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Neurohistochemical changes in the liver of guinea pigs following ligation of the common bile duct
EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
22,
29-34
(1975)
Neurohistochemical Changes in the Liver of Guinea following Ligation of the Common Bile Duct
Pigs
GY. UNGV~~RY AND T. DONA= Department of Experimental Pathology State Institute of Occupational Health, Budapest, Hungary and 1. Institute of Anatomy, Semmelweis Medical University, Budapest, Hungary. Received April 4, 1974, and in revised
form June26,
1974
The common bile duct of guinea pigs was ligated without injuring the nerves entering the liver. The innervation of the liver was studied for catecholamines and acetylcholinesterase with the methods of Falck et al. (1962) and Coupland and Holmes (1957), respectively, on the first, second, fourth, and fourteenth postoperative days. The results obtained were compared with the innervation pattern observed in the liver of control animals. Four days after the application of the ligature monoaminergic and acetylcholinesterase-positive innervation could only be observed in the hilus of the hepatic lobes. On the fourteenth day the histology of the liver indicated cirrhosis, the nerves, however, become demonstrable in a rearranged form within the expanded connective tissue areas. Views on the origin and plasticity of the reorganizing nerves are discussed.
Neurohistochemical experiments on autonomic nerve terminals have so far mostly been performed investigating the effect of direct stimuli like extirpation of ganglion, axotomy, electrical stimulation of nerves, enzyme inhibitors, etc. (Cannon and Lisdk, 1930; Euler and Ryd, 1963; Malmfors, 1965; Thomas, 1969; etc.). Studies dealing with morphological and histochemical changes of the nerves following alterations involving the whole innervated organ are far less numerous. The postganglionic terminals, either cholinergic or adrenergic, release different quantities of transmitters even under physiological conditions. Catecholamines, playing an important role in the maintenance of homeostasis are continuously produced, although with certain fluctuations. In a number of pathologic conditions (trauma, extensive surgical interventions, asphyxia, toxineffects, hypertension) catecholamine secretion is increased, while in others (orthostatic hypotension, effect of certain drugs) it is decreased. All these quantitative changes presumably involve acetylcholine as well. However, the proportions could not be stated in either case. For example, it is unknown how much of the catecholamines is released from the adrenal medulla and how much from the monoaminergic terminals. It would be important, therefore, to know to what extent the autonomic terminals take part in the changes of the organ innervated by them. The aim of this work has been to contribute to the solution of this complex problem by observing the neurohistochemical changes occurring after ligation of the common bile duct. 29 Copyright Q 1975 by Academic Press, Inc. All rights of repmduction in any form reserved.
UNGVARY
F ‘IG. 1. Liver of the control DO’ ups of specific fluorescence.
guinea Method
AND
DONATH
pig. Cross-section of of Falck et al. X 300.
preterminal
and
terminal
axon
GUINEA
MATERIALS
PIG LIVER
31
AND METHODS
Forty guinea pigs of both sexes were used; in 35 animals the common bile duct was ligated near Vater’s ampulla after median laparatomy. The hepatic artery and the portal vein were not injured. The animals were sacrificed 1, 2, 4, and 14 days after the ligation in groups of five. Five animals (pseudooperated) served as controls. (After the operation 15 animals died due to rupture of the common bile duct or of the gallbladder. ) The adrenergic and cholinergic nerves of the liver were examined with the methods of Falck et al. ( lQi32), and Coupland and Holmes (1957), respectively. Reserpin pretreatment was carried out as a control of Flack’s technique; some of the sections used for the Coupland-Holmes reaction were