Reply to "Commentaries on the publication entitled: 'Structure and distribution of an unrecognized interstitium in human tissues' by Benias et al. (2018)

We appreciate the time and attention paid to our paper by Prof. Mestres-Ventura and similarly appreciate the opportunity to respond to his concerns. We would like to address what we believe are several fundamental misunderstandings in his commentary.1. Scale: The most significant misunderstanding is one of scale. The schematic (Fig. 1) provided by Prof. Mestres-Ventura is (per personal communication) at the nano scale, while in vivo microscopy of extrahepatic bile duct and dermis shows that the collagen bundles we report are at the micron scale, each containing many individual fibrils at the nanometer scale. Indeed, examining the tissues described in our paper - submucosae, dermis and subcutaneous fascia - fresh in resected specimens or intraoperatively, we find that the structures we describe are visible at the macroscopic level (if one leans in closely enough). In other words, they are macroscopic, not microscopic. Prof. Mestres-Ventura, in summarizing the prior pCLE work of Wallace and Fockens, which he notes is similar to ours, states "the 'holes' shown under intravital microscopy and in cryofixed samples are huge (over 20 µm)" This is exactly our point - we were surprised as well at the scale of these structures, as this has not been well appreciated in the past

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Confocal endomicroscopic examination of malignant biliary strictures and histologic correlation with lymphatics.

BACKGROUND AND AIMS: Current methods to diagnose malignant biliary strictures are of low sensitivity. Confocal endomicroscopy is a new approach that may improve the diagnosis of indeterminate biliary strictures. The purpose of this study was to evaluate indeterminate biliary strictures using probe-based confocal laser endomicroscopy and to understand the histologic basis for the confocal images. METHODS: Fourteen patients with indeterminate biliary strictures underwent endoscopic retrograde cholangiopancreatography with examination of their common bile duct with fluorescein-aided probe-based confocal laser endomicroscopy. Standard brushings and biopsies were performed. In parallel, rat bile ducts were examined either with conventional staining and light microscopy or with multiphoton microscopy. RESULTS: Earlier published criteria were used to evaluate possible malignancy in the confocal images obtained in the 14 patients. None of the individual criteria were found to be specific enough for malignancy, but a normal-appearing reticular pattern without other putative markers of malignancy was observed in all normal patients. Multiphoton reconstructions of intact rat bile ducts revealed that the reticular pattern seen in normal tissue was in the same focal plane but was smaller than blood vessels. Special stains identified the smaller structures in this network as lymphatics. CONCLUSIONS: Our limited series suggests that a negative confocal imaging study of the biliary tree can be used to rule out carcinoma, but there are frequent false positives using individual earlier published criteria. An abnormal reticular network, which may reflect changes in lymphatics, was never seen in benign strictures. Better correlation with known histologic structures may lead to improved accuracy of diagnoses.

Electron microscopic detection of tin accumulation in biliopancreatic concrements after induction of chronic pancreatitis in rats by di-n-butyltin dichloride.

The organotin compound di-n-butyltin dichloride (DBTC) is able to induce an acute and later a chronic pancreatitis in rats. In previous papers the authors demonstrated this DBTC pancreatitis as a rat model for an interstitial pancreatitis with tendency to transduction to the chronic form. DBTC is excreted according to its lipophilic nature by liver and bile. Therefore, the bilio-pancreatic main duct is necrotized by the tin-loaded bile. The duct system is blocked by cell debris and later by epithelial proliferations. In the chronic phase, numerous rats develop concrements in the main duct. In the present paper, the authors report about bacterial growth in some bilio-pancreatic concrements. Whereas the electron microscopic detection of tin by energy-dispersive X-ray analysis (EDX) in SEM or electron energy loss spectroscopy (EELS) in TEM was negative in the parenchyma of pancreas and liver, some concrements with bacterial cells were positive for this element. Tin mapping with energy spectroscopic imaging (ESI) in TEM demonstrated the congruency of tin signals and electron-dense particles inside these bacteria and of electron-dense accumulations in the matrix of these concrements. The low content of tin in pancreatic and liver tissue and the higher quantity of tin inside the bacterial contaminated concrements were supported by atomic absorption spectrophotometry (AAS). The paper discusses the long time preservation of tin in the concrements as an action of heavy-metal- accumulating bacteria, which should be classified in the future by bacteriological methods.

