Neuropathological assessment of artemether-treated severe malaria.

In animals, high doses of intramuscular artemether and artemotil have been shown to cause an unusual pattern of selective damage to certain brainstem nuclei, especially those implicated in hearing and balance. We aimed to investigate whether a similar pattern arises in human adults. We examined the brainstems of adults who died after treatment with high dose artemether or quinine for severe falciparum malaria for evidence of a pattern of selective neuronal damage. Neuropathological findings were similar in recipients of quinine (n=15) and artemether (n=6; total artemether doses received 4-44 mg/kg). No evidence was recorded for artemether-induced neurotoxic effects.

Glycogenosomes in the aging rat brain: their occurrence in the visual pathways.

The significance of glycogenosomes (glycogen bodies), frequently seen in peripheral neurites of aging rats, is unknown and their occurrence elsewhere in nervous tissue is poorly documented. During the course of another study these bodies were observed by light microscopy in the visual pathways of aging rats where they have not previously been noted, and this report documents their occurrence, localisation and changes in density with age. Using the periodic acid-Schiff stain, small brightly red-staining bodies, digested by diastase and containing beta-glycogen particles, were seen in increasing numbers in the neuropil of the superior colliculi in brain sections from animals of 5 months of age onwards. From 1 year until more than 2 years of age they steadily became more numerous in the outer one third of the superior colliculus, but remained small, rarely exceeding 4 microm. They were also found at later times in small numbers lying singly in the optic tract, the optic chiasm and optic nerves, although rarely in lateral geniculate nuclei. Similar bodies were also found to accumulate with age in the retinal photoreceptor cell layer. Changes in their densities and size with age in both regions have been documented and it is suggested that, while their occurrence in retinal photoreceptor cells may be due to sustained light damage leading to mitochondrial oxidative stress, it is difficult to implicate this mechanism for their occurrence in retino-tectal nerve fibres. The role of physical trauma, suggested for the presence of these bodies in aging peripheral axons, can be excluded and they appear not to be related to polyglucosan bodies.

Corpora-amylacea and the family of polyglucosan diseases.

The history, characters, composition and topography of corpora amylacea (CA) in man and the analogous polyglucosan bodies (PGB) in other species are documented, noting particularly the wide variation in the numbers found with age and in neurological disease. Their origins from both neurons and glia and their probable migrations and ultimate fate are discussed. Their presence is also noted in other organs, particularly in the heart. The occurrence in isolated cases of occasional 'massive' usually focal accumulations of similar polyglucosan bodies in association with certain chronic neurological diseases is noted and the specific conditions Adult Polyglucosan body disease and type IV glycogenosis where they are found throughout the nervous system in great excess is discussed. The distinctive differences of CA from the PGB of Lafora body disease and Bielschowsky body disease are emphasised. When considering their functional roles, a parallel is briefly drawn on the one hand between normal CA and the bodies in the polyglucosan disorders and on the other with the lysosomal system and its associated storage diseases. It is suggested that these two systems are complementary ways by which large, metabolically active cells such as neurons, astrocytes, cardiac myocytes and probably many other cell types, dispose of the products of stressful metabolic events throughout life and the continuing underlying process of aging and degradation of long lived cellular proteins. Each debris disposal system must be regulated in its own way and must inevitably, a priori, be heir to metabolic defects that give rise in each to its own set of metabolic disorders.

Glutathione depletion increases brain susceptibility to m-dinitrobenzene neurotoxicity.

To test the hypothesis that glutathione (GSH) status in brain tissue plays an important role in the selective neurotoxicity of m-dinitrobenzene (DNB), the sensitivity to intoxication of three groups of male F344 rats were studied and correlated with brain tissue GSH levels. In Group I were young 6-8 week old rats with normal GSH levels, and in Group II were rats of the same age whose brain GSH levels had been reduced by intracerebroventricular (i.c.v.) injections of L-buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. In Group III were 6 month old rats that, as a result of normal aging, show GSH levels of 16-29% below those seen in younger animals. All three groups were subjected to a 1 to 4 dose schedule of dosing with DNB (7.5 mg/kg/day i.p.) and killed 1 day after the last dose of DNB. It was found that whereas Group I animals developed ataxia and brain stem lesions after 4 doses, Group III animals showed these changes after 3 doses, while Group II animals had brain stem lesions after only 2 doses of DNB. The timing of the onset of these changes correlated closely with the degree of reduction of brain tissue levels of GSH, this being greatest in those animals infused i.c.v. with BSO. This demonstration indicates that GSH status in brain tissue is likely to be an important factor in determining regional sensitivity to gliovascular damage from this agent.

