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Windup is a progressive increase in response of dorsal horn neurons to repetitive C-fiber stimulation that may underlie temporal summation of pain. We investigated the frequency- and intensity-dependency of windup, and the effects of isoflurane and N-methyl-d-aspartate (NMDA) receptor blockade, to determine if they parallel the influence of temporal and spatial summation of noxious stimuli on anesthetic requirements.We recorded responses of rat sacral dorsal horn neurons to 20-s trains of electrical tail stimulation at different frequencies (0.3-10 Hz) and intensities (0.8-5 x stimulus threshold) during delivery of 0.7 to 1.3 minimum alveolar anesthetic concentration isoflurane. Summed responses (area under the curve [AUC] windup), initial response, absolute windup (AUC minus 20 times the initial response), and slope of windup were quantified.Increases in stimulus intensity and frequency progressively increased AUC windup (P0.01 for both) and correlated with isoflurane concentrations required for immobility (R2 = 0.98 and 0.97, respectively). Increasing the isoflurane concentration significantly suppressed each measure of windup elicited by low-intensity and low-frequency, but not high-intensity and high-frequency stimulus trains. The initial response magnitude significantly correlated with slope of windup across stimulus intensities and isoflurane concentrations. The NMDA receptor antagonist MK801 significantly reduced windup (to 53%; P0.05) at 1 Hz.Windup of dorsal horn neurons at low stimulus intensities and frequencies increases isoflurane requirements for immobility via a NMDA receptor-dependent mechanism. At high stimulus intensities and frequencies, windup was resistant to isoflurane consistent with larger anesthetic requirements for immobility.

BACKGROUND: Because of the logistical obstacles to measurement under hyperbaric conditions, the effect of nitrous oxide (N 2 O) alone on spinal neuronal responses has not been tested. We hypothesized that, like other inhaled anesthetics, N 2 O would depress spinal neuronal responses to noxious stimulation. METHODS: The lumbar spinal cord was exposed in rats anesthetized with isoflurane. Mechanically ventilated rats were placed into a hyperbaric chamber and needle electrodes were inserted into the hindpaws. Isoflurane administration was discontinued and anesthesia converted to N 2 O by pressurizing the chamber with N 2 O. A microelectrode was inserted into the lumbar cord using computer-controlled motors and a hydraulic microdrive. Neuronal responses to electrical stimulation of the hindpaw were sought at 1.5, 2, and 2.5 atm N 2 O (0.8-1.3 minimum alveolar concentration). RESULTS: Increasing N 2 O partial pressures variably affected neuronal responses to a 2 s 100-Hz electrical stimulus. Neuronal depth and neuronal response were correlated, with superficial neurons tending to be facilitated, while deeper neurons were depressed; (overall responses were 1331 ± 408, 1594 ± 383, and 1578 ± 500 impulses/min at 1.5, 2, and 2.5 atm N 2 O, respectively; mean, standard error). N 2 O did not affect neuronal responses to a repetitive "windup" stimulus. Infusion of the N-methyl-D-aspartate blocker MK-801 into separate rats increased the neuronal response to the 100-Hz stimulus (from 781 ± 216 to 1352 ± 269 impulses/min, P < 0.05). CONCLUSIONS: N 2 O facilitated superficial spinal neuronal responses to noxious stimulation while depressing deeper neurons. These results suggest that anesthetic partial pressures of N 2 O have divergent effects on spinal neuronal responses to noxious stimulation, the specific responses depending on the depth of the spinal neurons.

The retrograde tracer cholera toxin beta-subunit (CTB) was used to trace long ascending propriospinal projections from neurons in the lumbosacral spinal cord to the upper cervical (C3) gray matter in adult male Sprague-Dawley rats. Following large 0.5 microl CTB injections restricted mainly to the upper cervical ventral horn (n=5), there were many lumbosacral CTB-positive neurons (14-17/section) in the intermediate gray and ventral horn (dorsal lamina VIII, medial VII extending into X) contralaterally, with fewer at corresponding ipsilateral locations. Labeled cells (4-8/section) were also observed in contralateral laminae IV-VI and the lateral spinal nucleus, with fewer ipsilaterally. Few labeled cells (2/section) were observed in superficial laminae I-II. Smaller (0.15 microl) microinjections of CTB restricted to the upper cervical ventral gray matter labeled cells in contralateral laminae VII-VIII (approximately 6-9/section) with fewer ipsilaterally. There were relatively fewer (2/section) in the intermediate dorsal horn and very few (1/section) in lamina I. Larger (0.5 microl) CTB injections encompassing the C3 dorsal and ventral gray matter on one side labeled significantly more CTB-positive neurons (6/section) in contralateral lamina I compared to ventral horn injections. These results suggest direct projections from ventromedially located neurons of lumbar and sacral segments to the contralateral ventral gray matter of upper cervical segments, as well as from neurons in the intermediate but not superficial dorsal horn. They further suggest that some lumbosacral superficial dorsal horn neurons project to the upper cervical dorsal horn. These propriospinal projections may be involved in coordinating head and neck movements during locomotion or stimulus-evoked motor responses.

