Granule Cell Necrosis Research Articles

Another umbrella murder? \u2013 A rare case of Minamata disease

We report a rare case of fatal intoxication in a 40-year-old man caused by injection of a fluid containing organic mercury, allegedly in an attack with a syringe fixed to the tip of an umbrella. The man suffered from severe neurological symptoms and progressive multiorgan failure and died 10 months later in refractory status epilepticus. Autopsy revealed severe brain atrophy and non-specific kidney damage. Neuropathological examination showed neuronal loss especially in the occipital lobe, distinct granule cell necrosis in the cerebellum and Wallerian degeneration in the brainstem. Postmortem toxicological analysis revealed extremely increased levels of mercury in liver and kidney tissue as well as methylmercury levels in peripheral blood.

Re-evaluation of Archival Material for Neuronal Cell Injury Produced by l-2-Chloropropionic Acid in the Rat Brain

Previous studies have shown that l-2-chloropropionic acid ( l-CPA) produces necrosis to cerebellar granule cells with some associated Purkinje cell damage in the rat [Neurotoxicology 17 (1996) 471]. We have re-evaluated the neuropathology using the original sections and fresh sections from archived brain material from rats treated with l-CPA at different ages, times after dosing and the following prior treatment with the N-methyl- d-aspartate (NMDA) receptor antagonist, MK-801. In addition we have determined the lobular distribution of cerebellar granule cell necrosis produced by l-CPA. Using Fluoro-Jade staining to detect degenerating neurons, we have identified three new brain regions that show neuronal cell necrosis as a result of exposure to l-CPA, these are the medial habenular nucleus, pontine gray and inferior olivary nucleus. The neuronal cell degeneration was confirmed in conventional haematoxylin and eosin stained sections and in some cases by glial fibrillary acidic protein staining for reactive gliosis. The neuronal cell necrosis at these new sites was both time and dose dependent; young 22-day-old rats, which are refractory to l-CPA-induced cerebellar granule cell necrosis, did however show some neuronal cell degeneration in the medial habenular, pontine gray and inferior olivary nuclei. Treatment of rats with MK-801 30 min prior to l-CPA, afforded complete protection against the neuronal cell injury in the medial habenular, pontine gray and inferior olivary nuclei, similar to that previously reported for the cerebellum, supporting an excitotoxic mechanism of neuronal cell death. In the cerebellum the lobular distribution of the granule cell loss was not uniform, more severe granule cell loss occurring in lobules 1–4 and 9a + b. This localization exactly mirrors that seen previously in the cerebellum of rats given l-CPA and examined by magnetic resonance imaging (MRI). The basis for the neuronal cell loss in the medial habenular nucleus, pontine gray and inferior olivary nucleus, in addition to the major site in the cerebellum, and the sensitivity of particular cerebellar lobes is not currently understood. Anatomical connections between the sites of injury and their likely neurotransmitter use are discussed.

Alterations in cyclin A, B, and D1 in mouse dentate gyrus following TMT-induced hippocampal damage.

The interactions of glia and neurons during injury and subsequent neurodegeneration are a subject of interest both in disease and chemical-induced brain injury. One such model is the prototypical hippocampal toxicant trimethyltin (TMT). An acute injection of TMT (2.0 mg/kg, i.p.) to postnatal day 21 CD-1 male mice produced neuronal necrosis and loss of dentate granule cells, astrocyte hypertrophy, and microglia activation in the hippocampus within 24 hrs. Neuronal necrosis and microglia differentiation to a phagocytic phenotype is temporally correlated with peak elevations in TNF-alpha, cyclin A2, cyclin B1 and cyclin D1 at 72 h post-TMT. TNF-alpha mRNA levels were significantly elevated in the hippocampus by 12 h and remained elevated for 72 h. mRNA levels for cyclin A2 and cyclin B1 were elevated by approximately 2-fold at 72 h. Immunohistochemistry suggested a cellular localization of cyclin A to microglia in the region of neuronal necrosis in the dentate, cyclin B in glial cells in juxtaposition to neurons in the hilus of the hippocampus and cyclin D1 to non-glial cells in the dentate. mRNA levels for cyclin D1 were elevated approximately 1.5-fold by 72 h as determined by RNase protection assay. No changes were seen in mRNA levels for cyclins E, F, G1, G2, H or I nor cyclin dependent kinases. These elevations are not associated with proliferation of microglia as determined by BrdU incorporation and Ki-67 immunohistochemistry. Upregulation of cell cycle genes was associated with cellular processes other than proliferation and may contribute to the differentiation of microglia to a phagocytic phenotype. These data suggest an integrated role for cell cycle regulation of neural cells in the manifestation of hippocampal pathophysiology.

