Distal effects in a model of proximal axonopathy: 3,3'-iminodipropionitrile causes specific loss of neurofilaments in rat vestibular afferent endings

3,3'-Iminodipropionitrile (IDPN) is a neurotoxic compound that causes both a proximal neurofilamentous axonopathy and loss of the vestibular sensory hair cells. We used immunocytochemistry to examine changes in the expression of heavy, medium and light neurofilament (NF-H, NF-M, NF-L) proteins in the afferent terminals of vestibular sensory epithelia after IDPN exposure in rats. Acute, repeated and subchronic IDPN exposure induced a marked loss of NFs in the nerve terminals. The effect of subchronic IDPN was specific, as demonstrated by comparison with the synaptic membrane protein SNAP-25. In addition, Western blot analysis indicated specific loss of NFs in the vestibular receptors. Ultrastructural analysis revealed that afferent endings in the vestibular receptors were significantly preserved in animals exposed to subchronic IDPN, but that these endings showed NF segregation from microtubules followed by NF loss. These effects were closely paralleled by ultrastructural changes in the nerve terminals, particularly in the afferent contacts with the hair cells, and preceded hair cell loss. Thus, distal NF loss and nerve terminal pathology occur in the IDPN model of proximal neurofilamentous axonopathy. Similar distal pathology could also occur in human diseases characterized by proximal axonal swellings, particularly in amyotrophic lateral sclerosis.     

Pathology of the rat vestibular sensory epithelia during subchronic 3,3'-iminodipropionitrile exposure: hair cells may not be the primary target of toxicity

3,3'-Iminodipropionitrile (IDPN) is a neurotoxic compound that causes proximal neurofilamentous axonopathy and loss of the vestibular sensory hair cells. During subchronic exposure, the hair cells are eliminated by extrusion of the virtually intact cell from the sensory epithelia towards the luminal cavity. We describe the alterations of the vestibular epithelia before and during hair cell extrusion. Adult male Long-Evans rats were exposed to 0.2% IDPN in the drinking water for 1, 3, 5, 8 or 14 weeks, or to 0.1% IDPN for 14 weeks. Protrusion and subsequent extrusion of hair cells were observed in the cristae and utricular maculae of rats exposed to 0.2% IDPN for at least 5 weeks. At earlier time points and at lower doses, we observed the following pathological signs: blebbing of hair cells, swelling, retraction and fragmentation of the afferent nerve terminals, detachment of hair cells from the surrounding structures and loss of the pre- and post-synaptic membrane thickenings between hair cells and their afferent terminals. Widespread enlargement of the intercellular spaces also preceded and accompanied the extrusion process. The present data challenge the hypothesis that IDPN specifically affects hair cells.     

Inhibitory effect of noradrenaline on acute liver injury induced by carbon tetrachloride in the rat.

The effect of exogenous noradrenaline (NA) on acute liver injury was investigated in rats receiving a single dose of carbon tetrachloride (CCl4). Animals were divided into the following groups: (I) no treatment, used for plasma catecholamine assay; (II) received CCl4 only; (III) treated with CCl4 plus noradrenaline (NA). Plasma levels of catecholamine (CA) were elevated in both groups II and III, particularly in NA: average value at 33 h after the exposure of CCl4 increased to 290-fold of the control in group II and to 513-fold in group III. Subsequently, the levels of NA decreased with time, and were comparatively well-preserved in the rats of group III. Hepatic changes observed in the animals of group II were as follows: destruction with reduced number in rough endoplasmic reticulum; destruction and disappearance of cristae in mitochondria, and numerous fat droplets (shown by electron microscopy); histologically observed marked centrilobular necrosis with steatosis followed by progression with time; and microangiographically demonstrated deranged intrahepatic microvasculature. By contrast, these changes were successfully prevented by NA treatment (group III). Furthermore, histologically observed centrilobular change was restored with time. It was concluded that, in a deranged state, NA takes a form quite dissimilar to ordinary state: Na exerts for hapatoprotective and is highly involved in liver injury.     

