Browsing by Author "AMBRÓSIO, Carlos Eduardo"

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Achado histopatológico de lipidose hepática em fetos de gambá (Didelphis sp.) lactantes
(Fundação de Ensino Octávio Bastos, 2008-11-30) GARCIA, Fernanda Figueiredo; MANÇANARES, Celina Almeida Furlanetto; MIGLINO, Maria Angélica; AMBRÓSIO, Carlos Eduardo; ROSA, Ricardo Alexandre; CARVALHO, Ana Flávia de
Opossums (Didelphis sp.) Are highly reproducible marsupials, have a gestation period of approximately 12 days and extensive lactation. Fetuses receive high levels of carbohydrates and lipids from the mother during lactation. The objective of this work was describe the macro and microscopic morphology of the livers of opossum fetuses (Didelphis sp.) at different stages of their postnatal development. An old animal (5 days, 15 days, 22 days and 35 days) for macroscopic and microscopic verification of liver structures and consequent comparison with other domestic species. The animals' livers were processed and included by paraffin embedding techniques. Each block was cut and the sections were stained with HE, picrosirius, PAS histochemical reaction with hematoxylin, toluidine blue and Masson's trichrome to observe the hepatic structures. The macro and microscopic results indicate a premature development of the species in the fetal stage, similar to domestic animals and interestingly there is presence of hepatic lipidosis in all animals.
Alterações no perfil esparmático e constituição histológica dos testículos de coelhos sob a influência do aumento transitório da temperatura testicular
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2004-09-30) MARTINS, João Flávio Panattoni; BARROS, Michele Andrade de; IVO, Marcos Alexandre; OLIVEIRA, Priscila Carvalho de; BALIEIRO, Cristiano de Carvalho; AMBRÓSIO, Carlos Eduardo; MIGLINO, Maria Angélica; CARVALHO, Ana Flávia de
This study was conducted to investigate the sperm profile of rabbits, evaluating concentration, motility vigor and incidence of sperm with changes in the compaction of genetic material (pathologies of the DNA protein complex), under normal conditions and under the influence of the transient increase in temperature testicular. Eight fertile rabbits were used evaluating the parameters of 3 ejaculates on alternate days and then the animals were subjected to an increase in testicular temperature for 3 days, collecting semen samples on Mondays, Wednesdays and Fridays for 38 days for analysis. . Every 5 days a rabbit underwent orchiectomy for histological evaluation of testicles and epididymis.
Análise biométrica de embriões, fetos e das membranas placentárias de equinos.
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2008-11-30) FERRAZ, Ana Cláudia Cristiane; MANÇANARES, Celina Almeida Furlanetto; MIGLINO, Maria Angélica; AMBRÓSIO, Carlos Eduardo; CARVALHO, Ana Flávia de
For this study, embryos, fetuses and attached membranes of 23 concepts of mares from Frigorífico Miramar - Pelotas RS were used. Embryos and fetuses were analyzed for size and external developmental characteristics to analyze gestational age. The attached membranes were weighed and measured for later correlation with gestational ages. The results obtained so far indicate that the embryonic and fetal development of horses is very fast in the early stages of development, this being a critical period of placental implantation and the beginning of the endocrine activities of the placenta. The biometric data related to the umbilical cord are not progressive according to development, having shown great variations among the animals studied.
Análise macro e microscópica do pâncreas e fígado de gambá
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2004-09-30) CASALS, Juliana; BELLATINE, Tatiana; AMBRÓSIO, Carlos Eduardo; CARVALHO, Ana Flávia de; MARTINS, João Flávio Panattoni; ROQUETO, Marco Antônio; MANÇANARES, Celina de Almeida Furlanetto; MIGLINO, Maria Angélica; HOFFMAN, Roberto
16 animals from natural death were used in different situations. The techniques used, often traumatic, were injection of 10% formaldehyde followed by injection of latex (blue for the veins and red for the arteries) in the jugular vein and abdominal aorta. The results showed that the liver was dome-shaped in the cranial abdominal region, being macroscopically composed of 5 lobes and histologically similar to other domestic animals. The pancreas is dispersed and has an irregular surface. Its vascularization consisted of pancreatic duodenal, cranial and caudal arteries. Histologically, it presented characteristics similar to the pancreas of domestic species.
