The slow Wallerian degeneration gene in vivo protects motor axons but not their cell bodies after avulsion and neonatal axotomy

The slow Wallerian degeneration gene (Wld(S)) delays Wallerian degeneration and axon pathology for several weeks in mice and rats. Interestingly, neuronal cell death is also delayed in some in vivo models, most strikingly in the progressive motoneuronopathy mouse. Here, we tested the hypothesis that Wld(S) has a direct protective effect on motoneurone cell bodies in vivo. Cell death was induced in rat L4 motoneurones by intravertebral avulsion of the corresponding ventral roots. This simultaneously removed most of the motor axon, minimizing the possibility that the protective effect toward axons could rescue cell bodies secondarily. There was no significant difference between the survival of motoneurones in control and Wld(S) rats, suggesting that the Wld(S) gene has no direct protective effect on cell bodies. We also tested for any delay in apoptotic motoneurone death following neonatal nerve injury in Wld(S) rats and found that, unlike Wld(S) mice, Wld(S) rats show no delay in cell death. However, the corresponding distal axons were preserved, confirming that motoneurone cell bodies and motor axons die by different mechanisms. Thus, Wld(S) does not directly prevent death of motoneurone cell bodies. It follows that the protection of neuronal cell bodies observed in several disease and injury models where axons or significant axonal stumps remain is most probably secondary to axonal protection.     

Axonal damage associated with enlargement of ventricles during hydrocephalus: a silver impregnation study

Motor and cognitive deficits are commonly associated with hydrocephalus. Although the mechanisms responsible for these impairments have not been confirmed, neuronal cell death and axon degeneration may play an important role, and have long lasting consequences on neuronal connectivity. The goal of this study was to determine if neural degeneration occurred during hydrocephalus in structures anatomically related to cognitive motor functioning, namely, the sensorimotor cortex, neostriatum, hippocampus and corpus callosum. Neural damage, as visualized by silver staining, was examined in adult rats 2-10 weeks after obstructive hydrocephalus was induced by kaolin injection into the cisterna magna. In mild or moderate hydrocephalus, mostly occurring 2-6 weeks after kaolin injections, silver-labeled axons were scattered in the white matter of the sensorimotor cortex, corpus callosum, neostriatum, and hippocampus. In severe hydrocephalus, 10 weeks after kaolin injections, axon degeneration was more extensive in these areas, as well as in layers IV through VI of the sensorimotor cortex. Axons in the subiculum and the fimbria were heavily labeled, suggesting damage to hippocampal afferent and efferent fibers. In contrast, neuron cell death was rarely observed at any stage of hydrocephalus. The major pathological change of brain regions involved in motor and learning functions during hydrocephalus is axon degeneration, and this degeneration is correlated with an enlargement of the cerebral ventricles.     

Axonal damage associated with enlargement of ventricles during hydrocephalus: A silver impregnation study

Abstract

Motor and cognitive deficits are commonly associated with hydrocephalus. Although the mechanisms responsible for these impairments have not been confirmed, neuronal cell death and axon degeneration may play an important role, and have long lasting consequences on neuronal connectivity. The goal of this study was to determine if neural degeneration occurred during hydrocephalus in structures anatomically related to cognitive motor functioning, namely, the sensorimotor cortex, neostriatum, hippocampus and corpus callosum. Neural damage, as visualized by silver staining, was examined in adult rats 2–10 weeks after obstructive hydrocephalus was induced by kaolin injection into the cisterna magna. In mild or moderate hydrocephalus, mostly occurring 2–6 weeks after kaolin injections, silver-labeled axons were scattered in the white matter of the sensorimotor cortex, corpus callosum, neostriatum, and hippocampus. In severe hydrocephalus, 10 weeks after kaolin injections, axon degeneration was more extensive in these areas, as well as in layers IV through VI of the sensorimotor cortex. Axons in the subiculum and the fimbria were heavily labeled, suggesting damage to hippocampal afferent and efferent fibers. In contrast, neuron cell death was rarely observed at any stage of hydrocephalus. The major pathological change of brain regions involved in motor and learning functions during hydrocephalus is axon degeneration, and this degeneration is correlated with an enlargement of the cerebral ventricles. [Neurol Res 2001; 23: 581-587]     

