| The liver has an huge and complex lymphatic system, and produces alarge volume of lymph, which is estimated to be25to50%of lymph flowingthrough the thoracic duct. The lymphatic system in the liver function as anessential transport and it fall into three categories depending on their locations:portal, sublobular, and superficial (or capsular) lymphatic vessels. Normally80%or more of hepatic lymph drains into portal lymphatic vessels, while theremainder drains mainly through sublobular lymphatic vessels. Sublobularlymphatic vessels exist in mang mammals, such as rabbits, cats, and human.The lymphatic networks around portal vessels and bile ducts extended as fardistally as terminal portal tracts in rabbits. The majority of lymph nodedrainage from the liver is to the hepatoduodenal ligament. From here to theceliac lymph nodes and then to the cisterna chyli. Sublobular lymphaticvessels lead into lymphatic vessels running along the inferior vena cava(1).Lymphatics perform tissue drainage system to maintain lipid homeostasis inthe liver. Although blood and lymphatic vessels function as parallel andintegrated systems, our understanding of lymphatic structure, regulation andfunctioning lags far behind that of the blood vascular system.Lymphostasis, with impaired tissue fluid flow, underlies or complicatesan indolent subclinical course with a long latent period and sporadic episodes.But the pathological changes on hepatic organizational structure and lipidhomeostasis after a long period of hepatic lymphostasis has not beenelucidated, this study was designed to elucidate some changes orcompensatory phenomenon on lymphatic vessels, hepatic cells and flat cells ofthe liver capsule, and investigated the underlying mechanisms of liver lipidhomeostasis. The experiment was divided into3parts, that is, establishingexperimental animal models through ligating abdominal thoracic duct in malehealthy New Zealand White rabbits to create liver lymphostasisi; observingthe tissue morphology of the liver of model animals and the controls with lightand electron microscopy; examining the apoE and LXRs on liver slices byradioimmunoassay; exploring the effects of lymphatic stasis on the tissuestructures and the function of the liver. The experimental contents and resultsare the followings:Part1The morphologcal changes on liver of lymphostasis induced byligation of thoracic ductObjective: To establish experimental animal model by abdominalsurgical ligation of rabbit thoracic duct, which leading lymph circumfluenceblock, and observed how the fat accumulated on the abdominal cavity andliver of experimental animals. And to test the method to make hepaticlymphostasis on New Zealand white rabbits.Methods:Twenty four,12-week-old, male healthy New Zealand White rabbits werekept in standard conditions and fed with water and rodent chow ad libitum.Their weight ranged from1500to1800g. The thoracic ducts of eight rabbitswere ligated (ligated group), while those of the others were not(sham-operated group,SO).Ligation of the thoracic duct: before the experiment the rabbits fasted fortwelve hours, drank water freely. All the experiment rabbits were anesthetizedwith10%chloral hydrate2.4ml/kg through marginal ear vein injection. Beforeanesthesia the rabbits were duck-stuffing fed10ml soybean oil by lavageneedle,1.5hrs later, anesthesia and operation were performed. Under strictaseptic operation principle, an incision was made at the abdominal wallmidline. The cisterna chyli and the thoracic duct in the upper part of theabdomen were filling ivory chyle, they were easy to recognize and clearlyrevealed. To avoid chyle leakage and ensure to achieve the effect of hepaticlymph stasis, We anatomically dissected the abdominal aorta, just up above the left adrenal gland1cm long, and aseptically ligated the thoracic duct alongwith its surrounding tissue. Just before close the abdominal cavity, we madean intraperitoneal injection of gentamicin5.0mg/kg. The rabbits recovered to agood condition1.5~2hrs later, then housed in temperature-andhumidity-controlled rooms with a12hr light/dark cycle and fed with waterand rodent chow ad libitum. Six month later, all rabbits, were processed foroperation. Liver tissue was fixed in4%formaldehyde solution. Just aftertaking tissue specimens, the rabbits were killed by a3-fold overdose