Análise macroscópica do aparelho reprodutor masculino do ouriço cacheiro
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2007-09-30) CASALS, Juliana Barbosa; BARREIRO, Juliana Regina; ROSA, Ricardo Alexandre; MANÇANARES, Celina Almeida Furlanetto; FAVARON, Phelipe Oliveira; CARVALHO, Ana Flávia de; MIGLINO, Maria Angélica; MARQUES, Leandro de Oliveira; ANDRADE, Filipe Ian Bindez de; NASCIMENTO, Priscila Leal do; FERRAZ, Ana Cláudia Cristiane; AMBRÓSIO, Carlos Eduardo
Rodents comprise most of the mammalian class. There are currently 2,021 species, organized into 443 genera and 29 families. Hedgehogs (Coendou villosus), are animals that in captivity, reproduce with some ease and become tame. In the urban and metropolitan region their presence is becoming common and this can be identified by the number of accidents involving dogs in the yard, registered in veterinary clinics. This work aimed to describe macroscopically the reproductive apparatus of Coendou villosus (hedgehog) to better clarify the anatomy of this important organ in the wild rodent model, since very little is known about this species. We compared the results obtained with other rodent species already studied. For the macroscopic description of the male reproductive system, 2 animals from donations were used, these animals ended up dying after attack by dogs.
Análise macroscópica dos anexos embrionários e fetais de equinos em até 107 dias de gestação
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2009-09-30) FERRAZ, Ana Cláudia Cristiane; MANÇANARES, Celina Almeida Furlanetto; MIGLINO, Maria Angélica; AMBRÓSIO, Carlos Eduardo; CARVALHO, Ana Flávia de
The horse (Equus caballus, Linneaus 1758) is a large ungulate mammal of the Equidea family, one of the seven modern species of the Equus genus. Domesticated for thousands of years, it is an unparalleled animal, of unusual beauty, grace, sensitivity and athletic ability; a fascinating animal for study, as described by Evans et al. (nineteen ninety); Ginther (1992) and Dantzer et al. (1998). Because it is not a simple model, it offers a comprehensive range for research and challenges scientists. Equine placentation and its embryonic development are some of the issues to be explored. Thus, this study aimed to describe microscopically the embryonic attachments of the embryo / fetus of horses in the first four months of pregnancy. In order to carry out this research, 37 placentas of adult, mixed breed mares were collected and divided into groups at the different stages of development, following the methodology employed by EVANS SACK (1973), until 107 days of gestation, and were subsequently destined for the microscopy of light. The membrane fragments (corium, allantois, amnion and yolk sac) were fixed in 10% formalin and Bouin followed by routine paraffin processing (TOLOSA et al; 2003). Serial slices with a thickness of 5μm were obtained in a microtome (Leica 2165®) which were submitted to staining by the methods of hematoxylin and eosin, picrosirius, Toluidine blue and Masson's trichrome (TOLOSA et al; 2003). In the four groups, the amnion presented with a simple pavement epithelium and mesenchyme without any vascularization, only vascularization was evident when the amnion fused with the allantois, which is also a simple pavement epithelium. The corium presented villi with a low columnar epithelium and brushed edges, uni and binucleated cells, highly vascularized, and the size and quantity of villi differed from the groups. The yolk sac presented an epithelium whose cells were large and globular, resting on the mesenchyme, forming vascular islands full of hemangioblasts, being non-existent in animals from 47 to 107 days of gestation.
Análise microscópica do estágio de ossificação do gambá (DIDELPHIS SP.) em diferentes estágios fetais através da diafanização e do exame radiográfico
(Fundação de Ensino Octávio Bastos, 2008-11-30) GARCIA, Fernanda Figueiredo; MANÇANARES, Celina Almeida Furlanetto; ALVES, Jeferson Douglas Soares; ROSA, Ricardo Alexandre; MIGLINO, Angélica; AMBRÓSIO, Carlos Eduardo; CARVALHO, Ana Flávia de
Opossums (Didelphis sp.) Are marsupials that have a very short gestation period, therefore, their offspring are born immature and will end their development in the mother's marsupium. Because they are very early in their development, in the fetal stage of these animals, they already have premature growth of their organs and tissues as a whole. The objective of this work was to describe the ossification stages of opossum fetuses (Didelphis sp.) At different postnatal stages, through diaphanization and radiographic examination. One animal of each age (5 days, 15 days, 22 days and 35 days) was used to verify ossification and consequent comparison with other domestic species. The results indicate a premature ossification of the species in the fetal stage, unlike domestic animals.