preincubated in 1W M iso-OMPA, a pseudocholinesterase inhibitor, for 30 min. RESULTS In the control animals the hepatic arteries and the portal veins were mostly of monoaminergic, the bile ducts, mostly of AChE positive innervation. The larger hepatic vein branches were, similarly to the other two intrahepatic blood vessels, innervated by noradrenergic fibers. In the preterminal part of the portal channels and along the vessels, in the interlobular and circumlobular areas (Fig. 1) nerves showing an intensive catecholamine-specific fluorescence were observed. These occasionally even enter the lobules. AChE-positive nerve fibers could be found in the preterminal part of the portal channels and they also entered, the lobules (Fig. 2). Microscopically leaf-shaped, triangular, or irregular areas imbibided with bile (well discernible even with the naked eye as yellow spots) appeared already 1 day after the ligation and became more marked on the second day. Their yellow flourescence and nonspecific ChE positivity were apparent (Figs. 3 and 4). The spots were occasionally situated in, or originating from, the portal areas and extended towards the centre of the lobules. In size several of the spots corresponded to the “simple aeinzls m acinzrs part of Rappaport.” Two days after applying the ligation the basic fluorescence of the hepatic tissue increased, whereas the number of nerve fibers demonstrable by histochemical means decreased. It was hard to estimate the AChE positivity because the localization of the reaction was vague. Neither monoaminergic nor AChE-positive terminals could be demonstrated in the hepatic tissue 4 days after the operation (Figs. 5 FIG. 2. Liver of the control guinea pig, AChE-positive nerve fibers in the portal areas (P) and among the laminae of liver cells. Method of Coupland-Holmes. 1W M iso-OMPA preincubation for 30 min. x 350. FIG. 3. On the second postoperative day, leaf-shaped territories of yellow autofluorescence imbibided with bile, could be observed in the perilobular areas. Nerve terminals could scarcely be found. Method of Falck et al. X 300. FIG. 4. A portion of the guinea pig liver, imbibided with bile penetrating from the portal area towards the middle of the lobulus, giving a slight nonspecific ChE reaction (x). Nerve fibers in the perilobular areas are hardly discernible (+) ( Coupland-Holmes reaction). FIG. 5. No nerve fibers of specific fluorescence could be traced on the fourth postoperative day in the liver of guinea pigs. Method of Falck et al. X 300. FIG. 6. No AChE-positive nerve fibers could be observed in the liver of guinea pigs on the fourth postoperative day. Method of Coupland-Hohnes. 10d M iso-OMPA preincubation. x 350.
32
Fro. 7. Within in the expanded
UNGVARY
AND
DONATH
a fortnight following the ligation, interstitial areas of the liver. X 300.
nerve
fibers
reappeared
in large
FIG. 8. Two weeks after the ligation a very rich AChE-positive plexus could in the areas between the pseudolobules. Numerous nerve fibers also entered Coupland-Holmes reaction after lad M iso-OMPA preincubation. X 140.
number
be observed the lobule.
and 6). Due to the ligation of the common bile duct, dilatation of the biliary ducts could be observed. On the fourth day, marked necrosis of the liver cells could still be observed frequently followed by cellular infiltration of the portal area, proliferation of the epithelium of the bile duct, eIongation of the winding biIe ducts, and by considerable connective tissue accumulation in the portal areas. In this way cirrhosis develops, localized primarily to the portal areas. The size of the lobules is thus reduced by the growing portal areas and a rearrangement of the lobules occurs. Two weeks after the ligation of the common bile duct numerous monoaminergic and AChE-positive fibers could be demonstrated (Figs. 7 and 8) in the expanded interlobular connective tissue. DISCUSSION The problem of monoaminergic innervation of the liver has been dealt with earlier (Ungvhry and Don&h, 1969). The additional AChE-positive innervation.