Relationship between insult intensity and mode of hair cell loss in the vestibular system of rats exposed to 3,3'-iminodipropionitrile

A variety of stimuli cause sensory hair cell loss in the mammalian inner ear. This loss occurs by several differing processes, the significance of which remains undetermined. This study examines the relationship between the intensity of the damaging stimulus and the mode of hair cell loss found in the vestibular sensory epithelia of the rat. The ototoxin 3,3'-iminodipropionitrile (IDPN) was administered to rats at three different intoxication rates: acute exposure to high doses, repeated exposure to intermediate doses, and subchronic exposure to low doses. The morphology of the vestibular epithelia was examined by light microscopy and by scanning and transmission electron microscopy (SEM and TEM). In addition, DNA fragmentation in the epithelia was assessed by terminal deoxynucleotidyl transferase (tdt)-dUTP-nick-end-label (TUNEL). One day after acute IDPN, necrosis of hair cells was observed. However, at day 4 with this dose, and 1 and 4 days after repeated exposure, apoptotic figures and positive TUNEL labeling predominated. Subchronic IDPN resulted in a slowly evolving extrusion of basically intact hair cells in the crista and utricle. The data demonstrate that extrusion is a major mechanism of hair cell demise in mammals, that necrosis, apoptosis, and extrusion form a continuum of modes of hair cell loss, and that the intensity of the damaging stimulus determines the prevalence of each mode: Necrosis was most evident when the intensity was at its highest, whereas extrusion predominated when the intensity was at the lowest end of the scale.     

Study of platelet-mediated neurotoxicity in rat brain.

BACKGROUND AND PURPOSE: The mechanism of ischemic neuronal injury is not fully resolved. The present view is that vascular occlusion per se does not fully account for the extent of neurological dysfunction. We previously hypothesized that platelet secretory products might contribute to neuronal injury in the central nervous system. Our preliminary studies using organotypic rat spinal cord cultures exposed to human platelet and its secretory products revealed that platelet products had neurotoxic properties. METHODS: Further studies, using the same methods, were conducted, with the addition of several refinements such as use of gel-filtered platelets (as opposed to washed platelets) and adding additional relevant controls including platelet membranes, red blood cells, and washed rat platelets. RESULTS: Exposure of spinal cord explant cultures to platelet secretory products resulted in reduced number of neurons per ventral horn compared with control. CONCLUSIONS: Our findings suggest that platelet secretory products have neurotoxic properties. This effect was seen with platelet secretion obtained from physiological platelet concentrations. It appears possible that more abundant release of platelet products at the site of thrombus formation could have pathological significance in vivo.     

Enhanced cell-to-cell contacts between activated microglia and pyramidal cell dendrites following kainic acid-induced neurotoxicity in the hippocampus

Microglia participate in immune responses in the brain. However, little is known about the contact-mediated interaction between microglia and neurons. We report here that the cell-to-cell contacts between microglial processes and dendrites of hippocampal CA1 neurons were dramatically increased in density and area following local injection of kainic acid (KA). A similar KA-induced increase in the degree of intercellular contacts was observed in mice lacking telencephalin (TLCN), a neuronal dendritic adhesion molecule of ICAM family. The results suggest that adhesive contacts independent of TLCN and contact-mediated interactions between microglia and dendrites were promoted by excitotoxic brain injury.     

Comparison of location, severity, and dose response of proximal axonal lesions induced by 3,3'-iminodipropionitrile and deuterium substituted analogs.

Administration of 3,3'-iminodipropionitrile (IDPN) to rats results in massive accumulation of tangled neurofilaments in the proximal axons of large neurons, such as in dorsal root ganglia (DRG) and ventral horns of the lumbar spinal cord (LSC). Clinically, rats develop hyperexcitability, circling, head bobbing, and retropulsion. The ultimate toxicant and the molecular mechanism are not known. In a study designed to explore potential activation and detoxification pathways, dose-related differences in location and severity of lesions were observed in rats treated with IDPN or deuterium substituted analogs, 2,2,2',2'-tetradeuterio-IDPN (2-d-IDPN) or 3,3,3',3'-tetradeuterio-IDPN (3-d-IDPN). The compounds or saline were administered intraperitoneally to three rats per group at dose levels of 3.0, 1.5, 1.0, and 0.0 mmole/kg/day for 3 days. One week after the initial dose, tissues from DRG and LSC were collected, prepared and evaluated histologically in zones extending from areas adjacent to the cell bodies, distally toward the DRG stalk or toward the lumbar spinal roots. In the low dose IDPN group, DRG and LSC lesions were most prominent in distal zones. As dosage increased, the lesions progressed in severity and in proximity to the cell bodies. At the high dose, lesions were prominent in all zones. The same general pattern occurred with both analogs, although 2-d-IDPN was less potent than IDPN and 3-d-IDPN was more potent than IDPN. The differences in potency from the secondary isotopic effect of deuterium suggest that the 3-position is important in detoxification while the 2-position is important in the bioactivation of IDPN.     

