Distribution of elements and water in peripheral nerve of streptozocin-induced diabetic rats.

Accumulating evidence suggests that alterations in Na, Ca, K, and other biologically relevant elements play a role in the mechanism of cell injury. The pathogenesis of experimental diabetic neuropathy is unknown but might include changes in the distribution of these elements in morphological compartments. In this study, this possibility was examined via electron-probe X-ray microanalysis to measure both concentrations of elements (millimoles of element per kilogram dry or wet weight) and cell water content (percent water) in frozen, unfixed, unstained sections of peripheral nerve from control and streptozocin-induced diabetic rats. Our results indicate that after 20 wk of experimental diabetes, mitochondria and axoplasm from myelinated axons of proximal sciatic nerve displayed diminished K and Cl content, whereas in tibial nerve, the intraaxonal levels of these elements increased. In distal sciatic nerve, mitochondrial and axoplasmic levels of Ca were increased, whereas other elemental alterations were not observed. These regional changes resulted in a reversal of the decreasing proximodistal concentration gradients for K and Cl, which exist in nondiabetic rat sciatic nerve. Our results cannot be explained on the basis of altered water. Highly distinctive changes in elemental distribution observed might be a critical component of the neurotoxic mechanism underlying diabetic neuropathy.

Mechanisms of calcium and sodium fluxes in anoxic myelinated central nervous system axons.

Electron probe X-ray microanalysis was used to measure water content and concentrations of elements (i.e. Na, K, Cl and Ca) in selected morphological compartments of rat optic nerve myelinated axons. Transaxolemmal movements of Na+ and Ca2+ were modified experimentally and corresponding effects on axon element and water compositions were determined under control conditions and following in vitro anoxic challenge. Also characterized were effects of modified ion transport on axon responses to postanoxia reoxygenation. Blockade of Na+ entry by tetrodotoxin (1 microM) or zero Na+/Li(+)-substituted perfusion reduced anoxic increases in axonal Na and Ca concentrations. Incubation with zero-Ca2+/EGTA perfusate prevented axoplasmic and mitochondrial Ca accumulation during anoxia but did not affect Na increases or K losses in these compartments. Inhibition of Na(+)-Ca2+ exchange with bepridil (30 microM) selectively prevented increases in intra-axonal Ca, whereas neither nifedipine (5 microM) nor nimodipine (5 microM) influenced the effects of anoxia on axonal Na, K or Ca. X-ray microanalysis also showed that prevention of Na and Ca influx during anoxia obtunded severe elemental deregulation normally associated with reoxygenation. Results of the present study suggest that during anoxia, Na+ enters axons mainly through voltage-gated Na+ channels and that subsequent increases in axoplasmic Na+ are functionally coupled to extra-axonal Ca2+ import. Na+i-dependent, Ca2+o entry is consistent with reverse operation of the axolemmal Na(+)-Ca2+ exchanger and we suggest this route represents a primary mechanism of Ca2+ influx. Our findings also implicate a minor route of Ca2+ entry directly through Na+ channels.

Elemental composition and water content of rat optic nerve myelinated axons during in vitro post-anoxia reoxygenation.