GUINEA
PIG LIVER
33
less abundant in other mammals, is highly characteristic for the liver of guinea pigs (Sutherland, 1964; Ungv&ry, 1971). For the present experiments guinea pigs were selected on account of this presumably double innervation. It should also be noted, however, that the AChEpositivity does not necessarily imply cholinergic transmission. The guinea pig produces the largest amounts of bile among laboratory animals (Quincke and Hoppe-Seyler, 1899) consequently, the described histological changes become apparent soon after the ligation of the duct. This was already described by Mac Mahon et al. ( 1929). In our previous studies (Ungvary et al., 1971, 1973) it was reported that the histochemical reactions of the monoaminergic and AChEpositive nerve fibres disappeared soon after the ligation. It was the increased pressure in the biliary ducts and gallbladder, resulting presumably in the degeneration of the fibers, that was held responsible for this phenomenon. Degeneration of nerves in the gallbladder has recently been demonstrated in electron microscopic studies (Ungv&y and L&&h, unpublished data). This degeneration, however, did not involve all the nerves of the gallbladder, even 5-7 days after applying the ligature, although the histochemical reactions were absent 24-40 hr (for catecholaminergic nerves demonstrable with the Falck-Hillarp technique) and approximately 48 hr (for AChE-positive nerves demonstrable with the Coupland-Holmes technique) after ligating the common bile duct. In the present experiments the effect of increased pressure could be ruled out as an exclusive factor, because no decrease in the pressure of the biliary ducts was evident after the fourth day when the restitution of neural function could already be demonstrated. It is more probable that the structure of the axonal membrane is altered due to the combined effect of a number of histological changes outlined in the foregoing (Malmfors, 1965; Csillik, 1970; etc.). It has to be emphasized, however, that the disappearance of the histochemical reaction does not necessarily signify the disappearance of the nerves. To clarify this problem further electronmicroscopic studies are necessary. The renewed demonstrability of the nerves and/or the appearance of the nerves deserves further attention. As reported by Mac Mahon et al. (1929) and by ourselves, parallel with the developing cirrhosis changes can be observed in the structure of the guinea pig liver. The expansion of the connective tissue areas on the expense of the hepatic tissue is considerable. The proliferation of the biliary ducts, accompanied by that of the concomitant artery branches, the so-called arterialization (Huh&r, 1966) is important from the point of view of innervation (Domini, 1959; Huh&r, 1966). These arterial branches and the monoaminergic and AChE-positive nerves supplying them are newly developed formations. However, our results are not informative as far as the mechanism of their development is concerned. The ability of intact autonomic nerves to grow and to develop new branches is well known. Intact nerve fibers of the iris can grow into grafts transplanted to the anterior chamber of the eye (Olson and Malmfors, 1970). Accordingly, two alternative mechanisms may be postulated: (i) the transiently degenerated fibers regenerate to suit the conditions of the new environment, or (ii) from intact nerves which cannot be demonstrated with the present methods, new branches develop to innervate the proliferating biliary ducts and blood vessels.
34
UNGVARY
AND
DONATH
The results obtained indicate that the change in the histology of the liver requires an adequate reorganization or neoformation of the autonomic innervation. The experiments published here support the statement of Szentagothai (1952) that the nerve centres of fixed physiological and anatomical organization are only capable of biologically expedient responses serving the survival of the animal under certain limited conditions, whereas the peripheral nervous system is able to ensure the adaptation of the organism by anatomical reorganization. REFERENCES CANNON,
W.
B.,
and
LISS~K,
K.
(1939).
Evidence
for
adrenaline
in
adrenergic
neurones.
Amer. J. Physiol. 125, 765-777. COUPLAND, R. E., and HOLMES, R. L. (1957). demonstration of peripheral nervous structures. CSILLIK, B. ( 1970). Cytochemistry of synapses
Th e use of cholinesterase Quart. J. Micr. Sci. 98, (in Hungarian). MTA
techniques
for
the
327-330. Biol. Oszt. Kb’zl. 12,
283-316. DOMINI, R. (1959). L’economia deIla circolazione intraepatical alIa lute de1 rapport0 fra diametro vascolare e angolo di ramificazione. Parte II. Studio sul fegato cirrotico. Lo Sperimentale 109, 296-307. V.EULER, U. S. (1972). Pathophysiological aspects of catecholamine production. Review. Clin. hh.StTy 18, 1445-1448. V.EULER, U. S., and Rxn, G. (1963). Effect of sympathetic denervation and adrenalectomy on the catecholamine content of the rat submaxillary gland. Actu Physiol. Stand. 59, 62-66. FALCK, B., HILLARP, N. A., THEME, G., and THORP, A. ( 1962). Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10,348-X4. HULT~N, 0. (1966). Arterialisation on the liver in cirrhosis. Scund. .Z. Clin. Lab. Invest. 18,
Suppl. 92, l-42. MAC MAHON, H. E., LAWRENCE, J. S., and MADDOCK, S. J. (1929). Experimental cirrhosis. Am. J. Path. 5, 631-643. MALMFORS, T. ( 1965). Studies on adrenergic nerves. Actu Physiol. Stand.
