Intrinsic Nerve Fibers in the Liver Parenchyma YOUSSEF MIKHAIL AND ABDEL LATIF SALEH Faculty of Medicine, E i n Shams University, Cairo, Egypt, U.A.R. The hepatic plexus which forms the largest offset from the coeliac plexus receives filaments from both vagi and right phrenic nerve. The nerves which enter the liver at the porta hepatis are reported to accompany mainly the blood vessels and bile ducts. Alexander ('40) could trace no nerve fibers into the parenchyma of the liver. Mitchell ( ' 5 3 ) claimed that the liver is supplied with terminal ganglia in or near the viscus. He also mentioned that intrinsic fibers in the liver are scanty and mainly confined to the vicinity of blood vessels, with few occasional groups found among the liver cells. It is surprising that in spite of the rich nature of the hepatic plexus the liver parenchyma is scarcely supplied by nerve fibers. Therefore this investigation was done to study the intrinsic nerve fibers of the liver and their mode of termination. The presence of terminal autonomic ganglia in the liver parenchyma was also verified. Holmes method. In the last series of animals, the portions of liver removed were fixed and stained by the Bodian protargol method. Several brands of protargol were used from different firms as DembachRoussel, Merck and Bayer. The best results were achieved by the Merck and B ayer preparations. OBSERVATIONS Rat The structure of the liver as revealed in the first series of experiments was identical with the normal histology of the liver which has been described in the literature before repeatedly and adequately. However few important points were noticed which deserved recording. The presence of spherical groups of cells at variable intervals in the liver parenchyma was peculiar. These collections of cells varied in their size and number of their constituent cells. They included clusters of small rounded cells with few triangular forms or even horse-shoe-shaped cells. Superficially MATERIAL AND METHODS these collections resembled terminal paraThe animals used in this investigation sympathetic ganglia. Only the cell bodies comprised normal adult male albino rats, were evident while their processes did not mice, guinea pigs, rabbits and dogs. The appear (fig. 1 ) . animals were divided into 4 series; each In sections stained with thionine similar series included at least two animals from collection of cells were stained with thioevery species investigated. All the animals nine appearing dark blue while the adjawere killed by gas and portions from the cent liver cells were stainless. However, different lobes as well as from the area the stain faded slowly and the appearance of the porta hepatis were removed. In the of the cells was not convincing. first series the portions of livers taken were In the third series of experiments the fixed in Bouin. After paraffin embedding, Holmes method helped to demonstrate the sections 15 p to 20 p in thickness were cut outline of nerve cells and nerve fibers in and stained with hematoxylin and eosin. the liver parenchyma. The nerve fibers In the second series the material was fixed were seen surrounding the branches of in 96% alcohol and after paraffin em- the portal vein and bile duct and even bedding sections were stained with 1% penetrating the liver parenchyma running thionine. In the third series the areas re- along the sinusoids of the liver (fig. 2). In the last series of experiments the outmoved were fixed in 5% formol saline, embedded in paraffin and stained by the line of nerve fibers and nerve cells was 317 318 YOUSSEF MIKHAIL AND ABDEL LATIF SALEH _I Fig. 1 - Ganglion Liver of rat stained by H & E. ( X 650) showing a large spherical ganglion. iwrve noer Fig. 2 Liver of rat stained by Holmes method ( x 320) showing nerve fibers extending along the sinusoids of the liver. very clear. Nerve fibers of varying thickness surrounded the branc:hes of the portal vein, hepatic artery and bile ducts. The nerves formed a meshwork of fibers which surrounded the vessels on all sides. Nerve fibers radiated from the big vessels to the various lobules of the liver. They extended between the cords of the liver cells for variable intervals being rather tortuous and confined mainly to the vicinity of the liver sinusoids. Terminal ganglia were found in the area of the porta hepatis and in the various lobes of