Persistent increase of blood lead level and suppression of δ-ALAD activity in northern bobwhite quail orally dosed with even a single 2-mm spent lead shot.

Birds that display grit ingestion behavior are potentially at risk of lead (Pb) poisoning from mistaken ingestion of spent Pb shot pellets. The majority of available studies designed to assess such risk have used unspent shot pellets rather than field-obtained spent shot, which is oxidized and otherwise changed by weathering. Available studies also often administered more or heavier shot pellets to a bird than it might be expected to ingest. The current study dosed northern bobwhite quail (Colinus virginianus) weighing 194.6 ± 23.1 g (female birds) and 199.3 ± 12.2 g (male birds) with one to three spent no. 9 Pb shot collected from a skeet range, with particular interest in the toxicity that may occur from ingestion of a single 2-mm, 50 mg shot. An 8 week post-dosing clinical observation period was employed, over which feed consumption, body weight, blood Pb levels, and a battery of blood physiological parameters were made. Weight loss occurred in the birds, including male birds dosed with one Pb pellet. Erythrocyte delta aminolevulinic acid dehydratase (δ-ALAD) levels were decreased for the duration of the study across exposures and to levels associated with injury in wild bird populations. Decreased ALAD was particularly severe in female birds dosed with one Pb pellet and was still 92 % decreased at 8 weeks after dosing. Together, these results suggest that inadvertent ingestion of a single no. 9 Pb shot pellet can adversely affect the health of northern bobwhite quail.

Anticonvulsant action of excitatory amino acid antagonists.

Compounds that antagonize neuronal excitation induced by dicarboxylic amino acids were tested in two animal models of epilepsy, namely sound-induced seizures in DBA/2 mice and threshold pentylenetetrazol seizures in Swiss mice. Sound-induced seizures could be prevented by intracerebroventricular injection of compounds that block excitation due to N-methyl-D-aspartic acid. The most potent such compound, 2-amino-7-phosphonoheptanoic acid, was anticonvulsant in both test systems when given either intraperitoneally or intracerebroventricularly. Specific antagonists of excitation that is caused by amino acids provide a new class of anticonvulsant agents.

Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain.

In rats ischemia of the forebrain induced by a 30-minute occlusion of the carotid artery, followed by 120 minutes of arterial reperfusion, produced ischemic lesions of selectively vulnerable pyramidal cells in both hippocampi. Focal microinfusion into the dorsal hippocampus of 2-amino-7-phosphonoheptanoic acid, an antagonist of excitation at the N-methyl-D-aspartate-preferring receptor, before ischemia was induced protected against the development of ischemic damage. It is proposed that excitatory neurotransmission plays an important role in selective neuronal loss due to cerebral ischemia.

Memory antibody responses of broiler and leghorn chickens as influenced by dietary vitamin E and route of sheep red blood cell administration.

Influences of dietary levels of vitamin E fed to hens and their progeny, and routes of SRBC inoculation on antibody responses of diverse populations of chickens were studied. Populations were a commercial broiler sire line (C), 2 commercial broiler dam lines (A and B), and Leghorn lines selected for high (H) or low (L) antibody response to SRBC. Dams from lines A and B were fed diets supplemented with either 10 or 300 IU/kg of vitamin E, whereas dams from lines H and L received only the diet with 10 IU of vitamin E/kg. Progeny from matings of C males with A and B females as well as H and L females mated to males from their respective lines were hatched on the same day and fed diets supplemented with either 10 or 300 IU/kg of vitamin E. Breeders were the same age and eggs were incubated in the same machine. Chicks were inoculated on d 14 intravenously with 0.1 mL of a 0.5% suspension of SRBC or intramuscularly with 0.1 mL of a 25% suspension of SRBC. Antibody response was measured 6 and 14 d later. Chicks received a booster i.m. inoculation of 0.1 mL of 25% SRBC on d 28. Titers were again measured 6 and 14 d later. Level of vitamin E fed to dams did not affect progeny BW or plasma vitamin E levels. Although titers were higher following i.v. than i.m. inoculation, the degree of difference varied among stocks. Dietary vitamin E level interacted with inoculation route with a greater response to the higher than lower level of vitamin E for i.v., but there was no difference for i.m. There were stock x level of vitamin E and stock x route of inoculation interactions for secondary responses to SRBC inoculation. Stock rankings after the first inoculation were not predictive of the rankings after the second inoculation. The 30-fold increase in dietary level of vitamin E resulted in >12-fold differences in plasma levels of vitamin E. Overall, there was a stock-dependent influence of dietary vitamin E on growth and humoral antibody response.