Neurotoxic potential of gadodiamide after injection into the lateral cerebral ventricle of rats.

PURPOSE: Results of a previous report showed that, if administered by intraventricular injection to access tissue normally protected by the blood-brain barrier, gadopentetate dimeglumine produced acute excitation, persistent ataxia, and widespread brain lesions in rats at 5-micromol/g brain but not at 3.8-micromol/g brain. The present study using gadodiamide was undertaken to see whether the effects were agent-specific. METHODS: Rats, surgically prepared with a lateral ventricular cannula, were administered a slow injection at 2 microL/min of gadodiamide into the lateral ventricle, and behavioral and neuropathologic changes were noted. RESULTS: Both gadodiamide and gadopentetate dimeglumine produced focal and generalized myoclonus over several hours. Gadodiamide did not produce the medium-term tremor or persistent ataxia seen after treatment with gadopentetate dimeglumine. Neuropathologic changes developed over 1 to 3 days and took three distinct forms: vacuolated thalamic lesions closely resembling those produced by gadopentetate dimeglumine; small but similar vacuolated symmetrical caudate lesions not produced by gadopentetate dimeglumine; and severe swelling and astrocytic hypertrophy and hyperplasia in the cerebellar vermis, again not produced by gadopentetate dimeglumine. Unlike gadopentetate dimeglumine, gadodiamide produced no spinal cord lesions. The cerebellar changes were seen at 1.25-micromol/g brain and above, behavioral changes at 2.5-micromol/g brain and above, and thalamic and caudate lesions at 10-micromol/g brain, the maximal dose used. Markedly reducing the rate of injecting the same volume over 28 hours prevented the acute excitation but did not reduce the severity of the morphologic effects. CONCLUSION: The acute excitatory effects of high intraventricular doses of gadopentetate dimeglumine and gadodiamide are similar and appear to be attributable to local action at the infusion site, but differences exist between the two agents in the character and topography of the distant morphologic changes. The cerebellum was the brain area most sensitive to gadodiamide in this experimental model. It is unlikely that gadodiamide would gain access to the brain at these tissue doses when used intravenously for conventional clinical imaging, but our experimental model suggested that it had some unexpectedly specific neuropathologic potential.

Spinal corpora amylacea and motor neuron disease: a quantitative study.

OBJECTIVE: To test the hypothesis that as there is growing evidence that corpora amylacea, or amyloid bodies, in the CNS are derived primarily from neurons, it might be expected that their numbers in the spinal cord would decline with loss of neurons in motor neuron degeneration as they do in the retina on destruction of ganglion cells by glaucoma. METHODS: The numbers of corpora amylacea were counted in PAS stained transverse sections of the lumbar cord from 27 patients with motor neuron disease and 21 control subjects of similar age and sex mix. The numbers and sizes of corpora amylacea were determined both in the anterior horn grey matter and in the submeningeal white matter regions in each case. RESULTS: In both groups the total numbers in the white matter and submeningeal regions ranged from 160 to more than 5000/section and there was minimal significant difference between the two groups. No relation with age was found in this narrow age range. The mean diameters of the corpora amylacea were significantly less in the grey matter of both groups than in the submeningeal regions. However, their densities in the grey matter of the anterior horn were significantly reduced in the spinal cord sections in the motor neuron disease group, but only where few motor neurons remained. CONCLUSIONS: These findings support the view that corpora amylacea may arise from neurons, and suggest that that there may be two compartments, one mobile and one static, the second most likely remaining in the periphery of the spinal cord for prolonged periods.

