Isoproterenol induces in vivo functional and metabolic abnormalities: similar to those found in the infarcted rat heart.

Chronic isoproterenol administration produces a rapid, highly reproducible rodent model of cardiac hypertrophy. Yet, despite widespread use of this model, the effects of isoproterenol on in vivo cardiac function and substrate metabolism are unknown. Isoproterenol (5 mg.kg(-1).day(-1)) was infused for 7 days in male Wistar rats (n = 22). In vivo magnetic resonance imaging (MRI) showed that left ventricular mass increased by 37% and end-diastolic and systolic volumes increased by 33% and 73%, respectively, following isoproterenol infusion. Cardiac function at the base of the left ventricle was normal, but apical ejection fraction decreased from 90% to 31% and apical free wall thickening decreased by 94%, accompanied by increased fibrosis and inflammation. Myocardial palmitate oxidation rates were 25% lower, and citrate synthase and medium chain acyl-coenzyme A dehydrogenase activities were reduced by 25% and 29%, respectively, following isoproterenol infusion. Fatty acid transporter protein levels were 11-52% lower and triglyceride concentrations were 55% lower in isoproterenol-infused rat hearts. Basal glycolysis and glycogen concentration were not changed, yet insulin stimulated glycolysis was decreased by 32%, accompanied by 33% lower insulin stimulated glucose transporter, GLUT4, protein levels in rat hearts following isoproterenol infusion, compared with controls. In conclusion, isoproterenol infusion impaired in vivo cardiac function, induced hypertrophy, and decreased both fatty acid and glucose metabolism, changes similar in direction and magnitude to those found in the rat heart following moderate severity myocardial infarction.

Longitudinally preserved psychomotor performance in long-term asymptomatic HIV-infected individuals.

BACKGROUND: Recent case reports have suggested that some asymptomatic HIV-infected individuals can develop CNS disturbances despite intact immunologic functioning and long-term suppression of plasma HIV concentrations to undetectable levels. This possibility has not yet been systematically studied longitudinally. METHODS: Using longitudinal data from the Multicenter AIDS Cohort Study, we investigated neuropsychological performance in long-term asymptomatic HIV-infected men who have sex with men. Performance over a 5-year period on the Symbol Digit Modalities test and the Trail Making Tests were compared in three HIV-positive asymptomatic groups [defined as 1) highly active antiretroviral therapy (HAART) treated with undetectable viral loads (n = 83), 2) AIDS-free for more than 15 years without HAART (n = 29), and 3) absence of clinical AIDS or CD4(+) lymphocyte count below 200 cells/muL at the beginning and end of the study period (n = 233)] and in HIV-negative controls (n = 237). Data were analyzed using linear mixed models and proportional odds logistic regression modeling with generalized estimating equations. RESULTS: There was no evidence of performance differences or performance declines over the 5-year period of study in any of the three long-term asymptomatic groups as compared with the HIV-negative group in the Symbol Digit Modalities test or the Trail Making Tests. Performance decrements were, however, observed with increasing age in each of the tests administered, demonstrating that performance declines could be detected by these methods. CONCLUSIONS: Regardless of how long-term asymptomatic status was defined immunologically or virologically, neuropsychological test performances remained stable. These findings suggest that psychomotor speed is preserved over many years in HIV-infected individuals with controlled HIV viremia.

Comparing contingent valuation and contingent ranking: a case study considering the benefits of urban river water quality improvements.

This paper contrasts applications of both the contingent valuation (CV) and contingent ranking (CR) methods as applied to a common issue, the valuation of improvements to the water quality of an urban river (the River Tame, running through the city of Birmingham, UK). Building upon earlier experimental work, the CV design used ensures that respondents are fully aware of all impending valuation tasks prior to undertaking any one of those tasks. Such an approach is directly comparable to the CR design for which full awareness of all options is a pre-requisite. Findings indicate that the CV responses exhibit strong internal consistency with expected relationships observed between values and theoretically expected parameters. External comparisons show that CR valuations are substantially larger than those elicited through CV (with protest votes excluded), and that the response rate for the CR survey is significantly higher than that for the CV survey.

A quantitative study of bioenergetics in skeletal muscle lacking utrophin and dystrophin.

