Methyl Green-pyronin Research Articles

Apoptosis in the transplanted canine transmissible venereal tumor during growth and regression phases

Twelve male, mongrel, adult dogs were subcutaneously transplanted with cells originated from two canine transmissible venereal tumors (TVT). The aim was to demonstrate and to quantify the occurrence of apoptosis in the TVT regression. After six months of transplantation, a tumor sample was obtained from each dog, being six dogs with TVT in the growing phase and six in the regression phase as verified by daily measurements. Samples were processed for histological and ultrastructural purposes as well as for DNA extraction. Sections of 4µm were stained by HE, Shorr, methyl green pyronine, Van Gieson, TUNEL reaction and immunostained for P53. The Shorr stained sections went through morphometry that demonstrated an increase of the apoptotic cells per field in the regressive tumors. It was also confirmed by transmission electron microscopy, which showed cells with typical morphology of apoptosis and by the TUNEL reaction that detected in situ the 3'OH nick end labeling mainly in the regressive tumors. The regressive TVTs also showed an intensified immunostaining for P53 besides a more intense genomic DNA fragmentation detected by the agarose gel electrophoresis. In conclusion, apoptosis has an important role in the regression of the experimental TVT in a way that is P53-dependent.

Appearance of Apoptotic Cells and Granular Cells in the Silkworm Midgut Lumen During Larval-Pupal Ecdysis

To study midgut degradation and programmed cell death, we performed methyl green-pyronin staining and Giemsa staining of the midgut of silkworms during metamorphosis. Midgut epithelial cells underwent pyknosis and cytoplasmic shrinkage on the second day of spinning. In the prepupal stage, all midgut epithelial cells desquamated into the midgut lumen, rapidly forming apoptotic bodies. The number of apoptotic bodies in the midgut decreased rapidly from the prepupal stage to the third day of the pupal stage. DNA fragmentation at the time of apoptotic body formation was confirmed by the comet assay. In the midgut lumen from the prepupal stage to the first through third days of the pupal stage in which apoptotic bodies were observed, granular cells were present. Their morphology was similar to that in the body fluid and, during the pupal stage, intracellular granules increased in size and number with time, giving the appearance of a foamy cell. In this stage, numerous granular cells were observed under the basement membrane of the midgut, and phagocytosed apoptotic bodies were seen within granular cells in the midgut lumen. Granular cells may be actively involved in the clearance of apoptotic bodies from the midgut during larval-pupal ecdysis.

Trichodinids (Ciliophora: Peritrichida) parasitic on gills of freshwater fishes, Carassius auratus and Aristichthys nobilis from China, with the description of Trichodina subtilihamata sp. nov.

Five trichodinid ectoparasites (Ciliophora: Peritrichida) were isolated from two freshwater fishes, Carassius auratus and Aristichthys nobilis collected from Chongqing, China. Trichodina subtilihamata sp. nov. is described from the gills of Carassius auratus and characterized by a slender hooked blade. The other four species of trichodinids are: Trichodina mutabilis Kazubski & Migala, 1968, Trichodina uniforma Van As & Basson, 1989, Trichodina nigra Lom, 1960 and Trichodina kazubski Van As & Basson, 1989. Comparative descriptions, with photomicrographs and morphometric data, are presented of these trichodinids to establish locality records. Descriptions presented here were obtained by examinations of specimens prepared using the dry silver nitrate impregation and the methyl green-pyronin staining. Comparisons with closely related species are provided for the new species.

