The equine teniae coli: An electrophysiological study

Refereed

THE EQUINE TENIAE COLI: AN ELECTROPHYSIOLOGICAL STUDY G.A. Burns, DVM, PhD; Claude A. Ragle, DVM; Michael P. Moore, DVM, MS

SUMMARY

Previous morphological studies of the equine teniae coli (intestinal bands) have shown them to be highly innervated. In this study, EMG electrodes were placed in the wall of the left ventral colon in order to determine whether intestinal bands serve as major conduits of myoelectrical activity. Specifically, electrodes were implanted in the lateral mesocolic band and the adjacent tenia-free bowel of 6 horses. In 3 of these horses, a 1 cm length of the intestinal band was excised to determine if a lesion of this size would ablate local waves of depolarization. Our resuits indicate that sequential EMG activity persisted despite this small, focal excision. The persistence of sequential EMG activity might reflect the importance of constantly regenerating stimuli to the intestinal motility of the horse. Whether making similar or somewhat larger lesions in all four teniae of the left ventral colon would more definitively disrupt normal pelvic flexure peristalsis will require further research.

gates of smooth muscle and connective tissue called"teniae" or intestinal bands. 1 Preliminary morphological studies indicate that the teniae coli are highly innervated. 2 Such a dense innervation might suggest that these bands serve as major conduits of myoelectrical activity. The nature of myoelectrical activity along the equine intestinal bands has not been studied. The dispersal pattern of such activity might be uniformly circumferential or, given their seemingly dense innervation, action potentials may be primarily transmitted along the tenial myenteric plexus with subsequent radiation into the adjacent teniafree bowel. This is an important consideration, as surgeons often use the substantial connective tissue component of the intestinal bands when placing stay sutures and performing enterotomies. 3 The effects of small, focal lesions of the tenial myenteric plexus (intrinsic nerve supply) on local intestinal motility are not known. In this study, we implanted EMG electrodes in the lateral mesocolic band of control horses and of those from which we excised a 1 cm segment to ascertain whether such an excision would ablate local myoelectrical activity.

MATERIALS A N D M E T H O D S INTRODUCTION

The tunica muscularis of the equine intestinal tract is composed of two layers of smooth muscle. Muscle fibers in the outer layer are longitudinally oriented, while those of the inner layer are circumferential. Modifications of the outer longitudinal layer exist at the cecum and colon. Here, the outer longitudinal layer is gathered into discrete aggreAuthors' address: Department of Veterinary Comparative Anatomy, Physiology, and Pharmacology, College of Veterinary Medicine, Room G-7 Wegner Hall, Washington State University, Pullman, Washington 99164-6520 Telephone: (509) 355-7645 FAX: (509) 335-4650 Acknowledgements: The authors wish to acknowledge the expertise of Mr. Larry Miller, whose assistance with the electrical recording devices was indispensable, This work was supported by the WSU Equine Research Fund.

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Six horses, 5 mares and 1 gelding, ranging in age from 2 to 14 years, were placed in a quarantine pasture and observed for at least 2 weeks prior to their use in this trial. None of these horses had a known history of enteric diseases or exhibited signs of an intestinal disorder either during the quarantine period or on routine admission physical examination. While in the Teaching Hospital, the horses were housed in stalls and provided 1.2 kg of sweet feed and 3.7 kg of hay at 7:00 AM and 7:00 PM and water ad libitum. Each horse was fasted for 24 hours prior to surgery. The horses were randomly assigned to treatment (n=3) and control (n=3) groups. Ten million units of procaine 111

