A Translational Model of Incomplete Catch-Up Growth: Early-Life Hypoxia and the Effect of Physical Activity.

Advances in therapies have led to prolonged survival from many previously lethal health threats in children, notably among prematurely born babies and those with congenital heart disease. Evidence for catch-up growth is common in these children, but in many cases the adult phenotype is never achieved. A translational animal model is required in which specific tissues can be studied over a reasonable time interval. We investigated the impact of postnatal hypoxia (HY) (12%O2 (HY12) or 10% O2 (HY10)) on growth in rats relative to animals raised in room air. Subgroups had access to running wheels following the HY period. Growth was fully compensated in adult HY12 rats but not HY10 rats. The results of this study indicate that neonatal hypoxia can be a useful model for the elucidation of mechanisms that mediate successful catch-up growth following neonatal insults and identify the critical factors that prevent successful catch-up growth.

Growth inhibition and compensation in response to neonatal hypoxia in rats.

BACKGROUND: Hypoxia (Hx) is an important disease mechanism in prematurity, childhood asthma, and obesity. In children, Hx results in chronic inflammation. METHODS: We investigated the effects of Hx (12% O2) during postnatal days 2-20 in rats. Control groups were normoxic control (Nc), and normoxic growth restricted (Gr) (14-pup litters). RESULTS: The Hx-exposed and Gr rats had similar decreases in growth. Hx increased plasma tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) levels and decreased insulin-like growth factor 1 (IGF-I) and vascular endothelial growth factor (VEGF) levels. Hx resulted in hypertrophy of the right ventricle (RV) but disproportionate decrements in limb skeletal muscle (SM) growth. miR-206 was depressed in the hypertrophied RV of Hx rats but was increased in growth-retarded SM. Hx resulted in decreased RV messenger RNA (mRNA) level for myostatin but had no effect on SM myostatin. The mRNA for Hx-sensitive factors such as hypoxia inducible factor-1α (HIF-1α) was depressed in the RV of Hx rats, suggesting negative feedback. CONCLUSION: The results indicate that Hx induces a proinflammatory state that depresses growth-regulating mechanisms and that tissues critical for survival, such as the heart, can escape from this general regulatory program to sustain life. This study identifies accessible biomarkers for evaluating the impact of interventions designed to mitigate the long-term deleterious consequences of Hx that all too often occur in babies born prematurely.

A meta-analysis of clinical improvements of general well-being by a standardized Lycium barbarum.

Four randomized, blind, placebo-controlled clinical trials were pooled to study the general effects of oral consumption of Lycium barbarum at 120 mL/day, as a standardized juice, GoChi(®) (FreeLife International, Phoenix, AZ, USA). A questionnaire consisting of symptoms graded 0-5 was given to the participants. For each question, the score changes in the questionnaire between pre- and postintervention were summarized by the standardized mean difference and associated SE to perform the meta-analysis. The change was also characterized into a binary outcome, improved or not, to derive odds ratio (OR) and associated SE derived by a binary outcome using the Mantel-Haenszel method. The meta-analysis and heterogeneity were evaluated with the R program using the rmeta package. Statistical significance was set at 5%. In total, 161 participants (18-72 years old) were included in the meta-analysis. Compared with the placebo group (n=80), the active group (n=81) showed significant improvements in weakness, stress, mental acuity, ease of awakening, shortness of breath, focus on activity, sleep quality, daydreaming, and overall feelings of health and well-being under a random effects model. A fixed effects model showed additional improvements in fatigue, depression, circulation, and calmness. The OR indicated significantly higher chance to improve fatigue, dizziness, and sleep quality. Three studies had statistically significant heterogeneity in procrastination, shoulder stiffness, energy, and calmness. The present meta-analysis confirmed the various health effects of L. barbarum polysaccharides-standardized L. barbarum intake found in the previous randomized, double-blind, placebo-controlled human clinical trials and revealed it resulted in statistically significant improvements in neurological/psychological performance and overall feelings of health and well-being compared with the placebo group under both the fixed and the random effects models of the R program.

Lycium barbarum increases caloric expenditure and decreases waist circumference in healthy overweight men and women: pilot study.