Halothane and isoflurane, in the peri-minimum alveolar anesthetic concentration (MAC) range, exert differential effects on spinal nociceptive neurons, whereby halothane further depresses their responses from 0.8 to 1.2 MAC, whereas isoflurane does not. We presently investigated if these anesthetics differentially affect windup, the progressive increase in neuronal responses to repetitive noxious stimuli, over a broad concentration range from 0 to 1.2 MAC. In decerebrated rats, single-unit recordings were made from dorsal horn neurons exhibiting windup to 20 1-Hz C-fiber strength electrical stimuli. Halothane and isoflurane (0, 0.4, 0.8, and 1.2 MAC) were tested in a counterbalanced crossover protocol. Increasing halothane and isoflurane from 0 to 1.2 MAC progressively suppressed the response to the first stimulus, as well as summed responses to all stimuli (to 34% 8% and 50% 8%, respectively; P 0.05). Absolute windup (summed response minus 20 the first response) was suppressed by both anesthetics from 0 to 0.8 MAC, with further depression by halothane but not isoflurane at 1.2 MAC. Responses of neurons isolated at 0 MAC were partially, but never totally, depressed at 0.8 MAC. The dose-dependent suppression of windup is consistent with reduced temporal summation of pain. Further depression at 1.2 MAC halothane, but not isoflurane, suggests different sites of immobilizing action for these two anesthetics. Immobility seems to not be mediated by severe anesthetic depression of a subpopulation of nociceptive neurons. (Anesth Analg 2006;103:753‐60)

We hypothesized that N-methyl-d-aspartate (NMDA) receptors mediate some or all of the capacity of inhaled anesthetics to prevent movement in the face of noxious stimulation, and that this capacity to prevent movement correlates directly with the in vitro capacity of such anesthetics to block the NMDA receptor. To test this hypothesis, we measured the effect of IV infusion of the NMDA blockers dizocilpine (MK-801) and (R)-4-(3-phosphonopropyl) piperazine-2-carboxylic acid (CPP) to decrease the MAC (the minimum alveolar concentration of anesthetic that prevents movement in 50% of subjects given a noxious stimulation) of 8 conventional anesthetics (cyclopropane, desflurane, enflurane, halothane, isoflurane, nitrous oxide, sevoflurane, and xenon) and 8 aromatic compounds (benzene, fluorobenzene, o-difluorobenzene, p-difluorobenzene, 1,2,4-trifluorobenzene, 1,3,5-trifluorobenzene, pentafluorobenzene, and hexafluorobenzene) and, for comparison, etomidate. We postulated that MK-801 or CPP infusions would decrease MAC in inverse proportion to the in vitro capacity of these anesthetics to block the NMDA receptor. This notion proved correct for the aromatic inhaled anesthetics, but not for the conventional anesthetics. At the greatest infusion of MK-801 (32 microg x kg(-1) x min(-1)) the MACs of conventional anesthetics decreased by 59.4 +/- 3.4% (mean +/- sd) and at 8 microg x kg(-1) x min(-1) by 45.5 +/- 4.2%, a decrease not significantly different from a 51.4 +/- 19.0% decrease produced in the EC50 for etomidate, an anesthetic that acts solely by enhancing gamma-amino butyric acid (GABA) receptors. We conclude that some aromatic anesthetics may produce immobility in the face of noxious stimulation by blocking the action of glutamate on NMDA receptors but that conventional inhaled anesthetics do not.

Studies using molecular modeling, genetic engineering, neurophysiology/pharmacology, and whole animals have advanced our understanding of where and how inhaled anesthetics act to produce immobility (minimum alveolar anesthetic concentration; MAC) by actions on the spinal cord. Numerous ligand- and voltage-gated channels might plausibly mediate MAC, and specific amino acid sites in certain receptors present likely candidates for mediation. However, in vivo studies to date suggest that several channels or receptors may not be mediators (e.g., gamma-aminobutyric acid A, acetylcholine, potassium, 5-hydroxytryptamine-3, opioids, and alpha(2)-adrenergic), whereas other receptors/channels (e.g., glycine, N-methyl-D-aspartate, and sodium) remain credible candidates.

