Development of biotissue training models for anastomotic suturing in pancreatic surgery.

BACKGROUND: Anastomotic suturing is the Achilles heel of pancreatic surgery. Especially in laparoscopic and robotically assisted surgery, the pancreatic anastomosis should first be trained outside the operating room. Realistic training models are therefore needed. METHODS: Models of the pancreas, small bowel, stomach, bile duct, and a realistic training torso were developed for training of anastomoses in pancreatic surgery. Pancreas models with soft and hard textures, small and large ducts were incrementally developed and evaluated. Experienced pancreatic surgeons (n = 44) evaluated haptic realism, rigidity, fragility of tissues, and realism of suturing and knot tying. RESULTS: In the iterative development process the pancreas models showed high haptic realism and highest realism in suturing (4.6 ± 0.7 and 4.9 ± 0.5 on 1-5 Likert scale, soft pancreas). The small bowel model showed highest haptic realism (4.8 ± 0.4) and optimal wall thickness (0.1 ± 0.4 on -2 to +2 Likert scale) and suturing behavior (0.1 ± 0.4). The bile duct models showed optimal wall thickness (0.3 ± 0.8 and 0.4 ± 0.8 on -2 to +2 Likert scale) and optimal tissue fragility (0 ± 0.9 and 0.3 ± 0.7). CONCLUSION: The biotissue training models showed high haptic realism and realistic suturing behavior. They are suitable for realistic training of anastomoses in pancreatic surgery which may improve patient outcomes.

Short-term potentiation of carotid chemoreflex: an NMDAR-dependent neural integrator.

Repetitive stimulation of the carotid sinus nerve (CSN) elicits a short-term potentiation (STP) of the reflex response in respiratory motor output in mammals. The input-output transformation approximates a leaky integrator with a time constant of several seconds. Here, we showed that STP induced by CSN stimulation in rats was manifested in the reflex response in the amplitude of rhythmic phrenic nerve activity as well as its duration. Moreover, pharmacological blockade of NMDA receptors (NMDAR) resulted in marked increases in the time constants of the equivalent neural integrator in both the STP induction phase (by 10- to 20-fold) and recovery phase (by 1- to 5-fold). Thus, NMDAR serves as a molecular switch that facilitates the integrative processing of CSN inputs by STP.

Phase resetting of the respiratory oscillator by carotid sinus nerve stimulation in cats.

1. Stimulation of the carotid sinus nerve causes an increase in inspiratory (I) and expiratory (E) neural activities. If central respiratory oscillation is generated by an attractor-cycle process, an increase in its activity can be caused by a centrifugal perturbation of state. We evaluated this hypothesis by comparing the respiratory oscillator's phase responses to carotid sinus nerve stimulations in cats to the phase responses of an attractor-cycle oscillator, the Bonhoeffer-van der Pol (BvP) equations, subjected to centrifugal perturbations. 2. We recorded phrenic activity in seven anaesthetized, vagotomized, glomectomized, paralysed and servo-ventilated cats. Carotid sinus nerve (CSN) stimulation with 0.5-0.8 s electrical pulse trains increased the immediate cycle period and delayed the onset of breaths after stimulation in a highly predictable manner, with the exception that strong stimuli (25 Hz, 0.25-0.90 V) caused unpredictable responses when given at the I-E or the E-I transitions. The resetting plots exhibited focal gaps corresponding to these unpredictable responses, and the size of the gaps increased with increases in the strength of CSN stimulation. Type 0 resetting was not achieved despite the large perturbations in rhythm induced by CSN stimulation. 3. Centrifugal perturbations of the BvP oscillator resulted in phase responses which were similar to those found in the animal experiments. The BvP cycle had two critical phases at which phase resetting was highly irregular and neighbouring state trajectories were highly divergent. The resetting plots had focal gaps that increased in size with increases in the strength of perturbation. The gaps did not represent true discontinuity because at higher computational resolution the resetting plots appeared to be steep but smooth portions of topological Type 1 resetting curves. 4. These studies support the concept that brief carotid sinus nerve stimulations cause a transient outward displacement of the central respiratory state away from its attractor cycle, in contrast to the unidirectional displacements that accompany midbrain reticular or superior laryngeal nerve stimulations. The findings define particular geometrical relationships between oscillatory state trajectories of the rhythm generator and perturbed state trajectories induced by inputs to the oscillator. These relationships provide a framework for developing and testing the validity of neural models of the respiratory oscillator.