BACKGROUND: Lycium barbarum (L. barbarum), a traditional Asian medicinal therapy for diabetes and other conditions, has been shown to increase metabolic rate and to reduce body-weight gains in rodent models, as well as to produce clinical improvements in general feelings of well-being including energy level. OBJECTIVE: To investigate the impact of L. barbarum consumption on (1) caloric expenditure and (2) changes in morphometric parameters (waist circumference) in healthy human adults. METHOD: Two separate randomized, double-blind, placebo-controlled, small clinical studies were conducted using a standardized L. barbarum fruit juice, GoChi, and assessing its effects on (1) resting metabolic rate (RMR) and postprandial energy expenditure (PPEE) as measured by indirect calorimetry after single-bolus intake of 3 doses of L. barbarum (30, 60, and 120 ml) and placebo; and (2) waist circumference and other morphometric changes in a 14-day intervention trial (120-ml daily intake) in the subjects (age = 34 years, body mass index = 29 kg/m(2)). RESULTS: (1) A single bolus of L. barbarum intake increased PPEE 1 through 4 hours postintake over the baseline level in a dose-dependent manner and was significantly higher than the placebo group by 10% at 1 hour postintake of 120 ml (p < 0.05). (2) In a 14-day intervention trial, L. barbarum was found to significantly decrease waist circumference by 5.5 ± 0.8 cm (n = 15) compared with the preintervention measurements and placebo group at postintervention day 15 (p < 0.01). By contrast, the changes in the placebo group (n = 14) from preinterventions was 0.9 ± 0.8 cm, which was not statistically significant. CONCLUSIONS: These results show that L. barbarum consumption increases metabolic rate and reduces the waist circumference, relative to placebo treated control subjects.

A rat model of exercise-induced asthma: a nonspecific response to a specific immunogen.

Exercise-induced bronchoconstriction (EIB) is common; however, key aspects of its pathogenesis are still unclear. We investigated the feasibility of adapting an established animal model of asthma to investigate the earliest stages of EIB. The hypothesis was that a single exposure to a normally innocuous, and brief, exercise challenge could trigger EIB symptoms in rats previously sensitized to ovalbumin (OVA) but otherwise unchallenged. Brown-Norway rats were sensitized by intraperitoneal injection of OVA at 0 and 2 wk. At week 3, animals were exposed to either aerosolized OVA (SS) or exercise (EXS). A trained, blinded, clinical observer graded EIB by respiratory sounds. Plasma and lung cytokine levels were analyzed. No control rats with or without exercise (EX, CON) showed evidence of EIB. Eighty percent of the SS group demonstrated abnormal breath sounds upon exposure to aerosolized OVA. Approximately 30% of EXS rats sensitized to OVA but exposed only to exercise had abnormal breath sounds. Lung tissue levels of TNF-α, IL-1α, growth-related oncogene/keratinocyte/chemoattractant, and IFN-γ were significantly higher (P < 0.001) in the SS group, relative to all other groups. Changes in most of these cytokines were not notable in the EXS rats, suggesting a different mechanism of EIB. Remarkably, IFN-γ, but not the other cytokines measured, was significantly elevated following brief exercise in both sensitized and unsensitized rats. Exercise led to detectable breathing sound abnormalities in sensitized rats, but less severe than those observed following classical OVA challenge. Precisely how this immune crossover occurs is not known, but this model may be useful in elucidating essential mechanisms of EIB.

Exercise and leukocyte interchange among central circulation, lung, spleen, and muscle.

Circulating leukocytes increase rapidly with exercise then quickly decrease when the exercise ends. We tested whether exercise acutely led to bidirectional interchange of leukocytes between the circulation and the lung, spleen, and active skeletal muscle. To accomplish this it was necessary to label a large number of immune cells (granulocytes, monocytes, and lymphocytes) in a way that resulted in minimal perturbation of cell function. Rats were injected intravenously with a single bolus of carboxyfluorescein diacetate succinamidyl ester (CFSE) dye which is rapidly and irreversibly taken up by circulating cells. The time course of the disappearance of labeled cells and their reappearance in the circulation following exercise was determined via flow cytometry. The majority of circulating leukocytes were labeled at 4h. post-injection and this proportion slowly declined out to 120 h. At both 24 and 120 h, running resulted in an increase in the proportion of labeled leukocytes in the circulation. Analysis of the skeletal muscle, spleen and lung indicated that labeled leukocytes had accumulated in those tissues and were mobilized to the circulation in response to exercise. This indicates that there is an ongoing exchange of leukocytes between the circulation and tissues and that exercise can stimulate their redistribution. Exchange was slower with muscle than with spleen and lung, but in all cases, influenced by exercise. Exercise bouts redistribute leukocytes between the circulation and the lung, spleen and muscle. The modulatory effects of exercise on the immune system may be regulated in part by the systemic redistribution of immune cells.

Increased rat neonatal activity influences adult cytokine levels and relative muscle mass.

