On the Rat Gastric Motility

The Japanese Journal of Physiology 16, pp.497-508, 1966

ON THE RAT GASTRIC MOTILITY

Takesi HUKUHARA AND Toshiaki NEYA

Department of Physiology, Okayama University Medical School, Okayama

From the results obtained in the experiments carried out on the automaticity of the motility of dogs small intestine, HUKUHARA, NAKAYAMA and FU- KUDA8) concluded that the origin of the intestinal motility was of neurogenic nature, that is, rhythmic contractions of the small intestine were maintained by acetylcholine which was spontaneously released from the intramural ganglion cells, including not only their cell bodies, but also their axons. This hypothesis is naturally expected to be applied to the gastric motility. Taking these facts and hypothesis into consideration, a series of experiments has been performed on the gastric motility. The experimental results here reported are concerned with the problems: the localization and specificity of the pacemaker, the difference of behavior of different regions of the stomach and the mechanism underlying these phenomena. As for the gastric peristalsis, the results obtained by investigators until 1924 were summarized by MCCREA et al.14) Since then there could be found only a few literatures4,6,10,11) related with the problems concerned.

METHODS

In order to observe the movement of the rat stomach in vivo, the well-fed animals weighing from 80 to 200 g were anesthetized with the intraperitoneal administration of 50 mg/kg pentobarbital sodium (Nembutal, ABBOT). It was characteristic that the movement of the rat stomach was not impaired despite administering such a large dose of the drug as described above. The animal was then set in supine position to the frames installed in the internal space of the double-walled trough, the lumen of the wall being irrigated with water appropriately warmed to keep the temperature of the space at about 37•Ž. After the abdominal cavity had been opened wide by the midline incision of the abdominal wall, the incised edges of the wall and the limb of the liver were connected at several spots to the frames by means of of cotton threads or serrefines in such a way that the ventral surface of the stomach was exposed to view. The abdominal cavity was then filled with Tyrode solution which was modified after TAKEMASA16)(1957) as follows: 0.221M-NaCl, 0.0028M-KCl, 0.0015M-CaCl2,

0.0024M-NaHCO3, 0.00032M-NaH2PO4 and 0.0101M-Glucose. For the purpose of observing the movements of the stomach in vitro the whole

Received for publication February 8, 1966. *福原武 ,祢屋俊昭

497 498 T. HUKUHARA AND T. NEYA

stomach was isolated from the animal, which was not fed for 24 to 48 hours before the

experiments, and immersed in the bath filled with Tyrode solution which was warmed

to the constant temperature of 37•Ž and bubbled with oxgen gas . Leaving the small amount of original gastric content, the glass cannulae were inserted into the esophageal and duodenal lumens, respectively. The former was connected by means of a vinyl

tube to a small, double-walled glass vessel which was filled with Tyrode solution and located as high as 5 cm H2O above the level of the stomach , while the latter was located at the level of the stomach. This Tyrode solution was kept at the temperature of

about 37•Ž by irrigating the lumen of the wall of the vessel with water appropriately

warmed. The gastric movement in vitro was observed sometimes on the dorsal side and sometimes on the ventral side of the stomach by means of a binocular dissecting

microscope with low magnifications. Occasionally, the gastric motility was recorded by means of levers assorted with strain gauge transducers , the levers being set up at the two different points of the gastric surface, several millimeters apart from each

other along its curved long axis. In order to study the features of the movements of various regions of the stomach

strips consisting of the whole gastric layers, about 15 mm long and 1.5 mm wide, were made from the various regions of the stomach and immersed in Tyrode solution cooled

to 3•‹ to 5•Ž (See FIG. 1): A) pacemaker strip which included the root of the left

gastric artery and the spot at which the 2nd and the 3rd branches of artery bifurcated, B) body strip which was made from the region of the body distal to the pacemaker region, C) pyloric strip which was made from the pyloric part of the stomach distal

to the angular incisure, D) sphincter strip which was made from the pyloric sphincter and E) fundus strip which was made from the pars proventricularis, to record their

movements. The strips were suspended in the Tyrode bath, the temperature of which

was kept constant at 37•Ž. Drugs used were physostigmine sulfate (Merck) and acetylcholine chloride (Daiichi

Seiyaku).

