Post-exposure treatment with nasal atropine methyl bromide protects against microinstillation inhalation exposure to sarin in guinea pigs

We evaluated the protective efficacy of nasal atropine methyl bromide (AMB) which does not cross the blood-brain barrier against sarin inhalation exposure. Age and weight matched male guinea pigs were exposed to 846.5 mg/m³ sarin using a microinstillation inhalation exposure technique for 4 min. The survival rate at this dose was 20%. Post-exposure treatment with nasal AMB (2.5 mg/kg, 1 min) completely protected against sarin induced toxicity (100% survival). Development of muscular tremors was decreased in animals treated with nasal AMB. Post-exposure treatment with nasal AMB also normalized acute decrease in blood oxygen saturation and heart rate following sarin exposure. Inhibition of blood AChE and BChE activities following sarin exposure was reduced in animals treated with nasal AMB, indicating that survival increases the metabolism of sarin or expression of AChE. The body weight loss of animals exposed to sarin and treated with nasal AMB was similar to saline controls. No differences were observed in lung accessory lobe or tracheal edema following exposure to sarin and subsequent treatment with nasal AMB. Total bronchoalveolar lavage fluid (BALF) protein, a biomarker of lung injury, showed trends similar to saline controls. Surfactant levels post-exposure treatment with nasal AMB returned to normal, similar to saline controls. Alkaline phosphatase levels post-exposure treatment with nasal AMB were decreased. Taken together, these data suggest that nasal AMB blocks the copious airway secretion and peripheral cholinergic effects and protects against lethal inhalation exposure to sarin thus increasing survival.     

Gene expression in TUNEL-positive neurons in human immunodeficiency virus-infected brain

Human immunodeficiency virus (HIV) infection of the central nervous system (CNS) results in neuronal damage and apoptosis, and both in vitro models and pathological studies suggest that a variety of neurotoxins released by HIV-infected and -activated macrophages/microglia selectively damage susceptible subsets of neurons. Confirmation of in vitro findings of mechanisms of neurodegeneration and neuronal cell dysfunction in vivo has been approached through detailed pathological analysis of regional structural damage, immunohistochemical detection of selected antigens in damaged cells, and, more recently, analysis of gene expression in whole tissue blocks or pooled populations (hundreds/thousands) of microdissected cells. Recently developed techniques of gene expression analysis through antisense mRNA amplification (aRNA) at the single-cell level may offer the potential to study pathways of neuronal cell death and to determine patterns of coordinated gene expression that may more specifically identify susceptible neuronal subclasses in vivo. Utilizing this unique technique, the authors have demonstrated, for the first time, RNA amplification and gene expression profiling in individual deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)-reactive neurons microdissected from fixed, archival human brain tissue. RNA amplification was successful in >80% of TUNEL-positive neurons, and quantitative aRNA/cDNA hybridization slot-blot analysis demonstrated similar levels of actin RNA but significant differences in caspase-2 RNA expression between TUNEL-reactive and -nonreactive neurons. Reliable quantitative comparisons were achieved with modest numbers of sampled neurons (～10). These studies suggest that analysis of coordinated gene expression in individual damaged neurons in vivo can be reliably used to identify neuronal subclasses that express certain susceptibility- or survival-promoting genes that may be targeted for more specific neuroprotective strategies against HIV.     

Aerosolized scopolamine protects against microinstillation inhalation toxicity to sarin in guinea pigs

Sarin is a volatile nerve agent that has been used in the Tokyo subway attack. Inhalation is predicted to be the major route of exposure if sarin is used in war or terrorism. Currently available treatments are limited for effective postexposure protection against sarin under mass casualty scenario. Nasal drug delivery is a potential treatment option for mass casualty under field conditions. We evaluated the efficacy of endotracheal administration of muscarinic antagonist scopolamine, a secretion blocker which effectively crosses the blood-brain barrier for protection against sarin inhalation toxicity. Age and weight matched male Hartley guinea pigs were exposed to 677.4 mg/m3 or 846.5?mg/?m3 (1.2?×?LCt??) sarin by microinstillation inhalation exposure for 4?min. One minute later, the animals exposed to 846.5?mg/?m3 sarin were treated with endotracheally aerosolized scopolamine (0.25?mg/kg) and allowed to recover for 24?h for efficacy evaluation. The results showed that treatment with scopolamine increased the survival rate from 20% to 100% observed in untreated sarin-exposed animals. Behavioral symptoms of nerve agent toxicity including, convulsions and muscular tremors were reduced in sarin-exposed animals treated with scopolamine. Sarin-induced body weight loss, decreased blood O? saturation and pulse rate were returned to basal levels in scopolamine-treated animals. Increased bronchoalveolar lavage (BAL) cell death due to sarin exposure was returned to normal levels after treatment with scopolamine. Taken together, these data indicate that postexposure treatment with aerosolized scopolamine prevents respiratory toxicity and protects against lethal inhalation exposure to sarin in guinea pigs.     