Background Production of retrograde amnesia by anesthetics would indicate that these drugs can disrupt mechanisms that stabilize memory. Such disruption would allow suppression of memory of previous untoward events. The authors examined whether isoflurane provides retrograde amnesia for classic (Pavlovian) fear conditioning. Methods Rats were trained to fear tone by applying three (three-trial) or one (one-trial) tone-shock pairs while breathing various constant concentrations of isoflurane. Immediately after training, isoflurane administration was either discontinued, maintained unchanged, or rapidly increased to 1.0 minimum alveolar concentration for 1 h longer. Groups of rats were similarly trained to fear context while breathing isoflurane by applying shocks (without tones) in a distinctive environment. The next day, memory for the conditioned stimuli was determined by presenting the tone or context (without shock) and measuring the proportion of time each rat froze (appeared immobile). For each conditioning procedure, the effects of the three posttraining isoflurane treatments were compared. Results Rapid increases in posttraining isoflurane administration did not suppress conditioned fear for any of the training procedures. In contrast, isoflurane administration during conditioning dose-dependently suppressed conditioning (P < 0.05). Training to tone was more resistant to the effects of isoflurane than training to context (P < 0.05), and the three-trial learning procedure was more was more resistant than the one-trial procedure (P < 0.05). Conclusions Isoflurane provided intense dose-dependent anterograde but not retrograde amnesia for classic fear conditioning. Isoflurane appears to disrupt memory processes that occur at or within a few minutes of the conditioning procedure.

UNLABELLED: The in vivo potencies of anesthetics correlate with their capacity to suppress the reaction of luciferin with luciferase. In addition, luciferin has structural resemblances to etomidate. These observations raise the issues of whether luciferin, itself, might affect anesthetic requirement, and whether luciferase resembles the site of anesthetic action. Because the polar luciferin is unlikely to cross the blood-brain barrier (we found that the olive oil/water partition coefficient was 100 +/- 36 x 10(-7)), we studied these issues in rats by measuring the effect of infusion of luciferin in artificial cerebrospinal fluid into the lumbar subarachnoidal space and into the cerebral intraventricular space on the MAC (the minimum alveolar anesthetic concentration required to eliminate movement in response to a noxious stimulus in 50% of tested subjects) of isoflurane. MAC in rats given lumbar intrathecal doses of luciferin estimated to greatly exceed anesthetizing doses of etomidate, did not differ significantly from MAC in rats receiving only artificial cerebrospinal fluid into the lumbar intrathecal space. MAC slightly decreased when doses of luciferin estimated to greatly exceed anesthetizing doses of etomidate were infused intraventricularly (P < 0.05). In contrast to the absent or minimal effects of luciferin, intrathecal or intraventricular infusion of etomidate at similar or smaller doses significantly decreased isoflurane MAC. Luciferin did not affect +-aminobutyric acid type A or acetylcholine receptors expressed in Xenopus oocytes. These results suggest that luciferin has minimal or no anesthetic effects. It also suggests that luciferin/luciferase may not provide a good surrogate for the site at which anesthetics act, if this site is on the surface of neuronal cells. IMPLICATIONS: In proportion to their potencies, anesthetics inhibit luciferin's action on luciferase, and luciferin structurally resembles the anesthetic etomidate. However, in contrast to etomidate, luciferin given intrathecally or into the third cerebral ventricle does not have anesthetic actions, and it does not affect +-aminobutyric acid or acetylcholine receptors in vitro. Luciferase may not provide a good surrogate for the site at which anesthetics act.

The anesthetic concentration just suppressing appropriate response to command (minimum alveolar anesthetic concentration awake [MAC-awake] for volatile anesthetics or plasma concentration to prevent a response in 50% of patients [Cp50]-awake for intravenous anesthetics) provides three important measures. First, along with pharmacokinetics, the ratio of the awakening concentration to the anesthetizing concentration (MAC-awake/MAC or Cp50-awake/Cp50) determines time to awakening. Second, a correlation between MAC-awake and the anesthetic concentration sufficient to prevent learning suggests MAC-awake provides a surrogate measure of amnestic potency. Third, population values for MAC-awake provide evidence for or against commonality in anesthetic mechanisms. We studied 22 male volunteers twice to determine both MAC-awake for desflurane (2.60% +/- 0.46%) and Cp50-awake for propofol (2.69 +/- 0.56 microgram/mL). Awakening with desflurane occurs at a concentration closer to its anesthetizing concentration (36% of MAC) than propofol (18% of Cp50); that is, 1) desflurane requires less of a decrement in anesthetic concentration at the effect site for arousal; and 2) if MAC-awake (Cp50-awake) values reflect the concentrations providing amnesia, propofol is a more potent amnestic. Of interest, the dose response curves of desflurane and propofol were equivalently steep, a finding consistent with a common mechanism of action. In contrast, sensitivity of each volunteer to desflurane did not correlate with sensitivity to propofol (r2 < 0.01, P = 0.98) arguing against a common mechanism.

