Mathematical modeling of marker influx and efflux in cells.

The tumor promoter, phorbol 12-myristate 13-acetate (PMA), affects the processing of fluid that enters a cell from the ambient medium. Previous work showed that marker accumulates to a higher level in PMA-treated than in untreated cells. Since PMA also affects the physical activity of the membrane and stimulates the normal process of taking up extracellular fluid, called endocytosis, it is important to learn whether the perturbations in fluid processing can be attributed entirely to a change in the cell's limiting membrane. To this end, a model for fluid uptake and processing was developed and applied to experiments in which a marker for extracellular fluid was added to cells. From previous work on marker accumulation, it was deduced that there were at least two functional compartments involved in fluid movement. Compartment I is a rapidly filling and rapidly recycling compartment. Compartment II is a slowly filling and emptying compartment. Three routes of vesicle traffic must be considered, one mediating influx from the ambient medium into compartment I, a second, efflux from compartment I to the medium, and a third efflux from compartment I into compartment II. Using earlier models for processing, workers found it difficult to estimate rates of movement through either of the latter routes, as well as the volume of compartment I. The difficulty arises from the fact that only one kinetic constant can be estimated directly from data, namely the instantaneous uptake rate. The remaining data depend on measuring the total mass of marker in the cells. Since the concentration of marker in the cell changes continuously, it is advantageous to employ differential equations to simulate the tracer movement. By applying the model to experimental values, we found estimates for all three rates of fluid movement and the volume of compartment I. It is thought that the model will enable us to determine whether apparent alterations in the time course of uptake arise solely from altered properties of the limiting membrane.

iNOS expression in human intestinal microvascular endothelial cells inhibits leukocyte adhesion.

Increased nitric oxide (NO) production by inducible nitric oxide synthase (iNOS) has been associated with intestinal inflammation, including human inflammatory bowel disease. However, NO can downregulate endothelial activation and leukocyte adhesion, critical steps in the inflammatory response. Using primary cultures of human intestinal microvascular endothelial cells (HIMEC), we determined the role of NO in the regulation of HIMEC activation and interaction with leukocytes. Both nonselective (NG-monomethyl-L-arginine) and specific (N-iminoethyl-L-lysine) competitive inhibitors of iNOS significantly increased binding of leukocytes by HIMEC activated with cytokines and lipopolysaccharide. Increased adhesion was reversible with the NOS substrate L-arginine and was not observed in human umbilical vein endothelial cells (HUVEC). Activation of HIMEC significantly upregulated HIMEC iNOS expression and NO production. NOS inhibitors did not augment cell adhesion molecule levels in activated HIMEC but did result in sustained increases in intracellular reactive oxygen species. In addition, antioxidant compounds reversed the effect of NOS inhibitors on HIMEC-leukocyte interaction. Taken together, these data suggest that after HIMEC activation, iNOS-derived NO is an endogenous antioxidant, downregulating leukocyte binding and potentially downregulating intestinal inflammation.

Dose-response relationship of clonidine in tetracaine spinal anesthesia.

The study was undertaken to define a dose-response relationship for clonidine in prolonging canine tetracaine spinal anesthesia. Using a randomized blind cross-over design, six mongrel dogs were given subarachnoid injections (1 ml) of the following solutions over an 8-week period: tetracaine 4 mg (control), or tetracaine 4 mg with clonidine in doses of 10, 25, 50, 100, 150, 200, and 300 micrograms. With clonidine doses equal to or exceeding 50 micrograms/ml, motor and sensory blockade were significantly (P less than 0.01) prolonged, when compared to the control times. Analysis of data by second order polynomial regression analysis produced a relationship defined by Y = 23.241 + 1.104(x) - 0.0023(x2) with r2 = 0.92 and P less than 0.001 for sensory blockade and Y = 38.7072 + 1.64425(x) - 0.004125(x2) with r2 = 0.90 and P less than 0.005 for motor blockade. From these curves, a plateau in clonidine dose-response for both sensory blockade and motor blockade occurred at 150 micrograms. The increase in duration of spinal anesthesia with clonidine may be related to a direct post-synaptic alpha 2 adrenoceptor arteriolar effect, a spinal cord pre- or post-synaptic alpha 2 antinociceptive action or supraspinal alpha 2 modulation of nociception. No animals showed evidence of neurologic dysfunction during the study. The authors conclude that a well-defined dose-response relationship exists for clonidine in canine tetracaine spinal anesthesia.