obstructive
64. Suppl.
248, l-93. OLSON, L., and MALMFORS, T. ( 1970). Growth characteristics of adrenergic nerves in the adult rat. Fluorescence histochemical and H*-nonadrenaline uptake studies using tissue transplantations to the anterior chamber of the eye. Actu Physiol. Scund. Suppl. 348, 1-112. QUINCKE, and HOPE-SEYLER ( 1899). Cited in Cameron, G. R., and Oakley, C. L. (1932). Ligation of the common bile duct. J. Path. Butt. 35, 769-798. SUTHERLAND, S. (1964). An evaluation of cholinesterase techniques in the study of the intrinsic innervation of the liver. I. Anut. (London) 98, 321-326. SZENTAGOTHAI, J. ( 1952). Experiment for natural classification of the tissular components of the nervous system (in Hungarian). M’I’A. Orv. Tud. Oszt. K&Z. 3, 365-412. THOIMAS, E. ( 1969). Histotopochemie und Histopathochemie des peripheren Nervensystems bei Verletzungen und Tumoren.” VEB. Gustav Fischer Verlag, Jena. UNWARY, GY. Functional morphology of the hepatic blood circulation and of the reguIation of the hepatic blood circulation. Thesis (in Hungarian). Budapest, 1971. UNWARY, GY., and DONATH, T. ( 1969). On the monoaminergic innervation of the liver. Actu
Anat. (Bused) 72, 446-1159. UNWARY, wall of
GY., DON~~TH, T. and NASZALY, S. A. ( 1973). Neurohistochemical changes the gall bladder injured by the ligation of the common bile duct. Actu Acud. Sci. Hung. 21,281-293. UNWARY, GY., DONATH, T., NASZ~~LY, S. A., and TAUBER, F. (1971). Effect of the mentally altered “millieu interieui on the histochemistry of the vegetative terminals. International Meeting of the International Society for Neurochemistry. Budapest, Abstracts p. 226. Akademiai Kiado, Budapest.
in the
Morph. experiThird 1971.
AND
MOLECULAR
PATHOLOGY
22,
29-34
(1975)
Neurohistochemical Changes in the Liver of Guinea following Ligation of the Common Bile Duct
Pigs
GY. UNGV~~RY AND T. DONA= Department of Experimental Pathology State Institute of Occupational Health, Budapest, Hungary and 1. Institute of Anatomy, Semmelweis Medical University, Budapest, Hungary. Received April 4, 1974, and in revised
form June26,
1974
The common bile duct of guinea pigs was ligated without injuring the nerves entering the liver. The innervation of the liver was studied for catecholamines and acetylcholinesterase with the methods of Falck et al. (1962) and Coupland and Holmes (1957), respectively, on the first, second, fourth, and fourteenth postoperative days. The results obtained were compared with the innervation pattern observed in the liver of control animals. Four days after the application of the ligature monoaminergic and acetylcholinesterase-positive innervation could only be observed in the hilus of the hepatic lobes. On the fourteenth day the histology of the liver indicated cirrhosis, the nerves, however, become demonstrable in a rearranged form within the expanded connective tissue areas. Views on the origin and plasticity of the reorganizing nerves are discussed.
Neurohistochemical experiments on autonomic nerve terminals have so far mostly been performed investigating the effect of direct stimuli like extirpation of ganglion, axotomy, electrical stimulation of nerves, enzyme inhibitors, etc. (Cannon and Lisdk, 1930; Euler and Ryd, 1963; Malmfors, 1965; Thomas, 1969; etc.). Studies dealing with morphological and histochemical changes of the nerves following alterations involving the whole innervated organ are far less numerous. The postganglionic terminals, either cholinergic or adrenergic, release different quantities of transmitters even under physiological conditions. Catecholamines, playing an important role in the maintenance of homeostasis are continuously produced, although with certain fluctuations. In a number of pathologic conditions (trauma, extensive surgical interventions, asphyxia, toxineffects, hypertension) catecholamine secretion is increased, while in others (orthostatic hypotension, effect of certain drugs) it is decreased. All these quantitative changes presumably involve acetylcholine as well. However, the proportions could not be stated in either case. For example, it is unknown how much of the catecholamines is released from the adrenal medulla and how much from the monoaminergic terminals. It would be important, therefore, to know to what extent the autonomic terminals take part in the changes of the organ innervated by them. The aim of this work has been to contribute to the solution of this complex problem by observing the neurohistochemical changes occurring after ligation of the common bile duct. 29 Copyright Q 1975 by Academic Press, Inc. All rights of repmduction in any form reserved.
