External quality assessment programmes for detection of HCV RNA, HIV RNA and HBV DNA in plasma: improved proficiency of the participants observed over a 2-year period.

BACKGROUND AND OBJECTIVES: Two External Quality Assessment Programmes (EQAPs) were run in 2008 and 2009 to evaluate the proficiency of blood centres in detecting, by nucleic acid amplification techniques (NAT), the possible contamination of plasma with hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis B virus (HBV). MATERIALS AND METHODS: In the EQAP-2008, three customized panels were designed; each containing positive samples with a viral nominal concentration for the three viruses of about three times the 95% DL of the respective commercial NAT assay. In the EQAP-2009, the proficiency of the participants was evaluated with a single panel, independently on the NAT method used. RESULTS: While 84% (102/122) of the participants in the EQAP-2008 correctly identified the positive and negative samples of the panels, in the EQAP-2009 the percentage of proficient laboratories increased to 97% (118/122). Most importantly, in this 2-year experience, we observed a decrease in the number of pre-/postanalytical errors, from 14 in 2008 to two in 2009. CONCLUSIONS: The design of these two EQAPs allowed participants to assess the performance of the NAT methods applied in their routine screening of blood donations, not only with respect to analytical errors but also to human errors that, despite the high level of automation reached by NAT methods, can still occur.

External quality assessment for the detection of HCV RNA, HIV RNA and HBV DNA in plasma by nucleic acid amplification technology: a novel approach.

BACKGROUND AND OBJECTIVES: In this EQA study a novel approach was used to assess the performance of blood centres and blood product manufacturers in detecting the possible contamination of plasma with HCV, HIV and HBV by NAT. MATERIALS AND METHODS: A panel of 12 samples, three negative and three positive for each virus, was distributed to the EQA participants. The positive samples were prepared, using the respective WHO standards, in order to obtain a viral concentration of about three times the 95% DL of the methods most commonly used by laboratories involved in blood screening by NAT. Participants were requested to test each sample of the panel on different days, possibly by different operators using their routine NAT assay. RESULTS: Overall, the participants' performance was satisfactory. In particular, 49 of the 59 participants (83%) were able to correctly identify all samples. Regarding the remaining 10 laboratories, in three cases a deviation from the laboratory's procedure that could be attributed to an operator's mistake was observed, in two cases a possible cross-contamination occurred while in the remaining five cases the failure to detect the positive samples couldn't be ascribed to any relevant deviation in the laboratory's procedure. CONCLUSIONS: The novel design of this EQA study allowed participants to verify their day by day activity as the study was carried out in the context of their routine testing. Under these conditions, it was demonstrated that, despite the high level of automation reached by NAT assays, human errors can still occur.

External quality assessment for the detection of blood-borne viruses in plasma by nucleic acid amplification technology: the first human immunodeficiency virus and hepatitis B virus studies (HIV EQA/1 and HBV EQA/1) and the fifth hepatitis C virus study (HCV EQA/5).

BACKGROUND AND OBJECTIVES: This External Quality Assessment (EQA) study was aimed at assessing the proficiency of blood centres and blood product manufacturers in detecting, by nucleic acid amplification technology (NAT), the possible contamination of plasma with hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis B virus (HBV). MATERIALS AND METHODS: Three independent panels, one for each virus, were prepared at the Istituto Superiore di Sanità (ISS) by diluting the respective reference preparations. NAT methods used by the EQA participants included polymerase chain reaction (PCR) assays by Roche, transcription-mediated amplification (TMA) assays by Chiron and in-house PCR assays. RESULTS: Forty-three of the 45 participants (95.6%) in the HCV EQA/5 who used a validated method were consistently able to detect a nominal concentration of 100 IU/ml for all six major genotypes. In the case of the HIV EQA/1, all 35 participants detected the samples containing 1000 IU/ml HIV, while five (14.3%) did not identify the samples containing 100 IU/ml HIV. With respect to the HBV EQA/1, all 16 participants correctly identified the positive samples containing either 1000 IU/ml or 100 IU/ml HBV. No false-positive results were observed with any of the three panels. CONCLUSIONS: The HCV EQA/5 showed an improved proficiency of laboratories as compared with the HCV EQA/4. In fact, HCV genotypes 1, 2, 3 and 5 were correctly identified in 100% of the assays and genotypes 4 and 6 in 97.8% of the assays. While most of the participants in the HIV EQA/1 showed a good level of proficiency, an excellent performance was shown by all participants in the HBV EQA/1.

