An international database and integrated analysis tools for the study of cancer gene expression.

Researchers working collaboratively in Brazil and the United States have assembled an International Database of Cancer Gene Expression. Several strategies have been employed to generate gene expression data including expressed sequence tags (ESTs), serial analysis of gene expression (SAGE), and open reading-frame expressed sequence tags (ORESTES). The database contains six million gene tags that reflect the gene expression profiles in a wide variety of cancerous tissues and their normal counterparts. All sequences are deposited in the public databases, GenBank and SAGEmap. A suite of informatics tools was designed to facilitate in silico analysis of the gene expression datasets and are available through the NCI Cancer Genome Anatomy Project web site (http://cgap.nci.nih.gov).

In silico analysis of cancer through the Cancer Genome Anatomy Project.

The Cancer Genome Anatomy Project (CGAP) was designed and implemented to provide public datasets, material resources and informatics tools to serve as a platform to support the elucidation of the molecular signatures of cancer. This overview of CGAP describes the status of this effort to develop resources based on gene expression, polymorphism identification and chromosome aberrations, and we describe a variety of analytical tools designed to facilitate in silico analysis of these datasets.

Blood chemistry changes in the rat induced by high doses of nitronyl free radical spin traps.

For greatest efficacy, it is desirable to use spin trapping agents in the highest concentrations possible. Fifty-four male Sprague-Dawley rats were used to explore the relative toxicity of four representative nitronyl spin traps at doses chosen on the basis of earlier lethality studies. Most studies were confined to the 3- to 6-h period following drug injection, because the behavioral signs of toxicity are most evident early after injection and because spin trapping studies would typically be performed within this time frame. Doses of spin trap were dissolved in a corn oil/buffer vehicle and injected intraperitoneally (i.p.). Toxic signs were recorded periodically, and at the time of euthanasia or spontaneous death a blood sample was collected by cardiac puncture for clinical chemistry analysis and a necropsy was performed. Both gross pathology and histopathological examination of the major organs were essentially negative in all cases, with no obvious evidence of cellular damage being observed. Neither DMPO (232 mg/100 g b.wt.) nor PBN (100 mg/100 g b.wt.) were lethal in the present study, while both M4PO (20 and 40 mg/ 100 g b.wt.) and PyOBN (100 and 200 mg/100 g b.wt.) were lethal. Abnormal clinical chemistry findings were generally confined to those animals that died spontaneously or were euthanized early for humane reasons. In most cases, death was associated with marked seizure activity and impaired respiration, and deaths occurred within a few min to a few hours. The mechanism of toxicity was unclear due to the lack of histopathological evidence and the wide range of abnormal serum analytes in those rats killed by either M4PO or PyOBN. In conclusion, during the first 6 h after IP administration there is little indication of tissue damage by the nitrone spin traps until the dose is increased to a lethal level, at which point an acute, rapidly occurring, wide-spread disruption of tissue integrity seems to occur.

Biological spin trapping. II. Toxicity of nitrone spin traps: dose-ranging in the rat.

To obtain the strongest possible free radical spin adduct signal using the electron paramagnetic resonance spectroscopy-spin trapping technique, it is desirable to load an animal with the highest dose of spin trap possible. One hundred and twenty six male Sprague-Dawley rats were used to establish the toxic dose range for PBN (alpha-phenyl N-tert butyl nitrone) and 18 other similar spin traps. The lethal dose of PBN was found to be approximately 100 mg/100 g BW (0.564 mmol/100 g. The 18 other compounds were then tested, and their toxicities were gauged in terms of molar equivalents to PBN. Of these spin traps, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was found to be the least toxic (no toxic signs at twice the lethal dose for PBN) while 2,6-difluoro-PBN and M4PO (3,3,5,5-tetramethyl-1-pyrroline-N-oxide) were the most toxic, both causing death at one eighth the PBN-equivalent lethal dose. Nine of the 18 nitrones appeared non-toxic at the 0.25 PBN-equivalent lethal dose level.

Oxygen metabolism changes and outcome in response to immediate colloid treatment in the endotoxaemic rat.