Poult performance as influenced by age of dam, genetic line, and dietary vitamin E.

An experiment was conducted to measure the effects of age of dam, genetic line, and dietary levels of vitamin E on growth and immunocompetence of poults. Age of dam was defined as younger (in early egg production) and older (past peak production); line consisted of a commercial sire and dam line; and dietary vitamin E was supplemented into the diet at 10 and 300 IU/kg of feed. Traits measured included body, liver, gizzard, and yolk sac weights at hatch; BW and feed conversion to 9, 28, and 42 d; response to SRBC, Phaseolus vulgaris agglutinin-P, and Escherichia coli administered at 28 d of age; and response to a cold stress on d 5 posthatch. Differences among genetic lines were evident with growth greater for poults from the sire than from the dam line. Performance of poults from older dams was generally superior to that of poults from younger dams. The higher level of vitamin E resulted in a greater than 7-fold increase in blood plasma vitamin E and reduced mortality. There were interactions among the main effects in which the fitness of poults from younger dams was enhanced by the higher level of vitamin E and the effect of breeder age differed among genetic lines.

Excitatory amino acid neurotoxicity and neurodegenerative disease.

The progress over the last 30 years in defining the role of excitatory amino acids in normal physiological function and in the abnormal neuronal activity of epilepsy has been reviewed in earlier articles in this series. In the last five years it has become clear that excitatory amino acids also play a role in a wide range of neurodegenerative processes. The evidence is clearest where the degenerative process is acute, but is more controversial for slow degenerative processes. In this article Brian Meldrum and John Garthwaite review in vivo and in vitro studies of the cytotoxicity of amino acids and summarize the contribution of such toxicity to acute and chronic neurodegenerative disorders.

Na+ channels as targets for neuroprotective drugs.

Drugs that block voltage-dependent Na+ channels are well known as local anaesthetics, antiarrhythmics and anticonvulsants. Recent studies show that these compounds also provide a powerful mechanism of cytoprotection in animal models of cerebral ischaemia, hypoxia or head trauma. In this article Charles Taylor and Brian Meldrum review evidence indicating that Na+ channel modulators are neuroprotective and describe recent ideas for the molecular sites of action of voltage-dependent Na+ channel blockers. Clinical trials with several compounds are now in progress for stroke and traumatic head injury, and the therapeutic potential for this group of compounds is discussed.

Noradrenergic regulation of growth hormone secretion in the baboon.

We have investigated the effects of iv administered noradrenergic agonists and antagonists on plasma GH concentration in the adolescent baboon, Papio papio, with the aim of defining the relative roles of adrenergic receptor subtypes (alpha 1, alpha 2, beta 1, and beta 2) in the regulation of GH release. Clonidine (0.02 mg/kg) or UK-14,304 (0.02 mg/kg), potent centrally acting alpha 2 noradrenergic agonists, were infused into 24 animals pretreated with either saline, or selective alpha 1 and alpha 2 noradrenergic antagonists. Both agonists potently augment plasma GH, producing peak levels of 30-60 ng/ml 15 min post infusion. These responses can be prevented by the prior infusion of the alpha 2 antagonist, piperoxane (1.0 mg/kg), but not by the alpha 1 antagonist, prazosin (2.0 mg/kg). Log dose response curves of the 2 agonists demonstrate a greater potency for UK-14,304 vs. clonidine on a molar basis. In animals pretreated with monoamine depleting agents (reserpine and alpha-methyl paratyrosine) the plasma GH response to an infusion of clonidine (0.02 mg/kg) is significantly enhanced (P less than 0.001). Beta-Adrenoreceptor antagonism by propranolol (0.02 or 1.0 mg/kg) or the more selective beta 2-adrenoreceptor antagonist, ICI 118,551 (0.02-1.0 mg/kg), results in a rapid and significant (P less than 0.01) increase in plasma GH. The beta 1-antagonist, practolol (0.2-2.0 mg/kg), does not alter plasma GH levels. It is proposed that in the baboon, noradrenaline acts on alpha 2-noradrenergic receptors to stimulate GH release and on beta 2-noradrenergic receptors to inhibit GH release.

