Serum sex hormone-binding globulin levels are reduced and inversely associated with intrahepatic lipid content and saturated fatty acid fraction in adult patients with glycogen storage disease type 1a.

PURPOSE: De novo lipogenesis has been inversely associated with serum sex hormone-binding globulin (SHBG) levels. However, the directionality of this association has remained uncertain. We, therefore, studied individuals with glycogen storage disease type 1a (GSD1a), who are characterized by a genetic defect in glucose-6-phosphatase resulting in increased rates of de novo lipogenesis, to assess the downstream effect on serum SHBG levels. METHODS: A case-control study comparing serum SHBG levels in patients with GSD1a (n = 10) and controls matched for age, sex, and BMI (n = 10). Intrahepatic lipid content and saturated fatty acid fraction were quantified by proton magnetic resonance spectroscopy. RESULTS: Serum SHBG levels were statistically significantly lower in patients with GSD1a compared to the controls (p = 0.041), while intrahepatic lipid content and intrahepatic saturated fatty acid fraction-a marker of de novo lipogenesis-were significantly higher in patients with GSD1a (p = 0.001 and p = 0.019, respectively). In addition, there was a statistically significant, inverse association of intrahepatic lipid content and saturated fatty acid fraction with serum SHBG levels in patients and controls combined (ß: - 0.28, 95% CI: - 0.47;- 0.09 and ß: - 0.02, 95% CI: - 0.04;- 0.01, respectively). CONCLUSION: Patients with GSD1a, who are characterized by genetically determined higher rates of de novo lipogenesis, have lower serum SHBG levels than controls.

Total regional and global number of synapses in the human brain neocortex.

An estimator of the total number of synapses in neocortex of human autopsy brains based on unbiased stereological principles is described. Each randomly chosen cerebral hemisphere was stratified into the four major neocortical regions. Uniform sampling with a varying sampling fraction in each region of neocortex was performed. The total volume of each neocortical region was estimated using point counting according to Cavalieri's principle. The ethanolic phosphotungstic acid staining technique was modified for synapses in human autopsy brains. The numerical density of synapses in each neocortical region studied was estimated using the disector at the electron microscopical level. The total number of neocortical synapses in each region was estimated as the product of the total volume of neocortex and the numerical density of synapses. The influence of the postmortem fixation delay on the number of synapses was investigated in five large mammals (one dog, one cow, and three pigs), the brains of which were kept under conditions similar to those under which human corpses are normally kept. The apparent decrease of 3.9% in the numerical density of synapses in the large mammals following a 2-day fixation delay was not significant. The average total number of synapses in the neocortex of five young male brains was 164 x 10(12) (CV = 0.17). An analysis of the precision of the estimate of the total number of synapses in neocortex indicates that blocks represent both the major source of variation and the largest workload. Using eight blocks per brain the imprecision of the estimate is, however, only 66% of the total variance.

Effects of thienylphencyclidine (TCP) on seizure activity and brain damage produced by soman in guinea-pigs: ECoG correlates of neurotoxicity.

The capacity of thienylcyclohexylpiperidine (TCP), a non-competitive blocker of the N-methyl-D-aspartate (NMDA) receptor, to counteract the convulsant, lethal, and neuropathological effects of 2 x LD50 of soman (an irreversible inhibitor of cholinesterase) was investigated in guinea-pigs treated by pyridostigmine and atropine sulphate. The effects of a weak dose of TCP (1 mg/kg) used in the present study globally reproduced those previously obtained with a higher dose (2.5 mg/kg; [Neurotoxicology 15 (1994) 837]): TCP was again most protective when given curatively within the first hour of soman-induced seizures. In this condition, (a) paroxysmal activity ceased in 10-20 min, (b) all the animals survived, (c) the majority of them recovered remarkably well and did not show any brain damage 24 h after the intoxication, and (d) the minimal duration of seizure activity normally required for producing soman-induced brain damage in other pharmacological environments was increased from 10 to 40 min to 80 min. Strikingly, when TCP was given 120 min after seizure onset, it failed to show any anticonvulsant activity but still provided neuroprotection in the hippocampus. The present study also gives additional evidence (see [Neurotoxicology 21 (4) (2000) 521]) that in soman poisoning, (a) the development of brain damage depends on the occurrence of ECoG seizures, (b) the topographical distribution of lesions depends on seizure duration, and (c) an increase of the relative power in the lowest (delta) frequency band might be a reliable marker of neuronal degradation. All these findings confirm that (a) glutamatergic NMDA receptors are involved in the mechanisms of soman-induced seizures and brain damage, (b) non-competitive antagonists of NMDA receptors might be promising candidates for post-treatment of soman poisoning, and (c) ECoG parameters from ECoG tracings and power spectrum might serve as useful external predictors for soman-induced neuropathological changes.

