Enzyme Histochemical Methods Research Articles

Porcine skeletal muscle typing in histochemical and in-situ RT-PCR analysis

Currently, there are plenty of histochemical methods to classify pig muscle fibers, which confused the naming and classification of muscle fibers. This study aims to analyze the difference and correlation of 6 different histochemical methods and select the most suitable method for muscle fiber classification at the molecular and histomological levels by in-situ RT-PCR and enzyme histochemical methods. Muscle fiber samples, including psoas (PM), semitendinosus (SM) and trapezius muscle (TM), were collected from Large Spotted (LS), Lantang (LT) and Landrace (LR) pigs at their market-ages (LS at 150 d, LT at 210 d, and LR at 150 d). 6 kinds of histochemical methods combining actomyosin adenosine triphosphatase (AM-ATPase) with succinate dehydrogenase (SDH) enzyme were conducted to differentiate fiber types. 2 types of fibers (I and II) were differentiated by acid 2-fibre (2-AC) or alkaline 2-fibre classification(2-AL), 3 types of fibers (βR, αR and αW) by 3-AC or 3-AL, and 4 types of fibers (I, IIa, IIx and IIb) by 4-AC, or 4-AL. Results showed that AC and AL muscle-fiber classification were consistent in reflecting the characteristics of muscle fibers(P > 0.05), but the color of each muscle fiber type was just opposite. AC methods may be superior to AL methods because of their clear staining background, the sensitivity to staining condition. But there were breed differences and tissue specificity in the optimal preincubation condition. The optimal acid preincubation condition for classifying muscle fibers was pH4.30 for LT, while pH 4.35 for the LS and LR pigs. Meanwhile the optimal acid preincubation condition was pH4.35 for PM, while pH4.40 for TM or SM. For further selection from 2, 3, 4-AC, in-situ RT-PCR was applied to detect the mRNA distribution of myosin heavy chain I (MyHC-I). By combining in-situ PCR with enzyme histochemistry methods, MyHC-I gene and its product – Type I fibrocytes were directly located in cells at both molecular level and morphological level. Compared with the cross-sectional area (CSA) of different muscle fibers (i.e. I, II, βR, αR, αW, IIa, IIx and IIb) identified by enzyme histochemistry, it was found that the CSAs with stronger mRNA expression signal of MyHC-Ⅰ were closer to those of the Type I muscle fiber measured by 4-AC enzyme histochemistry (P > 0.05). Therefore, 4-AC may be considered as the most proper muscle typing method to study muscle fiber typing as well as meat quality. And the combination of in-situ RT-PCR and histochemistry may help better understand porcine muscle fiber characteristics and meat quality in pigs.

Senescent tumor cells in colorectal cancer are characterized by elevated enzymatic activity of complexes 1 and 2 in oxidative phosphorylation.

Cellular senescence is defined as an irreversible cell cycle arrest caused by various internal and external insults. While the metabolic dysfunction of senescent cells in normal tissue is relatively well-established, there is a lack of information regarding the metabolic features of senescent tumor cells. Publicly available single-cell RNA-sequencing data from the GSE166555 and GSE178341 datasets were utilized to investigate the metabolic features of senescent tumor cells. To validate the single-cell RNA-sequencing data, we performed senescence-associated β-galactosidase (SA-β-Gal) staining to identify senescent tumor cells in fresh frozen colorectal cancer tissue. We also evaluated nicotinamide adenine dinucleotide dehydrogenase-tetrazolium reductase (NADH-TR) and succinate dehydrogenase (SDH) activity using enzyme histochemical methods and compared the staining with SA-β-Gal staining. MTT assay was performed to reveal the complex 1 activity of the respiratory chain in in-vitro senescence model. Single-cell RNA-sequencing data revealed an upregulation in the activity of complexes 1 and 2 in oxidative phosphorylation, despite overall mitochondrial dysfunction in senescent tumor cells. Both SA-β-Gal and enzyme histochemical staining using fresh frozen colorectal cancer tissues indicated a high correlation between SA-β-Gal positivity and NADH-TR/SDH staining positivity. MTT assay showed that senescent colorectal cancer cells exhibit higher absorbance in 600 nm wavelength. Senescent tumor cells exhibit distinct metabolic features, characterized by upregulation of complexes 1 and 2 in the oxidative phosphorylation pathway. NADH-TR and SDH staining represent efficient methods for detecting senescent tumor cells in colorectal cancer.