2-Halopropionic Acid-induced Cerebellar Granule Cell Necrosis in the Rat: In Vivo and In Vitro Studies

Daily oral administration of 2.3 mmol/kg l-2-chloropropionic acid ( l-2-CPA), dl-2-bromopropionic acid (2-BPA) or dl-2-iodopropionic acid (2-IPA) but not dl-2-fluoropropionic acid (2-FPA) produced cerebellar granule cell necrosis in the rat. Twenty four hours after three doses of l-2-CPA or two doses of 2-BPA, animals showed clinical signs of motor incoordination and reduced hindlimb function which was associated with marked cerebellar oedema and cerebellar granule cell necrosis. Biochemical analyses showed a marked increase in cerebellar water and Na + content, and a reduction in cerebellar glutamate and aspartate. 2-IPA at this dose was toxic, the animals not surviving a second dose, histopathology showed hepatic and renal necrosis with mild cerebellar granule cell necrosis. 2-FPA was not neurotoxic after four daily doses. A marked decrease in hepatic and cerebellar non-protein sulphydryl (NP-SH) content was observed 4 h after a single dose of 2.3 mmol/kg l-2-CPA, 2-BPA and 2-IPA but not 2-FPA. Daily doses of 2-BPA for 3 days produced a sustained 50% depletion in cerebellar NP-SH. In vitro, l-2-CPA, 2-BPA and 2-IPA produced glutathione (GSH) depletion in the presence of rat liver cytosol, while 2-FPA did not. Depletion of GSH in the presence of cerebellar cytosol was only observed with 2-IPA. Studies using primary cultures of rat cerebellar granule cells, showed that all analogues produced a concentration dependent loss of cell viability. Mean EC 50 values for 2-FPA, l-2-CPA, 2-BPA and 2-IPA toxicity were 1.7, >10, 0.5 and 0.3 mM, respectively, for 24 h continuous exposure. MK-801 and Vitamin E afforded protection against l-2-CPA-induced cytotoxicity but not against the other analogues. In summary, in addition to l-2-CPA, both 2-BPA and 2-IPA produce cerebellar granule cell necrosis in the rat. Depletion of GSH in the cerebellum may be contributory factor in the cascade of events leading to neurotoxicity.

The effect of postnatal age on L -2-chloropropionic acid-induced cerebellar granule cell necrosis in the rat

L-2-Chloropropionic acid (L-2-CPA) selectively damages the cerebellum in adult rats. The rat cerebellum continues to develop postnatally during the first 4 weeks of life. In this study we examined the neurotoxic effect on rats of increasing postnatal age. Daily oral dosing of rats aged 56 days with 250 mg/kg per day of L-2-CPA for 3 days produced necrosis to neurons in the cerebellar granule cell layer and to neurons in the medial/ventral region of the habenular nucleus. Rats aged 22 days were resistant to the cerebellar toxicity while rats aged 32 days and older were sensitive. A single large oral dose of 500 or 750 mg/kg L-2-CPA produced no clinical signs of neurotoxicity or lesions in the cerebellum 48 h after dosing in 22-day-old rats. Daily dosing of 22-day-old rats at 250 mg/kg per day L-2-CPA for 10 days also produced no signs of neurotoxicity or reduction in body weight gain, although histological examination of the brain revealed slight neuronal cell necrosis in the granule cell layer of the cerebellum with a minimal effect in the medial/ventral region of the habenular nucleus. In contrast, daily dosing of rats aged 32, 38, 48 and 58 days with 250 mg/kg per day of L-2-CPA for 3 days produced clear signs of neurotoxicity which were associated with reduced body weight gain and loss of hindlimb function. In these rats there was clear evidence of neuronal cell loss in the cerebellar granule cell layer and medial/ventral region of the habenular nucleus. This study showed that the postnatal developing cerebellum is resistant to L-CPA-induced injury in rats up to 25 days of age, but becomes vulnerable to the toxicity by 32 days of age. The basis for the resistance of the developing cerebellum to L-CPA is discussed.

