Biomedical subjects
Publications and source records attributed to K S Bedi.
Recently developed stereological methods have been used in experiments to examine the effects of two levels of undernutrition during early postnatal life on the total number of rat dentate gyrus granule cells. This study has shown that previously undernourished rats have significant deficits in the total number of this particular type of neuron.
Undernutrition during early life is known to affect the morphology of the hippocampal formation. Recent advances in stereological techniques have made it possible to make relatively unbiased estimates of total cell numbers in well-defined brain regions. It was decided to use these methods to determine the effects of different levels of undernutrition during early postnatal life on the granule cells of the rat dentate gyrus. Male hooded Long Evans rats were undernourished between the 16th day of gestation and 30 postnatal days of age to two different levels. The daily food intake of level-1 and level-2 rats represented about 60 and 40%, respectively, of that eaten by well-fed, age-matched controls. Nutritional rehabilitation of the rats was commenced when they had reached 30 days of age by placing them on an ad libitum diet. Groups of control and experimental rats were killed at 70 and 212 days of age. The Cavalieri principle was used to determine the granule cell layer volume within the dentate gyrus, and the "dissector" method was used to determine numerical densities of these granule cells. These estimates were used to calculate the total numbers of granule cells. There were between 260,000 and 320,000 granule cells within the dentate gyrus of 70-day-old control and experimental rats. By 212 days of age, well-fed controls had an average of about 834,000 granule cells. The level-1 and level-2 previously undernourished rats had about 515,000 and 595,000 granule cells, respectively. Two-way analysis of variance procedures showed significant main effects of nutrition and age as well as a significant interaction between them.(ABSTRACT TRUNCATED AT 250 WORDS)
It is known that removal of one eye in the rat in early postnatal life results in an aberrant ipsilateral projection from the remaining eye. We have estimated the number of optic nerve fibres in 36 days old Black and White Hooded rats that had been unilaterally enucleated at birth. There was no significant difference in the transverse cross-sectional area of the optic nerves of control and enucleated rats. Control animals had an average of about 109,000 myelinated fibres and about 11,000 unmyelinated fibres. The mean minimum diameter of the myelinated fibres was 0.73 microns. The corresponding values in the unilaterally enucleated animals were not significantly different from control animals. These results are discussed in the context of the previously reported increased ipsilateral projection in unilaterally enucleated rats.
Male rats were placed in complete darkness from birth until 30 days of age, followed in some cases by a 35 days period of rehabilitation in control lighting conditions. Groups of control and experimental animals were killed at 30 and 65 days of age by perfusion with buffered 2.5% glutaraldehyde. The right optic nerve was dissected out from each animal and processed for embedding in Epon. Quantitative stereological procedures were used to estimate the total number of both myelinated and non-myelinated optic nerve fibres and their mean minimum diameters. There were no significant differences in the total number of optic nerve fibres between dark- and light-reared rats. However dark-reared rats had myelinated and non-myelinated fibres with significantly larger fibre diameters than those in age-matched light-reared rats. The proportion of optic nerve fibres which were myelinated increased with age in both groups of animals. However by 65 days of age the degree of myelination was slightly but significantly greater in the previously dark-reared rats than in the light-reared controls. These results indicate that rats reared in complete darkness for the first 30 days of postnatal life show morphological changes in the optic nerves. The possible significance of these changes is discussed.
Rearing animals in dark conditions during early postnatal life has been shown to affect both the morphology and the normal functioning of the visual system. We have investigated the effects on the synapse-to-neuron ratios in the superior colliculi of rearing male rats in the dark from birth until 30 days of age, followed in some cases by a 35 day period of rehabilitation in control lighting conditions. Control lighting conditions consisted of a room on a 12 hour light/12 hour dark cycle. Synapse-to-neuron ratios were calculated from estimates of the numerical densities of neurons and synapses. These estimates were made using the 'disector' method at the light and electron microscopical levels. Neuronal nuclei were used as the counting unit for neurons and paramembranous densities for synapses. There were no significant differences in the numerical densities of neurons, synapses or synapse-to-neuron ratios between dark-reared and control rats at 30 days of age. Sixty five days old rats, previously raised in the dark, had a significantly smaller numerical density of neurons than light-reared controls. Two-way analysis of variance techniques showed significant effects of age on the estimates of the numerical densities of neurons and synapses. However, there were no significant main effects of the lighting conditions, nor any significant interaction for any of the measures.