A colloid (Hespan) fluid regimen in a 4 h rat model of endotoxaemia was used to prevent the development of the early hypodynamic phase of shock. Groups (N = 10 each) of isoflurane-anaesthetized, male Sprague-Dawley rats received either 1) E. coli endotoxin (E, 20 mg.kg-1 BW, i.v.), 2) 0.9% saline (S), 3) endotoxin + Hespan (E + H), or 4) saline + Hespan (S + H). After a 30 min baseline, 15 ml of 6% hetastarch (Hespan) were infused over 1 h beginning 1 min after endotoxin or saline. Pulmonary artery wedge pressures suggested no fluid overload in the E + H or S + H groups. By the end of the study, there were six spontaneous deaths in the E group vs. no deaths in the other groups. However, despite successful prevention of the early hypodynamic response together with increased cardiac output, increased oxygen delivery, decreased oxygen extraction, and sustained normal oxygen consumption in the E + H group, this fluid regimen failed to prevent significant and progressive acidaemia and hyperlactataemia. Also, by 4 h the E + H group exhibited declining blood pressures, marked hypoglycaemia, and significant small intestinal damage. Our results indicate that the early hypotensive, hypodynamic period is not crucial for the development of significant pathology in endotoxaemia, and that early flow-dependency of whole body oxygen uptake is not inherent to the early response to endotoxin in this model.

Effects of endotoxemia on the redox level of brain cytochrome a,a3 in rats.

We conducted the present study to determine the effect of endotoxin challenge on brain oxidative metabolism, after finding evidence in previous studies suggesting early uncoupling of mitochondrial oxidative phosphorylation in the rat small intestine during endotoxemia. Twenty male Sprague-Dawley rats were divided into two groups (N = 10 each) which received E. coli endotoxin (20 mg/kg BW) or an equal volume of 0.9% saline (1 ml/kg) by i.v. bolus. Catheter implantation and the subsequent data collection were conducted using isoflurane anesthesia with controlled ventilation. Hemodynamic and metabolic data were recorded for 30 min before and 60 min after endotoxin or saline injection. Tissue oxidative metabolism was monitored in vivo using differential multiwavelength near-infrared spectrophotometry. Optrodes were positioned on either side of the rat's head (transillumination mode) to monitor the redox state of mitochondrial cytochrome a,a3 (AA3) as well as the supply of oxygen to the brain as reflected by tissue oxyhemoglobin (HbO2). In contrast to our previous results for the small intestine, where the decrease in AA3 oxidation level was disproportionately greater than the concomitant HbO2 decrease, we found that the endotoxin-induced impairment in blood flow to the head was associated with a decrease in brain AA3 redox level, which was proportional to the decrease in tissue HbO2. This finding of an apparent oxygen-dependent AA3 redox shift in the brain during endotoxemia is similar to previous findings of others in hemorrhagic hypotension and hypoxic hypoxia. Possible mechanisms for the different mitochondrial AA3 redox responses to endotoxin in the brain and small intestine are discussed.

Rapid reduction of intestinal cytochrome a,a3 during lethal endotoxemia.

In vivo near-infrared spectrophotometry was used to determine whether lethal endotoxemia impairs small intestinal oxidative phosphorylation as reflected by the redox state of mitochondrial cytochrome a,a3 (AA3). Adult male Sprague-Dawley rats were anesthetized with 2.1% isoflurane in 30% O2:70% N2O, and the small intestine was partially exteriorized for spectrophotometric monitoring (OMNI-3). By 5 min after an iv bolus of Escherichia coli endotoxin (40 mg/kg, LD90, n = 7) a significant shift toward reduction in intestinal AA3 had occurred in association with hypotension and a marked fall in both superior mesenteric artery blood flow (SMAF) and cardiac output. In a separate group (n = 7) SMAF was kept at the baseline level by periodic infusions of donor rat plasma begun 1 min after endotoxin injection, and the reduction in AA3 was again found despite the fluid loading intervention which successfully maintained not only organ blood flow, but also cardiac output and mean arterial pressure in their normal ranges. Further experiments (n = 34) measuring SM vascular bed oxygen consumption indicated that intestinal VO2 remained unchanged during early endotoxemia. These findings suggest a rapid impairment of oxidative phosphorylation by endotoxin which seems to occur through direct (and/or indirect) toxic cellular effects rather than through impaired tissue perfusion.

