Intact Pancreatic Tissue Research Articles

Texture analysis of contrast enhancement СT in the differential diagnosis of mass-forming pancreatitis and pancreatic ductal adenocarcinoma

Purpose. Improving the efficiency of CT in the differential diagnosis of mass-forming pancreatitis (MFP) and pancreatic ductal adenocarcinoma (PDAC) making a diagnostic model based using a combination of texture features and contrast enhancement features.Methods and materials. 45 patients with histologically confirmed non-metastatic locally advanced PDAC and 13 patients with MFP where underwent CT examination with contrast enhancement. For each group, the ratio of the densities of intact pancreatic tissue and tumors, the relative tumor enhancement ratio (RTE) in all enhanced phases of CT, 94 texture features for each phase of the study were calculated and compared. The selection of predictors in the logistic model was carried out in 2 stages: 1) selection of predictors based on one-factor logistic models, the selection criterion was padj <0.2; 2) selection of predictors using LASSO-regression after standardization of variables. The selected predictors were included in a logistic regression model without interactions.Results. There were statistically significant differences in 14, 17, 4 out of 94 for the unenhanced, arterial, and venous phases of the study, respectively (p < 0.05). After selection, the final diagnostic model included the texture features CONVENTIONAL HUQ2 and DISCRETIZED HUQ1 for the unenhanced phase, DISCRETIZED HUQ1 and GLRLM RLNU for the arterial phase, DISCRETIZED Skewness for the venous phase, RTE for the delayed CT phase. The diagnostic model was built showed an accuracy of 81% in the diagnosis of MFP.Conclusion. We have developed a diagnostic model, including textural parameters and contrast enhancement features, which allows preoperatively distinguish MFP and PDAC, the developed model will increase the accuracy of preoperative diagnosis.

Texture analysis of contrast enhancement СT in the differential diagnosis of mass-forming pancreatitis and pancreatic ductal adenocarcinoma

Purpose. Improving the efficiency of CT in the differential diagnosis of mass-forming pancreatitis (MFP) and pancreatic ductal adenocarcinoma (PDAC) making a diagnostic model based using a combination of texture features and contrast enhancement features.Methods and materials. 45 patients with histologically confirmed non-metastatic locally advanced PDAC and 13 patients with MFP where underwent CT examination with contrast enhancement. For each group, the ratio of the densities of intact pancreatic tissue and tumors, the relative tumor enhancement ratio (RTE) in all enhanced phases of CT, 94 texture features for each phase of the study were calculated and compared. The selection of predictors in the logistic model was carried out in 2 stages: 1) selection of predictors based on one-factor logistic models, the selection criterion was p@adj <0.2; 2) selection of predictors using LASSO-regression after standardization of variables. The selected predictors were included in a logistic regression model without interactions.>Results. There were statistically significant differences in 14, 17, 4 out of 94 for the unenhanced, arterial, and venous phases of the study, respectively (p < 0.05). After selection, the final diagnostic model included the texture features CONVENTIONAL HUQ2 and DISCRETIZED HUQ1 for the unenhanced phase, DISCRETIZED HUQ1 and GLRLM RLNU for the arterial phase, DISCRETIZED Skewness for the venous phase, RTE for the delayed CT phase. The diagnostic model was built showed an accuracy of 81% in the diagnosis of MFP.Conclusion. We have developed a diagnostic model, including textural parameters and contrast enhancement features, which allows preoperatively distinguish MFP and PDAC, the developed model will increase the accuracy of preoperative diagnosis.

