Chronic Elevation Research Articles

Sex-specific density-dependent secretion of glucocorticoids in lizards: insights from laboratory and field experiments

Negative density feedbacks have been extensively described in animal species and involve both consumptive (i.e. trophic interactions) and non-consumptive (i.e. social interactions) mechanisms. Glucocorticoids are a major component of the physiological stress response and homeostasis, and therefore make a good candidate for proximate determinants of negative density feedbacks. Here, we combined laboratory and field experiments with enclosed populations to investigate the relationship between density, social stress and plasma corticosterone levels in the common lizard Zootoca vivipara. This species exhibits strong negative density feedbacks that affect females more than males, and its life history is sensitive to experimentally-induced chronic elevation of corticosterone plasma levels. We found that prolonged crowding in the laboratory can trigger a chronic secretion of corticosterone independent from food restriction. In the field experiments, corticosterone levels of females were not affected by population density. Corticosterone levels of males increased with population density but only during the late activity season in a first field experiment where we manipulated density. They also increased with density during the mating season but only in populations with a female-biased sex ratio in a second field experiment where we crossed manipulated density and adult sex ratio. Altogether, our results provide limited evidence for a role of basal corticosterone secretion in density feedbacks in this species. Context and density-dependent effects in males may arise from changes in behavior caused by competition for resources, male–male competition, and mating.

Characterization of the Circumlimbal Suture Model of Chronic IOP Elevation in Mice and Assessment of Changes in Gene Expression of Stretch Sensitive Channels.

To consider whether a circumlimbal suture can be used to chronically elevate intraocular pressure (IOP) in mice and to assess its effect on retinal structure, function and gene expression of stretch sensitive channels. Anesthetized adult C57BL6/J mice had a circumlimbal suture (10/0) applied around the equator of one eye. In treated eyes (n = 23) the suture was left in place for 12 weeks whilst in sham control eyes the suture was removed at day two (n = 17). Contralateral eyes served as untreated controls. IOP was measured after surgery and once a week thereafter. After 12 weeks, electroretinography (ERG) was performed to assess photoreceptor, bipolar cell and retinal ganglion cell (RGC) function. Retinal structure was evaluated using optical coherence tomography. Retinae were processed for counts of ganglion cell density or for quantitative RT-PCR to quantify purinergic (P2x7, Adora3, Entpd1) or stretch sensitive channel (Panx1, Trpv4) gene expression. Immediately after suture application, IOP spiked to 33 ± 3 mmHg. After 1 day, IOP had recovered to 27 ± 3 mmHg. Between weeks 2 and 12, IOP remained elevated above baseline (control 14 ± 1 mmHg, ocular hypertensive 19 ± 1 mmHg). Suture removal at day 2 (Sham) restored IOP to baseline levels, where it remained through to week 12. ERG analysis showed that 12 weeks of IOP elevation reduced photoreceptor (−15 ± 4%), bipolar cell (−15 ± 4%) and ganglion cell responses (−19 ± 6%) compared to sham controls and respective contralateral eyes (untreated). The retinal nerve fiber layer was thinned in the presence of normal total retinal thickness. Ganglion cell density was reduced across all quadrants (superior −12 ± 5%; temporal, −7% ± 2%; inferior −9 ± 4%; nasal −8 ± 5%). Quantitative RT-PCR revealed a significant increase in Entpd1 gene expression (+11 ± 4%), whilst other genes were not significantly altered (P2x7, Adora3, Trpv4, Panx1). Our results show that circumlimbal ligation produces mild chronic ocular hypertension and retinal dysfunction in mice. Consistent with a sustained change to purinergic signaling we found an up-regulation of Entpd1.

Advanced glycation end products induce brain-derived neurotrophic factor release from human platelets through the Src-family kinase activation