of chloralhydrate.Results:1. The liver and abdominal cavity before the models established: Theliver was bright red, soft texture, sharp edges, and smooth surface. There wereless abdominal fat in abdominal cavity and the mesentery was clear andtransparent. The changes of fat in liver and abdominal cavity after the modelsestablished (1) The study group: The liver became dark red, rough texture, andround edge. And its surface has a greasy feeling. It seemed that the liver wasmild fibrosis. The cracks on liver surface were width and darken, accompaniedby fatty deposits under the liver capsule. There were obviously increased fatdeposition in abdominal cavity. And there were obviously abundant of fatdeposited on the mesentery, and even fat gathered on the mesentery like astring of fat bubble and fat globules.(2) The sham operative group: The liverwas still bright red, soft texture, sharp edges, and smooth surface. There werestill less abdominal fat in abdominal cavity and the mesentery was still clearand transparent.2. Observation of paraffin sections：(1) On sham operative group, theliver lobular structure were complete and clear as normal, and hepatic cordswere also complete and clear as normal. And there was a small amount of fatcan be seen in the hepatic cells, little vacuolated and loose degenerationassociated with hepatic cells. The hepatic sinusoid was complete and clear asnormal, and its endothelial cells and macrophages in it were as normal. Thehepatic portal structure was complete and clear: interlobular veins, arteries and bile ducts, and there were some hepatic stellate cells and reticulum fibers inperisinusoidal space. And there were no necrosis. The squamouse epitheliumcells of liver capsule were complete and clear as normal, and there were nocells infiltration under capsule.(2) On study group, there were more hepaticcells which contained fat in liver, and which became larger in size, more onnumber. There were even some large fatty capsule formation and fattydegeneration. Some hepatic cells were necrosis, such as phenomenon of thenucleus pycnosis. And there were inflammatory cells infiltration aroundhepatic cells, which mainly lymphocytes. The closer hepatic cell was thehepatic lobule central vein and the more serious of steatosis. The closerhepatic cell was the hepatic lobule portal, the littler of steatosis. The liverlobular structure were damaged, and there was abnormal hepatic lobulestructure, but no false lobule. There were lymphocyte infiltration on hepaticportal, interlobular lymphangiectasia, interlobular bile ducts and interlobularvein, artery and the infiltration of lymphocytes, mild fibrosis. The epithelialsquamous cells on peritoneum, which covered in liver tissue, were arrangedlittle neatly, and there were lymphangiectasia under epithelium.Conclusions: The surgical ligation of New Zealand white rabbit thoracicduct abdominal segment, can create New Zealand white rabbit liver lymphaticstasis experimental animal model; New Zealand white rabbit ligation ofthoracic duct lymph stasis abdominal segment cause liver, liver fat depositionin cells, liver cells steatosis, liver steatosis, and even mild fibrosis.Part2The experimental liver lymphatic stasis induced changes on fineand ultra structure of liver tissueObjective: To discusses experimental liver lymphatic stasis in NewZealand white rabbit ultra structure of hepatic cells and squamous epitheliumon liver capsule.Methods:The selection, breeding, and methods of making animal models of NewZealand White rabbits were as above. Under deep anesthesia with10%chloralhydrate as above, the abdomen was opened also at midline. In the ligated group,10ml soybean oil were fed before anesthesia as above to confirmthat the thoracic duct lymph flow occluded at the ligated site. The part of liverthat near the sickle ligament was excised and cut into small pieces, whichwere washed with normal saline and then with2.5%glutaraldehyde in a0.1Mphosphate buffer (pH7.4), further immersed in the same fixative for scanningelectron microscopy (SEM). Other part of liver was excised and cut into smallpieces, which were washed with normal saline and then with4%glutaraldehyde in a0.1M phosphate buffer (pH7.4), further immersed in thesame fixative for transmission electron microscopy (TEM). The pieces werepostfixed in1%OsO4, dehydrated