SUN-654 Dynamic and Regional Variation of Pancreatic Innervation in Diabetes

Background: The pancreas is a highly heterogeneous organ, with regional anatomical, developmental and functional differences. The endocrine pancreas is densely innervated, and neural signals play a significant role in glucose regulation by modulating pancreatic hormone release. However, relatively little is known about the anatomical relationships between islets and nerves across the whole pancreas. Since thin filamentous structures, such as nerves, are difficult to quantify and trace over large volumes using thin section histology, there is a need for high resolution imaging and rendering of intact pancreatic tissue in 3D. Aim: To use optical clearing, whole organ imaging, and 3D rendering to quantify islets and innervation across the whole pancreas in healthy mice, in two mouse models of diabetes, and in pancreatic samples from nondiabetic and diabetic human donors. Methods: Whole-mount staining and clearing was performed using iDISCO+ to quantify innervation, defined by the neuronal marker NF200, and beta cells in pancreata from C57Bl/6 mice, non-obese diabetic (NOD) mice, streptozotocin (STZ)-treated mice, and in pancreatic samples from nondiabetic and diabetic human donors. Z-stacked optical sections were acquired with an Ultramicroscope II at 4x or 12x magnification. Imaris was used to create digital surfaces covering the NF200+ innervation and islets to automatically determine innervation density and islet/nerve interactions. Results: Beta cell volumes were 1-4% in the human pancreas, and 1-2% in the healthy mouse pancreas, with regional variations in islet volume and insulin intensity. There were also significant differences in islet biology between the diabetes models. Innervation of the endocrine pancreas was significantly enriched compared to the surrounding exocrine pancreas, with regional variation. Islets were closely associated with pancreatic innervation and decreased in size with increasing distance from nerves in both mouse and human pancreatic tissue. Innervated islets were relatively preserved in models of diabetes. Finally, islet innervation and expression of neural markers were higher in human samples from diabetic patients and in mouse models of diabetes, with temporal and regional differences. Conclusions: 3D imaging and unbiased analysis across the whole pancreas provides comprehensive measurement of pancreatic nerve volumes and distribution. It allows detailed analysis of the anatomical relationship between nerves and islets, and reveals a close association that is maintained across species. The relative enrichment of innervated islets in diabetes and dynamic changes in islet innervation during the development of diabetes suggest further work is needed to examine the role of pancreatic nerves in preserving and protecting beta cells.

Using Pancreas Tissue Slices for the Study of Islet Physiology.

Studies on islet of Langerhans physiology are crucial to understand the role of the endocrine pancreas in diabetes pathogenesis and the development of new therapeutic approaches. However, so far most research addressing islet of Langerhans biology relies on islets obtained via enzymatic isolation from the pancreas, which is known to cause mechanical and chemical stress, thus having a major impact on islet cell physiology. To circumvent the limitations of islet isolation, we have pioneered a platform for the study of islet physiology using the pancreas tissue slice technique. This approach allows to explore the detailed three-dimensional morphology of intact pancreatic tissue at a cellular level and to investigate islet cell function under near-physiological conditions. The described procedure is less damaging and faster than alternative approaches and particularly advantageous for studying infiltrated and structurally damaged islets. Furthermore, pancreas tissue slices have proven valuable for acute studies of endocrine as well as exocrine cell physiology in their conserved natural environment. We here provide a detailed protocol for the preparation of mouse pancreas tissue slices, the assessment of slice viability, and the study of pancreas cell physiology by hormone secretion and immunofluorescence staining.

Antarease-like Zn-metalloproteases are ubiquitous in the venom of different scorpion genera

BackgroundThe venoms of several scorpion species have long been associated with pancreatitis in animal models and humans. Antarease, a Zn-metalloprotease from Tityus serrulatus, is able to penetrate intact pancreatic tissue and disrupts the normal vesicular traffic necessary for secretion, so it could play a relevant role in the onset of acute pancreatitis. MethodsThe cDNA libraries from five different scorpion species were screened for antarease homologs with specific primers. The amplified PCR products were cloned and sequenced. A structural model was constructed to assess the functionality of the putative metalloproteases. A phylogenetic analysis was performed to identify clustering patterns of these venom components. ResultsAntarease-like sequences were amplified from all the screened cDNA libraries. The complete sequence of the antarease from T. serrulatus was obtained. The structural model of the putative antarease from Tityus trivittatus shows that it may adopt a catalytically active conformation, sharing relevant structural elements with previously reported metalloproteases of the ADAM family. The phylogenetic analysis reveals that the reported sequences cluster in groups that correlate with the geographical localization of the respective species. ConclusionsAntareases are ubiquitous to a broad range of scorpion species, where they could be catalytically active enzymes. These molecules can be used to describe the evolution of scorpion venoms under different ecogeographic constrains. General significanceFor the first time the complete sequence of the antareases is reported. It is demonstrated that antareases are common in the venom of different scorpion species. They are now proposed as targets for antivenom therapies.

Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

Patterns of Pathomorphological Changes in Acute Necrotizing Pancreatitis

Acinar necrosis is the basic microscopic sign of acute necrotizing pancreatitis (ANP). Microcirculation disorder is one of the major factors in the pathogenesis and morphogenesis of ANP besides free radicals and damage of enzymatic origin. This study is dedicated to the description of microscopic changes in the pancreatic stroma in ANP, which leads to destruction of the exocrine pancreas with a putative mechanism of endocrine function preservation. This study has been carried out on histological samples of pancreas from 224 patients with ANP. Histological staining was performed with hematoxylin-eosin (H&E), Masson, Gomori methods, and PAS. Microscopy was performed with magnifications of 40×, 100×, and 400×. Vascular endothelial desquamation, stasis, and sludge are typical changes in microcirculation observed in early stages of ANP. Initially, parietal circular intravascular microthrombosis accompanied by endothelial desquamation as early as stromal swelling occurs with no detectable necrosis. Residual stroma appears between areas of necrosis and intact pancreatic tissue. Mucoid swelling is first seen in the perivascular spaces extending to the parenchyma and changing into fibrinoid imbibition causing further necrosis. Reticulin argyrophilic backbone surrounding the pancreatic acini and small ducts decompose. Pancreatic structures, which may be preserved in necrotic tissue, include nerves, major ducts, and Langerhans islets.

Discrimination of metabolic profiles of pancreatic cancer from chronic pancreatitis by high‐resolution magic angle spinning 1H nuclear magnetic resonance and principal components analysis

The metabolic profiles of Sprague-Dawley rat pancreases were investigated by high-resolution magic angle spinning proton magnetic resonance spectroscopy ((1)H NMR) combined with principal components analysis (PCA) to discriminate pancreatic cancer from chronic pancreatitis. Intact pancreatic tissue samples were obtained from Sprague-Dawley rats with histologically proven pancreatic cancer (n = 5), chronic pancreatitis (n = 5), and two matched controls (n = 5 per group). Two (1)H NMR experiments, single-pulse and Carr-Purcell-Meiboom-Gill, were carried out separately. Increases in phosphocholine and glycerophosphocholine levels and decreases in leucine, isoleucine, valine, lactate and alanine levels were observed in chronic pancreatitis, whereas the opposite trends were observed in pancreatic cancer. Increasing taurine and decreasing betaine were found both in chronic pancreatitis and in pancreatic cancer. Additionally, the lipid content in pancreatic cancer was higher than that in chronic pancreatitis. PCA was carried out for the single-pulse and Carr-Purcell-Meiboom-Gill (1)H NMR spectra, respectively, to visualize separation among the samples and to extract characteristic metabolites of pancreatic cancer and chronic pancreatitis. Decreased phosphocholine and glycerophosphocholine were suggested as unique metabolite indicators of pancreatic cancer. Furthermore, even with the disturbance of various quantities of lipid contents pancreatic cancer and chronic pancreatitis could be differentiated well by the combination of high-resolution magic angle spinning (1)H NMR and PCA. Thus this combination was demonstrated to have the potential to improve magnetic resonance spectroscopy for positive early diagnosis of pancreatic cancer in clinical settings.