Effects of short periods of frequent milking on the persistency of milk yield and SCS in Turkish Saanen goats

ABSTRACTThe major purpose of this study was to investigate if increasing milking frequency for a short period of 3 weeks had any effects on the persistency of milk yield and the persistency of the somatic cell score (SCS). The methyl green–pyronin method was used for the direct microscopic SCS, converted to SCS for the analyses. Two groups were formed (control, n = 14 and treatment, n = 14). The treatment group was milked four times a day for 3 weeks after weaning. Both groups were milked twice a day outside this 3‐week period, throughout lactation. For the milk yield, persistencies were higher in the treatment group when the distance between the test days was equal to or less than 3 months. The exact opposite was true for the SCS; that is, the treatment group had higher persistencies when the distance between the test days was more than 3 months. Overall, persistencies related to milk yield were higher than persistencies related to SCS. For fat corrected milk yield, treatment persistencies were always higher than the control group persistencies, regardless of the distance between the tests. The same was true for the fat content. Increasing milking frequency for a short period of time (3 weeks) after weaning may help to increase the persistency of the milk yield, fat corrected milk yield and fat content. A 3‐D plot of least squares means indicated that the milk yield tended to show a classical lactation curve in lower SCS values while forming an unstable curve in higher SCS values.

Polyamines in spermatocytes and residual bodies of rat testis

Immunocytochemistry for polyamines in the rat testis revealed intense staining of small bodies close to the lumen of seminiferous tubules of spermatogenic stage VII and VIII as well as of spermatocytes. Methyl green-pyronin and propidium iodide staining combined with DNase or RNase predigestion showed that the small bodies contained RNA, but not DNA and double fluorescence staining showed that polyamines (PAs) colocalized with RNA in the bodies. Electron microscopy confirmed the absence of nuclei in the bodies and revealed that PA immunoreactivity was associated with ribosomes. These results strongly suggest that the small bodies correspond to the residual bodies, and agree with previous results showing localization of PAs to ribosomes in neurons and gastrointestinal epithelial cells. The accumulation of PAs in residual bodies may reflect a termination of their role in spermiogenesis with respect to protein synthesis.

Infusion of anti-nerve growth factor into the cisternum magnum of chick embryo leads to decrease cell production in the cerebral cortical germinal epithelium

There has been considerable recent progress in understanding the processes involved in cerebral cortical development. Several mitogenic and trophic factors have been implicated in the processes of cortical cell proliferation and differentiation. Anti-nerve growth factor (NGF) antibody was administered to 15 days chick foetuses through the cisternum magnum. Control group received phosphate buffered saline (PBS). To identify cells born in the cerebral cortex at the time of antibody or PBS injection, 5'-bromo-2'- deoxyuridine was administered to the foetuses by intravenous injection into an outlying vein using micromanipulation. After injection, the foetuses were re-incubated for another 3 days. All the foetuses were collected on day 18, the brains fixed in paraformaldehyde, cut with a microtome and stained with methyl green pyronin and anti-NGF antibody. Quantitative measurements showed that the thickness of the germinal epithelium (GE) and cerebral cortex in the anti-NGF antibody injected foetuses was decreased when compared with normal control embryos. The number of cells produced in the GE of antibody injected foetuses was decreased when compared with normal control embryos. The results from this study using neutralizing antibody suggests that NGF is an important factor in cerebral cortical development, stimulating neuronal precursor proliferation.

Diagnosis of chronic endometritis in biopsies with stromal breakdown

Plasma cells are the hallmark of chronic endometritis but are not specific for upper tract infection. Plasma cells have also been noted in hormonally mediated endometrial disorders in association with gland architectural changes ("disordered proliferative" and "anovulatory" patterns), and stromal breakdown. We reviewed benign endometrial biopsies diagnosed at Beth Israel Deaconess Medical Center over a 2-year period described as disordered/anovulatory patterns +/- stromal breakdown. Cases were excluded if tissue was not available; women were younger than 50 years where most diagnoses were atrophic or cancer; or diagnoses were secretory, menstrual endometrium, or polyps. The remaining 61 cases were compared to 33 samples of unremarkable proliferative endometrium. Plasma cells were quantified on hematoxylin and eosin-stained sections and using a histochemical stain methyl green pyronin. The indication for biopsy was an abnormal pattern of bleeding in 34 cases, infertility workup in 7, incidental part of workup for pain, or other findings in 5. The majority of disordered proliferative endometrium had plasma cells (61% grade 1, 17% grade 2) all seen on methyl green pyronin staining only. Two thirds of proliferative endometrium with breakdown showed plasma cells (19% grade 1, 39% grade 2, 10 % grade 3). Plasma cells were rare in inactive endometrium and noted in only 18% of unremarkable proliferative endometrium, all grade 1. Plasma cells are commonly present in the endometrium of women with dysfunctional uterine bleeding and focal stromal breakdown. Given the lack of clinical evidence for infection, the inflammation likely represents a physiologic process.