BackgroundBrain-derived neurotrophic factor (BDNF) exerts beneficial effects not only on diabetic neuropathies but also on cardiovascular injury. There is argument regarding the levels of serum BDNF in patients with diabetes mellitus (DM). Because BDNF in peripheral blood is rich in platelets, this may represent dysregulation of BDNF release from platelets. Here we focused on advanced glycation end products (AGEs), which are elevated in patients with DM and have adverse effects on cardiovascular functions. The aim of this study is to elucidate the role of AGEs in the regulation of BDNF release from human platelets.MethodsPlatelets collected from peripheral blood of healthy volunteers were incubated with various concentrations of AGE (glycated-BSA) at 37 °C for 5 min with or without BAPTA-AM, a cell permeable Ca2+ chelator, or PP2, a potent inhibitor of Src family kinases (SFKs). Released and cellular BDNF were measured by ELISA and calculated. Phosphorylation of Src and Syk, a downstream kinase of SFKs, in stimulated platelets was examined by Western blotting and immunoprecipitation.ResultsAGE induced BDNF release from human platelets in a dose-dependent manner, which was dependent on intracellular Ca2+ and SFKs. We found that AGE induced phosphorylation of Src and Syk.ConclusionsAGE induces BDNF release from human platelets through the activation of the Src-Syk-(possibly phospholipase C)-Ca2+ pathway. Considering the toxic action of AGEs and the protective roles of BDNF, it can be hypothesized that AGE-induced BDNF release is a biological defense system in the early phase of diabetes. Chronic elevation of AGEs may induce depletion or downregulation of BDNF in platelets during the progression of DM.

Measuring Deformation in the Mouse Optic Nerve Head and Peripapillary Sclera.

PurposeTo develop an ex vivo explant system using multiphoton microscopy and digital volume correlation to measure the full-field deformation response to intraocular pressure (IOP) change in the peripapillary sclera (PPS) and in the optic nerve head (ONH) astrocytic structure.MethodsGreen fluorescent protein (GFP)-glutamate transporter-GLT1 (GLT1/GFP) mouse eyes were explanted and imaged with a laser-scanning microscope under controlled inflation. Images were analyzed for regional strains and changes in astrocytic lamina and PPS shape. Astrocyte volume fraction in seven control GLT1/GFP mice was measured. The level of fluorescence of GFP fluorescent astrocytes was compared with glial fibrillary acidic protein (GFAP) labeled astrocytes using immunohistochemistry.ResultsThe ONH astrocytic structure remained stable during 3 hours in explants. Control strain—globally, in the central one-half or two-thirds of the astrocytic lamina—was significantly greater in the nasal-temporal direction than in the inferior-superior or anterior-posterior directions (each P ≤ 0.03, mixed models). The PPS opening (perimeter) in normal eye explants also became wider nasal-temporally than superior-inferiorly during inflation from 10 to 30 mm Hg (P = 0.0005). After 1 to 3 days of chronic IOP elevation, PPS area was larger than in control eyes (P = 0.035), perimeter elongation was 37% less than controls, and global nasal-temporal strain was significantly less than controls (P = 0.007). Astrocyte orientation was altered by chronic IOP elevation, with processes redirected toward the longitudinal axis of the optic nerve.ConclusionsThe explant inflation test measures the strain response of the mouse ONH to applied IOP. Initial studies indicate regional differences in response to both acute and chronic IOP elevation within the ONH region.

Manipulation of Amyloid Precursor Protein Processing Impacts Brain Bioenergetics and Glucose Metabolism

The amyloid precursor protein (APP) and one of its terminal cleavage products, amyloid beta (Aβ) are proposed to play a central role in the pathogenesis of Alzheimer's disease (AD). These proteins have also been shown to co-localise within mitochondria with Aβ directly inhibiting the electron transport chain. Cleavage of APP occurs down distinct pathways. Initial cleavage of the full-length protein is mediated by either α- or β-secretase (BACE1). Subsequent cleavage by γ-secretase releases p3 or Aβ protein fragments into the extracellular milieu. Whilst these interactions with mitochondria have been noted, the impact of manipulating APP processing by these distinct pathways on cellular metabolism and bioenergetics has yet to be elucidated. We utilised the human SH-SY5Y neuronal cell line stably overexpressing BACE1 and cortical slices from mice bearing inducible overexpression of APP harbouring the Swedish and Indiana mutations, favouring Aβ production. Protein expression and enzyme activity were determined via western blotting and enzyme-linked immunosorbance assays (ELISA). A Seahorse extracellular flux analyser and radio-labelled substrate assays were used to determine cellular bioenergetics in real-time. All data are expressed as mean ± standard error of the mean and statistical significance determined by Student's t-test. Manipulation of APP cleavage resulted in alterations of 2-deoxyglucose uptake into cells. Chronic elevation in BACE1 resulted in impaired functioning of a key fuel-partitioning enzyme, pyruvate dehydrogenase (PDH, activity reduced to 69 ± 8%, p < 0.05, n=6). This reduced substrate delivery to the mitochondria and increased reliance upon aerobic glycolysis for ATP generation (oxygen consumption rate (OCR) reduced to 65 ± 9%, p < 0.01, n=7 and extracellular acidification rate (ECAR) increased to 165 ± 16%, p < 0.01, n=7). Thus BACE1 overexpression impairs neuronal glucose oxidation resulting in an increased reliance upon aerobic glycolysis for ATP generation. Acute cortical overexpression of APP favouring amyloidogenic processing resulted in a significant reduction in PDH protein expression (reduced to 62 ± 6%, p < 0.01, n = 9-10) and activity of key tricarboxylic acid cycle enzymes, isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase (reduced to 62 ± 7%, p < 0.01, n=6 and 88 ± 2%, p < 0.001, n = 6). Therefore, manipulation of APP processing towards Aβ production in cells and tissue significantly impacts cellular metabolic pathways at the level of glucose uptake, substrate entry to the mitochondria and the TCA cycle.

