The exceptional longevity of the naked mole-rat may be explained by mitochondrial antioxidant defenses.

Naked mole-rats (NMRs) are mouse-sized mammals that exhibit an exceptionally long lifespan (>30 vs. <4 years for mice), and resist aging-related pathologies such as cardiovascular and pulmonary diseases, cancer, and neurodegeneration. However, the mechanisms underlying this exceptional longevity and disease resistance remain poorly understood. The oxidative stress theory of aging posits that (a) senescence results from the accumulation of oxidative damage inflicted by reactive oxygen species (ROS) of mitochondrial origin, and (b) mitochondria of long-lived species produce less ROS than do mitochondria of short-lived species. However, comparative studies over the past 28 years have produced equivocal results supporting this latter prediction. We hypothesized that, rather than differences in ROS generation, the capacity of mitochondria to consume ROS might distinguish long-lived species from short-lived species. To test this hypothesis, we compared mitochondrial production and consumption of hydrogen peroxide (H2 O2 ; as a proxy of overall ROS metabolism) between NMR and mouse skeletal muscle and heart. We found that the two species had comparable rates of mitochondrial H2 O2 generation in both tissues; however, the capacity of mitochondria to consume ROS was markedly greater in NMRs. Specifically, maximal observed consumption rates were approximately two and fivefold greater in NMRs than in mice, for skeletal muscle and heart, respectively. Our results indicate that differences in matrix ROS detoxification capacity between species may contribute to their divergence in lifespan.

The influence of sex and neonatal stress on medullary microglia in rat pups.

NEW FINDINGS: What is the central question of the study? Does neonatal stress, in the form of neonatal maternal separation, influence the maturation of microglial density, morphology and neuronal signalling in medullary regions regulating cardiorespiratory function in rat pups? What is the main finding and its importance? Using Iba-1 immunohistochemistry, we show that neonatal maternal separation augments microglial density and the proportion of cells with an amoeboid morphology in the medulla. Although the current understanding of the effect of early life stress on medullary development is relatively limited, these data show that within this area, microglia are affected by neonatal stress. Microglia could therefore be important effectors in cardiorespiratory disorders resulting from maternal separation. ABSTRACT: Neonatal stress has wide-ranging consequences for the developing brain, including the medullary cardiorespiratory network. In rat pups, the reflexive cardiorespiratory inhibition triggered by the presence of liquids near the larynx is augmented by neonatal maternal separation (NMS), especially in males. Sex-specific enhancement of synaptic connectivity by NMS might explain this cardiorespiratory dysfunction. Microglia influence the formation, maturation, activity and elimination of developing synapses, but their role in the wiring of medullary networks is unknown. Owing to their sensitivity to sex hormones and stress hormones, microglial dysfunction could contribute to the abnormal cardiorespiratory phenotype observed in NMS pups. Here, we first used ionized calcium-binding adapter molecule-1 (Iba-1) immunolabelling to compare the density and morphology of microglia in the medulla of male versus female rat pups (14-15 days old) that were either undisturbed or subjected to NMS (3 h day-1 ; postnatal days 3-12). Neonatal maternal separation augmented the density of Iba-1+ cells (caudal region of the NTS), increased the size of the soma and reduced the arborization area (especially in the dorsal motor nucleus of the vagus). Sex-based differences were not observed. Given that the actions of microglia are regulated by neuronal fractalkine (CX3 CL1 ), we then used western blot analysis to compare the expression of CX3 CL1 and its microglial receptor (CX3 CR1 ) in medullary homogenates from control and NMS pups. Although CX3 CR1 expression was 59% greater in males versus females, NMS had no effect on CX3 CL1 /CX3 CR1 signalling. Given that an amoeboid morphology reflects an immature phenotype in developing microglia, NMS could interfere with synaptic pruning via a different mechanism.

On the origins of sex-based differences in respiratory disorders: Lessons and hypotheses from stress neuroendocrinology in developing rats.

