Comparative Histo-Pathology of mouse models of human disease
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1 Spring 2013 Comparative Histo-Pathology of mouse models of human disease Practical Histopathology in mouse models of human disease: Guides to Phenotyping the Genetically Altered mouse (see schedule of lectures below) Course Organization: 1. Scope and Outline: The course is designed to guide investigators with the analysis of genetically altered mice. Lecture topics will emphazie use of histology, histochemistry, immunohistochemistry, in the interpretation of the histopathology of the various organ systems. Those who will benefit from attending the course will be graduate students, medical students, post-doctoral fellows and interested faculty. Lecture topics will include technical information and protocols, along with discussions on the various phenotyping methods available to help with the analysis of genetically altered mice, as compared with littermate controls. Identification of the different tissues and cell types in the body and the ability to detect abnormalities, will be the goal, thus enabling the optimal phenotypic analysis of genetically altered mice. Teaching methods: One two hour lecture/ demonstration per week for 8-10 weeks. Lectures, demonstrations, multi media and computer support Source Material Glass slides with microscope review, lectures, Textbooks: a) Pathology of the mouse. Ed. Robert R. Maronpot. Cache River Press ISBN: X b) di Fiores Atlas of Histology. Victor P. Eroschenko Lee and Febiger c) Histology. A text and Atlas. Ross, Romrell and Kaye. Williams and Wilkins ISBN: d) Human Histology. Stevens and Lowe. Mosby. ISBN: e) An Atlas of Histology. Zhang. Springer. ISBN: f) Pathologic Basis of disease. Robbins,Kumar and Collins. Saunders. ISBN: X g) The Laboratory Mouse. Suckow, Danneman, Brayton. CRC Press ISBN: h) A Color Atlas of Sectional Anatomy of the mouse. Iwaki, Yamashita, Hayakawa. Braintree Scientific Inc. ISBN: i) The Atlas of Mouse development. Kaufman. Academic Press. ISBN j) The House Mouse. Atlas of Embryonic development. Theiler. Springer-Verlag ISBN: k) Manipulating the mouse embryo, a laboratory manual. Hogan, Beddington, Costantini, Lacy. Cold Spring Harbor Laboratory Press ISBN: Evaluation: 1. Evaluation of Students: Quiz at end of course
2 2. Evaluation of Course: Evaluation forms to be filled out by students at the end of each lecture: a) title of lecture b) information obtained met objectives, did not meet objectives c) lecture content: too detailed, just enough detail, not enough detail d) other comments Classes PATH 234/MED 234 / BIOM 238/ BGGN 234 Practical Histopathology in mouse models of human disease: Guides to Phenotyping the Genetically Altered mouse Location: Basic Science Building: MDL lab-4 Time: Tuesday 1-3 pm Course Director MD Phone: nvarki@ucsd.edu Guest lecturers: Dzung Le MD PhD, Department of Pathology Michael Kalichman PhD, Department of Pathology Lars Eckman MD, Department of Medicine Maripot Corr MD, Department of Medicine Mark Fuster MD, Department of Medicine Kent Osborn DVM PhD, Department of Pathology Microscopes and slides will be available at every session for individualized review. Where applicable, protocols will be available Schedule of lectures for the Preclinical elective course Spring 2013 Practical Histopathology in mouse models of human disease: Guides to Phenotyping the Genetically Altered mousepath234, MED 234 / BIOM 234/ BGGN 234 Spring 2013 Elective PATH 234, MED 234, BGGN 234, BIOM234 Practical Histopathology in mouse models of human disease: Guides to Phenotyping the Genetically Altered mouse: Start date : Tuesday April 2, 2013 Time: 1 pm to 3 pm Location: Basic Science Building MDL lab#4 Microscopes and slides will be available at every session for individualized review Where applicable, protocols will be available