MRI studies of the neurotoxic effects of l-2-chloropropionic acid on rat brain

l-2-Chloropropionic acid (l-CPA) is selectively toxic to rat cerebellar granule cells; necrosis is first observed about 36 hours after administration of l-CPA (750 mg/kg p.o.) becoming more marked by 48 h. Parallel to the onset of cell death an increase in cerebellar water content and sodium concentration has been reported suggesting an oedematous reaction. In this study T2-weighted (T2WI) and diffusion weighted (DWI) imaging were used to detect the development of neuronal damage in the cerebellum of rats as a result of exposure to l-CPA. T2WI and DWI were not able to detect cerebellar abnormalities at 37 h post-dosing except for a slight swelling of the cerebellum. However, at 48 h post-dosing when cerebellar swelling and granule cell necrosis were marked, T2WI and DWI hyperintensities were observed in the cerebellum. Therefore, under the conditions of this study, MRI was not able to detect abnormalities in the cerebellum prior to the onset of the clinical signs of neurotoxicity or at the time of early histological changes. T2WI also suggested a marked increase in the amount of fluid in the ventricular system of rats 37 and 48 h after dosing; fluid accumulation was observed in all animals studied whether or not necrosis was detected. The occurrence of T2WI hyperintensity in the forebrain lead us to discover a new lesion in the habenular nucleus.

Neuroprotective effects of MK‐801 on l‐2‐chloropropionic acid‐induced neurotoxicity

L-2-Chloropropionic acid is selectively toxic to the cerebellum in rats; the granule cell necrosis observed within 48 h can be prevented by prior administration of MK-801. Short-term treatment (2 h) with L-2-chloropropionic acid has also been shown to activate the mitochondrial pyruvate dehydrogenase complex in fasted adult rats. This study aimed to investigate the effect of prior exposure to MK-801 on the biochemical and neurotoxicological effects of L-2-chloropropionic acid. Extracts were prepared from the forebrain and cerebellum of animals that had been treated with L-2-chloropropionic acid, with and without prior treatment with MK-801, and were analysed using magnetic resonance spectroscopy and amino acid analysis. Glucose metabolism was studied by monitoring the metabolism of [1-(13)C]-glucose using GC/MS. L-2-Chloropropionic acid caused increased glucose metabolism in both brain regions 6 h after administration, confirming activation of the pyruvate dehydrogenase complex, which was not prevented by MK-801. After 48 h an increase in lactate and a decrease in N-acetylaspartate was observed only in the cerebellum, whereas phosphocreatine and ATP decreased in both tissues. MK-801 prevented the changes in lactate and N:-acetylaspartate, but not those on the energy state. These studies suggest that L-2-chloropropionic acid-induced neurotoxicity is only partly mediated by the NMDA subtype of glutamate receptor.

The absence of cerebellar granule cell necrosis in the mouse following L-2-chloropropionic acid administration.

Oral administration of L-2-chloropropionic acid (L-CPA) to rats either as a single dose (750 mg/kg) or daily doses (250 mg/kg per day for 3 days) produces selective necrosis to the granule cell layer of the cerebellum. As part of a study to understand the mechanism of this selective toxicity, we investigated the toxicity of L-CPA and a related analogue, DL-2-bromopropionic acid to the mouse with particular emphasis on the brain. Following a single oral dose (up to 1000 mg/kg), or daily oral doses of 250 mg/kg per day L-CPA up to maximum tolerated doses, produced no evidence of neurotoxicity. Similarly, daily oral doses of DL-2-bromopropionic acid at 250 mg/kg per day produced no evidence of neurotoxicity. The basis for the lack of response was explored by examining the metabolism and disposition of L-[2-14C]-CPA in the mouse. Following a single oral dose of 250 mg/kg L-CPA, radioactivity was rapidly absorbed from the gastrointestinal tract into the blood stream. Peak plasma concentrations of radiolabel and L-CPA occurred within 2 h of dosing at about 1.8 mM, and were then lost from the plasma with a half-life of 1 h. The only metabolite detected in the plasma was 2-S-cysteinylpropanoic acid derived from the glutathione conjugate. About 39% of the dose was excreted in the urine in the first 24 h, mainly as 2-S-cysteinylpropanoic acid with only a small amount of unchanged L-CPA. The remaining radiolabel from L-CPA was excreted in the faeces (26%) and exhaled as carbon dioxide (about 14%) over 72 h. Radiolabel from L-[2-14C]-CPA was present in the cerebellum at a peak concentration of 1 mM 1-2 h after dosing and then was lost more slowly than from the plasma. Measurement of non-protein sulphydryl content in the brain, liver and kidneys showed a decrease in the liver and kidneys 4 h after dosing which recovered fairly rapidly, while a more prolonged decrease was found in the brain, especially the cerebellum. Our studies show that the mouse is refractory to cerebellar injury following treatment with L-CPA and DL-2-bromopropionic acid. The mouse appears to metabolize and excrete L-CPA as its glutathione-derived conjugate(s) more rapidly than the rat, thereby limiting the availability of L-CPA to the cerebellum, which may account for the absence of neuronal cell injury.

