Introduction to Histology and Anatomy

• Histology is a biomedical discipline that studies the structure and function of tissues, providing insights into the healthy and diseased organism
• Health relies on preventing disease and maintaining normal structure/function, while disease treatment aims to restore normal structure/function
• Studying the constituent parts (tissues) is essential for understanding organisms

Anatomy

• A biomedical science concerned with the external and internal structure of organisms
• Goes beyond mere description to understand the structural basis of function
• Subdivided based on component parts and study methods
• Gross anatomy involves studying structures via direct visual inspection, palpation, and dissection
• Histology studies structures not visible to the naked eye

Modern Histology

• A hybrid of anatomy, physiology, chemistry, and physics
• Concerned with tissue structure/function, using multidisciplinary approaches
• Includes subdisciplines like histochemistry/cytochemistry
• Utilizes techniques like autoradiography, fluorescence, immunocytochemistry, and cell culture

Organology

• The study of organ structure and function, typically using microscopy
• Comparative organology studies structural/functional differences/similarities among animal groups
• Discipline is essential for veterinary medical students

Technical Advancements

• Advances in physics/engineering lead to more powerful microscopes
• Transmission and scanning electron microscopes aid ultrastructural cytology studies

Integration

• Histology is a key part of medical education
• Provides insights into structure/function at the microscopic level
• A basic building block for understanding disease processes and histopathology

Tissues

• The term histology comes from Greek words "histos" (web) and "logos" (study)
• Tissues mean "texture or weave.", introduced to medical science by Marie F.X. Bichat in the 18th century
• There are four basic tissue types, sometimes five if adipose tissue is included
• Organs are built from these tissues in a logical, orderly way

Importance

• Understanding tissues clarifies organ structure/function
• Organs are the sum of tissues, and tissues are made of cells and extracellular components
• Logical approach to histology: from biochemistry to cells to tissues to organs
• A tissue is a group of similar/identical/dissimilar cells/extracellular products performing specific functions

Challenges

• Defining a tissue can be complex due to cellular dissimilarity
• Functional relationships of cells unify them as contributors to tissue types
• Tissues vary in the amount of extracellular material, but functional relationships remain important
• Grouping tissues can be subjective since classifications are human-made
• Basic tissues include epithelium, connective tissue, muscle, and nervous tissue, potentially with adipose tissue

Architectural Patterns

• Organs consist of various amounts/types of tissues in specific
• Basic tissues are building blocks for a complex organizational hierarchy
• Comprehensive knowledge of histology is essential for understanding health and disease

Methods of Histology

• Technological advances give histologists many ways to study cells and tissues
• Information needs to dictate which approach/combination of approaches to use
• Acquiring maximal data requires comprehension of a technique, the output, and its pros/cons

Cell Culture

• Enables direct observation of living cells
• Routine with antibiotics
• Allows continuous observation, manipulation, and testing of explanted cells without risk to the living organism
• Facilitates studying cell differentiation, transformations, cytogenetics, metabolism, interactions, and host-parasite interactions
• Indispensable in diagnostic virology, vaccine development, and production.
• Implantation of transparent viewing chambers and exteriorization/transillumination of organs/tissues complements direct observation
• Useful for observing microcirculation and responses

Viewing Chambers

• Transparent viewing chambers extend the observation period
• Neovascularization, cellular differentiation, movement, and vital processes are studied
• The anterior chamber of the eye is a naturally occurring viewing chamber

Stains

• Vital/supravital stains enhance understanding of cell function/structure
• Low toxicity, injected into a living organism (vital) or onto living cells (supravital)

Vital Stain Example

• Oxytetracycline is a broad-spectrum antibiotic vital stain
• Selectively stains surfaces where bone, cementum, and dentin form at therapeutic dosages
• Helps determine rates of bone formation, turnover, and maintenance

Additional Stains

• Lithium carmine and trypan blue are vital dyes to study phagocytosis
• Janus green B and neutral red are supravital stains for studying mitochondria/lysosomes
• Separating/purifying cell fractions via centrifugation aids in biochemical/metabolic studies

Microscopic Observations

• Direct observation of living cells requires removing them, placing them on a slide, and examining them with phase-contrast or dark-field microscopy
• Microscopic examination of living cells/tissues is difficult due to transparency and thickness
• Refractive indices are too similar
• Can be improved with thin tissue sections, staining, and examination

Paraffin Technique

• Allows the samples to be reliably accurate but results in modifications during sample preparation
• The most common method, the Paraffin technique, is used in histology courses, histopathology, and morphological research
• Sample acquisition is a critical step in studying the sample

Tissue Concerns

• Knowing gross anatomy is required for proper identification
• Removal of the sample must be atraumatic
• Tissues are fragile and easily altered
• Rapidly remove/defly tissue to fixatives to inactivate autolytic enzymes

Fixation

• Involves chemical fixation to stop postmortem autolysis by denaturing protein and deactivating enzymes
• Fixatives: formaldehyde, glutaraldehyde, ethyl alcohol, acetic acid, picric acid, potassium dichromate, mercuric chloride, chromic acid, and osmic acid

Coagulative Fixatives

• Alters macromolecular conformation, induces marked structural changes
• i.e. ethanol and methanol

