不同脱钙液对兔膝关节组织切片染色的影响

探讨不同脱钙液对新西兰大白兔膝关节切片染色效果的影响,为膝关节关节软骨及基质多糖的观察奠定试验基础。取正常新西兰大白兔膝关节,10%中性福尔马林溶液充分固定,流水冲洗后,分别浸入10%硝酸脱钙液、3%硝酸脱钙液、改良AF硝酸脱钙液及改良30%盐酸脱钙液中进行充分脱钙后,流水冲洗,制作石蜡切片,苏木精—伊红(HE)、奥尔辛蓝及沙红O染色,显微镜检观察并评价其效果。结果表明:①HE染色效果,3%硝酸组＞30%盐酸组＞10%硝酸组及改良AF硝酸组;②奥尔辛蓝及沙红O染色效果,3%硝酸组及改良AF硝酸组优于其他各组。     

Histological thin-sections: a method for the microscopic study of teeth in Spanish red deer (Cervus elaphus hispanicus)

A method to obtain thin histological sections (4-5 microm thick) from teeth, using a conventional microtome, is described for incisors, molars and canines from Spanish red deer (Cervus elaphus hispanicus). Decalcifying solutions of formic acid and nitric acid at different concentrations were ineffective. This was not the case for a solution of 14% hypochloric acid and polyvinylpyrrolidone (TBD-1(R) of Shandon). However, the time needed to complete decalcification was longer than the time estimated by other authors. Incisors required between 4 and 6 days, molars between 7 and 15 days, and canines between 8 and 12 days. The preparation of histological thin-sections from teeth with a conventional microtome, required some training or prior specialization, because most of defective or lost material occurred during the preliminary stage of the study. However this technique is inexpensive enough to be implemented in any histological laboratory. The preparations obtained are suitable for growth mark observation in dentine and in cementum for dating studies. Nevertheless, they are not suitable for enamel change studies, because the enamel is completely destroyed during the decalcifying process.     

Migration and development of Sarcocystis neurona in tissues of interferon gamma knockout mice fed sporocysts from a naturally infected opossum

Migration and development of Sarcocystis neurona was studied in 50 gamma interferon knockout mice fed graded doses of S. neurona sporocysts from the intestine of a naturally infected opossum. Mice were examined at necropsy 1-62 days after feeding sporocysts (DAFS). All tissue sections were reacted with anti-S. neurona-specific polyclonal rabbit serum in an immunohistochemical (IHC) test. Between 1 and 3 DAFS, organisms were seen mainly in intestines. Between 4 and 11 DAFS, organisms were seen in several visceral tissues. Beginning with 13 DAFS, schizonts and merozoites were present in sections of brains of all infected mice. All regions of the brain were parasitized but the hind brain was most severely affected. S. neurona was found in the spinal cord of all 10 mice examined 22-30 DAFS. Of the 28 infected mice examined 20-62 DAFS, S. neurona was found in the brains of all 28, lungs of 14, hearts of 8 and eyes of 3. More organisms were seen in IHC-stained sections than in sections stained with hematoxylin and eosin. Treatment of tissues with glutaraldehyde, Karnovsky fixative, and ethylene diamino tetra acetic acid (EDTA, used for decalcification) did not affect staining of organisms by IHC.     

Quantitative analysis of embryonic kidney impairment by confocal microscopy and stereology: effect of 1,2-dibromoethane in the chick mesonephros

1. Chick embryos in ovo were treated with a teratogenic dose of 1,2-dibromoethane (DBE) on embryonic day (ED) 3. On ED 6 and 10, histological sections of whole embryos were prepared for confocal microscopy. In parallel, mesonephroi of 10-d-old embryos were dissected for in situ staining with acridine orange (AO), a fluorescence probe for lysosomes. 2. DBE impaired differentiation of renal vessels which manifested as a delay in rearrangement of primitive renal vascular architecture on ED 6 and a significant reduction of the mesonephric vascularisation on ED 10. This was accompanied by delayed functional maturation of embryonic kidney, as suggested by staining with AO. 3. Renal vessels appeared to be more susceptible to DBE than tubules. Unequal growth of these renal components might be a cause of DBE-induced spatial disorganisation of tubular apparatus. 4. Nephrotoxic effects of DBE during the embryonic period are associated primarily with damage to the renal blood supply. 5. Confocal microscopy, stereological methods and three-dimensional reconstruction of developing tissues are useful tools to investigate pathogenic processes during embryonic development.     

