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Chemical denervation of the myenteric plexus of the muscular stomach of turkeys
Camp. Printed
Biochem. Physiol. in Great
Vol. 88C, No. 1, pp. 201-207,
1987 0
Britain
0306-4492/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
CHEMICAL DENERVATION OF THE MYENTERIC PLEXUS OF THE MUSCULAR STOMACH OF TURKEYS SUSAN B. CHAPLIN, GARY E. DUKE, HOLLY HUNT and LAUREL A. DEGERNES Department of Veterinary Biology, University of Minnesota, St Paul, MN 55108, USA. Telephone: (612) 624-5322 (Received 5 December 1986)
Abstract-1. The thin caudoventral muscle (TCM) of the muscular stomach of domestic turkeys was surgically exposed and painted with solutions of saline or 0.1, 0.25,0.5, and 1.O% benzalkonium chloride (BC), a cationic surfactant shown to irreversibly damage neurons but not muscle tissue in mammals. 2. Following fluoroscopic observations of gastric motility for 2 weeks, turkeys were euthanized, the entire muscular stomach was excised and weighed, and serial frozen sections of the TCM were taken for evaluation of the number and size of neurons in the myenteric plexus. 3. Treatment with 0.5 and 1.0% BC resulted in loss of motility in the TCM, significant hypertrophy (P < 0.001) of the CTM, a 70% decrease in number and 60% decrease in size of myenteric neurons, and a 4-fold increase in thickness of the serosa, compared with saline-treated controls.
In this study we have attempted to produce an experimental aganglionosis in the muscular stomach of the turkey. The primary aims of this study were to determine the concentration of BC which specifically destroys avian myenteric neurons and to investigate the consequences of neuronal loss on contractility of the thin caudoventral (TC) muscle of the muscular stomach. Data obtained from this study will form the basis for future studies aimed at isolating the hypothesized site or sites responsible for neuronal initiation and coordination of the gastroduodenal sequence in birds.
INTRODUCIXON Surfactants, such as benzalkonium chloride (BC) selectively damage nerve cell membranes causing cell death, but do not affect the anatomical or physiological integrity of muscle tissue. For these reasons they have been used to produce an experimental aganglionosis in mammals (Sato et al., 1978; Sakata et al., 1978). Fox et al. (1983) and Fox and Bass (1984) found that a concentration of 0.062% BC applied for 30min to the serosa of a segment of rat intestine significantly reduced the number of ganglion cells in the myenteric plexus. It also reduced the release of intestinal neuropeptides, abolished the propagation of spike potential associated with the migrating motor complex, and altered the basic electrical rhythm (BER). However, Fox and Bass (1986) found that BC treatment did not affect the contractility of the longitudinal muscle by direct electrical stimulus. In mammals the BER (or slow waves) is initiated in the interstitial cells of Cajal lying between the longitudinal and circular muscle layers (Thuneberg et al., 1982; Suzuki and Prosser, 1984). The BER estabof gastric and intestinal conlishes the “pace” tractions. In birds, contractions of the muscular stomach, glandular stomach, and duodenum are coordinated, but slow waves do not occur in the muscular and glandular stomachs (Duke, 1982). Although slow waves do occur in the avian duodenum they do not appear to be involved in the regulation of its contractions (Duke et al., 1975). A neurogenic pacemaker is believed to be responsible for coordination of the gastroduodenal cycle in birds (Nolf, 1937, 1938; Duke et aI., 1975). Supported in part by funds from the Minnesota Agricultural Experiment Station, Project 614; manuscript number 15,058.
MATERIALS AND
METHODS
1. Animal housing Commercially raised female turkeys (Meleugris gallopavo) were obtained at 4-6 weeks of age from Willmar Poultry and Jenny-O Foods in Willmar, Minnesota. They were paired until 8-10 weeks of age and then housed individually in cages with individual food and water pans located in rooms with automatic temperature, humidity, and photoperiod control. The birds were fed ad libitum a ration prepared by the Animal Science Department of the University of Minnesota (25% crude protein, 24% crude fat, 2.3% crude fiber). They were weighed once weekly to ascertain growth rate. 2. Surgery Solutions of benzalkonium chloride (BC) were prepared at concentrations of 0.1, 0.25, 0.5 and 1.00% in physiological saline just prior to surgery. Surgeries were performed at 5-8 weeks of age using aseptic techniques. The birds received a penicillin antiobiotic (i.m.) 24 hr prior to surgery (0.2 ml/kg Pen BP-48, Pfizer) and were fasted 18 hr prior to surgery. Gas anesthesia (4% isoflurane: 96% oxygen mixture for induction, 1.7% isoflurane for maintenance) or pentobarbital (40mg/kg) were used during surgery. A 34 cm longitudinal incision was made in the abdomen 1 cm to the left of midline, immediately ventral to the muscular 201
202
SUSAN B. CHAPLIN et al.
stomach. A suture placed through the craniodorsal thick muscle was used to lift the muscular stomach and externalize the TC muscle through the abdominal incision. The incision site was packed with sterile saline-dampened sponges, leaving the TC muscle protruding. Sponges soaked
in one of the 4 doses of BC or in 0.9% sterile saline were applied to the surface of the TC muscle (N = 48 in each treatment group); fresh solution was reapplied every 10 min for a total of 30min. The sponges were then removed and the TC muscle surface was carefully rinsed with sterile physiological saline, taking care to prevent run-off from entering the abdominal cavity. The muscular stomach was repositioned in the abdomen, 50,~O units of penicillin (Pfizerpen, Pfizer Pfipharmecs) were injected i.p., and the wound was closed. In some turkeys a wire marker sealed in a polyethylene capsule was sutured onto the TC muscle to help identify the particular site of BC application during radiographic observations. A separate group of control birds were not subjected to surgery nor were they treated with either BC or saline. 3. Radiography Observations of GI motility were made using image intensification radiography (IIR) with a Philips Super Ml00 RadioeranhiciFlurosconv unit (Phil&s Medical Systems, Inc., Shelion, ‘CT). This &as done once prior to surgery and twice weekly for 2 weeks thereafter on each treated bird. At the beginning of each observation period, a bird was restrained in lateral recumbency and 5-10~~ of barium sulfate was injected intragastrically per OS. Radiographic observations of the gastroduodenal contraction sequence were accomplished with the bird standing in a closed cardboard box. The frequency and duration of the TC muscle contractions were recorded and the normality of the contraction sequence and amplitude were noted. 4. Necropsy and histology At 2 weeks post surgery the turkeys receiving saline or BC treatments were euthanized. In addition, 16 turkeys weighing l-4 kg were euthan~z~ as controls. At the time of necropsy, the entire muscular stomach was excised, weighed (devoid of contents), and its anteroposterior (length) and transverse (width) dimensions were recorded. The TC muscle was then carefully dissected from the muscular stomach, weighed, and its length and width recorded. A_ median longitudinal section (0.5 x l.Ocm) of the TC muscle was trimmed and frozen in liquid nitrogen and then mounted for cryoscopy. Frozen sections were made for all turkeys which received saline or BC treatments and for 4 of the controls. Forty-micron longitudinal sections were cut at 25@-300pm intervals through each block of tissue. Two consecutive sections at each of l&l5 intervals were mounted on slides and air dried. To visualize the myenteric neurons with light microscopy, a histochemical reaction for NADH diaphorase activity using nitro-blue tetrazolium (nitro-BT) was employed (Gabella, 1969; Pearse, 1972). NADH diaphorase activity is most intense in neurons and less so in muscle and other tissue. The stain therefore produces sections with purple neurons, light blue muscle tissue, and magenta connective tissue and glia. The slides were treated with nitro-BT reagent for 30 min, rinsed in tap water, and immersed for 15 min in 10% buffered formalin to stop the reaction. They were then passed through a distilled water wash, and alcohol series (70% 95%, lOO%), and finally into xylene before mounting coverslips. Only the neurons localized beneath the serosal surface within ovoid cross-sections of plexus were counted (see Fig. 4). The total number of neurons were counted in each of 10 sections for each bird. Nuclear diameter, serosal thickness, and total plexus length were measured with an ocular micrometer.
