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.

Blood-brain barrier integrity and host responses in experimental metastatic brain tumours.

The effect of brain implants of Walker 256 carcinosarcoma tumour cells on the integrity of the blood-brain barrier was examined in rats using labelled albumin, horseradish peroxidase and trypan blue. Barrier integrity was intact within 1 hour of implantation but was gradually reduced within the tumour after 3.5 days. This was related to alterations in the fine structure of the tumour capillaries. Dissociated tight junctions were apparent within the tumour centre, but no fenestrated endothelium or gap junctions were observed by electron microscopy.

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.

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.

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.

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.

Spinal changes in the neuropathy of thallium poisoning. A case with neuropathological studies.

Thallium poisoning in a man of 32 years resulted in typical peripheral neuropathy, skin rash and alopecia. Post-mortem studies showed widespread loss of peripheral nerve fibres and chromatolysis in spinal neurones. The distal nature of the nerve fibre degeneration in thallium poisoning and the sensitivity of long fibres is emphasized in this case.

Morphometric effects of vincristine on nerve regeneration in the rat.

Administration of vincristine (200, 100 or 50 micrograms/kg/week) for 6 months during regeneration of the sciatic nerve after crush injury caused a dose-dependent reduction in nerve fibre size and failure of removal of myelin debris. Successfully regenerating neurites showed an unusual amount of shape distortion. The ratio of myelin sheath thickness to axon circumference was reduced, but the ratio of myelin sheath thickness to axon area was normal. Microtubule concentration was diminished in axons, but neurofilament density was unaffected. Unmyelinated axons were reduced in number but their axon diameter distribution was not affected. Fibres on the non-crushed side appeared normal. The toxicity of vincristine to regenerating nerves is probably related to increased blood-nerve permeability occurring both at the site of crush and along the degenerating nerve.

Vascular leakage in the dorsal root ganglia of the rat, studied with horseradish peroxidase.

Horseradish peroxidase (HRP) was found to pass from ganglionic blood vessels into the extracellular space of dorsal root and Vth nerve ganglia within 2 min of intravenous injection in the rat. By 5 min, the tracer had penetrated into perineuronal and periaxonal spaces. A brisk macrophage response occured, and these cells rapidly engulfed the tracer so that it has almost completely disappeared from the extracellular space 2-3 hr. Fenestrated blood vessels, with and without diaphragms were seen within ganglia. On the basis of the pattern of tracer distribution following post-fixation perfusion of HRP, it was concluded that the fenestrations and endothelial intercellular clefts were probably the most important route of tracer leakage. Because of the foreign and possibly toxic nature of the tracer, its movements and fate may not parallel that of normally extravasated proteins.

Lesion localisation: implications for the study of functional effects and mechanisms of action.

In some toxic neuropathies we see only distal "dying back" of longer and larger diameter axons, the perikaryon appearing to be intact. Some of these are the result of chronic energy deprivation, others are not. In other neuropathies sensory and autonomic neuron cell bodies are damaged without apparent selectivity. Toxic neuropathies often mimic the neurological effects of vitamin deficiencies by causing juxtaposed lesions in the same metabolic pathways. In acute energy deprivation toxic syndromes in the CNS, the pattern of damage is restricted to specific grey centres: there are variations in this pattern according to the site of the metabolic lesion, the species studied, the development of seizures, and other factors. Such toxic responses mimic human and animal disease states, such as Wernicke's encephalopathy and Leigh's disease, both of which are essentially acute energy deprivation syndromes.

Glial cell intrusions actively remove detritus due to toxic chemicals from within nerve cells.

It has been observed that when neurons are acutely damaged by toxic chemicals leading to accumulations of effete materials, glial supporting cells insert cytoplasmic processes into neuronal cytoplasm and appear to transfer this material into themselves. Essentially the same phenomenon has now been seen in several situations, namely in peripheral nerve axons in a number of experimental peripheral nerve intoxications, especially in spinal roots, as well as occasionally in normal axons in paranodal regions and more frequently above a nerve ligation. It has been seen, too, in cerebellar Purkinje cells after acrylamide intoxication and in hippocampal pyramidal neurons and in neurons of the pyriform cortex after triethyllead and trimethyltin intoxications. A similar process may also be taking place regularly both in normal and chemically damaged spinal ganglion cells through their satellite cell sheath. While probing of neurons by glia has also been noted normally in pre-synaptic regions of mammalian neurons as well as in the perikarya of certain goldfish neurons, the purpose for this is less apparent. Such findings in relation to removal of bulk residual material from neurons raise intriguing questions as to the signals required between cells to enable such evidently cooperative intercellular events to take place, and whether this process, that is apparently so inefficient in removing lipofuscin pigment from ageing neurons, may not, perhaps, be adversely influenced by environmental agents.

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.

Functional/metabolic modulation of the brain stem lesions caused by 1,3-dinitrobenzene in the rat.

To determine whether neuronal activity plays a role in the localisation of brain stem lesions in 1,3-dinitrobenzene intoxication we produced asymmetrical changes in auditory input by rupturing the left tympanic membrane in Fischer rats. This raised the auditory threshold on that side from 57-63 dB to 104-122 dB. It also decreased glucose utilisation in the ipsilateral cochlear nucleus and significantly increased utilisation in the contralateral nucleus, resulting in a relative deficit of 72 +/- 6%. Similarly, tympanic membrane rupture led to decreased glucose utilisation in the contralateral and increased utilisation in the ipsilateral inferior colliculus. Additional exposure to "white noise" prevented the decrease in glucose utilisation in the contralateral inferior colliculus. Dosing with dinitrobenzene (10 mg/kg in 4 doses over 48 hr) to otherwise normal rats produces symmetrical vasculonecrotic lesions in these regions, but in animals with left tympanic membrane rupture the severity of morphological changes in the ipsilateral cochlear nucleus and the contralateral inferior colliculus were substantially reduced. Additional exposure to "white noise" increased the degree of damage in the ipsilateral cochlear nucleus and contralateral inferior colliculus. These findings indicate that altered auditory function in rats, with its associated metabolic consequences exercises a significant role in the development of brain stem damage in auditory pathways following dinitrobenzene intoxication.

The 'dying back' process. A common denominator in many naturally occurring and toxic neuropathies.

The "dying back" process can be defined as a pathological changes affecting certain neurons in a number of systematized degenerative conditions. Examples exist to illustrate the nature of this process, which is unique to nervous tissue, and there is an association of this process with certain chronic vitamin-deficiency syndromes and some important neurotoxic chemicals. Albeit largely speculative, one can attempt to group the conditions showing the dying back process in terms of putative metabolic lesions. Although this attempt is admittedly only a first approximation, it enables us to look ahead to a future understanding of the metabolic problems of long neurons and how their selective degeneration comes about.

Chronic low-dose exposure of sodium nitrite in VM-strain mice: central nervous system changes.

1. There is suggestive evidence that nitrite may be a causative factor in cerebral glioma. 2. To test this hypothesis we selected the VM mouse strain, known for its susceptibility to spontaneous glioma formation, and exposed 300 animals to 0.2% sodium nitrite in their drinking water. One hundred of this group were exposed both in utero and throughout their adult lives. The remaining 200 animals received nitrite from the time of weaning. A further 200 mice were used as controls and received distilled water. 3. All animals were maintained until their natural death and were then subjected to autopsy and routine histological examination. 4. There was no excess of nervous system tumours in the experimental groups.

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.