Histological immunoenzyme techniques in canine tissues: evaluation of various methods and modifications

Several antigens including immunoglobulin light and heavy chains, C3, macrophage enzymes and various brain proteins were demonstrated immunohistologically in paraffin sections of canine tissues. The indirect immunoperoxidase, the unlabelled antibody enzyme (PAP), double bridging PAP, and biotinavidin-peroxidase (BAP) methods were compared. The influence of various histological procedures such as fixation and embedding, and other modifications such as enzyme treatment of sections was investigated. The best results were obtained with the PAP and BAP methods on formalin fixed tissues. Trypsin proved to be highly effective for demonstrating certain antigens but required firm adhesion of the sections. Prolonged incubation with the primary antisera improved the results considerably. Based on the results of this study a general strategy for solving immunohistological problems has been proposed.     

Demonstration of bovine herpesvirus type 1 and Mannheimia haemolytica antigens in specimens stored for up to 22 months in buffered formalin

Bovine herpesvirus type 1 (BHV-1) and Mannheimia haemolytica antigens were demonstrated in lung tissues that were stored in 10% neutral phosphate buffered formalin for 1 to 22 months using the immunoperoxidase method. There were no differences observed in terms of labelling intensity and distribution of M. haemolytica antigens between specimens stored for 1 and 22 months. The labeling intensity in sections from 2-cm thick specimens was comparable to those from 0.2-cm thick specimens. There was no difference observed between pronase-treated and -untreated sections. However, for BHV-1, the labeling intensity in untreated sections was reduced in tissues that had been stored from 12 to 22 months. Sections from thin specimens stored in neutral buffered formalin for 22 months exhibited a stronger staining intensity than those from thick specimens.     

PCR-based detection of canine Leishmania infections in formalin-fixed and paraffin-embedded skin biopsies: elaboration of a protocol for quality assessment of the diagnostic amplification reaction

Diagnosis of the cutaneous form of canine leishmaniosis is mostly performed by histological or immunohistological examination of skin biopsies. In modern histology, the polymerase chain reaction (PCR) has gained increasing importance as a complementary tool to directly demonstrate the presence of parasite DNA in the tissue sections. For the present study, a previously described Leishmania-PCR has been further developed and optimised in view of its practicability for routine histological application. Since formalin-fixation of histological specimens causes partial DNA-destruction, which may hamper diagnostic PCR analysis, primers specific for the highly conserved alpha-actin gene sequences were used to pre-diagnostically assess the isolated sample-DNA for its functionality in a PCR-reaction. This alpha-actin-specific PCR detects DNA from a large variety of mammalian species and thus exhibits relevance for both human and veterinary medical application. A recombinant internal positive control was introduced to monitor possible sample-related inhibitory effects during the amplification reaction. We performed a retrospective evaluative study with 18 formalin-fixed samples from dogs with suspected or proven leishmaniosis. Six samples were PCR-incompatible. In turn, 9 of the other 12 samples were PCR-positive, and immunohistochemical results matched these findings. Based on these technical achievements, the Leishmania-PCR proved to be a valuable tool to complement conventional histological and immunohistological methods for diagnosis of cutaneous leishmaniosis in formalin-fixed, paraffin-embedded skin biopsies.     

Differences in measurement of membrane thickness in hens' egg shells

The widely varying values given for the membrane thickness in hen's eggs suggest that a critical comparison of the methods of measurement is necessary. The membrane thickness was measured in pieces of isolated membranes, in decalcified sections and in ground sections. The first method gave the lowest values, probably owing to the impossibility of separating the whole of the membranes from the calcified shell. The other two methods showed an average difference in results of only 7.2–10.5 per cent. It was concluded that the actual thickness of dry egg shell membranes can only be measured in sections and that, without correction, such values should not be compared with those taken in isolated membranes.     