Black and white hooded Lister rats were undernourished for various times up to 150 days of age; some of them were nutritionally rehabilitated from 75 days. Undernourished rats weighed significantly less than well-fed controls at all ages studied. After embedding in resin, sections of cerebellar cortex were cut and examined at the light and electron microscopical levels using traditional morphometric methods. Undernourished rats showed significant deficits in synapse-to-neuron ratio, compared with controls, at 21 days of age. This deficit disappeared by 75 days despite continued undernutrition. Indeed, there was no alteration in this ratio even when undernutrition was extended up to 150 days even though the ratio for the controls decreased after that period. Rats undernourished from birth to 75 days and subsequently rehabilitated to 150 days had significantly more synapses per neuron than controls. The functional sequelae of these morphological changes remain unknown.
Stereological estimates of the numbers of Purkinje cell nucleoli in human cerebellar cortex have been obtained from systematic random samples of tissue by using the fractionator. The estimates are unbiased by fixation, section thickness, or sampling errors and are independent of any assumptions about cell shape, size, or spatial orientation. Twelve brains from aged subjects of both sexes were examined. The average complement of nucleoli in four female brains (age range 71-93 years) amounted to 14.8 millions (with an observed coefficient of variation between subjects of 29%). For three male brains (76-91 years), the corresponding estimates were 15.7 millions (10%). No significant sex differences were found for these small samples. Five brains of unknown sex and age yielded values of 15.8 millions (18%). For the twelve brains examined, the total number of Purkinje cell nucleoli per cerebellum was found to be 15.4 millions (19%). Estimated numbers showed a significant positive correlation with cerebellar weights. The number of nucleoli in an individual cerebellum was obtained with high precision in as short a time as 4 hours.
It is possible that the reported effects of early life undernutrition on brain morphology may be due to alterations in mother-infant interactions and not directly to undernutrition. We have investigated this possibility by comparing artificially reared with mother-reared rats. Four groups of black-and-white hooded male rats were reared. These consisted of mother reared control (MRC), mother reared undernourished (MRU), artificially reared control (ARC) and artificially reared undernourished (ARU). Artificially reared rats were raised in isolation away from their mothers from 5 to 21 days of postnatal age. They were fitted with a gastric cannula through which 'milk' was infused automatically. The period of undernutrition lasted from 5 to 25 postnatal days, following which the animals were fed ad libitum until 312 days of age. Rats from each group were then killed by perfusion with buffered 2.5% glutaraldehyde. Pieces of visual cortex from each rat were postfixed in osmium tetroxide and embedded in resin. Stereological procedures at the light and electron microscopical levels were used to estimate the synapse-to-neuron ratios in cortical layers II to IV. Both MRC and ARC rats had about 7000 synapses per neuron. However, this ratio was about 8300 in MRU rats whilst it was only about 5000 in ARU animals. The rearing x nutrition interaction was statistically significant at the 0.1% level. These changes in the synapse-to-neuron ratio were mainly due to alterations in the numerical densities of the synapses rather than that of neurons. These results demonstrate that environmental isolation, as a result of artificial rearing procedures, and concurrent undernutrition during the first three weeks of postnatal life, interact with one another to produce marked morphological changes in the adult rat brain. However, environmental isolation was not, by itself, sufficient to cause permanent changes in interneuronal connectivity.
Recent research has shown that a lengthy period of undernutrition during early postnatal life can cause alterations in the morphological structure of the dentate gyrus. As this region is involved in the control of spatial memory, we decided to investigate whether undernourished rats also showed any deficits in this aspect of behaviour. Rats were undernourished from about birth until either 30 or 60 days of age and then nutritionally rehabilitated for a lengthy period before testing. There were significant differences in the body weight of control and undernourished rats in each experiment. The testing procedure involved rats being placed in a large pool of opaque water. They were required to swim to find a hidden platform located just below the water surface onto which they could escape. Each rat had to perform this test 20 times over a period of 3 days. The time taken and the total distance swum by each rat during each trial was measured. In Experiment 1, rats were familiarised with the water before testing took place, whereas in Experiment 2 they were not. There were no significant differences in the latency to find the platform or the distance swum between well-fed control and previously undernourished rats in either experiment. In conclusion, in our experiments we could not demonstrate that undernutrition during early life caused any deficits in spatial memory performance.
One day old rats had their right eyes removed and together with non-enucleated controls were raised in either 'light' or 'dark' (red light) conditions from birth until 39 days of age. This resulted in four groups of animals: light-reared enucleated, light-reared non-enucleated, dark-reared enucleated and dark-reared non-enucleated. All animals were killed by intracardiac perfusion with 2.5% sodium cacodylate-buffered glutaraldehyde at 39 days of age. Pieces of visual cortex (Area 17) from both right and left cerebral hemispheres of each animal were dissected out and processed for electron microscopy. Stereological procedures were used to estimate the synapse-to-neuron ratios in Layers II-III of these cortices. Light-reared non-enucleated rats had about 16,000 synapses per neuron in both right and left visual cortices. Rearing non-enucleated rats in the dark reduced this value to about 11,500. The synapse-to-neuron ratio of unilaterally enucleated rats raised in the light was not significantly different from that observed in light-reared non-enucleated rats. Combined dark-rearing and unilateral enucleation did reduce the synapse-to-neuron ratio but not to any greater extent than did dark-rearing alone. However, there was a suggestion of a differential effect between the right and left cortices in these enucleated, dark-reared animals. These results demonstrate the importance of light stimulation for the normal development of interneuronal connectivity of the visual cortex. They also show that the effects of unilateral enucleation for the visual cortex are not as great as those previously observed for the superior colliculi in the same animals, at least as far as synapse-to-neuron ratios are concerned.