During transient hypoxic episodes, CNS nerve cells and their axons undergo structural and functional damage. However, additional injury occurs as a result of subsequent tissue reperfusion. To examine mechanisms of this secondary injury, we have characterized the temporal patterns of element (e.g. Na, K, Ca) and water deregulation in rat optic nerve myelinated axons and glia during in vitro exposure to post-anoxia reoxygenation. Isolated nerves were exposed to 1 h of anoxia followed by varying periods of reoxygenation (20, 40, 60 and 180 min). Changes in subcellular distribution of elements and water were determined using electron probe X-ray microanalysis. In response to reoxygenation, the majority of large and medium axons exhibited a progressive worsening of anoxia-induced elemental deregulation. Axoplasmic Na, Cl and Ca increased substantially while K concentrations remained at or slightly below anoxic levels. Respective mitochondria expressed similar elemental changes except that Ca levels increased dramatically. A limited number of large and medium axons and their mitochondria showed initial but transient improvements in elemental composition. In contrast, approximately 50% of small axons initiated early improvements in transmembrane elemental distribution that continued to advance throughout the reoxygenation period. Remaining axons of this group displayed severe elemental derangement similar to that of larger fibers. The elemental composition of reoxygenated glial cells and myelin remained comparable to that reported after 60 min of anoxia. These results indicate that while larger axons express eventual severe elemental deregulation in spite of reoxygenation, many small axons appear capable of re-establishing near-normal transmembrane ion gradients. Results of the present study suggest reoxygenation/reperfusion injury of CNS axons is mediated by exacerbation of Ca2+ entry and the generalized ion deregulation initiated during anoxic or ischemic episodes. These findings constitute basic information regarding damage induced by post-anoxia reoxygenation and could, therefore, contribute toward understanding the mechanism of reperfusion injury following hypoxic or ischemic episodes in CNS white matter. Furthermore, deciphering the route of Ca2+ influx during reoxygenation/reperfusion might provide a basis for rational design of effective pharmacotherapies.

Effects of ion channel blockade on the distribution of Na, K, Ca and other elements in oxygen-glucose deprived CA1 hippocampal neurons.

The pathophysiology of brain ischemia and reperfusion injury involves perturbation of intraneuronal ion homeostasis. To identify relevant routes of ion flux, rat hippocampal slices were perfused with selective voltage- or ligand-gated ion channel blockers during experimental oxygen-glucose deprivation and subsequent reperfusion. Electron probe X-ray microanalysis was used to quantitate water content and concentrations of Na, K, Ca and other elements in morphological compartments (cytoplasm, mitochondria and nuclei) of individual CA1 pyramidal cell bodies. Blockade of voltage-gated channel-mediated Na+ entry with tetrodotoxin (1 microM) or lidocaine (200 microM) significantly reduced excess intraneuronal Na and Ca accumulation in all compartments and decreased respective K loss. Voltage-gated Ca2+ channel blockade with the L-type antagonist nitrendipine (10 microM) decreased Ca entry and modestly preserved CA1 cell elemental composition and water content. However, a lower concentration of nitrendipine (1 microM) and the N-, P-subtype Ca2+ channel blocker omega-conotoxin MVIIC (3 microM) were ineffective. Glutamate receptor blockade with the N-methyl-D-aspartate (NMDA) receptor-subtype antagonist 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP; 100 microM) or the alpha-amino-3-hydroxy-5-methyl-4-isoazole propionic acid (AMPA) receptor subtype blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM/100 microM glycine) completely prevented Na and Ca accumulation and partially preserved intraneuronal K concentrations. Finally, the increase in neuronal water content normally associated with oxygen-glucose deprivation/reperfusion was prevented by Na+ channel or glutamate receptor blockade. Results of the present study demonstrate that antagonism of either postsynaptic NMDA or AMPA glutaminergic receptor subtypes provided nearly complete protection against ion and water deregulation in nerve cells subjected to experimental ischemia followed by reperfusion. This suggests activation of ionophoric glutaminergic receptors is involved in loss of neuronal osmoregulation and ion homeostasis. Na+ channel blockade also effectively diminished neuronal ion and water derangement during oxygen-glucose deprivation and reperfusion. Prevention of elevated Nai+ levels is likely to provide neuroprotection by decreasing presynaptic glutamate release and by improving cellular osmoregulation, adenosine triphosphate utilization and Ca2+ clearance. Thus, we suggest that voltage-gated tetrodotoxin-sensitive Na+ channels and glutamate-gated ionotropic NMDA or AMPA receptors are important routes of ion flux during nerve cell injury induced by oxygen-glucose deprivation/reperfusion.

Intraneuronal ion distribution during experimental oxygen/glucose deprivation. Routes of ion flux as targets of neuroprotective strategies.

