Cytophotometric analyses of mesencephalic reticular formation neuronal RNA in soman intoxicated rats

Quantitative azure B cytophotometry was used to monitor ribonucleic acid (RNA) responses of individual neurons within the nucleus cuneiformis (NC) and ventrotegmental nucleus (VTN) of the rat mesencephalic reticular formation following single subcutaneous soman (pinacolyl methylphosphonofluoridate) injections (0.5, 0.9 or 1.5 LD₅₀). The sub-lethal (0.5 LD₅₀) dosage of soman produced RNA accumulation in NC neurons, but VTN-RNA levels were not significantly altered. In contrast, both reticular nuclei exhibited prominent RNA depletion with higher soman dosages, the severity of which was greater with lethal (1.5 LD₅₀) than near-lethal (0.9 LD₅₀) dosages. These data indicate that metabolic correlates of enhanced activation of cholinergic reticular nuclei are present only with sub-lethal dosages, and that higher dosages produce characteristics of impaired activation of ascending cholinergic pathways. At present, mechanisms underlying soman-induced metabolic and neurologic deficits remain speculative.     

Cytophotometric analyses of thalamic neuronal RNA in soman intoxicated rats

Quantitative azure B-RNA cytophotometry was used to monitor metabolic responses of individual neurons within the ventrobasal nuclear complex (VBC) and nucleus reticularis (NR) of the rat thalamus following administration of soman (0.5, 0.9 or 1.5 LD₅₀, sc). A dose-dependent depression in brain acetylcholinesterase (AChE) was evidenced. With respect to thalamic RNA responses, a complex pattern of RNA alterations was evidenced, with these two regions generally exhibiting opposite patterns of dose-related RNA changes. With sub-lethal dosages of soman, RNA accumulation was evidenced in the acetylcholine (ACh) mediated excitatory VBC region and RNA depletion in the ACh mediated inhibitory NR neurons. With a lethal dose, an opposite RNA response pattern observed in both thalamic regions. It is postulated that the observed RNA response pattern with sub-lethal dosages of soman is what one would anticipate with cholinergic brainstem reticular formation activation. The absence of such a response with lethal doses strongly suggests some disruption of functional excitatory cholinergic activity and perhaps also an impairment of inhibitory cholinergic synaptic activity.     

Soman toxication in hypoxia acclimated rats: Alterations in brain neuronal RNA and survival

Effects of prior hypoxia acclimation (14-day at 380 mm Hg) on soman (pinacolyl methylphosphonofluoridate) induced brain neuronal RNA and acetylcholinesterase (AChE) depletion and lethality were monitored in rats following their return to ambient oxygenation. Quantitative cytochemical techniques were used to measure RNA and AChE changes in individual cerebrocortical (Layer III) and striatal (caudate plus putamen) neurons. In ambient P_{O 2} controls, soman eventuated in a moderate diminution of neuronal RNA in both brain regions and severe, dosedependent suppression of AChE activity. Hypoxia acclimation per se induced RNA alterations as manifested in cortical RNA depletion and increased variability of striatal neuron RNA contents. In hypoxia acclimated rats, the extent of neuronal RNA depletion following soman injection was attenuated in both brain regions, yet there were no discernible differences in saline control AChE levels or in the extent of soman-induced AChE inhibition in ambient control versus hypoxia acclimated treatment groups. Hypoxia acclimated rats, however, were found to be even more susceptible to lethal actions of soman as assessed using 24- and 48-hour survival following a three-point treatment regimen. These data indicate that while compensatory systemic and central metabolic adjustments associated with 14d acclimation to reduced oxygen availability may retard soman-induced neuronal RNA depletion, resistance to lethal or near-lethal soman exposure is not enhanced. It is postulated that hypoxia acclimation is associated with complex adaptive and maladaptive neurophysiological alterations influencing CNS responsiveness to soman toxication, and that detrimental consequences exceed protection afforded by metabolic adaptation.     