Biomechanical properties of canine cortical bone allografts: effects of preparation and storage

The effects of various preparation and storage procedures and of different storage times on structural properties of canine cortical bone allografts were determined by evaluation of the compressive load to failure of a whole diaphyseal segment, the ability of a screw to resist being pulled from a cortical segment, and the torque required to strip the threads of a screw hole in a cortical segment. Preparation and storage procedures evaluated were sterile collection and storage at -20 C; ethylene oxide sterilization and storage at room temperature (22 C); chemical sterilization (methanol and chloroform, then iodoacetic acid) and storage at -20 C; and chemical sterilization, partial decalcification, and storage at -20 C. Storage times were 1, 16, and 32 weeks for each procedure. After 1 week of storage, aseptically collected frozen bone and ethylene oxide-sterilized bone had an increase, compared with matched controls, in load to failure in compression, but pullout load or screw-stripping torque did not change. Chemically sterilized bone had not changed after 1 week of storage, whereas chemically sterilized and partially decalcified bone had a 40% to 60% decrease in compressive load to failure, pullout load, and screw-stripping torque. Chemically sterilized and partially decalcified bone remained weak after 16 and 32 weeks of storage. Significant structural alterations were not detected in aseptically collected bone after 16 or 32 weeks of storage. Ethylene oxide-sterilized bone had a reduced pullout load after 32 weeks of storage. Chemically sterilized bone had significantly reduced compressive load to failure and pullout load after 16 and 32 weeks of storage.(ABSTRACT TRUNCATED AT 250 WORDS)     

犬瘟热的微生物学诊断研究

对临床诊断为犬瘟热的１４例病犬，取其脑组织，运用细胞培养、合胞体检查、包涵体检查、免疫荧光试验、电镜观察等５种检测方法，就犬瘟热的微生物学诊断进行了系统的研究。结果表明，犬脑组织块与猫胚（ＦＥ）细胞或非洲绿猴肾（Ｖｅｒｏ）细胞共同培养，盲传的培养物出现不稳定的细胞病变，通过对不同代次的培养物检查包涵体，并作超薄切片或负染，电镜观察犬瘟热病毒（ＣＤＶ）粒子，证实分离到ＣＤＶ野毒。病犬脑组织切片经ＨＥ染色检查包涵体及用ＣＤＶ荧光抗体直接法检测脑抹片及接种犬脑的细胞培养物，均获得一定的阳性结果。建立的改良离子捕获电镜法，用于检测犬脑匀浆和犬脑接种细胞培养物，与离子捕获电镜法、免疫电镜法及直接电镜法相比，效果更为理想。１４例临床诊断为犬瘟热的病犬，综合运用上述微生物学手段检测，结果为１２例阳性，２例疑似。     

Video scanning for determination of the proportion of cortical tissue in the avian adrenal gland.

The use of a video scanning apparatus (Leitz, T.A.S.) for the determination of the corticomedullary proportion in histological sections of the avian adrenal gland is described and statistically evaluated. When the video scanning method was applied to material from groups of domestic hens, which had been exposed to different experimental conditions, the results were similar to those obtained through the integrating method as described by Siller et al. (1975). The mean values obtained by both methods did not differ significantly, and there was a highly significant correlation between the counts for both methods applied on the same sections. When applying the video scanning method to 16 sections from four adrenals, repeated measurements on each of the sections showed considerable variation. However, this variation was found to be significantly smaller than the variation among the sections. It is suggested that the video scanning method could be made more precise by improvement of the staining procedure. However, on relatively large samples it seems to give reliable results, and it has a great advantage in reducing the tedious work involved in other available methods.     

Validation of an immunohistochemical assay for bovine cysticercosis, with comparison to a standard histological method

The larval stage (syn Cysticercus bovis) of the human tapeworm Taenia saginata causes cysticercosis in cattle, which has both aesthetic and food safety implications to consumers of beef. A monoclonal antibody-based immunohistochemical (IHC) assay developed to improve postmortem diagnosis of this parasite and a standard histological method were assessed to determine their fitness for intended use. Sections from 169 known-positive specimens of T. saginata from experimentally or naturally infected cattle, and from 30 known-negative specimens and lesions of various etiologies from non-infected cattle, were tested. The IHC assay identified significantly more known positive bovine cysticerci than the histological method (91.7% and 38.5%, respectively). Positive IHC staining occurred on sections from other cestode species, but should not affect the diagnostic specificity of this assay for bovine cysticercosis, due to the different host and/or tissue preferences amongst these parasites. Use of the IHC assay should improve the reliability of diagnosing lesions caused by degenerated cysticerci, facilitating more effective and efficient control of bovine cysticercosis.     