RESULTS
1. Radiography We were able to observe and evaluate radiographically the effects of BC treatment on (1) contraction frequency of the muscular stomach, (2) whether the contraction sequence of the muscular stomach, or of the entire gastroduodenal apparatus, was normal, and (3) whether duration of contractions of the TC muscle and the timing of its contractions within the muscular stomach contraction sequence were normal. There were no significant differences in the rate of contractions of the muscular stomach between pre and post surgical conditions (ANOVA P = 0.65) or between treatment groups (ANOVA P = 0.44). The rate varied from 3-5 contractions per min (overall mean = 4.0, N = 25). Motility commenced almost immediately after the esophageal cannula was removed. In contrast, the contraction sequence of the muscular stomach was markedly affected by treatment of the TC muscle with BC. In control (non-treated) and saline-treated birds, the normal sequence of contraction began almost simultaneously at the medial surface of the thin craniodorsal muscle and the lateral margin of the TC muscle (Fig. I, top) and moved across the surfaces of these muscles to their opposite margins. The thin muscles did not relax until all of the muscle had contracted and their entire contraction lasted 4-6 sec. Only one bird in the 0. I % BC treatment group showed any departure from the normal. This bird had an extremely slow rate of contraction and the duration of the TC muscle contraction was shorter than normal. In birds treated with 0.25% BC, the TC muscle contracted somewhat asymetrically, i.e. often one side of the TC muscle contracted to a greater extent than the other, or one side relaxed more quickly than the other. Birds treated with 0.5% BC exhibited weak contractions with less movement of the TC muscle, relaxation of part of the muscle before all of it had contracted, and in one bird, no contraction at all. In all of the birds treated with 1.0% BC, the TC muscle failed to contract and became impacted with food by 7 days post surgery. In some turkeys the barium did not disperse through the impacted food in the pouch formed by the TC muscle, and its mucosal surface was only faintly outlined. This resulted in the abnormal radiographic appearance of the lumen of the muscular stomach shown in Fig. 1, bottom. Motility of the 1.O% BC-treated birds remained unchanged during the next 7 days of the trial, and the TC muscle remained impacted with food with uneven or no barium dispersal within. 2. Morphology ofthe muscular stomach Since the turkeys used in this study varied in age from 8 to 12 weeks and in body mass from 1 to 4 kg, we first had to determine the normal allometric changes in muscular stomach morphology. Muscular stomach mass (empty), length, and width, and TC muscle mass, length, and width were all strongly related to body mass (regression coefficients > 0.8 and P < 0.0001 in most cases) in 16 control turkeys ranging in body mass from 1.5 to 4.3 kg (Table 1). We
203
Denervation of turkey gizzard
THIN
THICK
CONTROL
THICK
THIN
1.0 % BC
Fig. 1. Radiographs taken by Image Intensification Radiography of the muscular stomachs (MS) of control (top) and 1.0% BC-treated (bottom) turkeys. The dark areas are the lumens of the muscular stomach as it appears during contractions of the pair of thin muscles (left) and of the pair of thick muscles (right).
The long tube running
around
obtained predicted values for these morphological parameters in the saline and BC-treated turkeys by using the allometric equations given in Table 1 for the control turkeys. The effect of high doses (0.5 and 1.0% BC) on the TC muscle were grossly visible at necropsy. The intact muscular stomach was greatly enlarged at the ventral aspect, and the TC muscle was enlarged 2-3-fold (Fig. 2). Muscular stomach mass and length, and TC muscle mass, width, and length of 0.5 and 1.O% BC-treated birds were consistently higher than predicted. For example, Fig. 3 illustrates the hypertrophy of gizzard mass caused by high doses of BC. Analyses of variance confirmed these findings. There were significant effects of BC treatment on muscular stomach mass (P = 0.0096) and length (P = 0.0014), and on TC muscle mass (P = 0.001) and length (P = 0.0007). In every analysis either the 0.5 or 1.0% BC or both treatment groups were significantly different from the saline-treated group (Table 2). Treatment with saline, 0.1 and 0.25% BC caused no significant changes in the means of mus-
the muscular
stomach
is the duodenum.
cular stomach mass or length, or TC muscle mass, length, or width compared to controls (Table 2). 3. Histology There were marked changes in the histology of the serosa and myenteric plexus of the caudal thin muscle as a result of BC treatment (Fig. 4 and Table 3). Saline-treated and untreated controls exhibited a thin serosal surface (1 W-200 pm), beneath which lay an extensive plexus, containing numerous large neuTable 1. Allometric equations for entire muscular stomach (MS) and thin caudoventral (TC) muscle mass and dimensions in 16 control
turkeys
Equation MS mass (g) MS length (cm) MS width (cm) TC mass (g) TC length (cm) TC width (cm)
= = = = = =
13.96 + 0.0138 x M 5.51 + 0.0005 x M 4.13 + 0.0006 x M 1.62 + 0.0016 x M 3.81 + 0.0005 x M 2.57 + 0.0003 x M
M is body mass (g), r is the regression of significance.
coefficient,
I
P
0.90 0.88 0.87 0.8 I 0.78 0.42
<0.0001 <0.0001 <0.0001
< 0.0001 < 0.0001 0.0426
and P is the level
&BAN
B. CHAPLIN ef a/. EFFECT
OF BC ON GIZZARD
MASS
90,
2s
D
z r”
70-
_..*
Lcl 2
60.