Ischemic neuronal injury appears to be mediated by disruption of subcellular ion distribution and, therefore, prevention of ion relocation might be neuroprotective. X-ray microanalysis was used to measure concentrations of Na, K, Ca and other elements in subcellular compartments (e.g., mitochondria) of CA1 neurons from oxygen/glucose-deprived (OGD) hippocampal slices. Results showed that OGD produced progressive loss of ion regulation in CA1 cells. Post-OGD reperfusion with normal media exacerbated the initial ion deregulation. To study neuroprotective mechanisms, we determined the ability of hypothermia (31 degrees C) or ion channel blockade to retard intraneuronal ion disruption induced by OGD/reperfusion. Whereas Ca2+ channel blockade (omega-conotoxin MVIIC, 3 microM) was ineffective, hypothermia and Na+ channel blockers (tetrodotoxin, TTX, 1 microM; lidocaine, 200 microM) reduced ion deregulation in subneuronal compartments. Blockade of glutamate receptors (AMPA, 10 microM; the non-NMDA receptor antagonist CNQX, 10 microM/100 microM glycine; the NMDA receptor antagonist CCP, 100 microM) during OGD/reperfusion provided nearly complete protection. These findings provide a foundation for identifying potential pharmacotherapeutic approaches and for discerning corresponding mechanisms of neuroprotection.

The effects of intrathecal sympathomimetic agents on neural activity in the lumbar sympathetic chain of rats.

Intrathecal injections of norepinephrine (0.30 mumol) and clonidine (0.035 mumol) produced decreases in neural activity recorded from the lumbar sympathetic chain and caused changes in rat thermoregulation consistent with a reduction in efferent sympathetic activity. These results support the conclusion that bulbospinal noradrenergic pathways inhibit sympathetic outflow. The data also indicate that the spinal cord is a potential site of action for clonidine.

Elemental composition and water content of myelinated axons and glial cells in rat central nervous system.

The distribution of elements (e.g. Na, Cl, K) and water in CNS cells is unknown. Therefore, electron probe X-ray microanalysis (EPMA) was used to measure water content and concentrations (mmol/kg dry or wet weight) of Na, Mg, P, S, Cl, K and Ca in morphological compartments of myelinated axons and glial cells from rat optic nerve and cervical spinal cord white matter. Axons in both CNS regions exhibited similar water content (approximately 90%), and relatively high concentrations (wet and dry weight) of K with low Na and Ca levels. The K content of axons was related to diameter, i.e. small axons in spinal cord and optic nerve had significantly less (25-50%) K than larger diameter axons from the same CNS region. The elemental composition of spinal cord mitochondria was similar to corresponding axoplasm, whereas the water content (75%) of these organelles was substantially lower than that of axoplasm. In glial cell cytoplasm of both CNS areas, P and K (wet and dry weight) were the most abundant elements and water content was approximately 75%. CNS myelin had predominantly high P levels and the lowest water content (33-55%) of any compartment measured. The results of this study demonstrate that each morphological compartment of CNS axons and glia exhibits a characteristic elemental composition and water content which might be related to the structure and function of that neuronal region.

Reoxygenation of anoxic peripheral nerve myelinated axons promotes re-establishment of normal elemental composition.

Previously we have shown that in vitro anoxia of rat peripheral nerve myelinated axons causes sequential deregulation of axoplasmic Na, K and Ca; i.e., an initial influx of Na and loss of K is coupled to subsequent Ca accumulation [7]. In the present study, we examined the ability of PNS axons to recover normal elemental composition following oxygen deprivation. Thus, electron probe X-ray microanalysis was used to determine the effects of post-anoxia reoxygenation on the concentrations of elements (i.e., Na, K, Cl, Ca, Mg, P and S) in rat posterior tibial nerve myelinated axons and Schwann cells. Results indicate that following 180 min of anoxia, peripheral nerve reoxygenation (60 and 120 min) promoted progressive recovery of normal elemental composition in axoplasm and mitochondria of small, medium and large diameter tibial nerve fibers. Our observations also indicate that small axons recovered normal elemental concentrations more rapidly than larger counterparts. Schwann cells and myelin exhibited only modest elemental disruption during anoxia from which reoxygenation promoted full reparation. The ability of peripheral nerve axons to restore normal elemental composition during post-anoxia reoxygenation is in marked contrast to the exacerbation of elemental deregulation which ensued during in vitro reoxygenation of anoxic rat CNS fibers [14]. This differential response to reoxygenation represents a fundamental difference in the pathophysiology of myelinated axons in the CNS and PNS.