UNGVARY
F ‘IG. 1. Liver of the control DO’ ups of specific fluorescence.
guinea Method
AND
DONATH
pig. Cross-section of of Falck et al. X 300.
preterminal
and
terminal
axon
GUINEA
MATERIALS
PIG LIVER
31
AND METHODS
Forty guinea pigs of both sexes were used; in 35 animals the common bile duct was ligated near Vater’s ampulla after median laparatomy. The hepatic artery and the portal vein were not injured. The animals were sacrificed 1, 2, 4, and 14 days after the ligation in groups of five. Five animals (pseudooperated) served as controls. (After the operation 15 animals died due to rupture of the common bile duct or of the gallbladder. ) The adrenergic and cholinergic nerves of the liver were examined with the methods of Falck et al. ( lQi32), and Coupland and Holmes (1957), respectively. Reserpin pretreatment was carried out as a control of Flack’s technique; some of the sections used for the Coupland-Holmes reaction were preincubated in 1W M iso-OMPA, a pseudocholinesterase inhibitor, for 30 min. RESULTS In the control animals the hepatic arteries and the portal veins were mostly of monoaminergic, the bile ducts, mostly of AChE positive innervation. The larger hepatic vein branches were, similarly to the other two intrahepatic blood vessels, innervated by noradrenergic fibers. In the preterminal part of the portal channels and along the vessels, in the interlobular and circumlobular areas (Fig. 1) nerves showing an intensive catecholamine-specific fluorescence were observed. These occasionally even enter the lobules. AChE-positive nerve fibers could be found in the preterminal part of the portal channels and they also entered, the lobules (Fig. 2). Microscopically leaf-shaped, triangular, or irregular areas imbibided with bile (well discernible even with the naked eye as yellow spots) appeared already 1 day after the ligation and became more marked on the second day. Their yellow flourescence and nonspecific ChE positivity were apparent (Figs. 3 and 4). The spots were occasionally situated in, or originating from, the portal areas and extended towards the centre of the lobules. In size several of the spots corresponded to the “simple aeinzls m acinzrs part of Rappaport.” Two days after applying the ligation the basic fluorescence of the hepatic tissue increased, whereas the number of nerve fibers demonstrable by histochemical means decreased. It was hard to estimate the AChE positivity because the localization of the reaction was vague. Neither monoaminergic nor AChE-positive terminals could be demonstrated in the hepatic tissue 4 days after the operation (Figs. 5 FIG. 2. Liver of the control guinea pig, AChE-positive nerve fibers in the portal areas (P) and among the laminae of liver cells. Method of Coupland-Holmes. 1W M iso-OMPA preincubation for 30 min. x 350. FIG. 3. On the second postoperative day, leaf-shaped territories of yellow autofluorescence imbibided with bile, could be observed in the perilobular areas. Nerve terminals could scarcely be found. Method of Falck et al. X 300. FIG. 4. A portion of the guinea pig liver, imbibided with bile penetrating from the portal area towards the middle of the lobulus, giving a slight nonspecific ChE reaction (x). Nerve fibers in the perilobular areas are hardly discernible (+) ( Coupland-Holmes reaction). FIG. 5. No nerve fibers of specific fluorescence could be traced on the fourth postoperative day in the liver of guinea pigs. Method of Falck et al. X 300. FIG. 6. No AChE-positive nerve fibers could be observed in the liver of guinea pigs on the fourth postoperative day. Method of Coupland-Hohnes. 10d M iso-OMPA preincubation. x 350.