Muscle energetics and function were investigated in the hindlimb of mice lacking dystrophin (mdx), utrophin and dystrophin (utr-dys) and controls (C57Bl/10) using 31P and 1H magnetic resonance techniques, electrical nerve stimulation and direct biochemical analysis. At rest, [adenosine triphosphate] and [total creatine] were lowest in utr-dys, while [inorganic phosphate] was elevated. Calculated [adenosine diphosphate] was 3-fold higher in mdx and 5-fold higher in utr-dys than in controls, consistent with an increased adenosine triphosphate requirement for ion pump activity. During stimulation, force production was low only in utr-dys, and this was reflected in the bioenergetic changes. Initial recovery rates of [phosphocreatine] and [adenosine diphosphate] after stimulation were rapid in all groups, indicative of normal mitochondrial adenosine triphosphate production in utr-dys and mdx. Recovery of pH was slow in utr-dys. The data indicate that the severe abnormalities which are present in the absence of utrophin and dystrophin leave basic muscle energetics intact and appear confined to processes involving the sarcolemma.

Acute exposure to a novel stressor further reduces the habituated corticosterone response to restraint in rats.

The present study sought to identify dishabituation of the hypothalamic-pituitary-adrenal(HPA) axis response to different psychological stressors. Young adult male Sprague Dawley rats were exposed to five, 1 h sessions of restraint stress on five consecutive days. On the sixth day, and 2 h before additional exposure to restraint, animals were subjected to 30 min of a small (27cm square), elevated open field stressor (pedestal), which served as the dishabituating stimulus. We predicted HPA axis response dishabituation in chronically restrained rats exposed to the novel pedestal. Rats which underwent five days of restraint stress showed significantly blunted plasma corticosterone levels to restraint (habituation) as compared to restraint-nai've rats. However, rats which underwent five sessions of restraint responded with an enhanced habituation response when confronted with restraint shortly after exposure to the novel pedestal. Instead of HPA axis response dishabituation, we observed enhanced habituation. Subsequent experiments determined that a 1.25 mgkg corticosterone injection could substitute for pedestal exposure to produce enhanced restraint habituation.Combined treatment with both the glucocorticoid receptor antagonist RU40555 (30 mgkg)and the mineralocorticoid receptor antagonist RU283 18 (50 mgkg) blocked the expression of enhanced habituation after pedestal exposure. Thus, the delayed corticosterone negative feedback produced by novel stress led to enhanced expression of corticosterone response habituation.

Selective blockade of the mineralocorticoid receptor impairs hypothalamic-pituitary-adrenal axis expression of habituation.

The present study investigated the role of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) in the expression of habituation of the hypothalamic-pituitary-adrenal (HPA) axis response to stress. Male rats were restrained for 1 h per day for six consecutive days. On day 6, 1 h prior to restraint stress, both restraint-naive and repeatedly restrained rats were injected s.c. with either vehicle (propylene glycol) or one of three corticosteroid receptor antagonist treatments: selective MR antagonist (RU28318 or spironolactone), selective GR antagonist (RU40555), or both MR and GR antagonists combined (RU28318 + RU40555). Blood samples were collected for corticosterone measurement at the beginning of stress, during stress, and 1 h after stress termination. Repeated restraint stress produced significant habituation of corticosterone responses. Acute treatment with the combined MR and GR antagonists prevented the expression of habituation. When tested alone, the MR antagonist also blocked the expression of corticosterone-response habituation, whereas the GR antagonist had no effect. Neither the MR, nor the GR antagonists alone, significantly altered the corticosterone response to restraint in rats exposed to restraint for the first time. The final experiment examined the corticosterone response to a corticotropin releasing hormone (CRH, 3 microg/kg i.p.) challenge. Neither previous exposure to restraint or acute pretreatment with the combined MR and GR antagonists (RU28318 + RU40555) altered the corticosterone response to CRH challenge. This result indicates that the expression of habituation and its blockade by corticosteroid receptor antagonists is not a result of altered pituitary-adrenal response to CRH. Overall, this study suggests that MR plays an important role in constraining the HPA axis response to restraint stress in restraint-habituated rats. The dependence of the HPA axis on MR-mediated corticosteroid negative feedback during acute stress may be an important mechanism that helps maximize the expression of stress habituation and thereby minimize exposure of target tissues to corticosteroids in the context of repeated stress.