The determination of the effect of in ovo administered monosodium glutamate on the embryonic development of thymus and bursa of Fabricius and percentages of alpha-naphthyl acetate esterase positive lymphocyte in chicken.

Monosodium glutamate (MSG) is a flavor enhancer commonly used in modern nutrition. In this study, it was aimed to determine the effect of in ovo administered MSG on the embryonic development of thymus, bursa of Fabricius, and percentages of alpha-naphthyl acetate esterase (ANAE) positive lymphocyte by using histological, histometrical, and enzyme histochemical methods in chickens. For this purpose, 410 fertile eggs were used. The eggs were then divided into five groups: group 1 (control group, n = 40 eggs), group 2 (distilled water-injected group, n = 62 eggs), group 3 (0.12mg/g egg MSG-injected group, n = 80 eggs), group 4 (0.6mg/g egg MSG-injected group, n = 90 eggs), and group 5 (1.2mg/g egg MSG-injected group, n = 138 eggs), and injections were performed via the egg yolk. On the 18th and 21st days of the incubation, the eggs were randomly opened from each group until six live embryos were obtained. The embryos of each group were sacrificed by decapitation, and blood, thymus, and bursa of Fabricius tissue samples were taken from the obtained embryos. The MSG-treated groups were found to be retarded embryonic development of thymus and bursa of Fabricius tissue compared to the control and distilled water groups. MSG treatment also resulted in reduced lymphoid follicles count and follicle diameters in bursa of Fabricius (P < 0.05). The percentage of peripheral blood ANAE positive lymphocytes was significantly lower in the MSG-treated groups than in the control and distilled water groups (P < 0.05). In conclusion, it has been found that in ovo administered MSG can adversely affect the embryonic development of thymus and bursa of Fabricius and decrease percentage of ANAE positive lymphocyte.

Side Effects of In ovo Given Sunset Yellow FCF on the Embryonic Development of the Spleen by Means of Histological and Enzyme Histochemical Methods

Background: E110 is one of the food colorants which is widely used in dairy products, fast foods, jam and dry beverage powders, aqueous drug solutions, tablets, capsules, toothpastes mouthwashes, hair care products and cosmetics. Doubts have accumulated in recent years that food additives might cause allergic reactions in humans or increase these ailments. In this study, side effects of Sunset yellow FCF (E110) on the embryonic development of chicken spleen were evaluated by means of histological, histomorphometrical and enzyme histochemical methods. Methods: In the study, 250 fertilized broiler eggs obtained from a commercial broodstock were used. Eggs were divided into 5 groups each having 50 eggs. Control eggs were either nontreated or distilled water injected via air sac. The eggs in the experimental groups were injected with 100ng/egg, 500ng/egg and 1.000ng/egg E110 prior to incubation. Blood and spleen samples were taken from randomly selected 10 eggs of each group at 11th, 15th, 18th and 21st days of incubation. Leukocyte formula, alpha-naphtyl acetate esterase-positive and acid phosphatase-positive lymphocyte percentages were determined in the blood samples and embriyonic development of the spleen was assessed in the tissue sections. Results: In the 500ng/egg and 1.000ng/egg E110 injected experimental groups, embryonic development of the spleen retarded, alpha-naphtyl acetate esterase-positive and acid phosphatase-positive lymphocyte rates were significantly depressed. Conclusions: Significant disturbances in the immune system functions of the affected animals might occure at post-natal period of their life.

The effects of bisphenol A given in ovo on bursa of Fabricius development and percentage of acid phosphatase positive lymphocyte in chicken.