Whether anesthetized patients register emotionally charged information remains controversial. We tested this possibility using subanesthetic concentrations of propofol or desflurane. Twenty-two volunteers (selected for hypnosis susceptibility) received propofol and desflurane (on separate occasions, and in a random order) at a concentration 1.5-2 times each individual's minimum alveolar anesthetic concentration (MAC)-awake (or equivalent for propofol). We gave vecuronium, intubated the trachea of each volunteer, controlled ventilation, and then presented a neutral (control) drama or a "crisis" drama stating that the oxygen delivery system had failed, assigning crisis and control dramas in a blinded, randomized, and balanced manner. One day later, interviewers blinded to the assigned drama conducted a 2-h structured interview (including hypnosis) to determine whether the contents of the interviews after crisis and control dramas differed. In addition, messages permitting subsequent assessment of learning of matter-of-fact information (Trivial Pursuit-type question task and a behavior task) were presented at the anesthetic concentration just sufficient to prevent response to command in each volunteer. No analyses of the tasks involving matter-of-fact information revealed learning except one which correlated hypnosis susceptibility with behavior task performance. Both propofol and desflurane suppressed memory of the crisis. Consistent with previous findings for isoflurane and nitrous oxide, propofol and desflurane suppressed learning of matter-of-fact information at concentrations just above MAC-awake, except that volunteers' susceptibility to hypnosis correlated with performance of a behavior suggested during anesthesia. Propofol and desflurane suppressed learning of emotionally charged information at anesthetic concentrations 1.5-2 times MAC-awake (less than MAC), a different result from that previously reported for ether.

The limited potency of nitrous oxide mandates the use of a hyperbaric chamber to produce anesthesia. Use of a hyperbaric chamber complicates anesthetic delivery, ventilation, and electrophysiological recording. We constructed a hyperbaric acrylic-aluminum chamber allowing recording of single unit activity in spinal cord of rats anesthetized only with N 2 O. Large aluminum plates secured to each other by rods that span the length of the chamber close each end of the chamber. The 122 cm long, 33 cm wide chamber housed ventilator, intravenous infusion pumps, recording headstage, including hydraulic microdrive and stepper motors (controlled by external computers). Electrical pass-throughs in the plates permitted electrical current or signals to enter or leave the chamber. In rats anesthetized only with N 2 O we recorded extracellular action potentials with a high signal-to-noise ratio. We also recorded electroencephalographic activity. This technique is well-suited to study actions of weak anesthetics such as N 2 O and Xe at working pressures of 4–5 atm or greater. The safety of such pressures depends on the wall thickness and chamber diameter.

Antagonism of N-methyl-d-aspartate (NMDA) receptors markedly decreases the minimum alveolar concentration (MAC) of inhaled anesthetics. To assess the importance of suppression of the temporal summation NMDA receptor component of MAC, we stimulated the tail of rats with trains of electrical pulses of varying interstimulus intervals (ISIs) and determined the inhaled anesthetic concentrations (crossover concentrations) that suppressed movement at different ISIs. The slopes of crossover concentrations versus ISIs provided a measure of temporal summation for each anesthetic. We studied five anesthetics that differ widely in their in vitro capacity to block NMDA receptors. To block NMDA receptor transmission and reveal the NMDA receptor component, the NMDA receptor antagonist, MK801, was separately added during each anesthetic. Halothane, isoflurane, and hexafluorobenzene did not appreciably suppress the NMDA receptor components of temporal summation, which contributed to 21% to 29% of MAC (P < 0.05 for each). Xenon and o-difluorobenzene suppressed these components to 8% to 0%, respectively, of MAC (neither significant), consistent with their greater NMDA receptor blocking action in vitro. NMDA receptor blockade may contribute to the MAC produced by inhaled anesthetics that potently inhibit NMDA receptors in vitro but not those that have a limited in vitro effect.

Previous reports suggest that the administration of epinephrine increases learning during deep barbiturate-chloral hydrate anesthesia in rats but not during anesthesia with 0.4% isoflurane in rabbits. We revisited this issue, using fear conditioning to a tone in rats as our experimental model for learning and memory and isoflurane and desflurane as our anesthetics. Expressed as a fraction of the minimum alveolar anesthetic concentration (MAC) preventing movement in 50% of rats, the amnestic 50% effective dose (ED(50)) for fear to tone in control rats inhaling isoflurane and injected with saline intraperitoneally (i.p.) was 0.32 +/- 0.03 MAC (mean +/- se) compared with 0.37 +/- 0.06 MAC in rats injected with 0.01 mg/kg of epinephrine i.p. and 0.38 +/- 0.03 MAC in rats injected with 0.1 mg/kg of epinephrine i.p. For desflurane, the amnestic ED(50) were 0.32 +/- 0.05 MAC in control rats receiving a saline injection i.p. versus 0.36 +/- 0.04 MAC in rats injected with 0.1 mg/kg of epinephrine i.p. We conclude that exogenous epinephrine does not decrease amnesia produced by inhaled isoflurane or desflurane, as assessed by fear conditioning to a tone in rats.