Bile canalicular barrier function and expression of tight-junctional molecules in rat hepatocytes during common bile duct ligation.

Tight junctions of hepatocytes form the intercellular barrier between the blood circulation and bile flow. We focused on early stages of common bile duct ligation to observe changes in tight junctions without the irreversible changes seen after lengthy ligation. Common bile ducts of 12-week-old male rats were ligated for 6 h because, at this time point, no histological changes were observed. Serum bilirubin and bile acid levels began to increase 3 h after ligation and were restored to the control level immediately after surgical removal of the ligation. To examine the barrier of hapatocytes, horseradish peroxidase was injected via the femoral vein, and bile was collected for the first 10 min. A four-fold elevation of the secretion and concentration was observed in the bile of ligated rats compared with that of control animals. We next examined lanthanum permeability by perfusion fixation of the liver. At 6 h after ligation, both dilation of the bile canaliculi and partial loss of microvilli were commonly observed. There were dense deposits of lanthanum in almost all bile canaliculi of ligated rats. In control animals, neither dilation of the bile canaliculi nor loss of microvilli was detected, and only 44% of bile canaliculi exhibited deposits. An apparent increase of occludin mRNA expression was detected in livers after 6 h ligation, whereas the expression of claudin-1, -2, and -3 was not influenced by ligation. These results indicate that regulation of occludin gene expression is different from that of claudin-1, -2, and -3. The early phase of bile stasis employed in this study is thought to be an indispensable approach for understanding the precise regulation of tight junctions.

[Clinical and pathogenetic significance of biliary tract abnormalities in a child with cholelithiasis]. / Kliniko-patogeneticheskoe znachenie anomalii zhelchnovyvodiashchikh putei u detei s kholelitiazom.

The clinical peculiarities of cholelithiasis in the children with biliary tract's anomalities are presented in this paper. The data of clinical, instrumental examinations and anamnesis are analysed. Although, there are some clinical peculiarities of cholelithiasis in the patients with biliary tract's anomalities, intrascopic methods of examination are preferable. Ultrasonography is used as quick method during before hospital examination. Retrogdade cholangiopancreatography in special hospital is the main diagnostic method in such patients, that defines the date, volume and kind of operation.

Mucus production of choledochal epithelial cells due to bile stagnation in the rat.

Mucus secretion is increased by various causative agents of inflammation in the mucosa. Although mucus production of the choledochal epithelial cells (except goblet cells) has never been noted in the rat in normal physiological conditions, it is seen as an inflammatory reaction in animals with choledochoenterostomy, which brings bacteria and active digestive enzymes into the common bile duct through regurgitation of intestinal contents. It is known that stagnant bile alone can cause inflammation in the mucosa of the gallbladder in patients with aseptic acalculous cholecystitis. In this study, aseptic bile stagnation was caused by choledochal dilation made by detaching the common bile duct from the hepatoduodenal ligament in five rats, and histological changes of the choledochal epithelium were observed by light and transmission electron microscopy 17 months after the operative procedure to determine whether stagnant bile could cause mucus production in choledochal epithelial cells. Mucus production was noted in two rats by light microscopy and in all the rats by transmission electron microscopy, and so it was demonstrated that stagnant bile could give rise to this phenomenon in the rat choledochal epithelium.

[Experimental study and clinical application of primary choledochorraphy after choledochotomy].

An animal model was established in dogs to compare primary choledochorraphy with the traditional T-tube method. Authors observed cholangic histologic changes under microscope and electron-microscope between the two groups. The result showed that primary choledochorraphy was better than the traditional method. The advantage of primary choledochorraphy lies in its simplicity, safety and reliability, which have been shown in 30 patients of cholelithiasis in clinical application. It is suggested that for patients primary choledochorraphy may clesirably be used for patients who are indicated for this approach.

