Effect of formalin-fixation and paraffin-embedded tissue storage times on RNAscope in situ hybridization signal amplification.

RNAscope in situ hybridization (ISH) detects target RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. Protocols suggest that prolonged FFPE storage and formalin fixation may impact signal detection, potentially limiting the utility of RNAscope ISH in retrospective studies. To develop parameters for RNAscope use with archived specimens, we evaluated the effect of formalin-fixation time by measuring the signal of a reference gene (16srRNA) in selected tissues fixed in 10% neutral-buffered formalin for 1, 2, 3, 5, 7, 10, 14, 21, 28, 60, 90, 180, and 270 d. The signal intensity and percent area of signal decreased after 180 d. Tissues had detectable signal at 180 d but not at 270 d of formalin fixation. To assess target detection in paraffin blocks, we qualitatively compared the signal of canine distemper virus (CDV) antigen via immunohistochemistry and CDV RNA via RNAscope ISH in replicate sections from blocks stored at room temperature for 6 mo, 1, 3, 6, 8, 11, 13, and 15 y; RNA was detected in FFPE tissues stored for up to 15 y. Our results demonstrate that RNAscope ISH can detect targets in tissues with prolonged paraffin storage intervals and formalin-fixation times.

Morphometric and histopathological evaluation of modified Elnady's plastinated tissue compared to non-plastinated tissue: Highlighting its relevance for teaching and research.

The present study aims to evaluate the morphometric and histopathological properties of Modified Elnady's plastinated tissue after a period compared to non-plastinated tissue. The plastination technique is utilized in research and teaching due to the potential health risks associated with prolonged exposure to formalin. The tissues and organs are permanently dried during plastination and can be used for further anatomical, histopathological and surgical educational purposes. This method involves drying tissue and allowing synthetic materials like glycerin to permeate it. The study compared non-plastinated and plastinated tissue post-plastination to determine if structural alterations differed from those linked to plastination. The study examined the histopathological examination of dogs' skin, muscles, liver, lung, and intestine using formalin-fixed organs for paraffin embedding and previously plastinated organs for a plastinated group. The study examined non-plastinated and plastinated tissues, their histological composition and biometric parameters revealing typical structures in the non-plastinated group. Plasmodiumted tissues exhibited a compacted appearance, volume changes, nuclear clarity, and cytoplasmic hypereosinophilia, with statistical differences between the two groups. The study reveals that plastinated tissues, after 5 years of plastination, maintain their histological architecture well, with some exceptions. Plastinated tissues can be utilized in future microscopic and immunological studies and will be beneficial for teaching and research.

Retrospective study of Bovine herpesvirus 5 meningoencephalitis in cattle from São Paulo State, Brazil / Estudo retrospectivo de meningoencefalite por herpesvírus bovino-5 em bovinos do estado de São Paulo, Brasil

Meningoencephalitis caused by Bovine herpesvirus 5 (BoHV-5) is an important neurological disease that affects Brazilian cattle herds. The present study investigated the presence of BoHV-5 DNA in cattle diagnosed with meningoencephalitis at Faculdade de Medicina Veterinária e Zootecnia, UNESP - Univ Estadual Paulista from 1980 to 2009. The records obtained from the Large Animal Internal Medicine Service and the Animal Pathology Service were reviewed to identify clinical and epidemiological data from cattle with neurological signs. Excluding rabies cases, we found 115 cases of cattle with neurological signs that had been necropsied. Non-suppurative meningoencephalitis was diagnosed in 28 animals of the 115 initially selected based on histopathological examination of brain tissues. Of these 28 animals, 15 (54%) were positive for BoHV-5 DNA by polymerase chain reaction (PCR) of formalin-fixed paraffin-embedded (FFPE) brain samples. PCR target was 159-bp fragment from the BoHV-5 glycoprotein C gene. The oldest case identified in the present study was from 1988. PCR was a good tool for the diagnosis of BoHV-5 DNA extracted from FFPE tissues, allowing retrospective studies of samples stored for more than 20 years.(AU)