Scanning cytophotometric analysis of myocardial nucleic acid and chromatin changes in soman toxicated rabbits

Myocardial nucleic acid responses were analysed in New Zealand White rabbits 20 min-1 h and 6-8 h following single subcutaneous injections of soman (20, 30, or 40 micrograms kg-1). Scanning-integrating microdensitometry was used to quantify Azure B-RNA and Feulgen-DNA (F-DNA) levels, and changes in the susceptibility of chromatin to Feulgen acid hydrolysis (F-DNA reactivity) of individual ventricular myocardial cells. With a dosage of 20 micrograms kg-1 soman, no RNA alterations were evidenced at 1 h whereas at 6-8 h myocardial cells exhibited higher RNA levels and an increase in F-DNA reactivity of chromatin. With dosages of 30 and 40 micrograms kg-1 soman there was an augmentation in RNA levels and in the acid hydrolysability of nuclear chromatin at both 20 min-1 h and 6-8 h. It is postulated that the observed cellular transformations represent a compensatory augmentation in myocardial metabolic functioning presumably in response to an increased functional demand on the ventricular myocardium. The absence of cytopathic or cytochemical evidence of impairment in nucleic acid metabolism is inconsistent with the premise that soman exerts direct cytotoxic effects on rabbit myocardium.     

Soman intoxication in hypothyroid rats: alterations in brain neuronal RNA,acetylcholinesterase and survival

Studies were conducted to investigate relationships among soman (pinacolyl methylphosphonofluoridate) induced seizure activity, central metabolic impairments and lethality in normal vs thyroid-deficient rats. Quantitative cytophotometric measurements of individual cerebrocortical (layer V) and striatal neuron RNA contents were made following dosages of 0.5, 0.9 and 1.5 LD₅₀ soman (LD₅₀ = 135 μg/kg, sc). Hypothyroidism was associated with a marked diminution of overt convulsive activity and reduced susceptibility to lethal actions of soman as indicated by enhanced 24- and 48-h survival rates at 0.9, 1.2 and 1.5 LD₅₀. Hypothyroidism per se produced RNA depletion in both cortical and striatal neurons. Soman treatment diminished cortical RNA to essentially the same extent in thyroid-deficient rats as in euthyroids, whereas there was no further reduction of striatal neuron RNA. It was found that amelioration of convulsive activity and lethal- ity in hypothyroid rats was accompanied by reduced cerebral acetylcholinesterase (AChE, EC 3.1.1.7) inactivation, and that plasma cholinesterase (EC 3.1.1.8) and aliesterase (EC 3.1.1.1) levels were significantly higher in hypothyroid than in euthyroid saline-control rats. The overall data indicate that soman- induced central metabolic impairments can occur independent of paroxysmal neural activity and lethal actions of the agent. Resistance to soman observed with thyroid deficiency may be due in large part to increased binding to plasma enzymes and diminished delivery of soman to AChE in vital cholinergic sites.     

Perikaryal RNA changes within neurons of the caudate-putamen and substantia nigra in soman intoxicated rats

Quantitative azure B cytophotometry was employed to monitor neuronal ribonucleic acid (RNA) metabolism within cholinergic and noncholinergic brain compartments following single sc injections of either 0.5, 0.9 or 1.5 LD₅₀ soman (pinacolyl methylphosphonofluoridate). Dose-dependent losses in neuronal RNA were observed within the cholinergic caudate-putamen (CP) and dopaminergic substantia nigra pars compacta (SNPC), whereas levels of RNA were generally maintained or elevated within the gabaergic substantia nigra pars reticulata (SNPR). CP acetylcholinesterase was inhibited in a dose-dependent fashion. These neuronal RNA changes are perhaps related to seizure activity. The overall data lend support to the hypothesis that alterations in noncholinergic activity contribute to certain manifestations of soman neurotoxicity, such as seizures, which probably stem from an impairment in the functional integrity of both excitatory and inhibitory neuronal elements.     

Effects of diazepam on soman-induced brain neuronal RNA depletion and lethality in rats

Studies were conducted to determine effects of the benzodiazepine anticonvulsant diazepam on soman induced brain neuronal RNA depletion and lethality in rats. Quantitative azure B-RNA cytophotometry was used to monitor RNA responses of cerebrocortical (layer V) and striatal neurons following dosages of 0.5, 0.9 and 1.5 LD50 soman (LD50 = 135 micrograms/kg, sc), whereas mean time of death and 24-h survival following 0.8, 1.2 and 1.5 LD50 were used to assess the antidotal efficacy of diazepam (2.2 mg/kg, im) pretreatment. Soman produced dose-dependent RNA depletion in both brain regions. This RNA impairment was almost completely prevented by diazepam, although neuronal RNA contents were generally slightly lower than corresponding control values. However, diazepam pretreatment was not associated with any change in mean time of death or in 24-h survival. The overall data suggest that excessive neural activity per se may underlie the genesis of soman-induced central metabolic impairments, but also appear to effectively dissociate epileptiform activity from lethal actions of soman.     

