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Interstitial cells of Cajal in the rat colon are damaged by mild hypoxia
Journal of the Autonomic Nervous System ELSEVIER
Journal of the Autonomic Nervous System 48 (1994) 175-180
Interstitial cells of Cajal in the rat colon are damaged by mild hypoxia James
Christensen,
Gary A. Rick
Gastroenterology Research Laboratories, Room 4548, JCP, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA
(Received 5 May 1993; revision received and accepted 3 November 1993)
Abstract We examined the hypothesis that vacuolation is a normal feature of interstitial cells of Cajal. With the zinc iodide-osmic acid stain, and using light microscopy, we examined the interstitial cells in Stach's plexus on the submucosal surface of the circular muscle layer of the colon in the rat. We subjected tissues to seven treatments designed to produce different degrees of damage from hypoxia. In the least-damaged colons (fixed immediately after dissection) about 20% of interstitial cells showed vacuoles. In the most damaged colons (immersed for 30 min in Krebs' solution equilibrated with 95% N2/5% CO 2 at 37°C) about 96% of cells showed vacuoles. Less drastic treatments produced fewer vacuoles, but even colons immersed for 30 min in Krebs' solution equilibrated with 95% 0 2 / 5 % CO 2 at 37°C showed vacuolation in about 28% of cells. The light microscopic impression was confirmed by electronmicroscopy, but cells subjected to even the most severe treatment retained a recognizable ultrastructure. Vacuolation in interstitial cells is probably artifactual, a consequence of hypoxia. Key words: Smooth muscle; Enteric nervous system; Myenteric plexus; Colonic motility
1. Introduction T h e p o l y m o r p h i c cells a s s o c i a t e d with axons in g a s t r o i n t e s t i n a l m u s c u l a t u r e , which C a j a l c a l l e d " i n t e r s t i t i a l cells", now a t t r a c t a r e n e w e d n o t i c e b e c a u s e t h e y s e e m to h e l p to p a c e r h y t h m i c cont r a c t i o n s [1]. V a r i o u s i n v e s t i g a t o r s d e s c r i b e d vacu o l e s as p a r t o f t h e n o r m a l u l t r a s t r u c t u r e of i n t e r s t i t i a l cells in s t o m a c h , small i n t e s t i n e a n d colon [2-7]. V a c u o l e s c o u l d result f r o m cell insult
* Corresponding author. Tel.: 319-356-2670; Fax: 319-3564552.
from, for e x a m p l e , hypoxia. W e set o u t to see if m i l d h y p o x i a c o u l d a c c o u n t for t h e v a c u o l a t i o n v a r i a b l y d e s c r i b e d as a p a r t o f t h e n o r m a l struct u r e of i n t e r s t i t i a l cells, o r if v a c u o l e s s h o u l d be c o n s i d e r e d a truly n o r m a l f e a t u r e o f t h e cells.
