C. Giuli

The aging of rat liver as revealed by electron microscopic morphometry--I. Basic parameters.

Abstract One-, 12- and 27-month-old female rats (5 in each group) were investigated by means of point-counting morphometry at light and electron microscopic sampling levels. Morphometric parameters are given for single hepatocytes, per unit volume of tissue and as specific values, i.e. per 100 g body weight. Many liver parameters showed age-dependent changes between the young and adult or between the adult and old ages. The most important stereologic manifestation of aging was the very strong reduction of surface area, surface density and specific surface density values of rough endoplasmic reticulum between the adult and old groups. This finding explains the decreased protein synthetic activity of old rat liver. Other parameters also changed, however, e.g. in case of mitochondria the interpretation is difficult because of a considerable swelling observed only in the old animals. Lysosomal elements showed no significant age-dependent changes. Some methodical problems of morphometry are also discussed.

Energy dispersive X-ray microanalysis of the electrolytes in biological bulk specimen: II. Age-dependent alterations in the monovalent ion contents of cell nucleus and cytoplasm in rat liver and brain cells

Energy dispersive X-ray microanalysis was carried out on the nucleus and cytoplasm of large brain cortical and liver cells of young, adult and old (1, 12 and 24 months of age, respectively) female Wistar rats, using a new method for preparation of freeze-dried bulk specimens. It was found that the K + , Na + and Cl − contents per unit dry mass in the nucleus in both tissues significantly increased except the K + content of the hepatocyte nucleus between 1 and 24 months of age. At the same time, in the cytoplasm some ions increased, others decreased or remained unchanged. There is an age-dependent intracellular water loss the extent of which is not known for the tissues studied, however, in human organism it averages about 10–14% between 20 and 99 years. Therefore, intranuclear and intracytoplasmic ionic strengths of the rat tissues studied were calculated assuming 2–6% water loss between 1 and 24 months of age. Already a water loss of 2% results in considerable increases of the ionic strength within the cell water even in those cases where the dry mass fraction values slightly decreased or remained unchanged. The values obtained in the old cell nuclei reach ranges of ionic strength where nonhistonic regulatory proteins are separated from the chromatin in vitro . The results are discussed in the light of recent biochemical observations.

Electron-microscopic morphometric analysis of mouse liver. II. Effect of ageing and thymus transplantation in old animals

The influence of age and thymus on liver cells has been investigated by performing a complete morphometric analysis. The age-dependence was tested in three groups of mice aged 2, 12, and 24 months, while the action of the thymus was studied on 24-month-old mice grafted with neonatal thymus 30 days before the analysis was performed. Among the investigated parameters, the most considerable changes were found in the mean nuclear volume, which displayed a significant increase during ageing. The thymus was capable of reversing such an age-dependent increase when grafted into old animals. The volume of single hepatocytes also displayed an age-dependent increase which was not corrected by thymus grafting. The data presented suggest that the thymus influences the mitotic activity of the hepatocytes and further support the hypothesis that the thymus also plays an important role in non-immunological processes of ageing.

The effect of acute and chronic centrophenoxine treatment on the synaptic plasticity of old rats

The cerebellar glomerulus was studied by electron microscopic morphometry in female Wistar rats. Age-dependent alterations have been revealed from 3 to 28 mth of age, and the effect of centrophenoxine (CPH) was analyzed in two different patterns of administration. First, 27-mth-old rats were treated daily for 6 wk (acute treatment), and second, 18-mth-old rats were treated 3 times per week for 5 months (chronic treatment). The dose was 100 mg CPH/kg body weight, injected intraperitoneally. The surface density (SV), the numerical density (NV) and the average length (L) of the synaptic junctions were calculated from data obtained on ethanol-phosphotungstic acid stained ultrathin sections. An age-dependent reduction of SV and NV of the synaptic contact zones was found, and the L increased in the oldest animals. CPH-treatment resulted in a marked increase of SV in both types of application, whereas the other two parameters behaved differently in the two groups. The chronic treatment resulted in a significant slowing down of the decrease of NV, whereas L remained invariate. On the contrary, the acute treatment increased L but did not alter significantly NV. The results and the differences between the treatment types are discussed in terms of synaptic plasticity and are interpreted as different manifestations of the same reactive synaptogenetic process.

Effect of chronic vitamin E deficiency on the synapses of cerebellar glomeruli in young rats

A morphometric investigation has been carried out on the synaptic junctions in the cerebellar glomeruli of young-adult rats chronically deprived of vitamin E for 10 months and control animals of the same age. The following parameters were evaluated: the average length of the synapses (L), the numerical (NV) as well as the surface (SV) density of the synaptic contact zones. The results from these experimental groups were compared with data from young, adult and old rats. The results obtained show a significant decrease of the surface density of the synaptic contact zones in old and alpha-tocopherol deprived young-adult (11-month-old) rats as compared to younger and normally fed animals. This reduction of the synaptic contact area seems to be due to the marked decline in the number of synapses found in both cases. The average size (L) of the synaptic junctions, on the other hand, was increased in alpha-tocopherol deficient rats as compared to normally fed littermates. The significant reduction of the synaptic contact area in old and vitamin E deprived young rats supports the hypothesis that a common denominator may be responsible to explain this alteration. Because of the recognized protective role of alpha-tocopherol against free radical attacks on plasma membranes, the present findings support an involvement of membrane structural alterations in aging as well as in vitamin E deficiency.

