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Histochemical studies of experimental fetal intestinal obstruction
H i s t o c h e m i c a l Studies of Experimental Fetal Intestinal Obstruction By Laurens R. Pickard, Salvatore Santoro, Robert G. Wyllie, and J. Alex Hailer, Jr. Baltimore, Maryland 9 Experimental intestinal atresia can be produced by mesenteric disruption in fetal lambs. In previous reports, a detailed histochemical study of the bowel in this atresia model demonstrated: (1) hyperplasia of ganglion cells in the dilated proximal segment, (2) involutional changes in the area of maximal distension, (3) decreased to absent adenosine triphosphatase (ATP-ase) production in the area of the atresia, (4) gradual increase of ATP-ase production to normal proximally, and (5) greater reduction of ATPase production along the antimesenteric border compared to the mesenteric border. In the present study, a model of fetal intestinal obstruction by simple ligation of the bowel has been created to observe the effects of pure obstruction of the lumen of the fetal bowel without the possible ischemic effects of any vascular interruption. Studies with this model reveal: (1) hyperplasia of ganglion cells in the dilated proximal segment, and (2) decreased ATP-ase production proximal to the obstruction, but (3) no involutional changes in the area of maximal distension. These findings show a pattern of disturbance of bowel morphology and function caused by obstruction of the fetal bowel that is similar to but less severe than that seen with intestinal atresia. INDEX W O R D S : Intestinal atresia; fetal intestinal obstruction.
A N Y P R O B L E M S remain to be solved in the area of small bowel atresia. Among those are disparity of proximal and distal bowel size that sets the stage for anastomotic dysfunction or may necessitate staged procedures. Marked motility dysfunction and malabsorption contribute to major management problems. To study some of the motility and malabsorption abnormalities, histochemical analyses of the bowel in fetal lambs with experimental intestinal atresia produced by mesenteric vascular disrup-
M
From the Division of Pediatric Surgery, the Johns Hopkins University School of Medicine, Baltimore, Md. Presented before the 29th Annual Meeting of the Surgical Section of the American Academy of Pediatrics, Detroit, Michigan, October 27-28, 1980. Address reprint requests to J. Alex Hailer, M.D., Division of Pediatric Surgery, The Johns Hopkins University School of Medicine, Baltimore, Md. 21205. 9 1981 by Grune & Stratton, Inc. 0022-3468/81/1603-0007501.0(9/0 256
tion has been p e r f o r m e d ) 2 These studies demonstrated: (1) hyperplasia of Auerbach's plexus ganglia in the dilated proximal bowel, (2) involutional changes in the areas of maximal distension, (3) decreased to absent adenosine triphosphatase (ATP-ase) production in the area of the atresia, (4) gradual increase of ATP-ase production to normal proximally, and (5) greater reduction of ATP-ase production along the antimesenteric border compared to the mesenteric border. Other findings in these studies revealed: (1) decreased acetycholinesterase (ACHE) in ganglia, (2) decreased ATP-ase in vascular smooth muscle and endothelium, (3) abnormal concentration and distribution of ATP-ase in the villous brush border, and (4) that some of the abnormalities were noted distal to the atresia as well as proximal to the atresia. The same histochemical studies were performed in clinical cases at our institution and the findings were similarJ Although we were convinced that some of these findings were due to the adjacent ischemic injury that produced the atresia, we could not exclude the role that simple mechanical obstruction of the fetal bowel lumen might play in producing the pathologic changes seen in intestinal atresia. The following experiment was designed to study the effects of obstruction only on fetal bowel. A fetal lamb model of intestinal obstruction was created by simple ligation of the bowel leaving the mesentery completely intact. These experimental preparations were studied in the same way that the atresia models were studied. MATERIALS AND METHODS Eight fetal lambs of ewes at 90-115 