A Mouse Model of Chronic Ocular Hypertension Induced by Circumlimbal Suture.

To develop a chronic ocular hypertension mouse model by inducing intraocular pressure (IOP) elevation using a suture technique previously developed for rats. C57BL/6 mice were given monocular circumlimbal suture (10/0) placement under anesthesia (ketamine/xylazine). The suture was left in place for 12 weeks (n = 10). A control group had the same treatment while it was removed after day 1 (n = 10). Intraocular pressure, electroretinogram (ERG), and optical coherence tomography (OCT) were measured in both groups for 12 weeks. At week 12, animals were euthanized and retina was harvested for histologic assessment. In the group with extended suture placement, IOP spiked from 14.3 ± 0.9 to 48.4 ± 4.8 mm Hg immediately after suture implantation. At day 1, it declined to 33.2 ± 3.7 mm Hg and remained elevated for 12 weeks. In the suture removal group, IOP normalized to baseline within a day following suture removal. In the group with prolonged IOP elevation, the retinal ganglion cell (RGC) mediated ERG responses continued to exacerbate and were significantly reduced by week 12 (P < 0.001). Progressive loss of retinal nerve fiber layer was found and it became significant from week 4 (P < 0.001). At week 12, significant loss of RGCs (P < 0.001) was noted. In the IOP normalization group, no alteration in ERG, OCT, and RGC counting was observed. The circumlimbal suture approach produces a mild chronic IOP elevation in mice. Functional and structural changes under model induction are largely independent of the initial IOP spike.

A chronic increase of corticosterone age-dependently reduces systemic DNA damage from oxidation in rats

Stress and depression are associated with an acceleration of brain and bodily aging; effects which have been attributed to chronic elevations of glucocorticoids. We tested the hypothesis that a three week administration of stress-associated levels of corticosterone (CORT, the principal rodent glucocorticoid) would increase systemic and CNS DNA and RNA damage from oxidation; a phenomenon known to be centrally involved in the aging process. We also hypothesized that older individuals would be more sensitive to this effect and that the chronic CORT administration would exacerbate age-related memory decline. Young and old male Sprague-Dawley rats were non-invasively administered CORT by voluntary ingestion of nut paste containing either CORT (25mg/kg) or vehicle for a total of 22 days. CORT increased the 24h urinary excretion of the hormone to the levels previously observed after experimental psychological stress and caused a downregulation of the glucocorticoid receptor in the CA1 area of the hippocampus. Contrary to our hypothesis, 24h excretion of 8-oxodG/8-oxoGuo (markers of DNA/RNA damage from oxidation) was reduced in CORT-treated young animals, whereas old animals showed no significant differences. In old animals, CORT caused a borderline significant reduction of RNA oxidation in CNS, which was paralleled by a normalization of performance in an object location memory test. To our knowledge, this is the first demonstration that chronic stress-associated levels of CORT can reduce nucleic acid damage from oxidation. These findings contradict the notion of elevated CORT as a mediator of the accelerated aging observed in stress and depression.

Study of the Characteristics of Pulmonary Trunk in Pulmonary Hypertension Secondary to Left Heart Disease Using Pressure-Velocity Loops (PU-Loops).