Response of the human triceps surae muscle to electrical stimulation during varying levels of blood flow restriction.

The changes in muscle force associated with varying degrees of lower-limb ischaemia were investigated. Isometric torque production by the triceps surae muscle was measured during a 5-min continuous train of 2-Hz electrical stimulation in six healthy young adults under different thigh cuff occlusion pressures. The reproducibility of this protocol when performed under complete ischaemia (tested five times over a 2-week period) was assessed as having a coefficient of variation (CV) for fatigue (end/initial force) of [mean (SEM) 12 (1)%; n = 5]. This compares favourably with that obtained for maximum voluntary contraction torque [CV 9 (1)%]. In six subjects, triceps surae muscle fatigue was assessed under thigh cuff pressures of 0, 6.7 kPa (50 mmHg, venous occlusion) and 28 kPa (210 mmHg, complete ischaemia), as well as two intermediate levels of occlusion that were established by cuff pressures of 13.4 (0.5) and 20.3 (1.1) kPa [103 (4) and 152 (8) mmHg, respectively]. These corresponded to ankle-brachial pressure indices of 1.3 and 0.8, respectively when subjects were seated, or 0.8 and 0.36 when supine. With undisturbed lower-leg circulation, force potentiated steadily over the 5 min of stimulation such that the final force was 135 (8)% of the initial value. With complete ischaemia, force fell to 47 (2)% of the initial value. Stimulation under thigh occlusion pressures of 6.7, 13.4 and 20.3 kPa elicited intermediate levels of reduction in force, graded according to the increasing restriction of perfusion. The results show that low-force twitch contractions, which themselves do not occlude blood flow, are extremely sensitive to impaired perfusion and may represent a viable alternative to established methods of muscle performance assessment in patients with blood flow insufficiency.

Dexamethasone suppression of corticosteroid secretion: evaluation of the site of action by receptor measures and functional studies.

A dose of dexamethasone was determined in rats (50 micrograms/kg s.c.) that suppressed the corticosterone response to restraint stress by 80%. Corticosteroid receptor occupancy estimates found that the 50 micrograms/kg s.c. dose of dexamethasone had no significant effect on available glucocorticoid receptor (GR) or mineralocorticoid receptor (MR) binding in brain regions (hypothalamus, hippocampus and cortex); on the other hand dexamethasone produced a selective and significant decrease in available GR in peripheral tissues (pituitary and spleen). Functional studies showed that the 50 micrograms/kg s.c. dose of dexamethasone completely blocked the effects of corticotropin-releasing hormone (CRH; 0.3-3.0 micrograms/kg i.p.) on corticosterone secretion, but did not inhibit the corticosterone response to an adrenocorticotropin hormone (ACTH; 2.5 I.U./kg i.p.) challenge. These studies indicate that this dose of dexamethasone exerts its inhibitory effects on the HPA axis primarily by acting at GR in the pituitary. The plasma dexamethasone levels produced by this dose of dexamethasone are similar to those present in humans the afternoon after an oral dexamethasone suppression test (DST), a time at which many depressed patients escape from dexamethasone suppression. These results support and extend other studies which suggest that the DST provides a direct test of the effects of increased GR activation in the pituitary on ACTH and cortisol secretion.

Antisense blockade of p21/WAF1 decreases radiation-induced G2 arrest in esophageal squamous cell carcinoma.

Introduction. The p21 cyclin-dependent kinase inhibitor arrests the cell cycle following DNA damage at the G1-S checkpoint. Recent literature has also demonstrated a role for p21 in G2 arrest. Studies with esophageal squamous cell carcinoma (ESSC) lines have shown that radiation-induced p21 protein induction is associated with G2 arrest. The aim of this study was to determine if p21 blockade would affect this G2 arrest pattern. Method. We transfected the ESSC line KYSE 170 with antisense p21 mRNA oligonucleotides or scrambled 20-mer p21 control oligonucleotides using a lipofectant reagent. Cells were exposed to 6 Gy or used as controls. p21 levels were determined by ELISA. Cell cycle arrest pattern was determined via flow cytometry. Student's t test and ANOVA were used to compare p21 levels and percentages of G2 arrest. Results. Irradiated/scrambled cells expressed 10.1 ng/ml of p21 protein compared to irradiated/antisense cells at 2.1 ng/ml (P < 0.05), demonstrating successful blockade of p21. Irradiated cells displayed prominent G2 arrest following 6 Gy doses, but there was a decrease from 65 to 44% G2 phase when p21 was blocked (P < 0.05). Conclusions. We have demonstrated that G2 arrest accompanying irradiation of ESSC cells decreases when p21 protein production is blocked via antisense oligonucleotides. These data support a role for p21 in mediating G2 arrest in these cells.