days gestation (average normal gestation is about 140 days) were operated upon according to the techniques developed in our research laboratory for intrauterine fetal surgery. Surital was used for induction of anesthesia and halothane was used for maintenance. The gravid uterus was exposed through a midline abdominal incision in the ewe. Hysterotomy was performed and the fetal abdomen was exposed taking care to avoid amniotic fluid loss. A left upper quadrant abdominal incision was made in the fetus and the fetal small bowel was eviscerated. After the ligament of Treitz was identified to orient the bowel, a single catgut ligature was placed around the jejunum, taking care to avoid any mesenteric vascular disrup-
Journal of Pediatric Surgery, Vol. 16, No. 3 (June), 1981
FETAL INTESTINAL OBSTRUCTION
tion. The fetal bowel was then gently replaced in the abdomen and the fetal abdominal wall, and the ewe's uterus and abdomen were all closed in a standard fashion. When twins were present only one fetus was operated upon and the other twin served as a control. Five newborn lambs were used as controls. The lambs were delivered at term by cesarean section and were sacrificed. Full circumference biopsies of the intestinal wall were obtained at the level of the proximal stump, 10 and 20 cm proximal to the intestinal obstruction and at level of the distal stump, 10 and 20 cm distally. The tissue samples were fresh frozen at minus 60~ in methyl butane and ice. Fresh frozen sections were cut at 6 #m on IEC model CTH cryostat. The ACHE activity of the autonomic ganglia of the intestinal wall was investigated according to Gerebtzoff's technique. 3 The ATP-ase activity of the villous brush border and the intestinal wall smooth muscle was studied by Wachstein's method. 4 RESULTS
All eight ewes operated upon survived the surgical procedures and all but two of the fetuses were delivered by C-section. The two fetuses born spontaneously died shortly after delivery and were excluded. All fetuses had marked abdominal distension and dilatation of the bowel proximal to the ligature (Fig. 1). Bowel speci-
257
Table 1. Summary of Histochemical Findings in Experimental Intestinal Obstruction Compared to Experimental Intestinal Atresia Atresia
Obstruction
Proximal hypertrophy of bowel wall muscle
-I-
+
ATP-ase in proximal bowel wall muscle
~
ATP-ase in vascular smooth muscle and endothelium Hyperplasia of proximal Auerbach's ganglia
~ +
+
ACHE in proximal ganglia Involutional changes in area of maximal distension
~ +
0
Abnormalities of villous brush border ATP-ase Distal abnormalities
+ +
0 0
+ -- Present, 0 = not present, ~ = diminished.
mens for histochemical studies were obtained from the six lambs sacrificed after C-section. The histochemical findings are summarized in Table 1. The findings in the obstruction model that were similar to the atresia model were: (1) proximal hypertrophy of bowel wall muscle, (2) reduced ATP-ase production in the proximal bowel wall muscle (Fig. 2), and (3) hyperplasia of proximal Auerbach's plexus ganglia (Fig. 3). Changes in the obstruction model that were similar to the atresia model but to a lesser degree were: (1) reduction in ATP-ase production in vascular smooth muscle and endothelium, and (2) reduction in A C H E in proximal ganglia (Fig. 3). Some findings in the obstruction model were distinctly different from the findings in the atresia model. Concentration and distribution of ATP-ase in the proximal villous brush border was normal (Fig. 4). Dilated and tortuous blood vessels and dilated lymphatics were prominent in the obstruction model but not in the atresia model. In the atresia model, enzyme depletion was noted to be more marked on the antimesenteric border than on the mesenteric border but this differential was only mild in the obstruction model. The intestine distal to the ligation did not show any difference at all from the normal pattern in the obstruction model (Figs. 3 and 5). DISCUSSION
Fig. 1. Appearance of lamb small bowel (jejunum) at term after intrauterine ligation (arrow).
The present experimental model of intestinal obstruction was designed primarily to investigate the role played by the increasing intraluminal
258
PICKARD ET AL.