Objectives: Although pulmonary hypertension (PH) caused by left heart disease (PH-LHD) is more common in PH, little is known about its properties of pulmonary artery (PA) in PH-LHD. The purpose of this study was to measure pulmonary regional pulse wave velocity (PWV) and to quantify the magnitude of reflected waves in patients with PH-LHD by the analysis of the pressure–velocity loops (PU-loop).Methods: High-fidelity PA pressure (Pm) and PA velocity (Vm) were measured in 11 subjects with PH-LHD (mean Pm>25 mmHg), 1 subject with atrial septal defect (ASD) without PH and 12 control subjects, using multisensor catheters. PWV was calculated as the slope of the initial part of the PU-loop in early systole. The similarity in the shapes of the pressure and flow velocity waveforms over one PU-loop was quantified as the magnitude of reflected wave by calculating the standard error of the estimate (Sy/x) from linear regression analysis between Pm and corresponding Vm. PWV and Sy/x during a Valsalva maneuver (VM) were also assessed in nine control subjects.Results: The contour of PU-loop was so characteristic between control and PH-LHD. Max. PWV (349 cm/s) was recorded in PH-LHD and min. PWV (111 cm/s) was recorded in ASD. VM increased Pm (12 [7–15] mmHg vs. 50 [18–110] mmHg; p=0.009) and PWV (200 [148–238] cm/s vs. 260 [192–306] cm/s; p=0.009) significantly without significant increase of Sy/x (19.6 [12.7–28.9]% vs. 28.2 [19.3–40.7]%; p=0.079). Although Sy/x was significantly higher in PH-LHD than in control and ASD (31.0 [14.3–36.3]% vs. 17.5 [8.4–28.9]%; p=0.009, ASD: 18.2%) , no significant difference was found in PWV between PH-LHD and control (269 [159–349] cm/s vs. 203 [154–289] cm/s; p=0.089).Conclusions: 1) The magnitude of wave reflection was elevated in PH-LHD significantly as compared with control and ASD. 2) Despite the significant increase in PA-PWV caused by abrupt elevation in Pm during VM in control, chronic elevation in Pm did not increase PA-PWV in PH-LHD significantly. It was hypothesized that the PA constituted a self-regulating system for maintaining the arterial stiffness stable against the chronic elevation in Pm in PH-LHD by a remodeling of increasing proximal pulmonary arterial crosssectional area gradually, which was compatible with the Moens–Korteweg equation. The PU-loop could provide a new simple and conventional method for assessing the pulmonary arterial properties, clinically. (This is a translation of J Jpn Coll Angiol 2016; 56: 45–53.)

Hair Cortisol and Perceived Stress in Health-studies Students during Summer Break and Fall Term

The objective of this study was to evaluate stress in health-studies students over time periods when classes are not in session (Summer) and are in session (Fall) using both a survey instrument and hair cortisol levels. Cortisol is produced by the adrenal cortex in response to stress. Chronic elevations in cortisol are associated with adverse effects on the cardiovascular, musculoskeletal, and central nervous system. As such, cortisol levels can serve as a biomarker for stress. Hair is a particularly useful matrix for measuring cortisol in the body since it grows at a uniform rate, about 1 cm/month, and thereby provides a time-averaged index of exposure to cortisol. A small bundle of hair (about 50 strands) was collected as close to the scalp as possible from Nursing and Pharmacy students at the beginning of the Fall semester (representing hair grown over the Summer when no classes were in session) and again at the end of Fall semester (representing hair grown during the semester, when classes were in session). A three cm-portion of each hair bundle was cut from the scalp end, ground, extracted, and analyzed by LC/MS/MS. The students also completed a survey to provide subjective feedback about perceived stress levels. These surveys were completed at the time of the hair collections. The analytical results showed no differences in hair cortisol concentrations between Summer and Fall or between Pharmacy and Nursing students. Perceived stress levels; however, were higher in Fall than Summer and somewhat higher in pharmacy students than nursing students during the Fall. The results of this study suggest that health-studies students experience psychological stress during the time classes are in session, but this perceived stress may not result in concomitant elevations in cortisol.

Pheochromocytoma and stress cardiomyopathy: Insight into pathogenesis.