2. Materials and methods M a t u r e rats ( > 150 gm) o f e i t h e r sex w e r e e x p o s e d to e t h e r until b r e a t h i n g just c e a s e d . T h e colon of e a c h was quickly r e m o v e d , o p e n e d , w a s h e d , a n d p u t into K r e b s ' s o l u t i o n e q u i l i b r a t e d with 95% 0 2 / 5 % C O 2 at 37 ° . A 1 cm s e g m e n t
0165-1838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-1838(93)E0141-Q
176
J. Christensen, G.A. Rick/Journal of the Autonomic Nerl'~ous System 48 (1994) 175-180
Z Christensen, G.A. Rick/Journal of the Autonomic Nervous System 48 (1994) 175-180
was taken from the mid-point of each colon, where the interstitial cells are most dense in the rat [8] and subjected to one of the following seven treatments (three rats in each treatment): (1) immediate fixation in 2% p a r a f o r m a l d e h y d e / 2.5% glutaraldehyde in 0.2 M phosphate buffer before initiation of the zinc iodide osmic acid (ZIO) stain; (2) immediate initiation of the ZIO stain without antecedent fixation; (3) incubation in Krebs' solution equilibrated with 95% 0 2 / 5 % CO 2 for 30 min before proceeding to the ZIO stain; (4) incubation in Krebs' solution, as above, for 60 min before ZIO staining; (5) incubation for 30 min in Krebs' solution equilibrated with 95% N 2 / 5 % CO 2 before ZIO staining; (6) devascularization and separation by ligature from the rest of the viscera but left in the abdominal cavity for 5 min before ZIO staining; and (7) devascularization and separation by ligature from the rest of the viscera but left in the abdominal cavity for 10 min before ZIO staining. These seven treatments were considered to constitute a graded series of hypoxic traumas which should indicate, through a graded degree of vacuole formation in response, whether vacuolation is wholly or partly artifactual in interstitial cells, a result of hypoxia. The ZIO staining method [9] involves slow fixation by osmic acid, a possible source of vacuolation which the first treatment, immediate fixation in Karnovsky's fixative, was meant to overcome. After the staining, the specimens were cut into 30/z serial tangential sections with a freezing sledge microtome, mounted in glycerine-gelatin, and examined by light microscopy. Three specimens were carried through each of the seven treatments. The observer examined the dense mat of interstitial cells on the submucosal surface of the circular muscle layer [9] where, together with a network of axons, they form the plexus described by Stach. Here, the observer (who was blinded)
177
counted at least 500 cells in each specimen, determining the proportion with vacuolation. In this process, he considered any visualized vacuole enough reason to call the cell a vacuolated cell. That is, size or number of vacuoles was not taken into account. Other studies were done to assess the effect by electronmicroscopy. Here, the tissues were subjected only to three of the treatments, immediate fixation, fixation after 30 min in Krebs' solution equilibrated with 95% 0 2 / 5 % CO 2 at 37 °, and fixation after 30 min in Krebs' solution equilibrated with 95% N 2 / 5 % CO 2 for 30 min. After fixation overnight in 2.5% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.2), specimens received three 20-min washes in 0.1 M cacodylate buffer and were immersed in 1% OsO 4 in 0.1 M cacodylate buffer containing 1.5% potassium ferricyanide for 1.5 h. After three 20-min rinses in 0.1 M cacodylate buffer and one 20-min wash in distilled water, they were exposed to 2.5% uranyl acetate in distilled water for 20 min, dehydrated in an alcohol series, put in 100% propylene oxide for 10 min, and embedded in Spurr's medium. Sections 60 to 100 nm thick were cut with a Reichert microtome, mounted on grids, and stained with uranyl acetate (10 min) and lead citrate (8 min) before examination on a Hitachi H-600 microscope.
3. Results
Light microscopy showed a major effect of hypoxia on the vacuolation of the interstitial cells. In tissues stained after immediate fixation in dilute Karnovsky's fixative, the proportion of cells with vacuoles was 19.9 + 5.2%. In these ceils, we saw only one or two very small vacuoles in the vacuolated cells. With the unavoidable delay involved in the slow fixation of the ZIO stain, the
Fig. 1. (A) A view of interstitial cells of Stach's plexus studied after prompt fixation to minimize hypoxic damage showing interstitial cells (arrows) and axons (a) stained black by the ZIO stain, and the pale-stained circular muscle (cm). Vacuolation of interstitial cells is not apparent here. The bar indicates 10/x. (B) A view of interstitial cells of Stach's plexus after 30 min exposure to Krebs' solution equilibrated with 95% 0 2 / 5 % CO 2 showing interstitial cells and axons (a) stained by the ZIO stain. The circular muscle (cm) is unstained. Many interstitial cells show vacuoles, some being quite large (arrows). The bar represents 10 ~.