Bisphenol A (BPA), one of the endocrine disrupting chemicals, is the object of great concern because of its widespread use throughout the world. In this study, it was aimed to determine the effects of in ovo administrated BPA on the bursa of Fabricius and percentage of acid phosphatase positive lymphocyte in peripheral blood by means of histological and enzyme histochemical methods. For this purpose, 310 fertile eggs of Isa Brown laying parent stock were used. The eggs were divided into 5 groups as control, vehicle control, 50, 100, and 250μg/egg BPA. At days 13, 18, and 21 of incubation, eggs were opened until 10 living embryos were obtained from each group. Tissue samples were taken from the obtained embryos and processed for enzyme histochemical methods in addition to routine histological techniques. It was observed that, in BPA-treated groups, embryonic development of bursa of Fabricius was retarded. It was also indicated that the percentage of peripheral blood ACP-ase positive lymphocytes was significantly decreased. These results suggested that a limited maternal transfer of BPA into the eggs might be lead to immunosuppression in chicks.

Determination of the changes on the small intestine of pregnant mice by histological, enzyme histochemical, and immunohistochemical methods.

The aim of the present study was to determine the changes on the small intestine in mice during pregnancy using histological, enzyme histochemical, and immunohistochemical methods. A total of 24 Swiss albino female mice were divided as non-pregnant/control, first week, second week, and third week of pregnancy (n=6). Tissue samples obtained from the duodenum, jejunum, and ileum were processed by means of routine histological techniques and stained with Crossmon's triple staining. Alkaline phosphatase (ALP) was demonstrated with the simultaneous azo-coupling method. Proliferating cell nuclear antigen (PCNA) was demonstrated with the streptavidin-biotin-peroxidase complex method. The numerical data of the parameters were obtained and analyzed statistically. Villus height, villus width, and the rate of villus height/crypt depth were decreased in the duodenum, jejunum, and ileum in the last week of pregnancy compared with the control group. Changes in the crypt depth of the duodenum, jejunum, and ileum in pregnancy were found. The muscle width increased in pregnancy. It was identified that the ALP reactivity statistically significantly increased in the duodenum, jejunum, and ileum in pregnancy. The percentage of PCNA-positive cells in the duodenum, jejunum, and ileum increased in the first and second weeks of pregnancy, whereas it decreased in the third week of pregnancy compared with non-pregnant control animals. In conclusion, villus parameters, ALP reactivity, and percentage of PCNA-positive cells in the small intestine were affected during pregnancy.

A novel histochemistry assay to assess and quantify focal cytochrome c oxidase deficiency

Defects in the respiratory chain, interfering with energy production in the cell, are major underlying causes of mitochondrial diseases. In spite of this, the surprising variety of clinical symptoms, disparity between ages of onset, as well as the involvement of mitochondrial impairment in ageing and age‐related diseases continue to challenge our understanding of the pathogenic processes. This complexity can be in part attributed to the unique metabolic needs of organs or of various cell types. In this view, it remains essential to investigate mitochondrial dysfunction at the cellular level. For this purpose, we developed a novel enzyme histochemical method that enables precise quantification in fresh‐frozen tissues using competing redox reactions which ultimately lead to the reduction of tetrazolium salts and formazan deposition in cytochrome c oxidase‐deficient mitochondria. We demonstrate that the loss of oxidative activity is detected at very low levels – this achievement is unequalled by previous techniques and opens up new opportunities for the study of early disease processes or comparative investigations. Moreover, human biopsy samples of mitochondrial disease patients of diverse genotypic origins were used and the successful detection of COX‐deficient cells suggests a broad application for this new method. Lastly, the assay can be adapted to a wide range of tissues in the mouse and extends to other animal models, which we show here with the fruit fly, Drosophila melanogaster. Overall, the new assay provides the means to quantify and map, on a cell‐by‐cell basis, the full extent of COX deficiency in tissues, thereby expending new possibilities for future investigation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Characterization of mast cell populations using different methods for their identification.

Mast cells are ubiquitous throughout the human tissues and play an essential role in physiology and pathology. For evaluation of patients with pathological conditions, mast cells were primarily detected using metachromatic staining with toluidine blue. In the last decades, the staining arsenal of pathologists was enriched with enzyme histochemical and immunohistochemical methods, and it was established that depending on species and tissue localization mast cells are not similar both in appearance and function. The aim of this study was to characterize different mast cell populations using the up-to-date methods of their identification. We compared standard metachromatic method for mast cells with enzyme histochemical detection of chloroacetyl esterase and with immunohistochemical detection of tryptase and chymase in human and rodent tissues. Combination of these methods allowed us to assay quantitatively mast cell populations in different organs of humans and rodents. Furthermore, we assessed the appropriate implementation of each of these methods for mast cell identification in diagnostic labs.