Excitotoxicity and selective neuronal loss in epilepsy.

The early stages of selective neuronal loss occurring in the hippocampus and other brain regions after prolonged epileptic activity have fine structural characteristics matching those induced by excitotoxic agents. NMDA receptor antagonists provide protection against such damage. The extracellular concentration of glutamate or aspartate may be transiently raised prior to or early in seizure activity but tends not to match the levels associated with hypothalamic damage in the original paradigm of excitotoxicity. Various aspects of the excitotoxic process are examined to see if they can account for particular details of the pattern of selective neuronal loss. A full explanation of selective vulnerability will take into account not only a range of characteristics of the vulnerable neuron but also its functional network during sustained activity.

Protection by NMDA antagonists against selective cell loss following transient ischaemia.

We have administered antagonists acting competitively or noncompetitively at the N-methyl-D-aspartate receptor after a short period of incomplete ischaemia and evaluated selective neuronal loss in the CA1 region of the rat hippocampus. The competitive antagonists D-(-)-2-amino-7-phosphonoheptanoate (2APH); 100 or 330 mg/kg; 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP); 3.3 or 10 mg/kg; and CGS 19755 (cis-4-phosphonomethyl-2-piperidine carboxylate) 3.3 or 10 mg/kg; and the noncompetitive antagonists MK801 [+)5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate), 0.3, 1, or 3 mg/kg, and dextrorphan, 2, 6, 18, or 54 mg/kg, were administered intraperitoneally 15 min and 5 h after a 10-min incomplete ischaemia period; additionally MK801 (1 or 3 mg/kg) and CGS 19755 (10 or 30 mg/kg) were administered 5 and 10 h postischaemia. Seven days after ischaemia, the brains were fixed by perfusion. CA1 pyramidal cell counts were performed on Nissl-stained sections using an ocular grid piece. Ventilated (no ischaemia) control animals had a mean of 406 +/- 13 CA1 neurones/3 grid lengths. Ischaemia reduced this mean to 157 +/- 23. A significant protective effect against this cell loss was seen after two injections (at 15 min and 5 h postischaemia) of 2APH, CPP (10 mg/kg), CGS 19755 (10 mg/kg), MK801 (1 mg/kg), and dextrophan (54 mg/kg). Delayed injection (5 and 10 h postischaemia) of CGS 19755 (10 and 30 mg/kg) and MK801 (1 and 3 mg/kg) did not provide any protection against pyramidal cell loss.

Long-term development of selective neuronal loss and the mechanism of protection by 2-amino-7-phosphonoheptanoate in a rat model of incomplete forebrain ischaemia.

Excitatory neurotransmission at the N-methyl-D-aspartate (NMDA) receptor is selectively blocked by 2-amino-7-phosphonoheptanoate acid (2-APH). Acute focal microinjection of 2-APH into the rat hippocampus partially protects against cytopathology developing in selectively vulnerable neurons after 30 min of ischaemia and 2 h of reperfusion. We show that this protective action of 2-APH does not involve alterations in local cerebral blood flow (CBF). Intermediate cytopathology and long-term neuronal survival has been assessed in rats receiving focal injections of (+/-) 2-APH, 20 micrograms in 1 microliter, into one dorsal hippocampus prior to and 4 and 10 h after a 10-min period of forebrain ischaemia. Cytopathology assessed 4 or 24 h after ischaemia shows no difference between the buffer and 2-APH-injected hippocampi. Assessment after 7 days survival shows a significant protection against neuronal loss in the CA1 zone of the 2-APH-injected hippocampus compared with the contralateral, buffer-injected hippocampus. Systemic injection of D(-)2-APH (675 mg/kg i.v. at 0 h, 4 h, and 10 h) affords significant protection to CA1 hippocampal neurones (as assessed after 7 days). These results suggest that maintained blockade of neurotransmission at the NMDA receptor in the postischaemic period can protect against delayed cell loss. The mechanism may be through antagonism of the excitotoxic action of an endogenous neurotransmitter acting in the postischaemic period.