in graded concentration of ethanol, andembedded in Epon-812resin Araldite. Semithin sections were stained withtoludine bule and examined under a light microscope. Ultrathin sections werewith both uranyl acetate and lead citrate. The sections were observed andtaken pictures by Hitachi H-7500type transmission electron microscope,80kV accelerating voltage.Results:1. Observation of paraffin sections：description as above.2. Observation under SEM:(1) On sham operative group, the flatepithelial cells on the liver membrane were arranged neatly, and theirmorphology were neat too. the squamous epithelium cells’ surface weresmooth, and their microvilli mainly distributed in the edge of the squamousepithelial cells. The gaps between each two squamous epithelial cells can beclearly seen, and the microvilli were sparse in there. On three scanningelectron microscope photes of liver surface, calculated the longth of eachrelatively clear gap between two squamous epithelium cells, and there wereabout20gaps. The meantime of gap between two squamous epithelium cellswas1.17um. Peritoneal hole is clearly visible on the surface of the livercapsule, each peritoneal hole exists among three adjacent epithelial cells, andthere was a small amount of phagocytes on its surface. On three scanningelectron microscope photes of liver surface, the longth of every peritoneumhole among three adjacent epithelial cells was calculated. The meantime of peritoneum hole, was2.79um.(2) On study group, the squamous epitheliumcells on liver surface were neat arrangement, and which were slightly flat andoval. The microvilli, which mainly exist on the squamous epithelium cellmarginal area, became longer and increased in the number. And the surface ofthe squamous epithelial cells, which was originally smooth, also covered alarge amount of microvilli. The whole flat epithelial cells on the number ofmicrovilli, not only with gap between squamous epithelium cells, aredistributed in the surface of the squamous epithelial cells, the gap betweensquamous epithelium cells become very not clear; The widening gap betweensquamous epithelium cells, the same method to calculate clearance size is2.08um, compared with the control group was statistically significant difference (P<0.05); Peritoneal hole is clearly visible on the surface of the liver capsule,exists between three adjacent epithelial cells, the same method to calculate theperitoneum hole size is3.98um, compared with the control group wasstatistically significant difference (P <0.05), its surface more than the numberof phagocytes, peritoneal visible around the hole diameter of about5to7umspherical vesicles, surface spines and microvilli, in addition, visible on thesurface of a large number of liver capsule diameter is about1um vesicles.3. Observation under TEM:(1) On sham operative group, theultrastructure of hepatocyte was complete and clear, and the cytoplasm wereabundant with rich in various kinds of organelles. The nucleus was round orsome and center in hepatic cell, structure is clear. And the euchromatin evenlydistributed in the nucleus with seldom was the heterochromatin. The nucleolusand nuclear membrane were complete and clear, and the nuclear pores wereclear. The Golgi complex, which were outside the nucleus, were complete andclear. The cytoplasm was abundant endoplasmic reticulum and mitochondria,and contained a lot of glycogen particles. And there were seldom lipid dropletin cytoplasm. there were some spherical lysosomes in cytoplasm. The sinusstructure was complete and clear. There was Kuffer cell around sinus. Theperisinusoidal space was normal and has a small of collagenous fiber. Therewere a lot of microvilli on hepatocyte membrane which was towards perisinusoidal space. The capillary bile duct between two hepatocyte wasnormal.