3 Week Date Topic(s) Instructor 1 Apr 2 Introduction to histology and histopathology (Cell injury/repair/death, inflammation, neoplasia, etc.) Histochemistry methods and protocols "Beyond Mouse Models in Biomedical Research: Approach and Response to Unexpected Morbidity and Mortality" 2 Apr 9 Hematopoietic system: Spleen, Thymus, Lymph nodes, Bone Marrow, Blood Coagulation, chemistries, hematology in mouse models, examples and protocols 3 Apr 16 Neural: Brain, cerebellum, Spinal cord, peripheral nerves Immunohistochemistry: principles, practice and protocols 4 Apr 23 Digestive system: Salivary Glands, Liver, Pancreas, Intestines: Esophagus, Stomach, Duodenum, jejunum, Ileum, colon, cecum, appendix Mouse models of Inflammatory bowel disease 5 Apr 30 Genitourinary: Kidney, Bladder, Histopathology and markers Female: Ovary, uterus, fallopian tube (human) Male: testis, epididymis, seminal vesicles, Prostate Mouse embryos, Placenta 6 May 7 Circulatory and Respiratory systems: Heart and blood vessels, skeletal muscle, smooth muscle, Trachea and lungs Lymphatic markers in cancer progression 7 May 14 Integument: Skin, Breast:Â non lactating, lactating, Bone, Cartilage, Joints Evaluation of mouse models of Joint disease 8 May 21 Endocrine: Pituitary, Thyroid, Parathyroid, Adrenal, Islets adipose tissue- fat, brown fat, 9 May 28 Responsible Conduct of Research Immunohistochemistry: principles, practice and protocols 10 June 4 Review and take home test Kent Osborn Dzung Le Lars Eckmann Mark Fuster Maripat Corr Mike Kalichman
4 Introduction: Although mice are now accepted as models to study effects of different genetic manipulations on the course of phenotypic changes related to human disease, we need to note that there are several differences in the organs. Because of inter-species differences, it is important to stay familiar with the different germ layers that make up the different cells in the different organs. Genetically altered mice may result as a consequence of integration of foreign DNA into a fertilized oocyte by random chromosomal insertion or after homologous recombination and introduction of embryonic stem cells into zygotes. The complete characterization of all of the resultant changes in gene expression in these altered animals then becomes a monumental task. Most investigators prefer therefore prefer to concentrate on changes that occur in specific organ systems. The comprehensive analysis usually includes assays using blood chemistry, hematology and immunology. Comparisons with wild type litter-mate animals that are matched for age and sex, forms the necessary control. The animals are then analyzed further using a number of histology methods. Routine histology of the adult mouse organs and of embryos (at different ages) may then lead to additional assays using either lectin histochemistry or immunohistochemistry. Use of a variety of markers thus helps to highlight changes that may not have been obvious using usual methods, and which may prove important to the characterization of the phenotypic changes. A phenotypic evaluation of animal models of human disease is critical in the understanding of the pathogenesis and the processes involved. A careful examination (always with the organs and tissues from littermate controls) of completely back-crossed genetically altered mice, at different ages, is required. A comprehensive histopathologic review, hematology analyses, chemistry panels and a variety of behavioral tests will help to accomplish some of these goals. A comprehensive study should plan to first look for systemic changes by doing basic screens with hematology and chemistry assays in serum. The Hematology/Chemistry section of the Mouse phenotyping core will get that organized. Then, if there are changes seen, we would plan a terminal experiment, with drawing more blood for coagulation assays and the organs would be fixed for histologic analysis. For an initial survey for abnormalities, one should plan to examine serum from about 32 animals and tissues from 24 animals. This would include 6 male and 6 female littermate controls, and then 6 male and 6 female test animals, as a minimal first run screen. Once organs are harvested, they are arranged in an organized manner such that interpretation and comparisons between control and mutant animals are possible. Tissues and organs from 6 wild type male mice that can then be compared with 6 mutant male mice and those from 6 wild type female mice which can be then compared with 6 mutant female mice.