High proficiency in detecting the six major hepatitis C virus genotypes of laboratories involved in testing plasma by nucleic acid amplification technology.

Since the introduction of mandatory HCV RNA testing of plasma pools for fractionation by nucleic acid amplification technology, we have organised External Quality Assessment studies (EQAs) addressed to blood products manufacturers and blood centres. Here we report the results of a new EQA, the first one to include all six major HCV genotypes. The results, reported by laboratories worldwide, showed that genotypes 1, 2 and 3 were correctly identified in 100% of the tests, genotype 4 in 96.7% and genotypes 5 and 6 in 98.3% of the assays. As detection of all HCV genotypes is critical for laboratories involved in testing plasma for HCV, all six genotypes should continue to be included in the next EQA studies.

Transient global brain hypoxia-ischemia in adult rats: neuronal damage, glial proliferation, and alterations in inositol phospholipid hydrolysis.

A model of ischemic-hypoxic brain injury which combines bilateral occlusion of common carotid arteries for 10 min and mild hypoxia (15% O2 for 10 min before and during occlusion) was developed. Global ischemia was assessed by a simplified EEG recording indicating isoelectric line, i.e. full arrest of cortical electrical activity. Histological examination of brain 7 days after ischemic insult showed from moderate to severe damage, mainly in the cerebral cortex (layers III, V and VI) and hippocampus (mainly CA1 subfield). The injury consisted of neuronal degeneration and necrosis with nuclear pyknosis and karyorrhexis. Immunohistochemical staining for gliofibrillar acidic protein showed a marked glial proliferation in the cerebral cortex and hippocampus. In the cortical slices, inositol phosphates accumulation stimulated by excitatory amino acid agonists (ACPD, ibotenate and quisqualate), as well as by norepinephrine and carbachol, was enhanced significantly (p < 0.01) with respect to sham-operated rats 7 days, but not 24 h, after the ischemic insult. The overall data show that the relatively simple transient brain hypoxia/ischemia rat model produces full arrest of cortical EEG, histopathological alterations and those relative to post-receptor neurochemical mechanisms characteristic of four-vessel occlusion model.

Ubiquitin-mediated stress response in a rat model of brain transient ischemia/hypoxia.

Ubiquitin (Ub) is a small 76-residue protein, involved in intracellular protein degradation through a specific ATP-dependent system, which uses Ub as a tag to label proteins committed to be hydrolyzed by a specific 26 S protease. PGP-9.5 is another important component of the Ub system, i.e. a neuron-specific carboxyl-terminal hydrolase, which recycles Ub from Ub-polypeptide complexes. We have investigated the expression of Ub and PGP-9.5 in rat hippocampal neurons in an early phase of reperfusion in a model of transient global brain ischemia/hypoxia (bilateral occlusion of common carotid arteries for 10 min accompanied by mild hypoxia-15% O2-for 20 min), by means of immunohistochemical methods using light and electron microscopy. The intensity of Ub and PGP-9.5 immunoreactivity was evaluated by image analysis. We have detected a marked increase of Ub immunoreactivity (UIR) in neurons of CA1, CA2, CA3, CA4, and dentate gyrus subfields 1 hr after ischemia/hypoxia (but not after hypoxia only), statistically significant as confirmed by image analysis. Such increase in immunoreactivity in ischemic/hypoxic rats was localized essentially in the nuclei of hippocampal neurons. There were no changes in PGP-9.5 immunoreactivity. The data suggest that in the present model of rat brain ischemia/hypoxia Ub is involved in the neuronal stress response.

Differential responsiveness of metabotropic glutamate receptors coupled to phosphoinositide hydrolysis to agonists in various brain areas of the adult rat.