Stable Golgi-Mitochondria Complexes and Formation of Golgi Ca2+ Gradients in Pancreatic Acinar Cells

We have determined the localization of the Golgi with respect to other organelles in living pancreatic acinar cells and the importance of this localization to the establishment of Ca(2+) gradients over the Golgi. Using confocal microscopy and the Golgi-specific fluorescent probe 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine, we found Golgi structures localizing to the outer edge of the secretory granular region of individual acinar cells. We also assessed Golgi positioning in acinar cells located within intact pancreatic tissue using two-photon microscopy and found a similar localization. The mitochondria segregate the Golgi from lateral regions of the plasma membrane, the nucleus, and the basal part of the cytoplasm. The Golgi is therefore placed between the principal Ca(2+) release sites in the apical region of the cell and the important Ca(2+) sink formed by the peri-granular mitochondria. During acetylcholine-induced cytosolic Ca(2+) signals in the apical region, large Ca(2+) gradients form over the Golgi (decreasing from trans- to cis-Golgi). We further describe a novel, close interaction of the peri-granular mitochondria and the Golgi apparatus. The mitochondria and the Golgi structures form very close contacts, and these contacts remain stable over time. When the cell is forced to swell, the Golgi and mitochondria remain juxtaposed up to the point of cell lysis. The strategic position of the Golgi (closer to release sites than the bulk of the mitochondrial belt) makes this organelle receptive to local apical Ca(2+) transients. In addition the Golgi is ideally placed to be preferentially supplied by ATP from adjacent mitochondria.

Long Distance Communication between Muscarinic Receptors and Ca2+ Release Channels Revealed by Carbachol Uncaging in Cell-attached Patch Pipette

We have investigated the characteristics of cytosolic Ca2+ signals induced by muscarinic receptor activation of pancreatic acinar cells that reside within intact pancreatic tissue. We show that these cells exhibit global Ca2+ waves and local apical Ca2+ spikes. This is the first evidence for local Ca2+ signaling in undissociated pancreatic tissue. The mechanism of formation of localized Ca2+ signals was examined using a novel approach involving photolysis of caged carbachol inside a patch pipette attached to the basal surface of an acinar unit. This local activation of basal muscarinic receptors elicited local cytosolic Ca2+ spikes in the apical pole more than 15 microm away from the site of stimulation. In some experiments, local basal receptor activation elicited a Ca2+ wave that started in the apical pole and then spread toward the base. Currently, there are two competing hypotheses for preferential apical Ca2+ signaling. One invokes the need for structural proximity of the cholinergic receptors and the Ca2+ release channels in the apical pole, whereas the other postulates long distance communication between basal receptors and the channels. Our intrapipette uncaging experiments provide definitive evidence for long distance communication between basal muscarinic receptors and apical Ca2+ release channels.

Heterogeneity of the human exocrine pancreas: Antidigestive enzyme antibody and lectin binding