Mechanisms of injury-induced calcium entry into peripheral nerve myelinated axons: in vitro anoxia and ouabain exposure.

In the present investigation, electron probe X-ray microanalysis was used to characterize the effects of in vitro ouabain (2 mM) or anoxia on elemental composition (e.g. Na, K, Ca) and water content of rat peripheral (tibial) nerve myelinated axons and Schwann cells. Results showed that independent of axon size, both ouabain and anoxia markedly increased axoplasmic Na and decreased K concentrations. However, only anoxia was associated with significant elevation of axonal Ca content. Mitochondrial areas from ouabain- or anoxia-exposed fibers exhibited changes in element and water contents that were similar to axoplasmic alterations. Schwann cells and myelin displayed small increases in Na and substantial losses of K in response to ouabain exposure. In contrast, these glial compartments were relatively resistant to anoxia as indicated by the modest and delayed nature of the elemental changes. Nonetheless, neither treatment significantly affected glial Ca concentrations. Our results suggest that Ca2+ accumulation in peripheral nerve axons is complex and involves not only deregulation of Na+ and K+ but other fundamental pathogenic changes as well. In addition to providing baseline information, we have identified an in vitro model (anoxia) which features Ca2+ build-up in PNS myelinated axons. Thus, the present study offers a foundation for investigation into mechanisms of Ca2+ entry following peripheral nerve injury.

Effects of acrylamide on subcellular distribution of elements in rat sciatic nerve myelinated axons and Schwann cells.

Electron probe X-ray microanalysis was used to determine whether experimental acrylamide (ACR) neuropathy involves deregulation of subcellular elements (Na, P, S, Cl, K, Ca and Mg) and water in Schwann cells and small, medium and large diameter myelinated axons of rat sciatic nerve. Results show that in proximal but not distal sciatic nerve, ACR treatment (2.8 mM in drinking water) was associated with an early (15 days of exposure), moderate increase in mean axoplasmic K concentrations (mmol/kg) of medium and small diameter fibers. However, all axons in proximal and distal nerve regions displayed small increases in dry and wet weight contents of axoplasmic Na and P. As ACR treatment progressed (up to 60 days of exposure), Na and P changes persisted whereas proximal axonal K levels returned to control values or below. Alterations in mitochondrial elemental content paralleled those occurring in axoplasm. Schwann cells in distal sciatic nerve exhibited a progressive loss of K, Mg and P and an increase in Na, Cl and Ca. Proximal glia displayed less extensive elemental modifications. Elemental changes observed in axons are not typical of those associated with cell injury and might reflect compensatory or secondary responses. In contrast, distal Schwann cell alterations are consistent with injury, but whether these changes represent primary or secondary mechanisms remains to be determined.

Hexanedione effects on protein phosphorylation in rat peripheral nerve.

Rats were treated with either 2,5-hexanedione (2,5-HD), 1,6-hexanediol (1,6-HDIOL), or saline for 7, 15 or 24 days. Protein phosphorylation was measured in proximal and distal sciatic nerve segments following incubation with [32P]orthophosphate. In proximal segments, 2,5-HD administration caused selective time-dependent increases in isotope incorporation in a 55 kDa protein, tentatively identified as tubulin, and a 180 kDa protein. Enhanced phosphorylation was highest at 24 days when motor function was most impaired. Administration of 1,6-HDIOL produced no consistent phosphorylation changes. Animals intoxicated with 3,4-dimethyl-2,5-hexanedione for 12 days showed proximal region increases in phosphorylation of the 55 and 180 kDa proteins and the major myelin proteins, Po and Pr.

