The Physiologic Stress Response During Mediation

Published in cooperation with the American Bar Association Section of Dispute Resolutio

Infant temperament predicts life span in female rats that develop spontaneous tumors

Abstract In a recent study, we found that male rats that minimally explored a novel environment as infants died significantly faster than their more exploratory brothers. At death, these males had various complex pathologies, precluding identification of specific hormonal mechanisms underlying adult disease progression and mortality. To minimize the variance of disease processes at the end of life, we conducted a longitudinal study with female Sprague-Dawley rats prone to high rates of spontaneous mammary and pituitary tumors. For females that developed either mammary or pituitary tumors, those that had been neophobic (least exploratory) as infants died approximately 6 months earlier than their neophilic (most exploratory) sisters. In the case of mammary tumors, both benign and malignant, neophobic females developed palpable tumors earlier than neophilic females, whereas the interval between first palpation and death was the same for all females, indicating psychosocial regulation of early rather than later stages of the disease. Neophobic females' ovarian function aged more rapidly than their neophilic sisters. Concomitantly, they had lower corticosterone responses to restraint in late adulthood, ruling out high estrogen or corticosterone levels during senescence as causal factors in their accelerated mortality. During puberty, when mammary tissue is proliferating and differentiating, neophobic females experienced more irregular cycles with prolonged "luteal" phases, suggesting a role for prolactin, prolonged progesterone and fewer estrogen surges during this sensitive period for mammary tumor risk. Thus, we identified prolactin, estrogen, progesterone and possibly corticosterone dynamics as candidates for neuroendocrine mechanisms linking infant temperament with onset of adult neoplastic disease. © 2006 Elsevier Inc. All rights reserved. Keywords: Temperament; Personality; Mammary tumors; Pituitary tumors; Reproductive cycles; Glucocorticoid dynamics; Longevity; Sibling differences Stable behavioral traits (e.g. temperament, personalities) are often associated with specific hormonal profiles (e.g. gonadal and/or adrenal function). Given the stability of certain behavioral/endocrine traits and the known costs and benefits of certain hormonal profiles Given the well-established bidirectional influence of ovarian steroids, prolactin and glucocorticoids on behavior and tumor cells, we focused on individual differences in the development of spontaneous tumors in a rodent model. Hormones have a wide array of influences on tumor development, with different effects depending on the kind of tumor, the stage of tumor development and the timing and duration of hormonal exposure. Tumor initiation may be regulated by both ovarian steroids and glucocorticoids. Dexamethasone decreases apoptosis in mammary cancer cells in vitro, increasing risk of spontaneous mutations and, if operable in vivo, accelerating spontaneous tumor Hormones and Behavior 50 (2006) initiation In a recent life span study of Sprague-Dawley rat brothers, we found that those reluctant to explore a new environment in infancy (i.e. neophobic) sustained that temperament into adulthood, had elevated corticosterone responses to novelty in adulthood and died earlier than their more exploratory brothers Much of the above work on mammary tumors has been done in vitro or with induced tumors or xenographs. Here, we tested the hypothesis that a neophobic temperament in infant females increases mortality rate as it did in males and extended the hypothesis to the onset and progression of spontaneous mammary tumor formation. We compared the following processes for females identified as neophobic or neophilic during infancy: frequency of spontaneous mammary and pituitary tumors, age when mammary tumors were first palpable, natural life span of females with tumors, ovarian function from late puberty to reproductive senescence and glucocorticoid responses during senescence. To minimize genetic variance among temperament types, we studied within family differences. In summary, we address the following questions: (1) is exploration of a novel environment a stable trait over the female life span? (2) Do infant female behavioral responses predict mortality rates in rats afflicted with spontaneous mammary or pituitary neoplasias? (3) Are ovarian function and/or glucocorticoid dynamics associated with infant temperament, providing potential mechanisms for differential disease progression and/or mortality rates among temperament types? If Sprague-Dawley sisters are like their brothers, we expect those with a neophobic temperament to have elevated glucocorticoid responses to novelty Methods Overall protocol Eighty-one female pups were selected from 14 independently bred SpragueDawley litters (parents from Charles River Laboratories, Wilmington, Massachusetts; selection criteria for exploratory temperament described below). Six sisters were selected for within-family comparisons because of known between-family differences in behavior, corticosterone