the liver. They were found near to the portal canals and even within the liver lobules. Occasionally the ganglia formed large spherical groups PERIPHERAL NERVES O F LIVER Nerve cell 3119 Nerve cell Fig. 3 Liver of rat stained by Bodian method ( X 950) showing a cluster of neurons and nerve fibers among the liver cells. scattered at wide intervals in the liver parenchyma. These ganglia were formed from large clusters of cells which were surrounded by numerous nerve fibers from all sides. The processes related to the cells in the middle of the ganglia were not evident while those related to the peripheral cells were apparent. Even post ganglionic fibers were often seen radiating from the cells and extending between the cords of liver cells. Frequently the terminal ganglia were formed from small clusters of cells which were found near the walls of the central veins of the liver lobules (fig. 3). The nerve fibers and the processes connected to these neurons extended for variable intervals between the cords of liver cells and along the sinusoids of the liver. Often isolated nerve cells one or more were seen in between the liver cells. They varied in their shape but usually they appeared either rounded, triangular or pyriform in shape (fig. 4). They were usually multipolar and their dendrites varied greatly in length. Cells which possessed long and short dendrites were frequently encountered while cells with pure short dendrites were scarcely seen. The dendrites extended in between the liver cells often interlacing with the dendrites of a neighbouring nerve cell forming a peripheral plexus. The short dendrites often ended in knob like swellings which were very close to the hepatic cells. In the terminal plexus it was often difficult to differentiate between long dendrites and post ganglionic fibers. Similarly the terminal nerve fibers in the plexus could not be identified as afferent and efferent by any of the techniques employed. Occasionally it was possible to differentiate the post ganglionic fibers from other processes as they sprouted from the nerve cells and extended for long distances among the liver cells. Since only fibers of vagus origin effected a synaptic connection with terminal ganglia, the numerous ganglia encountered in the liver of the rat were regarded as totally of parasympathetic nature. YOUSSEF MIKHAIL AND ABDEL LATIF SALEH 320 Multipolar nerve cell Fig.4 Liver of rat stainetd by Bodian method ( X 1200) showing a multipolar nerve cell. Mouse The parenchyma of the liver of the mouse was permeated by nerve fibers which extended along the portal canals. The terminal ganglia in the liver were however small, and scattered at wide intervals. Small collections of nerve cells were found near the walls of the central veins. Isolated nerve cells were often seen among the liver cells which appeared either triangular or oval in shape (fig. 5). Their processes often joined together forming a fine delicate plexus in the liver parenchyma. Guinea pig The distribution of nerve fibers and cells in the liver of the guinea pig was similar to the rat. The ganglia were however numerous, and scattered irregularly in the liver parenchyma. The ganglia were densely populated with neurons while the adjacent liver cells appeared far less crowded (fig. 6 ) . Rabbit The innervation of the liver of the rabbit showed the same variations recorded in the rat. The ganglia were however irregular in shape and distribution. Amidst the liver cells multipolar and occasionaIly bipolar forms of nerve cells were seen. The dendrites of the nerve cells ran tortuously in between the liver cells (fig. 7). Dog The parenchyma of the liver of the dog was richly supplied by nerve fibers. The terminal ganglia were large, spindle shaped 321 PERIPHERAL NERVES O F LIVER though detached clusters of neurons were seen (fig. 8). DISCUSSION Nerve cell Nerve cell Fig. 5 Liver of mouse (Bodian method) ( x 780) showing two multipolar nerve cells. The ultimate ramification of autonomic nerve fibers in glandular structures is one of the problems in neurology. The terminal intraglandular nerve fibers are notorious by their ability to evade all the methods of staining. In the present investigation the Bodian protargol method has given encouraging results specially after using the protargol of Merck and Bayer. The parenchyma of the liver contrary to the previous observations was richly supplied with nerve fibers which radiated along the vessels, and bile ducts and even extended along the sinusoids in the liver lobules. Terminal ganglia were seen in the livers of all animals investigated. Their cells were multipolar and they possessed long and short dendrites. There was no chance for confusing these terminal processes with connective tissue fibers as they were always traced to the cells from which they originated. AS the Ganglion Ganglion Fig. 6 Liver of guinea pig (Bodian method) ( X 450) showing two spherical ganglia. 