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Figure 1. EMG electrode implantation sites along the lateral mesocolic band (tenia mesocolica lateralis) and adjacent tenia-free bowel of the left ventral colon. The dots at each numbered site represent each member of the electrode pairs. The pairs are arranged sequentially in parallel rows. penicillin a was administered IM 2 hours prior to surgery and for the first post-operative week at 22 thousand units/ kg IM, every 12 hours. Each horse was tranquilized with xylazine, b General anesthesia was induced with ketamine c and maintained with halothane, d using an out-of-circle vaporizer. With the horse positioned in dorsal recumbency, the pelvic flexure was exteriorized through a routine ventral midline incision. The termination of the lateral mesocolic intestinal band was identified. This is the site at which this tenia blends with the tunica muscularis of the pelvic flexure. At a point 10 cm distal to this site, the first pair of EMG electrodes (EMG I) were implanted (Figure 1). These electrodes consisted of 28 Ga., stranded, stainless steel wire, insulated with clear, non-hygroscopic fluorocarbon e from which the last 5 cm had been stripped. Each electrode was deeply implanted into the tunica muscularis with a swaged-on suture needle and tied upon itself. The second member of the pair, a reference electrode, was positioned within 1 cm of its mate. A second pair of electrode wires (EMG II) was situdted at the termination of the lateral mesocolic band. Third and fourth Sets of electrodes were positioned at 10 cm intervals proximal to EMG II along this tenia. A fifth pair of electrodes was implanted in the tenia-free tunica muscularis approximately 3 cm medial and parallel to EMG II. Similarly, a sixth and seventh set of wires was positioned 3 cm medial and parallel to EMG III and EMG IV, respectively. An eighth electrode pair was implanted into the tunica muscularis of the tenia-free bowel approximately 3 cm medial and parallel to EMG V, while the ninth pair was similarly positioned adjacent to EMG VT. A ground wire was implanted in the rectus abdominis muscle near the celiotomy incision. Stay sutures were applied to aAgri-cillin, Agri-Pharm Incorporated; St. Joseph, MO. bRompun, Mobay Corporation; Shawnee, KS. CKetaset, Fort Dodge Laboratories; Fort Dodge, IA. dHalothane, Halocarbon Laboratories; North Augusta, SC. eBioflex wire, catalog number AS-816, CoonerWire Company; Chatsworth, CA.

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minimize both traction on the wires and the likelihood of incarceration of adjacent loops of intestine. EMG electrode placement was identical in those horses assigned to the treatment group except that, prior to the implantation of the third tenial pair, a segment of the tenial tunica muscularis and subjacent inner circular smooth muscle 1 cm in length was excised. The underlying submucosa and mucosa were left intact. The transverse ends of the defect were re-approximated with 2 or 3 interrupted 2-0 sutures. Following closure of the tenial resection, EMG III was implanted 10 cm proximal to the second set, as previously described. The pelvic flexure was returned to its normal position within the abdomen and the EMG wires were exited through a small flank incision located caudal to the last rib and approximately 30 cm ventral to the lumbar transverse processes. The ventral midline and flank incisions were closed in a routine fashion. The EMG wires were coiled within a protective fabric pouch which, in turn, was sutured to the skin. Over the first post-operative week, the animals were maintained at stall rest and gradually returned to a normal diet. Flunixin meglumine f was used at .25 mg/kg IM, once daily, to"control post-operative pain. In no instance were analgesics required after the fourth postoperative day. In each case, at 2:00 PM on post-operative day 10, the horses were placed in stocks. The EMG wires were connected to 18 input ports on a multichannel recorderg to yield 9 bipolar channels. The following recording parameters were used: paper speed 10 mm/sec, sensitivity 500 ~tV/cm, high frequency band pass 35 Hz, low frequency band pass 1.60 Hz, and a 60 Hz notch filter. In order to assess the quality of electrical contact, the impedance of each electrode was checked prior to beginning each recording session. In all cases, the animals had free access to any hay, remaining in their stalls from the morning feeding, prior to recording. Myoelectrical activity was recorded from each horse for 30 min in each of 3 separate recording sessions on post-operative days 10, 14, and 21. Upon completing the three recording sessions, the animals were euthanized and a necropsy was performed. The implantation sites of the EMG electrodes were carefully evaluated for evidence of residual inflammation. Biopsies of the surgical excision sites were examined histologically to ensure that the myenteric plexus and tunica muscularis had been completely removed and that no myocytes or axons had bridged the intervening surgical scar. The direction of conduction (retrograde, i.e., toward the oral cavity, or antegrade, i.e., in an anal direction), the conduction velocities, and the onset intervals were determined in each of the resulting tracings. A decision as to whether a given myoelectrical sequence was retrograde or 'fBanamine, Schering Corporation; Kenilworth, NJ gspectrum 32, Caldwelt Industries Incorporated; Kennewick, WA

JOURNAL OF EQUINE VETERINARY SCIENCE

Figure 2, (right) EMG tracings from a control horse. These discrete spike bursts, which first appeared in the most proximally positioned electrodes (EMG IV and VII) and finally arose in the electrode located most distally (EMG I), would be classified as an antegrade sequence. Spike bursts in the tenia-free electrodes (EMG V-VII, first parallel row; VIIXl, second parallel row) exhibited identical antegrade sequences. (Paper speed: 10 mm/ sec; dotted vertical lines = 0.6 sec intervals; solid vertical lines = 3.0 sec intervals.)