Lactate reduces glutamate-induced neurotoxicity in rat cortex.

Experiments were carried out to test the hypothesis that lactate reduces the neurotoxicity of glutamate in vivo. MAP2 immunohistochemistry was used to measure lesion size, and microdialysis to measure the changes in glucose and lactate in the extracellular compartment. After implantation of a microdialysis probe 100 mM glutamate with or without 6 mM lactate was added to the perfusion medium and infused into the cortex of unanesthetized rats. Infusion of 100 mM glutamate for a period of 30 min produced a lesion of 6.05 +/- 0.64 mm(3), an increase in lactate of 124 +/- 19% above basal and a 21 +/- 9% reduction of glucose below basal level. When 6mM L-lactate was perfused together with 100 mM glutamate there was a significant reduction in the size of the lesion and there was no reduction in dialysate glucose. When L-lactate was replaced with D-lactate the lesion size and the increase in dialysate lactate were greater than after glutamate alone. The neuroprotective role of L-lactate is attributed to its ability to meet the increased energy demands of neurones exposed to high concentrations of glutamate.     

Valproate pretreatment protects dopaminergic neurons from LPS-induced neurotoxicity in rat primary midbrain cultures: role of microglia

Parkinson's disease is a neurodegenerative disorder characterized by progressive degeneration of dopaminergic (DA) neurons in the substantia nigra. Accumulating evidence supports the notion that neuroinflammation is involved in the pathogenesis of this disease. Valproate (VPA) has long been used for the treatment of seizures and bipolar mood disorder. In vivo and in vitro studies have demonstrated that VPA has neuroprotective and neurotrophic actions. In this study, using primary neuron-glia cultures from rat midbrain, we demonstrated that VPA is a potent neuroprotective agent against lipopolysaccharide (LPS)-induced neurotoxicity. Results showed that pretreatment with 0.6 mM VPA for 48 h robustly attenuated LPS-induced degeneration of dopaminergic neurons as determined by [(3)H] dopamine uptake and counting of the number of TH-ir neurons. The neuroprotective effect of VPA was concentration-dependent and was mediated, at least in part, through a decrease in levels of pro-inflammatory factors released from activated microglia. Specifically, LPS-induced increase in the release of TNFa, NO, and intracellular reactive oxygen species was markedly reduced in cultures pretreated with VPA. These anti-inflammatory effects of VPA were time and concentration-dependent correlated with a decrease in the number of microglia. Thus, our results demonstrate that protracted VPA pretreatment protects dopaminergic neurons from LPS-induced neurotoxicity through a reduction in levels of released pro-inflammatory factors, and further suggest that these anti-inflammatory effects may be contributed by VPA-induced reduction of microglia cell number. Taken together, our study reinforces the view that VPA may have utility in treating Parkinson's disease.     

Prolonged antinociception following carbon dioxide anesthesia in the laboratory rat

In the laboratory rat, inhalation (30 s) of high (> 70%) CO₂ concentrations resulted in short-term (1–3 min) anesthesia, followed by a prolonged (up to 60 mn) mild antinociception. Exposure to 100% CO₂ resulted in significant thermal (hot-plate, 52°, and tail-flick) and mechanical (tail-pinch, 886 g force) antinociception. Control animals, placed in the same chamber filled with air, showed no such effects. Rats exposed to 70% CO₂ exhibited effects on the hot plate comparable to those seen after inhalation of 100% CO₂, indicating that the response is not due to CO₂-induced hypoxia. Additionally, recovery from halothane-induced anesthesia of comparable duration did not result in antinociception, confirming that anesthesia alone is not sufficient to produce the effect. Pretreatment with the opiate antagonist naltrexone (0.1–10 mg/kg i.p.) did not diminish the CO₂-induced antinociception, suggesting that endogenous opioids are not obligatory in the mechanism of this response. Furthermore, hypophysectomy abolished hot-plate antinociception in animals exposed to 100% CO₂ while sham-treated controls exhibited a pattern of hot-plate responses similar to that reported above. Taken together, these findings show that: (1) recovery from CO₂-induced anesthesia results in a prolonged mild antinociception, detectable with thermal and mechanical nociceptive tests; and (2) this response may represent a novel form of environmentally induced antinociception, mediated by a non-opiate hormonal substance.     