The use of in vivo fluorescence image sequences to indicate the occurrence and propagation of transient focal depolarizations in cerebral ischemia.

A method for the detection and tracking of propagated fluorescence transients as indicators of depolarizations in focal cerebral ischemia is described, together with initial results indicating the potential of the method. The cortex of the right cerebral hemisphere was exposed for nonrecovery experiments in five cats anesthetized with chloralose and subjected to permanent middle cerebral artery (MCA) occlusion. Fluorescence with 370-nm excitation (attributed to the degree of reduction of the NAD/H couple) was imaged with an intensified charge-coupled device camera and digitized. Sequences of images representing changes in gray level from a baseline image were examined, together with the time courses of mean gray levels in specified regions of interest. Spontaneous increases in fluorescence occurred, starting most commonly at the edge of areas of core ischemia; they propagated usually throughout the periinfarct zone and resolved to varying degrees and at varying rates, depending on proximity of the locus to the MCA input. When a fluorescence transient reached the anterior cerebral artery territory, its initial polarity reversed from an increase to a decrease in fluorescence. An initial increase in fluorescence in response to the arrival of a transient may characterize cortex that will become infarcted, if pathophysiological changes in the periinfarct zone are allowed to evolve naturally.

Calcium overload in selectively vulnerable neurons of the hippocampus during and after ischemia: an electron microscopy study in the rat.

Light and electron microscopy has been used to study the cytopathological changes in the rat hippocampus directly after a 30-min period of forebrain ischemia and after 30 or 120 min of reperfusion. The fine structural localization of calcium has been demonstrated using the oxalate/pyroantimonate procedure. Cellular changes considered typical of ischemia (swelling of astrocytic processes, distention of mitochondria, condensation of cytoplasm, "ischemic cell change") are most prominent after 30 min of reperfusion. At this time, dense calcium pyroantimonate deposits are evident in swollen mitochondria in pyramidal and hilar neurons. After 120 min of reperfusion, substantial restitution has occurred; most mitochondria appear normal and there are few calcium deposits. However, a small number of selectively vulnerable neurons (hilar and pyramidal neurons) show dense condensation (ischemic cell change) with multiple vacuoles containing calcium deposits. The role of excessive calcium entry and mitochondrial calcium overload during the reperfusion period in determining the death of selectively vulnerable neurons is discussed.

Cerebroprotective effect of the nitric oxide synthase inhibitors, 1-(2-trifluoromethylphenyl) imidazole and 7-nitro indazole, after transient focal cerebral ischemia in the rat.