Fig. 2. Bowel ATP-ase stain: (A) control; (B) 10 cm proximal to obstruction, decreased ATP-ase content; (C) 5 cm proximal to obstruction showing further decrease of ATP-asa content approaching obstruction point.
pressure and dilatation in the intestine proximal to an atresia in causing disturbance of bowel morphology and function. By the application of a ligature around the intestine taking great care to avoid any damage of mesenteric vessels, we assume that the pathologic changes were mainly related to the mechanical obstruction. Our findings demonstrated a disturbance of bowel morphology in experimental fetal mechanical obstruction that is similar to but less marked than that seen with experimental intestinal atresia. The differences between the atresia model and the obstruction model may result from residual ischemic injury to the adjacent bowel following the production of the atresia.
The greatest changes in both models have been observed in the segment immediately above the obstruction, where a pronounced hyperplasia of the ganglion cells of Auerbach plexus and a reduction of the normal content of A C H E enzyme occurred. Those changes that were similar in both models were thought to be primarily related to the consequences of mechanical obstruction. Mechanical obstruction produces: (1) reactive hypertrophy of the bowel muscle in an attempt to overcome the obstruction, and (2) concomitant hyperplasia of ganglia. As the obstruction is not relieved, hypertrophy continues and contractile efforts continue until enzymatic mediator depletion occurs when this
Fig. 3. Bowel ACHE stain: (A) 5 cm distal to obstruction exhibits no ganglia hyperplasia and normal ACHE content; (B) 20 cm proximal to obstruction, with hypertrophied ganglion cells; (C) 10 cm proximal to obstruction with hyperplasia of ganglion cells but reduced ACHE.
FETAL INTESTINAL OBSTRUCTION
259
Fig. 4. ATP-ase stain of villous brush border: (A) proximal to obstruction showing normal pattern; (B) abnormal concentration and distribution of ATP-ase as seen in atresia model.
Fig. 5. ATP-ase stain: (A) control; (B) 5 cm distal to obstruction showing normal pattern; (C) 5 cm distal to atresia showing reduction of ATP-ase.
260
PICKARD ET AL.
neuromuscular hyperactivity effect exceeds the organ's ability to adapt as proposed by Tepas et al. l In general, all of the derangements that were similar in quality seemed to be less severe in degree in the obstruction model than in the atresia model. The most distinctly less severe were: (1) reduction in ATP-ase production in vascular smooth muscle and endothelium, and (2) reduction in A C H E in proximal ganglia. We can hypothesize that most of the derangements that are similar in quality in both models are due to mechanical obstruction but that successful compensatory adaptation lasts longer without the atresia-producing ischemia before mediator depletion begins. The most prominent differences between these two models were: (1) absence of involutional changes in the obstruction model, and (2) absence of villous brush border changes of abnormal ATP-ase concentration and distribution in the area of maximal distension (Fig. 4). Involutional changes in the area of maximal distension, as seen in the atresia model, were not observed in the obstruction model, although some atrophy of the submucosal ganglia was observed in the obstruction model. Involutional changes are another derangement of the bowel morphology and function in congenital atresia that may be a sequelae of the primary etiologic ischemia of the atresia. The ability for the obstructed-only bowel to adapt somewhat better than atretic bowel may be accounted for by
better vascular adaptation suggested by the presence of more dilated and tortuous blood vessels in obstructed-only bowel. The distal bowel showed evidence of injury in the atresia model that is again most likely related to direct ischemic injury since the distal bowel in the obstruction model was normal except for smallness due to nonuse. The reason for less enzyme depletion differential between mesenteric and antimesenteric borders in the obstruction model as compared to the atresia model is unclear. These studies in experimentally obstructed fetal intestine may find analogy in human newborn intestinal obstruction from causes other than atresia as the analogy between experimental and human intestinal atresia has been demonstrated. CONCLUSION
( l ) This study suggests that the effect of mechanical obstruction of atretic bowel caused by obliteration of the lumen of the bowel during fetal life may account for most of the abnormalities of function of the bowel that may occur after correction of the atresia. (2) There is some evidence now, however, that the etiologic ischemia of the atresia produces damage both proximal and distal to the obstruction over-andabove the damage related to obstruction. (3) The changes shown in this study may account for some abnormalities of bowel function observed in conditions presenting as newborn bowel obstruction other than atresia.
