Rat brain glucose and energy metabolites: effect of +Gz (head-to-foot inertial load) exposure in a small animal centrifuge.

A unique small animal centrifuge with on-line physiological monitoring and brain tissue collection (in < 1 s) capability was used to investigate the effect of increasing +Gz levels, exposure duration, number of exposures, and time course of metabolic changes in the rat brain. To determine the +Gz tolerance, rats were exposed to +7.5 to 25 Gz (30 s each) and EEG was monitored. G-induced loss of consciousness (G-LOC) defined as isoelectric EEG (I-EEG) occurred only at +22.5 and 25 Gz within 14.5 +/- 3 s. To study the effect of increasing +Gz levels on metabolism, rats were exposed to either 0.5 (control) or +7.5 to 25 Gz (30 s each), and brains were collected 1 min postcentrifugation by freeze fixation. A significant increase in lactate (> or = +7.5 Gz) and a decrease in glucose, creatine phosphate (Cr-P), and ATP levels were observed at +15 Gz and higher. The effect of exposure duration was investigated by exposing the rats to +22.5 Gz for 15-60 s. Brain lactate levels increased six-fold while glucose decreased (75%) following the 60-s exposure. The level of Cr-P and ATP decreased significantly after the 15- and 30-s exposures with no further changes at longer +Gz exposures. For time course studies, brains were collected both during (5-35 s) and after (1-15 min) a +25 Gz exposure. A significant decrease in Cr-P occurred within 5 s, but changes in glucose, ATP, and lactate required 15 s. All metabolites returned to control levels within 3 min, except lactate and adenosine, which required 15 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of hypoxia and hyperoxia on human +Gz duration tolerance.

To determine the effects of varying inspired O2 on positive radial acceleration (+Gz; i.e., head-to-foot inertial load) duration tolerance, seven men were exposed to the +4.5- to +7.0-Gz simulated aerial combat maneuver (SACM) by use of the Armstrong Laboratory (Brooks Air Force Base) centrifuge. Exposures were repeated on different days while subjects breathed gas mixtures of fractional concentration of O2 in inspired air (FIO2) between 0.12 and 0.6. SACM duration tolerance was positively related to inspired O2 of FIO2 between 0.12 and 0.2 but was unchanged at FIO2 between 0.2 and 0.6. SACM exposure decreased arterial O2 saturation and increased heart rates; SACM-induced changes were additive to FIO2 effects. The positive relationship between blood lactate and SACM duration tolerance at all FIO2 indicated an anaerobic component. It is concluded that SACM duration tolerance is limited by reduced FIO2 but not enhanced by hyperoxia. Thus the aerobic component of +4.5- to +7.0-Gz SACM duration tolerance is much greater than previously believed.

c-fos and HSP70 gene expression in rat brains in high gravitation-induced cerebral ischemia.

Previous studies have shown that brief exposures of rodents to high gravitational forces (+Gz) in a specifically designed centrifuge cause global cerebral ischemia. In the present study, the effect of +Gz exposure to +22.5Gz for 15 to 60 s on c-fos and HSP70 gene expression was examined. Northern and RT-PCR analyses to total RNA isolated from brains of rats in different post-exposure times revealed a significant, time-dependent increase in the c-fos mRNA level which returned to near normal by 180 min. The HSP70 mRNA level was increased two-fold at 30 min post exposure, and remained elevated until 180 min. The transient stimulation of c-fos and HSP70 gene expression should serve as useful biomarkers for hypergravic stress on the brain. The present results should aid in design of future experiments in our understanding of the pathophysiology of the high +Gz challenges.

Differences in acetylcholine but not choline in brain tissue fixed by freeze fixation or microwave heating.

Among the many rapidly metabolized compounds in the brain, acetylcholine is one of the most challenging to sample effectively due to its rapid synthesis, degradation and sequestration. To ascertain problems that invalidate sampling procedures two methods of tissue fixation, microwave heat inactivation and freeze fixation, were used for obtaining mice and rat brain samples, respectively. The data show that acetylcholine levels obtained by microwave fixation were much higher than those obtained by freeze fixation. Choline levels were not affected by the fixation method used. Microwave fixation results in more accurate assessment of acetylcholine levels than the freeze fixation method, even though the tissue fixation time was less than 1 s in both methods, because tissue integrity is maintained in the microwave fixation, but not during freeze fixation.