FIG. 1. Ventral view of rat stomach. In the diagram are shown the arteries providing the blood supply to the stomach. The pacemaker area is hatched. The areas surrounded by broken lines show those from which the strip preparations were made. ON THE RAT GASTRIC MOTILITY 499

RESULTS

1) Observations of the rat stomach in vivo. Rat stomach was supplied with the left and right gastric arteries as shown in FIG. 1: At the site just distal to the cardia, the left gastric artery bran- ched into two main branches, the ventral and dorsal ones, each of which then diverged into small branches spreading radially over the corresponding surfaces of the stomach. In the present experiments they were, for convenience sake, named V1, V2-V6 and D1, D2-D6, respectively, according as to whether they supplied the ventral or dorsal surfaces, and were arranged from the small curvature to the pars proventricularis. In addition, two isolated branches came from the left gastric artery on its way to the cardia to supply the pars proventricularis, and the right gastric artery supplied the pyloric part as well as the region of the corps ventriculi adjacent to the the greater curvature. Observing the ventral surface of the stomach, it was noted that the gastric peristalsis always started from the cross-sectional area a few millimeters wide in which the root of the left gastric artery and the point where branch V3 of the artery ramified from V2 were included (hatched area shown in FIG. 1). This area would be conveniently referred to as the pacemaker area. It was characteristic that every spot involved in this band of contraction almost simultaneously contracted. Every point of the band then caudally propagated with the same speed along the long axis of the stomach until it terminated at the pylorus, without, however, propagating beyond the pylorus to the duodenum. The peristaltic waves repeatedly appeared with a regular time interval of about 10 seconds. As the cardia was about 12 mm apart from the pylorus and the time elapsed in a whole swing was estimated at about 12 seconds, the velocity of the peristalsis was estimated at about 1 mm/sec. The peristaltic wave was considerably strong in the whole course along the lesser curvature, while along the greater curvature it was weak until it arrived at the angular incisure but then the nearer the wave approached the sphincter the stronger it became. On the other hand, as soon as the peristaltic wave nearly reached the middle of the pyloric part, the next one appeared at the pacemaker area, two bands of contractions being thus distinctly seen at the same time on the stomach. In FIG. 2 is shown the course of the gastric peristalsis. The pars proventricularis was apparently motionless. In addition, so far as the site of initiation of peristaltic waves was concerned, the bilateral severance of vagus and/or splanchnic nerves had no effect.

2) The movements of the stomach in vitro. A) On the peristalsis of the whole isolated stomach. Within the first 10 500 T. HUKUHARA AND T. NEYA

. 2. Pictures indicating the course of the peristaltic waves of rat stomach in vivo. The stomach is view- ed from the ventral side. The pictures are taken with every 2 seconds intervals. Arrows show shallow de- pressions produced as the peristalsis sweeps down. Note that two depres- sions are seen at the same time in the pictures 1, 2, 5 and 6.

minutes after the lumen of the isolated stomach had been parfused, the peristaltic waves as usual appeared similarly to those observed in the stomach in vivo. And a minute amount of gastric content was expelled through the duodenal cannula, every time when the peristaltic wave reached the pylorus. On observing the stomach on its dorsal surface the gastric peristalsis showed the mirror image of that observed on the ventral surface. It was worthy to note that in this condition of the experiment the pacemaker shift occurred:

FIG ON THE RAT GASTRIC MOTILITY 501

the transverse band of contraction so faint as one might overlook appeared sometimes at the midportion and sometimes at the top of the pars proventricularis, propagating distally to reach the pylorus. By utilizing the device described in Chapter 'Methods', the change of motility associated with the peristaltic wave was recorded at two different points which were several millimeters apart from each other along the curved long axis of the stomach. These mechanograms allowed to estimate accurately the velocity of the conduction as well as the period of the peristaltic contractions. The example is shown in FIG. 3. In this case the period and the