Histochemical Demonstration of Tripeptidyl Aminopeptidase I.

Enzyme histochemical methods are valuable for the studies on the enzyme involvement in different pathological processes. Here we describe two protocols for chromogenic and fluorogenic histochemical demonstration of tripeptidyl aminopeptidase I (TPPI), a protease that is crucial for neuronal functions. The procedures are based on newly synthesized substrates for TPPI-glycyl-L-prolyl-L-metionyl-5-chloro-1-anthraquinonylhydrazide (GPM-CAH) and glycyl-L-prolyl-L-metionyl-4-hydrazido-N-hexyl-1,8-naphthalimide (GPM-HHNI). Using such protocols, precise enzyme localization can be obtained in tissue sections of mammalian organs.

The Use of Cytochrome C Oxidase Enzyme Activity and Immunohistochemistry in Defining Mitochondrial Injury in Kidney Disease

The renal biopsy is a dynamic way of looking at renal disease, and tubular elements are an important part of this analysis. The mitochondria in 20 renal biopsies were examined by immunohistochemical (electron transport chain enzyme: cytochrome C oxidase IV [COX IV]) and enzyme histochemical methods (COX), both by light and electron microscopy. The distal convoluted tubules and thick ascending limbs showed the greatest intensity in the COX immunostains and enzyme activity in controls. The degree of mitochondrial COX protein and enzyme activity diminished as the tubules became atrophic. With proximal hypertrophic changes, there was great variation in both COX activity and protein expression. In contrast, in three cases of systemic lupus erythematosus, biopsied for high-grade proteinuria, the activity was consistently upregulated, whereas protein expression remained normal. These unexpected findings of heterogeneous upregulation in hypertrophy and the dyssynchrony of protein expression and activity may indicate mitochondrial dysregulation. Functional electron microscopy showed COX activity delineated by the intense mitochondrial staining in normal or hypertrophic proximal tubules. With atrophic changes, residual small mitochondria with diminished activity could be seen. With mitochondrial size abnormalities (enlargement and irregularity, adefovir toxicity), activity persisted. In the renal biopsy, mitochondrial analysis is feasible utilizing immunohistochemical and enzyme histochemical techniques.

Evaluation of Muscle Fiber Types in German Shepherd Dogs of Different Ages.

The objective of this study was to determine and confirm the percentage of type I and type II muscle fibers that comprise the Gluteus Medius muscle in male and female canines of the German Shepherd breed, with standardized care, in different age groups, using the enzyme histochemical method. Muscle samples were collected from the Gluteus Medius muscles of forty clinically healthy dogs of the German Shepherd breed using the technique of percutaneous needle muscle biopsy. The samples were evaluated using histological and enzyme histochemical methods. The percentages of type I and II fibers and the ratio between the quantity of type I fibers/quantity of type II fibers were evaluated using the parameters of weight, age group, correlation between sex and age group, and between the sexes. It was found that there was no significant difference in relation to the types of fibers for the parameters of weight, age group, and age of the females. The correlation between the ages of the males suggested an increase in the percentage of type I fibers, a decrease in the percentage of type II fibers, or an increase in the ratio during the aging process. It was concluded that there was a decrease in the percentage of type II fibers with advancing age in male dogs, but without significant difference in the percentage of type I and type II fibers in relation to the weight. Anat Rec, 299:1540-1547, 2016. © 2016 Wiley Periodicals, Inc.

Histological and histochemical evaluations on the effects of high incubation temperature on the embryonic development of tibial growth plate in broiler chickens.