In vivo effects of vitamin E deficiency on the intracellular monovalent electrolyte concentrations in brain and liver of rat. An energy dispersive X-ray microanalytic study

Vitamin E is known to play a protective role for cell membranes against free-radical attacks. Vitamin E deficiency causes a rapid macroscopic ageing of rats. On the other hand, during normal ageing, cell membranes undergo functional alterations resulting in an increased intracellular potassium concentration in brain and liver cells. Therefore, is was of interest to study whether vitamin E deficiency produces similar alterations in young rats. Female Wistar rats were fed with a vitamin E deficient diet from 1 month of age for 10 months. The parietal brain cortex and the liver were analyzed by means of a quantitative energy dispersive X-ray microanalytic method using a JEOL JSM-35C-EDAX-711-NOVA-3 system. Monovalent electrolyte contents as well as the water content of the cells were determined in 5 treated and 5 control animals. Water content was measured by analyzing the potassium content in aqueous, frozen state, and again in the dry mass of the cells. On the basis of these data, a computer program calculated the water proportions. Average values for 200 or more cells of each organ per group revealed a significant increase in the intracellular potassium content of the brain cells, whereas the sodium and chloride contents increased to a much lower extent. There was a 2.6% loss of intracellular water in the brain cells in the vitamin E deficient group. The liver monovalent ions and water content remained unchanged. The results obtained are discussed in terms of the membrane hypothesis of ageing.

Chronic dietary choline modulates synaptic plasticity in the cerebellar glomeruli of aging mice

A morphometric investigation was carried out on ethanolic phosphotungstic acid (E-PTA) stained synaptic junctions in the cerebellar glomeruli of adult, old, old choline-deficient and old choline-supplemented mice. Numerical (Nv) and surface (Sv) density as well as average length (L) of the synapses were calculated on 100 pictures per group. A significant reduction of Nv and Sv, as well as an increase of L was found during aging. Choline deficient animals did not show any change as compared to old animals of the same age. In choline supplemented mice Nv and Sv were significantly increased and L significantly decreased, respectively, as compared to old control littermates. No difference was found between adult and choline supplemented mice. In the cerebellar glomeruli only a small fraction of fibers are cholinergic, therefore the present findings support the idea that dietary choline can influence systems other than cholinergic. The possible role of choline supplementation in the modulation of synaptic plasticity via the synthesis and/or turnover of neuronal membrane choline phospholipids, is discussed.

The aging of rat liver as revealed by electron microscopic morphometry-II. Parameters of regenerated old liver.

Abstract Two thirds hepatectomy was carried out on female Wistar rats of 24–25 months of age. The regenerated livers of 5 animals each were investigated 1, 5, 10, 20 days subsequent to hepatectomy using electron microscopic morphometry. The results show that liver cells of old animals display the same degree of aging even after regeneration, i.e. the surface area and surface density of the rough endoplasmic reticulum remain reduced to the same extent, as observed in the old control group. The regeneration caused a rejuvenation only of the mitochondria showing surface area and surface density of inner membrane + cristae corresponding to the values obtained in the young (1-month-old) animals. The results seem to support the idea that the accumulation of somatic mutations such as a strong reduction of the protein synthetic apparatus, may play an important role in the aging process.

Alterations in the numerical density of perichromatin granules in different tissues during ageing and cell differentiation.

Perichromatin granules (PG) were counted on electron micrographs of different tissues taken from young and old female Wistar rats as well as in the basal and spinous layers of adult human epidermis. A significant age-dependent decrease was observed in the following cell types: large brain cortical cells, cerebellar granular cells, hepatocytes, parotid gland cells. No significant decrease was found in the heart muscle nuclei and in the erythroblasts of identical maturation level. The number of PG decreased during the erythroblast maturation almost to zero, and increased in the cells of spinous layer of human epidermis as compared to the basal cells. There is a very wide variation in the number of PG per nucleus between the different tissues. The correlation between the transcriptional activity, the protein synthesis, ageing and cell differentiation is discussed.

Electron-microscopic morphometric analysis of mouse liver. I. Experimental studies on the morphogenetic significance of the thymus in nude and normal mice

Haired, nude, thymus-grafted nude and haired thymectomized Balb/c-nu mice 2 months of age were studied by electron-microscopic stereology. Each group consisted of 5 animals and a complete morphometric analysis was carried out on their livers. In the absence of the thymus there is a slowing down of the development of the whole organism. Among the liver parameters especially the nuclear ones displayed alterations. Namely, the volume of hepatocyte nuclei increased above the normal level and this phenomenon was reversed by thymus graft into the nude mice. The hepatocyte volume also increased significantly in the surgically thymectomized group, influencing all the morphometric parameters regarding mitochondria and endoplasmic reticulum, when measured per hepatocyte. On the basis of the results obtained, one can conclude that the thymus has a regulatory role in the development of hepatocyte morphology. The findings agree with the biochemical observations demonstrating non-immunological effects of the thymus on cellular development.