Figure 3, (right) EMG tracings from a control horse. These spike bursts occurred first in the most distally positioned electrode (EMG I) and finally arose in the two electrodes located most proximally (EMG VI and Vii). This would be considered a retrograde sequence. Again, note the close temporal relationship between the tenial and tenia-free electrodes. (Paper speed 10 mm/sec.)

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Neither the treatment nor control horses manifested post-surgical colic episodes. Discrete myoelectrical spike bursts were recorded from electrodes of both the tenial (those implanted directly into the lateral mesocolic band) and tenia-free (those placed in the adjacent area of the bowel wall devoid of bands) electrodes. These myoelectrical events occurred sequentially and nearly simultaneously (< 0.5 sec delay) in two of the control animals (Figures 2,3). Both retrograde and antegrade sequences were recorded from these animals. Of all the sequences evaluated, i.e., those in which the onset of myoelectrical activity was readily apparent in all leads (n=5 per horse), 71% were considered retrograde. The median conduction velocities for antegrade (2.47 cm/sec) and retrograde (2.45 cm/sec) sequences propagated along the lateral mesocolic band were nearly identical. Those for the adjacent tenia-free bowel, 2.36 and 2.38 cm/sec, were equally similar to previously recorded values .4-6 The median duration of the spike bursts was also similar in the tenial (7.6 sec) and

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antegrade was reached by carefully analyzing the time at which the event was recorded at each electrode. A typical retrograde sequence began with the onset of myoelectrical activity in EMG I followed by spike bursts in EMGs II, V, and VIII, EMGs III, VI, and XI, and EMGs IV and VII, respectively (see Figure 2). Antegrade sequences occurred in exaCtly the reverse order (see Figure 3). The term conduction velocity indicates the time required for a wave of depolarization to traverse the distance between the EMG electrodes (10 centimeters). Finally, the onset interval refers to the time lag between spike bursts in contiguous electrodes.

Volume 16, Number 3, 1996

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tenia-free bowel (8.3 sec). Rarely, longer events, lasting for approximately 60 secs, were recorded. These episodes were characterized by nearly constant spike burst activity in all the electrodes. The onset interval between myoelectrical sequences was not regular. The 42 intervals measured ranged from 3 to 43 seconds. In the 3 treatment and one of the control horses, the recorded spike bursts were sequential in the tenial, but not the tenia-free electrodes (see Figure 4). The tenial myoelectrical sequences were predominantly (58%) antegrade. The median conduction velocity of these antegrade sequences was more rapid (2.75 cm/sec) than that of the retrograde sequences (1.46 cm/sec). The onset ofmyoelectrical activity in the tenia-free electrodes was not concurrent with that in the contiguous tenial electrodes. The nonsequential depolarizations precluded calculating a median conduction velocity for the tenia-free bowel in these animals. The median tenial (10.4 sec) and tenia-free (9.4 sec) spike burst durations were nearly equal. The onset intervals were highly variable for both the control and treatment animals. Most importantly, the median onset interval for electrodes II and III in the treated horses (5.48 sec) was not greater than any other electrode pair (5.55 sec), despite the fact that a segment of tunica muscularis and the intercalated myenteric plexus had been excised between these two electrodes. At necropsy, remarkably little inflammation was found around any of the electrode wires. The local reaction was limited to a fibrin coat over each of the stay sutures, the stainless steel wire knots, and the wound by which the wires exited the flank. There was no histologic evidence of residual peritonitis. No gross evidence of intestinal parasites was found at necropsy.

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Figure 4. EMG tracings from a treatment home. Spike bursts in the tenial electrodes (EMG I-IV) occurred in a retrograde sequence pattern. Note the loss of continuity between the tenial and tenia-free electrodes. At the dark vertical line (arrow), the tracing at the end of one page was carefully joined to that of the next page in order to present them on a single page. (Paper speed 10 mm/sec.)