Infectivity rate and transmission potential of Hyalomma anatolicum anatolicum ticks for Babesia equi infection

The infectivity rate of Babesia equi in the salivary glands of Hyalomma anatolicum anatolicum was assessed. The hungry nymphs were fed on a donkey experimentally infected with B. equi. The engorged dropped-off nymphs were collected at different levels of parasitaemia and kept in BOD incubator. After ecdysis, the hungry adults were prefed on rabbits for different time intervals, thereafter the salivary glands were dissected out and acini were examined after methyl green pyronin (MGP) staining. A total of 134 male and 139 female ticks were dissected out. Average infected acini per tick were found to be significantly higher ( p < 0.05) in male as compared to the female ticks. Further, maximum infected acini in both male and female ticks were found at 24 h of prefeeding on rabbits and overall infected acini per tick increased with rise in parasitaemia. The release of infected ticks on susceptible donkeys resulted in development of clinical babesiosis.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA

BackgroundGene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays.ResultsThe MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases.ConclusionRNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

Potential role of multiple rectal biopsies in the diagnosis of equine grass sickness

Two samples were taken postmortem from the rectum of each of 14 horses with grass sickness and 10 control horses, and four sections stained with haematoxylin and eosin were examined....

Histotechnology as a Forerunner of Molecular Pathology Testing

Histotechnology essentiallv evolved from the doctrines of cellular pathology. Rudolph Virchow's doctrines of cellular pathology recognized microscopic pathology as a fundamental discipline in medicine which then led to the birth of Histotechnology. The term cellular then gives rise to minute Structures we refer to as cells, cellular meaning made of LIP cells that are essentially the basic unit of life. When the term molecular is defined, it refers to the composition of molecules which, in turn, are small masses of matter. One of the most important tasks for histotechnologists is to basically preserve tissues, tissue structures, and entities for microscopic examination. The art and science of staining is heavily used to then demonstrate the structures and entities at the cellular levels. And, in many cases, the cellular levels can be referred to as molecular. Most molecular testing, such as immunohistochemistry, in situ hybridization, apoptosis, and tissue microarrays, are built on histological techniques. Molecular techniques have almost always been reserved and thought of in regard to “futuristic” practices. However, many techniques in histotechnology have been focused on the molecular aspects of tissue handling and the end result. Histotechnologists also have been involved in the history of diagnostic testing from isolated procedures to more organized testing. Today, histopathology laboratories are staffed with professionals trailled in highly technical aspects of the theory and practice of tissue preservation and preparation. One of the most-used techniques that has been instrumental in demonstrating DNA and RNA is the methyl green-pyronin Y (MGP) stain. Performed for the most part by histotechnologists and used in both diagnostic patient care and research, the MGP stain has served as one of the first molecular tests in histotechnology. Because of increased interest in DNA and RNA in smears and tissues, the MGP stain has increased in use in the 1950s. Interestingly enough, this period also was during the time that James Watson and Francis Crick were able to propose a structure for DNA. In addition, many modifications of the original MGP techniques are implemented even today. (The J Histotechnol 28:249, 2005)Submitted November 15, 2005; accepted with revisions December 6, 2005