Inputs from upper airway affect firing of respiratory-associated midbrain neurons.

In 16 decerebrated unanesthetized cats, we studied effects of neural inputs from upper airway on firing of 62 mesencephalic neurons that also developed respiratory-associated (RA) rhythmic firing when respiratory drive was high [Z. Chen, F. L. Eldridge, and P.G. Wagner. J. Physiol. (Lond.) 437: 305-325, 1991] and on firing of 16 neurons that did not develop the rhythmic firing (non-RA neurons). Activity in RA neurons increased after mechanical expansion of pharynx (45% of those tested) or larynx (68%) and after stimulation of glossopharyngeal (50%) or superior laryngeal nerves (77%). The increased neuronal firing occurred despite decreases or abolition of respiratory activity (expressed in phrenic nerve). Neuronal firing also increased after mechanical stimulation of nasal mucosa (66%) or by jets of air directed into the nares (48%) and after light brushing of nasal skin ( approximately 40%). Most stimuli led to decreased firing in a smaller number of neurons, and some neurons showed no response. None of the non-RA neurons developed an increase of firing after any of the stimuli, although one had decreased firing after stimulation of the superior laryngeal nerve. We conclude that inputs from the upper airway and nasal skin have independent modulatory effects on the same mesencephalic neurons that are stimulated by ascending rhythmic RA input from the medulla. These findings may have relevance to generation of the sensation of dyspnea.

Relief of the 'air hunger' of breathholding. A role for pulmonary stretch receptors.

Fowler (Fowler, W.S., 1954, J. Appl. Physiol. 6:539-545) showed that rebreathing, despite worsening PCO2 and O2 saturation, relieved the distress of breathholding; he suggested a role for vagal input in the relief. We studied effects on respiratory sensation of breathholding and rebreathing in normals, patients with bilateral lung transplants (LT), who have a decrease in number of pulmonary stretch receptors (PSR), and heart transplant recipients (HT). Subjects held their breath until distress became intolerable, rebreathed various combinations of CO2 and O2, then performed another maximal breathhold. Respiratory distress was rated continuously (visual analog scale) by each subject. Both LT and HT had earlier onset of and more rapidly developing distress during breathholding, resulting in shorter breathhold times, than normals. Relief with rebreathing was neither as rapid nor as great in LT as in HT and normals. Our findings suggest that mechanisms that produce respiratory distress in HT and LT are similar, but differ from normals. However, reduction in distress on rebreathing is more rapid and greater in HT and normals than in LT. This is compatible with the loss during rebreathing of the inhibitory effect of PSR input on neural mechanisms that lead to respiratory distress.

Relief of distress of breathholding: separate effects of expiration and inspiration.

It is well known that rebreathing relieves the respiratory distress of maximal breathholding despite worsening blood gases, and it has been suggested that vagal input has a role in ameliorating this sensation via activation of pulmonary stretch receptors (PSR). However, it is believed by divers that expiration can lead to partial relief of distress of breathholding at total lung capacity (TLC) allowing a prolongation of breathholding. We studied the independent effects of an expiration and an inspiration on relief of respiratory distress of breathholding. Subjects held their breath at TLC until distress became intolerable, then exhaled to FRC and performed a second breathhold. When distress again became intolerable, subjects inspired to TLC a gas that resembled their exhaled gas and performed a third breathhold. Subjects noted partial relief with both an expiration and an inspiration. However, relief of distress was greater and the subsequent breathhold longer after an inspiration than after an expiration. We suggest that relief of distress after an inspiration is compatible with the inhibitory effect of PSR input; the mechanism of relief that occurs after an expiration is as yet uncertain.

Central integration of mechanisms in exercise hyperpnea.

