Comparative study of spermatogonial survival after x-ray exposure, high LET (HZE) irradiation or spaceflight.

Spermatogonial cell loss has been observed in rats flown on Space Lab 3, Cosmos 1887, Cosmos 2044 and in mice following irradiation with X-ray or with high energy (HZE) particle beams. Spermatogonial loss is determined by cell counting in maturation stage 6 seminiferous [correction of seminferous] tubules. With the exception of Iron, laboratory irradiation experiments (with mice) revealed a similar pattern of spermatogonial loss proportional to the radiation dose at levels less than 0.1 Gy. Helium and Argon irradiation resulted in a 5% loss of spermatogonia after only 0.01 Gy exposure. However, significant spermatogonial loss (45%) occured at this radiation level with Iron particle beams. The loss of spermatogonia during each space flight was less than 10% when compared to control (non-flight) animals. This loss, although small, was significant. Although radiation may be a contributing factor in the loss of spermatogonia during space flight, exposure levels, as determined by dosimetry, were not significant to account for the total cell loss observed.

Ultrastructure of phagocytosed Paracoccidioides brasiliensis in nonactivated or activated macrophages.

Transmission electron microscopy was used to study ultrastructures in Paracoccidioides brasiliensis yeast cells after ingestion by nonactivated or cytokine-activated murine peritoneal macrophages. Yeast cells ingested by nonactivated macrophages had typical bi- and trilayered cell walls, plasma membranes, mitochondria, nuclei, vacuoles, etc., which remained intact for 24 h of coculture. In contrast, yeast cells ingested by activated macrophages exhibited abnormal mitochondrial ultrastructures within 4 h of interaction. Subsequent events that occurred were the formation of several clear vacuoles per cell, disintegration of the cytoplasm, and development of empty cells with intact walls. These findings provide, for the first time, insights into stepwise damage to fungal cells by activated macrophages (of particular interest in this instance because of prior evidence that the damage is due to nonoxidative mechanisms) and give possible clues regarding fungicidal mechanisms.

Effects of spaceflight on the spermatogonial population of rat seminiferous epithelium.

Testes from rats flown on Cosmos 1887 were compared with vivarium control and synchronous control samples. The mean weights of flight testes, normalized for weight per 100 g, were 6.4% less when compared with the vivarium controls. Counts of spermatogonia from tissue sections (seminiferous tubules in maturation stage 6) from five animals in each group revealed 4% fewer spermatogonia in flight testes compared with synchronous controls and 11% fewer spermatogonia in flight samples compared with vivarium controls.

Morphological and biochemical examination of Cosmos 1887 rat heart tissue: Part I--Ultrastructure.

Morphological changes were observed in the left ventricle of rat heart tissue from animals flown on the Cosmos 1887 biosatellite for 12.5 days. These tissues were compared to the synchronous and vivarium control hearts. While many normal myofibrils were observed, others exhibited ultrastructural alterations, i.e., damaged and irregular-shaped mitochondria and generalized myofibrillar edema. Analysis of variance (ANOVA) of the volume density data revealed a statistically significant increase in glycogen and a significant decrease in mitochondria compared to the synchronous and vivarium controls. Point counting indicated an increase in lipid and myeloid bodies and a decrease in microtubules, but these changes were not statistically significant. In addition, the flight animals exhibited some patchy loss of protofibrils (actin and myosin filaments) and some abnormal supercontracted myofibrils that were not seen in the controls. This study was undertaken to gain insight into the mechanistic aspects of cardiac changes in both animals and human beings as a consequence of space travel (1). Cardiac hypotrophy and fluid shifts have been observed after actual or simulated weightlessness and raise concerns about the functioning of the heart and circulatory system during and after travel in space (2-4).

Effects of high energy particle (HZE) radiation on the distal lung.

Major sources of concern for manned space travel are the effects of high energy particle (HZE) radiation on various biological systems, and the consequences of major solar activity. To date, considerable attention has been directed toward HZE-induced alterations both on non-dividing systems, such as the retina, cornea and brain, and on dividing systems, such as the gut and testis. This paper is focused on the morphologically detectable late-occurring alterations in the distal lung, and toward a comparison of the changes with those induced by x-irradiation. Briefly, the salient alterations involve an increase in the width of the septal walls and the capillary and alveolar basal laminae, and the irregularity of the luminal surface of the capillaries, as exemplified by the presence of filipodial projections and blebbing. All alterations were focal in their localization, and no cells of any type (e.g., epithelial, endothelial or stromal) appeared to undergo damage, an observation quite unlike the cellular changes induced by x-irradiation.