FIG. 3. Mechanograms of the peristalsis of the whole isolated rat stomach. Utilizing the levers assorted with strain gauge transducers the gastric peristalses were recorded at pacemaker area (A) and pyloric area (B) which were 11 millimeters apart from each other. The contraction a in A which appeared in the pacemaker area was conducted to the pyloric area to produce the contraction a' in B. The contractions b, b' and c, c' were those belonging to another peristalses. P indicates the period of the peristalsis, and T the time elapsed when the peristalsis propagated from A to B. Time in seconds. velocity were 9.9 seconds and 1.1 mm/sec, respectively, hence the wave length of the peristalsis was estimated at 10.9 mm, being some millimeters shorter than the distance from the cardia to the pylorus. The period was not so constant as that observed in the stomach in vivo, being variable from individual to individual within the range from 9.9 to 13.0 seconds. And it was kept largely constant for ten and several minutes in the beginning stage of the experiment, but it was then gradually prolonged with the lapse of time. For instance, in one case the period which was in the mean 10.9 seconds at the beginnig of the experiment turned to be 12.3 seconds 100 minutes later. In other series of experiments the lumen of the stomach was perfused by means of a cannula inserted through the lumen of the pars proventricularis into the gastric body, distal end of the cannula being situated 2-3 mm distal to the pacemaker area described above. When the gastric body was ligated on the glass cannula by a thin cotton thread at the region just distal to the pacemaker area, the peristaltic waves did not propagate further beyond the ligated region. However, at the region just distal to the ligature, the con- 502 T. HUKUHARA AND T. NEYA

tractions rhythmically appeared and propagated distally to reach the pylorus, their period being always prolonged as compared with that observed before ligation. Some minutes after the stomach had been freed from the ligation the period was restored again to nearly the same as observed before the ligation. The example is shown in FIG. 4.

FIG. 4. The changes of the period of peristaltic contractions before and after the exclusion of the pacemaker. A and B: mechanograms of the gastric body recorded before and after the exclusion of the pacemaker, the period being in the mean 10.9 and 20.7 seconds, respectively. C indicates that the period wes restored soon after the stomach had been freed from the ligation, period being in the mean 13.2 seconds. Time in seconds.

It was then studied how the peristaltic waves behaved, when the gastric

wall was freed from stretching. The isolated stomach was, at first, incised

along the greater curvature and then spread out by pinning the cut edges

with the serosal surface upwards on a thin board which was immersed into

Tyrode solution warmed to 37•Ž and bubbled with oxgen gas, care being

taken not to stretch the gastric wall. In this condition of the experiment the contractions of the pars proventricularis could hardly be seen. The peristaltic

waves were repeatedly produced through the cross-sectional area including two spots, the one at which the ventral branch V3 of the left gastric artery

ramified from V2 and the other at which the dorsal branch D3 ramified from D2.

Along this area the contraction was produced almost simultaneously. When

with the lapse of time the preparation was deteriorated, the contraction had a

tendency to be confined within the region connecting the spots described above.

Any predominance of one of these spots over the other could not be ascertained.

From the results described above the spots are supposed to be the parts which

are most tenacious of life among any other parts of the stomach.

B) Automaticities of various strips from the various regions of the stomach. The results obtained in the experiments concerned with the exclusion of the

pacemaker raise the problem whether any differences exist or not in the automatic activities among the regions of the stomach. This problem was