The effects of the tremorgenic mycotoxin penitrem A on the rat cerebellum.

Within 10 minutes of intraperitoneal injection of penitrem A (3 mg/kg), rats develop severe generalized tremors and ataxia that persist for up to 48 hours. These are accompanied by a three- to fourfold increase in cerebellar cortical blood flow. Mitochondrial swelling occurs in cerebellar stellate and basket cells within 30 minutes of dosing and persists for more than 12 hours without leading to cell death. From 2 hours, Purkinje cell dendrites show early cytoplasmic condensation accompanied by fine vacuolation of smooth endoplasmic reticulum and enlargement of perikaryal mitochondria. From 6 hours, many Purkinje cells develop intense cytoplasmic condensation with eosinophilia that resembles "ischemic cell change," and from 12 hours, many other Purkinje cells show marked watery swelling. Astrocytes begin to swell from 0.5 hours after injection and show hypertrophy of organelles from 6 hours. Also from 6 hours onward, discrete foci of necrosis appear in the granule cell layer, while permeability of overlying meningeal vessels to horseradish peroxidase becomes evident at 8 hours. All changes are more severe in vermis and paravermis. Despite widespread loss of Purkinje cells, the animals' behavior becomes almost normal within a week. While tremor occurs with doses of 1.5 and 0.5 mg/kg, cellular damage is minimal. The tremor mechanism differs from that of harmaline since destruction of inferior olivary nuclei abolishes neither the tremor response to penitrem A nor the cellular damage. No morphological changes are found in other brain regions. The affinities of penitrem A for high-conductance calcium-dependent potassium channels and for gamma-aminobutyric acid receptors with the probability of resultant excitotoxity are considered to be important underlying factors for these changes.

Neuropathy target esterase: immunolocalization to neuronal cell bodies and axons.

Determination of the molecular mechanisms involved in organophosphate-induced axonopathy may help to elucidate those involved in normal axonal maintenance and in other neurodegenerative conditions. In this study we aimed to define the cellular distribution of neuropathy target esterase, the primary target protein for neuropathic organophosphates. A synthetic peptide corresponding to the sequence of a proteolytic fragment of neuropathy target esterase purified from chicken brain was used to raise a rabbit antiserum designated R28. The antiserum was shown by immunoprecipitation and western blotting of brain extracts to react with a polypeptide of the expected molecular size (155,000 mol. wt); this reaction was blocked by preincubating the antiserum with the immunizing peptide. Prominent intracellular immunostaining by R28 was seen in neuronal cell bodies and, in some cases, proximal axon segments in frozen sections of chicken brain cortex, optic tectum, cerebellum, spinal cord, and dorsal root ganglia. Cells with glial morphology were not immunostained, neither were normal sciatic nerve or motor end plates. However, 8-12 h following sciatic nerve ligation, immunoreactive material was seen to accumulate both proximal and, to a lesser extent, distal to the ligature, indicating that neuropathy target esterase undergoes fast axonal transport. No gross qualitative or quantitative changes in the above pattern of neuropathy target esterase immunoreactivity were detected in tissue obtained from chickens one or three days following treatment with a neuropathic organophosphate. The presence of neuropathy target esterase in essentially all neurons indicates that the selective vulnerability of long axons to neuropathic organophosphates is dependent on factors additional to the presence of the target protein.

Selective damage to the cerebellar vermis in chronic alcoholism: a contribution from neurotoxicology to an old problem of selective vulnerability.

The curiously consistent localization of cerebellar cortical damage in chronic alcoholism is re-evaluated in the light of selective damage, with a similar topography in the cerebellar vermal region, in superficial siderosis in man and in experimental animals exposed to certain toxic substances. Attention is drawn to the capacity for Purkinje cell dendrites and Bergmann glia to extract materials from the CSF, and to the close anatomical relationships of the susceptible lobules I-II, IX and X to the roof of the IVth ventricle and to the cistern of the great cerebral veins. This restriction of damage to vermis and paravermis may reflect some compartmentalization of CSF flow within leptomeninges, consistently increasing exposure of these cerebellar surfaces to materials circulating in the CSF. In other circumstances when this pattern of damage is encountered it raises the question as to whether other environmental agents, gaining access to the CSF, may be similarly distributed.