REFERENCES
1. Tepas J J, Wyllie RG, Shermeta DW, et al: Comparison of histochemical studies of intestinal atresia in the human newborn and fetal lamb. J Pediatr Surg 14:376-380, 1979 2. Inon A: Personal communication 3. Gerebtzoff MA: Recherches histochimiques sur les
acetylcholine et choline esterases. I. Introduction et technique. Acta Anat 19:366-379, 1953 4. Wachstein M, Meisel E: Histochemistry of hepatic phosphatases at a physiologicpH. Am J Clin Pathol 27:1323, 1957
A N Y P R O B L E M S remain to be solved in the area of small bowel atresia. Among those are disparity of proximal and distal bowel size that sets the stage for anastomotic dysfunction or may necessitate staged procedures. Marked motility dysfunction and malabsorption contribute to major management problems. To study some of the motility and malabsorption abnormalities, histochemical analyses of the bowel in fetal lambs with experimental intestinal atresia produced by mesenteric vascular disrup-
M
From the Division of Pediatric Surgery, the Johns Hopkins University School of Medicine, Baltimore, Md. Presented before the 29th Annual Meeting of the Surgical Section of the American Academy of Pediatrics, Detroit, Michigan, October 27-28, 1980. Address reprint requests to J. Alex Hailer, M.D., Division of Pediatric Surgery, The Johns Hopkins University School of Medicine, Baltimore, Md. 21205. 9 1981 by Grune & Stratton, Inc. 0022-3468/81/1603-0007501.0(9/0 256
tion has been p e r f o r m e d ) 2 These studies demonstrated: (1) hyperplasia of Auerbach's plexus ganglia in the dilated proximal bowel, (2) involutional changes in the areas of maximal distension, (3) decreased to absent adenosine triphosphatase (ATP-ase) production in the area of the atresia, (4) gradual increase of ATP-ase production to normal proximally, and (5) greater reduction of ATP-ase production along the antimesenteric border compared to the mesenteric border. Other findings in these studies revealed: (1) decreased acetycholinesterase (ACHE) in ganglia, (2) decreased ATP-ase in vascular smooth muscle and endothelium, (3) abnormal concentration and distribution of ATP-ase in the villous brush border, and (4) that some of the abnormalities were noted distal to the atresia as well as proximal to the atresia. The same histochemical studies were performed in clinical cases at our institution and the findings were similarJ Although we were convinced that some of these findings were due to the adjacent ischemic injury that produced the atresia, we could not exclude the role that simple mechanical obstruction of the fetal bowel lumen might play in producing the pathologic changes seen in intestinal atresia. The following experiment was designed to study the effects of obstruction only on fetal bowel. A fetal lamb model of intestinal obstruction was created by simple ligation of the bowel leaving the mesentery completely intact. These experimental preparations were studied in the same way that the atresia models were studied. MATERIALS AND METHODS Eight fetal lambs of ewes at 90-115 days gestation (average normal gestation is about 140 days) were operated upon according to the techniques developed in our research laboratory for intrauterine fetal surgery. Surital was used for induction of anesthesia and halothane was used for maintenance. The gravid uterus was exposed through a midline abdominal incision in the ewe. Hysterotomy was performed and the fetal abdomen was exposed taking care to avoid amniotic fluid loss. A left upper quadrant abdominal incision was made in the fetus and the fetal small bowel was eviscerated. After the ligament of Treitz was identified to orient the bowel, a single catgut ligature was placed around the jejunum, taking care to avoid any mesenteric vascular disrup-
Journal of Pediatric Surgery, Vol. 16, No. 3 (June), 1981
FETAL INTESTINAL OBSTRUCTION