AIMTo investigate the occurrence of cardiomyopathy (CMP) in a cohort of patients with histologically proven pheochromocytoma (pheo), and to determine if catecholamine excess was causative of the left ventricular (LV) dysfunction.METHODSA retrospective chart review spanning years 1998 through 2014 was undertaken and patients with a diagnosis of pheo confirmed with histopathologic examination were included. Presenting electrocardiograms and cardiac imaging studies were reviewed. Transthoracic echocardiography (TTE), ventriculography or single positron emission computed tomography imaging was evaluated and if significant abnormalities [left ventricular hypertrophy (LVH) or LV dysfunction] were noted in the pre operative period a follow up post-operative study was also analyzed. Multivariate analysis using logistic regression was used to investigate independent predictors for outcomes of interest, LV dysfunction and LVH.RESULTSWe identified 18 patients with diagnosis of pheo confirmed on pathology. Mean age was 54.3 ± 19.3 years and 11 (61.1%) patients were females. 50% of such patients had either resistant hypertension or labile blood pressures during hospitalization, which had raised suspicion for a pheo. Cardiac imaging studies were available for 12 (66.7%) patients at the time of inclusion into study and preceding the adrenalectomy. 7 (58.3%) patients with a TTE available for review had mild or more severe LVH while 3 (25%) patients had LV dysfunction of presumably acute onset. In a multivariate analysis, elevated catecholamine levels as assessed by urinary excretion of metabolites was not an independent predictor of development of LV systolic dysfunction or of presence of LVH on TTE. Two female patients with a preceding history of hypertension had marked LV hypertrophy and systolic anterior motion of the mitral valve. Prolongation of the QTc interval was noted in 5 (27.8%) patients but no acute arrhythmias were observed in any patient.CONCLUSIONThis study adds to the growing body of literature on the predilection of patients with pheochromocytomas to develop non-ischemic CMP. Degree of catecholamine excess as measured by urinary secretion of metabolites did not predict the development of CMP but 2 of 3 patients developed CMP in the setting of significant acute physiologic stress. Our findings provide support to the proposed etiologic role of elevated catecholamines in TC and other stress induced forms of CMP, however, activation of a brain-neural-cardiac axis from acute stress and local release of catecholamines but not chronic catecholamine elevations are likely to be responsible in pheo related CMP.

The Incidence and Risk Factors Associated with Chronic Liver Enzyme Elevation (cLEE) in HIV-Monoinfected Persons

The Incidence and Risk Factors Associated with Chronic Liver Enzyme Elevation (cLEE) in HIV-Monoinfected Persons

Mechanistic Links Underlying the Impact of C-Reactive Protein on Muscle Mass in Elderly

Background/Aims: Mechanisms underlying the relationship between systemic inflammation and age-related decline in muscle mass are poorly defined. The purpose of this work was to investigate the relationship between the systemic inflammatory marker CRP and muscle mass in elderly and to identify mechanisms by which CRP mediates its effects on skeletal muscle, in-vitro. Methods: Muscle mass and serum CRP level were determined in a cohort of 118 older women (67±1.7 years). Human muscle cells were differentiated into myotubes and were exposed to CRP. The size of myotubes was determined after immunofluorescent staining using troponin. Muscle protein synthesis was assessed using stable isotope tracers and key signalling pathways controlling protein synthesis were determined using western-blotting. Results: We observed an inverse relationship between circulating CRP level and muscle mass (β= -0.646 (95% CI: -0.888, -0.405) p<0.05) and demonstrated a reduction (p < 0.05) in the size of human myotubes exposed to CRP for 72 h. We next showed that this morphological change was accompanied by a CRP-mediated reduction (p < 0.05) in muscle protein fractional synthetic rate of human myotubes exposed to CRP for 24 h. We also identified a CRP-mediated increased phosphorylation (p<0.05) of regulators of cellular energy stress including AMPK and downstream targets, raptor and ACC-β, together with decreased phosphorylation of Akt and rpS6, which are important factors controlling protein synthesis. Conclusion: This work established for the first time mechanistic links by which chronic elevation of CRP can contribute to age-related decline in muscle function.

What Is Lipotoxicity?