Effects of acrylamide and 2,5-hexanedione on brain mitochondrial respiration.

The effects of acrylamide (ACR) and 2,5-hexanedione (2,5-HD) on brain mitochondrial respiration were assessed. Mitochondria were isolated from whole brains or brain regions of control and neurotoxicant-treated rats. Direct in vitro exposure of isolated brain mitochondria to ACR (1 mM final concentration) had no effect on respiration, whereas direct exposure to 2,5-HD (1 mM final concentration) inhibited state 3 respiration. Chronic treatment of rats with ACR (50 mg/kg/day x 10 days) did not affect respiration of mitochondria isolated from cortex or brainstem. However, in mitochondria from cerebellum of ACR treated rats, pyruvate + oxaloacetic acid (pyr/oaa) supported oxygen consumption was decreased significantly in both states 3 and 4. In addition, the ADP/O ratio was reduced in this brain structure. In all brain regions of 2,5-HD (400 mg/kg/day x 24 days) intoxicated rats, pyr/oaa supported state 3 respiration was reduced. Glutamate + malate (glu/mal) supported respiration was diminished only in mitochondria isolated from brain stem of 2,5-HD treated rats. In contrast, the non-neurotoxic analogs, 1,6-hexanediol and N,N'-methylene-bis-acrylamide did not alter mitochondrial respiration in parallel experiments. Thus, both ACR and 2,5-HD produce a substrate-dependent, toxicologically specific inhibition of brain mitochondrial respiration. This inhibition of mitochondrial energy production might play a role in the neurotoxic mechanisms of action for these chemicals.

The relevance of axonal swellings and atrophy to gamma-diketone neurotoxicity: a forum position paper.

Traditionally, gamma-diketone neuropathy is classified as a distal axonopathy and has been characterized by giant axonal swellings in CNS and PNS tissues. These swellings contain neurofilamentous masses and are associated with thinning and retraction of the myelin sheath. It has been proposed that this axonopathy is caused by direct gamma-diketone modification of neurofilaments (NFs) involving pyrrolation of epsilon-amino groups on NF lysyl residues and possibly secondary autoxidation of the pyrrole rings with creation of covalent NF-NF crosslinks. Neurofilaments are thought to undergo chemical modification as they progress along the axonal axis and, eventually, accumulate at distal nodes of Ranvier where their proximodistal movement is impeded. Development of swelling presumably initiates axonal degeneration and subsequent functional deficits. However, other research suggests that axonal swellings are a non-specific effect related to subchronic gamma-diketone exposure. Such evidence draws into question the mechanistic relevance of these swellings. In contrast, research conducted over the past decade indicates axonal atrophy is a specific morphologic component of gamma-diketone neuropathy which might have both functional and mechanistic importance. In this overview, the potential neurotoxicological significance of both axonal swellings and atrophy are evaluated critically. Based on the evidence to be presented, we propose that axonal atrophy is the morphological consequence of the molecular mechanism of gamma-diketone neuropathy. Accordingly, several mechanistic scenarios related to the development of atrophy will be discussed. It is hoped that this Forum will stimulate scientific debate and initiate laboratory investigations which will either confirm or refute the involvement of axonal atrophy in gamma-diketone neurotoxicity. Investigating gamma-diketone atrophy might provide insight into the mechanism of other toxic axonopathies which are also associated with reduced axon caliber; e.g., acrylamide and carbon disulfide neuropathies.

Acrylamide-induced distal axon degeneration: a proposed mechanism of action.

