unlikely to contribute to its occurrence. In the dorsal root ganglia the main abnormality was the presence of vacuolated neurons on the operated side. Nuclear eccentricity was also observed on the operated side in young animals in a proportion of the neurons; its frequency increased with age on the normal side and there was no difference in the older animals between operated and control sides. The possibility is discussed that growth factor deprivation secondary to axotomy is implicated in these changes. If so, there are age differences in its effect in giving rise to axonal atrophy and neuronal vacuolation.

Electrophysiological studies in Guillain-Barré syndrome: correlation with antibodies to GM1, GD1B and Campylobacter jejuni.

A retrospective study of 50 patients with Guillain-Barré syndrome (GBS) correlated analysis of serial motor nerve conduction studies with the presence of antibodies to Campylobacter jejuni, GM1 and GD1b, determined by ELISA. GBS patients with antibodies to C. jejuni (n = 8), GM1 (n = 4), or GD1b (n = 4) showed electrophysiological features suggestive of demyelination with prolonged distal motor latencies and temporal dispersion/conduction block similar to GBS patients without these specific antibodies. Three of 50 GBS patients had poor recovery with inability to walk at 1 year after onset of symptoms. All three patients had antibodies to C. jejuni, but not to GM1 or GD1b. Although later on in the clinical course distal motor responses were absent in two of these patients, reflecting extensive axonal degeneration, early nerve conduction studies showed findings suggestive of demyelination. We suggest that demyelination of peripheral nerve may be the initial disease mechanism in GBS independent of the presence of antibodies to C. jejuni, GM1 or GD1b.

The fate of motoneurons in the spinal cord after peripheral nerve repair: a quantitative study using the neural tracer horseradish peroxidase.

This study assessed the changes that occurred in the spinal motoneuron pool after the repair of a specific peripheral nerve by means of several clinically appropriate surgical techniques: nerve graft, muscle graft, and epineurial suture. The motoneuron pool relating to a single muscle was assessed at 50, 100, 200, and 300 days after repair via retrograde axonal transport of the neural tracer horseradish peroxidase. The results indicate that although a small portion of the motoneuron population dies following peripheral nerve surgery, this is not a significant number. The majority of the anterior horn cells appear to have the ability to both survive nerve transection and form new functional connections with the regenerated nerve after repair. The degree of cell loss is influenced by the nature of the injury and the method of repair implemented. Injuries involving neurotmesis result in the loss of a greater proportion of the cell population than less severe injuries involving axonotmesis. A greater proportion of the motoneuron population is preserved when the severed nerve has been repaired using a direct epineurial suture than when repair is achieved by means of a graft. The two methods of grafting produced comparable results, although the muscle graft tended to result in the preservation of a greater number of cells than the nerve graft, making it an acceptable alternative method for the surgical repair of short gaps in peripheral nerves.

Effect of isoproterenol on accessory pathways without overt retrograde conduction.

INTRODUCTION: Absence of overt retrograde accessory pathway conduction may be related to low resting sympathetic tone in patients with apparent unidirectional anterogradely conducting accessory pathways (UACAP). METHODS AND RESULTS: To test this hypothesis, we studied the effect of isoproterenol on accessory pathway function and tachycardia induction in 18 patients (12 men and 6 women, ages 34 +/- 16 years [mean +/- SD]) with UACAP. After baseline study in the drug-free state, electrphysiologic testing was repeated during infusion of isoproterenol (0.5 to 1.5 micrograms/min, titrated to increase heart rate by 20%). Isoproterenol shortened the anterograde effective refractory period (398 +/- 117 vs 305 +/- 63 msec; P < 0.01; basic drive cycle length 600 msec) of the accessory pathway. However, retrograde accessory pathway conduction and atrioventricular reentrant tachycardia were exposed in only 3 (17%) patients by isoproterenol infusion. All 3 patients with retrograde accessory pathway revealed after isoproterenol had clinically documented tachycardia (supraventricular tachycardia in 2, atrial fibrillation in 1) during exercise, while none of the patients with persistent absence of retrograde accessory pathway conduction had this symptom. CONCLUSIONS: We conclude that absence of overt retrograde conduction over accessory pathways may be related to low resting sympathetic tone in some individuals. Restoration of retrograde conduction with isoproterenol is unusual and most likely to be observed in patients with clinically documented paroxysmal supraventricular tachycardia related to exercise.