The novel neuronal nitric oxide synthase inhibitors, 1-(2-trifluoromethylphenyl)imidazole (TRIM) and 7-nitro indazole (7-NI), were used to investigate the role of nitric oxide in a model of transient focal cerebral ischemia in vivo. In halothane-anesthetized rats, the middle cerebral artery (MCA) was occluded for 2 hours using an intravascular thread and then reperfused for 22 hours before histologic evaluation. TRIM (10, 20, or 50 mg/kg), 7-NI (60 mg/kg), TRIM (50 mg/kg) plus L-arginine (300 mg/kg), or L-arginine (300 mg/kg) alone was administered intraperitoneally, either at 5 or 90 minutes after MCA occlusion. Immediate administration (5 minutes after MCA occlusion) of TRIM produced a dose-related reduction in lesion size, which was reversed with L-arginine coadministration. Similarly, delayed administration of TRIM (90 minutes after MCA occlusion, 50 mg/kg) decreased total lesion volume by 48.4% +/- 13.0% in comparison to a reduction of 39.3% +/- 10.9% when TRIM (50 mg/kg) was administered immediately (5 minutes) after occlusion. 7-NI (60 mg/kg) reduced the total lesion volume by 38.5% +/- 13.7% when administered immediately (5 minutes) after MCA occlusion, but had no effect when administration was delayed (90 minutes). Neither TRIM (50 mg/kg) nor 7-NI (60 mg/kg), administered 5 minutes after MCA occlusion, had any significant effect on mean arterial blood pressure throughout the ischemic period or for up to 10 minutes after reperfusion. These results indicate that immediate or delayed administration of the selective neuronal NOS inhibitor TRIM reduces the lesion volume after transient MCA occlusion. In contrast, only immediate administration of 7-NI reduces lesion volume.

A primate model for testing anticonvulsant drugs.

Senegalese baboons (Papio papio), with a natural syndrome of photosensitive epilepsy, consistently show generalized myoclonic jerks if stimulated stroboscopically at hourly intervals, two to eight hours after the intravenous administration of allylglycine, 200 mg/kg. This provides a model for testing the acute antiepileptic effects of established or new drugs. The relationship between concentration of drug, antiepileptic action, and acute neurological toxic effects can be studied. Pnehobarbital (15 mg/kg) and diazepam (0;5 to 1.5 mg/kg) were highly effective in the absence of signs of toxic reaction (plasma levels: phenobarbital sodium, 0.7 to 1.7 mg/100 ml; diazepam, greater than 0.5 mug/ml). After administration of carbamazepine (30 to 40 mg/kg) and diphenylhydantoin sodium (40 to 50 mg/kg), antiepileptic action was seen, but was accompanied by severe toxic signs (nystagmus and ataxia). Sulthiame (20 to 125 mg/kg) and ethosuximide (50 to 100 mg/kg) had little antiepileptic activity and no acute toxic effects. This primate model may aid the identification of new drugs that are active against grand mal seizures and status epilepticus.

The role of glutamate in epilepsy and other CNS disorders.

Glutamate is the principal excitatory neurotransmitter in the brain and, as such, it inevitably plays a role in the initiation and spread of seizure activity. It also plays a critical role in epileptogenesis. The process of "kindling" limbic seizures in rodents by repeated electrical stimulation is dependent on activation of N-methyl-D-aspartate (NMDA) receptors. The function of these receptors is enhanced in the hippocampus of kindled rats and in the cerebral cortex of patients with focal epilepsy. Microdialysis studies show an increase in the extracellular concentration of glutamate and aspartate before or during seizure onset, suggesting that either enhanced amino acid release or impaired uptake contributes to seizure initiation. Glutamate antagonists selective for NMDA or non-NMDA receptors are potent anticonvulsants when given systemically in a wide variety of animal models of epilepsy. They are of limited efficacy against kindled seizures in rats and (on the basis of preliminary evidence) in patients with drug-refractory complex partial seizures. Cognitive side effects appear to be a significant problem with competitive, as well as noncompetitive, NMDA antagonists. Glutamate receptor antagonists provide significant protection against brain damage following global or focal cerebral ischemia or acute traumatic injury in rodent models. Anticonvulsant compounds of the lamotrigine type, which act on sodium channels and reduce ischemia-induced glutamate release, are cerebroprotective in rodent ischemia models and are free from the cognitive side effects of NMDA-receptor antagonists.

Hyperglycemia does not augment neuronal damage in experimental status epilepticus.

The neuronal regions affected and the neuropathologic features of ischemia and status epilepticus are similar. Experimentally, elevated plasma glucose levels, increasing brain lactate, are associated with more severe neuropathologic damage from cerebral ischemia. We therefore studied the cytologic features and cerebral content of lactate and glucose in the selectively vulnerable neurons of rat hippocampus after 2 hours of L-allylglycine-induced status epilepticus in rats with mean plasma glucose concentrations of 65, 250, and 480 mg/100 ml. Brain lactate concentration was elevated in status and maximal in the high-glucose group, but the maximum levels (8 mumol/g) were less than those thought to augment cell death in ischemia. Using multiple linear regressions, only time-in-status predicted neuropathologic damage.