p21 expression is increased by irradiation in esophageal squamous cell carcinoma.

The p21 cyclin-dependent kinase inhibitor blocks cell cycle transition and replication in response to DNA damage. Although required for p53-mediated cell cycle arrest, p21 expression can also be initiated via p53-independent pathways. This study examines the postirradiation expression of p21 in squamous cell carcinoma (SCC) of the esophagus to determine whether this p21 production is p53-dependent or independent. We sequenced p53 exons 5-8 and the exon-intron junctions of four esophageal SCC lines, KYSEs 30, 150, 410, and 960. We exposed these same lines to increasing doses of radiation (3 to 24 Gy) and subsequently extracted their total protein. The p21 content of the protein was then determined via p21 ELISA. The same cell lines were also irradiated for determination of clonogenic survival over the course of 7 days. Cells were counted via a Coulter machine. KYSE 30 and 410 were found to have mutations in their p53 genes, while KYSEs 150 and 960 had wild-type p53 genomes. All cell lines produced basal levels of p21 (from 3.2 to 7.8 ng/ml) and all lines increased production in response to radiation (6.4 to 16.8 ng/ml at 3 Gy, P < 0.05 for all lines vs. their controls). Cells displayed dose-dependent mortality in response to radiation, with only minor differences in survival between two of the lines. All of the esophageal SCC lines studied produced basal p21 and increased p21 with irradiation. p21 production was independent of p53 status. Previous reports have failed to detect elevation of p21 expression in esophageal SCC, and this is the first report of radiation-induced p21 expression in esophageal cell lines.

Evidence for mineralocorticoid receptor facilitation of glucocorticoid receptor-dependent regulation of hypothalamic-pituitary-adrenal axis activity.

These studies further evaluated the relative role of mineralocorticoid (type I) and glucocorticoid (type II) receptors in mediating corticosteroid feedback regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Acute treatment of rats with the selective mineralocorticoid receptor antagonist, RU28318 (50 mg/kg sc), produced elevated basal corticosterone levels in the morning, but had no effect on basal corticosterone levels in the evening or on restraint stress corticosterone levels at either time of day. Acute treatment with the selective glucocorticoid receptor antagonist, RU40555 (30 mg/kg sc) had no effect on basal or restraint stress corticosterone levels at either time of day. However, combined treatment with RU28318 and RU40555 produced an elevation of evening basal corticosterone levels (and morning basal on one occasion) and produced an increase in corticosterone levels during and after stress at both times of day. In a separate experiment conducted in the morning, the combined RU28318 and RU40555 treatment also produced elevated ACTH responses during restraint stress. Based on available corticosteroid receptor measures, the RU28318 treatment was estimated to selectively occupy approximately 85% of mineralocorticoid receptors in rat brain, whereas the RU40555 treatment was estimated to selectively occupy approximately 50% of glucocorticoid receptors in rat brain. We conclude that mineralocorticoid receptor activation is necessary and sufficient to maintain low basal corticosterone levels during the circadian trough, whereas glucocorticoid receptor activation is necessary to constrain corticosterone secretion during the circadian peak or during acute stress. However, even during the circadian peak or acute stress, mineralocorticoid receptor activation plays an important role in facilitating the glucocorticoid receptor dependent regulation of HPA axis activity by corticosterone.

Evaluation of RU28318 and RU40555 as selective mineralocorticoid receptor and glucocorticoid receptor antagonists, respectively: receptor measures and functional studies.