I
~L
J. Christensen, G.A. Rick/Journal of the Autonomic Nervous System 48 (1994) 175-180
proportion of vacuolated cells rose to 27.8 _+ 13.7%. After incubation in oxygenated Krebs' solution for 30 min, the proportion was 50.7 +_ 8.7%, and after the same incubation for 60 min it was 34.6 _+ 12.0%. Incubation in equilibrated Krebs' solution with nitrogen replacing oxygen produced vacuoles in 96.5 _+ 2.3% of the cells, more than occurred in the segments which were devascularized for 5 min (63.1 _+ 9.5%) and for 10 min (64.4 + 16.5%). Examples of these interstitial cells, seen at light-microscopy, appear in Figs. 1A (immediate fixation), 1B (after 30 min incubation in oxygenated Krebs' solution), and 2A (after 30 min incubation in nitrogenated Krebs' solution). The increased proportion of cells with vacuoles was accompanied by an obvious increase in size of vacuoles and n u m b e r of vacuoles per cell. At all sizes and with all treatments, vacuoles showed no structural details beyond those evident in the illustrations. That is, we could not see any features to suggest their origin in mitochondria, endoplasmic reticulum, or Golgi structures. At electronmicroscopy the interstitial cells showed a few small vacuoles in the most rapidlyfixed tissues. Somewhat more vacuolation was apparent after 30 min incubation in oxygenated Krebs' solution, but even after 30 min incubation in nitrogenated Krebs' solution (Fig. 2B), the ultrastructure of the interstitial cells was recognizable.
4. Discussion Since vacuolation of interstitial ceils increased with increasing degrees of hypoxic insult, we conclude that vacuolation does not occur in normal interstitial cells but rather appears as a manifestation of the slight hypoxia induced in the usual preparation of tissues. Even the slight vacuolation of the cells in the tissues that were fixed immediately after dissection can be considered to be
179
artifactual since the sacrifice of the animals with ether and the time required for dissection involves an inevitable period of hypoxia. Though the interstitial cells seem to be sensitive in responding to hypoxia by forming vacuoles, they must still be capable of function because they exhibit their pacemaking function in vitro in oxygenated Krebs' solution for long periods. They may not function wholly normally, though. Anuras et al. [10] recording the slow waves from the cat colon in vitro, observed that slow wave frequency and amplitude diminished by about half when the colon was exposed to Krebs' solution in which nitrogen replaced oxygen in the equilibrating gas, but values reached these levels only after about 45 min, after which they were stable for 3 h. The depression of frequency and amplitude was immediately reversed with the reintroduction of oxygen. Dinitrophenol, like oxygen, reduced colonic slow wave frequency to about half with some reversal of effect after washing. The ease with which interstitial cells form vacuoles under conditions used in normal laboratory study of living tissues should warn investigators of over-interpretation of vacuolation. A recent report of the successful microelectrode penetration of an interstitial cell in vitro [11], for example, was possibly based on the misinterpretation of a vacuole as a puncture-hole. Either of two conclusions is possible about the discordance of the physiology and the morphology. First, the interstitial cells may retain the ability to generate pacemaking slow waves even after they have suffered considerable vacuole formation. Or, second, the interstitial cells may not be the sole source of the slow waves.
Acknowledgments Supported by Research G r a n t DK-11242 from the NIH.
Fig. 2. Interstitial cells of Stach's plexus after 30 min exposure to Krebs' solution equilibrated with 95% N2/5% C O 2. (A) A view of Stach's plexus en face showing some axons (a) and heavy vacuolation of interstitial cells (arrows). The bar indicates 10 tz. (B) An electron photomicrograph showing an interstitial cell with many large vacuoles (arrows) overlyingseverely damaged circular muscle (cm). The bar indicates 1 /x. 'sm' indicates the submucosa.