Histotechnology as a Forerunner of Molecular Pathology Testing

Histotechnology essentiallv evolved from the doctrines of cellular pathology. Rudolph Virchow's doctrines of cellular pathology recognized microscopic pathology as a fundamental discipline in medicine which then led to the birth of Histotechnology. The term cellular then gives rise to minute Structures we refer to as cells, cellular meaning made of LIP cells that are essentially the basic unit of life. When the term molecular is defined, it refers to the composition of molecules which, in turn, are small masses of matter. One of the most important tasks for histotechnologists is to basically preserve tissues, tissue structures, and entities for microscopic examination. The art and science of staining is heavily used to then demonstrate the structures and entities at the cellular levels. And, in many cases, the cellular levels can be referred to as molecular. Most molecular testing, such as immunohistochemistry, in situ hybridization, apoptosis, and tissue microarrays, are built on histological techniques. Molecular techniques have almost always been reserved and thought of in regard to “futuristic” practices. However, many techniques in histotechnology have been focused on the molecular aspects of tissue handling and the end result. Histotechnologists also have been involved in the history of diagnostic testing from isolated procedures to more organized testing. Today, histopathology laboratories are staffed with professionals trailled in highly technical aspects of the theory and practice of tissue preservation and preparation. One of the most-used techniques that has been instrumental in demonstrating DNA and RNA is the methyl green-pyronin Y (MGP) stain. Performed for the most part by histotechnologists and used in both diagnostic patient care and research, the MGP stain has served as one of the first molecular tests in histotechnology. Because of increased interest in DNA and RNA in smears and tissues, the MGP stain has increased in use in the 1950s. Interestingly enough, this period also was during the time that James Watson and Francis Crick were able to propose a structure for DNA. In addition, many modifications of the original MGP techniques are implemented even today. (The J Histotechnol 28:249, 2005)Submitted November 15, 2005; accepted with revisions December 6, 2005

A Causative Role of Stromelysin-3 in Extracellular Matrix Remodeling and Epithelial Apoptosis during Intestinal Metamorphosis in Xenopus laevis

The matrix metalloproteinases are a family of proteases capable of degrading various components of the extracellular matrix. Expression studies have implicated the involvement of the matrix metalloproteinase stromelysin-3 (ST3) in tissue remodeling and pathogenesis. However, the in vivo role of ST3 has been difficult to study because of a lack of good animal models. Here we used intestinal remodeling during thyroid hormone-dependent metamorphosis of Xenopus laevis as a model to investigate in vivo the role of ST3 during postembryonic organ development in vertebrates. We generated transgenic tadpoles expressing ST3 under control of a heat shock-inducible promoter. We showed for the first time in vivo that wild type ST3 but not a catalytically inactive mutant was sufficient to induce larval epithelial cell death and fibroblast activation, events that normally occur only in the presence of thyroid hormone. We further demonstrated that these changes in cell fate are associated with altered gene expression in the intestine and remodeling of the intestinal basal lamina. These results thus suggest that ST3 regulates cell fate and tissue morphogenesis through direct or indirect ECM remodeling.

The calyx fluid of Microplitis rufiventris parasitoid and growth of its host Spodoptera littoralis larvae

The morphology of the female reproductive system of pupal and adult stages of Microplitis rufiventris and the ultrastructure of the ovaries are described and illustrated. Two morphologically distinct types of particles of nuclear origin, i.e., polydnavirus (PDV) and a virus-like filamentous particle (VLFP) were detected in the ovarian calyx fluid of the female wasp. It is likely that these particles are injected into the host during oviposition. PDV initiated replication in the calyx of mid-aged pupae and in pharate adults and were present throughout adult life. VLFP were only seen in the calyx fluid of newly emerged adults, and therefore observed after the PDV. Feulgen and methyl-green pyronin staining revealed the presence of DNA in both types of particles. The effects of injection of Spodoptera littoralis larvae with a combination of parasitoid viruses and venom of M. rufiventris females (CxFV) were investigated and the results were compared with two control groups, i.e., larvae injected with Pringle's saline (PS) and naturally parasitized larvae. CxFV-larvae showed significant declines ( P < 0.05 ) in food consumption, weight of ejected faeces and weight gain when compared with PS-larvae. However, naturally parasitized larvae (parasitoid egg+CxFV+ovarian protein) displayed a high significance score ( P < 0.01 ) in comparison with those of PS-larvae. The approximate digestibility (AD) values of S. littoralis larvae were positively affected as early as day 2 post-treatment by either injection of CxFV or parasitization. However, a reduction in AD was observed in both PS- and CxFV-larvae on day 3–7 in comparison with naturally parasitized larvae. Other indices of food utilization were unchanged in CxFV-larvae when compared to saline treated or parasitized controls.