Physiologic bases of G-induced loss of consciousness (G-LOC).

Exposure of pilots to high sustained +Gz (head to feet) or rapid onset of +Gz can produce a variety of pathophysiologic effects ranging from the loss of peripheral vision to total blackout and, finally, G-induced loss of consciousness (G-LOC). A G-LOC research program divided into four phases has been organized at USAFSAM/Crew Technology Division. In contrast to previous studies in acceleration, this program will focus exclusively on the ultimate problem in G-LOC; namely, inadequate cerebral perfusion leading to impaired brain energy metabolism, structure and function. The primary objective of this research program is to identify and arrange chronologically the numerous physiological and biochemical alterations in the brain that comprise the mechanism of G-LOC.

Acceleration-induced effects on baboon blood chemistry.

Gravity-induced loss of consciousness (G-LOC) is known to have occurred in pilots since the early 1920's. Most of the research in this area has shown that G-LOC occurs due to a decrease in cerebral blood pressure and a concomitant reduction in brain perfusion. Since a reduction in cerebral blood flow can cause transient hypoxia, it is important to study the cerebral metabolism during high +Gz exposure. One component of these studies should include measurements of substrate availability and degradative products. In the present study, adult baboons were given multiple high +Gz exposures (2 to 6) using the Armstrong Laboratory human centrifuge. Venous blood was collected by an automatic syringe withdrawal pump before, during and after centrifuge exposures. The concentration of blood gases, glucose and lactate tended to decrease during the centrifuge exposure followed by an increase after the run. Total creatine kinase activity in serum was not significantly altered. These results suggest that during +Gz exposure, anaerobic glycolysis is stimulated resulting in elevated lactate production due to a reduction in cerebral blood flow (CBF). The elevated tissue lactate is released into the central circulation upon resumption of normal CBF (after the termination of centrifuge run). Therefore, the observed decrease in lactate concentration during the run may result from a lag in the release of tissue lactate into the blood due to a reduction in CBF. It is speculated that at high +6 Gz, G-LOC may occur as a protective response to reduce the brain metabolic rate, to maintain energy levels and to prevent severe cellular acidosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Endurance and performance during multiple intense high +Gz exposures with effective anti-G protection.

BACKGROUND: In many high-intensity wartime scenarios, pilots may be required to fly multiple, strenuous missions during the same day. HYPOTHESIS: New anti-G protection allows fighter pilots to endure multiple high +G, exposures during several sorties in a limited time interval. METHODS: Nine well-trained centrifuge subjects were exposed to tactical aerial combat maneuvers using balanced pressure breathing during G (PBG) and an extended coverage anti-G suit. The centrifuge exposures consisted of five simulated sorties during a 4-h period, each consisting of four engagements with rapid onset cycles (6 G x s(-1)) varying between +4 G, and -9 GC. The subjects executed a tracking task before, during, and after each engagement and verbally indicated their effort level. Neck muscle strength was measured before and after the test. RESULTS: Seven of the nine subjects could endure all five sorties during the 4-h period. On a scale from 0 (no effort) to 11 (maximum possible effort), highest effort level during runs varied from 5.5 to 11 units (mean 8.7). Maximal heart rate varied from 140-173 bpm (mean 159) and minimum finger oxygen saturation from 75-93% (mean 88). Maximal peripheral and central light-loss varied from 0 to 100% (mean 71 and 40, respeclively). Three G-LOCs (two in the same subject) and four cases of near loss of consciousness occurred. The general fatigue recovery time varied from 9 to 48 h (mean 21). The tracking tests showed that performance deteriorated significantly during all G exposures; the neck muscle contraction was impaired by 12% (p = 0.035) after the C exposures. CONCLUSION: It is possible to train subjects to withstand five simulated flight sorties during a 4-h period with a total of up to 80 peaks to +9 Gz and 80 peaks to +8 Gz using PBCG and an extended coverage anti-G suit.

Multiple +Gz exposures cause brain edema in rats.