5 Chapter 1: examination. Optimal harvesting and processing organs for histopathological When tissues are removed from the body, during surgery, or during an autopsy, prompt onset of autolysis occurs which can inhibit efforts to isolate nucleotides or certain enzymes and proteins for various investigative efforts. The tissue thus has to be flash-frozen for extracts, or frozen in cryoprotective agents, or fixed, using different proceduredependent fixatives, for analysis in histo-pathology. Freezing tissue for histopathology: Optimum Cutting Temperature Compound (OCT) is the commonly used cryoprotective agent to surround tissues prior to flashfreezing. If tissues are placed into the freezer without using cryoprotective agents, the water molecules freeze at different rates from the freezing of the tissue. When one then attempts to make frozen sections for use in histology or immunohistochemistry, the tissue will look broken and shattered with spaces where antibodies will congeal contributing to non-specific background. Figure 2 shows two panels of different mouse spleen samples, stained using hematoxylin and eosin. The panel of the left has recognizable splenic follicles but the panel on the right shows evidence of freeze artefact, with poor morphology of a poorly frozen mouse spleen. The tissue is harvested, blotted to remove tissue fluids, surrounded and immersed in freezing medium and flash frozen, either in liquid nitrogen or in a dry-ice/2-methyl butane slurry, and then stored at minus 80 until needed for frozen sections. Fixation of tissues for processing and paraffin embedding prior to morphologic examination: If tissues are fixed in one of the commonly used fixatives, they should be fixed for less than 24 hours, before being processed immediately, and embedded into paraffin for sectioning and staining. Hard structures such as bones will need to be softened by removing the calcium. For this there are various reagents that are used. Decalcification of Bones using one of various reagents is important before processing and embedding into paraffin blocks for sectioning and staining. Using EDTA for decalcification, takes longer but is advantageous in allowing the detection of antigens in immunohistochemistry assays, in paraffin sections of bone and cartilage. Brain: It is best to perfusion fix the mouse brain before removing for processing for histopathologic examination. Artefacts are introduced when the brain is removed from the skull, which may impede correct interpretation. Lungs: For the best morphological analyses it is best to inflate the lungs with fixative or with the freezing medium. Although human lungs are large in comparison to mouse lungs, if they are inflated with fixative, it is possible to analyze morphologic detail optimally. Since mouse lungs collapse when the thorax is opened it is important that, after identifying the cartilage-ringed trachea, the lungs are inflated in order to obtain the best histological detail.
6 Skin, Spleen, Thymus, Pancreas, Adipose tissue. These particular organs from mice, need to be flattened between histology sponges into cassettes, before fixing, in order to obtain the best sections for staining analyses. This flattening before fixation allows these tissues to be oriented, such that the entire section will show large areas for analysis.nsimilarly if skin is flattened before fixing the histotechnician will correctly orient while embedding it into paraffin wax, after processing. Examination of Tumors: Like most organs, tumors are also three dimensional, and so it is best to make thin slices, before fixing flat in correctly simply labeled cassettes, for fixing Specific requirements for mouse intestine: The intestinal mucosa is extremely sensitive to prolonged periods of drying and thus must be fixed with as much haste as possible. Small segments of small intestine and of the colon, are opened and rolled onto a stick, before immersion fixation, for processing, embedding, sectioning and staining.
7 Chapter 2: Identification of different Tissues and Organs, using the light microscope: The different cellular components within organs and tissues are derived from three embryonic layers and the identification of the different cell types become important in histo-pathological analyses. Thus, when examining tissue sections, it becomes important to be able to identify whether the tissue is epithelial in origin or is made up of stroma and other supporting cells as shown next. Epithelium is derived from different germ layers, and is an essential part of the impervious skin on the outer surface of the body, lines internal cavities of the gastrointestinal tract, respiratory tract, genitourinary tract, and comprises cells of the various organs, with different functions. Types of epithelium: Simple squamous epithelium is a single layer of cells which line the outside of many organs and also the inside of blood vessels. Stratified (layered) squamous epithelium is so named because when fully differentiated, the appearance on stained tissue sections is similar to stacks of plates of cells. The next figure shows a diagram and a photograph of stratified squamous epithelium from mouse esophagus. The uppermost layer is fully differentiated and is anuclear, and is the keratin layer, which sloughs