Robert L. Rountree

Neuronal degeneration in rat forebrain resulting from D-amphetamine-induced convulsions is dependent on seizure severity and age.

Neuronal damage and degeneration in the rat forebrain was characterized by B4 isolectin and Fluoro-Jade labeling techniques after 4 doses of 15 mg/kg amphetamine i.p. in 70- and 180-day-old Sprague-Dawley rats. In amphetamine-dosed rats some seizure activity occurred in all rats exhibiting pronounced hyperthermia but the degree of seizure activity varied greatly between individual rats. Over 90% of the rats in both age groups that showed behavioral signs of limbic seizures had somatic degeneration in the taenia tecta within 3 days of amphetamine exposure. Degenerating small star-shaped cells were seen in the septum and hippocampus in 70-day-old rats having extensive seizure activity. Although somatic degeneration only sporadically occurred in the piriform cortex of the younger rats, extensive B4 isolectin binding to activated microglia was observed in this area. In older rats prominent somatic degeneration was seen in the piriform cortex and orbital and insular areas of the frontal cortex of rats having seizures. Damage to the basal ganglia and related areas, including the thalamus, parietal cortex and dorsal medial striatum, occurred in rats with pronounced hyperthermia but only correlated with seizures in older rats. In the more severe cases of thalamic damage the highest density of neurodegeneration was localized perivascularly. Thus, amphetamine can produce notable damage to the limbic system when seizures occur and to the basal ganglia and related areas when hyperthermia occurs but the neurotoxicity profiles in these areas are age-dependent and not produced solely by hyperthermia. Further studies to determine whether neuronal damage is the result of or the cause of amphetamine-induced seizures are necessary.

Domoic acid: neurobehavioral and neurohistological effects of low-dose exposure in adult rats.

Adult rats treated IP with domoic acid at 0, 0.22, 0.65, or 1.32 mg/kg were tested for passive avoidance (PA), auditory startle (AS), or conditioned avoidance (CAR) behaviors. Clinical signs were observed only at the 1.32 mg/kg dose level. Within 24 h of dosing, rats surviving a dose of 1.32 mg/kg exhibited transient decreased body weight and exaggerated AS responding. Startle latency and habituation, PA, and CAR were not affected. Examination of brains from six rats per group revealed a subset (2/6) of animals receiving 1.32 mg/kg domoic acid with degenerating neurons in the hippocampal CA1/CA3 subregions and gliosis. The decreased body weight and increased startle suggest a hyperreactivity syndrome possibly related to neuronal degeneration in the hippocampus. In a separate experiment, domoic acid at an IP dose of 0.93 mg/kg was found to produce hypomotility in addition to a decrease in body weight. Both effects were reduced by pretreatment with scopolamine (2 mg/kg), but not with caffeine (30 mg/kg), indicating a possible cholinergic involvement in domoates toxicity.

3-Nitropropionic acid (3-NPA) produces hypothermia and inhibits histochemical labeling of succinate dehydrogenase (SDH) in rat brain.

Abstract3-nitropropionic acid (3-NPA) is a toxin sometimes produced on moldy crops (sugarcane, peanuts, etc.) in amounts sufficient to cause severe neurological disorders when consumed by humans. In vitro, 3-NPA irreversibly inactivates SDH, a Complex II respiratory enzyme required for mitochondrial energy production. A single dose of 3-NPA (30 mg/kg S.C.) was given to singly-caged adult male Sprague-Dawley rats. Rectal temperature was measured after dosing as a potential biomarker of exposure to 3-NPA, and animals were sacrificed at various times after 3-NPA exposure for histochemical visualization of SDH activity. 3-NPA-treated rats experienced a progressive hypothermia, which reached a loss of 3°C or more in core body temperature by 3 hours after dosing. The optical density of the SDH stain in brain was reduced according to a similar time-course, most prominently in the cerebellum and least sharply in the thalamus. The caudate nucleus had the greatest density of SDH staining that we measured in brain; it also has been reported to be the region most consistently lesioned by 3-NPA. However, within other areas of brain such as subdivisions of the hippocampus, neither endogenous SDH activity nor its sensitivity to inhibition by 3-NPA could predict the susceptibility to neurodegenerative changes. Although SDH activity remained significantly reduced in most areas of brain (except thalamus) for up to 5 days after dosing, core temperatures had returned to control values by 5 days suggesting that animals can utilize an alternate method of heat production to withstand insult by 3-NPA.