Exposure to acrylamide (ACR) monomer produces distal swelling and subsequent degeneration in central and peripheral myelinated axons of humans and laboratory animals. The molecular and cellular events leading to this type of axonopathy are currently unknown. Herein we describe a new mechanism of ACR axonopathy that represents a synthesis of recent research findings and prior hypotheses. According to this model, ion regulation in distal paranodal axon regions is compromised by diminished axolemmal Na/K-ATPase activity. It is suggested that decreased NA/K-ATPase activity is a consequence of aberrant cell body processing and/or deficient axonal transport. Reduced Na pump activity promotes membrane depolarization in conjunction with axoplasmic accumulation of Na and loss of K. Thermodynamically, this favors reverse operation of the Na/Ca-exchanger which permits axonal Ca entry in exchange for Na. The influx of Ca eventually overwhelms buffering mechanisms and leads to distal axon degeneration. Distal axons are predisposed to regulatory failure of this type due to a dependency on cell body output and the unique differential distribution of enzymes, ion channels and exchangers among nodal and internodal regions. This heuristic model might account for axon degeneration occurring as a result of exposure to other chemical neurotoxicants and following axotomy and other forms of mechanical injury.

Perturbation of axonal elemental composition and water content: implication for neurotoxic mechanisms.

The concentration and distribution of labile elements in nerve cells is tightly regulated by multiple membrane transport processes and by binding to lipids and proteins. The multifaceted nature of elemental regulation provides numerous sites at which toxicants or disease processes might act to disrupt this regulation. Such disruption can affect cytoskeletal integrity, macromolecular synthesis, energy production, osmoregulation and other cellular processes. The possible role of perturbed elemental homeostasis in the mechanism of nerve injury caused by certain chemicals (e.g., acrylamide, 2,5-hexanedione) and neuropathic diseases (e.g., diabetes) has not been determined. To investigate this possibility, we have used electron probe x-ray micro-analysis (EPMA) to measure the distribution of elements and water in cellular compartments of myelinated axons (axoplasm, mitochondria) and glial cells (cytoplasm, myelin) in normal rat central and peripheral nervous systems. Results indicate that each compartment exhibits a characteristic composition of elements and water which might reflect function of that anatomical region or organelle. Injury-induced changes in elemental content of PNS axons and Schwann cells have been identified using several neurotoxic models (i.e., acrylamide, axotomy, diabetic neuropathy). Each type of injury initiated early alterations in element and water composition of both axons and glial cells. Compositional changes were specific and developed sequentially instead of simultaneously. Results of these studies suggest that, rather than being an epiphenomenon, altered elemental regulation might represent a primary component of many neurotoxic mechanisms.

Nerve terminals as the primary site of acrylamide action: a hypothesis.

Acrylamide (ACR) is considered to be prototypical among chemicals that cause a central-peripheral distal axonopathy. Multifocal neurofilamentous swellings and eventual degeneration of distal axon regions in the CNS and PNS have been traditionally considered the hallmark morphological features of this axonopathy. However, ACR has also been shown to produce early nerve terminal degeneration of somatosensory, somatomotor and autonomic nerve fibers under a variety of dosing conditions. Recent research from our laboratory has demonstrated that terminal degeneration precedes axonopathy during low-dose subchronic induction of neurotoxicity and occurs in the absence of axonopathy during higher-dose subacute intoxication. This relationship suggests that nerve terminal degeneration, and not axonopathy, is the primary or most important pathophysiologic lesion produced by ACR. In this hypothesis paper, we review evidence suggesting that nerve terminal degeneration is the hallmark lesion of ACR neurotoxicity, and we propose that this effect is mediated by the direct actions of ACR at nerve terminal sites. ACR is an electrophile and, therefore, sulfhydryl groups on presynaptic proteins represent rational molecular targets. Several presynaptic thiol-containing proteins (e.g. SNAP-25, NSF) are critically involved in formation of SNARE (soluble N-ethylmaleimide (NEM)-sensitive fusion protein receptor) complexes that mediate membrane fusion processes such as exocytosis and turnover of plasmalemmal proteins and other constituents. We hypothesize that ACR adduction of SNARE proteins disrupts assembly of fusion core complexes and thereby interferes with neurotransmission and presynaptic membrane turnover. General retardation of membrane turnover and accumulation of unincorporated materials could result in nerve terminal swelling and degeneration. A similar mechanism involving the long-term consequences of defective SNARE-based turnover of Na+/K(+)-ATPase and other axolemmal constituents might explain subchronic induction of axon degeneration. The ACR literature occupies a prominent position in neurotoxicology and has significantly influenced development of mechanistic hypotheses and classification schemes for neurotoxicants. Our proposal suggests a reevaluation of current classification schemes and mechanistic hypotheses that regard ACR axonopathy as a primary lesion.