The numerical density of synapses in the molecular layer of the parietal cortex of normal 28-day-old rats has been estimated by using two stereological methods, namely an unfolding technique and the disector method. For the unfolding technique it was assumed that the synapses formed a polydispersed population of disc-shaped particles. The technique also relies upon various assumptions regarding the size, shape and projection of synapses in the plane of section, these assumptions producing a result that is biased to an unknown extent. Such simplifying assumptions are unnecessary for the disector method. The mean +/- S.E.M. numerical density of synapses was found to be 2.16 +/- 0.14 x 10(9)/mm3 using the disector method. This was not significantly different (paired t-test; P less than 0.05) from the value of 2.41 +/- 0.22 x 10(9)/mm3 obtained using the unfolding technique with correction for truncation ('lost caps'). When the unfolding method was used without a correction for lost caps the numerical density was 1.98 +/- 0.10 x 10(9)/mm3, a value also not significantly different from that obtained using the disector method. The mean projected height (Hs) of the synaptic particles was also determined using both procedures; the results were not significantly different. When the variances of the final estimates and the time taken to carry out the procedures described were taken into account, it was estimated that the disector method was about 86% more efficient than the unfolding method with capping correction. Although the unfolding method can give essentially similar values for the numerical density of synapses to those obtained by the disector method, it is biased and much less efficient. It is concluded, therefore, that the disector method is the superior method for the estimation of the number and size of particles such as synapses, particularly when they have complex spatial configurations.
Male rats were undernourished for various lengths of time between birth and 150 days of age, with some rats being nutritionally rehabilitated between 75 and 150 days of age. Eight control and eight experimental rats were anaesthetised and perfused with 2.5% glutaraldehyde at each of 21, 75 and 150 days of age. Stereological procedures were used to estimate granule-to-Purkinje cell ratios in lobes IV, V and VI, using 0.5 micron thick toluidine blue-stained sections. Undernourished rats had significantly lower body and cerebellar weights than controls at all ages examined. These deficits persisted even after a period of nutritional rehabilitation. The granule-to-Purkinje cells ratio did not differ between control and experimental groups at 21 or 75 days of age. However, at 150 days both undernourished and rehabilitated groups of animals had significant deficits in this ratio compared with age-matched controls. These results suggest that undernutrition can have profound effects on brain development in later life even if the effects are not apparent during the period of undernutrition.
Recent evidence showing alterations in spatial memory due to a period of undernutrition during early life has implicated the hippocampus as one of the brain centres that may be particularly adversely affected. However, there are very few quantitative morphological studies that have examined the neuronal and synaptic populations of the hippocampi from undernourished animals. We decided to carry out such experiments, paying particular attention to the granule cell of the dentate gyrus. Male rats were undernourished from the 18th day of gestation until 21, 75, or 150 days of age. Some of these previously undernourished rats were nutritionally rehabilitated between 150 and 250 days of age. Groups of well-fed control and experimental rats were killed by intracardiac perfusion with 2.5% sodium-cacodylate-buffered glutaraldehyde. The right hippocampus from each rat was dissected out and processed for electron microscopy. Stereological procedures at the light and electron microscopical levels were used to estimate the numerical densities of granular cell neurons and molecular layer synapses in the dorsal lip of the dentate gyrus. These estimates were used to calculate synapse: neuron ratios. There were 5,056 +/- 347 (mean +/- SE) and 5,002 +/- 190 synapses per neuron in 21-day-old control and undernourished rats, respectively. By 75 days these values had increased to 9,215 +/- 588 and 6,683 +/- 237. The difference was statistically significant. By 150 days of age the value for control animals had fallen once again to 6,518 +/- 209 whereas undernourished rats had increased slightly to 7,689 +/- 288 (P less than .01); 250-day-old rats, previously undernourished from birth to 150 days of age, showed a substantial increase in the synapse: neuron ratio during the period of nutritional rehabilitation. Thus these nutritionally rehabilitated rats had 9,407 +/- 365 synapses per neuron whereas age-matched controls had only 6,323 +/- 239 (P less than .01). These results indicate that the rat dentate gyrus is vulnerable to undernutrition even during the postweaning period and that a lengthy period of undernutrition can alter the developmental growth curve for synapse: neuron ratios.
