The effect of maternal thyroidectomy prior to conception on foetal brain development in sheep.

Merino ewes were surgically thyroidectomized, and mated 6 weeks later when their plasma thyroxine (T4) levels were negligible. Their foetuses were delivered by hysterotomy at 52, 71, 84, 98, 125, 140 days gestation or at term (150 days). Despite the very low levels of T4 in maternal plasma, the concentrations of T4 in foetal plasma were not significantly different after 71 days gestation from those of foetuses of sham-operated (control) ewes. Foetal brain and body weights, however, were reduced from 71 days compared to those of foetuses of sham-operated ewes. The foetal brain weights but not the body weights were restored to normal from 125 days to term. In addition to the weights, cell number (DNA) and cell size (protein:DNA ratio) appeared to be normal in the neonatal brain at parturition and this was confirmed by histological examination of the brains. Thus lack of maternal thyroid hormones in early pregnancy may cause a reduction in brain and body growth in the foetus which, in the case of the brain, appears to be restored to normal after the onset of foetal thyroid function.

Alcohol withdrawal causes a loss of cerebellar Purkinje cells in mice.

Mice received a liquid diet containing alcohol for 4 months, after which half of them were sacrificed and the others given a 4-month recovery period before being sacrificed. They were compared with similar mice receiving the diet with alcohol replaced isocalorically by sucrose. No damage was detected in the cerebellum during alcohol consumption, but the number of Purkinje cells was significantly reduced in the recovery period. The experiment was repeated twice with mice consuming a normal diet but exposed to alcohol vapor. The first group was exposed to alcohol vapor 24 hr/day for 3 weeks and then given alternating 1-week periods of recovery and exposure 24 hr/day until a total of 6 weeks of exposure to alcohol vapor and 4 one-week recovery periods had been experienced. They were compared with similar mice exposed to alcohol vapor 24 hr/day for 6 weeks without a recovery period. The second group was exposed to alcohol vapor 9 hr/day for 3 weeks, when part of the group was given a 3-week recovery period. In both experiments, damage was not detected in the cerebellum during alcohol exposure, but in mice withdrawn from alcohol, the number of Purkinje cells was reduced and qualitative evidence of neuronal degeneration was found with a silver stain. In a further group of mice, exposure to alcohol vapor was tapered off gradually, and no evidence of neuronal loss was found. Indications in the literature that withdrawal from alcohol can cause brain damage are briefly reviewed.

Restoration of brain growth in fetal sheep after iodized oil administration to pregnant iodine-deficient ewes.

Iodized oil was administered as a single intramuscular injection to pregnant iodine-deficient ewes at 100 days gestation and the subsequent growth of their fetuses compared with that of fetuses of severely iodine-deficient ewes and of iodine-replete ewes, all of which were fed the same low-iodine diet. The administered iodine produced a remarkable improvement in thyroid function and physical appearance of the fetuses, accompanied by an increase in brain growth and to a lesser extent in body growth, which at 140 days was only slightly (but significantly) less than that of the controls. There was restoration of the number of cells (DNA) and myelination (cholesterol/DNA) in the cerebellum and cerebral hemispheres which suggests a catch-up of neuroblast development during pregnancy. Histological examination, however, revealed that counts of synapses (density) in the cerebral cortex after iodized oil were still less than those of the control fetal brains. The relevance of these findings to the effects of iodine deficiency on human brain development is discussed.

Blood-brain barrier dysfunction in thiamine-deficient, alcohol-treated rats.

Rats maintained on a thiamine-deficient diet for 38 days loss weight and showed neurological symptoms. The PA value, representing the permeability of the blood-brain barrier to 14C-sucrose, was significantly increased whether urethane or ethanol was used as anaesthetic. This increase was prevented by giving rats on the same diet injections of thiamine twice weekly. Barrier function was normalised by injecting thiamine into deficient rats for just 3 days before biopsy. The brains of the thiamine-deficient rats were stained by the Fink-Heimer method but showed no degenerating axons except for silver grains in the glomeruli of the olfactory bulb. Other rats were maintained on the same diet for 38 days and additionally exposed to ethanol vapour for 16 h per day. This resulted in a similar loss of weight but a greater leakage of the blood-brain barrier. The latter was normalised by a thiamine injection only 24 h before biopsy, but was not reduced by withdrawal of ethanol for 3 days before biopsy. Axonal degeneration was present in the olfactory glomeruli. However, no lesions or extravasated blood cells were seen in any brains, there was no change in brain water indicative of oedema and no degeneration in retina, distal peripheral nerves or leg muscles. The relation of these and other experimental findings to alcohol-related brain damage is considered.

Organic lead and histological parameters of brain development.