..-..-* . . . . . ..-
0 . ..’ osi . . . . _.-. 50-
0
l Saline c Ol%BC * 0.25%BC n 0.5% BC 0 l.O%BC -- Predicted
‘I.....
T; 6
*..* 40-
f.G Op. .f.
30, 1.4
TC
.
00
0
1.8
2.2
2.6
3.0
BODY MASS
3.4
3.8
4.2
-I
(ko)
Fig. 3. Muscular stomach mass of turkeys treated with ~nzalkonium chloride (BC), compared with that predicted for turkeys of that body mass (predicted line derived from 16 controls). Symbols represent the concentrations of BC used on turkeys in this study (see box).
Fig. 2. Macroscopic
view of the muscular stomach of turkeys. Top is a line drawing illustrating aspects of gastroduodenal anatomy: Tn = thin muscle, Tk = thick muscle, GS = g~and~ar stomach, MS = muscular stomach, D = duodenum, TC = thin caudoventral muscle. Below are
photographs of the muscular stomachs of turkeys treated with saline (left) and a 1.0% solution of benzalkonium chloride (BC) (right). Note the elongation of the longitudinal axis of the BC-treated muscular stomach and the enlargement of the TC muscle.
rons, 26-30 irn in diameter. The plexus of birds treated with 0.1% BC had fewer neurons, and the neurons were smaller than those of controls. The plexus contained small, regularly-spaced vacuoles in some birds, which were interpreted as swollen axons,
but the tissue itself was intact and non-degenerative. The serosa was slightly thicker than in control birds, but the muscle layer beneath the plexus was unaffected and normal in appearance. Thus, even in the low dose group and in the absence of any demonstrable changes in the TC muscle motility by radiography, there were some structural changes occurring in the myenteric plexus. At the other extreme 1.0% BC-treated birds had few, scattered, small (10 pm) neurons in the plexus area. The plexus was incomplete in some sections, lacking a connective tissue boundary on the serosal side, and the interior of the plexus cross-section was disrupted and the cells disorganized, compared to the controls. The serosa was much thicker (SOO-1000~m) and was made up entirely of collagenous fibres and adipose cells. The muscle beneath the plexus exhibited a pocked appearance, probably due to the swollen axons permeating the tissue and interrupting the muscle fibers. Analysis of variance showed that there were significant effects of treatment on total number of neurons, neuron diameter, and serosal thickness (P < 0.001 for all analyses) but not on the length of plexus present. The 0.5 and 1.0% BC-treated groups were significantly different from both the untreated controls and the saline-treated birds in all histological characteristics except length of plexus (Table 3). The 0.25 and 0.1% BC-treated birds had significantly smaller neurons than the controls.
Table 2. Means (and standard deviations) of dimensional aarameters of muscular stomach and thin caudoventral muscle of control and -K-treated turkeys
Treatment Control Saline 0.1% EC 0.25% BC 0.50% BC 1.0% BC
N 16 4 4 4 4 5
~.._. Mass (G)
Muscular stomach
53.8 (12.7) 43.8 (6.9) 54.9 (9.4) 45.2 (9.8) 69.6 (11.6)* 69.2 (11.8)’
Thin caudoventral
Length (CM) Width (CM) 7.1 (0.6) 6.9 (0.3) 7.6 (0.5) 6.9 (0.4) 8.0 (O.s)* 8.1 (0.3j**
6.0 (0.8) 5.9 (0.6) 6.3 (0.3) 5.9 (0.3) 6.7 (0.3Y 6.5 (0.5j
Mass (G) 6.3f1.6) 4.9 (0.7) 7.6 (0.7) 5.7 (0.9) 8.6 (0.2Y 10.0(1.4j***
muscle ._~ Length (CM) Width (CM) -..--I____ 52. (0.6) 3.3 (0.5) 5.1 (0.1) 3.3 (0.8) 5.5 (0.1) 3.8 (0.5) 4.8 (0.5) 3.5 (0.5) 5.8 (0.5)’ 4.2 (0.5)’ 6.0 (0.6)* 4.6 (O.S)*
N is the number of turkeys sampled. Statistical significance between BC-treated birds and saline-treated birds was determined by Tukey’s method of pairwise comparison of means following analysis of variance. l=p
Denervation of turkey gizzard
SALINE
0.25%
-
BC
SER
- PL
0.5
% BC
1.0 % BC
-
SER
-PL*
MUS
Fig. 4. Photomicrographs of 40 pm sections of the thin caudoventral muscle of turkeys treated with saline versus those treated with 0.25, 0.5 and 1.O% solutions of benzalkonium chloride (BC). Neurons are the darkly stained cells in the plexus. SER denotes serosa, PL, plexus, and MUS, the smooth muscle layer. Note the thickening of the serosa and diminution of the plexus and neurons with BC treatment.
The total number of neurons and neuron diameter both decreased in dose-dependent manner (Table 3). Linear regression analysis revealed a strong linear relationship of these histological variables and BC
concentration (r > 0.66 and P < 0.0001). Serosal thickness, indicative of scar tissue formation, was positively correlated with increasing BC concentrations (r = 0.58 and P < 0.0001).
206
SUSAN B. CHAPLIN et al. Table 3. Histological
Treatment
N
Control Saline 0.1% BC 0.25% BC 0.5% BC 1.0% BC
4 4 5 5 8 7
effects of BC treatments on the thin caudoventral (mean and standard deviations)
Total neurons (No.) 37.7 38.9 22.4 23.3 15.5 10.9
(13.9) (10.6) (6.9) (7.1) (4.3)’ (8.0)**
Plexus length (mm) 6.1 6.0 5.0 6.6 6.7 6.3
(1.3) (I .9) (1.0) (2.2) (2.5) (4.6)
Neuron
diam.
(pm) 30.4 26.6 19.9 16.9 19.2 12.1
(5.5) (4.8) (6.5)* (4.3)‘* (5.8)” (2.2)**
muscle of turkeys Serosal width Olm) I56 (72) 200(174) 250 (337) 385 (246) 469 (248)** 750 (190)***
N is the number of turkevs in each samule. . but means and standard deviations represent evaluation of 10 sections for each turkey in the sample. Statistical significance between BC-treated birds and control birds was obtained by Tukey’s method of paired comparison of means following analysis of variance. * = P < 0.05; ** = P < 0.01; *** = P < 0.001.