32
Fro. 7. Within in the expanded
UNGVARY
AND
DONATH
a fortnight following the ligation, interstitial areas of the liver. X 300.
nerve
fibers
reappeared
in large
FIG. 8. Two weeks after the ligation a very rich AChE-positive plexus could in the areas between the pseudolobules. Numerous nerve fibers also entered Coupland-Holmes reaction after lad M iso-OMPA preincubation. X 140.
number
be observed the lobule.
and 6). Due to the ligation of the common bile duct, dilatation of the biliary ducts could be observed. On the fourth day, marked necrosis of the liver cells could still be observed frequently followed by cellular infiltration of the portal area, proliferation of the epithelium of the bile duct, eIongation of the winding biIe ducts, and by considerable connective tissue accumulation in the portal areas. In this way cirrhosis develops, localized primarily to the portal areas. The size of the lobules is thus reduced by the growing portal areas and a rearrangement of the lobules occurs. Two weeks after the ligation of the common bile duct numerous monoaminergic and AChE-positive fibers could be demonstrated (Figs. 7 and 8) in the expanded interlobular connective tissue. DISCUSSION The problem of monoaminergic innervation of the liver has been dealt with earlier (Ungvhry and Don&h, 1969). The additional AChE-positive innervation.
GUINEA
PIG LIVER
33
less abundant in other mammals, is highly characteristic for the liver of guinea pigs (Sutherland, 1964; Ungv&ry, 1971). For the present experiments guinea pigs were selected on account of this presumably double innervation. It should also be noted, however, that the AChEpositivity does not necessarily imply cholinergic transmission. The guinea pig produces the largest amounts of bile among laboratory animals (Quincke and Hoppe-Seyler, 1899) consequently, the described histological changes become apparent soon after the ligation of the duct. This was already described by Mac Mahon et al. ( 1929). In our previous studies (Ungvary et al., 1971, 1973) it was reported that the histochemical reactions of the monoaminergic and AChEpositive nerve fibres disappeared soon after the ligation. It was the increased pressure in the biliary ducts and gallbladder, resulting presumably in the degeneration of the fibers, that was held responsible for this phenomenon. Degeneration of nerves in the gallbladder has recently been demonstrated in electron microscopic studies (Ungv&y and L&&h, unpublished data). This degeneration, however, did not involve all the nerves of the gallbladder, even 5-7 days after applying the ligature, although the histochemical reactions were absent 24-40 hr (for catecholaminergic nerves demonstrable with the Falck-Hillarp technique) and approximately 48 hr (for AChE-positive nerves demonstrable with the Coupland-Holmes technique) after ligating the common bile duct. In the present experiments the effect of increased pressure could be ruled out as an exclusive factor, because no decrease in the pressure of the biliary ducts was evident after the fourth day when the restitution of neural function could already be demonstrated. It is more probable that the structure of the axonal membrane is altered due to the combined effect of a number of histological changes outlined in the foregoing (Malmfors, 1965; Csillik, 1970; etc.). It has to be emphasized, however, that the disappearance of the histochemical reaction does not necessarily signify the disappearance of the nerves. To clarify this problem further electronmicroscopic studies are necessary. The renewed demonstrability of the nerves and/or the appearance of the nerves deserves further attention. As reported by Mac Mahon et al. (1929) and by ourselves, parallel with the developing cirrhosis changes can be observed in the structure of the guinea pig liver. The expansion of the connective tissue areas on the expense of the hepatic tissue is considerable. The proliferation of the biliary ducts, accompanied by that of the concomitant artery branches, the so-called arterialization (Huh&r, 1966) is important from the point of view of innervation (Domini, 1959; Huh&r, 1966). These arterial branches and the monoaminergic and AChE-positive nerves supplying them are newly developed formations. However, our results are not informative as far as the mechanism of their development is concerned. The ability of intact autonomic nerves to grow and to develop new branches is well known. Intact nerve fibers of the iris can grow into grafts transplanted to the anterior chamber of the eye (Olson and Malmfors, 1970). Accordingly, two alternative mechanisms may be postulated: (i) the transiently degenerated fibers regenerate to suit the conditions of the new environment, or (ii) from intact nerves which cannot be demonstrated with the present methods, new branches develop to innervate the proliferating biliary ducts and blood vessels.