Cardiac muscle ultrastructure and cyclic AMP reactions to altered gravity conditions.

Morphological and biochemical analyses of heart muscle of rats subjected to microgravity on Spacelab 3 (SL-3) flight and rats born and reared under increased gravity (1.7 G) conditions were compared with 1-G controls. Electronmicroscopic studies showed an increase in the number of lipid droplets and in areas of glycogen storage. Distribution changes of microtubules and cytoskeletal elements from both SL-3 and 1.7-G groups were observed. The high Km cyclic AMP phosphodiesterase activity was lower (P less than 0.05) in SL-3 heart muscle, and low Km activity was lower in 1.7-G males but was unaltered in females. Cyclic AMP-dependent protein kinase (cA-PK) activity was decreased in subcellular fractions of heart muscle of SL-3 animals. Recompartmentalization of cA-PK activity occurred in particulate tissue fraction of 1.7-G animals (70.3% of total for 1.7 G vs. 35.9% for controls). Phosphorylation of endogenous low-mobility proteins increased in SL-3 heart-soluble fractions. Photoaffinity labeling (18 h, 4 degrees C) decreased in type II cA-PK regulatory (R) subunits in both SL-3 and in 1.7-G male heart tissue particulate fractions. The 1.7-G female heart R subunit distribution did not differ from controls. These findings indicate that in heart muscle altered gravity conditions influenced physiological reactions similar to catecholamine-induced receptor-mediated hormonal responses.

Axoglial contacts in the area postrema of the cat: an ultrastructural study.

Axoglial contacts were observed in an ultrastructural study of the area postrema of the cat. According to the disposition of the electron-dense projections attached to the adjoining membranes these contacts were classified as symmetrical or asymmetrical. The axon profiles contained aggregations of clear vesicles randomly distributed or grouped in cluster adjacent to the electron-dense projections. Dense core vesicles were occasionally seen. The neuroglial profiles were either astrocytic or ependymoglial in nature. The astrocytes showed a clear cytoplasm, polymorphous vesicles, mitochondria, glycogen granules, and bundles of filaments. The ependymal cells, in contrast, had a more electron-dense and granular appearance, tubular structures, irregular vesicular formations, profiles of smooth reticuloendoplasm, and filaments grouped in bundles or isolated in the cytoplasm. The possibility that these contacts might play a role in the chemical transfer from neurons to glial cells is discussed on the basis of existing biochemical data.

Long term effects of low doses of 56Fe ions on the brain and retina of the mouse: ultrastructural and behavioral studies.

Eight month old male C57BL6 mice were exposed without anesthesia to whole-body irradiation in circular holders. The mice were tested for behavioral decrements after 0.5 and 50 rads of Fe particle irradiation at 6 and 12 months post irradiation to obtain long term results. A standard maze was used and the animals were timed for completion thereof. A string test also was administered to the mice, testing their ability to grasp and move along a string to safety. The results from animals exposed to 50 rads were significantly different from [correction of fron] control results to p = < .001 in both systems of testing. The hippocampus (believed to be the location of environmental interaction in the brain) and the retina were examined for ultrastructural changes. The ultrastructural changes were similar to those we found in our Cosmos 782, 936 and in our Argon experiments. The mouse data indicate that iron particles were able to induce long term changes in the central nervous system which lead to behavioral impairment.

The effect of high energy (HZE) particle radiation (40Ar) on aging parameters of mouse hippocampus and retina.

Eight month old C57BL6 mice were exposed (head only) to 0.5 rad or 50 rads of Argon particles at the Lawrence Berkeley Radiation Facility, CA. Neuromotor performance was assessed monthly for six months beginning twelve weeks post-irradiation using a "string test". The decline in motor performance was dose-related and none of the animals was able to complete the task after four months of testing. Morphological changes were monitored six and twelve months post-irradiation by light and electron microscopy. The synaptic density in the CA-1 area of the hippocampus decreased six and twelve months after irradiation. The decrease after twelve months was less than after six months. The width of the outer nuclear layer (ONL) of the retina increased with increasing dose. The number of blood vessels between the ONL and the ganglion layer decreased twelve months after irradiation and this area did not show significant accumulation of age pigment.