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DISCUSSION

The results of this study indicate that the excision of a 1 cm segment of the tunica muscularis and myenteric plexus did not ablate the propagation of myoelectrical activity along the lateral mesocolic intestinal band. Bidirectional wave activity persisted despite the fact that an excision of this size would have removed an estimated 8,600 nerve cell bodies and their processes from the local myenteric plexus. 2 The discovery that a large number of nerve cell bodies, including all the abundant, complex interconnections,7 can be removed from a nerve plexus without interrupting the conduction of myoelectrical activity is an intriguing finding. This result differs from previous work which showed that conduction of peristaltic waves along the small intestine of the cat was completely prevented by a series of incisions into the myenteric plexus, a The nature of the two lesions would be quite different, however. In studies on the cat, 8 incisional lesions were circumferential and probably cell body-sparing, whereas our resection was focal and involved the deliberate removal of a large number of cell bodies from the local, intrinsic nerve supply. There are several possible explanations for the persistence of sequential electrical activity. Given the potential for bacterial contamination of the peritoneal cavity in the presence of abundant foreign material in the form of multiple recording devices, a full-thickness excision of the tenia was deemed ill-advised. Furthermore, excision of the tenial tunica muscularis left a relatively weak underlying submucosa and mucosa as the sole barrier to leakage of intestinal contents into the abdominal cavity. As an additional precaution, we elected to re-approximate the margins of the lesion. Smooth muscle cells may have bridged the surgical scar and established gap junctions and/or intact intrinsic enteric neurons may have reinnervated the area of the lesion. Microscopic examination of intestinal biopsies revealed substantial amounts of fibrous connective tissue intervening between what had been the margins of the previous excision. Thus, although the presence of abundant, intervening, fibrous connective tissue on the examined sections does not definitively prove that no smooth muscle cells or axons traversed the surgical scar, this scenario seems unlikely. Given the reticular configuration of the myenteric 114

plexus, nerve impulses may have circumvented the lesion via the adjacent tenia-free bowel. Waves of depolarization traveling along the tenia-free bowel might spread to the contiguous tenial myenteric plexus, with which it is continuous, and this electrical activity could be propagated along the tenia. Similarly, myoelectrical activity could be conducted from tenial to tenia-free smooth muscle cells via gap junctions. In this way, the lesion might be circumvented. However, the pronounced time disparity between tenial and tenia-free myoelectrical activity makes this implausible. Excision of the tunica muscularis would have transected axons coursing from the submucosal plexus to the myenteric plexus at the lesion site. 9 Hence, this would have eliminated the faciliatory influences of those submucosal neurons which lay directly beneath the excision. However, some submucosal fibers might extend cranially or caudally for a distance before projecting to the myenteric plexus, although, based upon studies in other species, 9-13 extending beyond a full centimeter seems unlikely. It could be argued that these faciliatory fibers might activate secondary neurons in distant submucosal ganglia, which, in turn, could project to the intact myenteric plexus, located proximal and distal to the excision site. In this way, the faciliatory influences of a relatively intact submucosal plexus might bypass the gap created in the my/enteric plexus. It appears that the persistence of conduction along the tenia despite focal transection of nerve and muscle fibers might be best explained by the importance, in this species, of constantly regenerating stimuli for the propagation of myoelectrical activity. In this paradigm, once initiated, a wave of depolarization might require additional factors, such as muscle stretch and serial mucosal afferent inputs, for its actual propagation. This explanation would suggest that the propagation of contraction waves over long distances is more complicated than mere cell-to-cell changes in membrane potential. By creating a more extensive tenial lesion or lesions in multiple teniae, one might assess the importance of submucosal inputs to the propagation of myoelectrical activity. It appears that waves of depolarization did not always emanate from the pelvic flexure in a strictly uniform, circumferential fashion. In two of the control horses, electrical activity appeared to be sequential and concurrent in both the tenial and tenia-free electrode arrays. This was not JOURNAL OF EQUINE VETERINARY SCIENCE