Soman intoxication-induced increase in the levels of mRNAs coding for acute phase reactants

The effect of lethal and repetitive sublethal soman intoxication on the composition of rat liver mRNA was examined by cell-free translation and hybridization. It was found that lethal as well as sustained sublethal soman poisoning of rats elicited a typical acute phase response as judged by a several-fold increase in levels of mRNAs coding for the major acute phase proteins and the vast number of systemic and metabolic changes creating the acute phase response should be taken into account when the metabolic events during the recovery from organophosphate intoxication are under consideration.     

Antecedent short-term central nervous system administration of estrogen and progesterone alters counterregulatory responses to hypoglycemia in conscious male rats

The aim of this study was to test the hypothesis that antecedent short-term administration of estradiol or progesterone into the central nervous system (CNS) reduces levels of neuroendocrine counterregulatory hormones during subsequent hypoglycemia. Conscious unrestrained male Sprague-Dawley rats were studied during randomized 2-day experiments. Day 1 consisted of an 8-h lateral ventricle infusion of estradiol (1 mug/mul; n = 9), progesterone (1 mug/mul; n = 9), or saline (0.2 mul/min; n = 10). On day 2, a 2-h hyperinsulinemic (30 pmol.kg(-1).min(-1)) hypoglycemic (2.9 +/- 0.2 mM) clamp was performed on all rats. Central administration of estradiol on day 1 resulted in significantly lower plasma epinephrine levels during hypoglycemia compared with saline, whereas central administration of progesterone resulted in increased levels of plasma norepinephrine and decreased levels of corticosterone both at baseline and during hypoglycemia. Glucagon responses during hypoglycemia were unaffected by prior administration of estradiol or progesterone. Endogenous glucose production following day 1 estradiol was significantly lower during day 2 hypoglycemia, and consequently, the glucose infusion rate to maintain the glycemia was significantly greater after estradiol administration compared with saline. These data suggest that 1) CNS administration of both female reproductive hormones can have rapid effects in modulating levels of counterregulatory hormones during subsequent hypoglycemia in conscious male rats, 2) forebrain administration of reproductive hormones can significantly reduce pituitary adrenal and sympathetic nervous system drive during hypoglycemia, 3) reproductive steroid hormones produce differential effects on sympathetic nervous system activity during hypoglycemia, and 4) reduction of epinephrine resulted in significantly blunted metabolic counterregulatory responses during hypoglycemia.     

Inhibition of high affinity choline uptake in the rat brain by neurotoxins: effect of monosialoganglioside GM1.

Mustard derivatives of ethyl-choline and hemicholinium-3 have been suggested as possible specific cholinergic neurotoxins. In this study a structural analog of hemicholinium-3, a,a'-bis[di(2-chloroethyl)amino]-4,4'-2-biacetophenone (toxin 7), was added to synaptosomes prepared from the cortex, striatum or hippocampus of rat brain. Synaptosomal high affinity choline uptake (HACU) was significantly decreased in a dose-dependent manner by addition of toxin 7, while synaptosomal uptake of GABA or dopamine was not changed. Incubation of cortical synaptosomes with the monosialoganglioside GM1 prevented the decrease in HACU seen following administration of toxin 7. This preventative effect of GM1 was greater if GM1 was added prior to or concomitant with toxin 7, than if GM1 was added following toxin 7. Two newly synthesized hemicholinium-3 analogs, 4-[3'-di(2-chloroethyl)aminopropionyl]biphenyl (toxin 5) and 4-[3'-di(2-bromoethyl)aminopropionyl]biphenyl (toxin 6) caused a large decrease in HACU when added to cortical synaptosomes, this decrease was significantly greater than that seen with the same dose of toxin 7 or ethyl-choline aziridinium (AF64A). Ultrastructural changes in the synaptosomal membrane following incubation with toxin 7 or toxin 7 with GM1 were examined by electron microscopy. Development of a compound which is both a potent neurotoxin, and is specific for cholinergic neurons will allow new insights into the normal function of the cholinergic system in the CNS and provide animal models of disease states in which cholinergic degeneration is an important element.     