Ultrastructural study on hyperplastic epithelium of rat common bile duct after choledochoenterostomy.

Hyperplasia of the choledochal epithelium occurs in rats after choledochoenterostomy. It is tubular or papillary and not like small intestinal mucosa in rats with choledochojejunostomy. It is, however, like large intestinal mucosa in rats with choledochocolonostomy. We investigated the ultrastructure of the hyperplastic epithelium in the two experimental groups of rats in this study. The main constituent cells of the hyperplasia of both the groups were like the principal cells of the control, but differed in that they had long microvilli and contained an aggregation of mucus droplets and an increased number of lysosomes. These findings were not seen in the control. The microvilli were sparse in the choledochojejunostomy group, but were dense in the choledochocolonostomy group. These findings suggest that the main function of the epithelium may be cytoprotection in the choledochojejunostomy group and both resorption and cytoprotection in the choledochocolonostomy group.

[A stereologic study on the influence of stent tube pressure on bilio-intestinal anastomotic scar formation].

Biliary tract injury results often in biliary scar stricture causing obstructive jaundice and biliary cirrhosis. Choledocho-jejunostomy with tube stent is the routine to prevent anastomotic stenosis, but how long the stent tube should be in place is controversial and little is known on the influence of the stent tube on anastomotic scar formation. In canine experimental models, bili-intestinal scar was sampled 3 weeks, 3, 6, 9 and 12 months after anastomosis for ultrastructure stereologic image analysis. It was found that the internal pressure of the stent tube exerting on anastomotic stoma inhibits scar formation, and the stent tube should be remained inside the stoma for not less than 9 months.

Effects of biliary endoprostheses on the extrahepatic bile ducts in relation to subsequent operation of the biliary tract.

Despite the widespread use of transpapillary biliary endoprostheses, little is known about their effect on the extrahepatic bile ducts. In an experimental study in dogs, we induced inflammatory changes in the bile ducts by stent insertion and studied the reversibility of these changes after stent removal. In addition, the consequences of a period of preoperative stenting for subsequent operation of the biliary tract and the eventual detrimental effects of stenting on the histologic factors of the liver were studied. Twenty-six mongrel dogs were randomly divided into four groups: group 1, stenting during four weeks; group 2, after four weeks stenting, construction of a hepaticojejunostomy; group 3, four days of common bile duct (CBD) ligation, four weeks stenting and hepaticojejunostomy, and group 4, four days of CBD ligation and hepaticojejunostomy. All dogs were sacrificed two months after the last procedure. Hepatic biopsies were obtained during each procedure and bile duct biopsies during hepaticojejunostomy and upon sacrifice. Four weeks of stenting of a normal or obstructed CBD resulted in fibrosed bile ducts, showing severe chronic inflammation with papillary hyperplasia of the epithelium. All bile cultures grew fecal bacteria. Two months after stent removal, inflammation was still present, albeit less severe. Stenting and subsequent surgical treatment resulted in a higher incidence of postoperative complications (54 percent) compared with the control group (14 percent), although this did not reach statistical significance. Hepatic histologic factors were not markedly changed after transpapillary endoprosthesis placement, but after hepaticojejunostomy cholangiolitis was observed. Whenever transpapillary biliary endoprostheses are used, the local effects on the extrahepatic bile ducts and the subsequent bacterial contamination of the bile should be considered.

Light and scanning electron microscopic studies of the extrahepatic bile duct of sheep with experimentally induced Fasciola hepatica infection.

Changes in the common bile duct that contained adult Fasciola hepatica of sheep were evaluated by light (LM) and scanning electron microscopy (SEM). Nine ewes were inoculated with F hepatica metacercariae and necropsied 18 weeks after inoculation. The proximal portion of the common bile duct (CBD) that contained adult flukes was recovered and examined by LM and SEM. The CBD from 2 noninoculated ewes were used for control. On gross examination, CBD were markedly large because of the adult flukes, which were free in the lumen of the ducts. Extensive hemorrhage was not found either in intrahepatic or in extrahepatic bile ducts of any sheep. Histologic examination revealed changes, such as villous hyperplasia and hypertrophy of the epithelium; cell infiltration, predominantly with eosinophils or macrophages; and arterial intimal proliferation. By SEM, the epithelial surface of the CBD appeared intact. Villous hyperplasia and hypertrophy of the epithelium observed by LM was clearly seen by SEM. Epithelial damage, seen as small areas of denuded surface by LM and SEM, was confined to a few areas of the mucosa. Lack of extensive hemorrhage and confined epithelial damage were evaluated relative to the mode of feeding of adult flukes.