the case in the treatment animals. It would be inappropriate to infer that the tunica muscularis excision caused the disarray of myoelectrical activity, since this may have been a pre-existing condition and a similar disarray was identified in one of the control horses. It is more likely that these non-phasic contractions were consistent with haustral, or mixing, movements in fed animals. Similar uncoordinated temporal patterns have been described in the human jejunum. 1~ In addition to monitoring the propagation of electrical activity along the longitudinal axis of the left ventral colon, a goal of this study was to record impulses traveling perpendicular to this axis. The electrode configuration was designed to record any electrical activity emanating from the tenia into the adjacent tunica muscularis and vice versa. A significant time disparity in the onset of electrical activity in adjacent electrode pairsmight suggest that waves of depolarization were traveling perpendicular to the long axis of the intestine. We did not detect any significant temporal differences in the onset of electrical activity between adjacent electrode pairs. The proximity of the adjacent rows of electrodes may have precluded recording subtle temporal differences. The span of tenia-free bowel between the lateral and medial mesocolic bands at the distal portion of the left ventral colon measured approximately 9 cm in the study horses. Thus, the rows of electrodes could only be separated by 3 cm. In addition, the myenteric and submucosal plexuses are not arranged perpendicularly, but, rather, follow a reticular configuration. TM Under these circumstances, waves of depolarization might proceed along a more acute vector, which would be more difficult to detect, particularly with closely placed electrode pairs. Also, action potenti',ds might be emanating to and from both the lateral and medial mesocolic bands. The recording device used in this study might not be capable of differentiating convergent waves of electrical activity. The predominance of retrograde waves of depolarization was expected in the control animals. A similar antiperistaltic predominance has been described in the proximal portion of the ascending colon in a variety of species. 12 The reason for the apparent shift toward a predominance of antegrade waves following excision of the tenia is unclear. A previous study of myenteric ganglionic topography reported considerable heterogeneity in the orientation of myenteric ganglia in the guinea pig. 13 Although axonal projections to most myenteric ganglia were predominantly retrograde, some ganglia received fibers predominantly or exclusively antegrade or circumferentially-oriented. Even if similar ganglionic heterogeneity exists in the equine myenteric plexus, it is still highly improbable that the surgical excision selectively obliterated the subset of enteric neurons charged with propagating retrograde waves of depolarization. The preponderance of antegrade waves in two of the treatment horses may simply be another manifestation of recording Volume 16, Number 3, 1996

from animals in the fed state. Finally, the EMG electrodes and stay sutures may have altered local motility. However, these changes were probably minor, given the minimal inflammatory responses elicited by these foreign materials. The stainless steel wires as well as the fluorocarbon insulation appear to be largely biologically inert. A substantial amount of each wire (at least 61 cm) remained in the abdominal cavity to allow relatively unrestricted movement of the pelvic flexure. In conclusion, the results of this preliminary study suggest that excision of a small segment of the tunica muscularis of the tenia mesocolica lateralis does not appear to eliminate sequential myoelectrical activity along its length, despite the fact that the excision entailed the removal of a large number of nerve cell bodies from the local myenteric plexus. This finding suggests that the intrinsic nerve supply to the horse' s intestine is an extremely plastic system, capable of remarkable adaptation to injury. Whether making similar or larger lesions in all four teniae of the left ventral colon would substantially disrupt normal pelvic flexure peristalsis will require further research.

REFERENCES 1. Nickel R, Schummer A, Seiferle E:The large intestine. In: Sack WO, (Ed). The Viscera of the Domestic Mammals. New York: Springer-Verlag, 1979. 2. Burns GA:The teniae of the equine intestinal tract. Come# Vet 1992;82:187-212. 3. Robertson JT: Intestinal enterotomy, resection, and anastomosis. In: White NA, (Ed). The Equine Acute Abdomen. Philadelphia: Lea and Febiger, 1990. 4. Sellers AF, Lowe JE, Drost CJ, Rendano VT, Georgi JR, Roberts MC: Retropulsion-propulsion in equine large colon. Am J Vet Res 1982;43:390-396. 5. Ross MW, Donawick WJ, Sellers AF, Lowe JE: Normal motility of the cecum and right ventral colon in ponies. Am J Vet Res 1986;47:1756-1762. 6. Lester GD, Boulton JR, Thurgate SM: Computer-based collection and analysis of myoelectrical activity of the intestine of the horse. Am J Vet Res 1992;53:1548-1552. 7. Furness JB, Costa M: Arrangement of the enteric plexuses. In: The Enteric Nervous System. New York: Churchill Livingstone, 1987. 8. Cannon WB: Peristalsis, segmentation and the myenteric plexus. Am J Physiol 1912;30:114-128. 9. Bornstein JC, Furness JB: Correlated electrophysiological and histochemical studies of submucous neurons and their contribution to understanding enteric neural circuits. J Auto Nerv Sys 1988;25:1-13. 10. Sarna SK, Soergel KH, Harig JM, Loo FD, Wood CM, Donahue KM, Ryan RP: Spatial and temporal patterns of human jejunal contractions. Am J Physiol 1989;257:G423-G432. 11. Burns GA, Cummings JF: The equine myenteric plexus with special reference to the pelvic flexure pacemaker. Anat Rec 1991 ;230:417-424. 12. Elliott TR, Barclay-Smith E: Antiperistalsis and other muscular activities of the colon. J Physiol London 1904;31:272304. 13. Takaki M, Wood JD, Gershon MD: Heterogeneity of ganglia of the guinea pig myenteric plexus: an in vitro study of the origin of terminals within single ganglia using a covalently bound fluorescent retrograde tracer. J Comp Neurol 1985;235:488-502.

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