Many hypotheses have been advanced to explain the hyperpnea of exercise and its close relations to the level of metabolic work, expressed as oxygen uptake (VO2) and carbon dioxide production (VCO2). Evidence is presented that a neural central command mechanism from the hypothalamus is important in the driving of both respiration and circulatory adjustments during locomotion or exercise, and that short-term potentiation of neurons in the medulla makes an important contribution. Both are probably augmented by receptors in working muscle and by the effects of increased [K+] acting on the carotid bodies. Feedback from "respiratory" mechanisms, including CO2 and O2 mediated mechanisms and inputs from the lungs, are important in stabilizing ventilation at the level primarily dictated by the major neural mechanisms.

The Fowler breathholding study revisited: continuous rating of respiratory sensation.

The respiratory distress of breathholding has been shown to be relieved by breathing, even without correction of worsening blood gases (Fowler, 1954). We repeated the study by having untrained normal subjects perform maximal breathholds which were followed by the rebreathing of a gas mixture containing 7.5% CO2 and 8.2% O2, and then by second breathholds. In addition, we had the subjects continuously rate their respiratory distress using a visual analog scale (VAS). The ratings were easy to perform and were highly reproducible on repeated trials in a given subject. Subjects experienced increasing distress during the breathhold, rapid and substantial relief upon rebreathing, and then were capable of performing second breathholds, all consistent with Fowler's results. The findings are consistent with animal studies in which a neural mechanism associated with stimulation of pulmonary stretch receptors inhibits the firing of midbrain neurons which may be involved in transmission to the cortex of sensory information about breathing.

Respiratory-associated rhythmic firing of midbrain neurons is modulated by vagal input.

We recorded phrenic nerve activities and single unit firings of mesencephalic neurons in 19 decerebrate, paralyzed and ventilated cats, in which the spinal cord had been transected at C7-T1 and carotid sinus nerves cut but vagus nerves left intact. After we had found neurons with respiratory-associated rhythmic activity, we tested the effect of changing pulmonary vagal input by (1) stopping and restarting the ventilator; (2) changing the ventilator's tidal volume; (3) progressively cooling the vagus nerves to 6-7 degrees C; and (4) vagal section. All methods of testing yielded results that showed that vagal input, probably from pulmonary stretch receptors, tonically inhibits the respiratory-associated firing of the mesencephalic neurons by a direct mechanism that is independent of a vagal effect on medullary respiratory drive. We have suggested that these neurons are involved in the mechanism that conveys information about respiration to the cortex where it may be interpreted as the sensation of dyspnea. If so, movement and increased expansion of the lungs can be expected to lessen the sensation.

Respiratory-associated thalamic activity is related to level of respiratory drive.

We recorded phrenic nerve activity and thalamic single unit firing in unanesthetized, suprathalamically decerebrated, paralyzed and ventilated cats, in which vagi and carotid sinus nerves (CSN) had been ablated. Seventy-six (14%) of 545 neurons in regions of the thalamus related to the ascending reticular system, which had been tonically firing at low respiratory drives, developed rhythmic increases of firing associated with each respiration when drive had been increased by CSN stimulation or hypercapnia. The increases of neuronal firing occurred in late inspiration/post-inspiration but sometimes lasted into expiration; the magnitude of change was graded according to the magnitude of respiratory activity. Thalamic neurons also fired with a rhythm related to ventilator-induced chest expansion, some units showing both the respiratory-associated and the ventilator-related rhythms. Simultaneously recorded mesencephalic and thalamic neurons developed similar rhythms when drive was increased. We suggest that these neuronal activities reflect the conveyance of information about respiration to the cortex, where it may lead to the sensation of dyspnea and perhaps to arousal.

Neural respiratory responses to cortically induced seizures in cats.