Overexpression of Wlds or Nmnat2 in mauthner cells by single‐cell electroporation delays axon degeneration in live zebrafish

Axon degeneration is supposed to be a therapeutic target for treating neurodegenerative diseases. Mauthner cells (M‐cells) are ideal for studying axons in vivo because of their limited numbers, large size, and long axons. In this study, we labeled M‐cells by single‐cell electroporation with plasmids expressing DsRed2 or EGFP. Injury‐induced axon degeneration in labeled M‐cell was imaged under a confocal microscope, and we found that the Mauthner axons started to degenerate about 24 hr after lesion. The Wld^S protein containing full‐length Nmnat1 is well‐known for its axon‐protective function in many systems. Overexpression of Wld^S in M‐cells also greatly delayed axon degeneration in live zebrafish. Nmnat2 is the only Nmnat highly expressed in brain. Here we demonstrated that overexpression of Nmnat2 in M‐cells significantly delayed axon degeneration in vivo, and disruption of the NAD synthesis activity of Nmnat2 markedly attenuated its axon‐protective function. All these data show that injury‐induced axon degeneration of M‐cell has a mechanism similar to that in mammalians and would be a valuable model for studying axon degeneration in vivo. © 2010 Wiley‐Liss, Inc.     

Ischemic axonal injury and its recovery after focal cerebral ischemia

After focal cerebral infarction by occluding the middle cerebral artery (MCA) of the rat, the neuronal death occurred in the ipsilateral thalamic neurons, because axons of the thalamic neurons were injured by infarction and retrograde degeneration occurred in the thalamic neurons. However, cortical neurons adjacent to the infarction survived despite their axons injured by ischemia. We employed immunohistochemical staining for 200 kilodalton (kD) neurofilament (NF), in order to study those responses of cortical and thalamic neurons against axonal injury caused by focal cerebral infarction. In the sham operated rats the immunoreactivity to the anti-200 kD NF antibody was only detected in the axon but not in the cell bodies and dendrites. At 3 days after MCA occlusion, axonal swelling proximal to the site of ischemic injury was found in the caudoputamen and internal capsule of the ipsilateral side. At 7 days after occlusion, cell bodies and dendrites of the neurons in the ipsilateral cortex and thalamus were strongly stained with anti-NF antibodies. At 2 weeks after occlusion these responses disappeared in the cortex, but lasted in the thalamus. These phenomena are caused by stasis of the slow axonal transport, because the NF is transported by slow axonal transport. In the cortical neurons impairment of slow axonal transport recovered in the early phase after injury, but in the thalamic neurons the impairment prolonged up to 3 weeks after occlusion. The early recovery of axonal transport from ischemia seemed to be essential for survival of neurons after ischemic axonal injury.     

The WldS gene delays axonal but not somatic degeneration in a rat glaucoma model

Glaucoma is a leading cause of blindness caused by progressive degeneration of retinal ganglion cells (RGCs) and their axons. The pathogenesis of glaucoma remains incompletely understood, but optic nerve (ON) axonal injury appears to be an important trigger of RGC axonal and cell body degeneration. Rat models are widely used in glaucoma research to explore pathogenic mechanisms and to test novel neuroprotective approaches. Here we investigated the mechanism of axon loss in glaucoma, studying axon degeneration in slow Wallerian degeneration (Wld(S)) rats after increasing intraocular pressure. Wld(S) delays degeneration of experimentally transected axons for several weeks, so it can provide genetic evidence for Wallerian-like degeneration in disease. As apoptosis is unaffected, Wld(S) also provides information on whether cell death results from axon degeneration or arises independently, an important question yet to be resolved in glaucoma. Having confirmed expression of Wld(S) protein, we found that Wld(S) delayed ON axonal degeneration in experimental rat glaucoma for at least 2 weeks, especially in proximal ON where wild-type axons are most severely affected. The duration of axonal protection is similar to that after ON transection and crush, suggesting that axonal degeneration in glaucoma follows a Wallerian-like mechanism. Axonal degeneration must be prevented for RGCs to remain functional, so pharmacologically mimicking and enhancing the protective mechanism of Wld(S) could offer an important route towards therapy. However, Wld(S) did not protect RGC bodies in glaucoma or after ON lesion, suggesting that combination treatments protecting both axons and cell bodies offer the best therapeutic prospects.     