studied in some details. Strips were prepared from the pars proventricularis, ON THE RAT GASTRIC MOTILITY 503 the pacemaker area and its adjascent part of gastric body, pyloric part and sphincter, respectively. The strips were prepared from pars proventricularis (fundic strip) always regularly contracted with the fastest rhythm among all kinds of strips, period ranging from 7.5 to 11.7 seconds. The pacemaker strips always contracted with fairly regular period, although the period was variable from individual to individual, within the range from 11.3 to 14.9 seconds. The body strips contracted in most cases with a regular but a little slower rhythm, period being variable from individual to individual within the range from 12.0 to 22.4 seconds. The pyloric and sphincter strips usually contracted with an irregular rhythm, the period ranging 12.2 to 37.5 and 16.4 to 53.9 seconds, respectively. The characteristic phenomena were observed when the excitability of the gastric muscle was raised by applying physostigmine in the concentration of 10-6 to 10-4 g/ml or acetylcholine in the concentration of 10-8 to 10-5 g/ml to the stomach. As shown in FIGS. 5A and 6A, physostigmine applied to the fundic and the pacemaker strips produced a remarkable rise of tone superimposed with rhythmic contracions of relatively small amplitude, period of contractions being hardly changed or only slightly shortened. And as shown in FIG. 5B, the body strip was in the character of response to the drug largely similar to that observed in the pacemaker strip; its rise of tone was remarkable and its period was shortened to be nearly the same as that of the latter. In striking contrast to the strips mentioned above the sphincter strip reacted characteristically to the drug: The tone rose in a certain degree, but persis- tently remained at a level much lower than that observed in the pacemaker strip, while rhythmic contractions had an enormously large amplitude and their frequency was so increased that it reached or surpassed that observed in the pacemaker strip (FIG. 6B). The pyloric strips were in the character of response to the drug similar to the sphincter strip, only difference being that the rise of tone is higher than that oberved in the latter.

FIG. 5. The effect of physostigmine sulfate upon the movements of the strips. A: Pacema- ker strip. B: Body strip. When at white rec- tangles the drug was added to Tyrode bath to give the final concentration of 10-5 g/ml, there occurred in both strips an enormous rise of tone superimposed with rhythmic contractions. P indicates the period of rhythmic contractions in seconds observed before and after the administration of the drug. The period of contractions of the pacemaker strip was only slightly shortened, whereas that of contractions of the body strip was considerably shortened. Time in 6 seconds. 504 T. HUKUHARA AND T. NEYA

FIG. 6. The effect of physostigmine sulfate upon the movements of the strips. A: Pace- maker strip. B: Sphincter strip. At white rec- tangles, the drug was added to Tyrode bath to give the final concentration of 10-4 g /ml. As to P the explanation is the same as that in FIG. 5. It was noted that the sphincter strip rhythmically contracted with much lower tone and much greater amplitude compared with those observed in the pacemaker strip, and that its period of contractions was remarkably shortened. Time in 6 seconds.

DISCUSSIONS

A. On the pacemaker. On the basis of the results obtained in the experiments in vivo it may be concluded that in the animal well fed the pacemaker of the gastric peristalsis is located in the transverse plane a few millimeters in its width which is projected through the region just distal to the cardia at right angle to its curved long axis. And the severance of extrinsic nerves has no influence upon the site of initiation of the peristalsis. It is characteristic that the propagation of contraction is along this transverse area so rapid that the whole area appears to contract at the same time, whereas the propagation of the contraction is pyloruswards much slower than that observed in the former area. The mechanism underlying the synchronous contraction of the pacemaker area described above is to be solved. The pacemaker area would contain far more cholinergic neurones, if our conclusion drawn from the work8) performed recently is correct. In addition, in the present experiment in which the stomach is full of contents the antiperistalsis could never been observed. Quite unexpectedly the pacemaker shift was observed in the experiments in vitro: The gastric peristalsis usually started at the top of the pars proventricularis and swept down as a band of wavelet to reach the pacemaker area described above, further course of the peristalsis being the same as observed in vivo. The reason why the site of initiation of the peristalsis in vivo and in vitro are not consistent with each other remains unex- plained. Some of the constituents of Tyrode solution may have an excitatory influence upon the mechanism involved in the automatic activity of the stomach. ON THE RAT GASTRIC MOTILITY 505