Background. Windup of spinal nociceptive neurones may underlie temporal summation of pain, influencing the minimum alveolar concentration (MAC) of anaesthetics required to prevent movement to supramaximal stimuli. We hypothesized that halothane and isoflurane would differentially affect windup of dorsal horn neurones. Methods. We recorded 18 nociceptive dorsal horn neurones exhibiting windup to 1 Hz electrical hindpaw stimuli in rats. Effects of 0.8 and 1.2 MAC isoflurane and halothane were recorded in the same neurones (counterbalanced, crossover design). Windup was calculated as the total number of C-fibre (100‐400 ms latency) plus afterdischarge (400‐1000 ms latency) spikes/ 20 stimuli (area under curve, AUC) or absolute windup (C-fibre plus afterdischarge‐20·initial response). Results. Increasing isoflurane from 0.8 to1.2 MAC did not affect AUC, but increased absolute windup from 429 (62) to 618 (84) impulses/20 stimuli (P

Windup is a progressive increase in responses of nociceptive spinal cord neurons to repeated electrical C fiber stimulation. We hypothesized that isoflurane would depress windup at approximately the minimum alveolar anesthetic concentration (MAC) required to suppress purposeful movement in response to noxious stimulation. We recorded windup responses in single lumbar spinal neurons (n = 17) to a series of 15 repetitive electrical stimuli delivered at 1 Hz to the hindpaw at C fiber strength; hindpaw withdrawal force was simultaneously recorded. The total number of action potentials per 15 stimuli (mean +/- sem as a percentage of each neuron's maximal response) was 83% +/- 5%, 84% +/- 5%, 67% +/- 7%, and 57% +/- 8% at 0.7, 0.9, 1.1, and 1.4 MAC, respectively. The 0.9 and 1.1 MAC values differed significantly from each other, whereas the 0.7 and 0.9 MAC values differed from the 1.4 MAC value (P < 0.05). The reduced firing was attributed to a depression of the initial C fiber-evoked responses in most units, and a reduction in windup slope over the initial 5 stimuli in 6 units. Muscle force was 67%, 11%, and 4% of the 0.7 MAC value at 0.9, 1.1, and 1.4 MAC, respectively. Isoflurane depressed excitability and variably affected windup of lumbar spinal cord neurons, while uniformly depressing windup of limb withdrawals in a concentration-dependent manner.

Background: General anesthesia may delay the onset of movement in response to noxious stimulation. The authors hypothesized that the production of immobility could involve depression of time-related processes involved in the generation of movement. Methods: The delays (latencies) between onset of tail clamp (n 16) or 50-Hz continuous electrical stimulation (n 8) and movement were measured in rats equilibrated at 0.1– 0.2% increasing steps of isoflurane. In other rats (n 8), the isoflurane concentrations just permitting and preventing movement (crossover concentrations) in response to trains of 0.5-ms 50-V square-wave pulses of interstimulus intervals of 10, 3, 1, 0.3, or 0.1 s during the step increases were measured. These measures were again made during administration of intravenous MK801, an N-methyl-D-aspartate receptor antagonist that can block temporal summation (n 6). Temporal summation refers to the cumulative effect of repeated stimuli. Crossover concentrations to 10- and 0.1-s interstimulus interval pulses ranging in voltage from 0.25–50 V were also measured (n 4). Results: The increase in concentrations from 0.6 to nearly 1.0 minimum alveolar concentration progressively increased latency from less tha n1st o 58 s.Shortening the interstimulus interval (50 V) pulses from 10 to 0.1 s progressively increased crossover concentrations from 0.6 to 1.0 minimum alveolar concentration. In contrast, during MK801 administration shortening interstimulus intervals did not change crossover concentrations, producing a flat response to change in the interstimulus interval. Increasing the voltage of interstimulus interval pulses increased the crossover concentrations but did not change the response to change in interstimulus intervals for pulses greater than 1 V. Conclusions: Increasing the duration or frequency (interstimulus interval) of stimulation increases the concentration of isoflurane required to suppress movement by a 0.4 minimum alveolar concentration MK801 blocks this effect, a finding consistent with temporal summation (which requires intact N-methyl-D-aspartate receptor activity) at concentrations of up to 1 minimum alveolar concentration isoflurane. THE mechanical or electrical stimulation used to determine MAC (the minimum alveolar concentration producing immobility in 50% of subjects receiving noxious stimulation) is applied continuously for up to 1 min. 1–5 This period allows for a delay between the onset of stimulation and the beginning of movement. The delay seems to increase as the anesthetic concentration approaches MAC, suggesting that time-related processes underlie the generation of the movement and that increasing anesthetic concentrations are required to suppress the response to increasing durations of stimulation. Such processes may be assessed by application of temporally graded electrical stimuli of the type used to create neuronal windup. 6 –10 Typically, these stimuli are trains of brief (0.5 ms) high-intensity square-wave pulses with relatively large interstimulus intervals (ISI), often up to 3 s. Such stimuli delivered to spinal cord afferents of spinal cord-transected rats can produce a cumulative depolarization of dorsal and ventral horn neurons. After several seconds, this progressive depolarization can reach a threshold, triggering a sustained burst of action potentials, giving rise to the term “windup.” N-methyl-Daspartate (NMDA) receptor activity underlies at least part of the cumulative depolarization and subsequent windup. 11,12 We hypothesized that temporal summation, the cumulative effect of repeated stimulation, might govern part of the MAC for isoflurane anesthesia. Although electrophysiologic and electromyographic effects of temporal summation have been investigated during isoflurane and halothane anesthesia in humans and rodents, 13–16 the involvement of temporal summation in the generation of movement during anesthesia has not been investigated. To document the time-related dose-dependent effect of isoflurane on the generation of movement responses, we measured the latency to movement after tail clamp and 50-Hz continuous stimulation. To test for temporal summation, we measured the effect of the ISI on the latency to movement responses, on the concentrations required to achieve immobility, and on the observed buildup of muscle tone and hindlimb electromyogram. To determine if disruption of temporal summation impaired the generation of movement, we measured the effect of the NMDA antagonist, MK801, on isoflurane concentrations required to achieve immobility during the ISI pulses. To study the dose-dependent effect of isoflurane on temporal summation, we studied the effect of ISIs of increasing voltage and the corresponding increasing concentrations required to achieve immobility.