180
J. Christensen, G.A. Rick/Journal of the Autonomic Nervous System 48 (1994) 175-180
References [1] Christensen, J., Commentary on the morphological identification of the interstitial cells of Cajal, J. Auton. Nerv. Syst., 37 (1992) 75-88. [2] Imaizumi, M. and Hama, K., An electron microscopic study on the interstitial cells of the gizzard in the love-bird (Uroloncha domestica), Z. Zellforsch., 97 (1969) 351-357. [3] Richardson, K.C., Electron microscopic observation on Auerbach's plexus in the rabbit, with special reference to the problem of smooth muscle innervation, Am. J. Anat., 103 (1958) 99-136. [4] Taxi, J., Sur la structure des trav~es du plexus d'Auerbach: confrontation des donn~es fournies par le microscope ordinaire et par le microscope filectronique, Ann. Sci. Natl. Zool., 1 (1959) 571-593. [5] Rogers, D.C. and Burnstock, G., The interstitial cell and its place in the concept of the autonomic ground plexus, J. Comp. Neurol., 126 (1966) 255-284. [6] Komuro, T., Three-dimensional observation of the fi-
[7] [8]
[9]
[10]
[11]
broblast-like cells associated with the rat myenteric plexus, with special reference to the interstitial cells of Cajal, Cell Tissue Res., 255 (1989) 343-351. Komuro, T., The interstitial cells in the colon of the rabbit, Cell Tissue Res., 222 (1982) 41-51. Christensen, J., Rick, G.A. and Lowe, L.S., Distributions of interstitial cells of Cajal in stomach and colon of cat, dog, ferret, opossum, rat, guinea pig and rabbit, J. Auton. Nerv. Syst., 37 (1992) 47-56. Christensen, J. and Rick, G.A., Intrinsic nerves in the mammalian colon: confirmation of a plexus at the circular muscle-submucosal interface, J. Auton. Nerv. Syst., 21 (1987) 223-231. Anuras, S., Chien, S.M. and Christensen, J., Metabolic dependence of the electromyogram of the cat colon, Am. J. Physiol., 239 (1980) G173-G176. Barajas-Lopez, C., Berezin, I., Daniel, E.E. and Huizinga, J.D., Pacemaker activity recorded in interstitial cells of Cajal of the gastrointestinal tract, Am. J. Physiol., 257 (1989) C830-C835.
Journal of the Autonomic Nervous System 48 (1994) 175-180
Interstitial cells of Cajal in the rat colon are damaged by mild hypoxia James
Christensen,
Gary A. Rick
Gastroenterology Research Laboratories, Room 4548, JCP, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA
(Received 5 May 1993; revision received and accepted 3 November 1993)
Abstract We examined the hypothesis that vacuolation is a normal feature of interstitial cells of Cajal. With the zinc iodide-osmic acid stain, and using light microscopy, we examined the interstitial cells in Stach's plexus on the submucosal surface of the circular muscle layer of the colon in the rat. We subjected tissues to seven treatments designed to produce different degrees of damage from hypoxia. In the least-damaged colons (fixed immediately after dissection) about 20% of interstitial cells showed vacuoles. In the most damaged colons (immersed for 30 min in Krebs' solution equilibrated with 95% N2/5% CO 2 at 37°C) about 96% of cells showed vacuoles. Less drastic treatments produced fewer vacuoles, but even colons immersed for 30 min in Krebs' solution equilibrated with 95% 0 2 / 5 % CO 2 at 37°C showed vacuolation in about 28% of cells. The light microscopic impression was confirmed by electronmicroscopy, but cells subjected to even the most severe treatment retained a recognizable ultrastructure. Vacuolation in interstitial cells is probably artifactual, a consequence of hypoxia. Key words: Smooth muscle; Enteric nervous system; Myenteric plexus; Colonic motility
1. Introduction T h e p o l y m o r p h i c cells a s s o c i a t e d with axons in g a s t r o i n t e s t i n a l m u s c u l a t u r e , which C a j a l c a l l e d " i n t e r s t i t i a l cells", now a t t r a c t a r e n e w e d n o t i c e b e c a u s e t h e y s e e m to h e l p to p a c e r h y t h m i c cont r a c t i o n s [1]. V a r i o u s i n v e s t i g a t o r s d e s c r i b e d vacu o l e s as p a r t o f t h e n o r m a l u l t r a s t r u c t u r e of i n t e r s t i t i a l cells in s t o m a c h , small i n t e s t i n e a n d colon [2-7]. V a c u o l e s c o u l d result f r o m cell insult
* Corresponding author. Tel.: 319-356-2670; Fax: 319-3564552.