The effects of metabotropic glutamate receptor (mGluR) agonists on inositol phosphates (IP) accumulation were investigated in slices of the cerebral cortex, hippocampus, striatum and cerebellum of adult Sprague-Dawley rats. EC(50) values for 1S, 3R-1-aminocyclopentane-1, 3-dicarboxylic acid (ACPD) did not differ significantly between various brain areas (range 10(-5) M), quisqualate was the most potent in all the brain areas (range 10(-7) - 10(-6) M), except the cerebellum (10(-5) M), ibotenate was the most potent in the striatum (range 10(-6) M) and the least potent in the cerebral cortex and hippocampus (range 10(-4) M). The efficacy in the four brain areas showed the following trend of ranking order for ACPD and quisqualate: hippocampus > striatum > cerebral cortex > cerebellum, and for ibotenate: hippocampus > cerebral cortex > striatum > cerebellum, although the observed differences reached the level of statistical significance only in the case of ACPD (hippocampus and striatum vs cerebellum) and ibotenate (hippocampus vs cerebellum). Co-incubation of the agonists at maximally effective concentrations in any pairwise combination resulted in no substantial additivity of IP accumulation. D,L-1-amino-3-phosphonopropionic acid (AP3) and D,L-2-amino-4-phosphonobutyric acid (AP4) at 0.5 mM concentration antagonized ACPD-induced IP accumulation by about 70 and 45 percent, respectively, without differences between brain areas. On the other hand, the antagonistic effects of L-serine-o-phosphate (SOP) at 1 mM concentration were the highest in the hippocampus (75 percent) and the lowest in the cerebellum (25 percent). The comparative data indicate considerable regional receptor heterogeneity, in terms of different ratios of response to the agonists (but not antagonists, except SOP). There is a robust responsiveness of mGluRs not only in the hippocampus and cerebral cortex, but also in the striatum which exhibits the highest affinity to both quisqualate and ibotenate.

Differential inhibition of rat brain acetylcholinesterase molecular forms by 7-methoxytacrine in vitro.

The effects of 7-methoxytacrine (7-MEOTA), a less toxic derivative of tetrahydroaminoacridine, on the activity of acetylcholinesterase (AChE) molecular forms were investigated in vitro. AChE molecular forms were separated by sucrose gradient sedimentation from homogenates of the frontal cerebral cortex prepared with buffer containing Triton X-100 (soluble + membrane-bound enzyme). Two molecular forms, namely 10S and 4S corresponding to globular tetrameric (G4) and monomeric (G1) forms, respectively, were detected; their molecular weights were 220,000 and 54,000 Da. A significantly higher sensitivity to 7-MEOTA of G4 than of G1 forms was observed. The Ki values were 0.21 +/- 0.07 microM for the former and 0.70 +/- 0.15 microM for the latter. The differential inhibition of AChE molecular forms by 7-MEOTA is discussed in relation to its possible clinical application for treatment of disorders such as Alzheimer's disease, in which a reduction of brain cholinergic neurotransmission is believed to play a role.

Differential reactivation by HI-6 in vivo of paraoxon-inhibited rat brain acetylcholinesterase molecular forms.

The effects of the cholinesterase reactivator HI-6, [1-(((4-(aminocarbonyl)-piridinio)methoxy)methyl-2-(hydroxy- imino)methyl pyridinium dichloride], on paraoxon-inhibited brain acetylcholinesterase (AChE) and its molecular forms were studied in rats. Treatment with paraoxon (0.25 mg/kg s.c.) caused approx. 60% inhibition of total AChE from frontal cerebral cortex, while that including HI-6 (140 mg/kg i.m.) and atropine (50 mg/kg i.m.) reduced such inhibition to only 25%. Two molecular forms of the enzyme, 10S and 4S, corresponding to globular tetrameric (G4) and monomeric (G1), were detected by sucrose gradient sedimentation. In paraoxon treated rats the G4 form was inhibited by approx. 65% while G1 only by 35%. The G4 form was considerably and selectively reactivated by HI-6 while the G1 form was not reactivated at all. The data show that HI-6 penetrates the blood-brain barrier and reactivates the molecular forms preferentially inhibited by paraoxon and involved in synaptic neurotransmission.

Age-related changes in acetylcholinesterase and its molecular forms in various brain areas of rats.