Effect of intrastriatal injection of soman on acetylcholine, dopamine and serotonin metabolism

Intrastriatal injection of soman (14.85 nmol) inhibits cholinesterase (ChE) activity in the striatum with much smaller decreases in ChE activity in other brain areas of the rat. As would be expected, there is a substantial increase in striatal acetylcholine (ACh) content shortly after soman injection. However, this increase is no longer significant 1 h following intrastriatal injection. There is no change in striatal KACh 20 min, 1 h or 24 h following soman injection. ACh content is not affected in the parietal cortex, hippocampus, or medulla/pons following intrastriatal soman injection. However, KACh and/or ACh turnover are reduced in these brain areas following soman injection. There is no consistent effect on dopamine (DA) metabolism in any of the brain areas studied. However, serotonin (5-HT) metabolism appears to be affected in the cortex, hippocampus and medulla/pons following intrastriatal injection of soman. Possible mechanisms of the actions of local injection of soman on brain Ach and 5-HT metabolism are discussed, as well as the differences observed between the effects of local and peripheral administration of soman on DA metabolism in the striatum.     

Adenosine mediation of presynaptic feedback inhibition of glutamate release

Conditions of increased metabolic demand relative to metabolite availability are associated with increased extracellular adenosine in CNS tissue. Synaptic activation of postsynaptic NMDA receptors on neurons of the cholinergic brainstem arousal center can increase sufficient extracellular adenosine to act on presynaptic A1 adenosine receptors (A1ADRs) of glutamate terminals, reducing release from the readily releasable pool. The time course of the adenosine response to an increase in glutamate release is slow (tau > 10 min), consistent with the role of adenosine as a fatigue factor that inhibits the activity of cholinergic arousal centers to reduce arousal.     

Critical role of Kupffer cells in the management of diet-induced diabetes and obesity

The aim of this study was to investigate the role of Kupffer cell in glucose metabolism and hepatic insulin sensitivity in mice. Both phagocytic activity and secretory capacity of Kupffer cells were blunted 24 h after GdCl₃ administration. Glucose tolerance - evaluated following an oral glucose tolerance test (OGTT) - was higher in GdCl₃-treated mice whereas fasting insulinemia and HOMA-IR index decreased. The improvement of glucose tolerance and hepatic insulin signalling pathway after inhibition of Kupffer cells was supported by a lower hepatic gluconeogenic enzyme expression and a higher phosphorylation of Akt upon insulin challenge. Moreover, fasting hyperglycemia, insulin resistance and impaired glucose tolerance - induced by high fat (HF) diet - were improved through chronic administration of GdCl₃. Interestingly, the inhibition of Kupffer cell exerted antiobesity effects in HF-fed mice, and lowered hepatic steatosis. Therefore, strategies targeting Kupffer cell functions could be a promising approach to counteract obesity and related metabolic disorders.     

Concomitant Increases in the Levels of Choline and Free Fatty Acids in Rat Brain: Evidence Supporting the Seizure-Induced Hydrolysis of Phosphatidylcholine

The main objective of this study was to determine whether the excitotoxic cholinesterase inhibitor soman increases the catabolism of phospholipids in rat brain. Injections of soman (70 micrograms/kg, s.c.), at a dose that produced toxic effects, increased the levels of both free fatty acids (175-250% of control) and free choline (250% of control) in rat cerebrum 1 h after administration. All fatty acids contained in brain phosphatidylcholine were elevated significantly including palmitic (16:0), stearic (18:0), oleic (18:1), arachidonic (20:4), and docosahexaenoic (22:6) acids. The changes observed were consistent with those reported to occur following ischemia and the administration of other convulsants. Pretreatment of rats with the anticonvulsant diazepam (4 mg/kg, i.p.) prevented both the signs of soman toxicity and the soman-induced increase of choline and free fatty acids. Diazepam alone did not affect the levels of choline or free fatty acids, cholinesterase activity, or soman-induced cholinesterase inhibition, suggesting that soman toxicity involves a convulsant-mediated increase in phosphatidylcholine catabolism. In addition, administration of the convulsant bicuculline, at a dose that produces seizures and increases the levels of free fatty acids in brain, significantly increased the levels of choline. Results suggest that excitotoxic events enhance the hydrolysis of phosphatidylcholine in brain as evidenced by a concomitant increase in the levels of choline and free fatty acids.     