Mammalian neuropathology: a paradigm for the study of teleost neurotoxicity?

In vertebrates, neurotoxicants are chemicals whose primary toxic effect is on the nervous system, either the central nervous system (CNS {brain and spinal cord}) or the peripheral nervous system (PNS {spinal roots, sensory ganglia, peripheral nerves}). Pathological aspects of neurotoxicity are well documented in experimental mammals. We illustrate the neurotoxicity of two chemicals, which both produce lesions in selective regions of the rat brain, a thiocarbamate herbicide (cycloate) and an industrial chemical, chloropropionic acid (L-CPA). Cycloate exposure results in neuronal cell death by apoptosis in specific areas of the forebrain, whereas L-CPA produces granule cell necrosis in the cerebellum. Our studies in rodents demonstrate the discriminative power of neuropathological tools for neurotoxicity. Many agrochemicals/industrial chemical products enter the aquatic environment, some with the potential for neurotoxicity. The teleost CNS is the most primitive among the vertebrates but has features in common with the CNS of more advanced forms. Even allowing for differences in phylogenetic organisation, neuropathological tools should have the potential to identify neurotoxicants that affect teleosts.

Multifocal cerebellar granular layer necrosis in traumatically head-injured lambs.

In a blunt, nonmissile, head impact model of traumatic brain injury in 4-5-week-old Merino lambs, multiple foci of internal granular layer necrosis were found in all 10 impacted animals. This lesion has not previously been reported after human or animal head injury. Temporal lobe impact contusions, predominantly microscopic (8/10) and contralateral contusions (2/10), parenchymal (10/10) and subarachnoid (10/10) hemorrhage, and widely distributed axonal injury were also observed. Although the precise pathogenesis of this focal granule cell necrosis and often attendant red cell change in Purkinje cells was unclear, an ischemic etiology due to trauma-related vascular damage is postulated.

Neuroprotection afforded by MK-801 against l-2-chloropropionic acid-induced cerebellar granule cell necrosis in the rat

Administration of a single oral dose of 750 mg/kg l-2-chloropropionic acid (L-CPA) to rats produces marked necrosis to the granule cell layer of the cerebellum by 48 h after dosing. Associated with the neuropathology the rats show locomotor impairment and a loss of body weight and a significant increase in cerebellar water and sodium content, indicating an oedematous reaction. Cerebellar aspartate and glutamate concentrations were reduced, while glycine and glutamine concentrations were increased after this treatment. Administration of the N-methyl-d-aspartate (NMDA) receptor channel antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), 30 min prior to L-CPA at a dose of 0.5, 1 or 5 mg/kg i.p. prevented the necrosis to the granule cell layer of the cerebellum and the signs of motor incoordination. Similarly there was no loss in cerebellar aspartate or glutamate concentration or increase in water or sodium content. Prior treatment with MK-801 at 0.1 mg/kg did not afford protection against the neurotoxicity. Post-treatment with 1 mg/kg MK-801 up to 1 h after administering L-CPA afforded complete neuroprotection, however if delayed until 2 or 6 h it gave only partial protection, and after 12 h it gave no protection. Administration of MK-801 alone at 5 mg/kg i.p., did not alter water content, sodium concentration, aspartate or glutamate concentrations in the cerebellum. In conclusion, we have shown that MK-801 given prior to and 1 h after L-CPA can afford complete neuroprotection, suggesting that a sub-population of NMDA receptors located on granule cells in cerebellum play a key role in mediating the selective toxicity of this chemical to the rat cerebellum.

Ultrastructural pathology and cytochemical investigations of L-2-chloropropionic acid-induced neurointoxication of the rat cerebellum.