DISCUSSION
The effects of benzalkonium chloride (BC) on myenteric neurons of the muscular stomach of turkeys were similar to those reported for ganglia of the myenteric plexus of mammalian intestine with some important exceptions. BC destroyed turkey myenteric neurons in a dose-dependent manner, but the concentrations of BC needed to ablate turkey myenteric neurons were much higher than those reported for rat jejunum or colon. For example, concentrations of 0.01% caused significant reduction in the number of neurons in rat jejunum (Fox et al., 1983) and rat colon (Sato et al., 1978). We were unable to detect any effect of concentrations within the range of O.OlM. 1% BC on caudal thin muscle in a pilot study. Fox et al. (1983) and Sato et al. (1978) used concentrations of O.l-0.25% BC to denervate rat jejunum and colon, respectively. We found that similar concentrations of BC had only slight to moderate effects on the number of neurons and neuronal diameter in the turkey myenteric plexus (Table 3). We were unable to completely denervate the TC muscle myenteric plexus, even at the highest BC concentration used in this study (1.0%) whereas Fox et al. (1983) and Sato et al. (1978) found complete absence of myenteric neurons in rat intestine at concentrations higher than 0.1%. However, since the turkey myenteric plexus itself appeared damaged (lacking its serosal margin) and those neurons present were only one-third the size of normal neurons and very sparsely distributed, this area was perhaps functionally denervated by treatment with 1.0% BC due to deterioration of neuronal elements. Treatment of rat intestine or colon with high concentrations of BC (0.5-1.0%) was fatal to the animal, causing perforation within a few days of treatment (Sato et al., 1978; Fox et al., 1983). In contrast, the turkeys appeared unaffected by 0.551.0% BC treatments to the muscular stomach. They ate normally, passed a normal amount of excreta, and gained weight at the same rate as controls. The only abnormality noted at necropsy was gaseous distension of the intestinal ceca and a change in the color of their contents from dark brown to yellow-brown. This may have been a result of the change in nutrient passage rate through the gut, due to alteration of motility of the muscular stomach by BC treatment. Conversely, the time course of effects of BC treat-
ment on the motility of the TC muscle in turkeys was similar to that observed in rats by Fox et aI. (1983), with the full effect of the treatment being reached within 2 weeks in both species. The marked hypertrophic response of the TC muscle due to BC treatment was also similar to that reported for rat intestine (Fox et al., 1983; Fox and Bass, 1986). Intestinal smooth muscle hypertrophy is a common neuropathological consequence of chronic disruption of the myenteric plexus (Smith, 1972), and hypertrophy occurs in other denervated smooth muscle preparations as well. For example, Ekstrom et al. (1984) found that dervated rat urinary bladder exhibited both hyperplasia and hypertrophy. We have not determined whether the increase in TC muscle mass was due to hypertrophy or hyperplasia of the muscle cells. Treatment of the TC muscle with high concentrations of BC (0.5-1.0%) induced the formation of extensive scar tissue and adhesions to the body wall, resulting in a greatly thickened serosal surface. The amount of scar tissue (serosal thickness) was directly proportional to the concentration of BC applied (Table 3) illustrating the irritant nature of this material. Although the scar tissue and adhesions could feasibly impede motility, we feel this was not the reason for the loss of motility in the TC muscle. Turkeys in which wire markers were sewn to the TC muscle following application of saline or 0.1% BC also developed extensive scar tissue on the serosal surface and thick adhesions around the wire markers, yet the TC muscle exhibited normal motility radiographically during the entire 2 weeks of the trial.
SUMMARY
The present study has demonstrated that benzalkonium chloride, a cationic surfactant, can selectively ablate the myenteric neurons in the muscular stomach of turkeys, just as it does in the mammalian intestine. Destruction of myenteric neurons by BC treatment of the caudoventral thin (TC) muscle of the muscular stomach resulted in cessation of motility and hypertrophy of the TC muscle, but did not change the rate of muscular stomach contraction. Thus, this site is not integral to establishing the pace or to coordinating the gastroduodenal contraction sequence. The technique should prove useful in determining whether a neurogenic “pace-maker” exists in
Denervation of turkey gizzard the avian muscular stomach (as proposed by Nolf 1937, 1938) and where this pacemaker is located. ~cknuwle~ge~enzs-We are grateful to Jenny-O Farms and Willmar Poultry Company for generously supplying the turkeys used in this study. We appreciate the assistance of Drs Al Beitz, Kathy Magnusson, and Jane Clements of our department in preparing the micrographs.
REFERENCES
Duke G. E., Kostuch T. E. and Evanson 0. A. (1975) Gastroduodenal electrical activity and intraluminal pressure changes in the lower small intestine of turkeys. Am. J. Digestive Dis. 20, 104&1058.
Duke G. E. (1982) Gastrointestinal motility and its reguiation. ~ou~~r~ Sci. 61, 12451256. Ekstrom J., Henningsson A. C., Henningsson S. and Malmberg L. (1984) Hyperplasia and hypertrophia in the denervated and distended rat urinary bladder. Acta physiol. stand. 122, 45.-48.
Fox D. A., Epstein M. L. and Bass P. (1983) Surfactants selectively ablate enteric neurons of the rat jejunum. J, Pharmuc. exp. Ther. 227, 538-544. Fox D. A. and Bass P. (1984) Selective myenteric neuronal denervation of the rat jejunum. Gastroenterology 87, 512-577.
207
Gabella G. (1969) Detection of nerve cells by a histochemical technique. Experientia 15, 218-219. Nolf P. (1937) On the existence in the bird of a system of intrinsic fibers connecting the stomach to the small intestine. J. ~~~~~o~,90, 53p54p. Nolf P. (1938) L’appareil nerveux de l’automatisme gastrique de l’oiseau. II. Etude des effects causes par une ou plusieurs sections de l’anneau nerveux du gesier. Arch. Znt. Phvsiol. Biochim. 46, 44-559. Pea&e A. G. E. (1977) Histochemistry: Theoretical and Applied, 3rd edn, pp. 1342-1343. Churchill Livingstone, Edinburgh. Sakata K., Kunieda T., Furuta T. and Sato A. (1979) Selective destruction of intestinal nervous elements bv local application of benzalkonium solution in the rat. Experientia 35, 1611-1612. Sato A., Yamamoto M., Imamura K., Kashiki Y., Kunieda T. and Sakata K. (1978) Pathophysiology of aganglionic colon and anorectum: an experimental study on aganglionosis produced by a new method in the rat. J. Ped. surg. 13, 399405. Smith B. (1972) The Neuropathology of the Alimentary Tract. Williams & Wilkins, Baltimore, MD. Suzuki N. and Prosser C. L. (1984) Interstitial cells in the intestine as pacemakers for electrical slow waves. Fedn. Proc. 43, 725 (abstract). Thuneberg L., Rumessen J. J. and Mikkelsen H. B. (1982) The interstitial cells of Cajal: intestinal pacemaker cells? In Mot&y of the Digest& Tract (Edited by M. Weinbeck), pp. 115-122. Raven Press, New York.
