Measuring plasminogen activator inhibitor activity in plasma by two enzymatic assays.

We compared two methods that measure plasminogen activator inhibitor (PAI) activity in plasma based on the ability of PAI to inhibit tissue plasminogen activator (tPA) or urokinase (uPA) in order to determine which method most accurately measures plasma PAI activity after stressors, like hemorrhage. Plasma PAI activity was significantly elevated after hemorrhage in both assays. Using standard curves derived from rhPAI-1, we found that the tPA-PAI assay was more sensitive than the uPA-PAI assay. However, we measured a 10-fold difference in PAI activity as measured between assays, suggesting that some endogenous plasma constituents (tPA, uPA, plasminogen or plasmin) may interfere with the accurate determination of PAI activity. Increasing the amount of plasma in each assay led to a progressive increase in PAI activity. However, removing either tPA or plasminogen from the tPA-PAI assay unmasked the presence of some endogenous tPA and plasminogen. Furthermore, increasing plasma volume in either assay increases measured plasma tPA, but not uPA. Finally, plasma tPA is elevated after hemorrhage, whereas plasma uPA is not. These results suggest that endogenous tPA and plasminogen may interfere with the measurement of plasma PAI activity in the tPA-PAI assay after hemorrhage or other stresses. The uPA-PAI assay does not have this confounding problem because endogenous uPA does not interfere with the assay, nor does it rise during hemorrhage.

Comparison of parathyroid imaging with technetium-99m-pertechnetate/sestamibi subtraction, double-phase technetium-99m-sestamibi and technetium-99m-sestamibi SPECT.

UNLABELLED: The ability of 99mTc-pertechnetate/sestamibi subtraction, double-phase 99mTc-sestamibi and 99mTc-sestamibi SPECT imaging to localize abnormal parathyroid tissue was compared. METHODS: Fifty-five consecutive patients had parathyroid imaging before surgery for hyperparathyroidism. Imaging consisted of 99mTc-pertechnetate pinhole images of the neck followed by 99mTc-sestamibi pinhole images of the neck and parallel-hole images of the neck and chest (early images). Within 2.5-4.0 hr later pinhole images of the neck, parallel-hole and SPECT images of the neck and chest were obtained (late images). Nodular foci of increased sestamibi activity were considered abnormal. RESULTS: The sensitivity for abnormal parathyroid glands by visual comparison of early images and pertechnetate images was 72%-75%, late images and pertechnetate images was 73%-78% and double-phase (early and late) sestamibi images was 62%-65%; computer subtraction of pertechnetate from early images was 71%-74%; and SPECT imaging was 79%. The sensitivity for parathyroid adenomas was 89%-98%, while the sensitivity for hyperplastic parathyroid glands was only 47%-58%. CONCLUSION: Late imaging, computer subtraction and SPECT may not be necessary since they provided only marginal improvements on visual comparison of early sestamibi with pertechnetate images. Double-phase sestamibi imaging was less sensitive, so baseline thyroid imaging with pertechnetate is recommended.

Impairment of blood volume restitution after large hemorrhage: a mathematical model.

A mathematical model tests possible mechanisms for the progressive failure of blood volume restitution seen after larger hemorrhages ( > 26%) with increasing changes in plasma osmolality. After 10% hemorrhage, the model requires a decrease in net hydrostatic capillary pressure, the release of solute into the extracellular space, and the release of Na+ and K+ from a bound pool in equilibrium with the interstitium to match the experimental data. The solute and released cations expand the interstitium to drive the restitution of volume and protein from 3 to 24 h. After 30% hemorrhage, the best prediction of the average experimental responses occurs when the Na(+)-K(+)-adenosinetriphosphatase (ATPase) in the cell membrane is inhibited by 38.7% from 0.8 to 3 h, and the proportionality between capillary pressure and blood volume is reduced by 68% from its value for 10% hemorrhage. When the change in plasma osmolality is doubled after 30% hemorrhage, an increase in the inhibition of the ATPase to 85% and extension of its duration to 24 h are necessary to match experimental findings. The associated defect in sodium transport may occur after large hemorrhage so that sodium and water move into cells. This response may oppose osmotically driven expansion of the interstitium and thus account for the failure of restitution.

Flexible endoscopy for the diagnosis of esophageal trauma.