Spinal cord blood flow following sub-arachnoid lidocaine.

Twelve mongrel dogs were randomized into two equal groups. Cervical, thoracic and lumbosacral spinal cord and spinal dural blood flows were measured using the radioactive microsphere technique. Blood flow determinations were made prior to and 20 and 40 minutes following lumbar subarachnoid injection of: two per cent lidocaine (100 mg) or two per cent lidocaine (100 mg) with 1/25,000 epinephrine (200 micrograms). Dogs receiving subarachnoid lidocaine demonstrated a decrease in mean arterial blood pressure of 23 per cent and 14 per cent (p less than 0.05), while dogs receiving lidocaine with epinephrine had a decrease of 38 and 34 per cent (p less than 0.05) at 20 and 40 minutes respectively. Cardiac index was not significantly changed in either group. Lumbar subarachnoid lidocaine (100 mg) produced a rapid regional dural hyperemia (observed at 20 minutes postinjection) and a delayed regional spinal cord hyperemia (observed at 40 minutes postinjection) which were not observed following the addition of epinephrine (200 micrograms).

Subarachnoid bupivacaine decreases spinal cord blood flow in dogs.

Eighteen mongrel dogs were randomized into two equal groups. Cervical, thoracic and lumbosacral spinal cord and spinal dural blood flows were measured using the radioactive microsphere technique. Blood flow determinations were made prior to, and 20 and 40 minutes following lumbar subarachnoid injection of: (1) 0.4 per cent bupivacaine (20 mg), or (2) 0.4 per cent bupivacaine (20 mg) with 1/25,000 epinephrine (200 micrograms). In dogs given subarachnoid bupivacaine or bupivacaine with epinephrine, the maximum decrease in mean arterial blood pressure (33 per cent) occurred at 40 minutes post-injection. Cardiac index decreased in dogs given subarachnoid bupivacaine (197 +/- 11 ml X kg-1 X min-1 control vs. 141 +/- 19 ml X kg-1 X min-1 at 40 minutes), while it increased in dogs given bupivacaine with epinephrine (201 +/- 11 ml X kg-1 X min-1 - control vs. 252 +/- 15 ml X kg-1 X min-1 at 40 minutes). Dogs receiving subarachnoid bupivacaine demonstrated a significant decrease in spinal cord blood flow to all regions. Dogs receiving subarachnoid bupivacaine with epinephrine demonstrated a significant decrease in thoracic and lumbosacral spinal cord blood flow; however, cervical cord blood flow remained unchanged. Thoracic and lumbosacral dural blood flows were significantly decreased in both groups following subarachnoid injection. Subarachnoid bupivacaine 0.4 per cent (20 mg) and 0.4 per cent with epinephrine 1/25,000 (200 micrograms) decrease spinal cord and spinal dural blood flow in dogs.

Ethanol disposition in newborn lambs and comparison of alcohol dehydrogenase activity in placenta and maternal sheep, fetal and neonatal lamb liver.

The kinetic disposition of ethanol was studied in neonatal lambs. The mean plasma clearance rate was 36 mg/l/h, 17% of that in near-term pregnant sheep. Activity increased slowly during the first 5 days of life. Hepatic alcohol dehydrogenase activity in the neonatal lamb was only 7% of that in adult sheep, but was similar to activity in the near-term fetus. Placental enzyme activity was even lower than that in the fetus and neonate, suggesting only a minor role for it in the metabolic disposition of ethanol during pregnancy. The pH optimum for alcohol dehydrogenase was higher in the fetus and neonate (9.6) than in maternal liver or placenta (9.0). The pharmacodynamic consequences of prolonged neonatal exposure to ethanol due to impaired metabolism remain to be explored.