Relatively little work has been done on the structural effects of organic lead in the central nervous system (CNS), although this form of lead may be a significant fraction of total brain lead. We tested a number of easily measured light-histological parameters of neuronal development in rats for sensitivity to (a) normal growth between 18 and 28 days of life and (b) the effect of weekly injections of tetramethyl lead (TML), administered from 1 week after conception until post-natal day 6. Several of the histological parameters were found to be sensitive to normal growth, but none showed any effect of organic lead treatment. This was despite a small but significant decrease in brain weight, and a significant increase in body/brain weight ratio, with tetramethyl lead treatment. The body/brain weight ratio was the parameter most sensitive to tetramethyl lead treatment. Possible reasons for the disparity between weight and histological parameters are discussed, with reference to previous workers' findings concerning the effects of organic lead on the development of myelin in the CNS and the availability of organic lead to brain tissue.

The effect of maternal and fetal thyroidectomy on fetal brain development in the sheep.

The combination of maternal and fetal thyroidectomy was found to have a significant influence on brain development in the fetal sheep at 140 days. There was reduced body weight (36%), brain weight (23%), DNA (26%) and protein (34%) content in five fetuses of ewes, subjected to thyroidectomy six weeks before mating and fetal thyroidectomy at 98 days gestation, compared with six sham operated controls. Cholesterol content was also reduced (36%) and water content increased (2.4%). The cerebellum was most severely affected and showed histologically an increased cell density associated with a significant reduction in the ratio of the molecular to granular cell layer area. The cell density was also significantly increased in the CA1 region of the hippocampus, but not in the CA4 region. It was also increased in the parietal layer of the cerebral cortex but not in the motor region. There was a significant reduction in the weight of heart (28.6%) and lungs (33.4%), while the kidneys and pituitary were enlarged (20.5% and 48.5% respectively) as a result of double thyroidectomy. The combined thyroidectomy was similar to iodine deficiency in its effect on fetal brain development, indicating that it is probable that iodine deficiency has its effects in the sheep by a combination of maternal and fetal hypothyroidism.

Chronic consumption of alcohol by adult mice: effect on hippocampal cells and synapses.

For 4 months C57 black mice were fed a nutritionally complete diet containing 9% alcohol or isocaloric sucrose and killed then or after 4 months recovery on standard food pellets. The number of cells in a thin plastic section of hippocampus was unchanged in field CA1 by alcohol exposure but was reduced 9% during withdrawal from alcohol. Electron microscopy was used to count synapses among the basal dendrites and no significant change was found in any treatment group. The spine heads were measured and found to be smaller in the alcohol group than in the sucrose group; many of the spines (in the alcohol group) were too small to be visible with the light microscope.

A change in susceptibility of rat cerebellar Purkinje cells to damage by alcohol during fetal, neonatal and adult life.

The sensitivity of rat cerebellar Purkinje cells to ethanol exposure during fetal, neonatal or adult life was assessed by histological techniques. Pregnant female rats were exposed to ethanol vapour during the last 2 weeks of gestation. Purkinje cells were counted 5 days after the pups were born. The number of Purkinje cells in lobe VIII was reduced by 45%, and the linear density of Purkinje cells in lobe I was 47% less than in controls not exposed to ethanol. Smaller reductions were found in other lobes. The weight of the cerebellum was reduced by 34%. Neonatal rats were exposed to ethanol vapour briefly during daylight hours on the third and fourth days after birth. Purkinje cells were counted on the fifth day after birth, and losses similar to those described above were found, with additional significant reductions of cell numbers in lobe I and of Purkinje cell density in lobe VIII. The weight of the cerebellum was reduced by only 4%. Adult male rats were exposed to ethanol vapour for 3 weeks and no Purkinje cell losses were subsequently found. The dura overlying the cerebellum of separate adult male rats was superfused with 100% ethanol for 1 h and no abnormalities were detected with electron microscopy in the exposed cortex 6 days later. It is remarkable that the brief neonatal treatment caused a more widespread loss of Purkinje cells than the 10 days of exposure to ethanol in utero, whereas the Purkinje cells present in adult animals show a great resistance to ethanol. The neonatal period seems to be a time of high susceptibility of Purkinje cells to ethanol.

Retarded fetal brain development resulting from severe dietary iodine deficiency in sheep.