The effects of perinatal hypoxia on the behavioral, neurochemical, and neurohistological toxicity of the metabolic inhibitor 3-nitropropionic acid

Abstract3-nitropropionic acid (3-NPA) neurotoxicity and long-term effects of perinatal hypoxia were evaluated in 18 adult rats. Hypoxia-insulted (I) and noninsulted (NI) rats were delivered by cesarean section. Hypoxic insult was effected by submerging dissected uterine horns in warmed saline for 15 min. NI rats were delivered from the adjacent nonsubmerged horns. At postnatal day 90, I and NI rats were trained to perform tasks thought to measure behaviors dependent upon aspects of time estimation (TE), motivation, and learning. At 12 months of age, rats were injected i.p. with escalating doses of 3-NPA (5 mg/kg/day to a maximum of 30 mg/kg/day) immediately after each test session and sacrificed at the end of treatment. Additional male rats were used as untreated controls. Although 3-NPA produced a dose-dependent impairment of performance in each task, the effects were qualitatively similar for each group. A significant difference between I and NI rats was, however, observed in the TE task where NI rats completed less of the task at high doses of 3-NPA compared to I rats. Compared to untreated controls, dopamine concentrations were decreased in caudate nucleus of both I and NI rats after 3-NPA. Specific areas most frequently damaged included cerebral cortex, hippocampal subfield CA1, thalamus, caudate nucleus, and the cerebellum. Lesions usually were less extensive in the I rather than NI members of a littermate pair, suggesting a possible protective effect of perinatal hypoxia against subsequent 3-NPA neurotoxicity.

Protective Effect of l‐Carnitine in the Neurotoxicity Induced by the Mitochondrial Inhibitor 3‐Nitropropionic Acid (3‐NPA)

ZBIGNIEW BINIENDA,a,c JOHN R. JOHNSON,a ALEXANDER A. TYLER-HASHEMI,a ROBERT L. ROUNTREE,a PHILIP P. SAPIENZA,b SYED F. ALI,a AND CHUNG S. KIMb aDivision of Neurotoxicology, National Center for Toxicological Research/ Food and Drug Administration (NCTR/FDA), Jefferson, Arkansas, USA bDivision of Toxicological Research, Center for Food Safety and Applied Nutrition/ Food and Drug Administration (CFSAN/FDA), Washington, DC, USA

Biomarkers of 3‐Nitropropionic Acid (3‐NPA)‐Induced Mitochondrial Dysfunction as Indicators of Neuroprotection

Abstract: In humans or animals, symptoms of mitochondrial energy dysfunction may be produced by mutations or inborn errors of the necessary enzymes, as well as by enzyme inhibitors or uncouplers of the oxidative phosphorylation process. 3‐Nitropropionic acid (3‐NPA) is a toxin that is sometimes produced on moldy crops (sugarcane, peanuts, etc.) in amounts sufficient to cause severe neuromuscular disorders when consumed by humans. In vitro, 3‐NPA irreversibly inactivates SDH, a Complex II respiratory enzyme important for mitochondrial energy production. We have been studying biomarkers of 3‐NPA exposure in the expectation that such markers may be useful in the screening process to identify neuroprotective agents against neurotoxicity produced by mitochondrial energy dysfunction. Animals were sacrificed at various times after 3‐NPA exposure for histochemical visualization of SDH activity and measurement of immediate postmortem rectal temperature. 3‐NPA‐treated rats experienced progressive hypothermia that reached a loss of 3°C or more in core body temperature by three hours after dosing. The optical density of the SDH stain in brain was reduced, following a similar time course, most prominently in the cerebellum and least sharply in the thalamus. Some rats were given injections of l‐carnitine (an enhancer of fatty acid transport) either alone, or as a pretreatment prior to a dose of 3‐NPA. Although l‐carnitine deficiency by itself can produce mitochondrial dysfunction, pretreatment with l‐carnitine was of limited efficacy at overcoming the effects of 3‐NPA on either body temperature or quantitative SDH histochemistry. Body temperature and SDH histochemistry may be useful biomarkers for evaluating the efficacy of neuroprotective agents against lower doses of 3‐NPA, against other pharmacological models of mitochondrial dysfunction, or even against genetic mitochondrial diseases.

Quantitating silver-stained neurodegeneration: the neurotoxicity of trimethlytin (TMT) in aged rats

This report describes the development of a histoanalytical procedure to measure the degree of neurodegeneration produced by the organometal toxicant trimethyltin (TMT). Based on a previous, non-quantitated experiment we hypothesized that the same dose of TMT would produce greater damage in animals of increasing age. Male rats aged 6, 12, 18, or 24 months at the time of dosing were given either 4.5 mg/kg TMT or saline (i.p.). One month after dosing, rats were perfused and their brains removed and processed to selectively silver-impregnate degenerating cell bodies as well as axon terminals and dendrites. Neurodegeneration was most prominent in the hippocampi (especially CA1 stratum radiatum) of TMT-treated rats, but not in the controls. Computer-assisted counting of the silver grains marking damage indicated greater neurotoxicity from the same dose of TMT when given to the older animals. Thus the grain density in the 6-month-old TMT-treated rats was not significantly elevated from the 6-month-old controls (P>0.10). The 12-month-old TMT-treated rats had significantly increased grain densities compared to their controls (P<0.05), but still larger increases of grain counts were observed in the 18- and 24-month-old rats (both P-values<0.01). Our findings with TMT are similar to previous, but nonquantitative, reports that the neurotoxic effects of kainic acid and methionine sulfoximine were also greater in older rats. An increased sensitivity to neurotoxicants might help explain the apparently spontaneous degeneration of cortical neurons in aging and in the neurological diseases of old age. The method we report here for quantitation of silver grains marking neurodegeneration should be adaptable to a wide range of histologically-based neurotoxicology investigations.