A time-dependent loss of retrograde transport ability in distally axotomized rubrospinal neurons.

Studies on the effect of axotomy on adult intrinsic central projection neurons have generally assumed that the severed proximal axonal stumps were still capable of retrogradely transporting tracer at varying times after injury. Failure of transport was interpreted as neuronal death, which is at odds with current understanding that central projection neurons survived distal axotomy. We used lumbar spinal cord-projecting rubrospinal neurons of the rat as a model to evaluate the ability of injured neurons to transport tracer retrogradely at different times after distal axotomy. We examined only the caudal part of the red nucleus, since rubrospinal neurons are concentrated here. In control animals, tracer applied to the rubrospinal tract at the T10 vertebral level labeled ventrolateral rubral neurons, while C3 application marked all rubral neurons. From 3 days after a T10 axotomy and tracer application, most ventrolateral neurons were no longer labeled by another tracer application at the C3 vertebral level via an axonal cut. The phenomenon was not caused by tracer toxicity, since a T10 tractotomy without tracer application also prevented these axotomized neurons from being labeled when treated similarly. Thus, neuronal retrograde transport capability was seriously retarded 3 days after a distal axotomy. Loss of retrograde transport may merely suggest that a mechanism no longer in service has been switched off, or perhaps it may insulate injured neurons from the effect of lesion site-derived factors. Using this property, we were able to localize cervical spinal cord-projecting rubrospinal neurons in the caudal red nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of apoptosis in mouse [correction of rat] olfactory neuroepithelium by synaptic target ablation.

The olfactory system provides a useful in vivo model for studying the influence of synaptic targets on the survival of relay neurones. The bipolar sensory neurones located in the olfactory mucosa project synaptically onto the ipsilateral olfactory bulb, and their survival depends on the integrity of this connection. We demonstrate here that the retrograde neuronal degeneration induced by olfactory bulb removal involves apoptosis. As revealed by typical nucleosome-sized fragmentations of the genomic DNA, the apoptosis rate reaches a maximum 32 h after bulbectomy. A transient c-fos mRNA accumulation was detected, peaking 16 h after bulbectomy, suggesting that c-fos is involved in the early steps of programmed cell death.

Immunohistochemical demonstration of neuron-specific enolase and microtubule-associated protein 2 in reactive astrocytes after injury in the adult forebrain.

Transformation of normal resting astrocytes to reactive astrocytes in the adult brain after injury has been well documented. Using double immunofluorescent labeling methods, we report that astrocytes in both the ischemically damaged and the retrogradely/anterogradely degenerating forebrain nuclei express not only the glial cell markers glial fibrillary acidic protein and vimentin, but also the neuronal markers neuron-specific enolase and microtubule-associated protein 2. Since these neuronal markers are expressed in glial precursor cells, these results suggest that one of the characteristic responses of astrocytes in the adult brain after injury may be re-expression of fetal trait(s) of early differentiating glial cells/neurons.

Ventral root avulsion: an experimental model of death of adult motor neurons.

The present study proposes a reproducible model of experimental degeneration of adult motor neurons in the rat. Avulsion of ventral roots in the adult lumbar cord transects motor axons at the root exit and leads to retrograde cell death of 80% of motor neurons 2 weeks later; this result follows a series of retrograde changes, including chromatolysis, loss of transmitter phenotype, and accumulation of phosphorylated neurofilaments in perikarya. Glial cells recruited at the site of retrograde injury express both microglia-specific epitopes (as exemplified by OX-42 immunoreactivity) and macrophage-specific markers (e.g., ED-1 immunoreactivity). Macrophage-specific markers become particularly intense 7 days postaxotomy and provide additional evidence of active phagocytosis of injured neurons. Ventral root avulsion is a very useful model for assessing mechanisms of motor neuron death and testing the ability of trophic factors and other agents to preserve the phenotype and promote the survival of adult motor neurons in vivo.

Axon-reflex reactions in affected and homologous contralateral skin after unilateral peripheral injury of thoracic segmental nerves in humans.