Análise microscópica e ultraestrutura do saco vitelino de equinos com até 47 dias de gestação
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2010-05-30) FERRAZ, Ana Claudia Cristiane; NASCIMENTO, Priscila Leal do; MANÇANARES, Celina de Almeida Furlanetto; FRANCIOLLI, André Luis Rezende; MIGLINO, Maria Angélica; AMBRÓSIO, Carlos Eduardo; CARVALHO, Ana Flávia de
This work aimed to describe microscopically the yolk sac of horses up to 47 days of gestation. To carry out this research, 37 placentas and embryos from 15 to 47 days of gestation were collected. The material was taken from adult, mixed breed mares in a slaughterhouse. Embryonic age was estimated according to the methodology used by EVANS SACK (1973). The embryos were destined for microscopic analysis. For analysis under light microscopy the fragments of the yolk sac membranes were fixed in 10% formaldehyde and Bouin followed by routine paraffin processing (TOLOSA et al; 2003). Serial slices with a thickness of 5μm were obtained in a microtome (Leica 2165®) which were submitted to staining by the methods of hematoxylin and eosin, picrosirius, Toluidine blue and Masson's trichrome (TOLOSA et al; 2003). Another part of the material was submitted to transmission and scanning electron microscopy. The yolk sac epithelium varied from globular to columnar, with uni or binucleated cells in a single layer, with a large amount of rough endoplasmic reticulum, distributed evenly, the epithelium supported on the mesenchyme forming vascular islands filled with hemangioblasts, being non-existent in animals older than 47 days of gestation; and characteristics of protein secretion.
Análise morfológica das células do aspirado medular de fetos caninos
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2011-05-30) MÁRIO, Lara Carolina; BORGHESI, Jéssica; MELLO, Ana Carolina Landentin; MIGLINO, Maria Angélica; AMBRÓSIO, Carlos Eduardo; MARTINS, Daniele; CARVALHO, Ana Flávia de; WENCESLAU, Cristiane Valverde
The adult bone marrow is a source of stem cells (CT), which include the hematopoietic stem cells (HSC) that originate the entire definitive hematopoietic lineage; and mesenchymal stem cells (MSC), and or stromal which are responsible for providing a favorable environment for hematopoiesis. In addition to these main cell types, bone marrow is composed of several parents, differentiated cells and an extracellular matrix. This environment where CT is found is called a niche, as it houses a large amount of CT and cellular progenitors that are kept quiescent, acting normally and or in the face of injuries. However, little is known about the fetal bone marrow niche. It is known that the bone marrow niche is derived during the ontogenesis of the yolk sac, which subsequently migrates to various hematopoietic organs such as the placenta and liver, ultimately settling in the bone marrow. Thus, the present work aims to describe morphologically the cell types of canine fetal bone marrow at 40 and 60 days of gestation. For this purpose, 6 pregnant female uteri were used to collect the medullary aspirate from the femur bone and later structural and ultrastructural morphological analysis. The results demonstrated that the canine fetal bone marrow at 60 days of gestation has cell types similar to that of the adult bone marrow as well as CT, thus being able to be used as a source of CT isolation.