34
UNGVARY
AND
DONATH
The results obtained indicate that the change in the histology of the liver requires an adequate reorganization or neoformation of the autonomic innervation. The experiments published here support the statement of Szentagothai (1952) that the nerve centres of fixed physiological and anatomical organization are only capable of biologically expedient responses serving the survival of the animal under certain limited conditions, whereas the peripheral nervous system is able to ensure the adaptation of the organism by anatomical reorganization. REFERENCES CANNON,
W.
B.,
and
LISS~K,
K.
(1939).
Evidence
for
adrenaline
in
adrenergic
neurones.
Amer. J. Physiol. 125, 765-777. COUPLAND, R. E., and HOLMES, R. L. (1957). demonstration of peripheral nervous structures. CSILLIK, B. ( 1970). Cytochemistry of synapses
Th e use of cholinesterase Quart. J. Micr. Sci. 98, (in Hungarian). MTA
techniques
for
the
327-330. Biol. Oszt. Kb’zl. 12,
283-316. DOMINI, R. (1959). L’economia deIla circolazione intraepatical alIa lute de1 rapport0 fra diametro vascolare e angolo di ramificazione. Parte II. Studio sul fegato cirrotico. Lo Sperimentale 109, 296-307. V.EULER, U. S. (1972). Pathophysiological aspects of catecholamine production. Review. Clin. hh.StTy 18, 1445-1448. V.EULER, U. S., and Rxn, G. (1963). Effect of sympathetic denervation and adrenalectomy on the catecholamine content of the rat submaxillary gland. Actu Physiol. Stand. 59, 62-66. FALCK, B., HILLARP, N. A., THEME, G., and THORP, A. ( 1962). Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10,348-X4. HULT~N, 0. (1966). Arterialisation on the liver in cirrhosis. Scund. .Z. Clin. Lab. Invest. 18,
Suppl. 92, l-42. MAC MAHON, H. E., LAWRENCE, J. S., and MADDOCK, S. J. (1929). Experimental cirrhosis. Am. J. Path. 5, 631-643. MALMFORS, T. ( 1965). Studies on adrenergic nerves. Actu Physiol. Stand.
obstructive
64. Suppl.
248, l-93. OLSON, L., and MALMFORS, T. ( 1970). Growth characteristics of adrenergic nerves in the adult rat. Fluorescence histochemical and H*-nonadrenaline uptake studies using tissue transplantations to the anterior chamber of the eye. Actu Physiol. Scund. Suppl. 348, 1-112. QUINCKE, and HOPE-SEYLER ( 1899). Cited in Cameron, G. R., and Oakley, C. L. (1932). Ligation of the common bile duct. J. Path. Butt. 35, 769-798. SUTHERLAND, S. (1964). An evaluation of cholinesterase techniques in the study of the intrinsic innervation of the liver. I. Anut. (London) 98, 321-326. SZENTAGOTHAI, J. ( 1952). Experiment for natural classification of the tissular components of the nervous system (in Hungarian). M’I’A. Orv. Tud. Oszt. K&Z. 3, 365-412. THOIMAS, E. ( 1969). Histotopochemie und Histopathochemie des peripheren Nervensystems bei Verletzungen und Tumoren.” VEB. Gustav Fischer Verlag, Jena. UNWARY, GY. Functional morphology of the hepatic blood circulation and of the reguIation of the hepatic blood circulation. Thesis (in Hungarian). Budapest, 1971. UNWARY, GY., and DONATH, T. ( 1969). On the monoaminergic innervation of the liver. Actu
Anat. (Bused) 72, 446-1159. UNWARY, wall of
GY., DON~~TH, T. and NASZALY, S. A. ( 1973). Neurohistochemical changes the gall bladder injured by the ligation of the common bile duct. Actu Acud. Sci. Hung. 21,281-293. UNWARY, GY., DONATH, T., NASZ~~LY, S. A., and TAUBER, F. (1971). Effect of the mentally altered “millieu interieui on the histochemistry of the vegetative terminals. International Meeting of the International Society for Neurochemistry. Budapest, Abstracts p. 226. Akademiai Kiado, Budapest.
in the
Morph. experiThird 1971.