A previous study conducted in this laboratory revealed a decrease in total cholinesterase (total ChE) in the cerebral cortex, hippocampus and striatum in aged rats (24 months) of various strains, as compared with young animals (3 months). The purpose of the present experiments was to extend the study to other brain areas (hypothalamus, medulla-pons and cerebellum) and to assess whether this decrease was dependent on the reduction of either specific acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) or both. By using ultracentrifugation on a sucrose gradient, the molecular forms of AChE were evaluated in all the brain areas of young and aged Sprague-Dawley rats. In young rats the regional distribution of total ChE and AChE varied considerably with respect to BuChE. The age-related loss of total ChE was seen in all areas. Although there was a reduction of AChE and, to somewhat lesser extent, of BuChE in the cerebral cortex, hippocampus, striatum, and hypothalamus (but not in the medulla-pons or the cerebellum), the ratio AChE/BuChE was not substantially modified by age. Two molecular forms of AChE, namely G4 (globular tetrameric) and G1 (monomeric), were detected in all the brain areas. Their distribution, expressed as G4/G1 ratio, varied in young rats from about 7.5 for the striatum to about 2.0 for the medulla-pons and cerebellum. The age-related changes consisted in a significant and selective loss of the enzymatic activity of G4 forms in the cerebral cortex, hippocampus, striatum, and hypothalamus, which resulted in a significant decrease of the G4/G1 ratio. No such changes were found in the medulla-pons or the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Size and charge isomers of acetylcholinesterase in the cerebral cortex of young and aged rats.

Previous studies in this laboratory showed an age-related decline of acetylcholinesterase (AChE) activity in the cerebral cortex of rats. In the present study the age-related differences in enzymatic activity were evaluated in terms of individual molecular forms. Extracts containing total, soluble and membrane-bound AChE were analyzed both by ultracentrifugation in sucrose gradient and by non-denaturing gradient polyacrylamide gel electrophoresis. By ultracentrifugation two molecular forms, namely 10S and 4S (corresponding to tetrameric-G4 and monomeric-G1 forms, respectively) were separated in extracts of total and soluble AChE, while only 10S forms were present in extracts of membrane-bound AChE. Electrophoresis of soluble AChE extracts revealed slowly- and fast-migrating bands, grouped in two clusters of at least three bands each; membrane-bound AChE contained only a single slowly-migrating band. Electrophoresis of the single forms isolated by ultracentrifugation showed that slowly- and fast-migrating bands corresponded to G4 and G1 forms, respectively. Therefore, in soluble AChE no one-to-one relationship between charge- and size-isomers was observed; on the contrary, such relationship has been shown for membrane-bound AChE. This implies that soluble G4 forms and membrane-bound-G4 forms are electrophoretically different, being heterogeneous the former and homogeneous the latter. The age-related decline of total AChE, accompanied by a decrease of G4/G1 ratio, depended mainly on a decrease of membrane-bound AChE while soluble AChE and its G4/G1 ratio was unchanged. The qualitative pattern of charge isomers was not modified by aging.

In vivo and in vitro effects of diisopropyl fluorophosphate and paraoxon on individual molecular forms of rat brain acetylcholinesterase.

Previous study in this laboratory showed that following a sc injection of an organophosphorus compound, diisopropyl fluorophosphate (DFP), into rats the inhibition of 10S molecular forms was considerably more pronounced than that of 4S forms of brain acetylcholinesterase (AChE). This could depend on different accessibility of the two forms or on their different intrinsic sensitivity to the antiChE compound. In the present study the effects of DFP and Paraoxon on 10S and 4S forms were evaluated in vivo, i.e., after systemic administration, and in vitro by adding the organophosphorus compounds to each of the two forms after extraction from brain of untreated rats, solubilization and separation. The in vivo preferential inhibition of 10S forms was confirmed. The 10S/4S ratios for control and DFP-treated rats were 9.05 and 5.01, respectively; these ratios were 8.46 and 3.33 for Paraoxon. On the other hand, in the in vitro experiments there were no significant differences between IC50 values for 10S and 4S forms both in the case of DFP (2.66 and 2.98 microM) and Paraoxon (32.4 and 42.4 nM, respectively). The overall data suggest that the preferential in vivo inhibition of 10S molecular forms with respect to 4S forms depends on their different accessibility probably due to different subcellular localization of the two forms and not on their different intrinsic sensitivity.

Alterations in the distribution of cholinesterase molecular forms in maternal and fetal brain following diisopropyl fluorophosphate treatment of pregnant rats.