Effect of l-Carnitine Pretreatment on 3-Nitropropionic Acid-Induced Inhibition of Rat Brain Succinate Dehydrogenase Activity

Abstract: l‐Carnitine (LC) plays an important regulatory role in the mitochondrial transport of long chain free fatty acids (FFA). 3‐Nitropropionic acid (3‐NPA) is known to induce cellular energy deficit and oxidative stress‐related neurotoxicity via an irreversible inhibition of mitochondrial succinate dehydrogenase (SDH). In the present study, activity of SDH was measured in order to evaluate neuroprotective effects of LC against the 3‐NPA‐induced neurotoxicity. Male, CD Sprague‐Dawley rats, three months old, were injected with either 50 or 100 mg/kg of LC, i.p., 30 min prior to 3‐NPA (30 mg/kg, s.c.) or with 3‐NPA alone. The activity of brain SDH was quantified spectrophotometrically in caudate nucleus (CN), frontal cortex (FC), and hippocampus (HIP) 60 min after the 3‐NPA injection. The SDH activity in the animals treated with 3‐NPA alone was 38% (CN), 50% (FC), and 36% (HIP) that of saline controls. Pretreatment with LC prior to 3‐NPA injection attenuated decreases of SDH activity by approximately 15 and 29% (LC low and high dose, respectively). Despite the attenuation of SDH inhibition, the activity of SDH in these regions remained significantly lower in treated than in control rats (p < 0.05). It appears that the protective effect of LC against 3‐NPA‐induced oxidative stress cannot be explained by the direct action of LC to interfere with the SDH inhibition but are rather achieved by LC actions downstream of the SDH inhibition.

Age and Dietary Factors in Hippocampal Sensitivity to Trimethyltin

Prolonged restriction of the total daily calories or protein fed to rodents slows the kidney damage, myocardial degeneration, and the rate of appearance of neoplasms, but not the decline of sensorimotor function that normally occurs with age.’ Because these dietary regimes are now being practiced by people in an effort to extend their lifespan, it is important to understand the effects of such dxts on the brain. Trimethyltin (TMT) neurotoxicity is in some respects similar to age-related neurodegeneration. Both TMT (FIG. la,b) and age damage hippocampal neurons, produce Alzheimer‘s Type I1 ghosis (both nuclei and cytoplasm of astrocytes are enlarged) (FIG. lc,d), and impair passive avoidance perf~rmance.~-~ Both TMT and age may be hypothesized to act on the hippocampus through release of endogenous “excitotoxins.” Therefore we used TMT to “challenge? the aging brain to determine how dietary factors might alter its resistance to neurotoxic (and by inkrence, aging) processes. We administered single i.p. doses of TMT (0 to 4.5 mgkg) to 7or 17-month-old male Fischer 344 rats that had been either fed ud libitum with a 21% (“medium”) protein diet, or restricted (“CR”) to 60% of the ud &turn caloric intake of the medium protein diet from 14 weeks of age. Other groups had been maintained ud libitum fbr at least 2 months on isocaloric 13% (“low”) or 2996 (“high”) protein diets. Three weeks after TMT dosing, we evaluated passive avoidance performance (as an indicator of behavioral impairment) and we carried out histological studies using a FinkHeimer stain (to find degenerating axons) and immunohistochemical studies (for &al fibrillary acidic protein [GFAP]). We also measured several amino acids and analyzed hippocampal kainic acid (KA) receptors. TABLE 1 summarizes our investigtions, which are reported in more detail elsewhere.6 Glutamine (Gln), which increases glutamate release when superfused

Evaluation of neurodegeneration in scrapie-infected animals by selective methods that detect cellular degeneration

Scrapie is a fatal neurodegenerative disease of sheep and goats. The precise details of neuronal and neurite degeneration in scrapie-infected animals remain unknown. Using specific silver staining methods, we compared the neurodegeneration caused by treatment of rats with kainic acid (KA) or ibogaine (IBO) to the neuropathology observed in mice infected with the C602 strain of scrapie. As reported previously, KA resulted in extensive silver labeling of neurons, especially in the cortex, putamen and hippocampus. IBO silver labeling was observed only in small clusters of Purkinje neurons in the paravermal region of the cerebellum. However, in scrapie-infected mice, a few silver stained neurons (differing from the dark degenerating neurons observed following neurotoxic exposure) were found in layer II of cortex, cingulate cortex, zona incerta, thalamus and hypothalamus. Some silver grains were observed in glial-like cells, especially those in the paraventricular region. Degenerating axons were positive for silver staining and were found in the cortex, cingulate cortex, corpus callosum, habenulae, septum, fornix, thalamus, caudate putamen and a few in fasciculus retroflexus and substantia nigra. Our results suggest that the limbic system is one of the important loci for the neurodegenerative effect of at least some scrapie strains.