Maternal and fetal ethanol pharmacokinetics and cardiovascular responses in near-term pregnant sheep.

The disposition and cardiovascular effects of ethanol were studied in near-term pregnant sheep and their fetuses after intravenous infusions either to the mother or the fetus. Placental transfer of ethanol from mother to fetus was rapid, but transfer from the fetal to maternal circulation was impaired when ethanol was administered directly to the fetus. Plasma clearance of ethanol was similar from the mother and fetus in utero. In the maternal infusion experiments, both maternal and fetal heart rate increased with plasma ethanol concentration. However, in the fetal infusion experiments, both maternal and fetal heart rate increased with plasma ethanol concentration. However, in the fetal infusion experiments, fetal heart rate was inversely related to plasma ethanol concentrations while maternal mean arterial blood pressure increased with maternal plasma ethanol concentration. Further studies in neonatal lambs are needed to determine if ethanol has adverse effects on hemodynamics when the neonate is separated from its protective intrauterine environment.

The effect of subarachnoid epinephrine and phenylephrine on spinal cord blood flow.

Eighteen mongrel dogs were divided into three equal groups. Spinal cord and spinal dural blood flow in the cervical, thoracic and lumbosacral regions were measured using the radioactive microsphere technique. Measurements were taken before and 10 and 40 minutes after lumbar subarachnoid injection of one of the following: physiologic saline; epinephrine 200 micrograms or phenylephrine 5 mg. No significant change in spinal cord blood flow occurred in any of the groups, nor was there any difference between the groups. Dogs receiving subarachnoid phenylephrine did demonstrate a significant reduction of thoracic dural blood flow at ten minutes. Dogs receiving intrathecal epinephrine or phenylephrine demonstrated a significant reduction in lumbo-sacral dural blood flow at ten minutes after injection. The reduction in dural blood flow was still evident at 40 minutes in dogs receiving phenylephrine. Subarachnoid epinephrine (200 micrograms) and phenylephrine (5 mg) do not effect spinal cord blood flow but do produce regional dural vasoconstriction.

The uptake of isoflurane by the foetal lamb in utero: effect on regional blood flow.

Because isoflurane has recently been approved for clinical use in anaesthesia, we have studied the effect of this agent in the foetus using the pregnant ewe as an animal model. Eight pregnant ewes of 120-125 days gestation were surgically prepared with indwelling catheters and tracheostomy. Prior to anaesthesia, labelled microspheres were injected into the foetal circulation to determine cardiac output and regional blood flows to all organs. The ewes were anaesthetized with a constant inspired concentration of 2.0 per cent isoflurane in oxygen. Blood samples were drawn to construct an uptake curve for both mother and foetus. At 60 and 96 minutes of anaesthesia, microspheres were injected into the foetal circulation to measure changes in organ blood flow from the control period. Isoflurane crossed the placenta and appeared in the foetal circulation within two minutes. By 96 minutes, maternal and foetal arterial levels were 116.3 +/- 9.9 and 99.3 +/- 9.1 mEq/L (0.98 vol% and 0.75 vol%). There were no significant changes in foetal blood pressure or pulse rate but foetal pH decreased significantly from 7.39 +/- 0.02 to 7.26 +/- 0.2 (mean +/- SEM) and base excess decreased from -1.1 +/- 1.5 to -6.2 +/- 0.7. Foetal cardiac index decreased from 390.8 +/- 26.9 ml X kg-1 X min-1 to 292.0 +/- 13.8 after 96 minutes of anaesthesia. There were no significant changes in any of the maternal cardiovascular or acid-base parameters.

Effect of halothane on cardiac output and regional flow in the fetal lamb in utero.