Sheep have been used to study the effect of dietary iodine deficiency on the development of the fetal brain. Severe iodine deficiency caused reduction in fetal brain and body weights and in brain DNA and protein from 70 days gestation to parturition. The lowered brain weight and brain DNA at 70 days gestation indicates a reduced number of cells, probably due to slower neuroblast multiplication which normally occurs from 40-80 days in the sheep, and the reduction in DNA and protein after 80 days implies that the development of neuroglia could be slowed also in iodine deficiency. Morphological changes were observed in both the cerebral hemispheres and the cerebellum. In the cerebral hemispheres of the iodine-deficient fetuses an increased density of neurons was apparent histologically in the motor cortex and visual cortex and in the CA1 and CA4 areas of the hippocampus in comparison with controls. In the cerebellum there was delayed migration of cells from the external granular layer to the internal granular layer and increased density of Purkinje cells in the iodine-deficient fetal brains. In addition, the molecular area was increased and the medullary area reduced in comparison with controls. These change are indicative of delayed brain maturation. Evidence of fetal hypothyroidism was provided by low fetal thyroid iodine and plasma T4 values, thyroid hyperplasia from 70 days gestation, significant reduction in body weight at the same time as the brain retardation, and absence of wool growth and delayed skeletal maturation near parturition. It is apparent from the biochemical and histological changes observed during iodine deficiency that iodine is an essential element for normal fetal brain and physical development in the sheep.

The blood-brain barrier in rats fed on diets high or low in saturated/unsaturated fat ratio.

The product of the permeability x vascular surface rate area (PA) of the blood-brain barrier to [14C]sucrose has been measured in rats raised on synthetic diets in which the saturated/unsaturated fat constitution was controlled at high or low levels. Gas-liquid chromatography demonstrated marked differences in brain fatty acid constitution between the dietary groups. No statistically significant differences was found between the permeability measurements in rats maintained on any of the synthetic diets, nor was there any difference from rats raised on a standard laboratory pellet food. The opportunity was taken to look at 3 other properties of brain that might be affected by lipid constitution. There was no change in the form of the membranous intracellular inclusions that can be induced by intracerebral injections of suramin, and Fink-Heimer staining of degenerating axons, which is inhibited by fat extraction, worked equally well on each diet group. The sleep time after an anaesthetic injection of alcohol was not significantly changed.

Weakening of the blood-brain barrier by alcohol-related stresses in the rat.

The product of the permeability x vascular surface area (PA) of the blood-brain barrier to [14C]sucrose has been measured in rats maintained for 3 weeks in a chamber, the air supply to which carried a controlled concentration of ethanol vapour. No statistically significant difference was found between the permeability measurements in rats inhaling ethanol vapour for 3 weeks and non-alcohol exposed rats. The PA value was found to be significantly increased (115%) in rats given the same ethanol exposure when additionally subject to starvation during the last 3 days of this treatment. If the ethanol supply was also withdrawn at the same time as the food, a similar significant increase (116%) in PA value was found. In the absence of any ethanol exposure, 3 days' starvation did not significantly alter the measured PA value. Finally, when rats are given 200 mg/kg disulfiram every second day during a 2-week period of ethanol inhalation, the PA value was not significantly altered, although the concentration of acetaldehyde in the blood was up to 129 microM. The results indicate that while ethanol or acetaldehyde alone do not cause a weakening in the blood-brain barrier, the additional stress of food withdrawal after alcohol exposure does reduce barrier function, and this could be significant in human binge drinking.

Comparison of extracellular space in the mature and aging rat brain using a new technique.

A new technique for measuring extracellular space in the rat brain has been developed. It involves opening the blood-brain barrier with a bolus of hyperosmotic sucrose followed by a high-pressure perfusion of the cerebral vasculature with an isotonic solution containing an impermeant radioactive tracer, [3H]sucrose. After allowing the concentration of tracer in the brain to reach a plateau, the amount of radioactivity/mg of brain tissue is expressed as a percentage of the amount of radioactivity/mg of perfusate to obtain a value for extracellular space. The addition of glutaraldehyde to the perfusate results in the brain being fixed simultaneously for electron microscopy. Reproducible estimates of extracellular space were obtained similar to those obtained by other methods (e.g. Levin et al. 1970). As it is impossible to be sure of the validity of the absolute value of extracellular space obtained by any method using perfused solutions we have used our method for comparative purposes. Extracellular space was measured in mature (control) and ageing rats to test the claim that the volume of space in the cerebral cortex is substantially reduced with ageing (Bondareff and Narotsky 1972). We found a consistent tendency for the extracellular space to increase with age in the 6 regions of brain examined. This was not statistically significant except in the group of ageing rats on a food-restricted diet. Therefore, these results do not support a generalisation that the extracellular space decreases in the ageing brain. In both control and ageing rats, extracellular space was shown to be unevenly distributed in the brain, the largest space being present in the cerebellum, olfactory bulb, inferior and superior colliculi and the least space in the white matter. These are the first measurements of extracellular space in which assessment is possible by both electron microscopy and by the measurement of a chemical tracer.

The peripheral and central changes resulting from cutting or crushing the afferent nerve supply to the whiskers.