Dose-related reduction of intestinal cytochrome a,a3 induced by endotoxin in rats.

Dose-related, endotoxin-induced changes in oxidative metabolism were investigated in vivo using near-infrared spectrophotometry. Sixty fasted male Sprague-Dawley rats were randomly allocated to groups (N = 10 each) receiving saline, 0.08, 0.3, 1.25, 5, or 20 mg/kg Escherichia coli endotoxin by i.v. bolus (1 ml/kg volume). Isoflurane anesthesia with controlled ventilation was used for surgery and the subsequent collection of hemodynamic and metabolic data. Approximately half the length of the small intestine was exteriorized for continuous spectrophotometric monitoring of tissue oxyhemoglobin (HbO2), deoxyhemoglobin (Hb), and mitochondrial cytochrome a,a3 (AA3) redox state. Hemodynamic parameters included arterial and central venous pressures, cardiac output, and superior mesenteric artery blood flow (SMAF). Intestinal AA3 exhibited a marked and sustained shift toward reduction in those groups (1.25, 5, and 20 mg/kg) which developed significant blood pressure and blood flow decreases. However, the AA3 reduction did not seem to reflect tissue hypoxia, since 1) the simultaneously measured tissue HbO2 levels remained essentially constant, 2) the AA3 redox shift did not reverse upon recovery of intestinal blood flow in the 1.25 mg/kg group, which exhibited only a transient decrease in SMAF, and 3) systemic oxygen consumption was unchanged in any group. Endotoxin-induced lethality was tested in six separate groups (N = 10 each) and was significant (60%) only in the 20 mg/kg group. These results suggest that endotoxin may induce a direct (or rapidly mediated indirect) impairment of cellular oxidative phosphorylation.

An assessment of plasma histamine concentrations during documented endotoxic shock.

Recent reviews of the literature involving histamine release during sepsis and endotoxemia have reported that the majority of the studies are inconclusive due to inadequate assays or experimental protocols. In a controlled experimental setting we have employed a specific and sensitive radioenzymatic assay to determine plasma histamine concentrations temporally during documented endotoxin-induced shock in the conscious rat. Cardiovascular and metabolic measurements for the control group (n = 7) were normal during the study period. Endotoxin (n = 8, LD/90-24 hrs.) induced an early transient hypotensive episode and increase in systemic vascular resistance and a sustained decrease in cardiac index and central venous pressure and increase in heart and respiratory rates. Hypoglycemia and hyperlacticemia were present at the end of the four-hour study period. The small intestine was severely hemorrhaged in all animals given endotoxin. Histamine concentrations for the control group were unchanged throughout the study period. Contrary to previous reports, this model of endotoxemia revealed unchanging histamine concentrations during the first 30 minutes which were temporally coincident with the characteristic early hypotensive episode evoked by endotoxin. The histamine concentrations at 60 and 240 minutes following endotoxin were increased two and three-fold, respectively, compared to the control group. Three of the 8 rats given endotoxin died before four hours; histamine concentrations in plasma taken when death appeared certain were 42, 91, and 174, compared to the control value of approximately 8 ng/ml. There was no clear association of the increases in plasma histamine with any of the parameters measured in this study: however, established histamine effects may have been masked by the pre-existing effects of other mediators known to be active during endotoxemia. In separate groups of animals endotoxin (n = 5) elicited early increases in plasma concentrations of norepinephrine (5-fold) and epinephrine (8-fold) that remained elevated for the 4-hour study period while the control group (n = 4) remained stable. This study establishes that a) plasma histamine concentrations are increased during endotoxemia, b) plasma histamine is not elevated during the initial hypotension episode following endotoxin, c) plasma histamine increases during the progression of endotoxic shock, and d) plasma histamine concentrations are extremely high prior to death.

Evaluation of thyrotropin releasing hormone as a therapeutic intervention for endotoxemia.