The role of flexible endoscopy in the diagnosis of esophageal trauma remains undefined. This study evaluates the use of immediate flexible fiberoptic esophagogastroduodenoscopy (EGD) as the primary diagnostic tool for detection of esophageal injury in trauma patients. Flexible EGD was performed on 31 patients for this purpose from August 1991 through January 1994. There were 28 males and 3 females with a mean age of 24.3 years (range, 16-54 years). Twenty-four of 31 patients (77%) were intubated at the time of the examination. Mechanism of injury was penetrating in 24 patients (20 gunshot wounds, four stab wounds) and blunt (motor vehicle crash) in seven patients. Penetrating injuries were located in the neck in 5 of 24 patients, in the chest in 15 of 24 patients, and in both the neck and chest in 4 of 24 patients. Upper gastrointestinal contrast studies were performed for 3 of 31 patients (10%), computed tomography was performed for eight patients (26%), bronchoscopy was performed for 13 patients (42%), angiography was performed for 17 patients (55%), and rigid esophagoscopy and laryngoscopy were each performed for one patient (3%). Evidence of esophageal trauma during EGD was seen in 5 of 31 patients. True-positive studies occurred for four patients, false-positive results occurred for one patient, true-negative results occurred for 26 patients (as demonstrated by exploration in five and clinical follow-up in 21), and no false-negative examinations occurred. Sensitivity of flexible EGD was 100%, specificity was 96%, and accuracy was 97%. No complications occurred related to the performance of EGD. Flexible fiberoptic endoscopy seems to be a safe and effective method for both detection and exclusion of esophageal trauma.

Plasminogen activator inhibitor-1 mRNA is induced by hemorrhage in endothelial and mesothelial cells of the rat liver.

We have recently shown that plasminogen activator inhibitor-1 (PAI-1) mRNA is elevated after hemorrhage in various tissues including liver. In this study, we set out to identify the cell types in the liver that are responsible for the increase in PAI-1 mRNA after hemorrhage using in situ reverse transcription-polymerase chain reaction (in situ RT-PCR). Male Sprague-Dawley rats were cannulated and subjected to a 20 ml/kg hemorrhage within 3 min or 300 micrograms/kg of endotoxin. Four hours later, the livers were harvested, fixed, frozen, and sectioned. RT-PCR showed an increase of PAI-1 mRNA in liver 4 h after hemorrhage or endotoxin-treatment. Standard in situ hybridization could not detect PAI-1 mRNA in the livers of either the hemorrhage, endotoxin, or control groups. However, in situ RT-PCR detected PAI-1 mRNA in vascular endothelial cells and capsular mesothelial cells, but not in hepatocytes, in both the hemorrhage and endotoxin groups. No signal was found in the control rats, or when the experimental protocol was modified to 1) omit the RT step, 2) precede the RT step with RNA digestion, or 3) use an irrelevant probe. These results demonstrate that hemorrhage induces PAI-1 mRNA in endothelial and mesothelial cells of liver.

Cell swelling and depolarization in hemorrhagic shock.

Although an increase in intracellular water volume (IWV) in hemorrhagic shock has been inferred from measured changes in transmembrane potential, it has not been measured directly. We have described the presence of a circulating protein that appears in hemorrhagic shock [circulating shock protein (CSP) 70] that depolarizes numerous cell types. To determine if this substance produced a concurrent increase in intracellular water, cells were incubated with CSP 70. Then we measured IWV as the difference between the 3H water space and the [14C]mannitol space. CSP 70 increased IWV 9% in rat red blood cells (RBCs) (n = 8, p < 0.05), 22% in rat H9c2 cells (n = 7, p < 0.05), 11% in dog RBCs (n = 10, p < 0.005), and 31% in dog white blood cells (n = 8, p < 0.005). The results indicate that a protein that circulates in hemorrhagic shock depolarizes cells and increases intracellular water. This suggests that the changes in transmembrane potential observed in hemorrhagic shock are accompanied by movement of extracellular fluid into cells and may account for the inability to restore blood volume after large hemorrhage.

Responses of immunoreactive ACTH and bioactive ACTH to large hemorrhage and resuscitation in conscious dogs.

We studied the effect of fluid resuscitation on immunoreactive adrenocorticotropic hormone (irACTH) and bioactive ACTH (bioACTH) after hemorrhage in conscious dogs. Animals (n = 7) were bled 30% (approximately 25 ml/kg) over 3 min and 30 min later were either resuscitated [43.3 ml/kg 0.9% NaCl (1.8 times hemorrhage volume) over 10 min] or not. Blood was reinfused after 210 min. Animals had both treatments (> 4 days apart). irACTH, bioACTH, cortisol, angiotensin II, and aldosterone increased rapidly after hemorrhage. Resuscitation increased blood volume and cardiac output to resting values, but arterial hypotension persisted. bioACTH and irACTH decreased 40-90 min after hemorrhage in both groups, but each decreased more rapidly after resuscitation. The elimination half-life of bioACTH was shorter than that of irACTH, but neither was affected by resuscitation. The ratio of bioACTH to irACTH followed the same pattern with or without resuscitation. Angiotensin II and aldosterone remained increased without resuscitation but decreased promptly after resuscitation. In conclusion, 1) saline infusion at 1.8 x hemorrhage volume provides effective cardiovascular resuscitation, with resolution of hormonal responses to hemorrhage; 2) although ACTH responses resolved with or without resuscitation, resuscitation produced more rapid resolution without changing the parameters of ACTH elimination; 3) the dynamics of the resolution of the ACTH response to hemorrhage are similar whether induced by stimulus removal or feedback inhibition.