responses and life span (Cavigelli and McClintock, unpublished data). Females lived throughout their lives in solid bottom plastic cages (43.5 × 23.5 × 20.5 cm) on a 14L:10D lighting schedule (lights on at 20:00 h) with food and water ad libitum. Cages were cleaned twice a week. Females were not allowed to breed. Until 22 days of age, females lived with their mother and littermates. Their mothers and brothers were then removed, and they continued living with their sisters until 27 days. As part of another study on peripubertal social experiences, housing conditions were manipulated during 28-46 days of age; 1/3 lived with two sisters, 1/3 with two unfamiliar females and 1/3 alone. This manipulation did not affect variables examined in this report and is therefore not discussed further. At 47 days of age, all females were housed in groups of 3 sisters per cage for the rest of their lives. Each trio included one low exploration, one intermediate exploration and one high exploration sister (based on testing at 20 days described below; trios were counterbalanced for peripubertal social experience). Daily vaginal cytology samples collected from each female from late-puberty through reproductive senescence were used to analyze ovarian cycle phase durations Temperament: behavioral response to a complex novel environment We measured rats' willingness to explore a complex novel environment at 20 days of age and again at 11 months of age. The first test was conducted just prior to typical weaning and moving above ground in the field For behavioral coding, nine square areas (940 or 1655 cm 2 for infant or adult arena) were defined by a 3× 3 grid. Exploration in the arena was quantified by the number of times a rat walked from one of the areas to an adjacent area (locomotion score). In a confirmatory factor analysis, this movement was associated with other exploratory behaviors in infancy, including sniffing and touching the objects, and in late adulthood with sniffing and climbing on them. It was not associated with escaperelated behavior such as inspecting or rearing up on the walls of the enclosure. Thus, the locomotion score in this arena probably reflects exploration and not high levels of undirected motor activity. Selection criteria Locomotion scores (our index of exploration) varied within and among litters (within litter ranges for the extremes: 0-34 vs. 0-76 squares entered). Females were categorized and selected based on their performance relative to the mean performance of all females within their family. The two most active sisters were identified as 'neophilic', the two least active were identified as 'neophobic' and those with values closest to the family mean as 'intermediate'. These categories are slightly different from those used in the previous study of males, in which we excluded the least active (i.e. nonresponding) males from each litter because they weighed less than their littermates. In this previous study, the male pups that showed intermediate levels of activity in the test arena were identified as 'neophobic' since the least exploratory were excluded. Pathology and life span Females were allowed to live their natural life span. Health of aging females was closely monitored by researchers and the institutional veterinarian. To preclude suffering, 47 of the 81 females were sacrificed when they displayed symptoms indicating that they were within 1 week of death Mammary tumors To determine age of mammary tumor detection, we used a comprehensive technique for repeatedly palpating all mammary tissue; three checkers could reliably detect mammary neoplasia as small as 3 mm in diameter. Regular checks began just prior to 10 months of age and were repeated every 2-3 weeks until death. We defined onset of tumor detection as the first date at which a growth was reliably palpable. Checkers were not informed as to which females had been palpated with tumors in the previous weeks. At necropsy, the ovoid mammary tumors were well encapsulated and easily excised. Mean (±SEM) mammary tumor size across all females was 78.4 ± 15.6 g, approximately 20% of the mean aged female body weight (i.e. approximately 400 g). Random samples revealed that they were histologically complex, with multiple tumor subtypes within a single solid tumor. Tumor diagnoses included malignant cancer (e.g. in situ ductal carcinoma (comedo and cribiform), invasive ductal carcinoma and carcinosarcoma) as well as benign tumors (e.g. fibroadenoma, lactacting adenoma and papillary cystadenoma). Histological diagnosis was validated by concordance of two surgical pathologists specializing in breast cancer pathology in the Department of Pathology at the University of Chicago. Pituitary tumors Necropsies were performed to determine presence of a pituitary tumor and to excise mammary tumors. Pituitary tumors were defined by pituitary gland enlargement (0.263 ± 0.017 g, as compared to normal range of pituitary gland: 0.009-0.074 g), extensive vasculature and confirmed histologically by concordance of the two surgical pathologists. Other gross morphological abnormalities were also noted indicating disease processes at the time of death (i.e. enlarged or abnormally small organs and other tumors). Hormonal function Ovarian cycles from puberty through