The experiments in vitro revealed that in the pacemaker area the pulsa- tions were ultimately abolished at the spot which was situated distally to the cardia. This fact suggests that pacemaker mechanism which is most tenacious among any other parts of the stomach involved in this spot. HOFMEISTER and SCHUTZ7), KEITH11), KLEIN12), MCCREA et al, ALVAREZ1) and KAWASAKI10)noted that the gastric peristalsis originated in the region just distal to the cardia, but they did not refer to such a characteristic transverse area as described above. On the other hand investigators such as CANNON5),BORCHERS3) and OPPENCHOWSKI15)who observed that the peristalsis started in the middle part of the gastric body did not pay so much attention. to the problem on the origin of the peristalsis. In addition, we could not observe the tonic constriction ring such as observed by OPPENCHOWSKI15)and others, so far as the stomach did not fall into the deteriorated condition. B. On the behavior of different regions of the stomach. If the frequency of contractions is taken as a criterion of automaticity, the fundic strip ranks among the highest class, the pacemaker strip being only a little inferior to the former, the sphincter strip among the lowest and the body strip among the middle. And it was noted that the strips made from upper regions. always contracted with a regular rhythm, whereas those made from the lower regions contracted with an irregular rhythm. The evidence that also in the whole stomach this gradient of excitability exists was clearly shown by ligating the stomach transversely: The contractions recurrently appeared at the region just anal and close to the ligated part always with a rhythm slower than that observed before the ligation. Furthermore, it is worthy to note that when physostigmine was applied, the frequency of contractions was hardly increased both in the fundic and pacemaker strips and only a little in the body strip, whereas in the sphincter and pyloric strips the frequency was so remarkably increased that it surpassed in some cases that observed in the former. On the basis of the facts described above and the results obtained in our work8) performed recently it may be concluded that the difference of automaticity of various regions of the stomach is attribut- able to the difference in the amount of acetylcholine released there: In the fundic and pacemaker regions the trigger substance, acetylcholine, is released, even in the normal condition, in an amount enough to stimulate the muscle cells located there to contract with maximal frequency, whereas in the sphincter it is released in an amount so small that the muscle cells located there contract with frequency far behind the former. From the results obtained in the rabbits small intestine MASLENNIKOVA13) postulated a hypothesis, in which he correlated the difference of frequency of contractions to the number of intramural ganglion cells. This hypothesis would, however, not apply in the stomach, because against this hypothesis the sphincter contains much more ganglion cells than the cardiac region (IR- 506 T. HUKUHARA AND T. NEYA

WIN)9). We are, therefore, of the opinion that the gradient of rate of contractions should be correlated to the number of excitatory (cholinergic) neurones. In addition, contrary to our results, ALVAREZ2) could not find the clear-cut gradient of rate of contractions in the experiments on the cat, dog and rabbit gastric strips, while on the frog GELLHORN and BUDDE6)found that the fundic strip contracted more frequently than the pyloric one. In the experiments upon the strips some important facts were found: As a result of application of physostigmine the pyloric and sphincter strips showed the lower tone superimposed with rhythmic contractions with a greater amplitude, while the pacemaker and body strips showed the high tone superimposed with rhythmic contractions with a smaller amplitude. The phenomena may be explained as follows: The pacemaker and body areas may mainly consist of excitatory neurones, their major role being in keeping the rhythm of gastric peristalsis fairly constant, while the pyloric part and sphincter may contain about an equal number of both the excitatory and inhibitory neurones, by whose mutual interplay the muscle alternately contracts and relaxes with an ample amplitude to effectively evacuate the gastric content. The facts sug- gesting that the regions of the stomach might have their characteristic nature, were observed by some investigators: BROWN and MCSWINEY4) who studied the motility of rabbit gastric strips said that in the upper regions pilocarpine caused a permanent shortening of the muscle fibers while in the distal region rhythmic movements only were augmented. The almost similar phenomena were also observed by GELLHORN and BUDDE6) on the frog fundic and pyloric strips affected by acetylcholine. In the human material ALVAREZ2)and TEZNER and TUROLD17)observed that the pyloric strip contracted much more powerful- ly than the strip made from the cardiac region.