We have previously reported the existance of fundamental heterogeneity of the exocrine pancreas in animals; binding of specific anti-digestive enzyme antibodies and lectins to intact pancreatic tissue and isolated acinar cells of rabbit and rat revealed highly heterogenous patterns of distribution of acinar cell digestive enzyme contents (J Histochem Cytochem 1995;44:215 and Gastroenterol 1997;112:A435). Aim: To determine if the adult human pancreas is heterogeneous. Methods: Areas of normal pancreas were obtained without alteration of the surgical procedure from the margins of resected specimens in 5 patients ages 56-76, two with serous cystadenoma, and three with pancreatic cancer. Tissue was fixed and sectioned by standard methods. Sections were permeabilized with saponin, and treated with commerciallyavailable anti-digestive enzyme antibodies employed singly or in pairs directed against amylase, lipase, chymotrypsin and trypsinogen, or with a panel of lectins. Vizualization was by both diaminobenzidine and fluoresence with FITC or Texas red. Routine hematoxylin and eosin staining was employed to ascertain tissue integrity, morphology and absence of pathology. Results: The human pancreas was distinctly heterogeneous at the morphological level as demonstrated by antidigestive enzyme antibody binding. Several patterns of heterogeneity were readily discerned: first, a pattern unique to the human wherein whole lobules were clearly labeled by one of a pair of antibodies -highly lipase-, chymotrypsinand amylasedominant lobules were noted to contrast with other areas where enzymes were co-localized. Second, an autumn leaves-type pattern was noted in which individual acini were found to contain either amylase or lipase predominantly. A third pattern wherein individual acinar cells differed in digestive enzyme content from neighboring cells within the same acinus, and a fourth pattern of anti-digestive enzyme antibody staining in halos of lipase-rich acinar cells surrounding the islets of Langerhans were also noted. Lectins LTL and UEA also strongly showed lobular patterns of heterogeneous binding. Lectins HPA, ACL, WFA, SBA and SWGA showed heterogeneous patterns from cell-tocell in the zymogen granule region. Lectin PHA showed distinct halos of acinar cells surrounding the islets. Numerous species differences in the patterns of heterogeneity were found; the human gland appeared even more broadly heterogeneous than rabbit or rat, and showed an extensive lobular pattern not seen as readily in the other species. Conclusion: The exocrine pancreas in the human adult is fundamentally heterogeneous with respect to the distribution of individual digestive enzyme species within acinar cells, acini, and pancreatic lobules. The heterogeneous interand intra-acinar patterns as well as the peri-insular halos have been found in all species examined; the highly lobular pattern for lipase, amylase and chymotrypsin is, to date, unique to the human.

Physiologic relevance of heterogeneity in the pancreatic beta-cell population

In vitro studies on purified rat beta cells have indicated a functional diversity among insulin-containing cells. Intercellular differences were found in the rates of glucose-induced insulin synthesis and release. They are attributed to differences in cellular thresholds for glucose utilization and oxidation, as can be caused by varying activities in rate limiting steps such as glucokinase-dependent phosphorylation. The percent of functionally active beta cells increases dose-dependently with the glucose concentration, making cellular heterogeneity and its regulation by glucose major determinants for the dose-response curves of the total beta-cell population. Beta cells which are already responsive to low glucose concentrations are characterized by a higher content in pale immature granules; their activated biosynthetic and secretory activity accounts for preferential release of newly-formed hormone by the total beta-cell population. At any glucose level, the amplitude of insulin release depends on the percent glucose-activated cells and their cyclic AMP content, an integrator of (neuro)hormonal influences. The in vitro described heterogeneity in beta-cell functions may bear physiological relevance as several of its characteristics are also detectable in intact pancreatic tissue; furthermore, in vitro signs of heterogeneity can be altered by prior in vivo treatment indicating that they express properties of the cells in their in situ configuration. Elevated basal levels of (pro)insulin may reflect the existence of an increased number of beta cells that are activated at low physiologic glucose concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Normal responses of rats with transplanted neonatal islets to stress, epinephrine, and cortisol.

Sympathetic responses were evaluated in rats transplanted with neonatal pancreatic tissue. Transplantation was by one of two methods: mild collagenase digestion with subsequent intraportal injection, or direct placement of intact pancreatic tissue under the renal capsule. Compared with control intact animals, both groups of transplanted rats reverted to normal fasting and postglucose load plasma glucose and insulin. The hyperglycemia characteristic of stress or of injected exogenous epinephrine was present and similar in all three groups. The response to an intragastric glucose load or to tolbutamide injection during stress was similar in transplanted and in control animals. All three groups of rats responded to sustained cortisol injection with a compensatory hyperinsulinemia but maintained normal plasma glucose, water intake, and urine volume. No evidence could be detected in this study of an exaggerated "denervated" response of the transplanted islets. The normal sympathetic responses of these neonatal islet preparations in which little or no collagenase was used, in comparison with the abnormal responses previously reported for collagenase-treated individually isolated adult rat islets, suggest differences in recuperative powers of the sympathetic innervation in islets, possibly related to age and to method of preparation of transplanted tissue.