The environment plays a critical role in shaping development and function of the brain. Stress, especially when experienced early in life, can interfere with these processes. In the context of respiratory control, perinatal stress can therefore alter the ability to achieve the "fine-tuning" necessary for proper detection of chemosensory stimuli and production of an adequate motor (respiratory) command. Depending on the timing, intensity, and duration, the detrimental consequences of perinatal exposure to adverse conditions on the respiratory network become manifest at various life stages and can persist into adulthood. During early life, respiratory diseases commonly associated with dysfunction of neural networks include apnea of prematurity (AOP) and cardio-respiratory failure leading to sudden infant death syndrome (SIDS). Sleep disordered breathing (SDB) can occur at various life stages, including adulthood. Regardless of age, a common element of these disorders is their greater prevalence in males. While this sexual dimorphism points to a potential role of sex hormones, our understanding of the neuroendocrine mechanisms remain poorly understood. In addition to their modulatory influence on breathing, gonadal hormones regulate sexual differentiation of the brain. Stress alters these effects, and over the years our laboratory has used various perinatal stress protocols to gain insight into the origins of sex-based differences in respiratory disorders. This review discusses our recent advances with a focus on the sex-specific impact of early life stress on O2-chemoreflex function both in newborn and adult rats. We conclude by discussing the basic principles emerging from this work, potential mechanisms, and clinical relevance.

Sex-Specific Consequences of Neonatal Stress on Cardio-Respiratory Inhibition Following Laryngeal Stimulation in Rat Pups.

The presence of liquid near the larynx of immature mammals triggers prolonged apneas with significant O2 desaturations and bradycardias. When excessive, this reflex (the laryngeal chemoreflex; LCR) can be fatal. Our understanding of the origins of abnormal LCR are limited; however, perinatal stress and male sex are risk factors for cardio-respiratory failure in infants. Because exposure to stress during early life has deleterious and sex-specific consequences on brain development it is plausible that respiratory reflexes are vulnerable to neuroendocrine dysfunction. To address this issue, we tested the hypothesis that neonatal maternal separation (NMS) is sufficient to exacerbate LCR-induced cardio-respiratory inhibition in anesthetized rat pups. Stressed pups were separated from their mother 3 h/d from postnatal days 3 to 12. At P14-P15, pups were instrumented to monitor breathing, O2 saturation (Spo2), and heart rate. The LCR was activated by water injections near the larynx (10 µl). LCR-induced apneas were longer in stressed pups than controls; O2 desaturations and bradycardias were more profound, especially in males. NMS increased the frequency and amplitude of spontaneous EPSCs (sEPSCs) in the dorsal motor nucleus of the vagus (DMNV) of males but not females. The positive relationship between corticosterone and testosterone observed in stressed pups (males only) suggests that disruption of neuroendocrine function by stress is key to sex-based differences in abnormal LCR. Because testosterone application onto medullary slices augments EPSC amplitude only in males, we propose that testosterone-mediated enhancement of synaptic connectivity within the DMNV contributes to the male bias in cardio-respiratory inhibition following LCR activation in stressed pups.

Consequences of maternal omega-3 polyunsaturated fatty acid supplementation on respiratory function in rat pups.

KEY POINTS: Incomplete development of the neural circuits that control breathing contributes to respiratory disorders in pre-term infants. Manifestations include respiratory instability, prolonged apnoeas and poor ventilatory responses to stimuli. Based on evidence suggesting that omega-3 polyunsaturated fatty acids (n-3 PUFA) improves brain development, we determined whether n-3 PUFA supplementation (via the maternal diet) improves respiratory function in 10-11-day-old rat pups. n-3 PUFA treatment prolonged apnoea duration but augmented the relative pulmonary surface area and the ventilatory response to hypoxia. During hypoxia, the drop in body temperature measured in treated pups was 1 °C less than in controls. n-3 PUFA treatment also reduced microglia cell density in the brainstem. Although heterogeneous, the results obtained in rat pups constitute a proof of concept that n-3 PUFA supplementation can have positive effects on neonatal respiration. This includes a more sustained hypoxic ventilatory response and a decreased respiratory inhibition during laryngeal chemoreflex. ABSTRACT: Most pre-term infants present respiratory instabilities and apnoeas as a result of incomplete development of the neural circuits that control breathing. Because omega-3 polyunsaturated fatty acids (n-3 PUFA) benefit brain development, we hypothesized that n-3 PUFA supplementation (via the maternal diet) improves respiratory function in rat pups. Pups received n-3 PUFA supplementation from an enriched diet (13 g kg-1 of n-3 PUFA) administered to the mother from birth until the experiments were performed (postnatal days 10-11). Controls received a standard diet (0.3 g kg-1 of n-3 PUFA). Breathing was measured in intact pups at rest and during hypoxia (FiO2  = 0.12; 20 min) using whole body plethysmography. The duration of apnoeas induced by stimulating the laryngeal chemoreflex (LCR) was measured under anaesthesia. Lung morphology was compared between groups. Maternal n-3 PUFA supplementation effectively raised n-3 PUFA levels above control levels both in the blood and brainstem of pups. In intact, resting pups, n-3 PUFA increased the frequency and duration of apnoeas, especially in females. During hypoxia, n-3 PUFA supplemented pups hyperventilated 23% more than controls; their anapyrexic response was 1 °C less than controls. In anaesthetized pups, n-3 PUFA shortened the duration of LCR-induced apnoeas by 32%. The relative pulmonary surface area of n-3 PUFA supplemented pups was 12% higher than controls. Although n-3 PUFA supplementation augments apnoeas, there is no clear evidence of deleterious consequences on these pups. Based on the improved lung architecture and responses to respiratory challenges, this neonatal treatment appears to be beneficial to the offspring. However, further experiments are necessary to establish its overall safety.

