Age-related telomere shortening occurs in lens epithelium from old rats and is slowed by caloric restriction.

We have investigated whether the average relative telomere length of lens epithelial cells (LECs) from brown Norway rats decreases with the age of the donor animal, and whether chronic caloric restriction (CR) of the rats delays the telomere shortening. Our previous studies have demonstrated that clonal proliferative potential of rodent LECs as well as the in vivo rate of DNA synthesis decreases with age and that this decrease is slowed by chronic lifelong caloric restriction (CR). In order to determine if telomeric shortening might be involved in this loss of proliferative potential, we examined relative telomeric lengths in young, old ad lib fed (AL), and old calorically restricted (CR) brown Norway rats. We used fluorescence in situ hybridization with a peptide nucleic acid probe (PNA) complementary to the telomeric repeat sequence to quantitate relative telomere lengths in LECs in lens sections (TELO-FISH). Control experiments demonstrated that the PNA probe binding was restricted almost entirely to the terminal portions of the rat chromosomes with less than 5% bound at interstitial sites in typical metaphase spreads. The relative telomere lengths of interphase human fibroblast standards, as determined by TELO-FISH, were in good agreement with terminal restriction fragment analyses of the same standards and with literature values for rat cells. The average telomere lengths of interphase nuclei in the old AL rat LECs were found to be 21% shorter than paired young AL controls (P < 0.01 by Wilcoxian signed rank test). The calorically restricted old rats had less telomere erosion (12%) than the old AL group (P < 0.05). Although it is not clear whether such moderate telomeric erosion can limit cell division in rodent LECs, the telomeric shortening correlated well with previous studies demonstrating reduced clonal, replicative potential, and reduced rates of in vivo DNA replication in LECs from old rodents and a delay in this attenuation in animals on chronic CR.

The effect of high and very low fluorescent light exposure levels on age-related cataract in a pigmented mouse strain.

This study examined the effect of fluorescent light on the timing and severity of age-related cataracts in a fully pigmented mouse strain, the (C57BL/6 x C3H)F1, that normally develops slowly progressing age-related cataracts only beyond middle age. Two groups of 56 animals each were exposed, respectively, either to a daily range of 66-222 foot candles (FC) or to 1 FC of standard fluorescent lighting for a period beginning at 5 weeks of age and ending at 33.5 months (by which time approximately 65% of the colony had died). Contrary to previous reports involving albino rats or mice and a strain of pigmented but cataract-prone transgenic mice, the two groups of animals in this experiment did not differ for cataract development in time of first occurrence, rate of advancement, or degree of severity. It was concluded that genetic predisposition, based on levels of oxidative free radical production vs antioxidant enzyme and repair enzyme protection in the lens, was probably the major factor governing the rate and degree of age-related cataract development in these animals. The effect of relatively intense life-long fluorescent light exposure was so minimal as not to be manifested in this strain of mice under the conditions of this experiment. Remarkably, maintaining the one group of mice in semi-darkness from 5 weeks of age to beyond their mean lifespans did nothing to delay or reduce the incidence or severity of their age-related cataracts.

Enhanced cell proliferation and biosynthesis mediate improved wound repair in refed, caloric-restricted mice.

Aged mice that have undergone long-term caloric-restriction (CR) have improved health and enhanced longevity in comparison to aged mice that are ad libitum-fed (AL). However, caloric-restriction does not benefit the impaired wound healing of aged mice. To test the hypothesis that CR mice have the capacity for enhanced wound repair, but require a short-term period of additional nutrient intake to show this advantage, we assessed wound healing in CR mice that had been refed (RF) an ad libitum diet for 4 weeks prior to wounding. Two strains of AL young (Y AL) (4-6 months), AL middle-aged (M AL) (15-17 months), and three different, matched cohorts of old mice (O) (30-33 months): O AL, O CR, and O RF were studied. Two full-thickness 4 mm diameter punch biopsy skin wounds were created on the dorsum of each mouse. Animals were sacrificed and wounds were harvested at 1,2,3,5, and 7 days post-wounding. Repair of wounds was slower in O AL and O CR mice compared to Y AL and M AL animals. In contrast, the O RF mice healed similarly to that of the Y AL and M AL mice, as assessed by measures of wound area and histologic criteria. O RF mice demonstrated enhanced synthesis of type I collagen mRNA in comparison to O AL and O CR mice. A greater number of endothelial cells and fibroblasts at the wound edge of the O RF mice exhibited replication in vivo as measured by uptake of BrdU. O RF mice had higher levels of insulin-like binding protein 3 (IGFBP-3). Furthermore, fibroblasts derived from the explant of the punch biopsy of O CR mouse skin revealed enhanced proliferation and contraction in vitro, in comparison to fibroblasts from the O AL mice. In conclusion, O RF mice demonstrate an enhanced capacity to undergo wound repair in comparison to O AL mice. This effect appears to be mediated, in part, by enhanced cell proliferation, contraction, and collagen biosynthesis. In addition, short-term refeeding induced an increase in the serum level of IGFBP-3, the major binding protein for IGF-1. These data confirm that cells from O CR animals have a preserved proliferative, biosynthetic, and contractile capacity, but that an adequate source of nutrients is necessary to demonstrate this advantage in wound healing.