The effects of experimentally induced high incubation temperature on the embryonic development of growth plate of the chicken were investigated by means of histological and enzyme histochemical methods. In the experiments, 250 fertile eggs of Ross-308 broiler strain were divided into two groups, the control eggs were maintained under optimal conditions (37.8°C and 65% ± 2% relative humidity, rh) during the whole incubation period. Heat-stress imposed eggs were maintained under normal conditions (37.8°C and 65% ± 2% rh) until the 10th day of incubation, and then, continuously (24 h per day) exposed to high temperature (38.8°C and 65% ± 2% rh). Tissue samples were taken from 10 animals of each group at the 11th, 13th, 15th, 18th, 21st days of incubation. Tissue samples were processed by enzyme histochemical methods in addition to routine histological techniques. The relative tibia weights and tibia length were lower in the heat-stress group compared to the control group. The results of the measurements of the growth plate showed that the proliferative zone was narrowed whereas, the transitional and hypertrophic zone were thickened in the heat stress group. Alkaline phosphatase (ALP) density was significantly decreased in the heat-stress group compared to the control group. In conclusion, bone formation and growth plate formation are crucial for embryo development and 1°C higher from optimum may increase the incidence of skeletal disorders and leg problems in broiler chickens which is one of the major animal welfare concerns for the poultry industry.

The role of histopathology in forensic practice: an overview

The role of forensic histopathology in routine practice is to establish the cause of death in particular cases. This is achieved on the basis of microscopic analysis of representative cell and tissue samples taken from the major internal organs and from abnormal findings made at autopsy. A prerequisite of this is adherence to the quality standards set out for conventional histological/cytological staining and enzyme histochemical and immunohistochemical methods. The interpretation of histological findings is performed by taking into account macroscopic autopsy findings and information on previous history. Histological analysis may prompt postmortem biochemical and chemical-toxicological investigations. The results of histological analysis need to be classified by experts in the context of the available information and the need to withstand the scrutiny of other experts.

Histological and enzyme histochemical investigation of the hemal nodes of the hair goat

We investigated the structure of the hemal node in six healthy hair goats using histological and enzyme histochemical methods. After processing, tissue sections were stained with Crossman's trichrome, Gordon-Sweet's silver and Pappenheim's panoptic stains. Alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACP-ase) were demonstrated in frozen sections. Hemal nodes were encapsulated by connective tissue and few smooth muscle cells. Several trabeculae originated from the capsule and extended into the hemal node. A subcapsular sinus was present beneath the capsule and was continuous with the deeper sinuses. Subcapsular and deep sinuses were filled with erythrocytes. The parenchyma consisted of lymphoid follicles, diffuse interfollicular lymphocytes and irregular wide lymphoid cords. Cortical and medullary regions were not distinct. ANAE (+) and ACP-ase (+) cells were located mainly in the germinal centers of the lymphoid follicles and also were scattered equally in the interfollicular region and lymphoid cords. Monocytes, macrophages and reticular cells displayed a diffuse positive reaction, whereas localized granular positivity was observed in lymphocytes. We demonstrated that the general structure of the hair goat hemal nodes is similar to that of other ruminant species.

Effects of Glucomannan on the Sacculus Rotundus and Peripheral Blood Lymphocytes in New Zealand Rabbits during Aflatoxicosis

This study was aimed to determine the effects of the glucomannan added to aflatoxin- (AF-) contaminated diet on the sacculus rotundus and peripheral blood lymphocytes of New Zealand rabbits by histological and enzyme histochemical methods. Twenty-four adult rabbits of both sexes were divided into four equal groups, namely, as control, glucomannan 0.2 g/day, AF 125 μg/kg/day, and glucomannan combined with AF. The animals in all groups were treated for 12 weeks by the above-mentioned diet. When compared to control, AF-treatment caused significant decrease in alpha-naphthyl acetate esterase- (ANAE-) positive peripheral blood lymphocyte (PBL) percentages. The addition of the glucomannan to AFcontaining diet recovered the adverse effects of AF on sacculus rotundus and increased the ANAE-positive PBL counts. These results suggested that glucomannan was effective against the negative effects of AF in rabbits.