The most serious effect of high sustained +Gz (head-to-foot inertial load) known to occur in pilots of high performance aircraft is +Gz-induced loss of consciousness (G-LOC), which may result in pilot incapacitation and subsequent loss of life. G-LOC is believed to occur due to a critical reduction in cerebral blood flow (CBF). Recently, using a small animal centrifuge (SAC), we showed that +Gz exposure causes global cerebral ischemia in a rodent animal model. Since ischemia, depending upon the severity and duration, has been associated with increased brain water content or edema, the present study was undertaken. Rats were exposed to six exposures of either +25 Gz (30 s each) or +10 Gz (2 min each) in the SAC at +20 Gz.s-1 G onset rate. The appearance of G-LOC was monitored by the flattening of the electroencephalography (EEG) brain wave recording. G-LOC was observed at 101 +/- 46 and 19.2 +/- 5 s during +10 and +25 Gz exposures, respectively. The brains from these animals were removed 15 min to 24 h after the +Gz exposure and analyzed for edema formation (increase in the percentage of tissue water), metabolites, and cerebral blood volume (CBV). A significant decrease in glucose and an increase in lactate concentration were observed during +Gz exposure. Edema formation was observed 15 min after six exposures of either +10 or +25 Gz. A slight but significant decrease in CBV was also observed in rats exposed to six +10 Gz exposures. Edema formation was transient and resolved within 24 h. We concluded that multiple exposures of either +25 Gz, short duration or +10 Gz, long duration, that resulted in G-LOC, can cause cytotoxic brain edema which probably results from tissue hyperosmolality due to metabolic changes and accumulation of lactate during ischemia.

Color vision with rapid-onset acceleration.

INTRODUCTION: Only sporadic information exists concerning perceived color shifts at increased G-loads. The purpose of this study was to investigate whether or not color vision is affected by rapid onset high G7-loads up to +9 Gz, and specifically whether perception of hue changes. METHODS: There were 10 male subjects, 9 with normal color vision and 1 with red-green protanomaly, all accustomed to Gz-loads in a human centrifuge. Each subject was tested on a total of 60 Gz-exposures with 10 s periods at +3, +5, +7, and +9 Gz in the centrifuge on three different days. G-onset rate was 6 G x s(-1). The subjects wore an anti-G suit and performed straining maneuvers if necessary to maintain vision. Five square color stimuli of medium saturation (yellow, red, blue, green, and gray) were projected one at a time on a screen in front of the subject, who gave his hue response orally. RESULTS: In 96.6% of exposures to various Gz-loads, the subjects responded by correctly naming colors. (The statistical analyses of the results were done for the subjects with normal color vision, with the protanomalous subject excluded.) Hue shifts occurred at the higher +Gz-levels, including 7.7% of the +9 Gz exposures. Yellow was the hue most frequently perceived as changed. Hue shifts were reported for yellow in 11% and 16% of the +7 and +9 Gz exposures, respectively. Hue shifts at +9 Gz occurred as frequently as blackout and G-LOC together. However, statistical analyses showed no significant effects for +Gz-load. CONCLUSIONS: Absolute identification of the color stimuli of medium saturation was stable and was not significantly affected by the rapid onset +Gz-loads up to and including +9 Gz.

Performance recovery following +Gz-induced loss of consciousness.

Seven male baboons (average weight, 20.6 kg) were trained in a simple shock-avoidance performance task on the USAFSAM centrifuge. A red light was presented to the baboon at approximate 2-s intervals. The animal was allowed 1 s to turn off the light or receive a 1-s shock. The shock could be abbreviated by a late trigger pull. Thus, the animal could avoid, escape, or accept the full shock. EEG was monitored from three transcranial stainless steel electrodes. Loss of consciousness (LOC) was induced by a rapid onset (4 or 6/s) exposure to 8 +Gz and sustained until LOC was identified by a near isoelectric EEG signal. Performance recovery time was measured from the return of EEG activity to the time when the animal resumed the performance task. These data were compared with previously obtained human data and found to be very similar. Also, it was found that time of performance recovery became significantly shorter after multiple LOC exposures and the performance recovery time significantly increased with increased time of unconsciousness. This study demonstrates the utility of the baboon as an animal model for G-induced LOC (G-LOC) research.