off as part of the normal process of differentiation. Columnar epithelium is named because the height of the cell is greater than the width of the cell, and the cells appear to form columns with nuclei at the base, as shown as a diagram and as a photograph of intestinal epithelium. Pseudo-stratified columnar epithelium is shown next, with photographs of human trachea as compared with mouse trachea. Pseudo-stratified columnar epithelium is found only in certain areas of the body and is characteristically seen lining the human trachea. It is termed pseudostratified, because it appears to have many layers under routine examination using light microscopy but detailed analysis shows that each cell rests on a basement membrane. Cuboidal epithelium has the nucleus occupying the middle of each cell. This type of epithelium is observed, lining thyroid follicles, as demonstrated here, or lining ducts exiting from glands, taking digestive enzymes from the pancreas or the salivary gland, lining bile ducts in the liver, etc. Transitional epithelium lines the bladder and ureters. The term transitional defines the epithelial cells that appear to be 3-4 layers when the bladder is empty, and appear to be flattened when the bladder is full. Examples are shown next, where the panel on the left depicts a section from an emptied mouse bladder and the section on the right is that from a full mouse bladder. Junctional zone epithelium is seen (as shown), where the squamous epithelium changes into columnar epithelium as seen at the ecto and endo cervix or esophagus and stomach in the human. In the mouse, the epithelial lining of the stomach is different from that of human in that the epithelium remains squamous before becoming columnar halfway into the stomach. Non-Epithelial or Supporting tissues:
8 The supporting tissues are primarily of mesodermal origin, they surround and support epithelial tissue, and are composed of different cell types. Some examples are shown next with details in specific chapters. As shown, smooth muscle and skeletal muscle support the upper esophagus in this photograph of a section of stained mouse esophagus. Muscle: There are 3 kinds of muscle as depicted next. Smooth muscle supports blood vessels, and the gastrointestinal tract. Skeletal muscle and cardiac muscle both have striations, but the difference is that cardiac muscle has central nuclei, whereas in skeletal muscle, nuclei are peripherally distributed. Bone and cartilage are also of mesodermal origin. Cartilage has abundant hyaluronic acids and thus changes the tinctorial characteristics which make it easy to recognize on stained tissue sections as shown. Other tissues of mesodermal origin include Adipose tissue, blood vessels, nerves, and cells comprising the different compartments of the hematopoietic system. Some examples are shown next with details in specific chapters. Lymph Nodes are part of the hematopoietic system and more details are described in that chapter. Shown is a photograph of a section from a lymph node taken from the hilum of the lung. The neuronal system has different areas and thus details of the cell types will be described further in that specific chapter. The correct orientation of organs becomes important when analyzing histology sections, and the way the sections are oriented allows for easier interpretation. Thus organs may be oriented sagittally, on coronal sections or after transverse orientation. Shown is a sagittal section of a mouse embryo at day 17 of development, and demonstrates various structures that are recognizable at these particular cuts, and decisions can then be made about the need for deeper sections, perhaps in order to be able to analyze the structure of the thymus, which is not yet visible, and which usually lies in the midline, above the heart.
9 Histochemical Stains that are commonly used to identify structures within tissues: As shown on this picture, multiple sections different organs can be placed on a slide. However, during examination under a microscope, without a histochemical stain, one can only recognize blood vessels but not much more. Thus a number of histochemical stains are applied to tissue sections in order be able to recognize the tissue and to determine if there are abnormalities present. of to Hematoxylin and Eosin The hematoxylin and eosin stain is the standard histochemical stain use to identify the different cell types within a tissue section before using other methods to further analysis them. Hematoxylin stains the nuclei in various shades of blue and eosin stains the cytoplasm in various shades of pink. When sections are made of tissue, whether they are frozen or paraffin sections, one cannot distinguish nuclei, cytoplasm or any details of the different structures that make up that tissue. Although on can discern different areas and can surmise what the composition is based on whether particular structures are recognizable, no further analysis is possible. Using hematoxylin alone may suffice in many cases to show the basic architecture and thus recognize the tissue type as shown, however adding the eosin generally increases the contrast and then one can distinguish small details and differences much better. The Giemsa stain or the Wright-Giemsa stain: Cells in the blood are stained with