Comparison of oxygenated perfluorocarbon and humidified oxygen for rewarming hypothermic miniswine.

This study examines a method to rapidly rewarm the core using total liquid ventilation with warmed, oxygenated perfluorocarbon. Yucatan miniswine were splenectomized and surgically implanted with telemetry devices to transmit electrocardiographic response, arterial pressure, and core temperature. Hypothermia (core temperature = 25.9 +/- 1.3 degrees C) was induced by placing cold-water circulating blankets over the animals. Control animals (N = 7) were rewarmed using warm (37.8 degrees C), humidified oxygen. Experimental animals (N = 6) were rewarmed with oxygenated perfluorocarbon liquid (37.3 degrees C). The time to rewarm was significantly shorter in experimental animals (1.98 +/- 0.5 vs. 8.61 +/- 1.6 hours, p < 0.0001), with almost no afterdrop in the experimental group. Lactate dehydrogenase and aspartate aminotransferase were significantly increased in the control animals compared with the experimental animals. All animals that survived being chilled to 25 degrees C survived rewarming. This method may provide a means of more rapidly rewarming profoundly hypothermic victims while reducing the risks associated with current methods.

Flow cytometric method using fluorescent microspheres to measure reticuloendothelial function or particulate translocation.

The reticuloendothelial system (RES) influences the outcome of vascular shock and environmental stress. We describe a procedure that employs flow cytometry and 1 microm fluorescent microspheres (FM) to study RES function. FM (2 x 10(10) beads/kg) were administered via a jugular cannula in Sprague-Dawley rats. After 15 min, blood and tissues were collected and digested in 15% KOH. Phycoerythrin 1 microm beads were added to each sample as an internal standard and analyzed by flow cytometry. FM were preferentially cleared by the spleen, liver and lung. Clearance was confirmed by fluorescent photomicroscopy. Addition of the internal standard to determine accurately aspiration volume enhanced precision. This procedure offers advantages over other RES clearance methods including bacterial, radioactive or carbon clearance assays. Moreover, this method could enhance accuracy, reproducibility and speed of data collection in particulate transport studies that are based on manual microscopic scanning and FM counting.

Model for environmental heat damage of the blood vessel barrier.

Environmental heat stress may result in loss of fluid from the vascular space, which can lead to circulatory shock. Since the endothelium serves as the blood vessel barrier between the vascular and interstitial spaces, direct heat damage to this tissue may contribute to such fluid loss. This study modeled heat influences on the actin cytoskeletal proteins that provide the tensile forces that sustain endothelial junctional integrity or barrier function. Heat effects on bovine aortic endothelial cell (BAEC) F-actin and F-actin stress fibers (FASFs) were correlated with intercellular permeability (IP). F-actin concentration and FASF distribution were analyzed by quantitation of the specific binding of rhodamine phalloidin (RP) to F-actin and by observing the fluorescence of RP-FASF complexes, respectively. Dextran fluorescein IP was determined. The IP was elevated (p < 0.05) at 43 degrees C, but not at 41 degrees C. At 43 degrees C, BAECs were rounded and had disrupted FASFs and diminished cell-to-cell apposition. Similar cells were seen at 41 degrees C, but these were interspersed among FASF-containing cells to sustain apposition. Thus, disruption of FASFs correlated with increases in IP. F-actin was increased (p < 0.05) after hyperthermia. Since G-actin is more susceptible to irreversible heat denaturation, F-actin sustainment may function to preserve the actin pool and prevent irrevocable loss of the blood vessel barrier after heat stress.

Thermoelectric technique to precisely control hyperthermic exposures of human whole blood.

The need in military research to avoid exposing humans to harsh environments and reduce animal use requires the development of in vitro models for the study of hyperthermic injury. A thermoelectric module (TEM) system was employed to heat human whole blood (HWB) in a manner similar to that experienced by heat-stroked rats. This system precisely and accurately replicated mild, moderate, and extreme heat-stress exposures. Temperature changes could be monitored without the introduction of a test sample thermistor, which reduced contamination problems. HWB with hematocrits of 45 or 50% had similar heating curves, indicating that the system compensated for differences in sample character. The unit's size permitted its containment within a standard carbon dioxide incubator to further control sample environment. These results indicate that the TEM system can precisely control temperature change in this heat stress in vitro model employing HWB. Information obtained from such a model could contribute to military preparedness.