Tissue Tropism and Bursal Transformation Ability of Subgroup J Avian Leukosis Virus in White Leghorn Chickens

In Experiment 1, a monoclonal antibody against the envelope glycoprotein (gp85) of subgroup J avian leukosis virus (ALV-J) was used to study the distribution of ALV-J in various tissues of White Leghorn chickens inoculated as embryos with the strain ADOL-Hcl of ALV-J. At 2 and 6 wk of age, various tissues from infected and control uninfected chickens were tested for the presence of ALV-J gp85 by immunohistochemistry. In Experiment 2, using the methyl green-pyronine (MGP) stain, sections of bursa of Fabricius (BF) from chickens of line 15I5 x 7(1), inoculated with ALV-J or Rous-associated virus-1 (RAV-1), a subgroup A ALV, at hatch were examined for transformation of bursal follicles at 4 and 10 wk of age. In Experiment 1, specific staining indicative of the presence of ALV-J gp85 was noted at both 2 and 6 wk of age in the adrenal gland, bursa, gonads, heart, kidney, liver, bone marrow, nerve, pancreas, proventriculus, spleen, and thymus. In Experiment 2, by 10 wk of age, transformed bursal follicles were detected in MGP-stained sections of BF in only one of five (20%) chickens inoculated with ALV-J at hatch, compared with five of five (100%) chickens inoculated with RAV-1. The data demonstrate distribution of ALV-J gp85 in various tissues of White Leghorn chickens experimentally inoculated as embryos with the virus. The data also confirm our previous observation that ALV-J is capable of inducing transformation of bursal follicles, albeit the incidence is less frequent than that induced by subgroup A ALV.

Assessment of contact allergens by dissociation of irritant and sensitizing properties

The human organotypic skin explant culture (hOSEC) model is a promising alternative in vitro model for screening contact allergens. In this model, the chemical-induced migration of Langerhans cells (LCs) out of the epidermis, evaluated after a 24-h exposure period, is used as a measure of sensitizer potential. As skin irritants can also induce LC migration it is essential that concentrations of test chemicals are used that are not even weakly irritant. Using the hOSEC irritation model chemicals are classified as weak irritants if they are toxic after a 48-h exposure period. Toxicity is determined by methyl green-pyronine (MGP) staining of hOSEC. We studied three frequently used non-sensitizing skin irritants and six potent or frequent human sensitizers in a dose–response. A complete discrimination between non-sensitizers and contact sensitizers was obtained for the chemicals tested when the concentrations used were lower than the weak irritant concentrations. Frequency of positive allergen reactions in patch test of human populations correlated with the difference between weak irritant concentrations and the lowest concentration inducing significant LC migration. Sensitizer potency correlated with chemical irritancy as determined by keratinocyte death. For the compounds tested, the hOSEC model predicted allergenicity in humans better than the guinea pig maximization test and the mouse local lymph node assay.