Histochemical and histological evaluations of the effects of high incubation temperature on embryonic development of thymus and bursa of Fabricius in broiler chickens

1. The effects of experimentally induced heat-stress on the embryonic development of bursa of Fabricius and thymus of the chicken were investigated by means of histological and enzyme histochemical methods. 2. In the experiments, 250 fertile eggs of the Ross 308 broiler strain were divided into two groups. The control eggs were maintained under optimal conditions (37⋅8°C and 65 ± 2% relative humidity, RH) during the whole incubation period. Heat stressed eggs were maintained under normal conditions (37⋅8°C and 65 ± 2% RH) until the 10th d of incubation and then exposed continuously (24 h per d) to high temperature (38⋅8°C and 65 ± 2% RH). Blood and tissue samples were taken from 10 animals of each group at d 13, 15, 18 and 21 of incubation and at d 2, 4 and 7 post-hatch. Tissue samples were processed for enzyme histochemical methods in addition to routine histological techniques. 3. The results revealed that egg temperatures were higher than incubator air temperature. Long-term heat-stress (40⋅1–40⋅6°C egg temperature) retarded development of thymus and bursa of Fabricius. Peripheral blood ACP-ase and ANAE-positive lymphocyte levels of heat-stressed animals were lower than in the controls. 4. These results give some morphological evidence for immunosuppression induced by high temperature exposure during the embryonic development. Temperature distribution and air circulation in incubator should be questioned in the case of lower broiler flock immunity.

Morphological and enzyme histochemical observations on alcohol induced disturbances in testis of two patients.

Testicular biopsies from two patients with alcohol induced fertility disturbances and one patient with intact spermatogenesis were investigated with the help of histological and enzyme histochemical methods. Main alcohol induced changes include: 1. loss of typical arrangement of elongated spermatids in the form of a "bunch of grapes", dominance of duplicated form of elongated spermatids, to a large extent loss of acrosomal formation: 2. thickening, hyalinosis, and sclerosis of lamina propria with nearly complete lack of Ca++-ATPase; 3. decrease of 3 beta-hydroxy steroid dehydrogenase and 17 beta-hydroxy steroid dehydrogenase activity in the Leydig cells, and 4. appearance of oval or spindle shaped mast cells in the interstitial tissue. Lamina propria seems to be the main target of deleterious effects of alcohol.

Muscle Biopsy: A Diagnostic Tool in Muscle Diseases

Muscle biopsy is an important and essential tool in the diagnosis and treatment of muscle disease. For several decades, histological staining of paraffin-embedded tissue was used in muscle pathology. In the past few decades, fresh-frozen enzyme histochemical techniques have become the standard for assessing muscle disease. They have been complemented by the use of electron microscopy to characterize ultrastructural changes. More recently, the discovery of certain muscle proteins and the development of antibodies made possible the combination of enzyme histochemical and immunohistochemical methods to better assess muscle changes. In this review, we discuss the roles of histochemistry, enzyme histochemistry, immunohistochemistry, and electron microscopy in the diagnosis of skeletal muscle pathology (The J Histotechnol 31:101, 2008).Submitted March 3, 2008; accepted with revisions May 24, 2008

Effects of N-acetylcysteine on mRNA expression of monocyte chemotactic protein and macrophage inflammatory protein 2 in acute necrotizing pancreatitis: experiment with rats

