Biomedical subjects
M A Brock
Publications and source records attributed to M A Brock.
Interactions of aging and annual rhythms on the recovery of cryopreserved murine splenic lymphocytes.
The physiological status of donor organisms is an often overlooked factor in cryopreservation experiments. Murine splenic lymphocytes exhibit systematic changes in function which are endogenous and influence recoveries of viable and functional frozen-thawed cells over the life span of mice. One of these changes is the decline in the performance of unfrozen cells as organisms age. Superimposed on the age-related decline in lymphocyte functions are circannual rhythms in T- and B-cell mitogenesis, and the properties of these rhythms also change with age. Splenocytes from young, 15-month-old and 23- to 27-month-old C57BL/6 mice were cryopreserved and tested for recovery of mitogenic responses to activation by the T-cell mitogens, phytohemagglutinin and concanavalin A, and the B-cell mitogen, lipopolysaccharide. Tritiated thymidine incorporation by activated, dividing cells was determined after 72 hr of in vitro culture. Seasonal patterns in recovery of viable and functional cryopreserved cells from young mice resembled those of their unfrozen controls (6). By 15 months of age, the responses after freeze-thaw stress decreased to the levels observed for cells obtained from senescent mice, and seasonal patterns were no longer observed. In these middle-aged mice, intracellular changes in lymphocytes that are equivalent to those in senescent animals resulted in irreparable structural-functional injury during cryopreservation.
Seasonal changes in recovery of cryopreserved murine lymphocytes resemble endogenous rhythms of unfrozen cells.
Seasonal changes in the resistance of C57BL/6 mouse splenocytes to cryopreservation stress were expressed in both the recovery of viable cells and the levels of responses of T and B lymphocytes to mitogens in vitro. Single cell suspensions in 10% Me2SO were cooled at 1 degree C/min, the optimum velocity which was determined by using a range of cooling rates during January and May, the months of minimum and maximum recoveries of viable cells, respectively. After rapid thawing and washing, ethidium bromide-fluorescein diacetate staining delineated viable and nonviable cells. Cultures containing 0.5 X 10(6) viable cells were stimulated with the T lymphocyte mitogens, phytohemagglutinin and concanavalin A, and the B lymphocyte mitogen, lipopolysaccharide. Tritiated thymidine was added to each culture for the last 18 hr of the incubation period, and its incorporation by activated dividing cells was determined. Recoveries of viable cells were high from March through July and then declined to minimum levels in January and February. During the seasons of low recoveries, greater numbers of cells lysed in response to the freeze-thaw cycle. Activation of both T and B lymphocytes by mitogens was maximal in the spring and summer and then declined to only 40% of unfrozen control levels in October. The patterns of activation resembled those of the previously documented endogenous seasonal rhythms in levels of blastogenesis of unfrozen cells. These seasonal differences in cryopreservation properties of lymphocytes from inbred mice living under constant conditions reinforce the previously reported endogenous annual rhythmicity in cellular functions.
Age-related changes in circannual rhythms of lymphocyte blastogenic responses in mice.
Blastogenic responses to T- and B-lymphocyte mitogens were tested in suspensions of splenocytes from 15- and 24- to 28-mo-old C57BL/6 mice and compared with analogous responses in young animals. The mice were housed under constant environmental conditions with alternating light-dark cycles (LD 12:12). Single cell suspensions were cultured in vitro with mitogens, and the induced incorporation of tritiated thymidine by dividing cells was determined. Increases in periodicity of responses to concanavalin A and phytohemagglutinin by T cells and to lipopolysaccharide by B cells and lower mean levels of activation characterized rhythms in cells from 15-mo-old and senescent mice compared with young animals. Amplitudes of the rhythms were unchanged at 15 mo, but by 24 mo of age rhythmic responses of T but not B cells were damped. The separable effects of age on expression of circannual rhythms by T and B lymphocytes suggest another mechanism for imbalance in the immune system. Phases of depressed responses that are extended for several months in populations of older mice could provide increased opportunities for environmental assaults.
A microprocessor-controlled rate controller for use in cryopreservation.
Explore the source record for details and available documents.
Comparative ultrastructure of chick fibroblasts in vitro at early and late stages during their growth span.
Explore the source record for details and available documents.
Temporal order vs. variability in activation of lymphocytes from aging mice.
Changes in patterns of circannual rhythms in the in vitro activation of splenic lymphocytes from aging C57BL/6 mice may account both for reports of conflicting results in tests of immune function and for the assumed increase in variability in data obtained using cells from older mice. Single cell suspensions from 4-6, 15 and 24-28-month-old mice living in presumably constant environments were cultured in vitro with mitogens, and the incorporation of tritiated thymidine by dividing cells was determined. Free-running periods of T and B lymphocyte rhythms of blastogenic responses increased in cells from both older groups; amplitudes of the T but not B cell rhythms were reduced in senescent mice. This results in continuously changing phase relationships of both T and B cell rhythms, without consistently higher levels of activation of cells from young animals, thus obscuring at intervals the age-related declines in function. Because data were collected for several years, it was clear from the patterns of the rhythms and also the combined data that the greatest ranges in levels of activation ("variability") characterized T and B cells from the youngest mice rather than older animals.