(2) On study group, the hepatocyte ultrastructure was abnormalobviously. There were a lot of lipid droplets in cytoplasm. And the nucleuswas abnormal, such as the gap between nuclear membrane was wider, therewere a lot of heterochromatin, and lumps of heterochromatin gathered undernuclear membrane. And there were seldom glycogen particles in cytoplasm.There was double-nucleus hepatocyte. The space around hepatocyte andperisinusoidal space were wider, and there was fat-storing cell inperisinusoidal space. There were more collagenous fiber in the space aroundhepatocyte and perisinusoidal space. The bile stasis in capillary bile duct.Conclusions: The liver lymphatic stasis, results in a fat deposition in theliver cells, liver cells steatosis, collect abbacy fibrosis; The hepatic lobulelymphocytes infiltration; Liver lymphatic stasis, causing the liver capsulesquamous epithelium cells increase in the number of microvilli, the longer, thewidening gap between squamous epithelium cells, exists between threeadjacent epithelial cells of peritoneal hole also widen, liver capsule is visibleon the surface of a large number of vesicles, about1um in diameter andlymphoid cells.Part3Hepatic lymphostasis induced the change of apolipoprotein E andliver X receptor on liver tissueObjective: To discusses experimental liver lymphatic stasis in NewZealand white rabbit squamous cell ultrastructure of liver cells and livercapsule.Methods:The selection, breeding, and methods of making animal models of NewZealand White rabbits were as above. Under deep anesthesia with10%chloralhydrate as above, the abdomen was opened also at midline. In the ligatedgroup,10ml soybean oil were fed before anesthesia as above to confirm thatthe thoracic duct lymph flow occluded at the ligated site. The part of liver wasexcised and cut into small pieces, which were washed with normal saline andthen with4%glutaraldehyde in a0.1M phosphate buffer (pH7.4), further immersed in the same fixative for transmission electron microscopy (TEM).The pieces were postfixed in1%OsO4, dehydrated in graded concentration ofethanol, and embedded in Epon-812resin Araldite. Semithin sections werestained with toludine bule and examined under a light microscope. Ultrathinsections were with both uranyl acetate and lead citrate. The sections wereobserved and taken pictures by Hitachi H-7500type transmission electronmicroscope,80kV accelerating voltage. Free floating immunofluorescentlabeling was conducted on a separate set of50μm slices.3slices from eachanimal. Secondary detection was conducted for mouse-anti GFAP usingCy5-conjugated donkey anti-mouse secondary antibody (1:500, JacksonImmunoresearch) and for Ib4using Cy5-conjugated streptavidin (1:250,Jackson Immunoresearch). Slices were incubated for2hrs at roomtemperature in the secondary antibody solution. After washing, all slices weremounted with PROLONG Antifade Gold with DAPI (Invitrogen).Results:1. Observation of paraffin sections：description as above.2. Observation under TEM: description as above.3. ApoE and LXRs immunofluorescence:(1) On sham operative group,the liver tissue immune fluorescence intensity is clear, focused, on behalf ofapolipoprotein E green fluorescent mainly concentrated in the portal area, andthe liver X receptor red fluorescence concentrated area, dual channel,according to the results of both the yellow area of dyeing overlap is not much,mainly concentrated in the portal area. Select three typical area, around theportal area, statistical immune fluorescence intensity and the control group onbehalf of apolipoprotein E green fluorescence intensity is67.31+12.23AU,the liver X receptor red fluorescence intensity was191.17+35.69AU.(2) Onstudy group, the liver tissue immune fluorescence intensity is weak,significantly lower than the control group, the central tendency is consistentwith the control group. Immune fluorescence intensity analysis, no matter onbehalf of apolipoprotein E green fluorescence, or the liver X receptor redfluorescence, strength are relatively less than control. Select three typical area, around the portal area, statistical immune fluorescence intensity, theexperimental group on behalf of apolipoprotein E green fluorescence intensityis49.56+22.85AU, the liver X receptor red fluorescence intensity was32.84+24.01AU, experimental fluorescence intensity are lower than the controlgroup, the difference was statistically significant.(P <0.05).Conclusions: The New Zealand white rabbit liver lymphatic stasiscaused the liver cells involved in lipid metabolism in apolipoprotein E andliver X receptor levels drop, lipoprotein can’t through lymphatic channels oftranshipment, New Zealand white rabbit blood lipid metabolic abnormalities,liver cells appear fat drops, even some fatty degeneration of liver cells. |
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