JOURNAL
OF SURGICAL
Action
RESEARCH
19,403-409
of Coherin
Propagation
(1975)
on the Basic Electrical
in the Isolated
Perfused
Rhythm
Canine
and
Jejunum
C. MENDEL, PH.D., D. JAECK, M.D., J. F. GRENIER, M.D. University Medical School and INSERM, Avenue Moliere, StrasbourgjHautepierre, France AND R. B. HIATT, M.D., I. GOODMAN, B. SANDLER, M.E.E.
PH.D.,
AND
Departments of Surgery and Biochemistry, Columbia University, New York, New York 10032 Submitted for publication June 10, 1975
Numerous studies support the view that quency decreases caudally from pylorus to the electrical activity in the smooth muscle ileum along the small intestine in stepwise fashion while successive frequency plateaus of the small intestine provides a regulatory mechanism for motility. Electrical activity are separated by boundary regions in which as recorded in the electroenterogram (ENG) BER potentials “wax and wane” [6, 9, 22, is composed of two main components. The 241. It is thought that the BER is myogenic in the longitudinal muscle from which it first is a cyclically repetitive slow fluctuation of potential called “slow wave” or “basic spreads passively to the circular muscle and electrical rhythm” (BER). The second that in some species, such as dogs and cats, component consists of a single fast os- spike potentials make the two muscle layers cillation or a burst of such oscillations that contract synchronously [l, 4,5]. are superimposed on the slow component When monopolar electrodes are uniand are known as “spike potentials.” The formly spaced along an intestinal segment shape of this composite electrical potential and if latencies of BER cycles recorded from varies with the speciesand the method of re- successive electrodes are uniform, the BER cording, whereas the frequency varies with is said to propagate along that segment at a the species and the region of the intestinal constant “conduction or propagation vetract. Spike potentials are said to be related locity,” In the dog, BER propagation veto smooth muscle contractions and occur on locity has been reported to decreaseaborally the average in only one-third of the BER along the intestine and to be normally in the cycles in the dog. They are regarded as the caudad direction [6,9, 19,221. electrical trigger that causes muscle conThe spike carrying BER cycles is tractions that in turn produce increases in considered responsible for eliciting muscular intraluminal pressure. If temporally coor- contractions and the BER to control vedinate, contractions can thus give rise to locity and direction of intestinal motor peristaltic propulsion along the gut. BER propagation. The BER thus functions as a potentials, on the other hand, are thought to pacemaking mechanism. A series of BER be spontaneous and present regardless of the pacemakers distributed along the intestine is absence or presence of smooth muscle assumed to control the overall electrical contractions (2, 3, 6-8, 18, 221. BER fre- activity of the gut. An electrical model based 403 Copyright o 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
404
JOURNAL
OF SURGICAL
RESEARCH
on multiple pacemakers is capable of explaining and simulating the main observed features of electrical activity in the intestine [9-11, 15,20,21,23]. The present report describes the results of electrophysiological experiments on perfused isolated segments of canine jejunum in vitro which extend earlier in vivo studies. It has been previously shown that coherin, a recently discovered peptide of the bovine posterior pituitary gland [12], has the capacity to coordinate disjunctive patterns of BER propagation as well as their concomitant mechanical activity in the intestine of the fasted dog [16, 171.Coherin, when injected iv in awake, trained, fasted dogs prepared with the Biebl loop, at doses from 0.1 to 1.Opg/kg body weight, was found to alter the character of the BER potentials from a random, uncoordinated pattern to a coherent BER. Although the intestinal action of coherin may be observed virtually instantaneously after the injection, its maximum effect on the BER occurs only after about 1 hr. To determine whether the target organ of coherin is the gastrointestinal tract and whether its action is direct or indirect, we have applied a perfusion method recently perfected for the study of canine intestinal electrical activity in vitro [ 13, 141. MATERIALS AND METHODS Mongrel dogs of either sex weighing from 12 to 20 kg were anesthetized with sodium pentobarbital and put on a respirator. The abdomens were opened through long midline incisions. A 15cm segment of jejunum was chosen 20-30 cm below the ligament of Treitz and six silver-silver chloride punctate wire electrodes were inserted into the longitudinal muscle to a depth of 2 mm in a straight line parallel to the mesenteric attachment with the tips uniformly spaced 2 cm apart. All but the O.Zmm-long wire tips were insulated. The segment was divided from the rest of the bowel, and the superior mesenteric artery and vein were exposed. All the branches of the superior mesenteric
VOL.