Retrograde cortical aand axonal changes following lesions of the pyramidal tract.

Following lesions of the pyramidal tract in hamsters, retrograde changes were studied in the sensorimotor cortex and in the pyramidal tract axons proximal to the lesion, at survival times ranging from 2 weeks to 14 months. Severe cell shrinkage occurred in layer 5 pyramidal neurons as early as 2 weeks, but there was no cell loss among these neurons even with long survival times. Use of the Fink-Heimer method for degenerating axons revealed that the pyramidal tract proximal to the lesion had undergone a retrograde axon degeneration which, in some respects, resembled anterograde degeneration. The retrograde axon degeneration began at the lesion site and advanced slowly rostralwards with time involving increasingly greater numbers of fibers. However, even at the longest survival times the degeneration fell off markedly at pontine levels. The results indicate that this process represents a true retrograde fiber degeneration (as opposed to an indirect Wallerian degeneration) which appears to reach a point of equilibrium such that a partially shrunken pyramidal cell is maintaining a partially degenerated axon.     

Clinical progression in Parkinson disease and the neurobiology of axons

Despite tremendous growth in recent years in our knowledge of the molecular basis of Parkinson disease (PD) and the molecular pathways of cell injury and death, we remain without therapies that forestall disease progression. Although there are many possible explanations for this lack of success, one is that experimental therapeutics to date have not adequately focused on an important component of the disease process, that of axon degeneration. It remains unknown what neuronal compartment, either the soma or the axon, is involved at disease onset, although some have proposed that it is the axons and their terminals that take the initial brunt of injury. Nevertheless, this concept has not been formally incorporated into many of the current theories of disease pathogenesis, and it has not achieved a wide consensus. More importantly, in view of growing evidence that the molecular mechanisms of axon degeneration are separate and distinct from the canonical pathways of programmed cell death that mediate soma destruction, the possibility of early involvement of axons in PD has not been adequately emphasized as a rationale to explore the neurobiology of axons for novel therapeutic targets. We propose that ongoing degeneration of axons, not cell bodies, is the primary determinant of clinically apparent progression of disease, and that future experimental therapeutics intended to forestall disease progression will benefit from a new focus on the distinct mechanisms of axon degeneration. ANN NEUROL 2010;67:715–725     

Light- and electron-microscopical study of phosphoprotein B-50 following denervation and reinnervation of the rat soleus muscle

The neuron-specific phosphoprotein B-50 was originally identified as a phosphoprotein in synaptic plasma membranes isolated from adult brain tissue. In this paper we study the reinnervation of the soleus muscle, a target muscle of sciatic nerve axons, using affinity-purified anti-B-50 antibodies. Light-microscopical evaluation of the reinnervation process revealed that the period of muscle fiber reinnervation corresponds closely with the time in which high B-50 immunoreactivity was observed in the nerve fibers that invade the muscle and in the newly formed neuromuscular junctions. Upon completion of reinnervation, B-50 immunoreactivity decreased. In the newly innervating terminals, B-50 was associated with presynaptic vesicular structures and with the presynaptic plasma membrane. In intact mature neuromuscular junctions, virtually no B-50 immunoreactivity could be detected with either light- or electron-microscopic procedures. These observations corroborate the association of high levels of B-50/GAP43 during axon outgrowth and support the concept that B-50 may be a key molecule in the reconstruction of axonal structures. We also observed an unexpected transient increase in B-50 immunoreactivity in the degenerating neuromuscular junctions. This observation cannot be explained in terms of increased neuronal synthesis of B-50, since the degenerating axon processes have been completely disconnected from their cell bodies. Thus, our evidence implies that a rise of B-50 immunoreactivity can be associated with stages of neuronal degeneration as well as with those of neuronal differentiation and axon outgrowth.     