tion. The fetal bowel was then gently replaced in the abdomen and the fetal abdominal wall, and the ewe's uterus and abdomen were all closed in a standard fashion. When twins were present only one fetus was operated upon and the other twin served as a control. Five newborn lambs were used as controls. The lambs were delivered at term by cesarean section and were sacrificed. Full circumference biopsies of the intestinal wall were obtained at the level of the proximal stump, 10 and 20 cm proximal to the intestinal obstruction and at level of the distal stump, 10 and 20 cm distally. The tissue samples were fresh frozen at minus 60~ in methyl butane and ice. Fresh frozen sections were cut at 6 #m on IEC model CTH cryostat. The ACHE activity of the autonomic ganglia of the intestinal wall was investigated according to Gerebtzoff's technique. 3 The ATP-ase activity of the villous brush border and the intestinal wall smooth muscle was studied by Wachstein's method. 4 RESULTS
All eight ewes operated upon survived the surgical procedures and all but two of the fetuses were delivered by C-section. The two fetuses born spontaneously died shortly after delivery and were excluded. All fetuses had marked abdominal distension and dilatation of the bowel proximal to the ligature (Fig. 1). Bowel speci-
257
Table 1. Summary of Histochemical Findings in Experimental Intestinal Obstruction Compared to Experimental Intestinal Atresia Atresia
Obstruction
Proximal hypertrophy of bowel wall muscle
-I-
+
ATP-ase in proximal bowel wall muscle
~
ATP-ase in vascular smooth muscle and endothelium Hyperplasia of proximal Auerbach's ganglia
~ +
+
ACHE in proximal ganglia Involutional changes in area of maximal distension
~ +
0
Abnormalities of villous brush border ATP-ase Distal abnormalities
+ +
0 0
+ -- Present, 0 = not present, ~ = diminished.
mens for histochemical studies were obtained from the six lambs sacrificed after C-section. The histochemical findings are summarized in Table 1. The findings in the obstruction model that were similar to the atresia model were: (1) proximal hypertrophy of bowel wall muscle, (2) reduced ATP-ase production in the proximal bowel wall muscle (Fig. 2), and (3) hyperplasia of proximal Auerbach's plexus ganglia (Fig. 3). Changes in the obstruction model that were similar to the atresia model but to a lesser degree were: (1) reduction in ATP-ase production in vascular smooth muscle and endothelium, and (2) reduction in A C H E in proximal ganglia (Fig. 3). Some findings in the obstruction model were distinctly different from the findings in the atresia model. Concentration and distribution of ATP-ase in the proximal villous brush border was normal (Fig. 4). Dilated and tortuous blood vessels and dilated lymphatics were prominent in the obstruction model but not in the atresia model. In the atresia model, enzyme depletion was noted to be more marked on the antimesenteric border than on the mesenteric border but this differential was only mild in the obstruction model. The intestine distal to the ligation did not show any difference at all from the normal pattern in the obstruction model (Figs. 3 and 5). DISCUSSION
Fig. 1. Appearance of lamb small bowel (jejunum) at term after intrauterine ligation (arrow).
The present experimental model of intestinal obstruction was designed primarily to investigate the role played by the increasing intraluminal
258
PICKARD ET AL.
Fig. 2. Bowel ATP-ase stain: (A) control; (B) 10 cm proximal to obstruction, decreased ATP-ase content; (C) 5 cm proximal to obstruction showing further decrease of ATP-asa content approaching obstruction point.
pressure and dilatation in the intestine proximal to an atresia in causing disturbance of bowel morphology and function. By the application of a ligature around the intestine taking great care to avoid any damage of mesenteric vessels, we assume that the pathologic changes were mainly related to the mechanical obstruction. Our findings demonstrated a disturbance of bowel morphology in experimental fetal mechanical obstruction that is similar to but less marked than that seen with experimental intestinal atresia. The differences between the atresia model and the obstruction model may result from residual ischemic injury to the adjacent bowel following the production of the atresia.