Little is known about the effect of physical activity in early life on subsequent growth and regulation of inflammation. We previously reported that exposure of muscles in growing rats to IL-6 results in decreased muscle growth apparently because of a state of resistance to growth factors such IGF-I and that running exercise could ameliorate this growth defect. Herein, we hypothesized that increased activity, for a brief period during neonatal life, would pattern the adult rat toward a less inflammatory phenotype. Neonatal rats were induced to move about their cage for brief periods from d 5 to d 15 postpartum. Additional groups were undisturbed controls (CONs) and handled (HAND). Subgroups of rats were sampled at the age of 30 and 65 d. Relative to CON and HAND groups, the neonatal exercise (EX) group demonstrated a decrease in circulating levels of TNFα, IL-6, and IL-1ß in adulthood, primarily in male rats. In addition, adult male EX rats had lower body mass and increased skeletal muscle mass suggesting a leaner phenotype. The results of this study suggest that moderate increases in activity early in life can influence the adult toward a more healthy phenotype with regard to inflammatory mediators and relative muscle mass.

Immunomodulatory effects of a standardized Lycium barbarum fruit juice in Chinese older healthy human subjects.

Lycium barbarum has been traditionally used in combination with several herbs for medicinal properties, but systematic modern clinical evaluation as a single herb has not been reported. To examine the systematic effects of L. barbarum on immune function, general well-being, and safety, we tested the effects of a standardized L. barbarum fruit juice (GoChi, FreeLife International, Phoenix, AZ, USA) at 120 mL/day, equivalent to at least 150 g of fresh fruit, the amount traditionally used, or placebo for 30 days in a randomized, double-blind, placebo-controlled clinical study in 60 older healthy adults (55-72 years old). The GoChi group showed a statistically significant increase in the number of lymphocytes and levels of interleukin-2 and immunoglobulin G compared to pre-intervention and the placebo group, whereas the number of CD4, CD8, and natural killer cells or levels of interleukin-4 and immunoglobulin A were not significantly altered. The placebo group showed no significant changes in any immune measures. Whereas the GoChi group showed a significant increase in general feelings of well-being, such as fatigue and sleep, and showed a tendency for increased short-term memory and focus between pre- and post-intervention, the placebo group showed no significant positive changes in these measures. No adverse reactions, abnormal symptoms, or changes in body weight, blood pressure, pulse, visual acuity, urine, stool, or blood biochemistry were seen in either group. In conclusion, daily consumption of GoChi significantly increased several immunological responses and subjective feelings of general well-being without any adverse reactions.

A randomized, double-blind, placebo-controlled, clinical study of the general effects of a standardized Lycium barbarum (Goji) Juice, GoChi.

BACKGROUND: This randomized, double-blind, placebo-controlled clinical trial is the first study reported from outside China that has examined the general effects of the orally consumed goji berry, Lycium barbarum, as a standardized juice (GoChi; FreeLife International LLC, Phoenix, AZ) to healthy adults for 14 days. METHODS: Based upon the medicinal properties of Lycium barbarum in traditional Asian medicine, we examined by questionnaire subjective ratings (0-5) of general feelings of well-being, neurologic/psychologic traits, gastrointestinal, musculoskeletal, and cardiovascular complaints as well as any adverse effects. Also, measures of body weight, body-mass index, blood pressure, pulse rate, and visual acuity were assessed before and after consuming 120 mL of GoChi/day or placebo control solution. Data were statistically analyzed for changes between day 1 and day 15. RESULTS: Significant differences between day 1 and day 15 were found in the GoChi group (N = 16) in increased ratings for energy level, athletic performance, quality of sleep, ease of awakening, ability to focus on activities, mental acuity, calmness, and feelings of health, contentment, and happiness. GoChi also significantly reduced fatigue and stress, and improved regularity of gastrointestinal function. In contrast, the placebo group (N = 18) showed only two significant changes (heartburn and happiness). No significant changes in musculoskeletal or cardiovascular complaints were observed in either group. All parametric data (body weight, etc.) were not significantly different between groups or between day 1 and day 15 for either group. CONCLUSIONS: These results clearly indicate that daily consumption of GoChi for 14 days increases subjective feelings of general well-being, and improves neurologic/psychologic performance and gastrointestinal functions. The data strongly suggest that further research is indicated to confirm and extend knowledge of the potential effects of Lycium barbarum upon human health.

Autonomic innervation and regulation of the immune system (1987-2007).