reproductive senescence Ovarian cycles were monitored daily from 55 to 450 days to measure cycle length and estimate the number of days each female spent in estrogenized or nonestrogenized "luteal" phases of each cycle (e.g. proestrus and estrus with cornified vaginal epithelium cells and no leukocytes followed by metestrus and diestrus with leukocytes and few cornified cells). To assess ovarian cycles, cells of the vaginal wall were collected by saline lavage during the middle of the dark (active) period. Samples were quantified according to the relative proportion of three cell types: cornified epithelial cells, nucleated epithelial cells and leukocytes. Rats were in the post-ovulatory ('luteal') phase when leukocytes represented more than 20% of the cells in a sample for two or more consecutive days Given potential sensitive periods for sex steroid influences on mammary tumorigenesis and progression At each age, we determined how many females were in one of four ovarian states: regular cycles, constant estrus, irregular cycles and persistent diestrus or anestrus (coding methods in Corticosterone response to restraint stressor at late middle age To determine if late adult corticosterone reactivity, following tumorigenesis, differed between neophobic and neophilic females, we collected repeated blood samples following brief physical restraint at 15 months of age. Restraint consisted of 30 min in a Plexiglas tube, which is considered a psychological stressor for these animals. Tube diameters were adjusted for individual female body weights, such that none was constricted. Blood sampling was performed at the end of the rats' active period (starting between 20:00 and 21:00 h). At this time, females were removed from their home cage in a pre-determined randomized order, carried to an adjacent room where blood sampling was conducted, placed into a restraint tube and bled within 4 min of home cage removal. After the first sample, the rats remained in the tube for 30 min, at which point a second sample was collected and the rats removed from the tube and placed in a clean solid-bottom plastic cage. Repeated samples were collected at 60, 90 and 150 min from the initial sample. The first sample was intended to capture unstimulated corticosterone concentrations. The 60-min sample, collected 30 min from the end of physical restraint, was timed to capture the near-maximal corticosterone response to this challenge. And the 150-min sample was collected to capture recovery levels-how quickly the corticosterone response is terminated and corticosterone removed from the system after a 30-min novelty. Females were processed on nine sequential evenings, with equal numbers of neophobic, intermediate and neophilic sisters bled each night and sampling time balanced across female temperaments. Blood was collected from the tip of the tail. Approximately 3 mm of the tail tip was removed with a scalpel blade and bleeding induced by tail palpation. Blood was collected into EDTA tubes (Microtainers from Becton Dickinson and Company) and kept on ice until centrifuged to collect plasma. Plasma was diluted (1:200 with assay kit diluent) and frozen at −80°C until assayed. Corticosterone was measured using a commercial radioimmunoassay kit (Rat and Mice Corticosterone kit, MP Biomedicals). All diluted samples were run in duplicate across nine assays. Sisters were included on the same assay. Intraassay and inter-assay variability for a low and high control were 9.8 and 8.7% and 13.3 and 12.7%. Consulting veterinarians concurred that cause of death in aging rats likely involves a complex interaction of multiple pathologies, and thus determining specific cause of death for all animals was beyond the scope of this study. To simplify analysis of the ongoing disease processes at the end of life and control for the two major types of neoplastic processes at the time of death, life span analyses were conducted within the two groups of females that had either mammary (N = 18) or pituitary tumors (N = 18) at death. Some of their sisters had both a pituitary and mammary tumors (N = 38), while others had rare types of pathology-e.g. invasive thoracic and/or abdominal cavity tumors (N = 5), cerebral hemorrhage (N =1), no detectable pathology at the time of death (N =1). To assess ovarian cycle patterns for each temperament, we compared relative representation in the four ovarian stages among neophobic, intermediate and neophilic females with χ 2 tests. To determine if baseline, reactivity or recovery levels of circulating corticosterone differed among the three temperament types, we used ANOVAs to compare corticosterone concentration at each sampling time, with post hoc paired t tests to compare neophobic and neophilic values. Because families had different overall corticosterone profiles, mean family corticosterone concentration at each time point was used as a covariate. Several females had died or were too ill for blood collection at 15 months; because temperament types were initially balanced within families, analyses of corticosterone data were limited to those sister trios in which all females were present for the blood collection (N = 54). Because estrous cycle profoundly affects corticosterone levels during the acrophase of the daily rhyth