The greatest changes in both models have been observed in the segment immediately above the obstruction, where a pronounced hyperplasia of the ganglion cells of Auerbach plexus and a reduction of the normal content of A C H E enzyme occurred. Those changes that were similar in both models were thought to be primarily related to the consequences of mechanical obstruction. Mechanical obstruction produces: (1) reactive hypertrophy of the bowel muscle in an attempt to overcome the obstruction, and (2) concomitant hyperplasia of ganglia. As the obstruction is not relieved, hypertrophy continues and contractile efforts continue until enzymatic mediator depletion occurs when this
Fig. 3. Bowel ACHE stain: (A) 5 cm distal to obstruction exhibits no ganglia hyperplasia and normal ACHE content; (B) 20 cm proximal to obstruction, with hypertrophied ganglion cells; (C) 10 cm proximal to obstruction with hyperplasia of ganglion cells but reduced ACHE.
FETAL INTESTINAL OBSTRUCTION
259
Fig. 4. ATP-ase stain of villous brush border: (A) proximal to obstruction showing normal pattern; (B) abnormal concentration and distribution of ATP-ase as seen in atresia model.
Fig. 5. ATP-ase stain: (A) control; (B) 5 cm distal to obstruction showing normal pattern; (C) 5 cm distal to atresia showing reduction of ATP-ase.
260
PICKARD ET AL.
neuromuscular hyperactivity effect exceeds the organ's ability to adapt as proposed by Tepas et al. l In general, all of the derangements that were similar in quality seemed to be less severe in degree in the obstruction model than in the atresia model. The most distinctly less severe were: (1) reduction in ATP-ase production in vascular smooth muscle and endothelium, and (2) reduction in A C H E in proximal ganglia. We can hypothesize that most of the derangements that are similar in quality in both models are due to mechanical obstruction but that successful compensatory adaptation lasts longer without the atresia-producing ischemia before mediator depletion begins. The most prominent differences between these two models were: (1) absence of involutional changes in the obstruction model, and (2) absence of villous brush border changes of abnormal ATP-ase concentration and distribution in the area of maximal distension (Fig. 4). Involutional changes in the area of maximal distension, as seen in the atresia model, were not observed in the obstruction model, although some atrophy of the submucosal ganglia was observed in the obstruction model. Involutional changes are another derangement of the bowel morphology and function in congenital atresia that may be a sequelae of the primary etiologic ischemia of the atresia. The ability for the obstructed-only bowel to adapt somewhat better than atretic bowel may be accounted for by
better vascular adaptation suggested by the presence of more dilated and tortuous blood vessels in obstructed-only bowel. The distal bowel showed evidence of injury in the atresia model that is again most likely related to direct ischemic injury since the distal bowel in the obstruction model was normal except for smallness due to nonuse. The reason for less enzyme depletion differential between mesenteric and antimesenteric borders in the obstruction model as compared to the atresia model is unclear. These studies in experimentally obstructed fetal intestine may find analogy in human newborn intestinal obstruction from causes other than atresia as the analogy between experimental and human intestinal atresia has been demonstrated. CONCLUSION
( l ) This study suggests that the effect of mechanical obstruction of atretic bowel caused by obliteration of the lumen of the bowel during fetal life may account for most of the abnormalities of function of the bowel that may occur after correction of the atresia. (2) There is some evidence now, however, that the etiologic ischemia of the atresia produces damage both proximal and distal to the obstruction over-andabove the damage related to obstruction. (3) The changes shown in this study may account for some abnormalities of bowel function observed in conditions presenting as newborn bowel obstruction other than atresia.
REFERENCES
1. Tepas J J, Wyllie RG, Shermeta DW, et al: Comparison of histochemical studies of intestinal atresia in the human newborn and fetal lamb. J Pediatr Surg 14:376-380, 1979 2. Inon A: Personal communication 3. Gerebtzoff MA: Recherches histochimiques sur les
acetylcholine et choline esterases. I. Introduction et technique. Acta Anat 19:366-379, 1953 4. Wachstein M, Meisel E: Histochemistry of hepatic phosphatases at a physiologicpH. Am J Clin Pathol 27:1323, 1957