Since 1987, only a few neuroanatomical studies have been conducted to identify the origin of innervation for the immune system. These studies demonstrated that all primary and secondary immune organs receive a substantial sympathetic innervation from sympathetic postganglionic neurons. Neither the thymus nor spleen receive any sensory neural innervation; however, there is evidence that lymph nodes and bone marrow may be innervated by sensory neurons located in dorsal root ganglia. There is no neuroanatomical evidence for a parasympathetic or vagal nerve supply to any immune organ. Thus, the primary pathway for the neural regulation of immune function is provided by the sympathetic nervous system (SNS) and its main neurotransmitter, norepinephrine (NE). Activation of the SNS primarily inhibits the activity of cells associated with the innate immune system, while it either enhances or inhibits the activity of cells associated with the acquired/adaptive immune system. Innate immune cells express both alpha and beta-adrenergic receptor subtypes, while T and B lymphocytes express adrenergic receptors of the beta2 subtype exclusively, except for murine Th2 cells that lack expression of any subtype. Via these adrenergic receptors, NE is able to regulate the level of immune cell activity by initiating a change in the level of cellular activity, which often involves a change in the level of gene expression for cytokines and antibodies.

Direct visualization of nitric oxide release by liver cells after the arrest of metastatic tumor cells in the hepatic microvasculature.

BACKGROUND: Our previous studies have shown that the injection of B16F1 melanoma cells into the mesenteric vein can induce the rapid local release of nitric oxide (NO) in the liver, causing apoptosis of the melanoma cells in the liver sinusoids and inhibiting the subsequent formation of hepatic metastases. In this study, we have investigated the distribution and cellular source of NO in this model. MATERIALS AND METHODS: In situ liver perfusion was established in both wild-type (wt) and endothelial nitric oxide synthase knockout (eNOS KO) C57BL/6 mice. A specific fluorescent NO probe, 4,5-diaminofluorescein diacetate (DAF-2 DA) (5 micromol/L), was perfused into the portal venous system to label the liver tissue. Then, a MitoTracker Orange labeled B16F1 melanoma cell suspension (2 x 10(6) cells/ml) was injected through a portal vein catheter by a peristaltic pump. Images of the liver tissue were taken by confocal microscopy from a selected area to determine the cellular source of NO. For quantification, the fluorescence intensity of this area was measured over time by Fluoview software. RESULTS: Diaminotriazolofluorescein (DAF-2T) fluorescence (indicating NO generation) was detected in hepatic parenchymal cells located in the periportal region in both wt C57BL/6 and eNOS KO C57BL/6 mice and was intensified by increased flow rate in the portal venous system. The B16F1 cells arrested in the periportal sinusoids, corresponding to zone 1 of the hepatic acinus. DAF-2T fluorescence was expressed by both sinusoidal lining cells and hepatocytes at the site of tumor cell arrest. The fluorescence intensity of these cells increased approximately 2-fold over a time of 500 s. In contrast, there was no increase in the fluorescence intensity of the sinusoidal lining cells and hepatocytes in mice perfused with buffer or in eNOS KO mice perfused with B16F1 cells. CONCLUSION: This study demonstrates that NO is produced by hepatic parenchymal cells mainly located in the periportal zones and that the arrest of the B16F1 melanoma cells causes an eNOS-dependent local burst of NO by the sinusoidal lining cells and hepatocytes in the periportal areas.

Stress-induced suppression of in vivo splenic cytokine production in the rat by neural and hormonal mechanisms.

The mechanisms mediating the effects of stress on immune function have yet to be fully described. In vitro studies have demonstrated a role for both the sympathetic nervous system (SNS) and the hypothalamic pituitary adrenal axis (HPAA) in regulating immune responses following exposure to various stressors. The purpose of the present set of experiments was to determine the in vivo contribution of the HPAA and SNS in regulating the effects of stress on lipopolysaccharide (LPS) induced splenic cytokine production. For this, rats with combinations of sham surgeries, splenic nerve cuts (SNC), and adrenalectomies (ADX) were exposed to 15 min of 1.6 mA intermittent footshock immediately following the intravenous (i.v.) injection of 0.1 microg of LPS. Although footshock was immunosuppressive to most indices of cytokine production, neither SNC nor ADX alone blocked the effects of stress on splenic immune function. However the combination of these two manipulations significantly abrogated the immunosuppressive effects of stress on cytokine production. Adrenal demedullation of animals with a SNC demonstrated that the SNS, not the HPAA, was primarily responsible for the immunosuppressive effects of stress.

Neuropeptide specificity of prostaglandin E2-induced activation of splenic and renal sympathetic nerves in the rat.