Acute high-dose methotrexate neurotoxicity in the rat

Intravenous high-dose methotrexate chemotherapy may produce acute, subacute, or chronic neurotoxicity in patients with cancer. Acute encephalopathies following high-dose methotrexate treatment are recognized with increasing frequency. This study describes a model of acute high-dose methotrexate neurotoxicity in the rat characterized by a profound dose-dependent depression of cerebral glucose metabolism in association with behavioral and electroencephalographic abnormalities. Alterations in the amino acid profile, similar to those described in cancer patients after high-dose methotrexate treatment, were observed in the absence of biochemical evidence of systemic organ toxicity. This model facilitates the study of the biochemical mechanisms of antifolate neurotoxicity in humans and permits the evaluation of potential therapeutic interventions.     

Irradiation neurotoxicity assessed in organotypic cultures of rat hippocampus.

The neurotoxic effects of irradiation on the developing nervous system were studied in organotypic cultures of hippocampus prepared from newborn Sprague-Dawley rats. Hippocampus slices of 7-day-old rats were irradiated at the day of explantation at doses of 1, 2 and 4 Gy, and cultured in a roller drum for a fortnight. Light and electron microscopy showed remarkable damage to neuronal cells following irradiation, oligodendrocytes and myelogenesis being also affected. In contrast to alterations in neuronal perikarya, no morphological changes in synapses were obvious, though their number seemed to be reduced after irradiation with 4 Gy. These results confirm that low dose radiation produces damage to the central nervous system not only pre-natally, but even in post-natal periods of differentiation and development.     

Potentiation of 3-hydroxyglutarate neurotoxicity following induction of astrocytic iNOS in neonatal rat hippocampal cultures

Neuronal damage in glutaryl-CoA dehydrogenase deficiency (GDD) has previously been addressed to N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity of the accumulating neurotoxic metabolite 3-hydroxyglutarate. However, acute encephalopathic crises in GDD patients are typically precipitated by febrile illness or even routine vaccinations, suggesting a potentiating role of inflammatory cytokines. In the present study we investigated the effect of interleukin-1beta and interferon-gamma on 3-hydroxyglutarate toxicity in rat cortical astrocyte cultures and neonatal rat hippocampal cultures. A cotreatment of both culture systems with interleukin-1beta and interferon-gamma induced the protein expression of astrocytic inducible nitric oxide synthase (iNOS), resulting in increased nitric oxide (NO) production. Cytokine pretreatment alone had no effect on cell viability but potentiated 3-hydroxyglutarate neurotoxicity. NOS inhibition by aminoguanidine and L-NAME prevented an iNOS-mediated potentiation of 3-hydroxyglutarate neurotoxicity but failed to protect neurons against 3-hydroxyglutarate alone. In contrast, superoxide dismutase/catalase as well as MK-801 prevented toxicity of 3-hydroxyglutarate alone as well as its potentiation by iNOS, supporting a central role of NMDA receptor stimulation with subsequently increased superoxide anion production. It is concluded that the potentiation of 3-hydroxyglutarate neurotoxicity is most probably due to an induction of astrocytic iNOS and concomitantly increased NO production, enabling enhanced peroxynitrite formation. Thus, we provide evidence for a neuroimmunological approach to the precipitation of acute encephalopathic crises in GDD by inflammatory cytokines.     

Dopaminergic neuronal injury in the adult rat brain following neonatal exposure to lipopolysaccharide and the silent neurotoxicity