The objectives of the studies described were to assess the ultrastructural neuropathology, blood-brain barrier (BBB) integrity and calcium status of the cerebellum of rats following a single dose of 750 mg.kg-1 L-2-chloropropionic acid (L-2-CPA). The first indications of intoxication appeared at 36 h when condensation of many granule cells associated with Purkinje cell degeneration and marked astroglial swelling were observed. Some electron-lucent granule cells were also noted lying amongst these condensed forms. Condensed granule cells had swollen, electron-lucent mitochondria, dilated Golgi apparatus and nuclear crenation. Occasionally, areas of the granule cell necrosis were also present at this time. Granule cell condensation probably represents a preliminary and irreversible stage in an excitotoxic process that leads to necrosis. At 48 and 72 h, most granule cells were necrotic, and occasionally, extravasation of both erythrocytes and leucocytes into the expanded extravascular space was observed. Evaluation of the BBB by ultrastructural cytochemical visualisation of horseradish peroxidase injected i.v. 2 min before killing by perfusion fixation showed substantial leakage. At 36 h post-dose, ultrastructural calcium localisation using oxalate/pyroantimonate precipitation demonstrated a substantial increase in calcium pyroantimonate precipitate in mitochondria and other membranous cytoplasmic organelles (especially the Golgi apparatus) in condensed granule cells, but with little in their nuclei. However, their immediate neighbours (of ostensibly normal ultrastructural appearances) contained greater amounts of intranuclear precipitate. Swollen astroglial cells (especially the Bergmann glia) contained considerable quantities of precipitate. A possible excitotoxic mechanism via L-2-CPA-induced NMDA receptor agonism leading to overwhelming calcium influx and disruption of cellular calcium homeostasis is proposed.

Calpain Activation and Not Oxidative Damage Mediatesl-2-Chloropropionic Acid-Induced Cerebellar Granule Cell Necrosis

Possible biochemical events involved inl-2-chloropropionic acid (L-CPA)-induced delayed cerebellar granule cell necrosis followingN-methyl-d-aspartate activation were studiedin vivo.We examined whether the calcium-sensitive proteolytic enzymes, the calpains, may be activated by L-CPA or whether the generation of excess quantities of cytotoxic free radicals may play a role in the neurotoxicity produced by oral administration of L-CPA (750 mg/kg, pH 7.0). Evidence for free radical-induced cellular damage was examined using biochemical approaches such as examining brains from L-CPA-treated rats for increased lipid peroxidation, DNA damage, or protein oxidation. Second, the ability of antioxidants to provide neuroprotective activity against L-CPA-induced neurotoxicity was examinedin vivo.Western blotting using antibodies against spectrin (α-fodrin) demonstrated evidence for calpain (EC 3.4.22.17) activation in the cerebellum, but not in the cerebral cortex of L-CPA-treated rats at 36 and 48 hr after L-CPA dosing. In contrast, there was no evidence for oxidative damage to cerebellar proteins or lipids in L-CPA-treated rat brains compared to controls. We also could not find evidence for DNA damage using the TUNEL method for the detection of single- and/or double-strand breakagein situin L-CPA-treated brains. We examined whether a number of reported antioxidants may be effective against L-CPA-induced neurotoxicity. The aminosteroids U74389G and U83836E, the free radical scavengers 3-methyl-1-phenylpyrazolin-5-one andN-tert-butylphenylnitrone, and the iron chelatorN-ethoxy-2-ethyl-3-hydroxypyridin-4-one were all ineffective in attenuating L-CPA neurotoxicity. We suggest that L-CPA-induced cerebellar necrosis is the result of calpain activation which results in the degradation of cytoskeletal proteins and other proteins necessary for cellular biochemistry. We could find no evidence of oxidative damage to cerebellar proteins, lipids, or DNA as a result of excess amounts of free radicals, and selective antioxidants were unable to provide neuroprotection against L-CPA neurotoxicity, suggesting that oxidative stress does not play a role in the granule cell necrosis.

Failure of glycine site NMDA receptor antagonists to protect against l-2-chloropropionic acid-induced neurotoxicity highlights the uniqueness of cerebellar NMDA receptors