Cerebral and spinal cord blood flow dynamics during high sustained +Gz.

This study had two purposes. First, the use of Transonic flowprobes placed on the common carotid and internal carotid arteries of seven male baboons was evaluated for measuring cerebral blood flow (BF) during +Gz stress. The approach was to compare BF's obtained with these flowprobes to microsphere measurements of total cerebral BF. The second purpose was to measure regional variations in cerebral and spinal cord BF during +Gz to test the hypothesis that +Gz produces a differential perfusion deficit throughout the central nervous system so that BF's at the superior portion of the brain are decreased more than in areas of the brain that are nearer to the heart. The results indicate that internal carotid artery and microsphere measurements of total brain BF were related so that the relative decrease in internal carotid artery BF was consistently comparable to that measured with the labeled microsphere technique. Thus, Transonic flowprobes placed on the internal carotid artery of the baboon give reliable estimates of cerebral BF during +Gz stress. The microsphere BF data demonstrated that there were no regional differences in the relative decrease in BF measured in the brain or spinal cord during +Gz. We conclude that our results do not support the hypothesis of a gradient of BF deficit within the brain or spinal cord during +Gz.

Total and regional cerebral blood flow during recovery from G-LOC.

INTRODUCTION: This study measured total and regional cerebral blood flow (BF) in baboons during +Gz-induced loss of consciousness (G-LOC) and during recovery from G-LOC. METHODS: Flowprobes (Transonic Inc., T201, Ithaca, NY) were placed on the common carotid and internal carotid arteries of five male baboons for continuous measurement of total cephalic and cerebral BF, respectively. Radiolabeled microspheres were used to measure regional central nervous system BF at discrete timepoints. G-LOC was determined from visual observations of the animals and from EEG recordings. RESULTS: Cerebral blood flow was maintained and animals remained conscious during 60 s exposure to +4 Gz. In contrast, G-LOC was observed during the first 16-25 s (mean = 20.3 +/- 3.7 s) of exposure to +8 Gz in all five animals. Internal and common carotid artery BF decreased rapidly to zero during the first few seconds of +8 Gz. BF always appeared to cease prior to the occurrence of G-LOC. During early recovery from G-LOC there was no hyperemic response recorded with flowprobes, whereas a hyperemic response was recorded following 60 s exposures to +4 Gz in which the animals did not experience G-LOC. Microsphere measurements of the regional distribution of BF are consistent with the hypothesis of a +Gz-induced differential perfusion deficit throughout the brain and central nervous system during G-LOC. CONCLUSIONS: We conclude that G-LOC is preceded by cessation of cerebral BF. The fact that the hyperemic response following +Gz exposure is less when G-LOC occurs than when G-LOC does not occur suggests CNS energy conservation during G-LOC.

Brief exposure to -Gz reduces cerebral perfusion pressure during subsequent +Gz stress in rats.

INTRODUCTION: In humans, +Gz exposure immediately preceded by exposure to zero or -Gz can result in unexpected incapacitation ("push-pull" effect). Our goals were to establish whether this phenomenon exists in rats and to evaluate the importance of varying the duration of -Gz exposure on magnitude of the push-pull effect on cerebral perfusion pressure. METHODS: Eight conscious male rats were studied in the transition from +5 Gz to +10 Gz imposed by centrifugation. This was done with (push-pull) or without (control) 2 s exposure to -5 Gz applied using a counterbalanced design. Seven isoflurane anesthetized rats were studied in the transition from 0Gz (+1Gy) to + 1Gz imposed by tilting. This was done with (push-pull) or without (control) 0.5, 1, 3, or 9 s exposure to -1Gz imposed immediately prior to the transition applied using a counterbalanced designed. RESULTS: Exposure to 2 s of -5 Gz significantly (p < 0.01) reduced carotid artery pressure in the 4th through 8th s of exposure to +10 Gz by an average of 15 mmHg compared with control. In the tilt experiments, a push-pull effect was found with mild Gz exposure (+/-1Gz) with as little as 0.5 s -Gz exposure. Varying the head-down dwell time did not alter the magnitude of the exaggerated hypotension induced by "push-pull" (p = 0.90). CONCLUSIONS: We conclude that rats express a "push-pull" effect similar to that observed in humans but that altering the duration of exposure to -Gz does not influence the magnitude of the "push-pull" effect.