this different stain, of which there are variations, based on the ability to be able to distinguish granules within cytoplasm etc. This stain is generally used on blood smears to identify the different kinds of circulating cells, although using specific cell type markers and flow cytometry helps to identify them more accurately. An example of a Giemsa stained blood smear is shown with a lymphocyte and a polymorphonuclear leukocyte with red blood cells and platelets. The Trichrome stain is used to identify collagen. When there has been an injury, the repair process involves making collagen and this is detected using a trichrome stain. A section of colon is used as a control because of the abundance of collagen under the epithelial layer. The PAS stain: A section of colon is often a good control for different kinds of histochemical and immunohistochemical stains, since it has a variety of cell types. The surface epithelium is composed of columnar cells, which have the capacity to produce mucins, which are digested during the processing of tissue and appears as clear spaces within the cells. These are termed goblet cells, since the cells are distended with mucus as though in a goblet. The mucus material, although not visible on a routine hematoxylin and eosin stain, can be highlighted using different
10 chemical treatments. One method is to treat the de-paraffinized and hydrated tissue sections with periodic acid and then allow the Schiff reagent to bind to the resultant aldehyde groups, which adds a bright pink hue to the mucus containing goblet cells. The PAS stain, which highlights basement membrane material, which is abundant with glycoproteins and also highlights the mucus containing cells. The mucus may also be stained using Alcian Blue at either ph2.5, (which picks up carboxylated and sulfated glycoproteins), and shows mucus within epithelial cells lower in the crypt, which do not stain with the PAS stain. Alcian Blue if used at ph 1.0, stains only the sulfated glycoproteins. The panel shows all of these stains on a section of mouse colon. Alizarin Red, Alcian Blue, Safranin O: Abnormalities in bone and cartilage may also be highlighted using special histochemical stains. For this it is technically easier to use either closeto-term embryos or young pups. The skin is removed from the pups and they are immersed in different concentrations of potassium hydroxide, to digest the soft tissues. The bones can then be stained with Alizarin red, which highlights calcium. Alcian Blue and Safranin O are used to stain cartilage. The image shows the appearance of mouse ribs, attached anteriorly to the sternum, which have been cleared before staining and the one on the right shows an image of ribs that were stained with Alizarin Red for calcium and Alcian Blue for cartilage. Oil Red O for lipids on Frozen sections: Another histochemical stain that is useful for identifying the presence of lipids within cells in the Oil Red O stain. The Oil Red O stain has to be used on frozen sections, because the processing and embedding into paraffin, destroys the lipids. The picture shows a frozen section of mouse liver, which is abnormal due to accumulation of lipid Finally, before proceeding on expensive mouse experiments in order to phenotype abnormalities, it is important to analyze serum chemistry values and hematology values, just as though one is examining human subjects. These screening tests are very sensitive and provide a road map towards the examination of particular organs, using histology, histochemistry, immunohistochemistry and even in-situ hybridization methods.
11 Chapter 3: Tissues and cell types screened during histopathologic analysis: The following is a list of tissues and cell types that are part of every histopathologic examination of the different organ systems: The segment labeled Tumors indicate studies of xenografts in mice that are often used as models of human disease, for assessment of the influence of therapies or gene alterations. In fact, studies in mice have allowed the understanding and interpretation of histopathology studies that use markers for different cell types, in the different organ systems of the body. Examination of tissues and organs to detect the presence of Histopathology: An abnormal appearing lesion may result due to: --Inflammation with or without an Abscess --scar tissue with or without a cyst formation --Infarct/Necrosis (death of tissue due to blocked blood vessels, recognized by the absence of nuclei) --hyperplasia due to compensatory mechanisms --neoplasm--new growth In a neoplasm, the nuclei are extra-large and occupy a major portion of the cells. A tumor forms a bulge and one has to determine if it is -BENIGN (encapsulated and contained, -MALIGNANT (invasive) A tumor that arises from Epithelial cells, if benign is named an Adenoma. A malignancy that arises from epithelial cells is called a Carcinoma and the malignant cells invade the basement membrane to enter surrounding supporting tissues and blood vessels. Tumors or malignancies arising from cells of Mesodermal origin are omas if benign, or sarcoma if malignant. For example, a hemangioma is a tumor arising from blood vessels, whereas a hemangiosarcoma is the malignant version.
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