Simultaneous visualization of G- and F-actin in endothelial cells.

We developed site-specific fluorescent probes that permit simultaneous microscopic observation of G- and F-actin in bovine endothelial cells. G-actin distribution was visualized with fluorescein-deoxyribonuclease I (DNAse I). F-actin was labeled with phalloidin conjugated to the new long-wavelength fluorophore BODIPY 581/591 (581-nm excitation, 591-nm emission), which is spectrally similar to Texas Red. The G-actin appeared as pervasive green fluorescence that was more intense in the nuclear region, where cell thickness is greater and stress fibers are less frequent. In addition, we observed a punctate fluorescein pattern around the nuclei and in other parts of the cells, suggesting that some G-actin is localized to small discrete sites. F-actin was observed as red fluorescent filaments. Unlabeled DNAse I effectively prevented staining of G-actin by the fluorescent DNAse I conjugates. The specificity of DNAse I for G-actin was confirmed by the presence of a single labeled band with molecular weight corresponding to actin in a Western blot of total cytoplasmic endothelial proteins reacted with biotin-DNAse I-streptavidin-alkaline phosphatase. Anti-actin antibody, which associates with both G- and F-actin, in conjunction with fluorescent secondary antibody produced a pattern similar to that obtained by simultaneous visualization with fluorescein-DNAse I and BODIPY 581/591- or rhodamine-phalloidin.

Comparisons of endothelial cell G- and F-actin distribution in situ and in vitro.

Numerous studies have described the F-actin cytoskeleton; however, little information relevant to G-actin is available. The actin pools of bovine aortic endothelial cells were examined using in situ and in vitro conditions and fluorescent probes for G-(deoxyribonuclease I, 0.3 microM) or F-actin (phalloidin, 0.2 microM). Cells in situ displayed a diffuse G-actin distribution, while F-actin was concentrated in the cell periphery and in fine stress fibers that traversed some cells. Cells of subconfluent or just confluent cultures demonstrated intense fluorescence, with many F-actin stress fibers. Postconfluent cultures resembled the condition in situ; peripheral F-actin was prominent, traversing actin stress fibers were greatly reduced and fluorescent intensity was diminished. Postconfluency had little influence on G-actin, with only an enhancement in the intensity of G-actin punctate fluorescence. When post-confluent cultures were incubated with cytochalasin D (15 min; 10(-4) M), F-actin networks were disrupted and actin punctate and diffuse fluorescence increased. G-actin fluorescence was not altered by the incubation. Although its unstructured nature may account for the minor changes observed, the stability of the G-actin pool in the presence of notable F-actin modulations suggested that filamentous actin was the key constituent involved in these actin cytoskeletal alterations. A separate finding illustrated that the concomitant use of actin probes with image enhancement and fluorescent microscopy could reveal simultaneously the G- and F-actin pools within the same cell.

Seasonal effects on human physiological adaptation factors, thermotolerance and plasma fibronectin.

Plasma fibronectin (PF) influences shock survival and basal levels increase with active conditioning that improves human physiological adaptation factors (PAF) and thermotolerance (TT). To evaluate further PF's relationship with PAF and TT, the effects of passive conditioning with seasonal change (spring vs. summer) in New England on PAF, TT, basal PF level and PF level during hot-humid exercise (HHE; bicycling; 40 +/- 4% VO2max; 35 degrees C; 70% rh; 45 min) were examined in male subjects (28.2 +/- 1.6 years; N = 7; values are means +/- SE). The spring and summer studies were separated by 2 months. In addition, 2 months prior to the spring study, a winter basal PF pre-screening was conducted. Winter (287 +/- 36 micrograms/ml), spring (272 +/- 21 micrograms/ml), and summer (278 +/- 19 micrograms/ml) basal PF levels were similar. The PF response during HHE was unremarkable with seasonal change. PAF were improved, since blood volume (6266 +/- 276 vs. 5895 +/- 251 ml), plasma volume (3896 +/- 198 vs. 3601 +/- 165 ml) and HHE sweat rate (18.7 +/- 5.5 vs. 12.9 +/- 6.4 ml/min) were elevated (p < 0.05) in the summer compared to the spring. However, this was not accompanied by improved TT, since spring and summer rectal temperatures during HHE were similar, while summer heart rate was elevated (p < 0.05) compared to the spring. In contrast to active conditioning, passively-induced improvements in PAF were not associated with elevations in TT or PF level. Unlike PAF, PF elevations might only occur when the conditioning resulted in increased TT, which suggests a potential for PF as a TT marker.