Previous work in this laboratory showed that during intoxication of rats with diisopropyl fluorophosphate at day 20 of pregnancy the recovery of ChE activity was faster in fetal than in maternal brain. In the present study the differences between recovery rats in dam and fetus brain were evaluated in terms of molecular forms and spontaneous reactivation. Using ultracentrifugation on sucrose gradient two molecular forms of ChE, namely 10S (tetrameric globular G4 form) and 4S (monomeric G1 form) were detected both in maternal and fetal brain of untreated rats. The ratios 10S/4S were about 5.0 and 0.75 for dams and 20-day fetuses, respectively. DFP administration (1.1 mg/kg sc) inducing at 90 min an about 80% inhibition of ChE in maternal brain caused a shift in its 10S/4S ratio to 1.63, and to 0.53 in fetal brain (in which overall inhibition was about 70%). This means that 10S forms were preferentially inhibited by DFP both in maternal and fetal brain. After 24 and 48 hr there was a negligible recovery of overall ChE in maternal brain with no shift in the ratio. On the other hand, complete recovery of ChE in fetal brain within 48 hr was accompanied by almost total normalization of the 10S/4S ratio. Rapid recovery of fetal ChE appeared not to depend on hydrolysis of DFP-inhibited ChE. In fact, maternal and fetal DFP-inhibited enzyme preparations following the addition of oximes (pralidoxime or obidoxime) in vitro showed similar rates of reactivation. The overall data indicate considerable differences in recovery rate of molecular forms between dams and fetuses, but not in reactivation by dephosphorylation.

Brain choline acetyltransferase and muscarinic receptor sites, brain and liver cholinesterases in precocial Acomys cahirinus and altricial rat during post-natal development.

Brain choline acetyltransferase, acetylcholinesterase with its molecular forms, and muscarinic receptor sites, as well as liver total cholinesterases were evaluated during the first postnatal month in pups of a precocial (Acomys cahirinus) and altricial (rat) murid species. At birth the levels of brain cholinergic markers were higher in the Acomys than in the rat, but in adulthood the differences were smaller or even reversed. The postnatal increase up in the markers to weaning was considerably more pronounced in the rat. However, substantial variations in the patterns of development of the three cholinergic markers within and between species were observed. Liver cholinesterases were considerably higher in Acomys than in rats at all ages investigated. These and literature data are discussed in relation to postnatal, post-conception and post-organogenesis age of pups belonging to the two species. The variability of the ontogenetic patterns between the enzymes suggests that there is some biological control of individual rates of maturation and that it is necessary to be careful in broadly interpreting growth patterns across organs within the same species and across species.

Change in the distribution of acetylcholinesterase molecular forms in frontoparietal cortex of the rat following nucleus basalis lesions with kainic acid.

The unilateral injection of kainic acid into the nucleus basalis magnocellularis (NBM) resulted in an alteration of the distribution of acetylcholinesterase (AChE) molecular forms in frontoparietal cortex ipsilaterally to the lesion. The G4/G1 ratio fell from 5.4 +/- 0.8 in contralateral to 3.0 +/- 0.5 in ipsilateral cortex. The NBM lesion effect thus, mimicks, the loss of tetrameric G4 form reported for various brain cortical areas of Alzheimer's disease (AD) patients. The data support the suggestion that G4 form is enriched in presynaptic nerve terminals.

Molecular forms of cholinesterases in CSF of Alzheimer's disease/senile dementia of Alzheimer type patients and matched neurological controls.

Acetylcholinesterase, pseudocholinesterase and their molecular forms were measured in the CSF of patients affected by Alzheimer's disease and of matched neurological controls. Three different molecular forms of ChE were found in the CSF of both groups of patients, but only two of them belonged to 'true' AChE. No differences were found between Alzheimer's disease patients and neurological controls in all the examined parameters.

Effects of diisopropylfluorophosphate on brain cholinergic systems of rats at early developmental stages.