Transection and regeneration of a rat peripheral nerve on one side reduces the ability of the contralateral nerve to evoke plasma extravasation after antidromic excitation of afferent C-fibers induced by electrical nerve stimulation (afferent axon reflex). An unknown transneuronal signalling substance was postulated. In humans, axon-reflex vasodilatation was studied using cutaneous iontophoresis of histamine for C-fiber stimulation and laser Doppler flowmetry for measuring vasodilatation. As a model of peripheral nerve lesion with regeneration, patients with severe unilateral zoster neuropathy of thoracic segmental nerves were examined. Axon-reflex reactions were considerably impaired within the affected dermatome compared with the unaffected side and compared with controls. In contrast, no significant differences could be found between the unaffected corresponding sites of patients and similar dermatomes of healthy controls, indicating that this type of nerve injury does not influence the ability of the contralateral nerve to evoke axon-reflex vasodilatation.

Microglial and astroglial reactions to anterograde axonal degeneration: a histochemical and immunocytochemical study of the adult rat fascia dentata after entorhinal perforant path lesions.

The reaction of microglial and a stroglial cells to anterograde axonal degeneration was studied in the fascia dentata of adult rats at various timepoints after removal of the entorhinal perforant path projection. Microglial cells were identified by histochemical staining for nucleoside diphosphatase (NDPase) at light and electron microscopical levels. Astroglial cells were stained immunocytochemically for glial fibrillary acidic protein (GFAP). Activated astroglial cells and some microglial cells also stained immunocytochemically for the intermediate filament protein vimentin. Phagocytotic activity was detected by histochemical staining for acid phosphatase. The postlesional connective reorganization of the cholinergic septohippocampal projection was monitored by histochemical staining for acetylcholinesterase. Twenty-four hours after entorhinal cortex ablation, microglial cells in the perforant path zones of the fascia dentata and the adjacent neuropil reacted by shortening and coarsening of processes and an increase in NDPase reactivity. These changes occurred prior to a noticeable increase in GFAP immunoreactivity and hypertrophy of astroglial cells (first evident on postlesional day 2) or sprouting of cholinergic septohippocampal fibres (first evident on day 3). There was evidence of an early, local proliferation of microglial cells in the denervated perforant path zones and migration into these zones of microglial cells from adjacent intact areas. The specific accumulation of strongly stained microglial cells within the denervated parts of the dentate molecular layer persisted for at least 4 weeks, while the astroglial reaction subsided at 3 weeks. The results demonstrate an early activation of microglial cells by axonal degeneration, and indicate that these cells may play a pivotal, inductive role in the subsequent glial and neural events.

Cerebellar terminations in the red nucleus of Macaca fascicularis: an electron-microscopic study utilizing the anterograde transport of WGA:HRP.

The red nucleus (RN) of the macaque monkey is divided into a rostral two-thirds, the parvicellularis (RNp), which projects to the cerebellum by way of the inferior olivary nucleus, and a caudal third, the magnocellularis (RNm), which projects to the spinal cord via the rubrospinal tract. The RNp and RNm receive afferents from two principal sources: the cerebral motor cortices and the deep cerebellar nuclei. The terminations of these two afferent projections tend to be spatially segregated on rubral neurons, in that most corticorubral afferents terminate on more distal dendrites, and those from the deep cerebellar nuclei terminate more proximally. The present electron-microscopic analysis of the cerebellar terminations in the macaque RN provides anatomical evidence for the presence of labeled afferents in both divisions of this motor nucleus, following injection of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) into the deep cerebellar nuclei and the anterograde transport of the tracer to the RN. The cerebellar terminal afferents are large; contain numerous mitochondria and primarily rounded synaptic vesicles; and form asymmetric synaptic contacts with rubral neurons. Unlike other terminals in the nucleus, they possess an electron-lucent cytoplasmic matrix and less densely packed synaptic vesicles. They are termed "large, round, pale" (LRP) terminals because of the morphological characteristics that distinguish them from other afferent terminal types found in RN. Labeled cerebellar afferents in RNp and RNm contact primarily neuronal somata, proximal dendrites emerging from the cell body, large-diameter dendrites, and the spines of rubral neurons that arise from somata and proximal dendrites.(ABSTRACT TRUNCATED AT 250 WORDS)