We studied the effect of halothane on the fetal cardiovascular system of six lambs in utero by measuring fetal heart rate and femoral arterial blood pressure and by injecting labeled microspheres during a control period and again after 60 and 90 min of halothane anesthesia administered to six pregnant ewes at an inspired concentration of 1.5%. There were no significant effects on maternal cardiovascular function or acid-base balance, but fetal blood pressure decreased significantly by 27% after 8 min of halothane anesthesia and remained at this level for the duration of the experiment. However, there were no significant changes either in fetal regional blood flow to the vital organs or in fetal cardiac output. Fetal oxygenation and acid-base status remained stable. We conclude that in normal fetal lamb in utero the decrease in mean fetal arterial blood pressure associated with maternal halothane anesthesia is due to a decrease in peripheral vascular resistance because regional blood flow and acid-base status are well maintained.

Fetal anesthetic requirement (MAC) for halothane.

We asked whether the anesthetic requirement (MAC) of fetal lambs is lower than that of pregnant ewes. In five pregnant ewes anesthetized with a subarachnoid block, a fetal foot was withdrawn through a hysterotomy. The ewe then breathed 1.5% halothane and a clamp was applied to the fetal foot at 2-min intervals. We concomitantly obtained arterial blood from previously implanted catheters. When fetal movement in response to clamping the foot ceased, halothane was discontinued and the stimulus and sampling continued until the fetus began to move. Anesthesia was again resumed and continued until movement stopped. Anesthesia was then deepened and MAC was determined in the mother (stimulus--ear clamp). The fetal blood concentrations of halothane at MAC were 48 +/- 28 mg/L; they were 133 +/- 5 mg/L in the mother. This difference was highly significant (P less than 0.001). Calculated end-tidal concentrations were 0.33% and 0.69%, respectively. In two animals delivered by cesarean section, MAC increased progressively over the first 12 h of life. Progesterone levels concomitantly decreased.

Cerebral vascular responses to ketamine and thiopentone during foetal acidosis.

Using a chronic sheep preparation, with induced foetal acidosis, the effects of low and high dose thiopentone and ketamine on maternal and foetal cardiovascular dynamics were studied. Ketamine 4 mg . kg-1 and thiopentone 10 mg . kg-1 were both associated with marked reductions in foetal cerebral blood flow and cerebral oxygen delivery. Blood pressure and cerebral blood flow in the acidotic foetus was better preserved, however, following the lower dose of ketamine 2 mg . kg-1 than following thiopentone 6 mg . kg-1.

Preservation of fetal brain blood flow relative to other organs during hypovolemic hypotension.

The asphyxiated newborn is particularly vulnerable to hypotension, which contributes to hypoxic brain damage by reducing cerebral perfusion. During asphyxia, cerebral blood flow (CBF)( is pressure passive, that is, CBF autoregulation is abolished. It is important to know if the nonasphyxiated fetus and newborn are similarly vulnerable to hypotension. In the present study, we have measured acute responses of organ blood flow to a hypovolemic/hypotensive stress in the normoxic near term sheep fetus. Changes in brain flow were compared to changes in other organs. Eight chronically prepared fetal lambs were studied. Organ blood flows were measured by the microsphere technique during a control period, after a 20% blood volume reduction, and again after reinfusion of that volume. Hypovolemia was accompanied by a 21% decrease in blood pressure and a 4 torr increase in PCO2; after reinfusion blood pressure increased 16% above control. Control measurements of organ perfusion were similar to those reported by other investigators. Cardiac output and flow to all organs, with the exception of the brain, were reduced 30-56% during hypovolemia. Brain blood flow was insignificantly reduced by 9%. If a correction is applied for the increase in PCO2, CBF corrected to control PCO2 would have have been significantly reduced by 18%. After reinfusion, flow to all organs increased to near control levels. We conclude that the normoxic fetal lamb shows evidence of CBF autoregulation, and is able to preserve relative constancy of CBF within a blood pressure range of +/- 20% of normal. However, the evidence presented in this study suggests that autoregulation may be less effective in response to a hypotensive stress, even though CBF is better preserved than flow to most other organs.