Effect of biliary infection on common bile duct healing in the rat.

It has been postulated that biliary infection plays a role in bile duct stricture formation. The aim of this study was to verify this hypothesis and to evaluate the effect of biliary infection on common bile duct healing. A 3-mm longitudinal choledochotomy was performed in 120 rats and closed with a continuous 11/0 Ethilon suture. Common bile duct division with end-to-end anastomosis using interrupted 11/0 Ethilon sutures was performed in another 30 rats. Biliary infection was achieved in half of the animals with retrograde injection of living 046:K1:H31 strain Escherichia coli recovered from the rat colonic content. All rats with choledochotomy, including those with biliary infection, showed no bile leakage at the suture line, and the bursting pressure at the site of choledochotomy exceeded 40 mmHg as early as 24 h. Rats with common bile duct anastomosis alone showed no stricture formation for up to 6 months after operation. All rats with biliary sepsis developed complete occlusion at the anastomosis. On scanning electron microscopy, the biliary epithelium was well preserved in all rats. The study suggests that in rats with biliary sepsis the risk of bile leakage after primary closure of the common bile duct is negligible, but biliary infection may play a critical role in common bile duct stricture formation.

[A comparative electron microscope study on the fine structure of the muscular coat in the common bile duct].

Little is known about the architecture of common bile duct, especially smooth muscle, except for Oddi's sphincter. The purpose of this study is to reveal the ultrastructural architecture of the upper part of the common bile duct from the viewpoint of comparative anatomy. Animals used in this study were the hamster, rat, dog, monkey and rabbit, and the results obtained were as follows. There were considerable differences in the structural appearance and distribution of smooth muscle cells in the upper part of the common bile duct among the animals examined. In the rat, a small number of smooth muscle cells were sparsely distributed in the subepithelial connective tissue of the duct. In the dog, smooth muscle cells showed a star-shaped appearance with many cytoplasmic processes, by which networks were formed. In the monkey, smooth muscle cells with fibroblast-like appearance were circularly distributed. In the monkey and rabbit, muscular coats consisting of typical smooth muscle cells were observed in the outermost layer of the duct. The muscular coat in the monkey was arranged longitudinally, and in the rabbit circularly. The muscular coat in the rabbit was thicker than that in the monkey. In the hamster, however, the duct wall was devoid of smooth muscle cells. The present study suggests that the occurrence of smooth muscular coat is not essential for transport of the bile.

Immunohistochemical localization of proteoglycans in interstitial elements of human pancreas and biliary system.

The immunohistochemical localization of large proteoglycan and small proteoglycan was observed, using antibodies 2B1 and 6B6 (Sobue et al., 1988, 1989a), in fetal and adult pancreas and biliary system as well as in tumour tissues, obtained from 11 autopsies and 74 biopsies. The distribution of chondroitin 4- and 6-sulphate side chains, type I and IV collagen and elastin were also studied. In adult pancreas and all the biliary tracts examined, periductal fibrous tissues consisted mainly of dermatan sulphate small proteoglycan with networks of fibrous elements, which were composed of large proteoglycan, elastin, type I collagen and type IV collagen. In the interstitial components of cystadenoma of pancreas and biliary duct carcinoma, similar small proteoglycan-rich components were relatively abundant, although large proteoglycan was present in much larger amounts than that in non-neoplastic adult tissues. In some cholangiomas, the extra- and intracellular hyaline globules formed by the carcinoma cells were found to contain chondroitin sulphate large proteoglycan, laminin and fibronectin. The distribution of proteoglycans was observed to be different in the arterial walls of the interlobular tissues of the adult and the fetal pancreas. The biological significance of large and small proteoglycans in the interstitial connective tissues was discussed.