Modulation of human plasma fibronectin levels following exercise.

Elevated reticuloendothelial function and plasma fibronectin (PF) level correlate with reduced rat heat shock mortality. Procedures that enhance human PF level may offer some advantage in dealing with the adverse effects of environmental stress. Both short- (STE) and long- (LTE) term exercise programs were evaluated for their ability to increase male human PF. STE (1 week; N = 14) consisted of treadmill running (0% grade) in a hot environment (41 degrees C, 39% relative humidity). The LTE (12 weeks) program was studied in two parts (LTE-1, N = 10; LTE-2, N = 19), with each part divided into four subject groups. Two groups performed the same running program combined with either full-body or upper-body weight training. The other two groups participated in only the running or full-body weight training. STE and LTE-2 had a significantly (p less than 0.05) higher mean initial PF level than LTE-1. STE (337.1 +/- 22.8 vs. 372.5 +/- 17.0 micrograms.ml-1). LTE-1 (266.0 +/- 13.0 vs. 348.0 +/- 18.8 micrograms.ml-1), and LTE-2 (370.9 +/- 13.8 vs. 413.6 +/- 12.5 micrograms.ml-1) all resulted in significant (p less than 0.05) increases in PF after program completion. Therefore, with diverse exercise training programs, PF elevations were attained even when initial concentration was high or the program was of short or long duration. However, PF level was suppressed in LTE-2 at 4 (323.2 +/- 10.8 micrograms.ml(-1)) and 8 (339.6 +/- 15.0 micrograms.ml(-1)) weeks before elevations occurred at 12 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of phospholipase A2 lytic activity by actin and myosin.

Prostacyclin (PGI2) production is closely coupled with endothelial cell shape and F-actin distribution in vitro. These findings may implicate cytoskeletal constituents in a mechanism regulating eicosanoid metabolism. To determine the potential for such a regulatory mechanism, cytoskeletal protein effects on the rate-limiting eicosanoid cascade enzyme (phospholipase A2; PLA2) were studied. Membrane phospholipid degradation was indirectly determined by spectrophotometric measurement of PLA2-induced rat red blood cell ghost (RBC-G) hemolysis. PLA2 was incubated with actin (skeletal, smooth, or nonmuscle cell) at a nonmuscle cell concentration (100 microM) and then exposed to the RBC-G. Comparisons in the presence or absence of actin revealed that F-actin stimulated whereas G-actin suppressed PLA2 lytic behavior significantly (P less than 0.05). When a 10: or 100:1 F-actin to myosin ratio was used, the F-actin stimulatory effect was significantly (P less than 0.05) reduced. These findings suggest that the in vitro correlation between PGI2 production and endothelial cell shape may be the result of PLA2 regulation by cytoskeletal elements that impart cellular form.

Ketanserin modulates rabbit foot cooling in the presence or absence of exogenous serotonin.

Since hypothermic conditions augment sensitivity to vasoactive amines like serotonin (5-HT) and 5-HT is associated with the etiology of Raynaud's phenomenon, this amine perhaps plays a role in cold-induced vasoconstriction. To determine if 5-HT participated in normal peripheral cooling and if ketanserin (KET), a 5-HT blocker, modulated such cooling, four groups of New Zealand white rabbits (N = 33) were studied. The femoral artery was cannulated to allow perfusion of a hindlimb. Thermistors were implanted in the footpad and rectum. The hindfoot was exposed to a 15 degrees C bath for 30 min, while footpad and rectal temperatures were recorded. During cold exposure, 5-HT (5 x 10(-2) M, group 1), KET (0.1 mg/kg) + 5-HT (group 2), KET (group 3), or saline (group 4) was perfused through the hindlimb. Groups 2 and 3 were also pretreated with KET (0.1 mg/kg perfused over 30 min). The rabbit footpad cooled rapidly when exposed to exogenous 5-HT (group 1). KET treatment in the presence of exogenous 5-HT (group 2) was associated with a significantly (P less than 0.05) reduced cooling rate. KET treatment in the absence of exogenous 5-HT (group 3) was also associated with a significantly (P less than 0.05) elevated limb temperature when compared to controls (group 4). This suggested that endogenous 5-HT participated in limb cooling. Therefore, as noted for Raynaud's disease, 5-HT may also influence peripheral cooling of tissues free of such pathologies.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation among endothelial cell shape, F-actin arrangement, and prostacyclin synthesis.