322 YOUSSEF MIKHAIL AND ABDEL L A T I F S A L E H Nerve cell __ Liver of rabbit stained by Bodian method ( x 950) showing isolated nerve cells. -I Fig. 7 ~~ Clusters of nerve cells Fig. 8 ~ Nerve fibers Liver of dog (Bodian method) ( x 550) showing detached clusters of neurons. 323 PERIPHERAL NERVES OF LIVER nerve cells were numerous and irregularly scattered in the liver parenchyma it was therefore not surprising to find that the processes connected to these cells were plentiful. They often joined together forming an intricate plexus in the liver parenchyma. These ganglion cells formed synapses with preganglionic fibers of the vagus nerve only. Therefore the terminal plexus in the liver contained preganglionic parasympathetic fibers, the intrinsic ganglion cells and their processes as well as the post ganglionic sympathetic fibers. The terminal plexus in the liver parenchyma as shown in this investigation is homologous with the Auerbach and Meissner plexuses in the alimentary canal and with the cardiac and pulmonary plexuses. The presence of terminal ganglia and the abundance of nerve fibers in the liver parenchyma may have a regulating effect on the hepatic function. The sympathetic fibers supplying the intra hepatic vessels has been shown to have a constricting effect upon them. Carnot et al. ('30) reported on the effects of vagus stimulation on intra hepatic circulation. Wakim ('42) found that parasympathetic stimulation did not elicit any appreciable effect on the flow of blood through the liver. Concerning the nervous regulation of biliary secretion, Hillyard ('30) failed to observe any change in the output of bile after carbohydrate, fat and protein meals in normal and completely denervated livers of dogs. Tanturi and Ivy ( ' 3 8 ) noticed a direct effect of vagus stimulation on the secretion of bile in the dog and the monkey but not in the cat or the rabbit. The latter investigators found that even after 5 days from sectioning the vagus nerve in the neck, stimulation of the peripheral end of the nerve had an excita- tory secretory effect. Stimulation of the central end of the divided nerve, while the other vagus was intact resulted in increase of biliary secretion. The various results showed the direct and indirect effects of vagus stimulation on bile production. The close association between the hepatic cell and the terminal nerve fibers and cells as shown in this investigation may support the possibility of a nervous regulation of the functional activity of the liver. SUMMARY The parenchyma of the liver of the rat, mouse, guinea pig, rabbit and dog was found to be richly supplied with nerve fibers. Terminal parasympathetic ganglia were found and they varied greatly in their size, shape and number of constituent cells. The neurons were either rounded or triangular in shape and were usually multipolar. Their dendrites and post ganglionic fibers interlaced together forming a terminal plexus in the liver parenchyma. LITERATURE CITED Alexander, W. F. 1940 The innervation of the biliary system. J. Comp. Neur., 72: 357-370. Carnot, P., R. Gayet and F. Merklen 1930 Exploration graphique des modification de la pression veineuse porte likes a des excitations vaso constrictives. C. R. SOC.Biol., 104: 12601266. Hillyard, L. V. 1931 The effect of denervation of the liver on secretion of bile. Amer. J. Physiol., 98: 612-614. Mitchell, G . A. 1953 Anatomy of the Autonomic nervous system. E. & s. Livingstone Ltd. Tanturi, C. A. and A. C. Ivy 1938 A study of the effects of vascular changes in the liver and the excitation of its nerve supply on the formation of bile. Amer. J. Physiol., 121: 61-74. Wakim, K. G. 1942 Effect of stimulation of autonomic nerves on intra heDatic circulation of blood i n intact animals. Proc. SOC. Exp. Biol., 49: 307-311.