from, for e x a m p l e , hypoxia. W e set o u t to see if m i l d h y p o x i a c o u l d a c c o u n t for t h e v a c u o l a t i o n v a r i a b l y d e s c r i b e d as a p a r t o f t h e n o r m a l struct u r e of i n t e r s t i t i a l cells, o r if v a c u o l e s s h o u l d be c o n s i d e r e d a truly n o r m a l f e a t u r e o f t h e cells.
2. Materials and methods M a t u r e rats ( > 150 gm) o f e i t h e r sex w e r e e x p o s e d to e t h e r until b r e a t h i n g just c e a s e d . T h e colon of e a c h was quickly r e m o v e d , o p e n e d , w a s h e d , a n d p u t into K r e b s ' s o l u t i o n e q u i l i b r a t e d with 95% 0 2 / 5 % C O 2 at 37 ° . A 1 cm s e g m e n t
0165-1838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-1838(93)E0141-Q
176
J. Christensen, G.A. Rick/Journal of the Autonomic Nerl'~ous System 48 (1994) 175-180
Z Christensen, G.A. Rick/Journal of the Autonomic Nervous System 48 (1994) 175-180
was taken from the mid-point of each colon, where the interstitial cells are most dense in the rat [8] and subjected to one of the following seven treatments (three rats in each treatment): (1) immediate fixation in 2% p a r a f o r m a l d e h y d e / 2.5% glutaraldehyde in 0.2 M phosphate buffer before initiation of the zinc iodide osmic acid (ZIO) stain; (2) immediate initiation of the ZIO stain without antecedent fixation; (3) incubation in Krebs' solution equilibrated with 95% 0 2 / 5 % CO 2 for 30 min before proceeding to the ZIO stain; (4) incubation in Krebs' solution, as above, for 60 min before ZIO staining; (5) incubation for 30 min in Krebs' solution equilibrated with 95% N 2 / 5 % CO 2 before ZIO staining; (6) devascularization and separation by ligature from the rest of the viscera but left in the abdominal cavity for 5 min before ZIO staining; and (7) devascularization and separation by ligature from the rest of the viscera but left in the abdominal cavity for 10 min before ZIO staining. These seven treatments were considered to constitute a graded series of hypoxic traumas which should indicate, through a graded degree of vacuole formation in response, whether vacuolation is wholly or partly artifactual in interstitial cells, a result of hypoxia. The ZIO staining method [9] involves slow fixation by osmic acid, a possible source of vacuolation which the first treatment, immediate fixation in Karnovsky's fixative, was meant to overcome. After the staining, the specimens were cut into 30/z serial tangential sections with a freezing sledge microtome, mounted in glycerine-gelatin, and examined by light microscopy. Three specimens were carried through each of the seven treatments. The observer examined the dense mat of interstitial cells on the submucosal surface of the circular muscle layer [9] where, together with a network of axons, they form the plexus described by Stach. Here, the observer (who was blinded)
177
counted at least 500 cells in each specimen, determining the proportion with vacuolation. In this process, he considered any visualized vacuole enough reason to call the cell a vacuolated cell. That is, size or number of vacuoles was not taken into account. Other studies were done to assess the effect by electronmicroscopy. Here, the tissues were subjected only to three of the treatments, immediate fixation, fixation after 30 min in Krebs' solution equilibrated with 95% 0 2 / 5 % CO 2 at 37 °, and fixation after 30 min in Krebs' solution equilibrated with 95% N 2 / 5 % CO 2 for 30 min. After fixation overnight in 2.5% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.2), specimens received three 20-min washes in 0.1 M cacodylate buffer and were immersed in 1% OsO 4 in 0.1 M cacodylate buffer containing 1.5% potassium ferricyanide for 1.5 h. After three 20-min rinses in 0.1 M cacodylate buffer and one 20-min wash in distilled water, they were exposed to 2.5% uranyl acetate in distilled water for 20 min, dehydrated in an alcohol series, put in 100% propylene oxide for 10 min, and embedded in Spurr's medium. Sections 60 to 100 nm thick were cut with a Reichert microtome, mounted on grids, and stained with uranyl acetate (10 min) and lead citrate (8 min) before examination on a Hitachi H-600 microscope.