Though many factors have been identified which modulate prostacyclin (PGI2) synthesis, there is little information on cellular mechanisms whereby endothelial cells (EC) regulate their basal eicosanoid metabolism. Using substrates of various adhesive capacities, bovine and porcine aortic EC shape and cytoskeletal F-actin arrangement could be modulated. Staining with rhodamine-phalloidin (R-P) permitted analysis of F-actin arrangement, while differences in cell shape were determined by measurement of cell perimeter surface area (CPSA). Spectrophotoflurometric measurements were used to quantitate the R-P binding capacity of the cultures. Cultures of reduced CPSA (225.2 +/- 13.5 mu2) generated the highest levels of basal PGl2 (6.14 +/- 0.51 pg/ug cell protein); had a diffuse arrangement of F-actin and an increased binding capacity for R-P (463.55 +/- 50.58 nmoles/ug cell protein). Cultures of enlarged CPSA (1399.3 +/- 148.3 mu2), with many actin cables and a significantly reduced (p less than 0.001) R-P binding capacity (74.941 +/- 11.79 nmoles/ug of cell protein) produced significantly smaller (p less than 0.001) basal quantities of PGl2 (1.33 +/- 0.14 pg/ug cell protein). Similarly, arachidonic acid stimulation of cultures of reduced CPSA resulted in an increased synthesis of PGl2 when compared to stimulated cultures of enlarged cells. These findings suggest a role for cell shape and the cytoskeleton in the mechanism controlling PGl2 production and indicate that alteration of the arrangement of F-actin may be of importance in regulation of EC eicosanoid metabolism.

Correlation between plasma fibronectin level and experimental rat heat stress mortality.

Reticuloendothelial system (RES) clearance function correlates with the mortality rate associated with stresses that can induce shock. Likewise, experimental rat heat stress (ERHS) mortality rate is altered by modulation of RES function. Since plasma fibronectin (PF) in many instances appears to mediate in vivo phagocytosis by the RES, the relationship between mean plasma fibronectin level (MPFL) and ERHS mortality was examined. A comparison of MPFLs prior to ERHS revealed that rats which ultimately comprised the survival group had a MPFL of 269.0 +/- 11.2 micrograms/ml, whereas that of the nonsurvivors was 252.9 +/- 11.9 micrograms/ml. Both groups had elevated MPFLs up to 12 h following ERHS. However, after this time, MPFL began to decline. The decline was more severe for the nonsurvivors, with MPFLs at 15, 18, and 20.3 h significantly (P less than 0.01) lower than the values for the survival group. Even the lowest MPFL (256.0 +/- 30.7 micrograms/ml) noted for the survival group was still significantly (P less than 0.01) higher than the value (159.3 +/- 13.3 micrograms/ml) determined for agonal samples collected from nonsurvivors. Furthermore, grouping rats according to their preheat PF level demonstrated that rats with levels exceeding 300 micrograms/ml had significantly (P less than 0.05) reduced mortality rates (12.5 vs. 51.3%) compared with rats with levels below this value. It was concluded that elevated PF levels prior to ERHS correlated with thermotolerance.

Relationship between rat heat stress mortality and alterations in reticuloendothelial carbon clearance function.