Seizure activity can lead to profound respiratory stimulation in spontaneously breathing animals with intact respiratory feedback mechanisms (Paydarfar et al., Am. J. Physiol. 260, R934, 1991). The present study was designed to test the hypothesis that peripheral respiratory feedback mechanisms are not important for the genesis of seizure-induced hyperpnea. Studies were performed in 16 anesthetized, vagotomized, glomectomized cats whose end-tidal PCO2 (PETCO2) was kept constant. Integrated phrenic nerve activity was used to represent respiration. Seizures were induced by injection of penicillin into the parietal cortex and electrocorticographic (ECoG) and biceps femoris nerve activities, arterial pressure, airway PCO2 and brain temperature were recorded continuously. Progressive seizure activity was associated with progressive increases of respiratory frequency and peak phrenic activity, despite constancy of PETCO2 and brain temperature. Patterns of entrainment were identified among ECoG spikes, biceps femoris nerve and phrenic nerve activities. Phrenic nerve activity became highly irregular during generalized ictal seizures and ceased to respond to changes of PETCO2. Acute intercollicular decerebration in all experiments resulted in normalization of respiratory rhythm even while ictal ECoG activity continued. We conclude that stimulation of breathing during seizures occurs in the absence of respiratory feedback mechanisms. The findings suggest that an important cause of the respiratory response is a feedforward mechanism, whereby activation of subcortical structures above medulla and pons results in stimulation of breathing.

Anesthesia affects respiratory and sympathetic nerve activities differentially.

Phrenic and cervical sympathetic nerve responses to hypercapnia were examined before and after anesthesia in twelve midcollicularly decerebrated, vagotomized, glomectomized, paralyzed and ventilated cats. We measured responses of integrated phrenic and cervical sympathetic nerve activities to increases in end-tidal PCO2 (PETCO2) from apneic threshold to approximately 30 torr above threshold. All cats were studied first in the unanesthetized state. Six cats were then restudied after a quarter of a usual dose of chloralose/urethane (10 mg/kg and 62.5 mg/kg, respectively) and then after half the usual dose of chloralose/urethane (20 mg/kg and 125 mg/kg). The other six animals were restudied after quarter of a standard dose of pentobarbital (9 mg/kg), after half the standard dose (18 mg/kg) and then after the full (35 mg/kg) dose. Both anesthetic agents led to significant increases in apneic thresholds for both phrenic and sympathetic nerve activities. These agents also caused dose-dependent decreases in peak, tonic and respiratory-related sympathetic nerve activities. Peak (tidal) phrenic nerve activities, in comparison, were much less affected by the anesthetic agents. CO2 response curves showed that both of these anesthetic agents depressed, at any given level of PETCO2, respiratory-related sympathetic nerve responses more than the responses found in the phrenic nerve. We conclude that the relations between peak, tonic (i.e. between phasic bursts) and respiratory-related sympathetic nerve activities and phrenic nerve activity can be altered by anesthesia.

Respiratory-associated rhythmic firing of midbrain neurones in cats: relation to level of respiratory drive.

1. We recorded phrenic nerve activities and single unit firing of mesencephalic neurones in unanaesthetized supracollicularly decerebrated, paralysed and ventilated cats, in which vagi and carotid sinus nerves had been ablated. We made these measurements first at low levels of respiratory drive associated with normal PCO2 levels, then with increased respiratory drive and levels of phrenic activity produced by hypercapnia or by carotid sinus nerve stimulation. 2. We found that at least a quarter of the neurones in the central tegmental field of the mesencephalon, which were irregularly tonic or silent at low respiratory drives, developed a rhythmic increase of firing associated with each respiration. There appeared to be a threshold at about 50% of maximum respiratory activity, below which the respiratory-associated rhythm did not occur. Above this level, neuronal firing increased in graded fashion with increasing magnitude of respiratory activity. The latency from onset of phrenic activity to onset of increased neuronal firing was quite long (1.0 s) at drives just above the threshold but shortened to as little as 0.3 s as drive increased towards its maximum. 3. Cutting the spinal cord at C1-C2 had no effect on the ability of increased respiratory activity to generate a respiratory-associated rhythm in mesencephalic neurones. 4. Short-lasting anaesthesia with the agent Saffan caused mesencephalic neurones to lose the respiratory-associated rhythm with little change in phrenic activity and no change in respiratory cycle timing. 5. We also found a mesencephalic response to ventilator-induced chest expansion. The latency of the response from onset of expansion, indexed by fall of airway PCO2, to onset of neurone firing was shorter (0.2 s) than that found with the respiratory-associated rhythm. In seventeen neurones we found both the respiratory-associated rhythm and the independent ventilator-associated rhythm. 6. We interpret our findings to show that the respiratory-associated rhythmic firing of midbrain neurones is not primarily involved in generation or modulation of the motor function of the respiratory oscillator. We believe, instead, that these neurones are part of a sensory pathway conveying information about the magnitude of central neural respiratory drive, as well as spinally transmitted information from receptors in the chest wall, to thalamus and cortex. We suggest that the sensation ultimately generated may be that of 'air hunger' or dyspnoea.