19, NO. 6, DECEMBER
1975
artery and vein were then divided except those going to the segment to be monitored. A major branch of the superior mesenteric artery adjacent to the mesenteric pedicle of the study segment was cannulated and attached to a pulsatile perfusion apparatus. Oxygenated heparinized donor blood was perfused in pulsatile fashion into the segment as the superior mesenteric vein and artery were divided proximally to avoid any period of ischemia. The jejunal segment with its mesentery and blood supply was removed from the dog and placed on a grounded stainless-steel grid in a chamber containing air saturated with water vapor at 38” C. The mesenteric vein was cannulated for determining blood flow rates. The pulsatile pressure was set to maintain a mean arterial pressure of 100 mm Hg. The pulse pressure was 40 mm Hg which is the same as that measured in the femoral artery of the intact dog. An averge flow of 35 ml/min/lOO g of intestine with a standard deviation of 7-8 ml was sufficient to support the extracorporeal jejunal segment. The venous blood coming from the isolated segment was discarded and not recirculated. Coherin was injected into the arterial perfusion tubing just proximal to the gut segment. Simultaneous ENG recordings were made from the six active punctate electrodes on a six-channel heat-writing recorder (LeichtiCardiopan 6) at a time constant of 1.8 set and an input impedance of 5 Mohms. The grounded steel grid was used as the reference electrode. Great care was taken to insure uniform operation on all channels for comparing preinjection and postinjection periods. The preinjection ENG was recorded continuously from the start of perfusion. Twenty minutes after 250 ng of coherin in 1 ml of isotonic saline solution was injected into the arterial blood perfusion tubing, from where the full dose reached the jejunal segment within 1 min. Post-coherin recordings were then made continuously or intermittently for a minimum of 70 min. The recording speed was 5 mm/set. Sevenjejunal test segments were investigated in that man-
MENDEL ET AL.; EFFECTS OF COHERIN ON THE JEJLJNUM
ner. For control purposes similar segments from fasting dogs were subjected to the same procedure except for the substitution of 1 ml of pure isotonic saline for the coherin in the saline solution. Five such control segments were investigated. A graphical method was employed to quantify the effect of coherin on the intestinal BER. A BER coherence index was defined as follows, analogous to that previously described for mechanical activity [12]. Reference points were assigned to the individual BER cycles. Minima were chosen for reference because they were the most clearly identifiable points. When the latenties between a set of BER cycles recorded sequentially from three or more electrodes were uniform, an oblique straight line could be drawn through the nearest corresponding reference points within a tolerance of & 1 mm, corresponding to + 0.2 sec. BER minima that can be connected by an oblique straight line are considered to be coherent, and the BER is said to propagate at constant velocity along the intestinal section spanned by this line (“primary propagation”). Since at least three points are required to form such a line, the degree of coherence is arbitrarily measured on the straight line by the number of intersected reference points minus two. The degree of coherence in a particular length of recording is the sum of the contributions of all straight lines in that
405
A BC a b c / ,’ I \.. \\ ‘\
OF SURGICAL
Action
RESEARCH
19,403-409
of Coherin
Propagation
(1975)
on the Basic Electrical
in the Isolated
Perfused
Rhythm
Canine
and
Jejunum
C. MENDEL, PH.D., D. JAECK, M.D., J. F. GRENIER, M.D. University Medical School and INSERM, Avenue Moliere, StrasbourgjHautepierre, France AND R. B. HIATT, M.D., I. GOODMAN, B. SANDLER, M.E.E.
PH.D.,
AND
Departments of Surgery and Biochemistry, Columbia University, New York, New York 10032 Submitted for publication June 10, 1975
Numerous studies support the view that quency decreases caudally from pylorus to the electrical activity in the smooth muscle ileum along the small intestine in stepwise fashion while successive frequency plateaus of the small intestine provides a regulatory mechanism for motility. Electrical activity are separated by boundary regions in which as recorded in the electroenterogram (ENG) BER potentials “wax and wane” [6, 9, 22, is composed of two main components. The 241. It is thought that the BER is myogenic in the longitudinal muscle from which it first is a cyclically repetitive slow fluctuation of potential called “slow wave” or “basic spreads passively to the circular muscle and electrical rhythm” (BER). The second that in some species, such as dogs and cats, component consists of a single fast os- spike potentials make the two muscle layers cillation or a burst of such oscillations that contract synchronously [l, 4,5]. are superimposed on the slow component When monopolar electrodes are uniand are known as “spike potentials.” The formly spaced along an intestinal segment shape of this composite electrical potential and if latencies of BER cycles recorded from varies with the speciesand the method of re- successive electrodes are uniform, the BER cording, whereas the frequency varies with is said to propagate along that segment at a the species and the region of the intestinal constant “conduction or propagation vetract. Spike potentials are said to be related locity,” In the dog, BER propagation veto smooth muscle contractions and occur on locity has been reported to decreaseaborally the average in only one-third of the BER along the intestine and to be normally in the cycles in the dog. They are regarded as the caudad direction [6,9, 19,221. electrical trigger that causes muscle conThe spike carrying BER cycles is tractions that in turn produce increases in considered responsible for eliciting muscular intraluminal pressure. If temporally coor- contractions and the BER to control vedinate, contractions can thus give rise to locity and direction of intestinal motor peristaltic propulsion along the gut. BER propagation. The BER thus functions as a potentials, on the other hand, are thought to pacemaking mechanism. A series of BER be spontaneous and present regardless of the pacemakers distributed along the intestine is absence or presence of smooth muscle assumed to control the overall electrical contractions (2, 3, 6-8, 18, 221. BER fre- activity of the gut. An electrical model based 403 Copyright o 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