Plasminogen activator inhibitor-1 rises after hemorrhage in rats.

Large hemorrhage leads to hypercoagulability, a phenomenon that has never been well explained. Because an elevation of plasminogen activator inhibitor (PAI)-1 increases procoagulant activity, we have determined whether plasma PAI activity and tissue PAI-1 mRNA are elevated after hemorrhage. Sprague-Dawley rats were bled (20 or 15 ml/kg) 4 days after cannulation. Plasma PAI activity was determined by the capacity of plasma to inhibit tissue-type plasminogen activator activity. Changes of PAI-1 mRNA in various tissues were detected by high-performance liquid chromatography after reverse transcription and polymerase chain reaction. Hemorrhage (20 ml/kg) significantly elevated plasma PAI activity at 0.5, 1, 2, 4, 6, and 8 h after hemorrhage and PAI-1 mRNA in liver at 1, 2, 4, and 6 h after hemorrhage. The PAI-1 message was also significantly elevated in lung, heart, and kidney at 4 h after hemorrhage. The increases of PAI-1 mRNA after 20 ml/kg hemorrhage were significantly greater than those after 15 ml/kg hemorrhage. These findings indicate that large hemorrhage can induce the increases in PAI activity and PAI-1 message and suggest that induction of PAI-1 may be involved in the thrombogenic responses observed after large hemorrhage.

Changes in regional vascular resistance and blood volume after hemorrhage in fed and fasted awake rats.

To determine whether fasting alters the response of blood flow to hemorrhage, blood flow was measured by radiolabeled microspheres before and after a 20 ml.kg-1.3 min-1 hemorrhage in fed and fasted chronically cannulated male Sprague-Dawley rats. Restitution of blood volume, as determined by dilution of hematocrit, was attenuated in fasted rats, although the responses of arterial blood pressure, heart rate, cardiac output, and total peripheral resistance were not significantly different. Fasting only affected resting blood flow in the bronchial artery and fat and had no effect on resting vascular resistance in any organ studied. In both fed and fasted rats, hemorrhage led to a significant fall in blood flow to the stomach, small intestine, cecum, colon, spleen, pancreas, kidney, bronchial artery, thymus, and muscle and a rise in blood flow to the adrenals. However, fasting did not significantly alter the response of flow or vascular resistance to these organs. Fasting did alter the blood flow response to hemorrhage in bone, fat, and the hepatic artery. These results demonstrate that 24 h of fasting does not affect the responses of blood flow and vascular resistance to hemorrhage in most organs, even though restitution of blood volume is attenuated.

Adrenal cortical carcinoma with adenosquamous differentiation. Report of a case with immunohistochemical and ultrastructural studies.

We report the case of an adrenal cortical carcinoma with glandular and squamous differentiation, demonstrated by light and electron microscopy as well as by immunohistochemical studies. The patient was a 63-year-old man presenting with a large adrenal mass and markedly increased 24-hour urine metanephrine, initially suggesting the diagnosis of pheochromocytoma. Upon histological examination of the surgically excised tumor, the presence of adenosquamous differentiation was most consistent with a metastasis to the adrenal gland. No other primary tumor was later found at autopsy, however. It thus becomes evident with this case that squamous and glandular differentiation can be observed in primary adrenocortical carcinomas, and therefore the conventional approach to the immunohistochemical and ultrastructural features of these tumors is challenged. This type of aberrant morphology in adrenocortical carcinomas makes the differentiation from a metastatic carcinoma particularly difficult for the surgical pathologist. Clinical correlation is absolutely necessary for an accurate diagnosis, and occasionally, as was the case with this patient, only with postmortem studies can another primary be ruled out with certainty.

Role of intestinal fluid in restitution of blood volume and plasma protein after hemorrhage in awake rats.