Pathology of the dentate nucleus in progressive supranuclear palsy: a histological,immunohistochemical and ultrastructural study

Summary Dentate nucleus pathology was studied histologically and immunohistochemically in four cases and ultrastructurally in three cases of progressive supranuclear palsy (PSP). In addition to neurofibrillary changes, there were ill-defined clumps of eosinophilic granular structures, named grumose degeneration (GD). GD was observed in three of the four cases; it was not seen in a case exhibiting severe Purkinje cell loss. In areas with prominent GD, neuronal loss was also marked and the remaining neurons were atrophic. GD, which was once believed to represent degenerated cell bodies of dentate neurons, could be histologically distinguished from perikarya of dentate neurons. Argentophilic rings and knobs were only a part of GD and the rest of GD was only faintly or not argentophilic. Immunostain for phosphorylated neurofilament proteins positively stained round structures of various sizes in GD, but a large part of GD did not react with the antibody. As described in other disorders, electron microscopy revealed that the GD consisted of clusters of numerous axon terminals and preterminal axons. Many appeared swollen to a varying extent and contained mitochondria, synaptic vesicles, neurofilaments, lamellar bodies, multivesicular structures, vacuoles or combinations of these organelles. A few were markedly swollen with accumulation of neurofilaments and other organelles, corresponding to the round structures which stained positively for phosphorylated neurofilament proteins. A considerable number of axon terminals with no apparent abnormal accumulations of organelles were found in areas of GD.     

Evidence for direct axonal toxicity in vincristine neuropathy

There is a long-standing debate concerning the localization of the primary insult that results in distal axonal degeneration, or 'dying back' neuropathy. To address this question, we created an in vitro model of vincristine neuropathy in rat dorsal root ganglia (DRG). DRGs were grown in compartmentalized chambers, allowing for isolated exposure of the cell body or the axon to vincristine. Initial dose-finding studies identified a dose of vincristine that showed differential effects on cell death when delivered to either the cell body or the axonal compartment. At this dose of 0.05 microM, exposure of the cell bodies had no effect on the growth of axons, whereas addition of vincristine to the axonal compartment caused axonal shortening without affecting the growth of unexposed 'sister' axons. Toxicity was seen only with exposure of the growing axonal tips. These data support localized axonal toxicity as a cause of distal axonal degeneration due to vincristine.     

The Survival of Transected Axonal Segments of Cultured Aplysia Neurons is Prolonged by Contact with Intact Nerve Cells

Axonal segments transected from their cell body in vivo commonly undergo degeneration within 3–4 days (Wallerian degeneration). In lower vertebrates and invertebrates, however, some transected axonal segments survive for long periods ranging between 30 and 200 days. To circumvent the technical complications of studying the mechanisms underlying long-term survival of transected axons in vivo , we developed an in vitro system. We found previously that isolated axonal segments of cultured Aplysia neurons preserved their morphological integrity for an average duration of 7.6 days (range 2–14 days) and maintain their passive and excitable membrane properties. This survival occurred in the absence of de novo protein synthesis. In the present study we examined the influence of homologous neurons on the survival of transected axonal segments. We found that the average survival time of transected axons was doubled when co-cultured in physical contact with intact homologous neurons (average 15.3 days, range 2–27 days). During this period, the transected axons extended neurites, maintained normal passive and excitable membrane properties, formed electrotonic junctions with the intact neurons and maintained normal free intracellular Ca²⁺ levels. Consistent with these observations, electron micrographs of the transected axon revealed that the cytoskeletal elements of the axon appeared normal even 20 days after transection. In contrast, the mitochondria and smooth endoplasmic reticulum appeared damaged. As the prolonged survival was conditional on physical contact between the transected axon and the surrounding intact neurons, we suggest that the prolongation of survival time is promoted by the direct transfer of material from the intact neurons to the transected axon. However, co-culture of transected axons with homologous neurons did not fully mimic in vivo conditions, in which transected axons can survive for several months.     