Thyrotropin releasing hormone (TRH) has been reported to reduce endotoxin-induced hypotension and mortality rate in conscious rats. Limited data are available to explain these effects. We evaluated hemodynamic parameters, metabolic function, tissue injury, and survival rate in three groups of instrumented conscious rats following intravenous endotoxin (20 mg/kg, LD/90-24 h) challenge. Pretreatment with TRH (2.0 mg/kg, i.v.) was administered 10 min before endotoxin (n = 10) and control (n = 10) animals were given an equivalent volume of saline. The post-treated group (n = 7) was given TRH at the nadir of the hypotensive response following endotoxin to duplicate published protocols. 5 min after endotoxin blood pressure and cardiac output were significantly higher in the post and pre-treatment groups, respectively, compared to the untreated group. There were no differences at other times. Systemic vascular resistance was not affected by either treatment mode at any time. TRH treatment following endotoxin resulted in transient increases in heart and respiration rates and decreased central venous pressure during the first 30 min. Metabolic function indicated by measurements of glucose, lactate, hematocrit, pH, PO2, and PCO2 at 60 and 240 min after endotoxin was not modified by TRH. The hemorrhagic small intestine characteristic of this model was not improved by either treatment mode. Mortality rates at 4 h after endotoxin were 20% for the untreated, 40% for the pre-treated, and 43% for the post-treated. These results suggest TRH exerts early transient effects on cardiovascular responses evoked by endotoxin in the conscious rat but no lasting beneficial effects were found to support the use of TRH as a mono-therapy for endotoxemia.

Respiratory and cardiovascular effects of thyrotropin-releasing hormone as modified by isoflurane, enflurane, pentobarbital and ketamine.

Thyrotropin-releasing hormone (TRH) possesses significant arousing and cardio-respiratory stimulant actions. The effects of a 2 mg/kg i.v. bolus dose of TRH on respiration and systemic hemodynamics were compared in conscious, freely-moving rats and during anesthesia with 4 different anesthetics. Fifty-four male Sprague-Dawley rats weighing 285 +/- 4 g (mean +/- S.E.M.) were divided into 5 groups: conscious, enflurane (2%), isoflurane (1.4%), pentobarbital (8 mg/kg/h i.v.), and ketamine (60 mg/kg/h i.v.). Anesthetized rats were intubated and breathed oxygen or anesthetic/oxygen spontaneously. Aortic blood pressure, heart rate, cardiac output, respiratory rate, arterial blood pH, blood gases, lactate and glucose were measured, and data were collected over a 20 min baseline period and for 130 min post-TRH. TRH increased respiratory rate in all groups; concomitant changes in arterial PCO2 indicated increased minute ventilation in the inhalation agent groups but not in the i.v. anesthetic groups or in the awake group. Significant respiratory depression in the enflurane group was rapidly reversed by TRH. The respiratory stimulant and arousing effects of TRH were smallest with ketamine anesthesia. The hemodynamic responses to TRH were consistent with a pattern of sympathoadrenalmedullary activation and were relatively uniform across groups despite anesthetic-induced alterations in baseline values. TRH or its analogues may prove useful as an analeptic in clinical anesthesia.

Improved techniques for cardiovascular monitoring in rats as applied during endotoxemia.

We describe a new combination of techniques for measurements of systemic blood pressure, central venous pressure, pulmonary arterial (PA) pressure, PA wedge pressure, and cardiac output in the rat. Application of the method to the conscious rat in a septic shock (Escherichia coli endotoxin iv injection) model demonstrated a response pattern of decreased cardiac output and stroke volume, increased total peripheral vascular resistance and heart rate, and transiently decreased systemic arterial pressure. In the pulmonary circulation, a very brief hypertension and a sustained increase in pulmonary vascular resistance were observed, but changes in PA wedge pressure were small. The soft PA catheter (0.3 mm ID, 0.6 mm OD) had no undue effects on cardiovascular function. We suggest that this combined technique could be useful for many cardiovascular studies in the rat, not only as related to shock research.

Choice of anesthetic alters the circulatory shock pattern as gauged by conscious rat endotoxemia.