A Morphological and Stereological Study on Cervical Segment of Spinal Cord of Quails

In this study, volume densities of white and grey matters of cervical segments of spinal cords of quail were investigated stereologically. In both sexes, mature, six quails were used as material of this study. All animals were fixed by perfusing in 10% buffered formalin. Tissue specimens were obtained from cervical spinal cords. These tissue specimens were cut every fiftieth section at 5 μm thickness by a microtome. And mean six or seven sections were examined from every block by this method at microscope. After that, these sections were stained by haematoxylin eosin and photographed. Densities of volumes of all tissues of cervical segments of whole spinal cords and white and grey matters were calculated with principle of Cavalieri. As a result, total volume of spinal cord, volumes of white and grey matters of cervical segment and volume rates of white and grey matters were calculated.     

The Accuracy of Intraoperative Diagnoses Based on Examination of Frozen Sections A Prospective Comparison with Paraffin-Embedded Sections

The accuracy of diagnoses based on examination of frozen sections was determined by comparing the results to those obtained by examination of tissues prepared using conventional methods (formalin fixation, paraffin-embedded tissue). One hundred ninety-four specimens were examined using the frozen section technique; 37 were examined to confirm a tentative diagnosis or to document lymph node metastasis and the remainder were examined to diagnose an unknown pathologic process. Of the 194 specimens examined, an accurate, specific diagnosis was obtained in 161 (83%); in 19 (10%), the pathologic process was correctly identified, but a specific diagnosis was not obtained; and in 2 (1%) the diagnosis was deferred. The remaining 12 (6%) were incorrectly diagnosed by the frozen section technique. When the number of specimens in which a specific diagnosis was obtained was combined with the number of specimens in which the pathologic process was correctly identified, the overall accuracy rate of the frozen section technique was 93%. There was no difference in the accuracy of the frozen section technique based on the reason for submission of the sample, source of tissue submitted, or the type of pathologic process (i.e., inflammatory or neoplastic). Of the 12 incorrect diagnoses, 4 (33%) were because of sampling errors and 8 (67%) were caused by interpretation errors. The proposed indications for the use of intraoperative frozen sections are: 1) to determine the nature of a pathologic process for which a preoperative diagnosis has not been established, 2) to determine the extent of spread of neoplastic tissue to lymph nodes and other organs, 3) to evaluate resection margins of a neoplastic process, and 4) to clarify situations where a discrepancy exists between the preoperative cytologic or histologic diagnosis and intraoperative gross pathology.     

Detection of Mycobacterium avium subsp paratuberculosis in formalin-fixed paraffin-embedded intestinal tissue by IS900 polymerase chain reaction

OBJECTIVES: To evaluate and compare methods for DNA extraction from formalin-fixed, paraffin-embedded tissues and methods for detection of Mycobacterium avium subsp paratuberculosis by IS900 PCR for confirmation of Johne's disease in ruminants. DESIGN: A laboratory study. PROCEDURE: Three methods of DNA extraction of differing complexity and two PCR protocols using different pairs of IS900 primers were compared. Sensitivity and specificity were assessed using samples from ruminants with and without histological evidence of Johne's disease. RESULTS: The simplest method of DNA extraction, which involved two cycles of boiling and freezing followed by centrifugation, gave more consistent results than two methods that required solvent extraction of paraffin, proteinase digestion and DNA purification. The sensitivity of detection of M avium subsp paratuberculosis in paraffin blocks stored for 1 to 6 years from 34 cases of Johne's disease in sheep, cattle and goats was 88% for a 229 bp IS900 PCR assay and 71% for a 413 bp assay, using the detection of acid-fast bacilli by Ziehl Neelsen staining of histological sections from the same blocks as the gold standard test. PCR results correlated with the abundance of acid-fast organisms in the tissues. No false positive reactions were detected. CONCLUSION: PCR for identification of M avium subsp paratuberculosis in formalin-fixed, paraffin-embedded intestinal tissues from ruminants is a rapid and useful method. A simple method of sample preparation is effective. Amplification of short fragments of IS900 is more effective than amplification of longer fragments.     