Beneficial effects of TCP on soman intoxication in guinea pigs: seizures, brain damage and learning behaviour.

Poisoning with the potent nerve agent soman produces a cascade of central nervous system (CNS) effects characterized by severe convulsions and eventually death. In animals that survive a soman intoxication, lesions in the amygdala, piriform cortex, hippocampus and thalamus can be observed. In order to examine the mechanisms involved in the effects of soman and to evaluate possible curative interventions, a series of behavioural, electrophysiological and neuropathological experiments were carried out in the guinea pig using the NMDA antagonist N-[1-(2-thienyl)cyclohexyl] piperidine (TCP) in conjunction with atropine and pyridostigmine. The NMDA antagonist TCP appeared to be very effective in the treatment of casualties who suffered from soman-induced seizures for 30 min: (i)Seizures were arrested within minutes after the TCP injection, confirmed by quantitative electroencephalogram (EEG), after fast Fourier analysis. Three hours after TCP the quantitative EEGs were completely normal in all frequency bands and remained normal during the entire 3-week intoxication period. The power shift to the lower (delta) frequency bands, indicative for neuropathology and found in control animals intoxicated only by soman, was not observed in the soman-TCP group. (ii)The gross neuropathology found in soman control animals within 48 h after soman was prevented in soman-TCP animals and was still absent in 3-week survivors. Instead, ultrastructural changes were observed, indicative of defense mechanisms of the cell against toxic circumstances. (iii)Twenty-four hours after soman, soman-TCP animals were able to perform in the shuttle box and Morris water maze. The beneficial effects of TCP on the performance in these tests during the 3-week intoxication period were very impressive, notwithstanding (minor) deficits in memory and learning. (iv)The increase in excitability after TCP was confirmed by an increase in the acoustic startle response. Taken together, these results confirmed the involvement of NMDA receptors in the maintenance of soman-induced seizures and the development of brain damage. They underline the current hypothesis that cholinergic mechanisms are responsible for eliciting seizure activity after soman and that, most likely, the subsequent recruitment of other excitatory neurotransmitters and loss of inhibitory control are responsible for the maintenance of seizures and the development of subsequent brain damage.

Effects of atropine sulphate on seizure activity and brain damage produced by soman in guinea-pigs: ECoG correlates of neuropathology.

The present study describes the effects of pyridostigmine (PYR; 0.2 mg/kg) and atropine sulphate (AS; 5 mg/kg) on guinea-pigs intoxicated by a high dose (2xLD50) of the organophosphate compound, soman, an irreversible inhibitor of acetylcholinesterase. The medication was shown to counteract the acute respiratory distress and lethality normally produced by the intoxication. Moreover, due to the central activity of AS, soman-induced electrocorticographic (ECoG) seizure activity was either totally prevented, or reduced in duration and overall intensity. In addition, as established in the 24-hr survivors, seizure-related neuropathology was either prevented, or reduced in topographical extent and severity. An attempt to correlate our electrographic and morphological findings gives evidence that (a), the occurrence of seizure activity is the primary factor necessary for the development of acute neuropathology; (b), the duration of ECoG seizures is a secondary factor, on which the topographical distribution of brain damage finally depends; (c), the minimal duration of seizures necessary to produce 24 hr-damage in the most sensitive areas (e.g. the amygdala) is less than 70 min; (d), the overall intensity/power of epileptiform discharges is a tertiary factor which influences the severity of damage; (e), in addition, ECoG power spectral analysis suggested that an acute increase of relative power in the lower (delta) frequency band might be a real-time external marker of the starting cerebral lesions and is thus predictive for their future installation. All these data confirm the tight relationships which exist between seizure activity and neuropathology in soman poisoning, and suggest that refined, standardized analysis of electrographic parameters drawn from ECoG tracings and power spectrum might serve as a useful tool to predict the presence, localization, and severity of soman-induced brain damage.