Modulation of experimentally induced epilepsy by intracerebral grafts of fetal GABAergic neurons.

Systemic administration of pilocarpine to rats induces seizures that resemble complex partial epilepsy in humans. Susceptibility to these seizures is increased by lesion of the GABAergic striatonigral projection. Transplantation of fetal GABAergic neurons, but also of control non-GABAergic tissue, to the deafferent substantia nigra can reduce such lesion-increased seizure susceptibility. These observations are consistent with prior evidence that GABAergic basal ganglia outflow plays an important role in controlling the spread of seizures, and raise the possibility that intracerebral grafts may be of use for therapy of medically-unresponsive epilepsies.

Decreased sensitivity to Group III mGluR agonists in the lateral perforant path following kindling.

The ability of the selective Group III mGluR agonist L-serine-O-phosphate (L-SOP) to inhibit lateral perforant path (LPP) evoked responses in the dentate gyrus was tested in hippocampal slices from commissurally-kindled rats 1-2 days after the last seizure, implanted controls, and fully-kindled rats rested for 28 days without stimulated seizures (28 days post-seizure, 28 dps). L-SOP was more potent in controls than kindled or 28 dps animals, decreasing the fEPSP slope with IC50s of 2.4 microM, 18.7 microM and 10.5 microM, respectively. Paired pulse facilitation (PPF, 50 ms) was comparable in control and kindled rats, but was markedly reduced in 28 dps rats, indicating increased release probability. Inhibition of the field excitatory postsynaptic potentials (fEPSP) by L-SOP was correlated with enhanced PPF in all groups, affirming a presynaptic site of action. At moderate levels of L-SOP-induced inhibition (20-60%), PPF showed significantly greater enhancement in 28 dps than in the other two groups. These results are interpreted as showing a functional reduction of the presynaptic inhibitory Group III mGluR (probably mGluR8) response in the LPP after kindling. Furthermore, PPF changes indicate that the kindled state may be associated with a long-lasting increase in the probability of release from LPP terminals, which may be temporarily masked or counterbalanced by recent seizures.

Anticonvulsant properties of flunarizine on reflex and generalized models of epilepsy.

The anticonvulsant activity of 1-bis(4-fluorophenyl)methyl-4-(3-phenyl-2-propenyl)-piperazine, flunarizine, was studied after intraperitoneal administration in DBA/2 mice (seizures induced by sound), intravenous administration in Papio papio (myoclonus induced by photic stimulation) and oral administration in Wistar rats (seizures induced by cefazolin). Protection against sound-induced seizures was observed after intraperitoneal administration of flunarizine (5-40 mg/kg). The ED50 for suppression of tonic, clonic and wild running phases of sound-induced seizures was 3.3, 9.8 and 17.5 mg/kg, respectively. This protective action was significantly reduced by pretreatment with aminophylline (50 mg/kg, i.p.). In photosensitive baboons flunarizine (0.5-1.0 mg/kg, i.v.) provided partial protection against myoclonic responses to stroboscopic stimulation. After flunarizine (2 mg/kg, i.v.) this protection lasted for more than 5 hr (and was complete at 2-3 hr). Cefazolin-induced seizures in rats were prevented by administration of flunarizine (20-40 mg/kg, orally). The ED50 for the suppression of tonic and clonic seizures evoked by subsequent intravenous administration of cefazolin was 25 mg/kg. The protective effects of flunarizine (40 mg/kg, orally) were maximal after 3-6 hr and were maintained for 16-24 hr. Behavioural effects of flunarizine included signs of sedation in both mice and rats. Tolerance to the antiepileptic effects of flunarizine was not seen after chronic treatment in rats. The role of purinergic receptors and of calcium entry blockade in the anticonvulsant action of flunarizine requires further study.