Rats whose right eyes were enucleated on day 1 after birth and nonenucleated rats were raised in either "light" or "dark" (red light) conditions from birth until 39 days of age. This resulted in 4 groups of animals: light-reared nonenucleated, light-reared enucleated, dark-reared nonenucleated, and dark-reared enucleated. At 39 days of age, the animals were killed by perfusion with 2.5% sodium cacodylate buffered glutaraldehyde. The superior colliculi were dissected out and processed for embedding in resin. Stereological procedures at the light and electron microscopical levels were used to estimate the synapse-to-neuron ratios in the superficial layers of these colliculi. Light-reared, nonenucleated rats had about 1,850 synapses-per-neuron in both the right and left superior colliculi. Rearing nonenucleated rats in the dark reduced this value to about 1,200. Enucleated rats reared in the light showed a differential response in the 2 colliculi. Thus, the contralateral (to the enucleated eye) colliculi showed a decrease, whereas the ipsilateral colliculi showed an increase in the synapse-to-neuron ratio compared with light-reared, nonenucleated rats. When enucleated rats were reared in the red light, there was a decrease in the ratio in both colliculi, although the extent of this decrease was more marked in the contralateral than the ipsilateral colliculi. However, the decrease in the contralateral colliculi was not significantly greater than that observed in the corresponding colliculi from dark-reared, nonenucleated rats. These results provide useful information on the combined and separate effects of unilateral enucleation at around birth and dark (red light) rearing during early life on the interneuronal connectivity of both the ipsi- and contralateral superior colliculi of rats. They also show the vast importance of visual stimulation for the normal development of the subcortical visual centers.
The exact measurement of the thickness of semi-thin and ultra-thin sections is of importance for some stereological methods. Although section thickness can be measured with great precision and accuracy using suitable microinterferometers, such instruments are expensive and not easily available to many laboratories. This paper describes a simple method of measuring the thickness of sections actually used for stereological counting procedures. It involves the division of any given section into two portions, one of which is used for the counting procedures. The other portion is re-embedded and re-sectioned through its thickness making it a simple task to measure its thickness.
Groups of pigmented (Black and White Hooded Lister) and albino (Sprague Dawley) rats were killed at 7, 15, and 25 postnatal days of age. Their optic nerves were embedded in resin suitable for both light and electron microscopy. Quantitative stereological procedures were used to estimate total fibre number and the degree of myelination in the optic nerves at the various ages. At 7 days of age, both albino and pigmented rats had about 220,000 optic nerve fibres. By 15 days, both strains showed a reduction of some 30,000 fibres. This fibre loss continued in both strains after 15 days of age, but more rapidly in the pigmented strain. At 25 days of age, pigmented rats had 72,371 +/- 7,244 fibres, whilst albino rats had 102,681 +/- 4,138 fibres (P less than .01). More than 99.5% of optic nerve axons in both strains were unmyelinated at 7 days of age. By 25 days of age about 90% of all remaining fibres were myelinated in both strains. The mean diameter of the myelinated axons was estimated to be between 0.59 and 0.65 micron in all animals, there being no significant age or strain differences. In contrast, the mean diameter of nonmyelinated axons increased significantly with age. This increase was greater for albino than pigmented rats, such that by 25 days of age the respective values were 0.49 micron and 0.42 micron (P less than .05).
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Black and white Lister hooded rats were undernourished from the 16th day of gestation until 25 postnatal days of age. These previously undernourished rats and a set of well-fed rats were later subjected to 30 days of environmental diversity, i.e., environmental enrichment or isolation. Two separate experiments were carried out. In experiment 1, the environmental diversity lasted from 85 to 115 days of age and in experiment 2, from 35 to 65 days of age. At the end of the period of environmental diversity, all rats were killed by perfusion with 2% phosphate-buffered glutaraldehyde. Small pieces of tissue from the right visual cortex were embedded in Spurr's resin. For each rat two blocks of resin-embedded tissue were randomly selected, and from these semithin sections (0.5 micron) were cut and stained with toluidine blue. Photomicrographs of cortical layers II and III were taken from these sections and used to estimate the numerical density of neurons. Ultrathin (ca. 70 nm) sections of the same region of the cortex were cut and stained with lead citrate. These sections were used to estimate the synaptic disc diameter and numerical density. Synapse-to-neuron ratios were calculated from the estimates of synaptic and neuronal numerical densities. In experiment 1, well-fed rats raised in enriched environments had a significantly smaller neuronal numerical density and a greater synaptic disc diameter than well-fed rats raised in an impoverished environment. In experiment 2, neither the well-fed nor previously undernourished rats showed significant effects of environmental treatment on any of the features studied. The statistical interaction between nutrition and environment was not significant for any of the features in either experiment.