To determine whether food and/or water in the gastrointestinal tract affects restitution of blood volume and plasma protein after hemorrhage, fed and 24-h-fasted awake rats received a 20 ml.kg-1 x 3 min-1 hemorrhage, and restitution of blood volume was measured by Evans blue dye and dilution of hematocrit. Restitution of blood volume and plasma protein in fed rats was complete by 2-4 h. In contrast, restitution was severely attenuated in fasted rats and was not complete by 24 h. Because initial blood volume was significantly lower in the fasted rats (55.4 +/- 1.7 vs. 64.9 +/- 2.5 ml/kg in fed), the percent blood lost during hemorrhage was significantly greater (36 vs. 31%). However, the attenuated restitution was not the result of the larger hemorrhage, as fed rats receiving a 36% hemorrhage also restored blood volume completely by 4 h. In fasted rats, complete restitution of blood volume did occur when either water or food and water were given 4 h after hemorrhage. Gastrointestinal water content fell (from 65.5 +/- 4.8 to 47.9 +/- 1.6 ml/kg) 2h after hemorrhage in fed but not in fasted rats (33.5 +/- 2.4 to 30.6 +/- 2.5 ml/kg). These data suggest that gastrointestinal fluid is essential for complete restoration of blood volume in the awake rat.

A circulating shock protein that depolarizes cells in vitro depresses myocardial contractility and rate in isolated rat hearts.

Previously, we identified the presence of a circulating shock protein (CSP) in the plasma of hemorrhaged rats that depolarizes a variety of cells in vitro. In isolated perfused rat hearts, partially purified CSP produced dose-dependent decreases in contractility and heart rate associated with an increase in coronary perfusion pressure (CPP). Electrical pacing failed to prevent the negative inotropic effects. Preventing the coronary vasoconstriction with nitroglycerin or attenuating it with a cyclooxygenase inhibitor also failed to prevent the inotropic or chronotropic effects of CSP. Carbocyclic thromboxane A2 (50ng/min) caused a similar increase in CPP to CSP but had no effect on contractility or rate during the first minute of infusion. These data indicate that the protein that appears in rat plasma after hemorrhage produces negative inotropic and chronotropic effects on the isolated heart that are independent of changes in CPP. Vasoactive arachidonic acid metabolites elicited by CSP are partially responsible for the increase in coronary vascular resistance.

Management of the trauma victim with pre-existing endocrine disease.

The clinical course of individuals after trauma largely is determined by their pretraumatic state. The endocrine system plays a major role in the response to injury, surgery, and sepsis, and endocrine dysfunction places the trauma victim at risk of greater morbidity and mortality. Further, chronic endocrine disease usually is accompanied by multiorgan dysfunction, which may compromise the physiologic reserve of the critically injured patient. Among patients with pre-existing endocrine disease, the severe stresses of multisystem trauma can lead to a further, often subtle, decompensation in endocrine function (Fig. 1). In the management of trauma victims with pre-existing endocrine disease, the role of the critical care specialist is three-fold--(1) to maintain a high index of suspicion for endocrine disease in all trauma victims, (2) to anticipate and prevent endocrine organ decompensation, and (3) to rapidly diagnose and institute therapy in those suspected of having endocrine disease.

Corticotropin-releasing hormone but not glutamate elicits hormonal responses from the parabrachial region in cats.

Corticotropin-releasing hormone (CRH)-containing processes were found by immunohistochemistry in the dorsal and lateral parabrachial nucleus extending medially over the dorsal aspect of the brachium and then along the lateral and medial aspects of the mesencephalic trigeminal tract. Reactivity of lesser density extended ventrally from the medial parabrachial nucleus into the locus ceruleus and subceruleus. To determine if CRH acts in these areas to modulate plasma adrenocorticotropic hormone (ACTH) and arginine vasopressin (AVP), acutely prepared, chloralose-anesthetized cats were tested with microinjections (100 nl/min, 2 min). Plasma ACTH increased significantly after injections of CRH (2 pmol) along the dorsal aspect of the brachium and in the locus subceruleus (P < 0.05 and P < 0.01, respectively). Plasma AVP increased significantly after injections of CRH into the medial parabrachial nucleus (P < 0.01). These responses of ACTH and AVP differed significantly from those to injections of either vehicle or glutamate at identical sites and from those to CRH injected in other areas. None of these latter responses was significant. CRH was without effect on arterial pressure even though glutamate (30 nmol) injected into the area ventral and medial to the brachium elicited a significant pressor response. We suggest that excitatory amino acids such as glutamate act in this area to activate neurons with descending projections that influence autonomic function. In contrast, CRH appears to activate other neurons with ascending projections that drive neuroendocrine release.