The effects of subchronic intoxication by diisopropylfluorophosphate (DFP) during pregnancy on development of brain cholinesterases (ChE) and [3H]quinuclidinyl benzilate (QNB) binding to muscarinic receptors were studied at the end of gestation and in offspring at 1, 5, 10, and 20 days after birth. Pregnant rats received on alternate days sc injections of DFP (first dose 1.1 mg/kg, subsequent doses 0.7 mg/kg) from Day 6 through Day 20. In spite of the considerable maternal toxicity of DFP, the level of brain ChE at birth and subsequent increases of enzymatic activity did not differ from those of controls. By contrast, significantly lower levels of QNB binding sites at birth were followed by a delay in development up to about 2 weeks. In the experiments using animals sacrificed on Days 20 and 21 of gestation, maternal brain ChE were consistently depressed, while in fetal brain an almost complete recovery occurred within 48 hr from the last DFP injection. Maximal number of QNB binding sites (Bmax) was decreased significantly both in maternal and fetal brain. These data indicate that tolerance mediated by receptor changes can be produced at early developmental stages, in spite of the accelerated recovery of brain ChE after treatment. In another experiment 7- to 27-day pups were treated on alternate days with sc doses of DFP (0.45 mg/kg from Day 7 to Day 19 and 0.70 mg/kg up to Day 27) and sacrificed on Day 7, 14, 20, 28, or 40. Brain ChE was reduced by 45-70%.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain acetylcholinesterase and its molecular forms in a precocial murid, Acomys cahirinus, and rat during post-natal development.

Brain acetylcholinesterase (AChE) and its molecular forms of a precocial murid, Acomys cahirinus, characterized by a large hippocampus, were measured during post-natal development and compared with rat. The activity of soluble AChE in Acomys increased slightly up to 4 weeks after birth. The total AChE activity increased somewhat more but, in rats, this increase was still greater. Three main molecular forms of AChE were separated by 7.5% polyacrylamide gel electrophoresis. Their close similarity to the rat AChE forms was assessed by gradient polyacrylamide gel electrophoresis and electrofocusing. Maturation of these forms, i.e., conversion of simple into more complex forms in the soluble fraction of AChE was, however, considerably delayed reaching only after 4 weeks the pattern comparable to that of rat.

Interactions between anticholinesterase agents and neuroleptics in terms of cholinesterase inhibition in brain and other tissues of rats.

Treatment of rats with chlorpromazine (CPZ, 15 mg/kg i.p. 60 min before sacrifice) did not modify cholinesterase (ChE) activity, but considerably enhanced the inhibition of total ChE induced by physostigmine (PhS, 0.5 mg/kg i.p. 40 min after CPZ) in brain, skeletal muscle, myocardium, lung, liver, and kidney. Additional experiments also showed a prolongation of PhS inhibition by CPZ in brain. The enhanced inhibition of total ChE due to CPZ depended in most peripheral organs on the effect on pseudoChE (as measured by a spectrophotometric method), except in the case of skeletal muscle in which potentiation of PhS effect was observed on true acetylcholinesterase (AcChE). The results indicate that the potentiation by CPZ of PhS inhibition occurs in all organs tested and is relatively non specific. CPZ was found to potentiate slightly the effects of Mevinphos but did not interact with Carbaryl, Diazinon or Azinphos. Furthermore, haloperidol did not potentiate the effects of physostigmine.

Development factors affecting brain acetylcholinesterase inhibition and recovery in DFP-treated rats.

The effect of a single dose of diisopropyl fluorophosphate (DFP; Isoflurophate, 1.1 mg/kg s.c.) administered to rats during pregnancy was evaluated by measuring postpartum maternal and newborn brain-soluble and total acetylcholinesterases (AChE) and their molecular forms at intervals of 1, 2, 3, 4 and 10 days between treatment and sacrifice. Subsequently, the effects of DFP were studied in 18-day-pregnant rats, fetuses and placentae at 90 min and 24 h after treatment. The inhibition of postpartum maternal enzymatic activity did not differ from that previously found in adult males, while inhibition was considerably less pronounced in newborns at all time intervals, with a nearly complete recovery already at 48 h after treatment. An even faster recovery of brain enzyme was observed in 18-day fetuses from DFP-treated mothers (24-hour interval between treatment and sacrifice). In this experiment, a comparable inhibition was observed at 90 min after treatment in the adult and the developing brain, excluding a major influence of disposition factors in the differential recovery phenomena. An experiment on weanling rats yielded intermediate results between those of newborn and those of adult animals. Finally, most data confirmed previous findings that the soluble portion of brain AChE and medium molecular weight enzyme forms may have special significance in the initial phases of recovery.