SUMMARY

1. On the rat stomach in vivo and in vitro the origin and the behavior of the peristalsis were studied in correlation with the automaticity of various regions of the stomach. 2. In the stomach in vivo the gastric peristalsis always started every 10 seconds as a ring of contraction which was projected through the root of the left gastric artery at right angle to the curved long axis of the stomach. This area was conveniently referred to as pacemaker area. The muscle con- tained in this area contracted almost synchronously, and this ring of contraction slowly propagated pyloruswards with the velocity of about 1 mm/sec. 3. If the frequency of contractions is taken as a criterion of automaticity, the fundic strip ranks among the highest class, the sphincter strip among the lowest and the body and pyloric strips among the middle. And it was noted that the strips made from the upper regions always contracted with a ON THE RAT GASTRIC MOTILITY 507 regular rhythm, whereas those made from the lower regions contracted with an irregular rhythm. 4. It was found that when physostigmine was applied, the pacemaker and its adjacent area were enormously increased in its tone, but the contractions remained in their frequency almost unchanged and were in their amplitude increased not so significantly, while the pyloric part and sphincter showed only a slight increase in its tone but an enormous rise both in its frequency and amplitude of contractions. 5. The phenomena described above revealed that the pars proventricularis and the body take a role of not only preserving the content but also keeping the rhythmicity constant, while the pyloric part and sphincter take a role of favorably evacuating the content.

REFERENCES

1) ALVAREZ, W. C.(1948). An introduction to gastroenterology. 4th ed. p.332. Paul B Hoeber Inc. N. Y. 2) ALVAREZ, W. C.(1948). ibid. p.366-368. 3) BORCHERS, E.(1921). Die Aussichten der Behandlung von Motilitatsstorung des Magens durch Vagusunterbrechung. Deutsch. Ztschr. Chir. 162: 19-28. 4) BROWN, G. F. AND MCSWINEY, B. A.(1926). Reaction to drugs of strips of the rabbit's gastric musculature. J. Physiol. 61: 261-267. 5) CANNON, W. B.(1911). The importance of tonus for the movements of the alimen- tary canal. Arch. Int. Med. 8: 417-426. 6) GELLHORN, E. AND BUDDE, W.(1923). Beitrage zur Physiologie der Magenmus- kulatur. I. Mitteilung. Studien am uberlebenden Magen des Frosches. Pflugers Arch. ges. Physiol. 200: 604-619. 7) HOFMEISTER, F. AND SCHUTZ, E.(1885). Ueber die automatischen Bewegungen des Magens. Arch. exp. Path. Pharmakol. 20: 1-33. 8) HUKUHARA, T., NAKAYAMA, S. AND FUKUDA, H.(1965). On the problem whether the intestinal motility is of a neurogenic or myogenic nature. Jap. J. Physiol. 15: 515-522. 9) IRWIN, D. A.(1930). The anatomy of Auerbach's plexus. Am. J. Anat. 49: 141-166. 10) KAWASAKI, G.(1939). On the movement of the isolated dog stomach whose blood vessels are perfused by the blood. J. Physiol. Soc. Jap. 4: 225-236.(in Japanese) 11) KEITH, A.(1915). The Cabendish lecture in a new theory of the causation of enterostasis. Lancet 93: 371-375. 12) KLEIN, E.(1926). Gastric motility. I. The origin and character of gastric peristalsis. Arch. Surg. 12: 571-582. 13) MASLENNIKOVA, L. D.(1962). On the relation between the motor function of the intestine and the gradient of its nervous elements. Bull. exp. Biol. Med. 52: 972- 976. 14) MCCREA, E. D., MCSWINEY, B. A., MORISON, J. W. AND STOPFORD, J. S. B.(1924). The normal movements of the stomach. Quart. J. exp. Physiol. 14: 379-397. 15) OPPENCHOWSKI, Th.(1889). Ueber die nervosen Vorrichtungen des Magens. Centralbl. Physiol. 3: 1-10. 508 T. HUKUHARA AND T. NEYA

16) TAKEMASA, K.(1957). On the inorganic salts in the artificial nourishing fluid for isolated intestine. J. Physiol. Soc. Jap. 19: 1217-1225.(in Japanese) 17) TEZNER, O. AND TUROLD, M.(1921). Pharmakologische and physiologische Stu- dien am uberlebenden menschlichen Magen. Ztschr. ges. Med. 12: 275-287.