Acrylamide neuropathy. I. Spatiotemporal characteristics of nerve cell damage in rat cerebellum.

Based on evidence from morphometric studies of PNS, we suggested that acrylamide (ACR)-induced distal axon degeneration was a secondary effect related to duration of exposure [Toxicol. Appl. Pharmacol. 151 (1998) 211]. To test this hypothesis in CNS, the cupric-silver stain method of de Olmos was used to define spatiotemporal characteristics of nerve somal, dendritic, axonal and terminal degeneration in rat cerebellum. Rats were exposed to ACR at either 50 mg/kg per day (i.p.) or 21 mg/kg per day (p.o.) and at selected times (i.p. = 5, 8 and 11 days; p.o. = 7, 14, 21, 28 and 38 days) brains were removed and processed for silver staining. Results demonstrate that intoxication at the higher ACR dose-rate produced early (day 5) and progressive degeneration of Purkinje cell dendrites in cerebellar cortex. Nerve terminal degeneration occurred concurrently with somatodendritic argyrophilia in cerebellar and brainstem nuclei that receive afferent input from Purkinje neurons. Relatively delayed (day 8), abundant axon degeneration was present in cerebellar white matter but not in cortical layers or in tracts carrying afferent fibers (cerebellar peduncles) from other brain nuclei. Axon argyrophilia coincided with the appearance of perikaryal degeneration, which was selective for Purkinje cells since silver impregnation of other cerebellar neurons was not evident in the different cortical layers or cerebellar nuclei. Intoxication at the lower ACR dose-rate produced simultaneous (day 14) dendrite, axon and nerve terminal argyrophilia and no somatic Purkinje cell degeneration. The spatiotemporal pattern of dendrite, axon and nerve terminal loss induced by both ACR dose-rates is consistent with Purkinje cell injury. Injured neurons are likely to be incapable of maintaining distal processes and, therefore, axon degeneration in the cerebellum is a component of a "dying-back" process of neuronal injury. Because cerebellar coordination of somatomotor activity is mediated solely through efferent projections of the Purkinje cell, injury to this neuron might contribute significantly to gait abnormalities that characterize ACR neurotoxicity.

Acrylamide neuropathy. II. Spatiotemporal characteristics of nerve cell damage in brainstem and spinal cord.