Análise ultra estrutural das membranas embrionárias e fetais de equinos
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2008-11-30) NASCIMENTO, Priscila Leal do; MANÇANARES, Celina Almeida Furlanetto; MIGLINO, Maria Angélica; AMBRÓSIO, Carlos Eduardo; CARVALHO, Ana Flávia de
To develop this research, 13 pregnant uteri were used, of which 13 placentas were obtained divided into groups at different stages of development. The material was taken from adult, mixed breed mares and at different gestation periods of up to 120 days and was destined for ultrastructural analysis. The results obtained from SEM (scanning electron microscopy) were similar to those found by ASSIS-NETO (2005), in bovine membranes. The allantoic showed cells whose surface was bordered by microvilli, forming polyhedral images, protruding “button” shaped structures were also observed. At the amnion, a surface similar to that of the allantois was observed, with its entire length covered by microvilli. In the corium, the apical surface of the cells are rounded and covered by microvilli. The yolk sac showed droplets of secretion at the apex of the cells indicating exocytosis. So far it has been concluded that the mare's placenta is chorioallantoic, epithelial, diffuse, non-deciduous and villous.
Características de células fibroblast-like derivadas do cultivo do saco vitelino em diferentes períodos gestacionais
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2007-09-30) WENCESLAU, Cristiane Valverde; MARTINS, Daniele dos Santos; AMBRÓSIO, Carlos Eduardo; FRANCIOLLI, André Luiz Resende; AGRESTE, Fernanda R.; MORINI, Adriana Caroprezo; MORINI JÚNIOR, João Carlos; CARVALHO, Ana Flávia de; MIGLINO, Maria Angélica
Based on the knowledge regarding the erythropoietic activity of the vitelline membrane, which is considered the first hematopoietic and vascular site that appears in mammals during organogenesis, added to the erythropoiesis support function performed by mesenchymal stem cells, the present work aims to verify the cell expansion of the yolk sac using the explant technique, in view of the cell niches found in the fetal annex. For the cultivation of the yolk sac, 5 bitches with gestational ages of 30, 40 and 60 days were used, and were checked by ultrasound and Crow - Rump of each fetus according to Evans & Sack (1979). The bitches were submitted to ovariosalpingohisterectomy to collect the fetal attachment. All material was collected at the Faculty of Veterinary Medicine and Animal Science at the University of São Paulo - USP. The yolk sac was fragmented manually (mechanical fragmentation), with the aid of a scalpel blade until obtaining a homogenate of tissue fragments and cell groups, which were implanted in the 5 ml culture bottle with the modified Eagle Dulbeco medium - DMEM High with 10% fetal serum, streptomycin and penicillin. The cells were incubated at 38.5 degrees Celsius, at 100% humidity and 5% CO 2. The morphological description of the cell culture was performed periodically using the Nikon TS100 phase contrast microscope. The cells maintained in these conditions grew in the form of monolayer, with different fusiform cell types and large cells with cytoplasmic extensions like fibroblast-like.
Caracterização da morfologia externa de fetos de Paca
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2007-09-30) FRANCIOLLI, André Luis Rezende; AMBRÓSIO, Carlos Eduardo; MARTINS, Daniele dos Santos; MANÇANARES, Celina Almeida Furlanetto; CARVALHO, Ana Flávia de; WENCESLAU, Cristiane Valverde; COSTA, Gerlane M.; MORINI JÚNIOR, João Carlos; MORINI, Adriana Caroprezo; MIGLINO, Maria Angélica
The present scientific investigation aimed to study the embryonic development of pacas (Agouti Paca), with a view to the external morphological description of fetuses. Five fetuses were used, three of which were donated from the donation maintained by the Faculty of Agricultural and Veterinary Sciences of Jaboticabal - UNESP and two donated from the collection of biological material from FMVZ - USP. Fetuses 1 and 2 with 5.7 and 6.8 cm of Crow Rump, respectively, presented as morphological characteristics: marked facial immaturity, with the eyes covered by a prominent lens; the earlobes had a low level of development. The mouth had a small opening, where the formation of the incisor teeth was observed. Fetuses 3 and 4 with 9.6 and 10.5 cm of Crow Rump, respectively, showed well-developed ears, thoracic and pelvic limbs were in an equal degree of development with the presence of claws and development of facial features, like vibrissae around the nasal edges, the eyes remain protected by a prominent lens and the mouth contained the incisor teeth in an intermediate stage of development. Fetus 5, with 15.5 cm of Crow Rump, presented as a striking feature, the body covered by hair, thoracic and pelvic limbs in an equal degree of development with claws, development of facial characteristics, such as vibrissae, prominent eyes and well-developed ears. In all fetuses, the genital tubercle and the anal rim can be seen in the perineal region.