Cultured cerebellar granule cells and cerebellar slices from neonatal rats have been widely used to examine the biochemistry of excitatory amino acid-induced cell death mediated in part by the activation of NMDA receptors. However, the NMDA subunit stoichiometry, producing functional NMDA receptors is different in cultured granule cells, neonatal and adult rat cerebellum as compared to the NMDA receptors in forebrain regions. We have used the l-2-chloropropionic acid ( l-CPA) (750 mg/kg) model of NMDA-medialed selective cerebellar granule cell necrosis in vivo to examine the role of the glycine binding site and possible effect of the NR2C subunit (which is largely expressed only in the cerebellum) on granule cell necrosis. The abilities of various NMDA receptor antagonists were examined in vivo to determine the relative contribution of both glutamate and glycine sites involved in the l-CPA-induced neurotoxicity. The potent neuroprotective, non-competitive NMDA receptor antagonist dizocilpine (MK-801) was compared with glutamate and glycine site NMDA antagonists. We have examined a number of markers for the l-CPA-induced granule cell necrosis. The l-CPA-induced reduction in cerebellar aspartate and glutamate concentrations were used as markers of granule cell necrosis. We also measured the cerebellar water content and sodium concentrations as measures of the l-CPA-induced cerebellar edema that accompanies the granule cell necrosis. Finally the ability of the NMDA antagonists to attenuate the l-CPA-induced reductions in body weight gain and the prevention of the loss in hindlimb function using a behavioral measure of hindlimb retraction were examined. The potent glutamate antagonists, CPP and CGP40116 and dizocilpine prevented the l-CPA-induced locomotor dysfunction and granule cell necrosis as measured by their ability to prevent l-CPA-induced reductions in aspartate and glutamate concentrations. CPR CGP40116 and dizocilpine also prevented the appearance of cerebellar edema following l-CPA administration. In addition, dizocilpine, CPP and CGP40116 were able to partially prevent the l-CPA-induced loss in body weight over the 48 h experimental period. In contrast, none of the glycine partial agonists or antagonists, namely (±)HA-966, d-cycloserine, MDL-29951, DPCQ, MNQX or L-701 252 were able to prevent the l-CPA-induced loss in body weight, l-CPA-induced granule cell necrosis and behavioral disturbances when administered to rats. None of the NMDA antagonists had any effect on the cerebellar neurochemistry when injected alone or had any effect on animal behavior except for dizocilpine, CPP, CGP401 l6 and (±)HA-966 which resulted in a transient sedation for between three and five hours immediately following their administration. In conclusion, we demonstrate that NMDA open channel blockade and glutamate antagonists can provide full neuroprotection against the l-CPA-induced granule cell necrosis. The failure of the glycine partial agonists and antagonists to provide any neuroprotection against l-CPA-induced neurotoxicity in the cerebellum contrasts with their neuroprotective efficacy in other animal models of excitatory amino acid-induced cell death in forebrain regions in vivo. We therefore suggest that the glycine site plays a lesser role in modulating NMDA receptor function in the cerebellum and may explain why cells expressing NMDA receptors composed of NR1/NR2C subunits are particularly resistant to excitatory amino acid-induced neurotoxicity.

The role of glutathione in L-2-chloropropionic acid induced cerebellar granule cell necrosis in the rat.

The role of glutathione (GSH) in the neurotoxicity produced following a single oral dose of 750 mg/kg L-2-chloropropionic acid (L-CPA) has been investigated in rats. L-CPA-induced neurotoxicity was characterised by up to 80-90% loss in cerebellar granule cells and cerebellar oedema leading to locomotor dysfunction. Neurochemically, L-CPA-induced neurotoxicity produced a reduction in the concentration of aspartate and glutamate in the cerebellum and a reduction in the density of NMDA receptors in the cerebellar cortex, whilst there was an increase in cerebellar glycine, glutamine and GABA concentrations. Treatment of rats with buthionine sulfoximine (BSO) at 1 g/kg, i.p., an inhibitor of GSH synthesis, potentiated the toxicity of L-CPA, such that many of the neurochemical markers were significantly different from controls at earlier time points, compared to animals which had received L-CPA alone, and toxicity was also seen in the kidney of BSO plus L-CPA treated rats. In contrast, supplementing GSH concentrations by administration of the isopropyl ester of glutathione (i.p.-GSH) at 1 g/kg, s.c., was able to protect rats against L-CPA neurotoxicity and prevent many of the neurochemical changes. In order to assess whether the depletion of GSH in the rat cerebellum following L-CPA treatment was related to the delivery of cysteine or cystine, the accumulation of [14C] cystine into cerebellar slices was characterised and found to be energy dependent, Na+ independent and obey saturation kinetics with an apparent Km of 77 microM and an apparent Vmax of 450 nmol/g wet weight per h. The accumulation of cystine into cerebellar slices was non-competitively inhibited by the cysteine conjugate of L-CPA with an apparent K(i) of approximately 60 microM, whilst glutamate only inhibited cystine accumulation at doses which were cytotoxic to cerebellar slices. Hence the depletion of GSH in the rat cerebellum, following L-CPA administration, may be due to a reduction in the delivery to the brain of cysteine or cystine, one of the components required for GSH synthesis, by the cysteine conjugate of L-CPA. Our studies show the pivotal role GSH plays in cerebellar granule cell necrosis induced by L-CPA in the rat, indicating that a marked and sustained reduction in cerebellar GSH content by L-CPA may leave granule cells vulnerable to cytotoxic free radical damage leading to cell death, possibly mediated through excitatory amino acids.