Caloric restriction: conservation of in vivo cellular replicative capacity accompanies life-span extension in mice.

In male mice of a long-lived hybrid strain (B6D2F1), long-term 40% caloric restriction (CR) extended both mean and maximum life spans by 36 and 20%, respectively, over that of ad libitum fed (AL) controls. Measurements of entry into S-phase were made in vivo of six different cell types in five different organs using 2-week exposures to BrdU. The labeling index (L.I.) in all organs studied was lower in young CR mice than in young AL fed mice. In most cases, the L.I. in AL mice fell to the levels of that in the CR mice by 13 months of age, and the two groups then remained so through old age. However, when the L.I. was measured in old CR mice which had been placed on the AL diet for a period of 4 weeks (this was termed refeeding (RF), it was found to be above that of similar age AL or CR mice and almost at the level of young AL mice. This was still true, but to a lesser degree, in a repeat study using an 8-week period of RF. In a separate but parallel in vitro study (companion paper, this volume), the superiority of CR over AL for retention of cellular replication capacity was confirmed by clone size distribution measurements made in several cell types in mice of several age groups. These results indicate that: (1) the rate of cell replication in AL diet mice diminishes greatly by early middle age in all organ sites studied and then plateaus or declines much more slowly; (2) CR broadly preserves in vivo cellular replicative capacity but often requires the energy levels provided by a switch to AL feeding to demonstrate this late in life; (3) accordingly, the replicative deficit in AL fed mice appears to be cumulative and is significant only in old age. The mechanism(s) involved is yet to be discovered but may be related to, or even the same as, that which extends life spans in CR animals. Correspondingly, and with corroborative data from our in vitro companion study, (W. R. Pendergrass et al., 1995. Exp. Cell. Res. 217, 309-316), we suggest that cell populations sustain an accrual of biochemical damage or physiological alterations which increasingly limit their replicative capacity as the animal ages, and that CR reduces the accrual of this damage.

Dissecting the hematopoietic microenvironment. IX. Further characterization of murine bone marrow stromal cells.

We have previously shown the adherent nontransformed, nonimmortalized murine bone marrow stromal cell (BMSC) population to consist of phagocytic macrophage and endothelial-like cells and nonphagocytic fibroblasts. Both colonial and near confluent growth of each cell type was obtained following magnetic bead separation, subsequent passaging, and sustained culture with fetal bovine serum and cytokines. Monoclonal antibody staining of antigenic determinants was used to characterize the phenotype of the stromal cell population in primary platings of murine colony-forming unit fibroblast and long-term bone marrow cultures. The antibodies MECA-99, MECA-32, and MJ7-18, raised against murine vascular endothelial antigenic determinants, and von Willebrand's factor all stained selectively for the rounded endothelial-like cells. Endothelial-like cells as well as macrophages expressed the myeloid surface antigens F4/80, 7/4, and Mac-1 under our culture conditions. The cytoskeleton of the stromal fibroblasts in culture was shown to express smooth muscle-specific actin isoforms, as evidenced by positive staining of stress fibers for alpha smooth muscle-1, CGA-7 (alpha/gamma isoforms), and HHF-35 (recognizes all muscle-specific actins). Under culture conditions, stromal fibroblasts were also found to be positive for a polyclonal smooth muscle myosin. It was found that these fibroblasts stained for collagens type I, III, and IV in our cultures. Although collagen type IV is considered a by-product of endothelial cells, endothelial-like cells in our cultures did not stain for any of the collagen types. We propose a classification listing for murine BMSCs as macrophages, endothelial-like cells, and fibroblasts that display smooth muscle-like characteristics in culture.

Simultaneous concept analysis of spiritual perspective, hope, acceptance and self-transcendence.

This paper seeks to clarify four previously elusive concepts of concern to nurses: spiritual perspective, hope, acceptance and self-transcendence. Simultaneous concept analysis (SCA), a method developed by the authors, was used to generate a process model of antecedents, critical attributes and outcomes of all four concepts. This resulted in refined theoretical definitions for each concept. Interrelationships that became apparent among the concepts are discussed and implications of these interrelationships for theory and measurement are presented.

A method to establish pure fibroblast and endothelial cell colony cultures from murine bone marrow.