3. Results
Light microscopy showed a major effect of hypoxia on the vacuolation of the interstitial cells. In tissues stained after immediate fixation in dilute Karnovsky's fixative, the proportion of cells with vacuoles was 19.9 + 5.2%. In these ceils, we saw only one or two very small vacuoles in the vacuolated cells. With the unavoidable delay involved in the slow fixation of the ZIO stain, the
Fig. 1. (A) A view of interstitial cells of Stach's plexus studied after prompt fixation to minimize hypoxic damage showing interstitial cells (arrows) and axons (a) stained black by the ZIO stain, and the pale-stained circular muscle (cm). Vacuolation of interstitial cells is not apparent here. The bar indicates 10/x. (B) A view of interstitial cells of Stach's plexus after 30 min exposure to Krebs' solution equilibrated with 95% 0 2 / 5 % CO 2 showing interstitial cells and axons (a) stained by the ZIO stain. The circular muscle (cm) is unstained. Many interstitial cells show vacuoles, some being quite large (arrows). The bar represents 10 ~.
I
~L
J. Christensen, G.A. Rick/Journal of the Autonomic Nervous System 48 (1994) 175-180
proportion of vacuolated cells rose to 27.8 _+ 13.7%. After incubation in oxygenated Krebs' solution for 30 min, the proportion was 50.7 +_ 8.7%, and after the same incubation for 60 min it was 34.6 _+ 12.0%. Incubation in equilibrated Krebs' solution with nitrogen replacing oxygen produced vacuoles in 96.5 _+ 2.3% of the cells, more than occurred in the segments which were devascularized for 5 min (63.1 _+ 9.5%) and for 10 min (64.4 + 16.5%). Examples of these interstitial cells, seen at light-microscopy, appear in Figs. 1A (immediate fixation), 1B (after 30 min incubation in oxygenated Krebs' solution), and 2A (after 30 min incubation in nitrogenated Krebs' solution). The increased proportion of cells with vacuoles was accompanied by an obvious increase in size of vacuoles and n u m b e r of vacuoles per cell. At all sizes and with all treatments, vacuoles showed no structural details beyond those evident in the illustrations. That is, we could not see any features to suggest their origin in mitochondria, endoplasmic reticulum, or Golgi structures. At electronmicroscopy the interstitial cells showed a few small vacuoles in the most rapidlyfixed tissues. Somewhat more vacuolation was apparent after 30 min incubation in oxygenated Krebs' solution, but even after 30 min incubation in nitrogenated Krebs' solution (Fig. 2B), the ultrastructure of the interstitial cells was recognizable.