In vitro duct-like structure formation after isolation of bile ductular cells from a murine model.

The epithelium of the biliary tree is involved in the response to numerous liver disease processes including immunologic destruction during liver transplant rejection and liver graft-versus-host disease after bone marrow transplantation. Furthermore, very little is known concerning the function of this previously inaccessible epithelium, because attempts to isolate and culture biliary cells have been unsuccessful until recently. We present a method for the isolation and culture of bile ductular cells from mice with external bile duct obstruction. Over 85% of the isolated cells stain positive for cytokeratin 19, which is characteristic of murine biliary epithelium. Incubation with recombinant murine gamma-interferon resulted in increased class II antigen expression on the isolated cell surface. When these cells were placed, on a basement membrane matrix, they formed duct-like structures composed of cells that had the morphologic characteristics of bile ductular epithelium when examined by transmission electron microscopy. The ability to isolate murine biliary epithelium that forms duct-like structures will be useful for the in vitro study of biliary epithelial characteristics and injury.

A scanning electron microscopic study of the common bile duct epithelium in the rabbit during experimental biliary tract obstruction.

Complete biliary obstruction was induced in rabbits by distal ligation of the common bile duct (CBD). The epithelial surface of the CBD was studied with scanning electron microscopy (SEM) at 1-6 days post-ligation. Concomitant biochemical assessments and pressure measurements in the CBD were performed. SEM of non-ligated CBD showed that the epithelium is arranged in folds and clefts, the latter displaying interdigitating cellular processes. At 2 days post-ligation, the epithelial clefts were focally distended showing marked dehiscence of the cellular processes with exposure of the subepithelial connective tissue layer. At 4-6 days post-ligation, extensive defects in the epithelium were observed, frequently with a sharp demarcation between intact epithelium and exposed connective tissue stroma.

The fine structure and histochemistry of human bile duct in obstruction and choledocholithiasis.

Biopsies from the common bile ducts from seven patients undergoing surgery for biliary obstruction due to stones or malignancy were studied histochemically and electron microscopically. The surface of the bile duct is lined by a tall epithelium which extends into diverticula. Apically, they contain some neutral and sialated mucosubstances. Fucosyl residues were found in the Golgi apparatus and along the apical cell membrane. The latter is lined by microvilli. There was a well-developed rough endoplasmic reticulum and Golgi apparatus and a small number of apical secretory droplets. Large numbers of lipid droplets were present basally in some cells. Lipid-containing macrophages were also seen intra-epithelially and in the lamina propria. This suggests a possible pathway for lipid transport. The glands were lined by cuboidal cells, some containing much mucus--sulphated, sialated, and neutral with a basal nucleus. A well-developed endoplasmic reticulum and Golgi apparatus were found with abundant secretory droplets. The glandular epithelium contained lysozyme, alpha-1-antitrypsin, and alpha-1-antichymotrypsin. These may play a protective role. The lamina propria contained scattered smooth muscle cells amongst the fibroblasts and inflammatory cells.

[Ultrastructures of smooth muscles of the choledochus at the choledocho-duodenal junction].

The inner circular muscle (ICM) of the cat duodenum at the choledocho-duodenal junction is thickened, surrounding the choledochus at its proximal portion within the duodenal wall. Such a thickened portion of ICM is considered to be a main part which serves as a sphincter of the choledochus. This portion of the ICM was referred to as 'sphincter of choledochus' in the present study. The sphincter of choledochus and the adjacent ICM were studied by freeze-fracture and conventional thin sectioning methods by electron microscopy in order to clarify the morphological characteristics of smooth muscle cells in the sphincter of choledochus in comparison with ICM. Smooth muscle cells of the sphincter of choledochus were smaller (3-5 microns in diameter) in size, and had less developed dense patches and dense bands. However, they were richly innervated and had numerous gap junctions for electric coupling between cells. These findings indicate that sphincter of choledochus, though its contraction ability may be weaker than the ICM, can exhibit a highly synchronized contraction needed for the sphincter function.