Increasing or decreasing nervous activity modulates the severity of the glio-vascular lesions of 1,3-dinitrobenzene in the rat: effects of the tremorgenic pyrethroid, Bifenthrin, and of anaesthesia.

To test the hypothesis that altered neuronal activity may influence the extent and severity of the glio-vascular lesions produced by 1,3-dinitrobenzene (DNB), rats were either given the tremorgenic pyrethroid, Bifenthrin, or anaesthetised during various dosing schedules of DNB. When compared with controls dosed only with DNB, Bifenthrin tremor made both the ataxia and other functional effects caused by DNB more pronounced. Lesions in the brain stem were made significantly more severe and widespread across three dose levels of DNB. Centres such as facial nuclei, motor nuclei of fifth nerve, subthalamic nuclei and mamillary bodies, not damaged by DNB alone, were also affected in some animals. In contrast, general anaesthesia by either isoflurane ur urethane decreased the severity of the lesions, this being more pronounced with urethane. The character of the tissue changes, however, was not altered by these additional procedures. These findings support the suggestion that neuronal activity is one important determinant of the selective vulnerability of sensitive brain stem nuclei to glio-vascular damage from DNB intoxication.

Neurotoxic effects of gadopentetate dimeglumine: behavioral disturbance and morphology after intracerebroventricular injection in rats.

PURPOSE: To determine the neurotoxic potential of gadopentetate dimeglumine in an animal model that allowed the agent to avoid the blood-brain barrier. Gadopentetate dimeglumine is known to produce functional changes when injected into the cerebrospinal fluid, and we hypothesized that such changes might be associated with morphologic damage. METHODS: Conscious rats, surgically prepared with a lateral ventricular cannula, were given a slow injection of gadopentetate dimeglumine into the lateral ventricle, and behavioral and neuropathologic changes were noted. RESULTS: Gadopentetate dimeglumine produced signs of acute neurotoxicity over several hours (stereotyped movements and myoclonus), medium-term signs over several days (ataxia and tremor), and neuropathologic changes over 24 hours, with reactive changes persisting for 42 days. All of the above were dose-dependent over the range of 2.5 to 15 mumol/g brain. The lowest dose producing morphologic or behavioral changes was 5 mu mol/g brain. Iso-osmotic, isovolumetric injections of sucrose produced no such effects. Focal lesions occurred within the thalamus, brain stem, and spinal cord, with necrosis of glia, loss of myelin, and, usually, sparing of neurons and nerve fibers. Persisting ataxia was always associated with brain stem or spinal cord lesions. CONCLUSION: Intraventricular administration of contrast medium allows toxicity to be evaluated in areas such as the spinal cord that are not accessible by osmotic opening. While it is unlikely that these toxic effects would be seen at the doses used for clinical imaging by the intravenous route, gadopentetate dimeglumine clearly has some neurotoxic and neuropathologic potential. Although the acute excitation could be attributed to a transiently high local concentration of the agent at the injection site, the lesions were widely distributed through the brain and spinal cord and may reflect a region-specific neurotoxic action, possibly related to central pontine myelinolysis.

The topography, structure and incidence of mineralized bodies in the basal ganglia of the brain of cynomolgus monkeys (Macaca fascicularis).

Whole coronal slices from 6 levels of the brain of 16 cynomolgus monkeys (8 control and 8 treated by daily gavage with a novel pharmaceutical agent for one year) were examined histologically. Mineralized bodies were identified only in coronal sections passing through the optic chiasma and mammillary bodies. Identical mineralized structures were present in the basal ganglia of both control and treated animals. The majority were seen in the globus pallidus, occasionally in the putamen and once in the nearby caudate nucleus. These structures were partially ferruginated and also partially calcified. They appeared to arise in relation to small vessels. They are part of the naturally occurring background pathology of several species of non-human primates and the incidence in this study (3/8 control and 5/8 treated) was approximately what might be expected from reports in the literature. Mineralized bodies of the basal ganglia of primates represent a spontaneous lesion with a characteristic distribution. They may cause confusion in interpretation of toxicological studies if their natural occurrence is not appreciated.