Biochem. Physiol. in Great
Vol. 88C, No. 1, pp. 201-207,
1987 0
Britain
0306-4492/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
CHEMICAL DENERVATION OF THE MYENTERIC PLEXUS OF THE MUSCULAR STOMACH OF TURKEYS SUSAN B. CHAPLIN, GARY E. DUKE, HOLLY HUNT and LAUREL A. DEGERNES Department of Veterinary Biology, University of Minnesota, St Paul, MN 55108, USA. Telephone: (612) 624-5322 (Received 5 December 1986)
Abstract-1. The thin caudoventral muscle (TCM) of the muscular stomach of domestic turkeys was surgically exposed and painted with solutions of saline or 0.1, 0.25,0.5, and 1.O% benzalkonium chloride (BC), a cationic surfactant shown to irreversibly damage neurons but not muscle tissue in mammals. 2. Following fluoroscopic observations of gastric motility for 2 weeks, turkeys were euthanized, the entire muscular stomach was excised and weighed, and serial frozen sections of the TCM were taken for evaluation of the number and size of neurons in the myenteric plexus. 3. Treatment with 0.5 and 1.0% BC resulted in loss of motility in the TCM, significant hypertrophy (P < 0.001) of the CTM, a 70% decrease in number and 60% decrease in size of myenteric neurons, and a 4-fold increase in thickness of the serosa, compared with saline-treated controls.
In this study we have attempted to produce an experimental aganglionosis in the muscular stomach of the turkey. The primary aims of this study were to determine the concentration of BC which specifically destroys avian myenteric neurons and to investigate the consequences of neuronal loss on contractility of the thin caudoventral (TC) muscle of the muscular stomach. Data obtained from this study will form the basis for future studies aimed at isolating the hypothesized site or sites responsible for neuronal initiation and coordination of the gastroduodenal sequence in birds.
INTRODUCIXON Surfactants, such as benzalkonium chloride (BC) selectively damage nerve cell membranes causing cell death, but do not affect the anatomical or physiological integrity of muscle tissue. For these reasons they have been used to produce an experimental aganglionosis in mammals (Sato et al., 1978; Sakata et al., 1978). Fox et al. (1983) and Fox and Bass (1984) found that a concentration of 0.062% BC applied for 30min to the serosa of a segment of rat intestine significantly reduced the number of ganglion cells in the myenteric plexus. It also reduced the release of intestinal neuropeptides, abolished the propagation of spike potential associated with the migrating motor complex, and altered the basic electrical rhythm (BER). However, Fox and Bass (1986) found that BC treatment did not affect the contractility of the longitudinal muscle by direct electrical stimulus. In mammals the BER (or slow waves) is initiated in the interstitial cells of Cajal lying between the longitudinal and circular muscle layers (Thuneberg et al., 1982; Suzuki and Prosser, 1984). The BER estabof gastric and intestinal conlishes the “pace” tractions. In birds, contractions of the muscular stomach, glandular stomach, and duodenum are coordinated, but slow waves do not occur in the muscular and glandular stomachs (Duke, 1982). Although slow waves do occur in the avian duodenum they do not appear to be involved in the regulation of its contractions (Duke et al., 1975). A neurogenic pacemaker is believed to be responsible for coordination of the gastroduodenal cycle in birds (Nolf, 1937, 1938; Duke et aI., 1975). Supported in part by funds from the Minnesota Agricultural Experiment Station, Project 614; manuscript number 15,058.
MATERIALS AND
METHODS
1. Animal housing Commercially raised female turkeys (Meleugris gallopavo) were obtained at 4-6 weeks of age from Willmar Poultry and Jenny-O Foods in Willmar, Minnesota. They were paired until 8-10 weeks of age and then housed individually in cages with individual food and water pans located in rooms with automatic temperature, humidity, and photoperiod control. The birds were fed ad libitum a ration prepared by the Animal Science Department of the University of Minnesota (25% crude protein, 24% crude fat, 2.3% crude fiber). They were weighed once weekly to ascertain growth rate. 2. Surgery Solutions of benzalkonium chloride (BC) were prepared at concentrations of 0.1, 0.25, 0.5 and 1.00% in physiological saline just prior to surgery. Surgeries were performed at 5-8 weeks of age using aseptic techniques. The birds received a penicillin antiobiotic (i.m.) 24 hr prior to surgery (0.2 ml/kg Pen BP-48, Pfizer) and were fasted 18 hr prior to surgery. Gas anesthesia (4% isoflurane: 96% oxygen mixture for induction, 1.7% isoflurane for maintenance) or pentobarbital (40mg/kg) were used during surgery. A 34 cm longitudinal incision was made in the abdomen 1 cm to the left of midline, immediately ventral to the muscular 201
202
SUSAN B. CHAPLIN et al.
stomach. A suture placed through the craniodorsal thick muscle was used to lift the muscular stomach and externalize the TC muscle through the abdominal incision. The incision site was packed with sterile saline-dampened sponges, leaving the TC muscle protruding. Sponges soaked
in one of the 4 doses of BC or in 0.9% sterile saline were applied to the surface of the TC muscle (N = 48 in each treatment group); fresh solution was reapplied every 10 min for a total of 30min. The sponges were then removed and the TC muscle surface was carefully rinsed with sterile physiological saline, taking care to prevent run-off from entering the abdominal cavity. The muscular stomach was repositioned in the abdomen, 50,~O units of penicillin (Pfizerpen, Pfizer Pfipharmecs) were injected i.p., and the wound was closed. In some turkeys a wire marker sealed in a polyethylene capsule was sutured onto the TC muscle to help identify the particular site of BC application during radiographic observations. A separate group of control birds were not subjected to surgery nor were they treated with either BC or saline. 3. Radiography Observations of GI motility were made using image intensification radiography (IIR) with a Philips Super Ml00 RadioeranhiciFlurosconv unit (Phil&s Medical Systems, Inc., Shelion, ‘CT). This &as done once prior to surgery and twice weekly for 2 weeks thereafter on each treated bird. At the beginning of each observation period, a bird was restrained in lateral recumbency and 5-10~~ of barium sulfate was injected intragastrically per OS. Radiographic observations of the gastroduodenal contraction sequence were accomplished with the bird standing in a closed cardboard box. The frequency and duration of the TC muscle contractions were recorded and the normality of the contraction sequence and amplitude were noted. 4. Necropsy and histology At 2 weeks post surgery the turkeys receiving saline or BC treatments were euthanized. In addition, 16 turkeys weighing l-4 kg were euthan~z~ as controls. At the time of necropsy, the entire muscular stomach was excised, weighed (devoid of contents), and its anteroposterior (length) and transverse (width) dimensions were recorded. The TC muscle was then carefully dissected from the muscular stomach, weighed, and its length and width recorded. A_ median longitudinal section (0.5 x l.Ocm) of the TC muscle was trimmed and frozen in liquid nitrogen and then mounted for cryoscopy. Frozen sections were made for all turkeys which received saline or BC treatments and for 4 of the controls. Forty-micron longitudinal sections were cut at 25@-300pm intervals through each block of tissue. Two consecutive sections at each of l&l5 intervals were mounted on slides and air dried. To visualize the myenteric neurons with light microscopy, a histochemical reaction for NADH diaphorase activity using nitro-blue tetrazolium (nitro-BT) was employed (Gabella, 1969; Pearse, 1972). NADH diaphorase activity is most intense in neurons and less so in muscle and other tissue. The stain therefore produces sections with purple neurons, light blue muscle tissue, and magenta connective tissue and glia. The slides were treated with nitro-BT reagent for 30 min, rinsed in tap water, and immersed for 15 min in 10% buffered formalin to stop the reaction. They were then passed through a distilled water wash, and alcohol series (70% 95%, lOO%), and finally into xylene before mounting coverslips. Only the neurons localized beneath the serosal surface within ovoid cross-sections of plexus were counted (see Fig. 4). The total number of neurons were counted in each of 10 sections for each bird. Nuclear diameter, serosal thickness, and total plexus length were measured with an ocular micrometer.