Caracterização das glândulas salivares maiores do gambá
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2004-09-30) MARTUCCI, Mariane; MALEK, Carolina; AMBRÓSIO, Carlos Eduardo; CARVALHO, Ana Flávia de; MIGLINO, Maria Angélica
6 animals from natural death were collected and collected in different situations such as being run over, attacks by other animals on neighboring farms, etc. Which were donated to the Anatomy Laboratory of the Faculty of Veterinary Medicine of the Fundação de Ensino Octávio Bastos; São João da Boa Vista, SP. The anatomical techniques used were injection of 10% aqueous solution of formaldehyde and dissection of the glands. The parotid gland was found between the mandibular gland and the ear, the mandibular gland and the sublingual gland. No findings similar to the dog's palatine and zygomatic gland were observed.
Caracterização e fórmula dentária de Kerodon rupestris
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2004-09-30) THOMAZ, Juliana Mantovani; CARVALHO, Ana Flávia de; MIGLINO, Maria Angélica; AMBRÓSIO, Carlos Eduardo; MANÇANARES, Celina de Almeida Furlanetto
The dentition of mammals varies according to species, although it is formed by the same structural components. Keredon rupestris presents the dental formula 1 1/1, C 0/0, P 1/1, M 3/3, its teeth being hypsodonts, whose layers, from the external to the internal region are: cementum, enamel, dentin and pulp. In his oral cavity, the presence of a region known as diastema was noticed, which is responsible for the displacement of the cutter apparatus away from the face.
Caracterização macroscópica do funículo umbilical de búfalos
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2001-11-30) FERREIRA, Guilherme J. B.C.; CARVALHO, Ana Flávia de; AMBRÓSIO, Carlos Eduardo; MIGLINO, Maria A.; ROSA, Ricardo Alexandre; ESTEVES, Alessandra; THOMAZ, Juliana M.
The umbilical funiculus is the organ that connects the fetus to the placenta where blood and excreta from the fetus pass. Arterial blood flows from the placenta to the fetus through the umbilical veins and venous blood through the arteries, the excretion is carried through the allantoic duct. In this study, nine fetuses were used, which had their funicles measured and dissected, in order to establish developmental correlations and describe their morphology. The funnel was rod-shaped, a parallel arrangement of the vessels in the fetal just portion and a standard ruminant arrangement in the just portion - placental and middle.
Desenvolvimento das membranas fetais em carnívoros (Canis familiaris, Linnaeus, 1758)
(Fundação de Ensino Octávio Bastos, 2004-09-30) WENCESLAU, Cristiane V.; MIGLINO, Maria Angélica; AMBRÓSIO, Carlos Eduardo; CARVALHO, Ana Flávia de; OLIVEIRA, Priscila C. de; FIORAVANTE FILHO, Nivaldo; ROSA, Ricardo Alexandre; MANÇANARES, Celina Almeida Furlaneto
The present study aims to provide morphological subsidies that can contribute to research in the processes of biotechnology of reproduction, as well as in vitro fertilization, cloning and obtaining stem cells, since this process in carnivores is complex and complicated. Therefore, our proposal aims at describing the structures and evolution of fetal membranes in dogs, including the process of involution of the yolk sac.
O efeito da própolis e da membrana amniótica no tratamento de queimaduras provocadas em ratos: um estudo macro e microscópico
(Fundação de Ensino Octávio Bastos, 2008-11-30) PESSOLATO, Alícia Greyce Turatti; MARTINS, Daniele dos Santos; MANÇANARES, Celina Almeida Furlanetto; AMBRÓSIO, Carlos Eduardo; CARVALHO, Ana Flavia de
The present work aimed to study macro and microscopically the dermal and epidermal response in the regenerative or healing repair process of rats treated daily with different and alternative local dressings. After anesthesia, a burn with a plate heated to 130 ° C for five seconds in 29 rats that were divided into three groups, being a control group treated with sterile saline (n = 9), group treated with propolis ointment (n = 10) and group treated with amniotic membrane (n = 10). The animals were euthanized at seven, 14 and 21 days of treatment. Macro and microscopically on the seventh day after injury, animals treated with propolis already showed significant acceleration of the healing process. The granulation tissue was evident and chronologically more advanced than in the control group, where the granulation tissue was more evident on the 21st day after burning, while the amnion-treated group, despite not accelerating the healing process, did not show inflammatory cells. since the first days of application. Both treatments studied were effective in treating burns compared to the control group. The 5% propolis ointment accelerated the local tissue repair process and therefore anticipated healing in the initial period macroscopically. The amniotic membrane stimulated tissue cell regeneration at the injured site and inhibited the inflammatory reaction on all days following the injuries.