UNLABELLED The nonimmobilizer 1,2-dichlorohexafluorocyclobutane (2N, also termed F6) does not suppress movement to noxious stimuli but does suppress learning of fear-potentiated startle. The mechanism whereby 2N suppresses this learning is unknown. Herein, we report the effect of 2N on suppression of two other forms of learning, fear conditioning to context and to tone. Because 2N does not cause sedation, we could study the effect of 2N on short-term memory (memory for fear conditioning measured during or immediately after training) as well as on long-term memory (measured 24 h after training). The EC(50) for suppression of long-term memory (the concentration decreasing memory by 50%) of fear conditioning to context was 2.00% plus/minus 0.01% (mean plus/minus SEM), and for fear conditioning to tone was 3.45% plus/minus 0.26%, (P < 0.05). The EC(50) for suppression of short-term memory of fear conditioning to context was 2.59% plus/minus 0.21% (P < 0.05, compared with long-term memory of context conditioning), whereas short-term memory of fear conditioning to tone was not suppressed by 3.5%, the largest concentration studied. Thus, short-term memory resists the depressant effect of 2N more than long-term memory, fear conditioning to tone is less vulnerable to the effect of 2N than fear conditioning to context, and 3.5% 2N does not preclude transmission of tone and shock signals to the site where tone-shock associations are formed. IMPLICATIONS The nonimmobilizer 1,2-dichlorohexafluorocyclobutane has a greater depressant effect on long-term memory than short-term memory, suggesting that it impairs the processes responsible for the retention of memory more than for the formation of memory itself.

We previously demonstrated that intrathecal administration of the noncompetitive gamma-aminobutyric acid type A (GABA(A)) receptor antagonist picrotoxin increased isoflurane MAC (the minimum alveolar concentration of anesthetic producing immobility in 50% of animals) by a maximum (ceiling effect) of approximately 40%. We also found that IV administration of picrotoxin increased MAC by more than 60%, without evidence of a ceiling effect. The larger increase with IV administration suggested a role of cerebral GABA(A) receptors. Accordingly, in this study we examined the effect of intracerebroventricular administration of picrotoxin in rats, finding that picrotoxin infusion into the third ventricle increased isoflurane MAC by a maximum of approximately 40%, without finding a ceiling effect. In addition, we concurrently infused picrotoxin into the intrathecal and intracerebroventricular spaces, producing an increase in MAC in excess of 70%, also with no evidence of a ceiling effect. The dose-response relationship for the intrathecal-intraventricular infusion paralleled that of the IV infusion but was shifted to the left by an order of magnitude. We conclude that both cerebral and spinal GABA(A) receptors modulate the capacity of inhaled anesthetics to produce immobility. Because other studies have shown that the spinal cord, and not the brain, mediates the capacity of inhaled anesthetics to produce immobility, these results call into question the relevance of GABA(A) receptors to the immobilizing action of isoflurane.

UNLABELLED General anesthetics cause surgical immobility and oblivion (unconsciousness and amnesia). A class of compounds known as "nonimmobilizers" were predicted to be anesthetic, based on their physiochemical properties, but found to cause only amnesia. In humans, cerebrocortical electrical activity after auditory stimulation is depressed by concentrations of anesthetics which impair auditory recall. We sought to use these evoked responses to characterize the effects of the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (2N) and conventional inhaled anesthetics on early sensory processing in rats. Unrestrained rats with chronically implanted epidural silver screw electrodes were put into a chamber. On separate days, the same population of rats were exposed to isoflurane, desflurane, nitrous oxide, or 2N, each at several subminimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus concentrations. After equilibration at each concentration, auditory-evoked responses were obtained. The behavioral state (activity and righting reflex) and electroencephalogram were also examined. 2N did not significantly change the middle latency auditory-evoked response, whereas the anesthetics all slowed conduction and depressed amplitude in a dose-dependent fashion. 2N neither depressed the righting reflex, nor induced epileptiform activity. IMPLICATIONS Although the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (2N) suppresses learning, we find that 2N does not depress middle latency auditory-evoked responses. This suggests that 2N may suppress learning by depressing transmission through rostral subcortical structures, such as the amygdala, rather than by acting on the brainstem or neocortical structures.