Aerosolized delivery of oxime MMB-4 in combination with atropine sulfate protects against soman exposure in guinea pigs

We evaluated the efficacy of aerosolized acetylcholinesterase (AChE) reactivator oxime MMB-4 in combination with the anticholinergic atropine sulfate for protection against respiratory toxicity and lung injury following microinstillation inhalation exposure to nerve agent soman (GD) in guinea pigs. Anesthetized animals were exposed to GD (841?mg/m(3), 1.2 LCt(50)) and treated with endotracheally aerosolized MMB-4 (50 μmol/kg) plus atropine sulfate (0.25?mg/kg) at 30?sec post-exposure. Treatment with MMB-4 plus atropine increased survival to 100% compared to 38% in animals exposed to GD. Decreases in the pulse rate and blood O(2) saturation following exposure to GD returned to normal levels in the treatment group. The body-weight loss and lung edema was significantly reduced in the treatment group. Similarly, bronchoalveolar cell death was significantly reduced in the treatment group while GD-induced increase in total cell count was decreased consistently but was not significant. GD-induced increase in bronchoalveolar protein was diminished after treatment with MMB-4 plus atropine. Bronchoalveolar lavage AChE and BChE activity were significantly increased in animals treated with MMB-4 plus atropine at 24?h. Lung and diaphragm tissue also showed a significant increase in AChE activity in the treatment group. Treatment with MMB-4 plus atropine sulfate normalized various respiratory dynamics parameters including respiratory frequency, tidal volume, peak inspiratory and expiratory flow, time of inspiration and expiration, enhanced pause and pause post-exposure to GD. Collectively, these results suggest that aerosolization of MMB-4 plus atropine increased survival, decreased respiratory toxicity and lung injury following GD inhalation exposure.     

Gene expression in TUNEL-positive neurons in human immunodeficiency virus-infected brain

Human immunodeficiency virus (HIV) infection of the central nervous system (CNS) results in neuronal damage and apoptosis, and both in vitro models and pathological studies suggest that a variety of neurotoxins released by HIV-infected and -activated macrophages/microglia selectively damage susceptible subsets of neurons. Confirmation of in vitro findings of mechanisms of neurodegeneration and neuronal cell dysfunction in vivo has been approached through detailed pathological analysis of regional structural damage, immunohistochemical detection of selected antigens in damaged cells, and, more recently, analysis of gene expression in whole tissue blocks or pooled populations (hundreds/thousands) of microdissected cells. Recently developed techniques of gene expression analysis through antisense mRNA amplification (aRNA) at the single-cell level may offer the potential to study pathways of neuronal cell death and to determine patterns of coordinated gene expression that may more specifically identify susceptible neuronal subclasses in vivo. Utilizing this unique technique, the authors have demonstrated, for the first time, RNA amplification and gene expression profiling in individual deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)-reactive neurons microdissected from fixed, archival human brain tissue. RNA amplification was successful in >80% of TUNEL-positive neurons, and quantitative aRNA/cDNA hybridization slot-blot analysis demonstrated similar levels of actin RNA but significant differences in caspase-2 RNA expression between TUNEL-reactive and -nonreactive neurons. Reliable quantitative comparisons were achieved with modest numbers of sampled neurons (approximately 10). These studies suggest that analysis of coordinated gene expression in individual damaged neurons in vivo can be reliably used to identify neuronal subclasses that express certain susceptibility- or survival-promoting genes that may be targeted for more specific neuroprotective strategies against HIV.     

An in vitro and in vivo evaluation of the efficacy of recombinant human liver prolidase as a catalytic bioscavenger of chemical warfare nerve agents

In this study, we determined the ability of recombinant human liver prolidase to hydrolyze nerve agents in vitro and its ability to afford protection in vivo in mice. Using adenovirus containing the human liver prolidase gene, the enzyme was over expressed by 200- to 300-fold in mouse liver and purified to homogeneity by affinity and gel filtration chromatography. The purified enzyme hydrolyzed sarin, cyclosarin and soman with varying rates of hydrolysis. The most efficient hydrolysis was with sarin, followed by soman and by cyclosarin {apparent k_cat/K_m [(1.9?±?0.3), (1.7?±?0.2), and (0.45?±?0.04)]?×?10^5?M^?1?min^?1, respectively}; VX and tabun were not hydrolyzed by the recombinant enzyme. The enzyme hydrolyzed P (+) isomers faster than the P (?) isomers. The ability of recombinant human liver prolidase to afford 24 hour survival against a cumulative dose of 2?×?LD₅₀ of each nerve agent was investigated in mice. Compared to mice injected with a control virus, mice injected with the prolidase expressing virus contained (29?±?7)-fold higher levels of the enzyme in their blood on day 5. Challenging these mice with two consecutive 1?×?LD₅₀ doses of sarin, cyclosarin, and soman resulted in the death of all animals within 5 to 8?min from nerve agent toxicity. In contrast, mice injected with the adenovirus expressing mouse butyrylcholinesterase, an enzyme which is known to afford protection in vivo, survived multiple 1?×?LD₅₀ challenges of these nerve agents and displayed no signs of toxicity. These results suggest that, while prolidase can hydrolyze certain G-type nerve agents in vitro, the enzyme does not offer 24 hour protection against a cumulative dose of 2?×?LD₅₀ of G-agents in mice in vivo.