In neonatal rats, crushing or cutting the infraorbital nerve, the sensory nerve supply to the whiskers, has been found to prevent cortical barrel formation. However, both procedures are followed by regeneration of one-third to one-half of the nerve fibres and reinnervation of the whiskers. By counting fibres in individual whisker follicle nerves, it has been shown that 29-67% (mean 45%) of the myelinated fibres regenerate to the whiskers after a crush compared to 24-56% (mean 39%) after a cut. Further differences between the crush and cut lesions were indicated by studies on the time course of regeneration. Counts of the regenerating fibres at various ages as well as recordings of cortical evoked potentials in normal, nerve-crushed and nerve-cut animals showed that recovery was 3-4 days earlier in the nerve-crushed, compared with the nerve-cut animals. In normal and nerve-crushed animals the evoked potential was first detectable 2-3 days after birth while the response after nerve cut could not be recorded until day 7. Even after 60 days the amplitude of responses on both crushed and cut pathways was only about one-third of normal, while the latency was prolonged (normal 5.8 +/- 0.25 ms, crush 6.5 +/- 0.26 ms, cut 7.7 +/- 0.67 ms). Central changes occurring as a result of nerve cut or crush have been studied by microelectrode recordings from the trigeminal nucleus (the first synaptic level) and the somatosensory cortex. These also indicate clearly the greater severity of the cut lesion. Thus, in crushed animals, all levels of the trigeminal nucleus as well as the cortex show only minor modifications. The whiskers occupy the same total area and responses from all whiskers are present at their normal sites. However, after nerve cut, the responses from both the trigeminal nucleus and cortex show clear abnormalities. The total whisker area is reduced with a concomitant expansion of responses from the nose, check, lower jaw, and whiskers by the eye and ear. In addition, only one-third to one-half of the whiskers give responses. The site of these abnormalities is localized to the trigeminal nucleus since all whiskers show innervation in the peripheral nerve. It is suggested that the longer recovery time as well as the reduced accuracy of reinnervation may contribute to the poorer central recovery after a nerve cut.

Effect of suramin on the activities of degradative enzymes of sphingolipids in rats.

Three to nine days after administration of suramin, 500 mg/kg intravenously in rats, a small amount of the drug (about 0.25 micromoles/g tissue) was retained by the liver and spleen, and a larger amount (about 1.2 micromoles/g tissue) was retained by the kidneys. The activities of the sphingolipid hydrolases beta-hexosaminidase and GM3-sialidase were strongly inhibited by suramin in vitro. The activity of beta-hexosaminidase was inhibited 70% by 10(-5M) and 85% by 10(-4M) suramin, and the activity of GM3-sialidase was inhibited 80% by 10(-4M) suramin. The activities of sphingomyelinase and beta-galactosidase were also inhibited by suramin but at higher concentrations of the drug. Suramin, in vitro is a weak inhibitor of glucocerebrosidase, galactocerebrosidase, alpha-galactosidase and arylsulfatase A (less than 50% inhibition at 10(-3M) concentration of the drug). The inhibition of beta-hexosaminidase by suramin was non-competitive. Inhibition of beta-hexosaminidase and GM3-sialidase may explain the accumulation of GM2 and GM3 gangliosides in the brains of rats treated intracerebrally with suramin (Constantopoulos et al, 1980).

The short-term toxicity of ethanol to neurons in rat cerebral cortex tested by topical application in vivo, and a note on a problem in estimating ethanol concentrations in tissue.

In rats anesthetized with ethanol 4.0 g/kg i.p. the dura overlying the parietal cortex was exposed and superfused with 100% ethanol for 1 h. After 6 days survival the underlying cortex was stained with a silver method that is selective for degenerating axons and their terminals. No degeneration was found in the superfused cortex, although heat-lesioned tissue stained concurrently showed axonal degeneration and so validated the technique. Electron microscopy after 3-20 days survival did not show any degeneration, and synapses of normal appearance were present immediately beneath the cortical surface. In other rats the ethanol concentration in the superfused tissue was assayed in 0.4 mm thick discs sectioned with a vibratome from a 4-mm diameter core cut with a trocar from the cortex immediately after 1 h of superfusion. The ethanol was eluted in 2% TCA, and an aliquot assayed enzymatically. A second elution of the tissue disc contributed a further 5% of the ethanol content indicating a partition coefficient for ethanol between wet brain tissue and 2% TCA of about 10. The total concentration of ethanol in the superficial cortex was found to be about 0.82 M or 3.8%. This estimation was confirmed by superfusion with 14C-labelled ethanol and scintillation counting. Thus neurons in the cerebral cortex did not degenerate after exposure for 1 h to a concentration of ethanol that was 3 times greater than the concentration that causes death in a rat by paralysis of the respiratory centre (1.2%).