Previous studies of acrylamide (ACR) neuropathy in rat PNS [Toxicol. Appl. Pharmacol. 151 (1998) 211] and cerebellum [NeuroToxicology 23 (2002) 397] have suggested that axon degeneration was not a primary effect and was, therefore, of unclear neurotoxicological significance. To continue morphological examination of ACR neurotoxicity in CNS, a cupric silver stain method was used to define spatiotemporal characteristics of nerve cell body, dendrite, axon and terminal degeneration in brainstem and spinal cord. Rats were exposed to ACR at a dose-rate of either 50 mg/kg per day (i.p.) or 21 mg/kg per day (p.o.), and at selected times brains and spinal cord were removed and processed for silver staining. Results show that intoxication at the higher ACR dose-rate produced a nearly pure terminalopathy in brainstem and spinal cord regions, i.e. widespread nerve terminal degeneration and swelling were present in the absence of significant argyrophilic changes in neuronal cell bodies, dendrites or axons. Exposure to the lower ACR dose-rate caused initial nerve terminal argyrophilia in selected brainstem and spinal cord regions. As intoxication continued, axon degeneration developed in white matter of these CNS areas. At both dose-rates, argyrophilic changes in brainstem nerve terminals developed prior to the onset of significant gait abnormalities. In contrast, during exposure to the lower ACR dose-rate the appearance of axon degeneration in either brainstem or spinal cord was relatively delayed with respect to changes in gait. Thus, regardless of dose-rate, ACR intoxication produced early, progressive nerve terminal degeneration. Axon degeneration occurred primarily during exposure to the lower ACR dose-rate and developed after the appearance of terminal degeneration and neurotoxicity. Spatiotemporal analysis suggested that degeneration began at the nerve terminal and then moved as a function of time in a somal direction along the corresponding axon. These data suggest that nerve terminals are a primary site of ACR action and that expression of axonopathy is restricted to subchronic dosing-rates.

Acrylamide neuropathy. III. Spatiotemporal characteristics of nerve cell damage in forebrain.

Previous studies of acrylamide (ACR) neuropathy in rat PNS [Toxicol. Appl. Pharmacol. (1998) 151:211-221] and in spinal cord, brainstem and cerebellum [NeuroToxicology (2002a) 23:397-414; NeuroToxicology (2002b) 23:415-429] have suggested that axon degeneration was not a primary effect and was, therefore, of unclear neurotoxicological significance. To conclude our studies of neurodegeneration in rat CNS during ACR neurotoxicity, a cupric silver stain method was used to define spatiotemporal characteristics of nerve cell body, dendrite, axon and terminal argyrophilia in forebrain regions and nuclei. Rats were exposed to ACR at a dose-rate of either 50 mg/kg per day (i.p.) or 21 mg/kg per day (p.o.) and at selected times brains were removed and processed for silver staining. Results show that intoxication at either ACR dose-rate produced a terminalopathy, i.e. nerve terminal degeneration and swelling were present in the absence of significant argyrophilic changes in neuronal cell bodies, dendrites or axons. Exposure to the higher ACR dose-rate caused early onset (day 5), widespread nerve terminal degeneration in most of the major forebrain areas, e.g. cerebral cortex, thalamus, hypothalamus and basal ganglia. At the lower dose-rate, nerve terminal degeneration in the forebrain developed early (day 7) but exhibited a relatively limited spatial distribution, i.e. anteroventral thalamic nucleus and the pars reticulata of the substantia nigra. Several hippocampal regions were affected at a later time point (day 28), i.e. CA1 field and subicular complex. At both dose-rates, argyrophilic changes in forebrain nerve terminals developed prior to the onset of significant gait abnormalities. Thus, in forebrain, ACR intoxication produced a pure terminalopathy that developed prior to the onset of significant neurological changes and progressed as a function of exposure. Neither dose-rate used in this study was associated with axon degeneration in any forebrain region. Our findings indicate that nerve terminals were selectively affected in forebrain areas and, therefore, might be primary sites of ACR action.

Evaluation of the ability of d-penicillamine to protect rats against the neurotoxicity induced by zinc pyridinethione, acrylamide, 2,5-hexanedione and p-bromophenylacetylurea.

Dietary exposure of rats to three different concentrations of zinc pyridinethione (ZPT; 166, 332, 498 ppm) caused delayed onset failure in a treadmill test and, at the higher concentrations (332 and 498 ppm), death. Daily treatment with d-penicillamine (d-PEN) increased the latency period for treadmill failure and lethality. Comparable levels of toxicity were achieved only after d-PEN treated rats had consumed 2-3 times more ZPT than rats not treated with d-PEN. In contrast to ZPT, administration of d-PEN did not affect the onset of treadmill failure associated with acrylamide, p-bromophenylacetylurea or 2,5-hexanedione. Thus, d-PEN provided protection which was selective for ZPT.