Estabelecimento das condições de isolamento e cultivo das células tronco mesenquimais da médula óssea em fetos de cães
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2011-05-30) BORGHESI, Jéssica; MARIO, Lara Carolina; MELLO, Ana Carolina Landentin; MIGLINO, Maria Angélica; AMBRÓSIO, Carlos Eduardo; MARTINS, Daniele; CARVALHO, Ana Flávia de; WENCESLAU, Cristiane Valverde
Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into multiple differentiated terminal strains, including bone, cartilage, muscle, nerve, adipose and marrow stromal cells in vivo and in vitro. These cells are in addition to being isolated from the adult bone marrow and can be isolated from the fetal bone marrow. However, there is little study regarding the isolation technique of MSCs from fetal bone marrow. In this way, the work proposed to isolate MSC from canine fetuses based on the established methodology for obtaining MSC from adult human bone marrow. For this purpose, 12 pregnant female uteri were used to collect the medullary aspirate from the femur bone. The culture medium of choice was alpha-MEM and 15% fetal bovine serum, which provided the isolation of positive CTM-CD44.
Estudo da morfologia e vascularização do rim de avestruz
(Fundação de Ensino Octávio Bastos, 2001-11-30) MARTINS, Daniele dos Santos; AMBRÓSIO, Carlos Eduardo; MIGLINO, Maria Angélica; FINAZZI JÚNIOR, Renato; FERREIRA, Guilherme J. B.C.; BARREIRO, Rodrigo H.
The main objective of this work is to describe the morphology and vascularization of the ostrich kidney, making it possible to compare it with the domestic rooster, duck and goose kidney with that bird. The morphological study shows that these kidneys have a cranial lobe, a middle lobe and a caudal lobe. sciatic artery. The venous system corresponds to the renal portal system, which presents two amastomoses, in the ventral view; one occurs at the interposition of the middle lobe with the caudal lobe, and another located in the cranial lobe; the veins that drain the lobes are: cranial renal vein, middle renal vein and caudal renal vein.
Estudo da pluripotencialidade do fígado fetal canino nos diferentes períodos gestacionais
(Centro Universitário da Fundação de Ensino Octávio Bastos, 2007-09-30) VIEIRA, Eduardo; MARTINS, Daniele dos Santos; WENCESLAU Cristiane Valverde; AMBRÓSIO, Carlos Eduardo; CARVALHO, Ana Flávia de; ROSA, Ricardo Alexandre; MIGLINO, Maria Angélica
The study of the differentiation capacity of some cells, called stem cells or '' sistem '' cells represents an important tool for understanding and developing new research, as well as an enormous potential for discovering the treatment of numerous diseases. These embryonic stem cells are pluripotent, capable of multiplication and differentiation, and can proliferate in several cell types depending on the culture conditions. The identification of factors that lead to the direction of the cell differentiation process will allow, from embryonic stem cells, the culture to be carried out in a controlled manner among the most different cell types, making bioengineering possible. In this work, cells of the canine fetal liver were studied. The liver performs an important hematopoietic function during fetal development, while in the adult it has a multiplicity of functions such as: bile excretion, storage, synthesis and biotransformation. The livers, for study, were obtained from fetuses with gestational age with an average of 25, 35, 45 and 55 days, from females of mixed breed dogs that underwent ovariosalpingohesterectomy. These canine fetal liver cells were analyzed histologically to characterize cell differentiation, using previously established histological methods.