Mild traumatic brain injury to the infant mouse causes robust white matter axonal degeneration which precedes apoptotic death of cortical and thalamic neurons

The immature brain in the first several years of childhood is very vulnerable to trauma. Traumatic brain injury (TBI) during this critical period often leads to neuropathological and cognitive impairment. Previous experimental studies in rodent models of infant TBI were mostly concentrated on neuronal degeneration, while axonal injury and its relationship to cell death have attracted much less attention. To address this, we developed a closed controlled head injury model in infant (P7) mice and characterized the temporospatial pattern of axonal degeneration and neuronal cell death in the brain following mild injury. Using amyloid precursor protein (APP) as marker of axonal injury we found that mild head trauma causes robust axonal degeneration in the cingulum/external capsule as early as 30 min post-impact. These levels of axonal injury persisted throughout a 24 h period, but significantly declined by 48 h. During the first 24 h injured axons underwent significant and rapid pathomorphological changes. Initial small axonal swellings evolved into larger spheroids and club-like swellings indicating the early disconnection of axons. Ultrastructural analysis revealed compaction of organelles, axolemmal and cytoskeletal defects. Axonal degeneration was followed by profound apoptotic cell death in the posterior cingulate and retrosplenial cortex and anterior thalamus which peaked between 16 and 24 h post-injury. At early stages post-injury no evidence of excitotoxic neuronal death at the impact site was found. At 48 h apoptotic cell death was reduced and paralleled with the reduction in the number of APP-labeled axonal profiles. Our data suggest that early degenerative response to injury in axons of the cingulum and external capsule may cause disconnection between cortical and thalamic neurons, and lead to their delayed apoptotic death.     

Long-term consequences of impaired regeneration on facial motoneurons in the C57BL/Ola mouse

Peripheral nerves of the C57BL/Ola mouse mutant undergo markedly slowed Wallerian degeneration following injury. This is associated with impaired regeneration of both sensory and motor axons. Following a crush lesion of the facial nerve, there was no cell loss in facial nuclei of normal (C57BL/6J) adult mice, but 40% cell loss occurred in Ola mice and the survivors increased in size during the period when functional reinnervation was established. These results are interpreted as a result, first, of prolonged deprivation of target-derived trophic factor in the slowly regenerating Ola motoneurons and second, increased peripheral field size of the survivors. Within the regenerated facial nerve, there was marked heterogeneity of myelinated fibre size in Ola mice. Some Ola axons, both proximal and distal to the lesion site, had areas over twice as great as the largest 6J axons when measured 1 year following injury. A population of small diameter fibres, not observed in 6J nerves, persisted distal to the crush site in Ola nerves, and this was associated with an increase in the total number of myelinated axons in the distal nerve: on average, each parent Ola axon retained three persistent daughter axons. The delayed Wallerian degeneration in Ola mice not only impairs immediate axon regrowth, but also results in a breakdown of the normal mechanisms which regulate axon number and size in regenerating nerve.     