Background Suppression of response to command commonly indicates unconsciousness and generally occurs at anesthetic concentrations that suppress or eliminate memory formation. The authors sought midlatency auditory evoked potential indices that successfully differentiated wakeful responsiveness and unconsciousness. Methods The authors correlated midlatency auditory evoked potential indices with anesthetic concentrations permitting and suppressing response in 22 volunteers anesthetized twice (5 days apart), with desflurane or propofol. They applied stepwise increases of 0.5 vol% end-tidal desflurane or 0.5 microg/ml target plasma concentration of propofol to achieve sedation levels just bracketing wakeful response. Midlatency auditory evoked potentials were recorded, and wakeful response was tested by asking volunteers to squeeze the investigator's hand. The authors measured latencies and amplitudes from raw waveforms and calculated indices from the frequency spectrum and the joint time-frequency spectrogram. They used prediction probability (PK) to rate midlatency auditory evoked potential indices and concentrations of end-tidal desflurane and arterial propofol for prediction of responsiveness. A PK value of 1.00 means perfect prediction and a PK of 0.50 means a correct prediction 50% of the time (e.g., by chance). Results The approximately 40-Hz power of the frequency spectrum predicted wakefulness better than all latency or amplitude indices, although not all differences were statistically significant. The PK values for approximately 40-Hz power were 0.96 during both desflurane and propofol anesthesia, whereas the PK values for the best-performing latency and amplitude index, latency of the Nb wave, were 0.86 and 0.88 during desflurane and propofol (P = 0.10 for -40-Hz power compared with Nb latency), and for the next highest, latency of the Pb wave, were 0.82 and 0.84 (P < 0.05). The performance of the best combination of amplitude and latency variables was nearly equal to that of approximately 40-Hz power. The approximately 40-Hz power did not provide a significantly better prediction than anesthetic concentration; the PK values for concentrations of desflurane and propofol were 0.91 and 0.94. Changes of 40-Hz power values of 20% (during desflurane) and 16% (during propofol) were associated with a change in probability of nonresponsiveness from 50% to 95%. Conclusions The approximately 40-Hz power index and the best combination of amplitude and latency variables perform as well as predictors of response to command during desflurane and propofol anesthesia as the steady-state concentrations of these anesthetic agents. Because clinical conditions may limit measurement of steady-state anesthetic concentrations, or comparable estimates of cerebral concentration, the approximately 40-Hz power could offer advantages for predicting wakeful responsiveness.

The number, size, and site of formation of particulate emboli produced by three different extracorporeal perfusion systems were studied in three groups of 10 patients each. Systems that contained a bubble oxygenator and stainless steel filter (400 μ pores), a bubble oxygenator and stratified polyester urethan filter (27 μ pores in final filtration), and a spiral-coil membrane oxygenator and stratified polyester urethan filter were compared. The membrane oxygenator produced fewer emboli than did the bubble oxygenator, but the venous reservoir of the membrane oxygenator produced more emboli than did the reservoir used with the bubble oxygenator. The total number of platelet aggregate emboli produced by the two oxygenator systems did not differ. The stainless steel filter did not remove or generate platelet aggregate emboli. The stratified polyester urethan filter removed 90 per cent of emboli greater than 150 μ and 60 per cent of emboli between 50 and 150 μ in diameter but often produced emboli less than 50 μ in diameter. The perfusion systems studied did not produce significant differences in platelet counts, fibrinogen, and fibrin split product concentrations during and up to 24 hours after bypass.

We examined the formation of platelet aggregate emboli and factors that affect platelet aggregate formation and embolization in prototype hollow-fiber membrane oxygenators used to perfuse dogs. Platelet aggregates formed on the inlet surface of the oxygenator. Many aggregates subsequently formed emboli which passed through the fibers and sometimes lodges in them. Aggregates also developed on the outlet surface. Aggregates that adhered to the oxygenator surfaces contained tightly packed platelets having multiple pseudopodia extracellular granules, and occassional fibrin strands and white cells. The number of platelet aggregate emboli found downstream to the oxygenator varied between 0 and 4 emboli/ml blood. These emboli did not contain fibrin and extracellular granules. Platelet aggregate formation and embolization was reduced or eliminated by increasing the dose of heparin or by using a circuit with a large priming volume of albumin solution and a large surface area oxygenator.