Membrane progesterone receptor-ß, but not -α, in dorsal brain stem establishes sex-specific chemoreflex responses and reduces apnea frequency in adult mice.

We tested the hypothesis that membrane progesterone receptors (mPR) contribute to respiratory control in adult male and female mice. Mice were implanted with osmotic minipumps for continuous infusion of small interfering RNA (siRNA) directed against mPRα, mPRß, or a control solution in the fourth ventricle (to target brain stem respiratory areas) for 14 days. We then performed respiratory and metabolic recordings by whole body plethysmography at rest and in response to hypoxia (12% O2) or hypercapnia (5% CO2, 5 min each). For each treatment, we have verified with immunohistochemistry that the staining intensity of mPRα or mPRß in the brain stem is decreased. At rest, the siRNA against mPRα and mPRß increased respiratory frequency in males only. The siRNA against mPRß almost tripled the frequency of apneas in male and in female mice, while the siRNA against mPRα had no effect. Regarding respiratory chemoreflex, the siRNA against mPRß suppressed the response to hypoxia in male and female mice and reduced by ∼50% the response to hypercapnia, while the siRNA against mPRα had more limited effects. Interestingly, control females had higher ventilatory response to hypoxia and hypercapnia than males, and these sex-specific effects were suppressed by the siRNA against mPRß, whereas they were still present after treatment with the siRNA against mPRα. We conclude that mPRß reduces apnea frequency in male and female mice and establishes sex-specific ventilatory chemoreflex.

Neonatal stress affects the aging trajectory of female rats on the endocrine, temperature, and ventilatory responses to hypoxia.

Human and animal studies on sleep-disordered breathing and respiratory regulation show that the effects of sex hormones are heterogeneous. Because neonatal stress results in sex-specific disruption of the respiratory control in adult rats, we postulate that it might affect respiratory control modulation induced by ovarian steroids in female rats. The hypoxic ventilatory response (HVR) of adult female rats exposed to neonatal maternal separation (NMS) is ∼30% smaller than controls (24), but consequences of NMS on respiratory control in aging female rats are unknown. To address this issue, whole body plethysmography was used to evaluate the impact of NMS on the HVR (12% O2, 20 min) of middle-aged (MA; ∼57 wk old) female rats. Pups subjected to NMS were placed in an incubator 3 h/day for 10 consecutive days (P3 to P12). Controls were undisturbed. To determine whether the effects were related to sexual hormone decline or aging per se, experiments were repeated on bilaterally ovariectomized (OVX) young (∼12 wk old) adult female rats. OVX and MA both reduced the HVR significantly in control rats but had little effect on the HVR of NMS females. OVX (but not aging) reduced the anapyrexic response in both control and NMS animals. These results show that hormonal decline decreases the HVR of control animals, while leaving that of NMS female animals unaffected. This suggests that neonatal stress alters the interaction between sex hormone regulation and the development of body temperature, hormonal, and ventilatory responses to hypoxia.