Further Studies on Motor and Sensory Nerve Regeneration in Mice With Delayed Wallerian Degeneration

The axons of both peripheral and central neurons in C57BL/Wld ^s (C57BL/Ola) mice are unique among mammals in degenerating extremely slowly after axotomy. Motor and sensory axons attempting to regenerate are thus confronted with an intact distal nerve stump rather than axon-and myelin-free Schwann cell-filled endoneurial tubes. Surprisingly, however, motor axons in the sciatic nerve innervating the soleus muscle regenerate rapidly, and there is evidence that they may use Schwann cells associated with unmyelinated fibres as a pathway. If this is so, motor axon regeneration might be impaired in C57BL/Wld ^s mice in the phrenic nerve, which has very few unmyelinated fibres. We found that as long as the myelinated axons in the distal stump of the phrenic nerve remained intact (up to 10 days), regeneration of motor axons did not occur, in spite of vigorous production of sprouts at the crush site. In contrast to motor axons, myelinated sensory axons regenerate very poorly in C57BL/Wld ^s mice, even in the presence of unmyelinated axons. We showed that this was also due to adverse local conditions confronting nerve sprouts, for the dorsal root ganglion cell bodies responded normally to injury with a rapid induction of Jun protein-like immunoreactivity and when the saphenous nerve was forced to degenerate more rapidly by multiple crush lesions sensory axons regrew much more successfully. The findings show that motor and sensory axons in C57BL/Wld ^s mice, although very atypical in the way that they degenerate, are able to regenerate normally but only in an appropriate environment. The results also give support to the view that intact peripheral nerves either fail to encourage or actively inhibit axon growth, and that an unsuitable local environment can prevent regeneration even if the cell body is reacting normally to injury.     

Thalamic retrograde degeneration in the congenitally hydrocephalic rat is attributable to apoptotic cell death

Congenitally hydrocephalic HTX rats develop ventricular dilatation with extensive damage of the cerebral white matter. Recently, we have reported that neuronal cell death also occurs in the thalamus of HTX rats. To investigate the mechanism underlying this thalamic degeneration in these animals, we carried out a histopathological study of the brain at different phases of postnatal development. Eosinophilic neurons with condensed chromatin or fragmented nuclei were observed in the thalamus from postnatal day 17 onward. The incidence of cell death in the thalamus increased with the progression of hydrocephalus. Ultrastructurally, thalamic neurons occasionally had apoptotic features including nuclear chromatin condensation and marginalization. Immunohistochemically, single‐stranded DNA‐positive neuronal nuclei were found in the thalamus. They were also positively stained with the TUNEL method. Marked loss of myelin and axons with many TUNEL‐positive oligodendrocytes were found in the cerebral white matter. These findings suggest that the neuronal cell death observed in the thalamus in hydrocephalic HTX rats is retrograde degeneration due to extensive damage of axons in the cerebral white matter and that the thalamic retrograde degeneration is attributable to apoptotic cell death.     

Swellings of proximal axons in a case of motor neuron disease

Serial semithin sections of lumbar anterior horn cells from a patient with rapidly progressive sporadic motor neuron disease were searched for direct connections between swellings of neuronal processes and perikarya. Focal swellings of the proximal axons were not uncommonly seen to be connected directly to perikarya. These swellings varied in shape and size and some were identified as spheroids. Most of the cell bodies connected with swellings showed otherwise normal architecture. These observations suggest that the focal swellings of proximal axons, particularly the distal portion of the initial segment and the first internode of the myelinated axon, indicate an early pathological change, and may represent functionally and morphologically vulnerable sites.     

Traumatic brain injury in the immature mouse brain: characterization of regional vulnerability