A circulating shock protein depolarizes cells in hemorrhage and sepsis.

OBJECTIVE: A study to determine if both septic and hemorrhagic shock lead to the appearance of a substance that depolarizes cells in plasma was performed. SUMMARY BACKGROUND DATA: Transmembrane potential decreases in skeletal muscle, hepatocytes, and red blood cells early in the development of both hemorrhagic and septic shock. The associated movement of ions and water into cells leads to extracellular fluid loss and exacerbates shock. METHODS: Adult male Sprague-Dawley rats with indwelling arterial and venous cannulae were bled 20 mL/kg or received intravenously 2 x 10(10) Escherichia coli suspended in 400 mL of 0.9% saline. Blood samples were taken after hemorrhage and induction of sepsis to determine the presence of a plasma factor that depolarized red blood cells. Control rats were not injected with E. coli or bled. Plasma from bled and septic rats was processed by sequential precipitation with ammonium sulfate and subjected to gel filtration. RESULTS: Depolarizing activity was highest 20 minutes after hemorrhage and 60 minutes after E. coli injection, decreasing to control levels by 2 (hemorrhage) and 4 (sepsis) hours. Control rats showed no significant change in depolarizing activity. Tryptic and chymotryptic digestion eliminated the depolarizing activity, indicating that the active substance is, at least in part, a protein. Depolarizing activity from bled and septic processed plasma was confined essentially to the 70% ammonium sulfate fraction and the activity migrated with an apparent molecular mass of 200 kD after gel filtration. Separation of the complex by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) produced an identical pattern of bands in both bled and septic animals. CONCLUSIONS: A circulating plasma protein complex of high molecular weight causes cellular depolarization in both hemorrhage and sepsis and may be responsible for the associated increases in cell sodium and water seen in both hemorrhagic and septic shock.

Ferric iron potentiates cell depolarization by a circulating shock protein.

OBJECTIVE: To determine whether or nor iron affects the depolarizing activity of a circulating shock protein that appears in plasma after hemorrhage. DESIGN: Randomized design. SETTING: University laboratory. ANIMALS: Healthy male Sprague-Dawley rats weighing 300 to 400 g with femoral artery and vein cannulas placed 4 days before hemorrhage. INTERVENTION: A 20-mL/kg hemorrhage and plasma collection. MAIN OUTCOME MEASURES: Depolarizing activity was measured as the increased fluorescence of an oxonol dye in the presence of Fe3+, Fe2+, or the iron chelator deferoxamine mesylate and was titrated against increasing concentrations of circulating shock protein or iron. Circulating shock protein was derived from plasma and was purified in two steps: stepwise ammonium sulfate precipitation followed by denaturing ion-exchange chromatography and refolding. RESULTS: At physiologic concentrations, Fe3+ but not Fe2+ potentiated the depolarizing activity of plasma after ammonium sulfate. Addition of deferoxamine abolished activity. Denaturing chromatography removed nearly all the depolarizing activity; however, Fe3+ restored activity to this fraction. Fe3+ increased total activity and decreased the concentration at which 50% activity was observed. CONCLUSION: These data indicate that physiologic concentrations of Fe3+ may act to modulate the depolarizing activity of circulating shock protein that in turn mediates the intracellular accumulation of salt and water in shock.

Reno-colic fistula following primary repair of the colon: case report.

Reno-colic fistula is a rare entity that to our knowledge has not previously been reported as a complication of penetrating abdominal trauma. We report a case of reno-colic fistula complicating primary repair of a colonic wound from a gunshot to the abdomen. Causes of previously reported reno-colic fistulas include primary renal and colonic pathologic states involving infectious, malignant, or other inflammatory processes. The occurrence of this complication may have been related to the primary repair of the colon. Although recent reports support primary colonic repair, perhaps renal injuries deserve special consideration when patients are evaluated for primary versus staged repair of penetrating colon injuries.

Alterations of locus coeruleus noradrenergic activity in relation to pituitary secretion after hemorrhage in cats.

Noradrenergic (NA) activity in the cat ventral locus coeruleus (vLC), measured either by voltammetry or by push-pull perfusion, increased in response to hemorrhage. This stimulus also elicited plasma adrenocorticotropin (ACTH) and vasopressin responses. Increased vLC NA activity following the initial hemorrhage (InHem) persisted even after plasma hormone levels returned toward prestimulus values. Upon stimulus repetition, the peak increase in vLC NA activity was similar to that observed during InHem while the hormone responses were of greater magnitude (i.e. potentiated). Hence, it is suggested that the LC may exert a modulatory role in the hemodynamic control of hypothalamic-pituitary axis function.