Respiratory responses to focal and generalized seizures in cats.

We studied the effects on breathing of seizures induced by focal injection of penicillin G into the parietal cortex in 13 anesthetized cats. Electrocorticograms, ventilation, end-tidal PCO2, and intrapleural and arterial pressures were monitored; changes of these variables were related to the stages of motor seizure. The first respiratory responses, tachypnea and hyperpnea, usually occurred before any peripheral muscular contractions developed. Progression of the seizure was always accompanied by further tachypnea and hyperpnea. The hyperpnea associated with all stages of seizure activity resulted in hypocapnia, which was sustained even during prolonged tonic-clonic motor convulsions that caused a threefold increase of metabolic rate. The extreme tachypnea of tonic generalized convulsions led to increased end-expiratory lung volume because of dynamic hyperinflation associated with very short expiratory durations in the tonic phase. We suggest that the profound effects of seizures on respiration are by feedforward mechanisms from the cortical focus itself and from subcortical circuits, such as hypothalamus, that become involved during seizure propagation and generalization. Peripheral respiratory feedback mechanisms are not important for the genesis of seizure-induced hyperpnea.

Development of short-term potentiation of respiration.

Development of short-term potentiation (STP) of respiration, which leads to the respiratory 'afterdischarge', was studied in anesthetized, paralyzed, vagotomized and glomectomized cats. Phrenic nerve activity was used as an index of respiratory output. Respiratory output was increased and the potentiating mechanism activated by electrical stimulation of a carotid sinus nerve (CSN). Development of STP was determined from the magnitude of potentiation after various durations (0 to 60 sec) of stimulation. The average time constant (TC) for the development of the potentiation was 9 sec, whereas the TC for its decay (afterdischarge) was 46.1 +/- 3.9 sec. The magnitude of potentiation is dependent upon the number of pulses in the stimulus train. We conclude that the development of short-term potentiation of respiration is relatively slow but much faster than the decay, or afterdischarge. We suggest that the slow increase of respiration during a stimulation and the decay afterwards are due to a common mechanism, short-term potentiation of neural activity in respiratory control pathways.

Enrichment of bovine X- and Y-chromosome-bearing sperm with monoclonal H-Y antibody-fluorescence-activated cell sorter.

Bovine spermatozoa were assessed indirectly for the presence of a Y chromosome by monitoring expression of the H-Y antigen. Spermatozoa labeled with a monoclonal H-Y antibody (MoAb) and fluorescein-conjugated goat antibody to mouse F(ab)2 were counted with both a fluorescent microscope and a fluorescence-activated cell sorter (FACS). Of ejaculated spermatozoa, 40% to 60% fluoresced by this procedure compared to 1% to 15% of sperm reacted with nonimmune serum. Semen from three bulls was exposed to nonimmune serum (control) or MoAb, sorted by FACS, and analyzed for DNA content with a scanning microdensitometer. Control samples showed two distinct peaks with a mean difference in DNA content of 3.95%; these peaks were assumed to represent Y- and X-chromosome-bearing spermatozoa populations, respectively. DNA analyses of the MoAb-treated sperm of three bulls that sorted positively for H-Y antigen (fluorescent sperm) yielded ratios of Y- to X-chromosome-bearing spermatozoa of 76 : 24, 88 : 12, and 77 : 23, and those sorted negatively for H-Y antigen (nonfluorescent sperm) yielded ratios of 26 : 74, 35 : 65, and 23 : 77. The proportions of Y- and X-chromosome-bearing spermatozoa in nonsorted samples were not different from 50 : 50. Suitable MoAbs can be used in conjunction with FACS to enrich the proportion of Y- or X-chromosome-bearing spermatozoa in bovine semen.