Studies on the effect of the microenvironment on hematopoiesis would benefit from the availability of pure populations of nontransformed cells of each of the stromal cell types. The adherent murine bone marrow stromal cell population in this study consisted of fibroblasts, endothelial cells, and macrophages. Fibroblasts were segregated from the phagocytic endothelial cells and macrophages by allowing the phagocytic cells to ingest magnetic beads, with subsequent exposure to a magnetic field, effecting cell separation. Pure colony cultures of fibroblasts and endothelial cells were formed by varying the bead-to-cell ratio and incubation period of the cells. For complete purification of the fibroblasts, subsequent passaging was also necessary. Near confluent growth of each type was obtained with subsequent passages and sustained culture. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to enhance endothelial cell growth. We were not able to obtain pure populations of bone marrow macrophages in near confluent culture. The three cell types were identified by cellular morphology, acid and alkaline phosphatase staining, binding with the lectins Ulex europaeus and Bandeiraea simplicifolia, and the capacity to stain for the factor VIII-related antigen (von Willebrand's Factor).

Intervention factors for promoting adjustment to nightwork and shiftwork.

This chapter reviews a broad range of factors that, if controlled, might promote adaptation to nightwork, shiftwork, and extended workshifts. Systematic study has begun in four of the areas reported here: work schedule design, napping, bright light stimulation, and drugs. Physical activity, ambient temperature, diet, and individual behaviors have been studied only superficially.

Neurochemical and operant behavioral studies of a strain of alcohol-preferring rats.

The levels of serotonin (5-HT), 5-hydroxyindoleacetic acid, tyrosine (TYR), norepinephrine (NE), acetylcholine, GABA, glutamate (GLU), aspartate (ASP), alanine, glycine (GLY) and taurine were measured in the CNS of adult male rats selectively inbred to the F8 generation for alcohol preference (P) and non-preference (NP). With respect to the values found in the NP group, higher levels of 5-HT, GABA, GLU and GLY and lower levels of ASP were found in the diencephalon-mesencephalon (D-M) and higher levels of NE were found in the telencephalon (TEL) of the P group. The animals in the P and NP strains were further subdivided into two additional groups, one given only H2O (W) and the second given 10% ethanol (A) during a one week period, thereby producing four groups (NP-W, NP-A, P-W and P-A). With these conditions, the level of (a) TYR in the D-M was higher in the P-A and NP-A animals than in the P-W and NP-W groups respectively; (b) 5-HT in the TEL was higher in the NP-A group than NP-W group; and (c) GABA in the TEL was higher in the P-A than P-W animals. No differences were observed in the cerebellum between the two strains or between the subgroups within each strain. The present study also demonstrated that the P animals will work in an operant situation to obtain 10% ethanol, even when H2O is freely available, and will voluntarily bar-press up to 6-7 times for each ethanol reinforcement.

Effects ofp-chlorophenylalanine on the metabolism of serotonin from 5-hydroxytryptophan.

The effects ofD,L-ϱ-chlorophenylalanine methyl ester (PCPA-methyl ester) and two of its metabolites, 2-(ϱ-chlorophenyl)-ethylamine (PCPEA) and ϱ-chlorophenylacetic acid (PCPAA), on the metabolism of serotonin (5-HT) fromD,L-5-hydroxytryptophan (5-HTP) ware studied in vitro and in vivo using the telencephalon and brainstem of the rat. For in vivo studies and some in vitro experiments, rats were injected with either 100 mg/kg PCPA-methyl ester or saline alone on days 1, 2, and 3, and were killed on day 15. When the in vivo metabolism of 5-HT was to be studied, the saline group and the PCPA group of animals were injected with 75 µg/kg [(3)H]D,L-5-HTP 20 min before sacrificing. With respect to the values found for the saline-injected animals, the specific activity (S.A.; dpm/nmol) of 5-HIAA was significantly greater in the telencephanol and brainstem of the animals injected with PCPA-methyl ester. The S.A. of 5-HTP was the same in both groups; the S.A. of 5-HT was lower in the telencephalon of the PCPA group than in the saline group; in the brainstem, there was no difference. In both the saline- and PCPA-injected animals, the S.A. of 5-HIAA was greater than the S.A. of 5-HT. There was no difference between the saline- and PCPA-injected animals with regard to: (1)L-5-HTP decarboxylase activity; (2)L-5-HTP-induced release of [(3)H]5-HT in vitro from crude nerve ending fractions (P2); or (3) in vitro uptake of [(3)H]D,L-5-HTP and its conversion to [(3)H]5-HT using the P2 fraction. In vitro studies demonstrated that the PCPEA could directly cause a large increase in the release of [(3)H]5-HT from the P2 fraction, whereas PCPA and PCPAA had little or no apparent effect. The data were interpreted to suggest that in the telencephalon of the animals treated with PCPA-methyl ester, there was a higher turnover of 5-HT than was found in the saline-treated group.