Enlarged fat cells in obese adipose tissue diminish capacity to store fat and are resistant to the anti-lipolytic effect of insulin. Insulin resistance (IR)-associated S-nitrosylation of insulin-signaling proteins increases in obesity. In accordance with the inhibition of insulin-mediated anti-lipolytic action, plasma free fatty acid (FFA) levels increase. Additionally, endoplasmic reticulum stress stimuli induce lipolysis by activating cyclic adenosine monophosphate/Protein kinase A (cAMP/PKA) and extracellular signal-regulated kinase ½ (ERK1/2) signaling in adipocytes. Failure of packaging of excess lipid into lipid droplets causes chronic elevation of circulating fatty acids, which can reach to toxic levels within non-adipose tissues. Deleterious effects of lipid accumulation in non-adipose tissues are known as lipotoxicity. In fact, triglycerides may also serve a storage function for long-chain non-esterified fatty acids and their products such as ceramides and diacylglycerols (DAGs). Thus, excess DAG, ceramide and saturated fatty acids in obesity can induce chronic inflammation and have harmful effect on multiple organs and systems. In this context, chronic adipose tissue inflammation, mitochondrial dysfunction and IR have been discussed within the scope of lipotoxicity.

Optic neuropathy and increased retinal glial fibrillary acidic protein due to microbead-induced ocular hypertension in the rabbit.

To characterize whether a glaucoma model with chronic elevation of the intraocular pressure (IOP) was able to be induced by anterior chamber injection of microbeads in rabbits. In order to screen the optimal dose of microbead injection, IOP was measured every 3d for 4wk using handheld applanation tonometer after a single intracameral injection of 10 µL, 25 µL, 50 µL or 100 µL microbeads (5×106 beads/mL; n=6/group) in New Zealand White rabbits. To prolong IOP elevation, two intracameral injections of 50 µL microbeads or phosphate buffer saline (PBS) were made respectively at days 0 and 21 (n=24/group). The fellow eye was not treated. At 5wk after the second injection of microbeads or PBS, bright-field microscopy and transmission electron microscopy (TEM) were used to assess the changes in the retina. The expression of glial fibrillary acidic protein (GFAP) in the retina was evaluated by immunofluorescence, quantitative real-time polymerase chain reaction and Western blot at 5wk after the second injection of microbeads. Following a single intracameral injection of 10 µL, 25 µL, 50 µL or 100 µL microbead, IOP levels showed a gradual increase and a later decrease over a 4wk period after a single injection of microbead into the anterior chamber of rabbits. A peak IOP was observed at day 15 after injection. No significant difference in peak value of IOP was found between 10 µL and 25 µL groups (17.13±1.25 mm Hg vs 17.63±0.74 mm Hg; P=0.346). The peak value of IOP from 50 µL group (23.25±1.16 mm Hg) was significantly higher than 10 µL and 25 µL groups (all P<0.05). Administration of 100 µL microbead solution (23.00±0.93 mm Hg) did not lead to a significant increase in IOP compared to the 50 µL group (P=0.64). A prolonged elevated IOP duration up to 8wk was achieved by administering two injections of 50 µL microbeads (20.48±1.21 mm Hg vs 13.60±0.90 mm Hg in PBS-injected group; P<0.05). The bright-field and TEM were used to assess the changes of retinal ganglion cells (RGCs). Compared with PBS-injected group, the extended IOP elevation was associated with the degeneration of optic nerve, the reduction of RGC axons (47.16%, P<0.05) and the increased GFAP expression in the retina (4.74±1.10 vs 1.00±0.46, P<0.05). Two injections of microbeads into the ocular anterior chamber of rabbits lead to a prolonged IOP elevation which results in structural abnormality as well as loss in RGCs and their axons without observable ocular structural damage or inflammatory response. We have therefore established a novel and practical model of experimental glaucoma in rabbits.

A Period of Controlled Elevation of IOP (CEI) Produces the Specific Gene Expression Responses and Focal Injury Pattern of Experimental Rat Glaucoma.