Sacroiliac Joint of the Horse 3. Histological appearance

To establish a baseline for the histological appearance of the sacroiliac joint of the horse, joint specimens were collected from 41 horses from late fetal life to the age of 14 years. Sagittal sections from the joints were radiographed and sectioned for histological examination.
There was a striking difference in structure between the sacral and iliac articular cartilages, the former being hyaline and the latter predominantly fibrous. Degenerative changes were seen even in young horses and were more markec on the iliac side. The degeneration of the articular cartilage showed a progressive, age-related pattern.     

Immunohistological demonstration of erythroid cells in canine bone marrow

In an immunohistological/cytological study of canine bone marrow, the aim was to demonstrate canine erythroid cells with the help of various commercially available antibodies against human antigens (monoclonal antibody against glycophorin A, polyclonal antibodies against haemoglobin and spectrin). In order to preserve possible cross-reacting epitopes various fixation methods (cross-linking, precipitating and dehydrating fixing agents, partly in combination with unmasking measures), decalcification techniques [acid or ethylenediaminetetraacetic acid (EDTA) decalcification] and tissue-embedding methods (paraffin embedding, cryostat sectioning technique) were used. Alternative methods, such as the preparation of cell smears and immunoblotting, were also employed. The only result that was of use for routine diagnostic procedures (paraffin sections) was that obtained by using polyclonal antibodies against haemoglobin. Best results were achieved when tissue was fixed in a formaldehyde-glutaraldehyde mixture, decalcified in EDTA and treated with microwave irradiation. The primary antibody was used in a dilution of 1:500 and incubated for 16 h. With the exception of mature red blood cells and proerythroblasts, different stages of erythrocytopoietic cells in canine bone marrow were shown to be arranged in erythrons. The polyclonal antibody against spectrin also showed clear cross-reactivity, but was only employable in other systems (immunoblotting). The monoclonal antibody against glycophorin A reacted only when used on human tissue or cells.     

Technical note: preservation of tissues and gastrointestinal tract portions by plastic coating or plastination.

Two methods to preserve gastrointestinal tract (GIT) organs and tissues, plastic coating (PC) and plastination (PN), were investigated and compared. Specimens to be preserved were removed from animals within 2 h of death and immediately cleaned with water. Digesta contents were removed by flushing desired portions of GIT with water until the exiting water was clear. In the PC method, cleaned specimens were dehydrated by immersion in an isopropanol solution, dried with forced air after positioning and orientation as in situ, and finally coated on the outer and inner surfaces with a clear plastic material. In the PN procedure, specimens were filled with, and submerged in, a low-formaldehyde fixative, then dehydrated by immersion in a cold acetone solution. Dehydrated specimens were immersed in silicone and placed in a freeze drier for impregnation under low vacuum, followed by overnight gas curing with a silicone crosslinker. Finally, viewing windows were cut out with a scalpel in GIT preserved by both methods. Preserved GIT and tissues had an appearance similar to their appearance in vivo. The PC method was simple and inexpensive. Plastinated specimens were more flexible, durable, and lifelike than those preserved by the PC method. In addition, many body parts, such as muscles, nerves, bones, ligaments, and central nervous system specimens, were preserved by PN. Both methods were found to be useful tools for postmortem studies of tissues and GIT organs.     

禽流感群特异性间接原位RT-PCR检测方法

从GenBank下载禽流感病毒(Auian in fluenza virus,AIV)NP基因序列,通过比对选取NP基因中较为保守的片段,设计1对引物,通过RT-PCR方法扩增270 bp的目的片段,经测序确认目的片段序列正确后,用地高辛标记扩增产物制备探针.在BSL-3实验室,用AIV人工感染鸡胚成纤维细胞和SPF鸡后,制作细胞涂片和组织石蜡切片,然后在经前处理后的细胞涂片和石蜡切片上进行原位RT-PCR,再与地高辛标记的探针进行原位杂交,对细胞和组织中的AIV进行检测和定位.研究结果表明,本方法能检测出约1μg/L质粒的DNA,并能特异性地在细胞涂片和石蜡组织切片中检测和定位AIV,且成纤维细胞感染病毒8 h后即可检测到阳性信号,具有良好的特异性和较高的敏感性,为动物组织中AIV的检测定位和致病机理研究提供了敏感、特异和更为直观的方法.