Guinea pig pancreatic acini prepared with purified collagenase

Dispersed guinea pig pancreatic acinar cells have been used to investigate several aspects of stimulus-secretion coupling but possess the disadvantage that they are less sensitive and less responsive to secretagogues than in vitro preparations of intact pancreatic tissue (lobules). To overcome the poor responsiveness of isolated acinar cells, we have developed a new procedure for preparing dispersed, intact pancreatic acini whose sensitivity to secretagogues and morphological characteristics are similar to those of pancreatic lobules. Dispersed acini can be manipulated as suspensions of cells and full access of macromolecular probes to apical and basolateral plasmalemmal domains is obtained. Acini were prepared in good yields (~70% on a DNA basis) using only purified collagenase and mild mechanical shear in medium containing 2.0 mM Ca 2+. Morphologically, acinar cells in the preparations retained intact junctional complexes, asymmetrical distribution of intramembranous particles between apical and basolateral plasmalemmal domains, and polarized distribution of intracellular organelles as found in intact pancreas. Dose-response curves of acini and mechanically prepared lobules to caerulein, carbachol, and bombesin were similar though acini were more sensitive to the C-terminal octapeptide of cholecystokinin. Net stimulated secretory protein discharge was ~36% over 2 h. Crude collagenase was purified for use in preparation of acini by Sephadex G-75 column chromatography which resolved collagenase from clostripain and a non-sulfhydryl-requiring protease. The purified collagenase contained at least four proteins with molecular weights between 85 000 and 110 000. Collagenase with <0.14 units of protease per unit of collagenase produced highly responsive acini; collagenase with >0.9 units of protease per unit of collagenase yielded unresponsive acini. Acini incubated with crude collagenase, chymotrypsin, or the non-sulfhydryl-requiring protease showed depressed secretory response to caerulein. Freeze-fracture electron microscopy of protease-treated acini indicated that the intramembranous particles aggregated and that many of the tight junctions had undergone a proliferation of non-cross-linked sealing strands which extended far down the basolateral plasma membrane and encircled gap junctions. Acini incubated with purified collagenase or with a clostripain-containing fraction from the Sephadex G-75 column appeared unaltered. This procedure produces acini which are morphologically and biochemically similar to the in situ pancreas and overcomes the poor response to secretagogues by isolated pancreatic acinar cells.

Transplantation of pancreatic beta-cells prevents development of hypothalamic obesity in rats.

The present experiments have tested the hypothesis that ventromedial hypothalamic (VMH) lesions enhance insulin secretion by neural mechanisms. Rats were made diabetic by injecting streptozotocin to destroy their own pancreatic beta-cells. Subsequently, transplants of fetal pancreatic tissue were placed under the renal capsule. VMH lesions were placed in rats whose diabetes was cured with transplants as well as sham-transplanted animals. The animals were followed for 4 wk. The lesioned rats with pancreatic transplants gained no more weight than the sham-operated controls. There was no significant rise in insulin in the transplanted rats after VMH lesioning, but the VMH lesioned rats with intact pancreatic tissue showed the expected rise in insulin. Food intake rose 71% in the VMH lesioned rats with intact beta-cells, but only 23% in the VMH lesioned rats with transplants. Hypertrophy of the pancreatic islets was also observed in the VMH lesioned rats with an intact pancreas, but was not found in the VMH lesioned rats with a transplanted pancreas. Thus, transplantation of pancreatic tissue beneath the renal capsule of diabetic rats prevented the characteristic hyperphagia, hyperinsulinemia, and obesity in VMH lesioned rats whose pancreas was free from intact innervation. The results support the hypothesis that neural mediation of the rise in insulin is the primary factor in the development of hypothalamic obesity.