RESULTS
1. Radiography We were able to observe and evaluate radiographically the effects of BC treatment on (1) contraction frequency of the muscular stomach, (2) whether the contraction sequence of the muscular stomach, or of the entire gastroduodenal apparatus, was normal, and (3) whether duration of contractions of the TC muscle and the timing of its contractions within the muscular stomach contraction sequence were normal. There were no significant differences in the rate of contractions of the muscular stomach between pre and post surgical conditions (ANOVA P = 0.65) or between treatment groups (ANOVA P = 0.44). The rate varied from 3-5 contractions per min (overall mean = 4.0, N = 25). Motility commenced almost immediately after the esophageal cannula was removed. In contrast, the contraction sequence of the muscular stomach was markedly affected by treatment of the TC muscle with BC. In control (non-treated) and saline-treated birds, the normal sequence of contraction began almost simultaneously at the medial surface of the thin craniodorsal muscle and the lateral margin of the TC muscle (Fig. I, top) and moved across the surfaces of these muscles to their opposite margins. The thin muscles did not relax until all of the muscle had contracted and their entire contraction lasted 4-6 sec. Only one bird in the 0. I % BC treatment group showed any departure from the normal. This bird had an extremely slow rate of contraction and the duration of the TC muscle contraction was shorter than normal. In birds treated with 0.25% BC, the TC muscle contracted somewhat asymetrically, i.e. often one side of the TC muscle contracted to a greater extent than the other, or one side relaxed more quickly than the other. Birds treated with 0.5% BC exhibited weak contractions with less movement of the TC muscle, relaxation of part of the muscle before all of it had contracted, and in one bird, no contraction at all. In all of the birds treated with 1.0% BC, the TC muscle failed to contract and became impacted with food by 7 days post surgery. In some turkeys the barium did not disperse through the impacted food in the pouch formed by the TC muscle, and its mucosal surface was only faintly outlined. This resulted in the abnormal radiographic appearance of the lumen of the muscular stomach shown in Fig. 1, bottom. Motility of the 1.O% BC-treated birds remained unchanged during the next 7 days of the trial, and the TC muscle remained impacted with food with uneven or no barium dispersal within. 2. Morphology ofthe muscular stomach Since the turkeys used in this study varied in age from 8 to 12 weeks and in body mass from 1 to 4 kg, we first had to determine the normal allometric changes in muscular stomach morphology. Muscular stomach mass (empty), length, and width, and TC muscle mass, length, and width were all strongly related to body mass (regression coefficients > 0.8 and P < 0.0001 in most cases) in 16 control turkeys ranging in body mass from 1.5 to 4.3 kg (Table 1). We
203
Denervation of turkey gizzard
THIN
THICK
CONTROL
THICK
THIN
1.0 % BC
Fig. 1. Radiographs taken by Image Intensification Radiography of the muscular stomachs (MS) of control (top) and 1.0% BC-treated (bottom) turkeys. The dark areas are the lumens of the muscular stomach as it appears during contractions of the pair of thin muscles (left) and of the pair of thick muscles (right).
The long tube running
around
obtained predicted values for these morphological parameters in the saline and BC-treated turkeys by using the allometric equations given in Table 1 for the control turkeys. The effect of high doses (0.5 and 1.0% BC) on the TC muscle were grossly visible at necropsy. The intact muscular stomach was greatly enlarged at the ventral aspect, and the TC muscle was enlarged 2-3-fold (Fig. 2). Muscular stomach mass and length, and TC muscle mass, width, and length of 0.5 and 1.O% BC-treated birds were consistently higher than predicted. For example, Fig. 3 illustrates the hypertrophy of gizzard mass caused by high doses of BC. Analyses of variance confirmed these findings. There were significant effects of BC treatment on muscular stomach mass (P = 0.0096) and length (P = 0.0014), and on TC muscle mass (P = 0.001) and length (P = 0.0007). In every analysis either the 0.5 or 1.0% BC or both treatment groups were significantly different from the saline-treated group (Table 2). Treatment with saline, 0.1 and 0.25% BC caused no significant changes in the means of mus-
the muscular
stomach
is the duodenum.
cular stomach mass or length, or TC muscle mass, length, or width compared to controls (Table 2). 3. Histology There were marked changes in the histology of the serosa and myenteric plexus of the caudal thin muscle as a result of BC treatment (Fig. 4 and Table 3). Saline-treated and untreated controls exhibited a thin serosal surface (1 W-200 pm), beneath which lay an extensive plexus, containing numerous large neuTable 1. Allometric equations for entire muscular stomach (MS) and thin caudoventral (TC) muscle mass and dimensions in 16 control
turkeys
Equation MS mass (g) MS length (cm) MS width (cm) TC mass (g) TC length (cm) TC width (cm)
= = = = = =
13.96 + 0.0138 x M 5.51 + 0.0005 x M 4.13 + 0.0006 x M 1.62 + 0.0016 x M 3.81 + 0.0005 x M 2.57 + 0.0003 x M
M is body mass (g), r is the regression of significance.
coefficient,
I
P
0.90 0.88 0.87 0.8 I 0.78 0.42
<0.0001 <0.0001 <0.0001
< 0.0001 < 0.0001 0.0426
and P is the level
&BAN
B. CHAPLIN ef a/. EFFECT
OF BC ON GIZZARD
MASS
90,
2s
D
z r”
70-
_..*
Lcl 2
60.