To explore the potential role of monocyte chemotactic protein 1 (MCP-1) and macrophage inflammatory protein 2 (MIP-2) in the pathogenesis of acute necrotizing pancreatitis (ANP), and to study the effect of N-acetylcysteine (NAC) on the mRNA expression of MCP-1 and MIP-2. Thirty-five SD rats were randomly divided into 3 groups: sham-operation (SO) group (n = 5), acute necrotizing pancreatitis (ANP) group (n = 15), and NAC-pretreated group (n = 15), ANP were induced by retrograde injection of 4% sodium taurocholate into the biliopancreatic duct. The NAC- groups underwent intraperitoneal injection of NAC 500 mg/kg 30 minutes before the induction of sodium taurocholate. The ANP and NAC groups were re-divided into 3 equal subgroups respectively: 3 h, 6 h, and 12 h subgroups. 3, 6, and 12 hours after the establishment of models heart blood samples were collected from 5 rats from each model group to detect the serum amylase. Then the rats were killed by blood letting with their pancreases taken out. HE staining and microscopy were used to observe the pathological changes of the pancreas. The wet/dry ratio of pancreas was determined. Enzyme histochemical method was used to detect the activity of myeloperoxidase (MPO) in the pancreas. RT-PCR was used to examine the mRNA expression of MCP-1 and MIP-2. The levels of serum amylase, wet/dry ratio of pancreas, pancreatic MPO activity, and histological score of pancreas of the ANP group increased time-dependently, all the levels at different time-points were significantly higher than those of the SO group (all P < 0.01). The expression levels of MCP-1 mRNA at the time points 3, 6, and 12 h of the ANP group were 0.3653 +/- 0.0213, 0.5890 +/- 0.0225, and 0.7164 +/- 0.0275 respectively, all significantly higher than that of the SO group (0.1492 +/- 0.0036, all P < 0.01). The expression levels of MIP-2 mRNA at different time points of the ANP group were 0.3871 +/- 0.0286, 0.6040 +/- 0.0448, and 0.7692 +/- 0.0620 respectively, all significantly higher than that of the SO group (0.1593 +/- 0.0117, all P < 0.01). The intrapancreatic MPO levels at the time points 3 h, 6 h, and 12 h of the NAC group were 0.63 +/- 0.03, 0.88 +/- 0.05, and 1.31 +/- 0.09 respectively, all significantly lower than those of the ANP groups (0.89 +/- 0.03, 1.42 +/- 0.14, and 1.94 +/- 0.07, all P < 0.05). The MCP-1 mRNA expression levels at different time points the NAC group were 0.2497 +/- 0.0168, 0.4457 +/- 0.0097, and 0.6306 +/- 0.0423 respectively, and the MIP-2 mRNA expression levels at the time points 3 h, 6 h, and 12 h of the NAC group were 0.2436 +/- 0.0099, 0.4312 +/- 0.0221, and 0.6302 +/- 0.0288 respectively, all significantly lower than those of the ANP group (all P < 0.05). The pancreatic histological scores at different time points of the NAC group were 3.50 +/- 0.61, 5.60 +/- 0.65, and 7.50 +/- 0.79, all significantly lower than those of the ANP group (5.10 +/- 0.42, 7.50 +/- 0.50, and 9.90 +/- 0.96, all P < 0.05). The MCP-1 mRNA expression and MIP-2 mRNA expression were both correlated with the severity of pancreatic injury (r = 0.76 and 0.82, both P < 0.05). The chemokines of MCP-1 and MIP-2 are overexpressed at the early stage of acute pancreatitis. Both of them may play an important role in the pathogenesis of AP. NAC may have beneficial effects on AP through downregulation of the expression of MCP-1 and MIP-2.

Effect of advanced gastric cancer on vagniae vasorum of gastric vessels

To explore the effect of advanced gastric cancer on vagniae vasorum of gastric vessels. To provide evidence for the surgical treatment of gastric cancer. The study included 107 specimens of left and right gastric arteries (55 left and 52 right ) from 59 patients who underwent radical gastrectomy for carcinoma. All specimens were dealt with frozen section method, then they were stained with HE, enzyme histochemical method and immunohistochemical method, respectively. Metastatic cancer cells or tubercles were found inside vagina vasorum in some stage IV and III specimens. Cytokeratin positive of tumor cells in or on vagina vasorum was showed on 26 slices from 14 tumors. Among them, 4 slices from 2 tumors belonged to stage III, and 22 slices from 12 tumors belonged to stage IV. Observed under light microscope, the lymphatic capillaries within vagina vasorum were dark brown with 5'-nucleotidase staining in 107 specimens, the capillary tubes within vagina vasorum were blue with alkaline phosphatase staining in 101 specimens. The two structures changed with the development of gastric carcinoma. Average area and bulk density of lymphatic vessel vaginae vasorum were associated with TNM staging of gastric cancer (P=0.001 and P=0.004). Vaginae vasorum dissection, which may not be applied for early gastric cancer, is recommended when clearing lymph nodes around arteries in radical gastrectomy for carcinomas in stage II and above.