Fine-structural changes in the midgut of old Drosophila melanogaster.

Variations of the mitochondrial structure with increasing age are of particular interest because the production of ATP is necessary for most cellular activities. Therefore, a senescent decrease of mitochondrial activity could jeopardize cell survival and function. We combined a statistical with a morphological approach to investigate these changes in mitochondrial size and structure of Drosophila melanogaster. The midgut epithelium is of special interest because previous research shows striking aging changes. Investigations of the mitochondria of flies of three age groups (10, 39, and 65 days) reveal an increase in size as well as in its standard deviation. This is linked to the fact that mitochondrial enlargement occurs especially in the luminal region. Ultrastructural studies show that with increasing age the mitochondria become more osmiophilic, their cristae disarranged, cristae-free areas can also be found, and there is an accumulation of homogeneous, granular and lamellar dense bodies. In addition, an accumulation of virus-like particles, fiber-shaped structures, reticular structures, and dense bodies without a surrounding membrane was found.

Response of the seminiferous epithelium of the mouse exposed to low dose high energy (HZE) and electromagnetic radiation.

This study was undertaken to observe the response of a rapidly dividing cell population (spermatogonial) to Helium and Argon ions as compared to x-rays. Low doses (below 100 rads) were used to more nearly simulate radiation encountered during space missions. The methods used proved compatible for both light and electron microscope studies. The average relative biological effectiveness (RBE) for Helium ions is 1, while Argon is twice as effective in killing spermatogonial cells. Part of this mixed population of cells exhibits a higher sensitivity to radiation below 15 rads. Quantitation of the radiation effects by counting the necrotic cells is not feasible because of their rapid removal, therefore all measurements were done using the surviving fraction (S/So) of spermatogonial cells.

Effects of simulated increased gravity on the rate of aging of rats: implications for the rate of living theory of aging.

Ever since Pearl proposed the rate of living theory of aging numerous studies have demonstrated its validity in poikilotherms. In mammals, however, satisfactory experimental demonstration is still lacking because an externally imposed increase of basal metabolic rate of these animals (e.g. by placement in the cold) is usually accompanied by general homeostatic disturbance and stress. The present study was based on the finding that rats exposed to slightly increased gravity are able to adapt with little chronic stress but at a higher level of basal metabolic expenditure (increased 'rate of living'). The rate of aging of 17-mth-old rats that had been exposed to 3.14 times normal gravity in an animal centrifuge for 8 mth was larger than of controls as shown by apparently elevated lipofuscin content in heart and kidney, reduced numbers and increased size of mitochondria of heart tissue, and inferior liver mitochondria respiration (reduced 'efficiency': 20% larger ADP: 0 ratio, P less than 0.01; reduced 'speed': 8% lower respiratory control ratio, P less than 0.05). On the other hand, steady-state food intake per day per kg body weight, which is presumably proportional to 'rate of living' or specific basal metabolic expenditure, was about 18% higher than in controls (P less than 0.01) after an initial 2-mth adaptation period. Finally, though half of the centrifuged animals lived only a little shorter than controls (average about 343 vs. 364 days on the centrifuge, difference statistically nonsignificant), the remaining half (longest survivors) lived on the centrifuge an average of 520 days (range 483-572) compared to an average of 574 days (range 502-615) for controls, computed from onset of centrifugation, or 11% shorter (P less than 0.01). Therefore, these results show that a moderate increase of the level of basal metabolism of young adult rats adapted to hypergravity compared to controls in normal gravity is accompanied by a roughly similar increase in the rate of organ aging and reduction of survival, in agreement with Pearl's rate of living theory of aging, previously experimentally demonstrated only in poikilotherms.

Spontaneous mutant of Blastomyces dermatitidis attenuated in virulence for mice.