Effect of D-tagatose on liver weight and glycogen content of rats.

D-tagatose is an incompletely absorbed ketohexose (stereoisomer of D-fructose) which has potential as an energy-reduced alternative sweetener. In an earlier 90-day toxicity study, rats fed diets with 10, 15 and 20% D-tagatose exhibited increased liver weights, but no histopathological alterations. To determine whether there might be any toxicological relevance to this effect, three studies were conducted in male, adult Sprague-Dawley rats. In the first study, four groups received Purina diet (group A), Purina diet with 20% D-tagatose (group B), SDS diet (group C), or SDS diet with 20% D-tagatose (group D). For groups A and B, the 28-day treatment period was followed by a 14-day recovery period (Purina diet). Food remained available to all animals until the time of sacrifice. Groups of 10 rats were killed on days 14 (groups A and B), 28 (groups A-D), and 42 (groups A and B). Body weights, as well as weights of wet and lyophilized livers, were determined. The lyophilized livers collected on day 28 from groups A and B were analyzed for protein, total lipid, glycogen, DNA, and residual moisture. By day 14, relative wet liver weights had increased by 23% in group B. On day 28, the increase was 38% in group B and 44% in group D. At the end of the recovery period, the increase had diminished to 14% in group B. On day 28, liver glycogen content (in %) was significantly increased, and liver protein, lipid, and DNA contents were significantly decreased in group B compared to group A. Total amounts per liver of protein, total lipid, glycogen, and DNA were significantly increased. In the second study, four groups of 20 rats each received SDS diet with 0, 5, 10, and 20% D-tagatose for 29-31 days. The food was available until the time of sacrifice. At termination, plasma was obtained from 10 rats/group for clinicochemical analyses. Five rats/group were subjected to whole-body perfusion, followed by processing of livers for qualitative and quantitative electron microscopic examination. Livers of 6 rats/group were analyzed for acyl-CoA oxidase and laurate 12-hydroxylase (cytochrome P450 4A1) activity, DNA synthesis (Ki-67 index), and number of nuclei per unit area of tissue. Liver weights were significantly increased in linear relation to the D-tagatose intake. Plasma transaminases (but not glutamyl transferase and alkaline phosphatase) were increased in the high-dose group. Except for glycogen accumulation, no ultrastructural changes were seen on electron microscopic examination of livers of the control and high-dose groups. Morphometric analysis confirmed the increase of glycogen and the absence of alterations of endoplasmatic reticulum, mitochondria, and Golgi apparatus. The Ki-67 index did not differ between the groups. A dose-related decrease of the number of nuclei per unit area signified some hepatocellular hypertrophy. Acyl-CoA oxidase and CYP4A1 activity were significantly increased in the mid- and high-dose groups, but these increases were small and not accompanied by electron-microscopic evidence of peroxisome proliferation. In the third study, four groups received SDS diet (groups A and C) or SDS diet with 5% D-tagatose (groups B and D). All animals were killed on day 28. Groups A and B were fasted for 24 h before sacrifice; groups C and D had food available until sacrifice. Liver weights and liver composition were measured as in Study 1. Relative wet and dry liver weights were increased in response to the treatment in rats killed under the fed condition, but not in rats killed under the fasted condition. The livers of the treated rats (group D) had an increased glycogen content in comparison to the controls (group C). Taken together, these results demonstrate that D-tagatose at dietary levels of 5-20% increases liver glycogen deposition and relative liver weights in nonfasting rats. In fasted rats the 5% dose level is the no-effect level. (ABSTRACT TRUNCATED)

Search for a therapy against soman-intoxication.

This mini-review mainly describes a part of the pharmacological research carried out in our laboratory during the past decades, aimed at finding a therapy against intoxication by cholinesterase-inhibiting organophosphates, in particular against the nerve agent soman. In particular soman, because this is one of the nerve agents that consistently appears to be very resistant to treatment. Various experimental approaches are described. Yet, even after all these years of research an adequate (pre)treatment against poisoning by soman is still not available.

Behavioral performance, brain histology, and EEG sequela after immediate combined atropine/diazepam treatment of soman-intoxicated rats.