Corticosterone regulates a wide range of physiological parameters. Two receptors for corticosterone have been identified, the mineralocorticoid (type I) receptor (MR) and the glucocorticoid (type II) receptor (GR). To determine the relative role of these two receptors in mediating the effects of endogenous corticosterone, many studies have relied on the use of putative selective corticosteroid receptor antagonists. This study further examined the in vivo receptor selectivity of two compounds, RU28318 and RU40555 that are believed to be selective antagonists for MR and GR, respectively. Acute treatment of adrenalectomized rats with RU28318 (10-50 mg/kg) selectively decreased ex-vivo available MR binding in the hippocampus, whereas acute treatment with RU40555 (10-30 mg/kg) selectively decreased available GR binding in the hippocampus and pituitary. These receptor binding measures suggest that RU28318 in vivo selectively occupied MR, and that RU40555 in vivo selectively occupied GR. In functional studies, RU28318 (50 mg/kg) blocked the normalizing effect of aldosterone (120 microg/kg) on saline intake of adrenalectomized rats. RU40555 (30 mg/kg) blocked the suppressive effect of dexamethasone (50 microg/kg) on acute stress-induced corticosterone secretion. These studies further support the in vivo corticosteroid receptor selectivity of these two compounds and confirms their effective corticosteroid antagonistic properties.

Diazepam attenuation of restraint stress-induced corticosterone levels is enhanced by prior exposure to repeated restraint.

Prior research has demonstrated that diazepam decreases hypothalamic-pituitary-adrenal cortex (HPA) axis activity in stressful contexts but, paradoxically, acts as a stimulator of basal axis activity. Also, several investigators have reported that low doses of diazepam are not effective in reducing stress-induced corticosterone (CORT) levels, yet similar doses typically produce anxiolytic effects on behavioral measures of fear and anxiety. We have examined the effects of diazepam on plasma CORT levels in male Sprague-Dawley rats utilizing a repeated restraint paradigm. Consistent with most literature, diazepam administered IP (1.5, 3.0, or 6.0 mg/kg) 1 h prior to restraint increased non-stress, baseline plasma CORT levels in a dose-dependent fashion. During the first exposure to the 1 h restraint-stress procedure, CORT levels of diazepam-injected rats did not differ from the stress levels of controls except at the 60-min stress time point in those subjects receiving 6.0 mg/kg. However, diazepam at all three doses was able to attenuate the stress-induced increase in CORT following 5 days of diazepam+restraint treatment. Using the 3.0 mg/kg dose as a probe, it was found that this effect was not dependent on the repeated administration of diazepam, but rather on repeated exposure to restraint. These results suggest that repeated restraint produces a change in neural sensitivity to benzodiazepines.

Adaptation to arterial wall hypoxia demonstrated in vivo with oxygen microcathodes.

Iliofemoral arteries of 9 rabbits were balloon de-endothelialized resulting in subintimal thickening. Contrary to expectation, enzyme and lactate determinations did not indicate arterial wall hypoxia when compared with arteries of 10 control rabbits. The explanation came from in vivo measurement of oxygen tension profiles across the de-endothelialized and control femoral arteries and from the subsequent histological findings. They showed that the impaired oxygen supply of the de-endothelialized arteries with subintimal thickening was counteracted by a centripetal oxygenation of the arterial wall obviously induced by proliferation of newly formed nutrient vessels in the adventitia. Such adaptation is an important mechanism against hypoxia induced by arterial injury and may be an essential protective factor in atherogenesis.

Metastatic rat adenocarcinoma: histochemical evaluation of fibroblasts and extracellular matrix.

This study was based on the hypothesis that after tumour transplantation, fibroblast metabolism increases adjacent to a tumour and this increase correlates with an increase in certain components of the extracellular matrix. A serial histochemical study of the cellular metabolism and extracellular matrix in a fast-growing mammary rat carcinoma was designed. The model used was the N-nitrosomethylurea-induced adenocarcinoma. At 24, 48, 72 or 96 h after transplantation, tumours and surrounding tissues were excised and processed. Ribonucleic acid and succinate dehydrogenase stains were used to indicate cellular metabolism; the extracellular matrix was stained for collagen, elastin, acid mucopolysaccharides, mucoproteins, glycoproteins and glycolipids. The results of this histology were compared with the histology of nonneoplastic transplants. In subcutaneous tissue adjacent to neoplasia, fibroblasts were abundant and showed an increase in metabolism between 24-96 h; this was correlated with an increase in collagen. For nonneoplastic transplants, fibroblasts were present only at 96 h, and collagen increases did not occur. It is inferred from the results that the tumour transplant is responsible for the increase in fibroblast metabolism in vivo which in turn increases fibre production.