A standardized rat endotoxin shock model was used to assess the differential effects of four commonly used anesthetics upon hemodynamics before and during endotoxin shock. Forty-nine male Sprague-Dawley rats weighing 307 +/- 4 g were divided into five groups:freely-moving conscious, enflurane (2%), isoflurane (1.4%), pentobarbital (6 mg.kg-1.h-1 i.v.), and ketamine (45 mg.kg-1.h-1 i.v.). Anesthetic doses were chosen as 1.0 MAC equivalent. Anesthetized rats were intubated and ventilated with oxygen. The right carotid artery was cannulated with a thermocouple-catheter for aortic blood pressure, heart rate, and thermodilution cardiac output measurements. The right jugular vein was cannulated for measurement of central venous pressure and for i.v. injections. Data were collected over a 30-min baseline period and for 4 h after an i.v. bolus of endotoxin (40 mg.kg-1, LD100 within 24 h). Gross small intestinal pathology was rated on a 0-4 scale. Anesthetic effects were judged in terms of significant deviations from the awake data on each parameter. Despite differing patterns, total deviations were similar for enflurane, ketamine, and pentobarbital, although the latter drug was the least preferable due to particularly high systemic vascular resistance. Significantly less hemorrhagic small intestinal pathology occurred with enflurane. Ketamine offered no advantage over the inhalation anesthetics. Among the four anesthetics tested, results were closest to the awake pattern with isoflurane anesthesia, thus making it the logical choice for hemodynamic studies in experimental shock research.

Dose-related effects of isoflurane on superior mesenteric vasoconstriction induced by endotoxemia in the rat.

To investigate the interplay between endotoxin-induced circulatory shock and the cardiovascular effects of different doses of isoflurane, mean aortic pressure (MAP), central venous pressure (CVP), mean pulmonary arterial pressure (MPAP), heart rate (HR), cardiac output and superior mesenteric artery flow (SMAF), were monitored in rats anesthetized with either 1.4% or 2.0% isoflurane in oxygen. Cardiac index (Cl), total peripheral vascular resistance (TPR) and superior mesenteric vascular resistance (SMVR) were derived. During continuous administration of isoflurane, endotoxin (LD90, 40 mg X kg-1 iv) was given after a 30-min baseline period, and data were collected for an additional 2-h period. Sham-challenged (saline) animals served as controls. The response to endotoxin in the systemic circulation showed a decrease in Cl and MAP, while HR and TPR increased. MPAP and CVP were essentially unchanged. There were no significant differences in the systemic circulation variables between endotoxin groups, apart from a more pronounced HR increase during 1.4% isoflurane. Regionally, however, SMAF was lower and SMVR was higher in the 2.0% versus the 1.4% isoflurane group following endotoxin. To conclude, the degree of mesenteric vasoconstriction during endotoxemia was dependent on the dose of isoflurane. This dose-related effect seems to be mediated through interaction with intrinsic vascular control, a higher dose allowing a more pronounced local blood flow reduction.

The effects of H1 and H2 histamine receptor antagonists on the development of endotoxemia in the conscious, unrestrained rat.

This study was designed to use the H2 antagonist cimetidine and the H1 antagonist diphenhydramine alone and in combination to clarify the role of histamine in the development of endotoxin shock. The jugular vein and the carotid artery of male Sprague-Dawley rats were cannulated during enflurane anesthesia. After recovery, blood pressure, heart rate, and respiration rate were continuously monitored. Animals were pretreated with saline, a combination of the H1 and H2 receptor antagonists diphenhydramine (20 mg/kg) and cimetidine (80 mg/kg), or individual doses of diphenhydramine or cimetidine. After pretreatment an endotoxin bolus (40 mg/kg) was given. Arterial blood samples (0.35 ml) were taken before endotoxin and after endotoxin at 60 and 240 min for measurement of pH, PO2, PCO2, hematocrit, glucose, and lactate. Pathological examinations were made at 240 min. Four additional groups of animals (N = 10) were studied for the effect of each of the treatment modes on 24-hr survival rate. Treatment with cimetidine plus diphenhydramine prevented the endotoxin-induced blood pressure fall, increase in heart rate, and hypoglycemia; increased the 24-hr survival rate from 10 to 60%; and inhibited the small intestinal pathology found in control rats. Treatment with diphenhydramine alone produced similar results except that there was a gradual blood pressure decrease later in shock. The results obtained from the cimetidine-treated groups were much the same but there was a slight, transient decrease in blood pressure early after endotoxin and the survival rate was increased to 90%. These results demonstrate that in the conscious rat antagonism of the H1 and/or H2 receptors modifies hemodynamic and metabolic responses and the subsequent pathology, altering the course of endotoxin shock and survival. This study provides substantial evidence to implicate histamine as one of the key vasoactive mediators in the development of endotoxin shock.