PurposeWe determine if several hours of controlled elevation of IOP (CEI) will produce the optic nerve head (ONH) gene expression changes and optic nerve (ON) damage pattern associated with early experimental glaucoma in rats.MethodsThe anterior chambers of anesthetized rats were cannulated and connected to a reservoir to elevate IOP. Physiologic parameters were monitored. Following CEI at various recovery times, ON cross-sections were graded for axonal injury. Anterior ONHs were collected at 0 hours to 10 days following CEI and RNA extracted for quantitative PCR measurement of selected messages. The functional impact of CEI was assessed by electroretinography (ERG).ResultsDuring CEI, mean arterial pressure (99 ± 6 mm Hg) and other physiologic parameters remained stable. An 8-hour CEI at 60 mm Hg produced significant focal axonal degeneration 10 days after exposure, with superior lesions in 83% of ON. Message analysis in CEI ONH demonstrated expression responses previously identified in minimally injured ONH following chronic IOP elevation, as well as their sequential patterns. Anesthesia with cannulation at 20 mm Hg did not alter these message levels. Electroretinographic A- and B-waves, following a significant reduction at 2 days after CEI, were fully recovered at 2 weeks, while peak scotopic threshold response (pSTR) remained mildly but significantly depressed.ConclusionsA single CEI reproduces ONH message changes and patterns of ON injury previously observed with chronic IOP elevation. Controlled elevation of IOP can allow detailed determination of ONH cellular and functional responses to an injurious IOP insult and provide a platform for developing future therapeutic interventions.

Associations of acute and chronic stress hormones with cognitive functions in autism spectrum disorder

Extensive studies have reported cognitive abnormalities in neurodevelopmental disorders, such as autism spectrum disorder (ASD). Another line of evidence suggests that stress also affects cognitive functions. In this study, we investigated whether there were associations between stress hormones and cognitive functions in ASD and typically developing (TD) children. Cognitive functions in ASD and TD children were evaluated with a battery of psychological tests for working memory, behavioral flexibility, and social cognition for emotional assessments of others. ASD children exhibited higher hair and salivary cortisol, which reflects chronic and acute stress hormone levels of subjects, respectively, than TD children. Autism-spectrum quotient (AQ) was positively correlated with hair cortisol and the scores of Spence Children's Anxiety Scale in ASD children. In addition, a negative correlation was present between spatial working memory performance and hair cortisol in ASD, but not in TD, children. These results suggest that chronic stress hormone elevation may have relationships with some aspects of cognitive dysfunction in ASD subjects.

Tryptophan hydroxylase 1 Inhibition Impacts Pulmonary Vascular Remodeling in Two Rat Models of Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease defined by a chronic elevation in pulmonary arterial pressure with extensive pulmonary vascular remodeling and perivascular inflammation characterized by an accumulation of macrophages, lymphocytes, dendritic cells, and mast cells. Although the exact etiology of the disease is unknown, clinical as well as preclinical data strongly implicate a role for serotonin (5-HT) in the process. Here, we investigated the chronic effects of pharmacological inhibition of tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in peripheral 5-HT biosynthesis, in two preclinical models of pulmonary hypertension (PH), the monocrotaline (MCT) rat and the semaxanib (SUGEN, Medinoah, Suzhou, China)-hypoxia rat. In both PH models, ethyl (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1'-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate and ethyl (S)-8-(2-amino-6-((R)-1-(3',4'-dimethyl-3-(3-methyl-1 H-pyrazol-1-yl)-[1,1'-biphenyl]-4-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate, novel orally active TPH1 inhibitors with nanomolar in vitro potency, decreased serum, gut, and lung 5-HT levels in a dose-dependent manner and significantly reduced pulmonary arterial pressure, and pulmonary vessel wall thickness and occlusion in male rats. In the MCT rat model, decreases in lung 5-HT significantly correlated with reductions in histamine levels and mast cell number (P < 0.001, r2 = 0.88). In contrast, neither ambrisentan nor tadalafil, which are vasodilators approved for the treatment of PAH, reduced mast cell number or 5-HT levels, nor were they as effective in treating the vascular remodeling as were the TPH1 inhibitors. When administered in combination with ambrisentan, the TPH1 inhibitors showed an additive effect on pulmonary vascular remodeling and pressures. These data demonstrate that in addition to reducing vascular remodeling, TPH1 inhibition has the added benefit of reducing the perivascular mast cell accumulation associated with PH.

Islet adaptations in fetal sheep persist following chronic exposure to high norepinephrine.