A spontaneous mutation occurred in cultures of a virulent strain of Blastomyces dermatitidis during serial passage at 35-37 degrees C. The mutant strain was shown to be 10,000-fold less virulent for mice than the parent strain, and this was independent of the challenge dose, the age of the challenged host, and the route of challenge (although attenuation was greater for pulmonary than for peritoneal challenge). The mutant grows as well as the parent in fungal media or in the presence of murine substrates. Studies of the chronology of pulmonary infection indicate the mutation affects an early event (first week after challenge) in establishment of infection and interaction with host defenses. Scanning electron microscopy showed that the mutant lacks deep irregular surface ridges and grooves present in the parent. It had increased resistance to crystal violet in vitro, increased susceptibility to bile, and could hydrolyze esculin. The latter findings are of interest because they place the mutant in an intermediate position with respect to these properties between the virulent parent and a previously studied avirulent strain. Lipid analyses indicated a marked increase in fatty acids in the mutant. Studies of four B. dermatitidis strains have also associated increasing palmitic acid and phospholipid content with increasing virulence.

Accelerated aging of fasted Drosophila. Preservation of physiological function and cellular fine structure by thiazolidine carboxylic acid (TCA).

Thiazolidine carboxylic acid (TCA) is a natural liver metabolite whose Mg-salt increased lifespan of flies and mice (Miquel and Economos, 1979, Exp. Geront. 14: 279). We studied the physiological and cellular fine structural effects of various concentrations of TCA in the food of male Drosophila. Flies on 0.3% TCA at 27 degrees C had a reduced oxygen consumption rate (about 20% less than controls) at 3 wks of age while their mating capacity and speed of mating were preserved; the flies lived in various experiments 20-30% longer than controls. Apparently TCA improved the metabolic efficiency of the flies (possibly from less "waste" of energy due to improved mitochondrial coupling). However, flies on 0.9% TCA had a reduced mating capacity and lifespan (:toxicity) while at 0.1% TCA was ineffective. A similar dose-response relationship was found in young flies treated with TCA for 1 week and then deprived of food and water, a procedure found to induce accelerated physiological aging. TCA at the 0.3% and 0.6% level reduced the speed of development and the size of the enclosed flies. Electron microscopic investigation of wing muscle showed that 0.3% TCA had a protective effect on cellular fine structure. Though in starved controls (40% survivors after 24 hours of starvation) there was a total absence of glycogen granules, and a striking shrinkage and densification of mitochondria, TCA to a large extent protected muscle cells from these effects of starvation.

Virulence of fungi: correlation of virulence of Blastomyces dermatitidis in vivo with escape from macrophage inhibition of replication in vitro.

Strains of Blastomyces dermatitidis which are virulent (V), attenuated (A), or avirulent (AV) in mice have been identified. Virulence of V has been reported to be associated with its ability to replicate in vivo, causing lethal infections in the lungs or in the peritoneal cavity. We report here that V, but not A or AV, has the ability to replicate in vivo in nonlethal subcutaneous lesions. The possibility that V was able to replicate in vivo by evading primary host defenses was investigated. We studied the interaction of macrophages and strains of B. dermatitidis in vitro. In 24-h cocultures, the replication of all three strains of B. dermatitidis was inhibited by macrophages from: (i) untreated mice (20 to 40%), (ii) mice that had recovered from nonlethal blastomycosis (40 to 60%), (iii) concanavalin A-treated mice (50 to 70%), or (iv) peritoneal exudates elicited by thioglycolate (65 to 80%). However, at 72 h in the first three types of cocultures A and AV remained inhibited, whereas V was not inhibited (0 to 4%). Only the most inhibitory macrophages, those induced by thioglycollate, inhibited V at 72 h, though V was again less inhibited than A or AV at that time. Thus, we show a correlation between virulence of V in vivo and its ability to escape from macrophage inhibition of replication in vitro.

Retinal changes in rats flown on Cosmos 936: A cosmic ray experiment.

Ten rats, five centrifuged during flight to simulate gravity and five stationary in flight and experiencing hypogravity, orbited the Earth. No differences were noted between flight-stationary and flight-centrifuged animals, but changes were seen between these two groups and ground controls. Morphological alterations were observed comparable to those in the experiment flown on Cosmos 782 and to the retinal cells exposed to high-energy particles at Berkeley. Affected cells in the outer nuclear layer showed swelling, clearing of cytoplasm, and disruption of the membranes. Tissue channels were again found, similar to those seen on 782. After space flight, preliminary data indicated an increase in cell size in montages of the nuclear layer of both groups of flight animals. This experiment shows that weightlessness and environmental conditions other than cosmic radiation do not contribute to the observed damage of retinal cells.