Isoflurane: an anesthetic for the eighties?

The introduction of isoflurane to clinical practice follows the search for a nonflammable, potent inhalation anesthetic which, above all, is chemically stable so as to resist biodegradation or attack by other chemicals. These attributes characterize isoflurane (Table 2). The hoped for freedom from hepatic and renal toxicity and from carcinogenic and mutagenic properties is a reality with this drug. Other favorable characteristics include relatively low solubility in blood in relation to anesthetic dose, lack of arrhythmogenic effect, provision of good muscle relaxation, and the absence of central nervous system excitation. Its moderate pungency detracts slightly from the ease of inhaled induction. Disadvantages include respiratory depression, reduced arterial blood pressure, uterine relaxation, decreased uteroplacental blood flow, and likely ability to trigger malignant hyperpyrexia. The frequency and/or significance of tachycardia and dilation of muscle blood vessels in clinical practice remain to be established. We believe isoflurane is a significant improvement over earlier potent inhalation anesthetics.

The "milieu interieur"-a model of brain physiology and pathophysiology.

The "milieu interieur" or "internal environment" of the brain is determined by the glial cells, the cerebrospinal fluid, the blood-brain barrier, the cerebral blood flow, the central regulation of respiration, and the intracranial pressure. Anaesthetic agents and anoxia-ischaemic insult can disrupt this important but vulnerable neuronal environment. Head injury is used as an example of a common insult to the neuronal environment and the principles of management are discussed, using a model of brain physiology and pathophysiology which can be modified to include other clinical situations.

Regional cerebral blood flow changes during severe fetal asphyxia produced by slow partial umbilical cord compression.

We studied the effects of severe partial asphyxia on regional cerebral blood flow and arterial blood pressure in the unanesthetized, physiologically stable fetal lamb. Cerebral blood flow was measured by the microsphere technique before and during partial umbilical cord compression. Asphyxia sufficient to decrease pH from 7.40 to 7.04 and reduce oxygen saturation from 50% to 19% increased cerebral blood flow to all areas of the brain with the largest increases going to the brain stem (275% of control) and deep cerebral structures (240% of control). Fetal arterial blood pressures increased from a mean of 58 mm Hg to a mean of 71 mm. Hg during asphyxia. The blood pressure increases correlated closely with the regional cerebral blood flow increases. There was a poor correlation between cerebral blood flow increases and changes in Paco2' pH, or oxygen saturation. We conclude that during severe fetal asphyxia arterial blood pressure is the critical factor in determining cerebral blood flow.

Effect of anaesthetic agents on the ionic composition of cerebrospinal fluid following total cerebral ischaemia.

Total cerebral ischaemia in rats caused a marked increase in the cisternal CSF potassium concentration but little change in CSF sodium or chloride concentration. The anaesthetic techniques studied (pentobarbitone, halothane/oxygen and nitrous oxide/oxygen/relaxant) did not effect the potassium increase following cerebral ischaemia. We conclude that the mechanism of barbiturate protection following cerebral ischaemia is different from that of hypothermia.

Halothane depresses baroreflex control of heart rate in man.

Baroreflex control of heart rate was determined during three awake control situations and during two depths of halothane anesthesia in man. Baroreflex function was quantiated by calculating the pressor test slope from the R-R interval change on the ECG produced by a pharmacologically induced pressor response. During the three awake control situations the subjects breathed room air or 100 per cent O2 spontaneously or 100 per cent O2 with ventilation controlled. Mean (+/- SD) slopes obtained were 15.1 +/- 4.5, 15.6 +/- 6.8 and 18.4 +/- 9.9, respectively. No significant difference in baroreflex function slope was observed. During light halothane anesthesia (0.7 per cent endtidal) baroreflex function was significantly depressed (mean slope = 2.5 +/- 1.5), and it was abolished at 1.1 per cent end-tidal halothane (mean slope = 0.03 +/- 0.04). It is concluded that halothane anesthesia produces depression of baroreflex control of heart rate in man.