The right ventricular working heart preparation.

An isolated working rat heart preparation was modified to study right ventricular (RV) performance. All hearts were perfused with a Krebs-Henseleit bicarbonate buffer via a Langendorff column at 90 mm Hg. Right atrial filling (preload) was varied by raising a buffer reservoir from 5 cm below to 10 cm above the right atrium while pulmonary artery outflow resistance remained fixed. RV systolic pressure and the maximum rise and decrease in pressure development (+/- dP/dt) were measured via a catheter in the RV. Cardiac output was collected with a catheter placed in the pulmonary artery. One group of hearts, monitored at a fixed preload (0 cm H2O) for 2 hr, and another group of hearts, in which two ventricular function curves were performed, demonstrated the stability and reproducibility of the preparation. Additionally, the ability of this preparation to measure changes in inotropy was studied. A negative inotropic effect was measured after verapamil (5 X 10(-8) M) treatment. Positive dP/dt showed the greatest depression (30%) and was significantly lower at every preload. A positive inotropic effect was demonstrated by reducing the buffer Ca2+ concentration to 1.9 mM for the first work curve followed by an addition of Ca2+ (2.8 mM final concentration) or ouabain (5 X 10(-5) M) for the second work curve. Again, the greatest effect was found in the dP/dt measurements (elevated by 20 and 30%, respectively). Thus, this preparation manifests qualities similar to those used in studying the left ventricle and allows investigation of various cardiac diseases which may affect RV pump function.

Right ventricular performance after monocrotaline-induced pulmonary hypertension.

Chronic pulmonary hypertension leads to a compensatory hypertrophy of the right ventricle (RV). Performance of the hypertrophied heart has been shown to vary depending on the type and severity of the overload, the species, age, and sex of the animal in which the hypertrophy is induced and the ventricle to which the overload is applied (left ventricle vs. right ventricle). In this study we employed two novel approaches to examine the performance of the hypertrophied right ventricle in male Sprague-Dawley rats. First, monocrotaline (MCT), a pyrrolizidine alkaloid isolated from the plant Crotalaria spectabilis, was used to noninvasively induce chronic pulmonary hypertension, RV pressure overload, and RV hypertrophy. After 5 wk the RV-to-(left ventricle + septum) ratio was increased by 94% over control. The volume of isolated right ventricular myocytes from MCT-treated rats was increased due primarily to an increase in cell cross-sectional area. Second, a stable, isolated, working heart preparation, normally used to study left ventricular function, was modified to study right ventricular function. In vitro ventricular performance of severely hypertrophied hearts was elevated compared with control hearts at all preloads. Significant elevations in positive and negative maximum pressure development (dP/dtmax) suggested that both the rate of pressure development and the rate of relaxation were enhanced. Coronary flow and RV-diastolic pressure were similar in the MCT and control group. Thus RV hypertrophy caused by a chronic pressure overload induced by MCT resulted in enhanced ventricular performance with no evidence of failure.

G-force induced alterations in rat EEG activity: a quantitative analysis.

A major physical limitation affecting pilots is G-force (+Gz, head-to-foot inertial load) induced loss of consciousness. Previous studies have shown that +Gz produces qualitatively similar effects on human and rat EEG activity. The present study sought to quantitatively correlate changes in rat EEG activity with increasing +Gz levels. A frontal-parietal differential electrode recorded rat EEG data during +Gz exposures (30 s) ranging from +0.5 to +25.0 Gz. Acceleration levels < or = +10 Gz had little effect on EEG activity. Acceleration levels of +15 to +20 Gz were associated with increased EEG slowing, depression and sharp waves. Acceleration levels > or = +17.5 Gz evoked burst suppression followed by isoelectric activity. Times to first onset of delta, depressed, and isoelectric EEG activity were approximately 12, 14 and 18 s, respectively. Acceleration effects on delta (1-4 Hz), theta (5-8 Hz), alpha (9-12 Hz), beta (13-30 Hz) and total (1-30 Hz) EEG powers were examined using Fourier transform analysis. EEG measures with the most predictive value at the following post-acceleration onset times (PAOT) were as follows (in s); increasing theta power: PAOT 0-2, decreasing delta power: PAOT 3-9, and decreasing beta power: PAOT > or = 12. This study provides a quantitative description of +Gz-induced alterations in EEG magnitude, time course and spectral content. Additionally, several EEG measures were identified which correlated with acceleration level at specific post-acceleration onset times.