Complications in pregnancy elevate fetal norepinephrine (NE) concentrations. Previous studies in NE-infused sheep fetuses revealed that sustained exposure to high NE resulted in lower expression of α2-adrenergic receptors in islets and increased insulin secretion responsiveness after acutely terminating the NE infusion. In this study, we determined if the compensatory increase in insulin secretion after chronic elevation of NE is independent of hyperglycemia in sheep fetuses and whether it is persistent in conjunction with islet desensitization to NE. After an initial assessment of glucose-stimulated insulin secretion (GSIS) at 129 ± 1 days of gestation, fetuses were continuously infused for seven days with NE and maintained at euglycemia with a maternal insulin infusion. Fetal GSIS studies were performed again on days 8 and 12. Adrenergic sensitivity was determined in pancreatic islets collected at day 12. NE infusion increased (P < 0.01) fetal plasma NE concentrations and lowered (P < 0.01) basal insulin concentrations compared to vehicle-infused controls. GSIS was 1.8-fold greater (P < 0.05) in NE-infused fetuses compared to controls at both one and five days after discontinuing the infusion. Glucose-potentiated arginine-induced insulin secretion was also enhanced (P < 0.01) in NE-infused fetuses. Maximum GSIS in islets isolated from NE-infused fetuses was 1.6-fold greater (P < 0.05) than controls, but islet insulin content and intracellular calcium signaling were not different between treatments. The half-maximal inhibitory concentration for NE was 2.6-fold greater (P < 0.05) in NE-infused islets compared to controls. These findings show that chronic NE exposure and not hyperglycemia produce persistent adaptations in pancreatic islets that augment β-cell responsiveness in part through decreased adrenergic sensitivity.

Reversal of functional loss in a rat model of chronic intraocular pressure elevation.

This pilot study considered whether intraocular pressure (IOP) lowering could reverse ganglion cell dysfunction in a rat model of chronic ocular hypertension. A circumlimbal suture was applied in one eye to induce ocular hypertension (n = 7) in Long-Evans rats. The contralateral eye served as an untreated control. After 8 weeks of IOP elevation the suture was removed to lower IOP for the remaining 7 weeks. Electroretinogram (ERG) and optical coherence tomography (OCT) were measured at baseline, 2, 4, 8, 12 and 15 weeks. Retinae were collected for histology at week 15. In sutured eyes, IOP was elevated by 7-11 mmHg above control eyes (12 ± 0.2 mmHg [standard error of the mean]). Eight weeks of chronic IOP elevation resulted in a reduction of the ganglion cell mediated positive Scotopic Threshold Response (pSTR, -25 ± 7% of baseline), as well as smaller photoreceptor (-7 ± 4%) and bipolar cell mediated responses (-6 ± 5%). After suture removal, IOP recovered to normal. By 15 weeks the a-wave (0 ± 6%), b-wave (-2 ± 6%) and pSTR had recovered back to baseline (from -25 ± 7% to -4 ± 6%). The retinal nerve fiber layer was thinned by -9 ± 3% at week 8 and showed no further decline at week 15 (-10 ± 2%). Cell numbers in the ganglion cell layer were similar between suture removal and control eyes at week 15 (3543 ± 478 vs 4057 ± 476 cells mm-2 ). The circumlimbal suture model might be a useful platform to study the reversibility of neuronal dysfunction from chronic IOP challenge.

Morphine Protects Spinal Cord Astrocytes from Glutamate-Induced Apoptosis via Reducing Endoplasmic Reticulum Stress.

Glutamate is not only a neurotransmitter but also an important neurotoxin in central nervous system (CNS). Chronic elevation of glutamate induces both neuronal and glial cell apoptosis. However, its effect on astrocytes is complex and still remains unclear. In this study, we investigated whether morphine, a common opioid ligand, could affect glutamate-induced apoptosis in astrocytes. Primary cultured astrocytes were incubated with glutamate in the presence/absence of morphine. It was found that morphine could reduce glutamate-induced apoptosis of astrocytes. Furthermore, glutamate activated Ca2+ release, thereby inducing endoplasmic reticulum (ER) stress in astrocytes, while morphine attenuated this deleterious effect. Using siRNA to reduce the expression of κ-opioid receptor, morphine could not effectively inhibit glutamate-stimulated Ca2+ release in astrocytes, the protective effect of morphine on glutamate-injured astrocytes was also suppressed. These results suggested that morphine could protect astrocytes from glutamate-induced apoptosis via reducing Ca2+ overload and ER stress pathways. In conclusion, this study indicated that excitotoxicity participated in the glutamate mediated apoptosis in astrocytes, while morphine attenuated this deleterious effect via regulating Ca2+ release and ER stress.