Additive Fixatives

• Fixes by chemically reacting with cellular components without severe morphological changes
• i.e. Aldehydes like formaldehyde and glutaraldehyde
• Neutral-buffered formalin (10%) is the most common fixative

Actions of Fixatives

• Prevents autolysis, facilitates sectioning, enhances staining, minimizes leaching, stabilizes structure, protects histologists.
• Immediate immersion in fixative is essential
• Trim tissue blocks to millimeters thick to allow penetration of the fixative to the entire sample
• The ratio of the volume of fixative to tissue volume must be 30:1

Time Requirements

• Time for fixation depends on fixative diffusion and tissue density
• Most formaldehyde fixation is achieved within 24 hours

Dehydration and Clearing

• Require chemicals
• When infiltrating a sample with the paraffin to permit sectioning, water must be removed
• Alcohol is added
• Washed in water to remove excess fixative

Dehydrating Agents

• Ethanol, butanol, dioxane, isopropanol
• Ethanol is the most common
• Dehydration occurs until total dehydration with absolute ethanol occurs
• Because dehydrated alcohol and reagents don't mix, clearing agents must be added

Embedding

• Cleared specimens are processed through solutions containing increasing paraffin waxes
• Maintained at their melting points through the infiltration process
• Can produce morphological alterations in tissues
• Mixtures of purified paraffin and polymers can be effective
• Sections are placed into molds

Sectioning

• Hardens
• Blocks are trimmed and mounted on a microtome
• Thin shavings of samples are removed
• Sections form a collection or ribbon after sample removal
• Ribbons are floated in warm water to be prepped for placement on slides

Rotary Microtome

• Microtome (unit of measurement, one micrometer) allows for thin sectioning
• Specimens for histological examination are sectioned between 5 and 7 µm

Staining and Mounting

• Sections are placed on glass and affixed that have paraffin in them
• Paraffin is not soluble to water and alcohol- based stains
• Slides are covered with a medium that is miscible and a thin layer of coating

Principles of Staining

• Help visualize sections that have been set on slides
• Components of the tissue are similar and can't be easily identified without staining
• Some stains/combinations highlight reactivity for tissue
• Some stains are specific for cells and tissues
• Hematoxylin (H) & Eosin (E) are frequently used together

Stain Components

• Stain's effectiveness is enhanced by understanding how it works
• Some chemical reactions in staining are not completed and maximize if the principles are understoo
• Are usually described as acidic or base stains for their water-soluble properties
• If the radial of the components are in the acidic side it will be a basic stain and vice versa
• Is dependent on the an/cation charges associated with tissues/cell

Cells

• Basic cells react to stains/tissues containing saline to color the cells and water
• Acidic components (cells) do basically the same thing with the reaction for coloring
• Eosin and Hematoxylin reaction using H&E stain through basic dying by applying in blue/purple
• Color is secreted in the acidic contents/secretory regions
• Applied after dying

Techniques

Periodic acid-Schiff stain

• Is useful
• The procedure helps achieve reaction and the results of oxidation and amino help groups or alcohol by addition
• Then exposed by Schiff (basic reagent) to remove color and added back by magenta

Selected Preparation Techniques

• Amount of data available can be limited by the approach that you can choose
• If Structural relationships are only the concern
• There are limitations to this system, the main focus is on the technique
• If you have a concern for altered function then less harsh methods can be used
• The use of frozen section is an alternative

Freezing Techniques

• Involve biopsies that give a frozen section in a short time period, usually with liquid nitrogen temp
• No heat or fixatives are used
• Then embedded and stained
• Helpful with enzymes and insights
• Samples prepared afforded some advantages but some occur in freezing the sample with limited thinness

Histochemistry and Cytochemistry

• These techniques focus on location and characteristics of cells
• Simple process in that a stain is added to enhance with easy to read results
• Used for enzymes and stability
• Must not alter during the procedure

Methacrylate Technique

• Is Easy and quick
• Has disadvantages: cannot resolve the fine details of the composition with a thin or thick sample. and heat is required
• Electron microscopes have more enhanced resolution
• Must use chemicals for the procedures

GMA Embedding

• Must use the method to the advantage of light microscopes
• Some chemicals for staining can be avoided altogether
• Chemicals can be determined more efficiently is chemicals are reduced

Autoradiography

• Helpful technique
• Tissue samples need radioactive material for the process to work for the soft Beta emitters
• Latent imaged is developed and is routinely used
• Allows for visualization and staining of the silver grains
• Has advantages for the study of cell synthetics and mitosis for methods

Immunocytochemistry

• For electron/light methods
• Injection a purified substance to gain the desire properties to inject into an animal
• Responses are determined by the type of animal, treatment etc

TEM

• Preparation is almost the same to that of paraffin
• Samples must be obtained to prevent bad reactions
• Heavy stains are used to determine the compositions

SEM

• Almost the same to TEM, but you do not stain or section
• Specimens used give a real or artificial surface
• To minimize surface tension distortion is acquired fixed and dry technique

Freeze Fracture

• Freezes samples with Nitrogen fast and does not use the chemicals to fix or remove water
• Thin plates are prepped of these methods and fractured
• Then surface layers can show images from the method