Phase resetting of respiratory rhythm: effect of changing respiratory "drive".

We studied the effect of changing drive on resetting of respiratory rhythm in anesthetized cats and in a model (Van der Pol) of a limit-cycle oscillator. In cats, rhythm was perturbed by brief mesencephalic stimuli. Stimulus time in the cycle (old phases) and times of onset of rescheduled breaths (cophases) were measured. Previous study [Paydarfar and Eldridge, Am. J. Physiol. 252 (Regulatory Integrative Comp. Physiol. 21): R55-R62, 1987] showed distinct types of phase resetting that depended on strength of stimuli. In this study, stimulus strength was kept constant, but respiratory drive was changed by increasing PCO2, by stimulating carotid sinus nerve, or by cooling intermediate areas of ventral medulla. Type 0 (strong) resetting occurred when respiratory drive was low, type 1 (weak) resetting when drive was high, and a phase singularity when drive was intermediate. Phase-resetting patterns generated by the model showed the same behavior when a drive parameter was changed. The findings support the idea that continuous limit-cycle dynamics underlie generation of respiratory rhythm. Increased respiratory drive, by increasing size of the limit cycle, reduces functional effectiveness of the same perturbing stimulus in causing phase resetting.

Buspirone, an anxiolytic drug that stimulates respiration.

The recently released drug buspirone is an anxiolytic agent that appears not to have the sedating effects of barbiturates and benzodiazepines, both known to have respiratory depressant effects. Because of its increasing clinical use, we desired to study the effects of buspirone on respiratory control. We therefore determined central neural respiratory responses, measured from phrenic nerve activity, after intravenous administration in paralyzed, vagotomized, and glomectomized cats whose end-tidal PCO2 and body temperature were kept constant. The responses were compared to the effects of the sedating tranquilizer diazepam. Buspirone had a dose-dependent stimulatory effect on respiratory output primarily through an increase of tidal activity but with an increase of frequency in some animals. Associated with this was a shift of the apneic threshold to a lower level of PCO2 without a change of slope or shape of the CO2 response curve. In contrast, diazepam led to a depression of respiration and a shift of the apneic threshold to a higher PCO2. The findings indicate that buspirone does not have the typical neural respiratory depressant actions of diazepam but instead stimulates respiration. Although the findings will need to be shown to apply to human beings, they suggest that buspirone may be a useful drug to treat anxiety in patients without causing undesirable respiratory depression.

Desynchronized respiratory rhythms and their interactions in cats with split brain stems.

1. The effects on activities and rhythms of the two opposing phrenic nerves (C5 roots) of mid-line sagittal splitting of the medulla were determined in anaesthetized or decorticate, vagotomized, paralysed and ventilated cats. 2. Splitting the medulla above the obex led to marked decreases of phrenic activity on both sides, but no desynchronization of the two phrenic rhythms occurred. Further splitting to more than 3 mm below the obex led to desynchronized phrenic rhythms in fourteen of the fifteen animals that survived the necessary surgery, although it was often necessary to increase respiratory drive by means of hypercapnia, stimulatory drugs or electrical stimulation of the mesencephalon to cause the rhythms to occur. 3. When only the brain stem had been split, the two desynchronized rhythms showed interactions that led to modulations of amplitude of phrenic bursts, both being larger when in phase than when out of phase. In addition each side modulated the rhythm of the opposite side, demonstrating a 'magnet' effect. 4. Both types of modulation were eliminated after additional splitting of the spinal cord at the level (C5-C6) of the phrenic motoneurone pools. 5. Potential explanations for the amplitude modulations include cross-over of activity from one phrenic motoneurone pool to the opposite side and cross-over from the medulla of one side to the opposite phrenic motoneurone pool at the phrenic level. 6. Since the rhythm generators were independent in our preparation and located in the split halves of the medulla and since peripheral sensory feed-back was not important in these paralysed animals, we propose that the phase modulations must be due to a corollary discharge, an afferent feed-back driven by phrenic motoneurone activity that crosses the mid-line at C5-C6, ascends to the brain and affects respiratory rhythm in the opposite medullary half.