It is known that rats poisoned with near-lethal doses of pinacolyl methylphosphonofluoridate (soman) develop brain lesions, particularly when convulsions are induced. When rats were intoxicated with a LD50 of soman and treated immediately thereafter with a combination of low doses of atropine and diazepam (LOW AS/DZ treatment), large decrements in performance of an earlier acquired shuttle-box task were found 6 days after intoxication. In contrast, no such decrements were found in soman-intoxicated animals treated similarly with a combination of high doses of these drugs (HIGH AS/DZ treatment). Surprisingly, surviving LOW AS/DZ animals acquired the same task again at a speed that was almost as fast as before intoxication. Similarly treated animals were examined light-microscopically 24 h after intoxication; in LOW-AS/DZ-treated animals, neuropathology was only observed in animals that had exhibited convulsions, whereas in HIGH AS/DZ animals neither convulsions nor brain damage were observed. Power spectra, obtained from electroencephalograms (EEGs) 6 days after intoxication, revealed significant differences between both treatment groups, particularly in the delta-, theta-, and beta-frequencies. After the HIGH AS/DZ treatment, a significant increase in delta activity was found compared to control values, suggestive of neuropathology. It is concluded that, in contrast with the LOW AS/DZ combination, HIGH AS/DZ prevents active avoidance deficits, convulsions, and light-microscopically detectable neuropathology after soman intoxication. However, the results of EEG measurements suggest that some aberrations may still remain even after the HIGH AS/DZ treatment.

Comparison of methods for the estimation of the thickness of ultrathin tissue sections.

For a number of quantitative electron microscopical techniques it is relevant to obtain an estimate of the thickness t' of the section which, in general, will differ from the actual distance t between the two cuts that generate the section. To estimate t' of ultrathin sections, several techniques have been adopted in the past, both with and without the aid of the electron microscope and additional equipment, which are summarized in an appendix. In the present study five methods have been evaluated experimentally using sections of ten different interference colours: (a) the 'small-fold' technique, (b) the 'electron scattering' method, (c) interference microscopy with (A) the Vicker's M86 scanning microinterferometer and (B) the Jenoptik Amplival Interphako interference microscope and (d) the 're-embedding' method. Reliable, reproducible and comparable results were obtained with the small-fold technique, with the Vickers M86 scanning microinterferometer and with the electron scattering method. For the last method, standard test lines for the different settings of the electron microscope were developed. The results obtained with the Jenoptik Amplival Interphako interference microscope are reproducible, but show a constant difference, i.e. a factor of 1.36, in thickness compared with the other three techniques. The possible cause of this 'systematic error' is discussed. The re-embedding method proved to be more laborious and slightly less reliable than the other techniques. The variation in t' between sections of a particular interference colour (inter-section variation) was found to be larger than the variation in t' within a section (intra-section variation).

A critical evaluation of methods for estimating the numerical density of synapses.

Several methods for estimating the numerical density (NV) of particles are described. The usefulness and the limitations of different methods with respect to the estimation of synaptic densities are discussed. These methods are: the discrete unfolding technique, the serial section technique and the disector technique. From the results it is concluded that it is not advisable to use an unfolding technique to estimate the number and size of synapses since all sorts of assumptions regarding the shape, truncation and overprojection are hazardous. Consistently lower values for NV were obtained with the disector technique compared with the results of the serial section technique. This difference, obtained with two unbiased techniques, is discussed. The main conclusion with respect to this point is that both techniques can be used to estimate synaptic densities, provided a reliable estimate of the section thickness is obtained and an appropriate sampling procedure is used.

The complex-shaped 'perforated' synapse, a problem in quantitative stereology of the brain.

Failure to appreciate the consequences for stereological work of the simultaneous presence of complex-shaped perforated and disc-like non-perforated synapses in brain tissue results in underestimation of synaptic profile length and overestimation of synaptic density when measured in randomly selected ultrathin E-PTA slices. This problem can be solved by using serial slices and a calculation method which makes no assumptions about synaptic size and shape. A three-dimensional reconstruction is unnecessary.

Effects of extracts of denervated muscles on the morphology of cultured muscle cells.

Previously, trophic effects of extracts from whole chick embryos and from innervated muscles on cultured muscle cells were described. The present study demonstrated similar effects of extracts from 10-days denervated chick muscles. Extracts from innervated as well as from denervated muscles exsanguinated in vivo with saline prior to dissection showed only marginal trophic activity, suggesting a major contribution of serum components to the trophic effects of tissue extracts. Indeed, serum of adult chicks appeared to have a trophic action similar to that of chick embryo extract.