Evaluation of cardiac output, total peripheral vascular resistance, and plasma concentrations of vasopressin in the conscious, unrestrained rat during endotoxemia.

To eliminate the influence of anesthesia while investigating the role of vasoactive hormones during shock, we have developed an unanesthetized rat model that provides information on key cardiovascular parameters pertinent to shock. Enfluane anesthesia was used while the animals were being catheterized. After recovery from anesthesia, endotoxin (40 mg/kg) was given, and after 4 hr of measurements, animals (11) were killed. Cardiac index (CI) fell significantly 5 min after endotoxin and remained depressed for 4 hr. Mean blood pressure (MBP) decreased from 121 +/- 5.7 mmHg to 79 +/- 5.6 at 5 and 15 min, and increased to 113 +/- 3.0 at 180 min. Central venous pressure (CVP) was decreased from 30 min to the end of the study. Heart rate (HR) increased from 357 +/- 13 to a high of 448 +/- 14 at 5 min and remained at well over 400 for the 4-hr monitoring period. Total peripheral vascular resistance (TPVR) was twice that of the control at 30 min and remained elevated. Stroke volume decreased to 37% of that of the control at 15 and 30 min and remained at 50% of that of the control until sacrifice. Plasma concentrations of vasopressin measured by radioimmunoassay increased from 14 pg/ml (+/- 2.2, SEM) to 144 (+/- 56) and 144 (+/- 66) at 15 and 240 min after endotoxin. Pathological examination at the end of the study revealed extensive hemorrhage in all areas of the small intestine. We conclude that in the conscious endotoxic rat decreases in CI, MBP, and CVP are accompanied by compensatory increases in HR and TPVR; plasma vasopressin concentrations are greatly elevated; and severe small intestinal hemorrhage occurs. The control animals (12) were stable throughout the entire study period, establishing the conscious, instrumented, unrestrained rat presented here as an attractive model for the study of shock without the bias of anesthesia.

Laryngoscopic endotracheal intubation of rats for inhalation anesthesia.

Endotracheal intubation of the rat under direct vision is described together with the details of procedures and apparatus for conducting inhalation anesthesia in this species. Our intubation method requires no special manufacture of equipment, because it employs the human laryngoscope equipped with an infant blade (size 0). Using inhalation anesthetics such as enflurane or halothane for induction, clear laryngoscopic visualization of the glottis is reliably obtained, allowing rapid and routine intubation of the rat in a highly predictable amount of time. In contrast, the injected anesthetics such as ketamine or pentobarbital sodium seem unsuited to laryngoscopic intubation as a result of problems of variable induction times, copious oral secretions, and strong pharyngeal-laryngeal reflexes.

Anesthetic-induced changes in cardiovascular and small intestinal responses to endotoxin in the rat.

The influence of anesthesia on endotoxin shock patterns in the rat was examined. Blood pressure, heart rate, arterial blood gases, glucose, lactate, and pathology of the small intestine were measured before and after endotoxin (40 mg/kg) challenge in male Sprague-Dawley rats. The three groups studied were: (1) enflurane anesthetized (N = 10), (2) ketamine anesthetized (N = 10), and (3) awake and unrestrained (N = 13). Measurements were made during 30 min prior to endotoxin injection (IV) and for 240 min afterward. Reflex tachycardia, which occurred in the awake group simultaneously with the initial endotoxin-induced hypotension, was not present during either enflurane or ketamine anesthesia. Significantly less gross intestinal pathology was found at 4 hr after endotoxin in the anesthetized groups compared to the awake group. These results suggest that the response of the sympathoadrenal system to the hypotension following endotoxin may be blunted in the anesthetized animal.