404
JOURNAL
OF SURGICAL
RESEARCH
on multiple pacemakers is capable of explaining and simulating the main observed features of electrical activity in the intestine [9-11, 15,20,21,23]. The present report describes the results of electrophysiological experiments on perfused isolated segments of canine jejunum in vitro which extend earlier in vivo studies. It has been previously shown that coherin, a recently discovered peptide of the bovine posterior pituitary gland [12], has the capacity to coordinate disjunctive patterns of BER propagation as well as their concomitant mechanical activity in the intestine of the fasted dog [16, 171.Coherin, when injected iv in awake, trained, fasted dogs prepared with the Biebl loop, at doses from 0.1 to 1.Opg/kg body weight, was found to alter the character of the BER potentials from a random, uncoordinated pattern to a coherent BER. Although the intestinal action of coherin may be observed virtually instantaneously after the injection, its maximum effect on the BER occurs only after about 1 hr. To determine whether the target organ of coherin is the gastrointestinal tract and whether its action is direct or indirect, we have applied a perfusion method recently perfected for the study of canine intestinal electrical activity in vitro [ 13, 141. MATERIALS AND METHODS Mongrel dogs of either sex weighing from 12 to 20 kg were anesthetized with sodium pentobarbital and put on a respirator. The abdomens were opened through long midline incisions. A 15cm segment of jejunum was chosen 20-30 cm below the ligament of Treitz and six silver-silver chloride punctate wire electrodes were inserted into the longitudinal muscle to a depth of 2 mm in a straight line parallel to the mesenteric attachment with the tips uniformly spaced 2 cm apart. All but the O.Zmm-long wire tips were insulated. The segment was divided from the rest of the bowel, and the superior mesenteric artery and vein were exposed. All the branches of the superior mesenteric
VOL.
19, NO. 6, DECEMBER
1975
artery and vein were then divided except those going to the segment to be monitored. A major branch of the superior mesenteric artery adjacent to the mesenteric pedicle of the study segment was cannulated and attached to a pulsatile perfusion apparatus. Oxygenated heparinized donor blood was perfused in pulsatile fashion into the segment as the superior mesenteric vein and artery were divided proximally to avoid any period of ischemia. The jejunal segment with its mesentery and blood supply was removed from the dog and placed on a grounded stainless-steel grid in a chamber containing air saturated with water vapor at 38” C. The mesenteric vein was cannulated for determining blood flow rates. The pulsatile pressure was set to maintain a mean arterial pressure of 100 mm Hg. The pulse pressure was 40 mm Hg which is the same as that measured in the femoral artery of the intact dog. An averge flow of 35 ml/min/lOO g of intestine with a standard deviation of 7-8 ml was sufficient to support the extracorporeal jejunal segment. The venous blood coming from the isolated segment was discarded and not recirculated. Coherin was injected into the arterial perfusion tubing just proximal to the gut segment. Simultaneous ENG recordings were made from the six active punctate electrodes on a six-channel heat-writing recorder (LeichtiCardiopan 6) at a time constant of 1.8 set and an input impedance of 5 Mohms. The grounded steel grid was used as the reference electrode. Great care was taken to insure uniform operation on all channels for comparing preinjection and postinjection periods. The preinjection ENG was recorded continuously from the start of perfusion. Twenty minutes after 250 ng of coherin in 1 ml of isotonic saline solution was injected into the arterial blood perfusion tubing, from where the full dose reached the jejunal segment within 1 min. Post-coherin recordings were then made continuously or intermittently for a minimum of 70 min. The recording speed was 5 mm/set. Sevenjejunal test segments were investigated in that man-
MENDEL ET AL.; EFFECTS OF COHERIN ON THE JEJLJNUM
ner. For control purposes similar segments from fasting dogs were subjected to the same procedure except for the substitution of 1 ml of pure isotonic saline for the coherin in the saline solution. Five such control segments were investigated. A graphical method was employed to quantify the effect of coherin on the intestinal BER. A BER coherence index was defined as follows, analogous to that previously described for mechanical activity [12]. Reference points were assigned to the individual BER cycles. Minima were chosen for reference because they were the most clearly identifiable points. When the latenties between a set of BER cycles recorded sequentially from three or more electrodes were uniform, an oblique straight line could be drawn through the nearest corresponding reference points within a tolerance of & 1 mm, corresponding to + 0.2 sec. BER minima that can be connected by an oblique straight line are considered to be coherent, and the BER is said to propagate at constant velocity along the intestinal section spanned by this line (“primary propagation”). Since at least three points are required to form such a line, the degree of coherence is arbitrarily measured on the straight line by the number of intersected reference points minus two. The degree of coherence in a particular length of recording is the sum of the contributions of all straight lines in that
405
A BC a b c / ,’ I \.. \\ ‘\