4. Discussion Since vacuolation of interstitial ceils increased with increasing degrees of hypoxic insult, we conclude that vacuolation does not occur in normal interstitial cells but rather appears as a manifestation of the slight hypoxia induced in the usual preparation of tissues. Even the slight vacuolation of the cells in the tissues that were fixed immediately after dissection can be considered to be
179
artifactual since the sacrifice of the animals with ether and the time required for dissection involves an inevitable period of hypoxia. Though the interstitial cells seem to be sensitive in responding to hypoxia by forming vacuoles, they must still be capable of function because they exhibit their pacemaking function in vitro in oxygenated Krebs' solution for long periods. They may not function wholly normally, though. Anuras et al. [10] recording the slow waves from the cat colon in vitro, observed that slow wave frequency and amplitude diminished by about half when the colon was exposed to Krebs' solution in which nitrogen replaced oxygen in the equilibrating gas, but values reached these levels only after about 45 min, after which they were stable for 3 h. The depression of frequency and amplitude was immediately reversed with the reintroduction of oxygen. Dinitrophenol, like oxygen, reduced colonic slow wave frequency to about half with some reversal of effect after washing. The ease with which interstitial cells form vacuoles under conditions used in normal laboratory study of living tissues should warn investigators of over-interpretation of vacuolation. A recent report of the successful microelectrode penetration of an interstitial cell in vitro [11], for example, was possibly based on the misinterpretation of a vacuole as a puncture-hole. Either of two conclusions is possible about the discordance of the physiology and the morphology. First, the interstitial cells may retain the ability to generate pacemaking slow waves even after they have suffered considerable vacuole formation. Or, second, the interstitial cells may not be the sole source of the slow waves.
Acknowledgments Supported by Research G r a n t DK-11242 from the NIH.
Fig. 2. Interstitial cells of Stach's plexus after 30 min exposure to Krebs' solution equilibrated with 95% N2/5% C O 2. (A) A view of Stach's plexus en face showing some axons (a) and heavy vacuolation of interstitial cells (arrows). The bar indicates 10 tz. (B) An electron photomicrograph showing an interstitial cell with many large vacuoles (arrows) overlyingseverely damaged circular muscle (cm). The bar indicates 1 /x. 'sm' indicates the submucosa.
180
J. Christensen, G.A. Rick/Journal of the Autonomic Nervous System 48 (1994) 175-180
References [1] Christensen, J., Commentary on the morphological identification of the interstitial cells of Cajal, J. Auton. Nerv. Syst., 37 (1992) 75-88. [2] Imaizumi, M. and Hama, K., An electron microscopic study on the interstitial cells of the gizzard in the love-bird (Uroloncha domestica), Z. Zellforsch., 97 (1969) 351-357. [3] Richardson, K.C., Electron microscopic observation on Auerbach's plexus in the rabbit, with special reference to the problem of smooth muscle innervation, Am. J. Anat., 103 (1958) 99-136. [4] Taxi, J., Sur la structure des trav~es du plexus d'Auerbach: confrontation des donn~es fournies par le microscope ordinaire et par le microscope filectronique, Ann. Sci. Natl. Zool., 1 (1959) 571-593. [5] Rogers, D.C. and Burnstock, G., The interstitial cell and its place in the concept of the autonomic ground plexus, J. Comp. Neurol., 126 (1966) 255-284. [6] Komuro, T., Three-dimensional observation of the fi-
[7] [8]
[9]
[10]
[11]
broblast-like cells associated with the rat myenteric plexus, with special reference to the interstitial cells of Cajal, Cell Tissue Res., 255 (1989) 343-351. Komuro, T., The interstitial cells in the colon of the rabbit, Cell Tissue Res., 222 (1982) 41-51. Christensen, J., Rick, G.A. and Lowe, L.S., Distributions of interstitial cells of Cajal in stomach and colon of cat, dog, ferret, opossum, rat, guinea pig and rabbit, J. Auton. Nerv. Syst., 37 (1992) 47-56. Christensen, J. and Rick, G.A., Intrinsic nerves in the mammalian colon: confirmation of a plexus at the circular muscle-submucosal interface, J. Auton. Nerv. Syst., 21 (1987) 223-231. Anuras, S., Chien, S.M. and Christensen, J., Metabolic dependence of the electromyogram of the cat colon, Am. J. Physiol., 239 (1980) G173-G176. Barajas-Lopez, C., Berezin, I., Daniel, E.E. and Huizinga, J.D., Pacemaker activity recorded in interstitial cells of Cajal of the gastrointestinal tract, Am. J. Physiol., 257 (1989) C830-C835.