Our previous studies have shown that neonatal exposure to lipopolysaccharide (LPS) resulted in motor dysfunction and dopaminergic neuronal injury in the juvenile rat brain. To further examine whether neonatal LPS exposure has persisting effects in adult rats, motor behaviors were examined from postnatal day 7 (P7) to P70 and brain injury was determined in P70 rats following an intracerebral injection of LPS (1 mg/kg) in P5 Sprague-Dawley male rats. Although neonatal LPS exposure resulted in hyperactivity in locomotion and stereotyped tasks, and other disturbances of motor behaviors, the impaired motor functions were spontaneously recovered by P70. On the other hand, neonatal LPS-induced injury to the dopaminergic system such as the loss of dendrites and reduced tyrosine hydroxylase immunoreactivity in the substantia nigra persisted in P70 rats. Neonatal LPS exposure also resulted in sustained inflammatory responses in the P70 rat brain, as indicated by an increased number of activated microglia and elevation of interleukin-1β and interleukin-6 content in the rat brain. In addition, when challenged with methamphetamine (METH, 0.5 mg/kg) subcutaneously, rats with neonatal LPS exposure had significantly increased responses in METH-induced locomotion and stereotypy behaviors as compared to those without LPS exposure. These results indicate that although neonatal LPS-induced neurobehavioral impairment is spontaneously recoverable, the LPS exposure-induced persistent injury to the dopaminergic system and the chronic inflammation may represent the existence of silent neurotoxicity. Our data further suggest that the compromised dendritic mitochondrial function might contribute, at least partially, to the silent neurotoxicity.     

Enhanced nociceptive behaviour following conditioning injection of formalin in the perioral area of the rat

The possible existence of long-term modifications in response to a transient nociceptive conditioning stimulation was investigated in the rat in three experiments. (1) A nociceptive conditioning stimulus was delivered in the form of a s.c. formalin injection (conditioning injection) in the left upper lip. Evaluation of the nociceptive behaviour triggered by another formalin injection (testing injection) made in the controlateral right upper lip was carried out in distinct groups of rats 7, 14 or 28 days after the conditioning. An enhanced nociceptive response at days 7 and 14 and a return to the baseline at day 28 were observed. (2) A similar protocol was developed with formalin used for both conditioning and testing but an anaesthetic blockade of the infraorbital nerve was performed just before the conditioning injection to suppress the initial barrage. The change observed at day 7 was suppressed by the nerve block. (3) A conditioning nociceptive stimulus was applied either ipsilaterally to the right lower lip or to the tail. An increased nociceptive response was observed when the conditioning stimulus was applied to the same side as the test stimulus but no increase in the formalin test response was detected when the conditioning stimulus was applied to the tail. These results indicated that, after a single formalin injection in the left upper lip, a hyperexcitability developed that depended on the initial barrage, lasted for at least 2 weeks, was no longer present at 4 weeks and might rely on a segmental mechanism. The hypothesis of a central sensitization triggered by an initial barrage and maintained by an ongoing input induced from the periphery is discussed.     

The morphology of carbon disulfide neurotoxicity

The morphology of carbon disulfide induced peripheral neuropathy was studied in rats exposed to three concentrations of carbon disulfide by inhalation for 90 days. Rats exposed to 800 ppm developed neurofilamentous axonal swellings in the distal portions of long fibers, including the dorsal ascending sensory and corticospinal tracts of the spinal cord. In peripheral nerve the predominant effect was seen at the level of the posterior tibial nerve. Teased fiber preparations of the muscular branch of the posterior tibial nerve showed numerous paranodal and internodal swellings as well as Wallerian degeneration. Ultrastructurally the swellings were characterized by neurofilament accumulations, decreased numbers of microtubules and thin myelin. Other features included segregation of axoplasmic organelles and cytoskeletal components, intrusion of Schwann cell processes into the axoplasm, Schwann cells with increased cytoplasmic contents, and Schwann cell proliferation around many swollen and demyelinated axons. These features draw important parallels between the morphology of carbon disulfide neuropathy and the neurofilamentous neuropathies induced by hexacarbons and beta,beta' iminodipropionitrile (IDPN).     

Enhanced localization of amyloid β precursor protein in the rat hippocampus following long-term adrenalectomy

Using various antibodies to the amyloid ß precursor protein (APP) associated with Alzheimer's disease, we investigated changes in the distribution of APP in the hippocampus and neocortex of adrenalectomized (ADX) rats. In contrast to sham-operated controls, ADX rats euthanised after a survival period of 5 months showed striking APP reactivity in the CA1–CA4 fields and in the surviving cells in the dentate gyrus. Our results suggest the enhanced APP reactivity in hippocampal neurons may pertain to previous observations on the accumulation of APP fragments in the neocortex during ischemic or traumatic injury. Thus, long-term hormone deprivation would be another factor, which may influence the expression of APP in brain.