Performance evaluation of recessed microcathodes: criteria for tissue PO2 measurement.

Measurement of local tissue PO2 using recessed microcathodes has again been criticized. Therefore, we reexamined electrode performance. Sharply beveled electrodes (3 micron external diameter) were fabricated with several tip recess lengths (4-10 micron), and some recesses were filled with hydrated polymer. In vitro, 2-mm agar (3%) sheets were equilibrated with solution of known PO2 (continuously flowing). Electrode currents at 100-micron intervals through the agar and of convected superfusion solution were compared. At the longest recess lumen length-to-diameter ratio of 10, minimum response midagar (1 mm) averaged 98%. Performance improved with the use of recess polymer and increased recess length. For in vivo studies, microcathodes (ratio approximately 10) were fluid calibrated, and PO2 was measured at 10-20 micron through canine femoral artery walls. PO2 distribution fit a model for radial diffusion with medial O2 consumption. After local cyanide application to the femoral wall, PO2 fit a model for radial diffusion without tissue O2 consumption. Carefully designed microcathodes and experiments measure accurate tissue PO2.

Methods in laboratory investigation. Identification and evaluation of histopathology at microelectrode puncture sites.

Tissue puncture techniques using microelectrodes for various measurements have been criticized for producing undetermined degrees of tissue damage. Therefore, a method permitting routine identification of puncture tracks was developed to determine local microelectrode-induced injury. Rabbits were anesthetized and the femoral arteries surgically exposed. A 3-ml mixture of saline-India ink suspension was introduced through an ear vein. Oxygen-sensitive (pO2) microcathodes were advanced into and through the arterial wall at 10- or 20-micron intervals using a stepping microdrive to 150 to 450 micron and then withdrawn. The arteries were fixed in 10% formalin and gelatin embedded, and serial frozen sections (less than or equal to 15 micron) of the microelectrode puncture area were made. We observed within 5 minutes of microcathode withdrawal a dark, punctate, microscopic discoloration within the arterial wall. Histologically, ink distribution within the arterial wall demonstrated an acute permeability change: puncture depths generally less than 300 micron showed ink-lined microelectrode tracks (generally less than 2 micron wide) in the media, and greater puncture depths showed local hemorrhage and focal laminar accumulation of ink which extended from the track. The immediate adjacent area to microelectrode puncture depths less than 300 micron showed an apparent intact internal elastic lamina and media. Therefore, microelectrode damage has been shown to be primarily limited to microelectrode tissue tracks.

Correlation of regional disease and in vivo PO2 in rat mammary adenocarcinoma.

A knowledge of the distribution of oxygen tension (PO2) and vascularization in neoplasia has been fundamental to understanding relationships between tumor growth, hypoxia, and therapy. We have combined recessed oxygen microcathode and freeze-substitution techniques to correlate in situ PO2 profiles and morphologic features in 7,12-dimethylbenz(a)anthracene (DMBA) tumors in rats. Overlying connective tissue of transplanted tumor was exposed by a 1-2 mm incision and a cross-stitch pattern demarcated electrode puncture sites for histologic reference. Three buffered salt solutions (BSS) with different PO2 were each allowed to flow through a well over the tumor where electrodes were placed for calibration. Zero electrode oxygen current was recorded from a buffered yeast-agar mixture of zero torr. PO2 was recorded at 5-mu intervals to approximately 1-2 mm. Atmospheric contamination was eliminated by continuous well flow of BSS, 30 torr. Finally, the tumor and surrounding tissues were quick-frozen in vivo with Freon 22 and liquid nitrogen. The tissue block was freeze-substituted and sectioned. PO2 profiles were superimposed onto correspondingly scaled photomicrographs. A viable periphery with a PO2 range of 50-82 torr and a transition to necrotic areas of PO2, 2-13 torr were observed. This transition was characterized by PO2 gradients within distances of 50-300 mu at variable puncture depths. This technique should be useful in further studies of growth, necrosis, and therapy.