Encephalomyelopathy in young cats.

Nineteen cats, aged three to 16 months, developed neurological signs including hindleg paralysis, head shaking, nystagmus, defective vision and reduced proprioception. Most of the animals were in cat colonies in research centres and were derived from specific pathogen-free stock. One was referred from veterinary practice. Over 40 per cent of litters could be affected constituting a serious commercial loss. Wallerian degeneration affected long tracts in the spinal cord and variously in the brain stem and cerebral white matter. In seven animals there was loss of Purkinje cells in the cerebellum and in eight there was neuronal loss in the spinal cord. Gliosis accompanied all changes. Although no viral agent was isolated the clinical pattern of the disease and evidence from other cases reported in the literature suggest an infectious cause.

Pathogenic factors underlying the lesions in Leigh's disease. Tissue responses to cellular energy deprivation and their clinico-pathological consequences.

In a search for pathogenic factors that might play roles in the selective vulnerability of brain regions to the lesions of Leigh's disease, archival material from 20 cases of this condition, dying between 1975 and 1992 and aged from 4 days to 11.75 years at death, have been examined. Attention was paid to the topography of the lesions, their nature and timing in the evolution of the disease, the clinico-pathological correlations and the ages of the subjects at onset and at death. The following observations would appear to be explicable in terms of the present understanding that impairment of cellular energy generation is known to be defective in some, and probably all, cases. (i) The characteristic lesion of this disease is symmetrical vasculonecrotic damage affecting several brainstem centres, the topography of which is variable and may partly depend upon the age of the individual. (ii) Early features of this lesion are indistinguishable from a small partial infarction and progress similarly. The size of the damaged area is generally related to the size of the region affected. There is no haemorrhagic component and haemosiderin is not at any time found, unlike the lesions of Wernicke's disease. (iii) The process is episodic and total tissue damage is thus cumulative. More than one episode of damage may be seen in a region, changes of clearly different ages being often present together. (iv) In some regions the lesions appear to be age dependent, e.g. inferior olivary nuclei, and may be related to behavioral development and neuronal activity. Other regions show damage at any age, e.g. substantia nigra. (v) Myelin and sometimes axon loss in optic pathways is usually central, the periphery being spared. This occurred in more than half the cases and may represent a partial infarct-like change. (vi) The characteristic dorsal spinal column degeneration is always associated with focal necrosis of central grey and white matter; this also resembles a partial infarction with secondary ascending degeneration. (vii) Massive myelin loss in the centra semiovalia occurred in one-third of the cases, with or without cavitation, often in association with spongy myelin changes elsewhere. A mild general spongy change in myelin alone occurred in two cases. The massive lesions are focal, infarct-like and analogous to Binswanger's disease. (viii) Selective neuronal loss, common in some mitochondrial disorders, is not a major feature of Leigh's disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Is Purkinje cell loss in Leigh's disease an excitotoxic event secondary to damage to inferior olivary nuclei?

In a series of 17 cases of Leigh's disease it has been observed that there is a close correlation between damage to the inferior olivary nuclei by vasculo-necrotic change and loss of Purkinje cells in the cerebellar cortex. It is suggested that this association may be explained on the basis of the selective loss of climbing fibres causing increased firing activity of Purkinje cells with consequent excessive entry of calcium ions. In these circumstances control of calcium ion regulation in the presence of reduced energy production, which is the basis of this metabolic disease, would be expected to put these cells' survival seriously at risk.

beta-Amyloid precursor protein fragments and lysosomal dense bodies are found in rat brain neurons after ventricular infusion of leupeptin.