Effects of pretreatment with 8018 on the toxicokinetics of soman in rabbits and distribution in mice

The effects of 8018 [3-(2'-phenyl-2'-cyclopentyl-2'-hydroxyl-ethoxy)quinuclidine] on the elimination of soman in rabbits blood and distribution in mice brain and diaphragm were investigated using the chirasil capillary gas chromatographic analysis method. In all experiments, the concentration of P(+)soman was below the detection limit (<0.1 ng x mL(-1)). 8018 (1 mg x kg(-1), im, 10 min pre-treated) could significantly reduce the concentration of P(-)soman in rabbit blood from 53.6 +/- 13.3 to 26.2 +/- 9.70 ng x mL(-1) blood as compared to soman-treated control animal at 15 s following soman injection (43.2 microg x kg(-1), iv). Toxicokinetic parameters showed 8018 could increase clearance (CL((S))) from 20.8 +/- 1.54 to 38.2 +/- 15.3 mLx kg(-1) x s(-1) and reduce AUC of P(-)soman from 2.08 +/- 0.151 to 1.30 +/- 0.564 mg x s x L(-1). 8018 could reduce the concentration P(-)soman in diaphragm from 74.7, 70.5, 88.7 ng x g(-1) to 54.5 45.6, 50.0 ng x g(-1) at the time of 30, 90, 120 s after intoxication of soman subcutaneously vs. soman control respectively, but it had no influence on the concentration of free P(-)soman in brain. Isotope trace experiments showed that it could significantly increase the distribution amount of bound [3H]soman in mice plasma and small intestine during 0-120 min after mice received [3H]soman (0.544 GBq.119 microg x kg(-1), sc) compared to soman control group.     

Evidence that Alterations in γ-Aminobutyric Acid and Acetylcholine in Rat Striata and Cerebella Are Not Related to Soman-Induced Convulsions

Many reports have suggested that gamma-aminobutyric acid (GABA) may play a role in organophosphate-induced convulsions. The balance between GABA and acetylcholine (ACh) in the brain also has been suggested by some investigators to be related to brain excitability. We examined these questions by studying the levels of GABA and ACh and the ratios of GABA to ACh in rat striata and cerebella (two major motor control areas in the CNS) after the administration of soman, an organophosphate acetylcholinesterase inhibitor also known as nerve gas. Male Sprague-Dawley rats weighing 250-300 g were injected subcutaneously with three different doses of soman: a subconvulsive dose of 40 micrograms/kg (approximately 30% of the ED50 for convulsions in rats), a convulsive dose of 120 micrograms/kg (approximately one ED50 for convulsions), and a higher convulsive dose of 150 micrograms/kg (approximately 120% of the ED50 for convulsions). The incidence and severity of convulsions were monitored in individual rats until they were sacrificed by focused microwave irradiation of the head at the following time points after soman administration: 4 min, a time prior to the onset of convulsions; 10 min, the time of onset of convulsions; 1 h, the time of peak convulsive activity; and 6 h, a time at which rats were recovering from convulsions. Results showed that in rat striata and cerebella, neither changes in levels of GABA and ACh nor changes in ratios of GABA to ACh were related to soman-induced convulsions, i.e., none of the changes in either levels or ratios of these two neurotransmitters were related to the initiation of, maintenance of, or recovery from soman-induced convulsions.     

Effects of soman and its antidotes on tracheal mucociliary transport of ferrets

The purpose of this study was to examine the role of acetylcholinesterase on mucociliary transport by use of a potent anticholinesterase agent, soman, and potential antagonists, atropine (muscarinic antagonist) and pralidoxime (acetylcholinesterase reactivator). Initial measurements of mucociliary transport rate were obtained in anesthetized ferrets at 30-min intervals for 5.5 h. These rates remained constant at a mean of 18.2 +/- 1.0 (SE) mm/min. We studied the effects of intravenously administered soman (1-8 micrograms/kg) and observed a dose-related change in the rate of mucociliary transport [-1.1 +/- 2.7 (SE) mm/min after 1 microgram/kg, 9.8 +/- 2.9 mm/min after 5 micrograms/kg, and 14.4 +/- 4.3 mm/min after 8 micrograms/kg of soman]. Pretreatment with atropine completely prevented the response to soman, whereas pretreatment with pralidoxime did not significantly alter the response. We postulate that soman's effect on mucociliary transport relates directly to its cholinergic activity. Failure of pralidoxime to inhibit the effects of soman may relate to pralidoxime's inability to reactivate acetylcholinesterase successfully.