It has recently been reported that, although bacterial endotoxins of intestinal origin are not associated with death after experimental rat heat stress, a state of endotoxin tolerance significantly decreases the heat stress mortality rate. To determine if this phenomenon were associated with the ability of endotoxins to stimulate clearance by the reticuloendothelial system (RES), the relationship between rat heat stress mortality and carbon clearance by the RES was examined. RES carbon clearance was stimulated by prior treatment with endotoxin, zymosan, or sublethal heat stress, as indicated by the significantly reduced (p less than 0.05) blood carbon concentrations 15 min after carbon injections. Prior treatment with injections of gelatin blocked RES carbon clearance. Rats subjected to endotoxin or sublethal heat treatment were significantly (p less than 0.05) resistant to the experimental heat stress, whereas zymosan treatment had no effect. Blockade of the RES with gelatin significantly (p less than 0.05) increased the heat stress mortality rate. These data compare favorably with previously reported studies evaluating RES function and mortality after experimental injury and shock and indicate that the RES may play a fundamental role in the pathogenesis of, and tolerance to, experimental heat stress.

Role of bacterial endotoxins of intestinal origin in rat heat stress mortality.

Using unanesthetized rats, the effect on heat stress mortality of endotoxin tolerance or zymosan treatment was determined. In addition, the incidence of invasion by gram-negative bacteria and their endotoxins was studied to evaluate the role of gut-derived bacterial endotoxins after heat stress. Endotoxin tolerance resulted in heat stress resistance. The estimated mean total thermal area, which induced an LD50 in endotoxin-tolerant rats (61.85 degrees C . min) was significantly greater (P less than 0.001) than that for non-tolerant rats (44.03 degrees C . min). Rats were significantly (P less than 0.005) more sensitive to endotoxin after zymosan treatment, but this treatment did not alter the heat stress mortality rate. The Limulus amoebocyte lysate test indicated that endotoxemia did not occur as a result of heat stress. Though a significantly increased incidence of high gram-negative bacterial count in the duodenum was noted, extraintestinal invasion was not found. It was concluded that resistance to heat stress may not be due to protection from gut-derived bacterial endotoxins, but resistance may possibly be associated with the ability of endotoxin tolerance to protect from shock syndromes. Thus bacterial endotoxins of intestinal origin did not appear to have a significant role in rat heat stress mortality.

Comparison of plasma extraction techniques in preparation of samples for endotoxin testing by the Limulus amoebocyte lysate test.

Due to the presence of inhibitory and possible mimicking substances in plasma difficulties have occurred in the use of the Limulus amoebocyte lysate test. Currently, there are a variety of extraction techniques discussed in the literature which are used to remove these interfering substances, but there is little information comparing these techniques. Five such procedures were compared in their ability to provide an extracted plasma sample in which low levels of endotoxin could be detected by the Limulus amoebocyte lysate test. Results indicated that some procedures adversely affected endotoxin detection. The dilution + heating extraction method was found to be as effective as the widely used chloroform extraction method. Comparison of Limulus amoebocyte lysate test results from healthy human plasma samples extracted by these two methods indicated that lysate type and not extraction procedure was associated with previously reported questionable positive tests. Thus, ambiguities associated with Limulus amoebocyte lysate tests of plasma samples may be due not only to extraction method but also the lysate type employed.

Isolation of Salmonella spp. from dead gulls (Larus californicus and Larus delawarensis) from an Idaho irrigation reservoir.

An epizootic of unknown etiology resulting in the death of about 500 sea gulls (Larus californicus and Larus delawarensis) in 24 hr occurred on an irrigation reservoir in southwestern Idaho in April 1975. Salmonella spp. were isolated from necropsy specimens from 2 of 6 gulls examined. No Salmonella spp. were isolated from fecal specimens or dead gulls collected at a nesting site.

Effect of oral inoculation of Escherichia coli on colostral antibody production in cattle.

Oral inoculation of approximately 1.2 x 10(9) viable Escherichia coli to pregnant cows resulted in increased blood serum and colostral whey titers to the "O" antigen. The antibody titers were more pronounced in colostral whey and were correlated with the inoculum strain of Escherichia coli. There was no correlation between antibody titers of the colostrum ingested and the resulting serum antibody titers of the calves. The incidence of diarrhea in calves did not correlate with the antibody titer in the colostrum. The occurrence of diarrhea was significantly greater in calves that did not ingest colostrum until they were 12 hours old, compared with calves that had free access to their dams and suckled within an hour of birth.