..-..-* . . . . . ..-
0 . ..’ osi . . . . _.-. 50-
0
l Saline c Ol%BC * 0.25%BC n 0.5% BC 0 l.O%BC -- Predicted
‘I.....
T; 6
*..* 40-
f.G Op. .f.
30, 1.4
TC
.
00
0
1.8
2.2
2.6
3.0
BODY MASS
3.4
3.8
4.2
-I
(ko)
Fig. 3. Muscular stomach mass of turkeys treated with ~nzalkonium chloride (BC), compared with that predicted for turkeys of that body mass (predicted line derived from 16 controls). Symbols represent the concentrations of BC used on turkeys in this study (see box).
Fig. 2. Macroscopic
view of the muscular stomach of turkeys. Top is a line drawing illustrating aspects of gastroduodenal anatomy: Tn = thin muscle, Tk = thick muscle, GS = g~and~ar stomach, MS = muscular stomach, D = duodenum, TC = thin caudoventral muscle. Below are
photographs of the muscular stomachs of turkeys treated with saline (left) and a 1.0% solution of benzalkonium chloride (BC) (right). Note the elongation of the longitudinal axis of the BC-treated muscular stomach and the enlargement of the TC muscle.
rons, 26-30 irn in diameter. The plexus of birds treated with 0.1% BC had fewer neurons, and the neurons were smaller than those of controls. The plexus contained small, regularly-spaced vacuoles in some birds, which were interpreted as swollen axons,
but the tissue itself was intact and non-degenerative. The serosa was slightly thicker than in control birds, but the muscle layer beneath the plexus was unaffected and normal in appearance. Thus, even in the low dose group and in the absence of any demonstrable changes in the TC muscle motility by radiography, there were some structural changes occurring in the myenteric plexus. At the other extreme 1.0% BC-treated birds had few, scattered, small (10 pm) neurons in the plexus area. The plexus was incomplete in some sections, lacking a connective tissue boundary on the serosal side, and the interior of the plexus cross-section was disrupted and the cells disorganized, compared to the controls. The serosa was much thicker (SOO-1000~m) and was made up entirely of collagenous fibres and adipose cells. The muscle beneath the plexus exhibited a pocked appearance, probably due to the swollen axons permeating the tissue and interrupting the muscle fibers. Analysis of variance showed that there were significant effects of treatment on total number of neurons, neuron diameter, and serosal thickness (P < 0.001 for all analyses) but not on the length of plexus present. The 0.5 and 1.0% BC-treated groups were significantly different from both the untreated controls and the saline-treated birds in all histological characteristics except length of plexus (Table 3). The 0.25 and 0.1% BC-treated birds had significantly smaller neurons than the controls.
Table 2. Means (and standard deviations) of dimensional aarameters of muscular stomach and thin caudoventral muscle of control and -K-treated turkeys
Treatment Control Saline 0.1% EC 0.25% BC 0.50% BC 1.0% BC
N 16 4 4 4 4 5
~.._. Mass (G)
Muscular stomach
53.8 (12.7) 43.8 (6.9) 54.9 (9.4) 45.2 (9.8) 69.6 (11.6)* 69.2 (11.8)’
Thin caudoventral
Length (CM) Width (CM) 7.1 (0.6) 6.9 (0.3) 7.6 (0.5) 6.9 (0.4) 8.0 (O.s)* 8.1 (0.3j**
6.0 (0.8) 5.9 (0.6) 6.3 (0.3) 5.9 (0.3) 6.7 (0.3Y 6.5 (0.5j
Mass (G) 6.3f1.6) 4.9 (0.7) 7.6 (0.7) 5.7 (0.9) 8.6 (0.2Y 10.0(1.4j***
muscle ._~ Length (CM) Width (CM) -..--I____ 52. (0.6) 3.3 (0.5) 5.1 (0.1) 3.3 (0.8) 5.5 (0.1) 3.8 (0.5) 4.8 (0.5) 3.5 (0.5) 5.8 (0.5)’ 4.2 (0.5)’ 6.0 (0.6)* 4.6 (O.S)*
N is the number of turkeys sampled. Statistical significance between BC-treated birds and saline-treated birds was determined by Tukey’s method of pairwise comparison of means following analysis of variance. l=p
Denervation of turkey gizzard
SALINE
0.25%
-
BC
SER
- PL
0.5
% BC
1.0 % BC
-
SER
-PL*
MUS
Fig. 4. Photomicrographs of 40 pm sections of the thin caudoventral muscle of turkeys treated with saline versus those treated with 0.25, 0.5 and 1.O% solutions of benzalkonium chloride (BC). Neurons are the darkly stained cells in the plexus. SER denotes serosa, PL, plexus, and MUS, the smooth muscle layer. Note the thickening of the serosa and diminution of the plexus and neurons with BC treatment.
The total number of neurons and neuron diameter both decreased in dose-dependent manner (Table 3). Linear regression analysis revealed a strong linear relationship of these histological variables and BC
concentration (r > 0.66 and P < 0.0001). Serosal thickness, indicative of scar tissue formation, was positively correlated with increasing BC concentrations (r = 0.58 and P < 0.0001).
206
SUSAN B. CHAPLIN et al. Table 3. Histological
Treatment
N
Control Saline 0.1% BC 0.25% BC 0.5% BC 1.0% BC
4 4 5 5 8 7
effects of BC treatments on the thin caudoventral (mean and standard deviations)
Total neurons (No.) 37.7 38.9 22.4 23.3 15.5 10.9
(13.9) (10.6) (6.9) (7.1) (4.3)’ (8.0)**
Plexus length (mm) 6.1 6.0 5.0 6.6 6.7 6.3
(1.3) (I .9) (1.0) (2.2) (2.5) (4.6)
Neuron
diam.
(pm) 30.4 26.6 19.9 16.9 19.2 12.1
(5.5) (4.8) (6.5)* (4.3)‘* (5.8)” (2.2)**
muscle of turkeys Serosal width Olm) I56 (72) 200(174) 250 (337) 385 (246) 469 (248)** 750 (190)***
N is the number of turkevs in each samule. . but means and standard deviations represent evaluation of 10 sections for each turkey in the sample. Statistical significance between BC-treated birds and control birds was obtained by Tukey’s method of paired comparison of means following analysis of variance. * = P < 0.05; ** = P < 0.01; *** = P < 0.001.