We characterized the regional and temporal patterns of neuronal injury and axonal degeneration after controlled cortical impact of moderate severity in mice at postnatal day 21. Animals were euthanized at 1, 3, or 7 days after injury or sham operation. The brains were removed and prepared for immunolocalization of neurons and microglia/macrophages or subjected to Fluoro-Jade and silver stains, indicators of irreversible neuronal cell injury and axonal degeneration. There was significant neuronal loss in both the ipsi- and the contralateral cortices, ipsilateral hippocampus, and ipsilateral thalamus by 7 days post injury compared to sham-operated animals. Activated microglia/macrophages were most prominent in regions of neuronal loss including the ipsilateral cortex, hippocampus, and thalamus. Neuronal injury, as evidenced by Fluoro-Jade labeling, was not apparent in sham-operated animals. In injured animals, labeling was identified in the ipsilateral cortex and hippocampus at 1 and 3 days post injury. Silver- and Fluoro-Jade-labeled degenerating axons were observed in the ipsilateral subcortical white matter by 1 day post injury, in the ipsilateral external capsule, caudate putamen, and contralateral subcortical white matter by 3 days post injury, and in the internal capsule, pyramidal tracts, and cerebellar peduncles by 7 days post injury. Our findings demonstrate that controlled cortical impact in the developing brain generates neuronal loss in both the ipsilateral and the contralateral cortex, a temporally distinct pattern of subcortical neuronal injury/death, and widespread white matter damage. These observations serve as an important baseline for studying human brain injury and optimizing therapies for the brain-injured child.     

Degeneration of neuronal cell bodies following axonal injury in Wld(S) mice

The phenotype of Wld(S) ("slow Wallerian degeneration") mice demonstrates prolonged survival of injured axons. However, whether the Wld(S) mutation delays degeneration of the neuronal cell body following axonal injury is unclear. We used a retrograde model of axonal transport failure in Wld(S) mice to test whether the mutant Wld(S) protein has any beneficial effect on the neuronal cell body. Retrograde axonal transport was physically blocked by optic nerve crush and confirmed by the absence of Fluoro-Gold labeling in wild-type and in Wld(S) mice. After this axonal injury, there was marked protection of axonal degeneration in the Wld(S) phenotype, as confirmed by immunohistochemistry and electron microscopy. However, the Wld(S) protein, localized in the nucleus of retinal ganglion cells, did not prevent or delay degeneration of the retinal ganglion cell body, confirmed by TUNEL staining and Fluoro-Gold labeling. These results imply that, after axonal injury, Wallerian degeneration of axons and degeneration of the neuronal cell body have different mechanisms, which are autonomous and independent of each other. Although the Wld(S) phenotype can be used to demonstrate stable enucleate axons, the mutation is unlikely to protect neurons in neurodegenerative diseases in which there is failure of retrograde transport.     

Domoic acid-treated cynomolgus monkeys (M. fascicularis): effects of dose on hippocampal neuronal and terminal degeneration

Domoic acid is a tricarboxylic amino acid (structurally related to kainic acid and glutamic acid) that is found in the environment as a contaminant of some seafood. To determine the nature of any neurological damage caused by domoate, as well as the minimum neurotoxic dose, juvenile and adult monkeys were dosed intravenously with domoate at one of a range of doses from 0.25 to 4 mg/kg. When animals were perfused one week later, histochemical staining using a silver method to reveal degenerating axons and cell bodies showed two distinct types of hippocampal lesions. One lesion, termed ‘Type A’, was a small focal area of silver grains restricted to CA2 stratum lucidum, the site of greatest kainic acid receptor concentration in the brain. Type A lesions occurred over a dose range of 0.5 to 2.0 mg/kg in juvenile animals and 0.5 to 1.0 mg/kg in adult animals. No mortality occurred in any of the juvenile monkeys, but one juvenile animal that received 4.0 mg/kg sustained a second type of lesion, termed ‘Type B’, characterized by widespread damage to pyramidal neurons and axon terminals of CA4, CA3, CA2, CA1, and subiculum subfield of the hippocampus. Doses of more than 1.0 mg/kg in the adult monkeys either proved lethal or resulted in Type B lesions. Induction of c-fos protein had occurred in the hippocampal dentate gyrus and CA1 regions of moribund animals perfused within hours of their initial dose. Our results show that relatively low doses of domoic acid selectively effect mossy fiber terminals, that larger (near lethal) doses damage hippocampal pyramidal neurons and their axons, that some moribund monkeys had experienced limbic seizures prior to death, and that adult animals are more sensitive to domoic acid than juveniles.