Response of White Leghorn Chickens of Various Genetic Lines to Infection with Avian Leukosis Virus Subgroup J

In Experiment 1, chickens from various white leghorn experimental lines were inoculated with strain ADOL-Hcl of subgroup J avian leukosis virus (ALV-J) either as embryos or at 1 day of age. At various ages, chickens were tested for ALV-J induced viremia, antibody, and packed cell volume (PCV). Also, at 4 and 10 wk of age, bursal tissues were examined for avian leukosis virus (ALV)-induced preneoplastic lesions with the methyl green-pyronine (MGP) stain. In Experiment 2, chickens harboring or lacking endogenous virus 21 (EV21) were inoculated with strain ADOL-Hcl of ALV-J at hatch. All embryo-inoculated chickens in Experiment 1 tested positive for ALV-J and lacked antibody throughout the experimental period of 30 wk and were considered viremic tolerant, regardless of line of chickens. By 10 wk of age, the incidence of ALV-J viremia in chickens inoculated with virus at hatch varied from 0 (line 0 chickens) to 97% (line 1515); no influence of ALV-J infection was noted on PCV. Results from microscopic examination of MGP-stained bursal tissues indicate that ALV-J can induce typical ALV-induced transformation in bursal follicles of white leghorn chickens. Lymphoid leukosis and hemangiomas were the most common ALV-J-induced tumors noted in chickens in Experiment 1. At termination of Experiment 2 (31 wk of age), 54% of chickens harboring EV21 were viremic tolerant compared with 5% of chickens lacking EV21 after inoculation with ALV-J at hatch. The data indicate that genetic differences among lines of white leghorn chickens, including the presence or absence of EV21, can influence response of chickens to infection with ALV-J.

Feline plasma cell pododermatitis: a study of 8 cases.

Eight cases of feline plasma cell pododermatitis, collected over a 3-year period, were studied using histological (haematoxylin and eosin), histochemical (methyl green-pyronin) and immunohistochemical (antibody against lambda light chains of immunoglobulins) techniques. No sex, breed or age predisposition, or paw predilection was observed. The disease began as a swelling of the footpads, followed by ulceration. Histologically, lesions were characterized by the presence of a large number of plasma cells in a predominantly perivascular pattern. Binucleated plasma cells and mitotic figures were observed. Clinical follow-up for over 1 year revealed total remission of the lesions both after glucocorticoid therapy in four cases, and total surgical excision in two animals. Follow-up studies were not possible in one case.

Ocorrência de apoptose em leucócitos no esfregaço de sangue periférico e em sincícios na infecção in vivo pelo vírus da cinomose canina

The aim of this paper is to report the occurrence of apoptosis in peripheral blood leukocytes and in syncytia induced by experimental infection of dogs with canine distemper virus. Blood was collected from nine puppies that were inoculated with the Snyder Hill strain of CDV, and stained with May Grunwaldt-Giemsa. Dogs were necropsied and retropharyngeal lymph nodes were collected and processed for paraffin embedding. Five micrometers thick sections were stained with hematoxylin-eosin and methyl green pyronin. Other sections were submitted to TUNEL reaction for in situ detection of apoptosis, and immunohistochemistry to detect CDV antigen. Immunohistochemistry confirmed the presence of viral antigen. Syncytia were present in all retropharyngeal lymph nodes. Several syncytia contained apoptotic nuclei as detected by hematoxylin-eosin and methyl green pyronin stains. Apoptotic nuclei were labeled inside syncytia by the TUNEL reaction. Blood smear analyses indicated higher proportion of apoptotic leukocytes in the peripheral blood of infected dogs.

Methyl green-pyronin Y staining of nucleic acids: studies on the effects of staining time, dye composition and diffusion rates

Since the introduction of the methyl green-pyronin Y procedure as a differential histological stain more than 100 years ago, the method has become a histochemical procedure for differential demonstration of DNA and RNA. Numerous variants of the procedure have been suggested, and a number of hypotheses have been put forward concerning kinetics and binding mechanisms. Using both filter paper models containing DNA, RNA or heparin and histological sections, we have attempted to evaluate the kinetics of staining and the role of staining time for methyl green and pyronin Y by applying the dyes individually, simultaneously and sequentially. The results are presented as color charts approximating the observed staining patterns using a computerized palette. Our results indicate unequivocally that the differential staining is not time-dependent, but that it is dictated by the relative concentrations of methyl green and pyronin Y and by the pH of the staining solution.