Possible role of nitric oxide in the development of L-2-chloropropionic acid-induced cerebellar granule cell necrosis.

1. L-2-Chloropropionic acid (L-CPA) produces selective neuronal cell necrosis in rat cerebellum when administered orally at 750 mg kg-1 that is mediated in part through activation of N-methyl-D-aspartate (NMDA) receptors. Cerebellar granule cell death occurs between 30 and 36 h following L-CPA administration exhibiting a number of features in common with excitatory amino acid-induced cell death. We have used this in vivo model to examine the neurochemical processes following L-CPA-induced activation of NMDA receptors leading to neuronal cell death in the rat cerebellum. 2. The effects of a number of compounds which potently block nitric oxide synthase in vitro were examined on L-CPA-induced neurotoxicity 48 h following L-CPA dosing, to discover whether the neuronal cell death is mediated in part by excessive nitric oxide generation. Four inhibitors were studied, NG-nitro-L-arginine (L-NOARG), NG-nitro-L-arginine methyl ester (L-NAME), NG-iminoethyl-L-ornithine (L-NIO) and 3-bromo-7-nitroindazole (BrNI). 3. L-NAME (50 mg kg-1, i.p. twice daily) and BrIN (50 mg kg-1, i.p. twice daily) administration prevented the L-CPA-induced loss of granule cells which can reach up to 80-90% of the total cell number in rats treated with L-CPA alone. L-NOARG (50 mg kg-1, i.p. twice daily) and L-NIO administered at either 25 or 100 mg kg-1, twice daily did not produce any significant protection against L-CPA-induced neurotoxicity. 4. Both L-NAME and BrIN also prevented the L-CPA-induced increase in cerebellar water content and sodium concentrations. L-NIO when administered at the highest doses prevented the increase in cerebellar sodium concentration but not water content. L-NIO and L-NOARG were ineffective in preventing the L-CPA-induced increases in cerebellar water and sodium concentrations. 5. L-CPA-induced reductions in cerebellar aspartate and glutamate concentrations and increases in glutamine and GABA concentrations were prevented by L-NAME and BrIn, but not by L-NIO or L-NOARG. Also reductions in L-[3H]-glutamate binding to glutamate ionotrophic and metabotrophic receptors in the granule cell layer of rat cerebellum was prevented by L-NAME and BrIN, but not L-NIO or L-NOARG. 6. In conclusion, the neuroprotection offered by L-NAME and BrIN suggests that L-CPA-induced cerebellar granule cell necrosis is possibly mediated by or associated with excessive generation of nitric oxide. The inability of nitric oxide synthase inhibitors, L-NOARG and L-NIO to afford protection may result from their limited penetration into the brain (L-NIO) or rapid dissociation from the enzyme.

Glutathione depletion in the liver and brain produced by 2-chloropropionic acid: relevance to cerebellar granule cell necrosis.

L- and D-2-chloropropionic acid (L-CPA and D-CPA) produce selective damage to granule cells of the rat cerebellum by a mechanism that is not currently understood. We have demonstrated that both L- and D-CPA produce a rapid, dose and time dependent depletion of liver non-protein sulphydryl (NP-SH) content, mainly glutathione (GSH), while in the cerebellum and forebrain, there is a slower, dose and time dependent decrease in NP-SH. Twenty-four hours after a single dose of 750 mg/kg L-CPA (a dose sufficient to produce cerebellar toxicity, but a time prior to the onset of cellular necrosis), the content of total GSH was depleted by 85% in the cerebellum and to a lesser degree in the forebrain, while no increase in oxidised glutathione was observed in either tissue. In vitro both L- and D-CPA caused a marked reduction in GSH concentration when incubated with hepatic cytosol but not hepatic microsomes or brain cytosol. The hepatic cytosolic depletion appeared to be due to a direct reaction catalysed by a theta class glutathione S-transferase. A GSH adduct of L-CPA was isolated by high pressure liquid chromatography and identified by mass spectrometry as 2-S-glutathionyl propanoic acid, confirming a direct substitution reaction. No GSH adducts were formed by cerebellar or forebrain cytosol, suggesting that the particular isoform of glutathione S-transferase catalysing the reaction may not be present in the brain. We suggest that the marked and sustained CPA-mediated GSH depletion in the granule cells of the cerebellum may render these cells more vulnerable to oxidative free radical damage.