Fourteen 1.4 m 2 hollow fiber and 23 1.5 m 2 spiral coil membrane oxygenators that were designed for total cardiopulmonary bypass in infants were evaluated. Operating procedures and toxicity were studied during total cardiopulmonary bypass and one hour of circulatory arrest at 15° C (rectal) in four monkeys. Oxygen and carbon dioxide transfer, water loss, production of emboli, transmission of pulse waves, and pressure-flow relationships were studied at three different blood flow rates, at hematocrits of 25% and 40%, and at 25° C and 37° C during partial venoarterial bypass in 33 dogs. Linear regression equations were calculated for oxygen and carbon dioxide transfer and for pressure-flow relationships at the different flows, temperatures, and hematocrits. Rated flow (Rf), defined as the blood flow rate at which inlet O 2 saturation of 65% increases to an outlet O 2 saturation of 95%, was calculated for each oxygenator at hematocrit 40 (Rf 40) or hematocrit 25 (Rf 25). The hollow fiber oxygenator had a priming volume of 140 ml, an Rf 40 of 800 ml/min, and an Rf 25 of 1200 ml/min. The spiral coil oxygenator had a priming volume of 208 ml, an Rf 40 of 1400 ml/min, and an Rf 25 of 1800 ml/min. At Rf 40 and inlet pCO 2 50 torr, the hollow fiber oxygenator transferred 8.9 ml CO 2 /100 ml and the spiral coil 5.7 ml CO 2 /100 ml. The performance of the two oxygenators differed, but both functioned very satisfactorily.

By reflected light microscopy, the thrombi formed on a variety of foreign surfaces have been shown to be composed of isolated cellular aggregates and an interaggregate red cell fibrin mesh. The thrombi viewed by this method were seen in two dimensions as they formed at the foreign surface blood interface. Standard histological evaluation of the thrombi in the third dimension, that is in the depth dimension, revealed columnar shaped cellular aggregates extending from the foreign surface into the blood and a red cell fibrin mesh surrounding the aggregates. The thrombi formed at Epon plate-blood interfaces were viewed with an electron microscope. Plates made of Epon embedding epoxy were used as the foreign surface exposed to blood, and the plates and fixed initial thrombi formed on them were embedded in more Epon. A layer of heavy metal staining, noncellular material was found covering the surface of the plates exposed to blood, with platelets deposited on this layer. Since platelets were not found adherent to the foreign surfaces directly, it is our postulate that the intervening film “conditions” the surface preliminary to platelet adhesion.

The nature of the events occurring immediately after exposing foreign surfaces to fresh flowing blood was assessed using a combination of MAIR infrared spectroscopy, ellipsometry, and contact angle measurements. Within the first 5 sec after contact with blood issuing from the jugular veins of lightly anesthesized dogs, germanium prisms were uniformly coated with strongly adherent proteinaceous filsm having an average optical thickness equivalent to 2 layers of stearic acid (approx. 50 A) and having critical surface tensions of about 36 dyne/cm. A contact time of 60 sec led to a less uniform coating of average optical thickness equivalent to 5 layers of stearic acid (approx. 125 A), but with similar MAIR spectrum and wettability. The internal refelection spectra were consistent with the presence of either alpha-helical or random-chain configurations, but not with the extended chain beta-structure for proteins. Films cast from purified fibrinogen on platinum foils gave similar spectra and exhibited similar wetting properties; these observations are consistent with the hypotehsis that initial adsorptive events are dominated by fibrinogen deposition. The involvement of small amounts of lipid or other protein remains a distinct possibility, however.

Thromboresistance of the alloy most often used in artificial heart valves depends upon a hitherto unrecognized erosion resistant hydrocarbon coating. Contact angle measurements show the actual surface to be dominated by closely packed methyl groups as occur with adlineated aliphatic chains. The likely source for this layer is a tallow-based compound from which adventitious components become tightly bound at the metal/air interface during final polishing. Here, as in other systems, the exposure of CH3 terminal groups correlates with significant blood compatibility.

Our problem can be stated simply: What surface properties are required for candidate biomedical materials to be as nonthrombogenic as possible? This symposium paper relates recent progress along three converging paths we have taken while attempting to solve this problem. First, we report qualitative results from in vitro and in situ surface chemical measurements on blood vessels which were carried out by Dutton and Baier at the National Heart Institute. A more quantitative treatment of these data will be published separately. Second, we report at some length the results of Gott, Dutton and Baier -- obtained during two years of collaborative effort -- which suggest an explanation for the excellent thromboresistance observed with prosthetic implants of the metallic alloy Stellite. Third, we briefly review the surface chemical investigation of candidate biomaterials now being prosecuted by Gott and Baier with the support of the Artificial Heart Program of the National Institutes of Health.

Experiments on clotting, both in vitro and in vivo, showed that a colloidal graphite surface, when rinsed with a cationic, surface-active agent, was capable of bonding heparin. The resistance of this graphite-heparin surface to the formation of clots was far greater than plastic or silicone surfaces in comparable studies.