Sympathetic activation occurs rapidly following intracerebroventricular (icv) injection of prostaglandin E2(PGE2). This study examined whether neuropeptides mediate PGE2-induced sympathetic nerve activation in urethane/chloralose-anesthetized Sprague-Dawley rats. Animals were pretreated (20.0 microg, icv) with the following receptor antagonists; CRF ([D-Phe12,Nle21,38,Calpha-MeLeu37]CRF12-41), AVP-V1 (Des-Gly-[Phaa1, D-Tyr(Et)2,Lys6,Arg8]-vasopressin), or OT (OT+V1, [d(CH2)5,Tyr(Me)2,Orn8]-vasotocin) followed 20 min later by PGE2 (2.0 microg, icv). Pretreatment with the CRF antagonist attenuated the increase in renal nerve activity induced by PGE2 when measured 10 and 30 min post-injection. PGE2-induced renal nerve activity was also inhibited at both time points by the AVP antagonist and, to a similar extent, the OT antagonist. The AVP antagonist did not effect splenic nerve responses to PGE2 whereas the CRF antagonist produced an incomplete and transient reduction in PGE2-induced activation of the splenic nerve. However, the OT antagonist completely blocked the activation of the splenic nerve after central injection of PGE2. ICV injections of AVP and OT produced immediate changes in splenic and renal nerve activity whereas CRF failed to alter the activity of either nerve in anesthetized or conscious animals. Thus, PGE2 acts through neuropeptide-specific pathways to initiate sympathetic outflow and OT is a specific component of the sympathetic pathway innervating the spleen.

Contribution of the adrenal glands and splenic nerve to LPS-induced splenic cytokine production in the rat.

Both the hypothalamic pituitary adrenal axis (HPAA) and the sympathetic nervous system (SNS) can inhibit immune function and are regarded as the primary efferent pathways for neural-immune interactions. To determine if this relationship is maintained in vivo in response to an inflammatory stimulus, rats were injected intravenously (iv) with various doses of lipopolysaccharide (LPS) and splenic cytokine mRNA and protein levels were measured at several dose and time intervals post-injection. The spleen was chosen as the target organ because both the neural and hormonal inputs to the spleen can be selectively removed by splenic nerve cut (SNC) and adrenalectomy (ADX), respectively. Data from our dose response studies established that maximum levels of splenic cytokines were induced in response to relatively low doses of LPS. Minimal changes in LPS-induced splenic cytokine levels were observed in response to ADX, SNC, or a combination of the two procedures across several doses of LPS. These results suggest that there are aspects of immune regulation that are functionally removed from these central modulatory systems and that the counter-regulatory responses induced by LPS have minimal impact on the concurrent induction of cytokines by this inflammatory stimulus. The conceptual model of neural-immune regulation as an inhibitory feedback system, at least with regards to the early activational effects induced by an inflammatory stimulus, was not supported by these studies.

Arrest of B16 melanoma cells in the mouse pulmonary microcirculation induces endothelial nitric oxide synthase-dependent nitric oxide release that is cytotoxic to the tumor cells.

Metastatic cancer cells seed the lung via blood vessels. Because endothelial cells generate nitric oxide (NO) in response to shear stress, we postulated that the arrest of cancer cells in the pulmonary microcirculation causes the release of NO in the lung. After intravenous injection of B16F1 melanoma cells, pulmonary NO increased sevenfold throughout 20 minutes and approached basal levels by 4 hours. NO induction was blocked by N(G)-nitro-L-arginine methyl ester (L-NAME) and was not observed in endothelial nitric oxide synthase (eNOS)-deficient mice. NO production, visualized ex vivo with the fluorescent NO probe diaminofluorescein diacetate, increased rapidly at the site of tumor cell arrest, and continued to increase throughout 20 minutes. Arrested tumor cells underwent apoptosis with apoptotic counts more than threefold over baseline at 8 and 48 hours. Neither the NO signals nor increased apoptosis were seen in eNOS knockout mice or mice pretreated with L-NAME. At 48 hours, 83% of the arrested cells had cleared from the lungs of wild-type mice but only approximately 55% of the cells cleared from eNOS-deficient or L-NAME pretreated mice. eNOS knockout and L-NAME-treated mice had twofold to fivefold more metastases than wild-type mice, measured by the number of surface nodules or by histomorphometry. We conclude that tumor cell arrest in the pulmonary microcirculation induces eNOS-dependent NO release by the endothelium adjacent to the arrested tumor cells and that NO is one factor that causes tumor cell apoptosis, clearance from the lung, and inhibition of metastasis.