Infusion of the serine and thiol protease inhibitor, leupeptin, is known to cause a reduction of fast axoplasmic transport, and accumulation of lysosomal dense bodies in neuronal perikarya. We have found these dense bodies in hippocampal and cerebellar neurons contain ubiquitin conjugated proteins. We now demonstrate that these accumulated neuronal lysosomes are labeled by antisera to the cytoplasmic, transmembrane and extracellular domains of beta-amyloid precursor protein (APP) and also that lysosomal APP is fragmented. This in vivo model confirms that neurons can process APP via a lysosomal pathway and that neuronal lysosomes in vivo contain both N-terminal and potentially amyloidogenic C-terminal fragments of APP. We also show that increased APP immunoreactivity after leupeptin treatment is seen first in neurons and later in astrocytes. On recovery from infusion, APP N-terminal immunoreactivity diminishes whilst C-terminal reactivity remains in neurons. These findings are consistent with production in whole brain of potentially amyloidogenic fragments of APP within neuronal lysosomes in perikarya and dendrites implying that neurons may play a role in forming the beta-amyloid of plaques.

Selective vulnerability in acute energy deprivation syndromes.

The topography and cellular events in the experimental lesions caused by chlorosugars, 6-aminonicotinamide, dinitrobenzene and tribromoimidazole in animals are considered in relation to those features in human acute thiamine deficiency (Wernicke's) encephalopathy and for comparison in Leigh's disease. The topography and cellular changes when closely examined are different and particular to each condition, although there is a basic cellular process common to all. The pathogenesis of each condition must be considered as multifactorial and a search for the factors responsible for the neuronal and cellular selective vulnerability of different regions of the neuraxis will lead us to understanding the pathogenesis of the disease process in each instance. The experimental models offer much for the understanding of the human conditions, particularly in the search for satisfactory therapeutic strategies.

The neurotoxicity of alpha-chlorohydrin in rats and mice: II. Lesion topography and factors in selective vulnerability in acute energy deprivation syndromes.

Mice and rats have been found almost equally susceptible to (R, S)-alpha-chlorohydrin neurotoxicity, but in rats the distribution of lesions in the neuraxis is less widespread. The topography of the brain lesions shows an incomplete relationship to the regional hierarchy of local glucose utilization in rats and local cerebral blood flow in mice, suggesting that other, unknown, factors also play roles in determining this. Evidence suggesting progressive tonotopic selective vulnerability was found in inferior colliculi in rats given five doses of 50 mg/kg/day. Distinct differences in the patterns of damage to brain stem centres found with chlorohydrin by comparison with other acute energy deprivation syndromes, despite the proximity of the metabolic lesions along the energy generation pathway, suggests there are other unrecognized factors that play a role in determining whether a neuronal centre is at risk or not.

Routes of excretion of neuronal lysosomal dense bodies after ventricular infusion of leupeptin in the rat: a study using ubiquitin and PGP 9.5 immunocytochemistry.

To determine the rate and routes of removal of lysosomal, lipofuscin-like dense bodies from neurons, the protease inhibitor, leupeptin, was infused into the lateral ventricle of rats for up to nine days. After seven days a number of animals were then allowed to recover. The formation and later disappearance of dense bodies was followed by morphology and immunocytochemistry. After 48 h of infusion lysosomal dense bodies in large numbers appeared in cortical, hippocampal and cerebellar neurons, which also showed increased ubiquitin immunoreactivity, as well as in other cell types. By 3-4 days ubiqutin-immunoreactive dense bodies were equally distributed between neurons and astroglia. After seven to nine days of infusion ubiquitin immunoreactive dense bodies filled neuronal perikarya, dendrites and expanded initial segments of many axons and were abundant in glial processes. All dense bodies studied by electron microscopy were ubiquitin immunoreactive. After four days of recovery dense bodies were markedly fewer in neuronal perikarya, and virtually all were now within glial processes. From 7 to 28 days of recovery, when most neurons appeared normal, lipofuscin bodies remained in axon initial segments and in reduced numbers in glial processes, particularly around blood vessels and beneath the pia of hippocampus and of cerebellar cortex. Thus, neurons probably have a steady passage of short lived proteins through the lysosomal excretory pathway. The observed temporal sequence of events on recovery suggests that secondary lysosomes probably pass rapidly from neuronal perikarya and dendrites to astrocytes and thus to the vascular bed or pia-arachnoid. The mechanism of cell-to-cell transfer is not clear from this study.