Chloropropionic acid-induced alterations in glucose metabolic status: possible relevance to cerebellar granule cell necrosis.

We have examined the effect of L- and D-2-chloropropionic acid (L-CPA and D-CPA) on the concentrations of pyruvate, lactate, glucose and beta-hydroxybutyrate in the blood at various times after doses which produce cerebellar granule cell necrosis. Blood pyruvate and lactate concentrations were reduced in these animals 4 h after dosing and remained below those of controls for up to 48 h. No changes were seen in concentrations of plasma glucose of beta-hydroxybutyrate at any time point. Similarly, no changes in cerebellar glucose, pyruvate or lactate were seen 24 h after 750 mg/kg L-CPA. Oxidation of [14C] pyruvate or [14C] glucose to [14CO2] by cerebellar slices from control rats was not altered by the presence of L- or D-CPA (1-10 mM for 2 h). Nor were these parameters affected in cerebellar slices from rats killed 24 h after a dose of 750 mg/kg L-CPA. We conclude that the activation of the pyruvate dehydrogenase complex is unlikely to be a critical factor in the selective toxicity of CPA to the cerebellum.

Synergistic Neurotoxic Effects of Styrene Oxide and Acrylamide: Glutathione-Independent Necrosis of Cerebellar Granule Cells

Conjugation with glutathione (GSH) is a mechanism of detoxification of acrylamide (ACR); hence, prior depletion of GSH might be expected to exacerbate ACR′s neurotoxicity. GSH levels in female rats were reduced by ip administration of styrene oxide (SO; 250 mg/kg), diethylmaleate (DEM; 0.5 ml/kg), or 2-vinylpyridine (VP; 100 mg/kg) 1.5 or 2 hr prior to a single dose of ACR (100 mg/kg). The time course of GSH depletion following treatment with SO/ACR, DEM/ACR, or VP, ACR showed that all three regimens were equally effective in reducing GSH in liver, cerebellum, cerebral cortex, and hippocampus. GSH levels in the liver were reduced to 4-22% of control levels between 2 and 4 hr after treatment and to 38-57% of control levels in all brain regions between 4 and 8 hr. ACR alone (100 mg/kg) reduced both brain and liver GSH to about 60% of normal. The administration of a second dose of ACR (also 100 mg/kg) 12 hr later further depleted brain and liver GSH to 33% of control. Brains were examined 2, 4, 7, 14, and 30 days after treatment by light and electron microscopy. The administration of SO plus ACR (in either order) produced lesions consisting of pyknotic granule cells confined to the anterior portions of the cerebellum and some of the small neurons of lamina II and Ill of the cerebral cortex. Electron microscopy revealed condensation of the granule cell chromatin and dissolution of the cytoplasm with the formation of large pericellular spaces. The granule cell lesion was not produced when the time between SO and ACR injections was either 4 or 24 hr. No pathology was observed following treatment with DEM/ACR, VP/ACR, ACR/ACR, vehicle (peanut oil), SO, or ACR alone. It appears that the neurotoxicity in animals treated with SO plus ACR is not directly the result of reduced cellular GSH levels per se, but may involve other detoxification pathways of ACR and SO.

Injections of fluid or septal cell suspension grafts into the dentate gyrus of rats induce granule cell degeneration

This study was originally aimed at investigating the effects of intragyral cell suspension grafts which had been enriched in basic fibroblast growth factor (bFGF) before being implanted into the rat hippocampus denervated by aspiration of the septohippocampal pathways. Whether treated with vehicle alone, vehicle + bFGF, cell suspension with or without bFGF, and irrespective of the surgical treatment (sham-operation, lesions or lesions + grafts), we unexpectedly found approximately 80% of the rats to show morphological alterations in the dentate gyrus (20 weeks post-grafting). These alterations consisted of loss of a part of the granule cells; this loss was most often located in the dorsal leaf of the dentate gyrus. Also, in the close vicinity of the degeneration area, we found severe shrinkage of the molecular layer and disappearance of the typical laminae pattern of acetylcholinesterase distribution. These observations confirm previous findings which showed that fluid injections into the dentate gyrus, a widely used technique for intracerebral administration of drugs, trophic factors or neural grafts, may induce undesirable granule cell necrosis.