Olfactory Thresholds of the U.S. Population of Home-Dwelling Older Adults: Development and Validation of a Short, Reliable Measure

Current methods of olfactory sensitivity testing are logistically challenging and therefore infeasible for use in in-home surveys and other field settings. We developed a fast, easy and reliable method of assessing olfactory thresholds, and used it in the first study of olfactory sensitivity in a nationally representative sample of U.S. home-dwelling older adults. We validated our method via computer simulation together with a model estimated from 590 normosmics. Simulated subjects were assigned n-butanol thresholds drawn from the estimated normosmic distribution and based on these and the model, we simulated administration of both the staircase and constant stimuli methods. Our results replicate both the correlation between the two methods and their reliability as previously reported by studies using human subjects. Further simulations evaluated the reliability of different constant stimuli protocols, varying both the range of dilutions and number of stimuli (6–16). Six appropriately chosen dilutions were sufficient for good reliability (0.67) in normosmic subjects. Finally, we applied our method to design a 5-minute, in-home assessment of older adults (National Social Life, Health and Aging Project, or NSHAP), which had comparable reliability (0.56), despite many subjects having estimated thresholds above the strongest dilution. Thus, testing with a fast, 6-item constant stimuli protocol is informative, and permits olfactory testing in previously inaccessible research settings

Social Isolation and Adult Mortality: The Role of Chronic Inflammation and Sex Differences

The health and survival benefits of social embeddedness have been widely documented across social species, but the underlying biophysiological mechanisms have not been elucidated in the general population. We assessed the process by which social isolation increases the risk for all-cause and chronic disease mortality through proinflammatory mechanisms. Using the 18-year mortality follow-up data (n = 6,729) from the National Health and Nutrition Examination Survey (1988–2006) on Social Network Index and multiple markers of chronic inflammation, we conducted survival analyses and found evidence that supports the mediation role of chronic inflammation in the link between social isolation and mortality. A high-risk fibrinogen level and cumulative inflammation burden may be particularly important in this link. There are notable sex differences in the mortality effects of social isolation in that they are greater for men and can be attributed in part to their heightened inflammatory responses

A Human Chemosignal Modulates Frontolimbic Activity and Connectivity in Response to Emotional Stimuli

Evidence suggests the putative human pheromone 4,16-androstadien-3-one (androstadienone), a natural component of human sweat, increases attention to emotional information when passively inhaled, even in minute amounts. However, the neural mechanisms underlying androstadienone’s impact on the perception of emotional stimuli have not been clarified. To characterize how the compound modifies neural circuitry while attending to emotional information, 22 subjects (11 women) underwent two fMRI scanning sessions, one with an androstadienone solution and one with a carrier control solution alone on their upper lip. During each session, participants viewed blocks of emotionally positive, negative, or neutral images. The BOLD response to emotional images (relative to neutral images) was greater during exposure to androstadienone in right orbitofrontal and lateral prefrontal cortex, particularly during positive image blocks. Androstadienone did not impact the response to social images, compared to nonsocial images, and results were not related to participant sex or olfactory sensitivity. To examine how androstadienone influences effective connectivity of this network, a dynamic causal model was employed with primary visual cortex (V1), amygdala, prefrontal cortex, and orbitofrontal cortex on each side. These models indicated that emotional images increased the drive from V1 to the amygdala during the control session. With androstadienone present, this drive to amygdala was decreased specifically for positive images, which drove downstream increases in orbitofrontal and prefrontal activity. This evidence suggests that androstadienone may act as a chemical signal to increase attention to positively valenced information via modifications to amygdala connectivity

Olfactory function and the social lives of older adults : A matter of sex

Social factors play a critical role in a panoply of health processes, including, as recently demonstrated, olfaction. Here, we investigated sex-dependent differences in the relationship between social lives and ability to identify odors in a large sample of nationally representative older US adults (n = 3005, National Social Life and Aging Project (NSHAP)). Social life was measured by the number of friends and close relatives as well as frequency of socializing. We here confirm the association between social lives and olfactory function and extend the notion by showing specifically that olfactory identification ability is modulated by sex in older adults. The connection between olfactory performance and social lives could reflect social modulation of aging as has been reported for health in general. Future studies are necessary to elucidate the precise mechanisms underlying this association and sex difference