Expression of c-fos, c-jun and HSP70 mRNA in rat brain following high acceleration stress.

Rats exposed to high +Gz forces in a small animal centrifuge (SAC) exhibit loss of neuronal function (isoelectric EEG), termed G-induced loss of consciousness (G-LOC). This phenomenon is presumably due to a reduction in cerebral blood flow (CBF) or ischemia. Ischemia induces various metabolic and physiologic changes including expression of immediate early genes (IEGs) in the brain. Expression of IEGs have been suggested to be reliable markers for neuronal response to external stimuli or stress. In the present study expression of IEGs c-fos, c-jun and stress response gene HSP70 were measured in the brains of rats subjected to six 30 s exposures of +22.5Gz in a small animal centrifuge. The level of c-fos, HSP70 and beta-actin mRNA were measured by both Northern blot and RT-PCR. Expression of c-jun was measured only by RT-PCR. Expression of c-fos and c-jun was significantly stimulated at 0.5, 15, 30 and 60 min post-centrifugation. The level of HSP70 mRNA was significantly higher only at 60 and 180 min post-centrifugation. Measurement of metabolities showed a significant increase in lactate and a decrease in Cr-P level at 30 s and 15 min post-centrifugation, respectively. Lactate, but not Cr-P and ATP levels were restored to control levels by 60 min post-centrifugation. It is concluded that the transient expression of c-fos, c-jun and HSP70 mRNA is stimulated by repeated ischemic/reperfusion episodes induced by high acceleration stress.

Changes in morphology of endocytotic vesicles in cardiac capillary endothelial cells and myocytes of rats after treatment with adenine and AMP.

Isolated rat hearts were perfused with media containing no additions (sham), adenine, or AMP to determine if adenine or AMP would affect post-ischaemic myocyte ultrastructure. The most significant change induced by both adenine and AMP was a dramatic increase in the number and size of endocytotic vesicles apparent in capillary endothelia and myocytes. This observation suggests endocytosis as a possible transport mechanism for AMP or adenine, or other large polar molecules including purine nucleotide precursors, from the coronary circulation across capillary endothelia and into myocytes.

In vivo evaluation of the effects of gravitational force (+Gz) on over-the-wire stainless steel Greenfield inferior vena cava filter in swine.

This study was done to determine the effect of exposure to gravitational force (acceleration stress) on in vivo over-the-wire stainless steel Greenfield inferior vena cava filters. Fifteen pigs underwent venous cut down and placement of a stainless steel Greenfield filter. A 4-week observation period simulated realistic convalescence and allowed sufficient time for epithelialization. Ten pigs were exposed to acceleration stress in a centrifuge (3G run for 15 sec followed by rest until return to baseline heart rate, then a 9G run for 15 sec), with inertial loading in a head-to-tail direction (+Gz). Fluoroscopy during acceleration stress allowed assessment for filter migration. Five pigs were not exposed to acceleration stress. AP and lateral abdominal radiographs were obtained at post-filter placement, convalescence, and centrifuge exposure to determine the position and integrity of the filter. All 15 IVCs were resected and evaluated for gross or histological injury to the vessel wall. IVC filter placement was technically successful in all 15 pigs. Radiographic measurements were limited secondary to differences in pig positioning. Fluoroscopy showed no filter migration. All filters were securely attached to the vena cava by the hooks without gross evidence of perforation or hemorrhage. There were varying degrees of fibroplasia involving the hooks and tip of the filters in both the control and experimental groups. Histologically, there was evidence of prior hemorrhage at the level of the hooks, which was similar between the control and experimental groups. It is concluded that Greenfield filter position and vena caval integrity at the implantation site is unaffected by high acceleration stress.