DISCUSSION
The effects of benzalkonium chloride (BC) on myenteric neurons of the muscular stomach of turkeys were similar to those reported for ganglia of the myenteric plexus of mammalian intestine with some important exceptions. BC destroyed turkey myenteric neurons in a dose-dependent manner, but the concentrations of BC needed to ablate turkey myenteric neurons were much higher than those reported for rat jejunum or colon. For example, concentrations of 0.01% caused significant reduction in the number of neurons in rat jejunum (Fox et al., 1983) and rat colon (Sato et al., 1978). We were unable to detect any effect of concentrations within the range of O.OlM. 1% BC on caudal thin muscle in a pilot study. Fox et al. (1983) and Sato et al. (1978) used concentrations of O.l-0.25% BC to denervate rat jejunum and colon, respectively. We found that similar concentrations of BC had only slight to moderate effects on the number of neurons and neuronal diameter in the turkey myenteric plexus (Table 3). We were unable to completely denervate the TC muscle myenteric plexus, even at the highest BC concentration used in this study (1.0%) whereas Fox et al. (1983) and Sato et al. (1978) found complete absence of myenteric neurons in rat intestine at concentrations higher than 0.1%. However, since the turkey myenteric plexus itself appeared damaged (lacking its serosal margin) and those neurons present were only one-third the size of normal neurons and very sparsely distributed, this area was perhaps functionally denervated by treatment with 1.0% BC due to deterioration of neuronal elements. Treatment of rat intestine or colon with high concentrations of BC (0.5-1.0%) was fatal to the animal, causing perforation within a few days of treatment (Sato et al., 1978; Fox et al., 1983). In contrast, the turkeys appeared unaffected by 0.551.0% BC treatments to the muscular stomach. They ate normally, passed a normal amount of excreta, and gained weight at the same rate as controls. The only abnormality noted at necropsy was gaseous distension of the intestinal ceca and a change in the color of their contents from dark brown to yellow-brown. This may have been a result of the change in nutrient passage rate through the gut, due to alteration of motility of the muscular stomach by BC treatment. Conversely, the time course of effects of BC treat-
ment on the motility of the TC muscle in turkeys was similar to that observed in rats by Fox et aI. (1983), with the full effect of the treatment being reached within 2 weeks in both species. The marked hypertrophic response of the TC muscle due to BC treatment was also similar to that reported for rat intestine (Fox et al., 1983; Fox and Bass, 1986). Intestinal smooth muscle hypertrophy is a common neuropathological consequence of chronic disruption of the myenteric plexus (Smith, 1972), and hypertrophy occurs in other denervated smooth muscle preparations as well. For example, Ekstrom et al. (1984) found that dervated rat urinary bladder exhibited both hyperplasia and hypertrophy. We have not determined whether the increase in TC muscle mass was due to hypertrophy or hyperplasia of the muscle cells. Treatment of the TC muscle with high concentrations of BC (0.5-1.0%) induced the formation of extensive scar tissue and adhesions to the body wall, resulting in a greatly thickened serosal surface. The amount of scar tissue (serosal thickness) was directly proportional to the concentration of BC applied (Table 3) illustrating the irritant nature of this material. Although the scar tissue and adhesions could feasibly impede motility, we feel this was not the reason for the loss of motility in the TC muscle. Turkeys in which wire markers were sewn to the TC muscle following application of saline or 0.1% BC also developed extensive scar tissue on the serosal surface and thick adhesions around the wire markers, yet the TC muscle exhibited normal motility radiographically during the entire 2 weeks of the trial.
SUMMARY
The present study has demonstrated that benzalkonium chloride, a cationic surfactant, can selectively ablate the myenteric neurons in the muscular stomach of turkeys, just as it does in the mammalian intestine. Destruction of myenteric neurons by BC treatment of the caudoventral thin (TC) muscle of the muscular stomach resulted in cessation of motility and hypertrophy of the TC muscle, but did not change the rate of muscular stomach contraction. Thus, this site is not integral to establishing the pace or to coordinating the gastroduodenal contraction sequence. The technique should prove useful in determining whether a neurogenic “pace-maker” exists in
Denervation of turkey gizzard the avian muscular stomach (as proposed by Nolf 1937, 1938) and where this pacemaker is located. ~cknuwle~ge~enzs-We are grateful to Jenny-O Farms and Willmar Poultry Company for generously supplying the turkeys used in this study. We appreciate the assistance of Drs Al Beitz, Kathy Magnusson, and Jane Clements of our department in preparing the micrographs.
REFERENCES
Duke G. E., Kostuch T. E. and Evanson 0. A. (1975) Gastroduodenal electrical activity and intraluminal pressure changes in the lower small intestine of turkeys. Am. J. Digestive Dis. 20, 104&1058.
Duke G. E. (1982) Gastrointestinal motility and its reguiation. ~ou~~r~ Sci. 61, 12451256. Ekstrom J., Henningsson A. C., Henningsson S. and Malmberg L. (1984) Hyperplasia and hypertrophia in the denervated and distended rat urinary bladder. Acta physiol. stand. 122, 45.-48.
Fox D. A., Epstein M. L. and Bass P. (1983) Surfactants selectively ablate enteric neurons of the rat jejunum. J, Pharmuc. exp. Ther. 227, 538-544. Fox D. A. and Bass P. (1984) Selective myenteric neuronal denervation of the rat jejunum. Gastroenterology 87, 512-577.
207
Gabella G. (1969) Detection of nerve cells by a histochemical technique. Experientia 15, 218-219. Nolf P. (1937) On the existence in the bird of a system of intrinsic fibers connecting the stomach to the small intestine. J. ~~~~~o~,90, 53p54p. Nolf P. (1938) L’appareil nerveux de l’automatisme gastrique de l’oiseau. II. Etude des effects causes par une ou plusieurs sections de l’anneau nerveux du gesier. Arch. Znt. Phvsiol. Biochim. 46, 44-559. Pea&e A. G. E. (1977) Histochemistry: Theoretical and Applied, 3rd edn, pp. 1342-1343. Churchill Livingstone, Edinburgh. Sakata K., Kunieda T., Furuta T. and Sato A. (1979) Selective destruction of intestinal nervous elements bv local application of benzalkonium solution in the rat. Experientia 35, 1611-1612. Sato A., Yamamoto M., Imamura K., Kashiki Y., Kunieda T. and Sakata K. (1978) Pathophysiology of aganglionic colon and anorectum: an experimental study on aganglionosis produced by a new method in the rat. J. Ped. surg. 13, 399405. Smith B. (1972) The Neuropathology of the Alimentary Tract. Williams & Wilkins, Baltimore, MD. Suzuki N. and Prosser C. L. (1984) Interstitial cells in the intestine as pacemakers for electrical slow waves. Fedn. Proc. 43, 725 (abstract). Thuneberg L., Rumessen J. J. and Mikkelsen H. B. (1982) The interstitial cells of Cajal: intestinal pacemaker cells? In Mot&y of the Digest& Tract (Edited by M. Weinbeck), pp. 115-122. Raven Press, New York.