A meningoencefalite por herpesvírus bovino-5 (BoHV-5) é uma doença neurológica importante no rebanho bovino brasileiro. Este estudo tem por objetivo verificar a presença do DNA de BoHV-5 em bovinos diagnosticados com meningoencefalite na Faculdade de Medicina Veterinária e Zootecnia da Universidade Estadual Paulista, entre os anos de 1980 e 2009. Foram revisados os arquivos do Serviço de Clínica de Grandes Animais e da Patologia Animal em busca dos dados clínicos e epidemiológicos de bovinos com sinais neurológicos. Excluídos os casos de raiva, foram encontrados 115 casos de bovinos com sinais neurológicos, que foram necropsiados. O exame histopatológico realizado nos tecidos encefálicos desses animais constatou lesões de meningoencefalite não supurativa em 28 animais. Destes, em 15 (54%) casos foi identificada a presença do DNA de BoHV-5 por meio de PCR realizada em amostras de tecido encefálico fixadas em formalina e incluídas em parafina (FFPE). O alvo da PCR foi um fragmento de 159 pb do gene da glicoproteína C do BoHV-5. O caso mais antigo identificado neste estudo foi de 1988. A PCR apresentou-se como boa ferramenta para o diagnóstico do DNA de BoHV-5 extraído de tecidos FFPE, possibilitando estudos retrospectivos e diagnóstico de amostras com mais de 20 anos de armazenamento.(AU)

Morphological study of the male genital organs of Gracilinanus microtarsus / Estudo morfológico dos órgãos genitais masculinos em Gracilinanus microtarsus

Gracilinanus microtarsus is one of the smallest marsupials on earth. Since it spreads seeds, it has great ecological relevance. However, its reproduction data, especially those related to the anatomy of its reproduction apparatus, are scarce in the literature. Current analysis describes the male genital organs of six adult specimens of G. microtarsus. Macroscopic studies were undertaken on dissected organs, whereas histological studies were performed by inclusion technique in paraffin and by hematoxylin and eosin and Masson trichrome staining. The male genital organs of G. microtarsus consist of a penis with bifid glans, two testicles within a pendular scrotum, placed cranially to the penis, featuring a histology consisting of seminiferous tubules with spermatogonic cells, spermatozoa and Sertoli cells, and a peritubular region with Leydig cells. Testicles are closely associated with epididymis with head, body and tail, with histological differences between the different regions. Deferent ducts, spermatic funicles and annexed glands were reported. The latter were composed of prostate glands divided into three distinct segments and bulbourethral glands. Results show that the male reproduction system of G. Microtarsus is anatomically similar to that of Didelphis sp. and other marsupials groups, with slight details such as the site of each organ...

Gracilinanus microtarsus é um dos menores marsupiais do mundo. Possui grande potencial dispersor de sementes e por esta razão apresenta grande importância ecológica. No entanto, dados em relação à reprodução desses animais são escassos na literatura, principalmente, aqueles relacionados à anatomia dos órgãos do aparelho reprodutor. Por essa razão, objetivamos com este estudo realizar uma descrição dos órgãos genitais masculinos em seis exemplares de G. microtarsus adultos. Para tanto, realizamos estudos macroscópicos por dissecação dos órgãos e estudos histológicos por técnica de inclusão em parafina e coloração em hematoxilina e eosina, e tricromo de Masson. Encontramos, que os órgãos genitais masculinos e glândulas acessórias em G. microtarsus são representados por dois testículos localizados dentro de um escroto pendular, posicionados cranialmente ao pênis, cuja histologia revelou túbulos seminíferos com espermatogônias, espermatozoides e células de Sertoli e região peritubular com células de Leydig; testículos em íntima associação com os epidídimos, divididos em cabeça, corpo e cauda, com diferenças histológicas entre as diferentes regiões; ductos deferentes; funículo espermático; glândulas anexas compostas pelas glândulas prostáticas divididas em três distintos segmentos e glândulas bulbouretrais; uretra pélvica e peniana e um pênis com glande bífida. Concluímos que os órgãos genitais e as glândulas acessórias masculina de G. microtarsus, anatomicamente são muito semelhantes aos dos Didelphis sp. e outros grupos de marsupiais, diferenciando-se apenas em alguns detalhes, como a localização de cada órgão...