Microanatomy and Embryology Course Guide

Course Administration and Personnel

• Instructor Information:     * Matthew J Valentine, BVMS, MRCVS, PhD, Diplomate ACVP.     * Affiliation: Ross University School of Veterinary Medicine (RUSVM).
• Lecture History and Contributions:     * Lecture modified from Dr. M. Smith (RUSVM, September $2007$).     * Dr. M. Zibrin and Dr. L. Bogdanovic (RUSVM, $2010$, $2011$).     * Dr. C. Fuentealba (September $2011$).     * Callanan and Bolfa (May $2015$).
• Copyright Notice: ©$2021$ Ross University School of Veterinary Medicine. All rights reserved.

Course Navigation and Laboratory Resources

• Course Structure:     * Topics generally consist of $2-3$ Lectures.     * Each topic includes $1$ guided digital slide review and demonstration of laboratory objectives via virtual slide sessions.
• The Canvas Modules Page: This serves as the primary hub for access to:     * Lectures.     * Laboratory objectives.     * Course resources.
• Course Home Section Resources:     * Exam dates and question distribution.     * Required and additional reading lists.     * Course Objectives.     * Laboratory Exercises.     * Virtual E-slides Access for self-directed laboratory exercises.     * Aperio ePathViewer for iPad.     * Microscopy resources, including a Practical Laboratory Exercise for the use of the microscope and recommended instructional videos.
• Course Materials Section: Lecture PDFs are arranged by topic and week, including categories such as Introduction & Methods of Histology, Cytology, Epithelium, Female Reproductive System, Male Reproductive System, Embryology, Early Embryology, Cardiovascular, Respiratory, Nervous System, Blood and Hematopoiesis, Lymphatic System, Endocrine System, Digestive System, Ear, Integument, Eye, Urinary System, Muscle Tissue, Cartilage, and Bone.
• Laboratory Facilities:     * Digital slides are accessible $24/7$.     * The Multi-Purpose Lab (MPL) is accessible $24/7$ (when not otherwise in use) for glass slides and microscopes.     * Crucial Rule: Laboratory exercises can be completed using the cloud-based digital slide server at any time, but MUST NOT be used during the guided slide review to prevent a server crash.

Assessment, Grading, and Examinations

• Weekly Quizzes (Weeks $2-12$):     * $12$ questions per quiz, except for Quiz $1$, which contains $6$ questions.     * $8$ questions are based on the topic studied the previous week.     * $4$ questions are cumulative from topics covered the two weeks prior.     * Question distribution is approximately equal across all topics for both quizzes and finals.
• Final Examination:     * Counts for $40\%$ of the final grade.     * The focus is primarily on topics that have not yet been examined, though it can cover the whole course.
• Direct Observed Preclinical Procedural Skill (DOPPS):     * Focuses on the use of the light microscope.     * Training occurs in small groups during weeks $1$ through $6$.     * Assessments occur during weeks $7$ through $12$.     * Counts for $3\%$ of the final grade.     * Provides immediate feedback during the assessment.     * Support resources include pre-training videos and PDFs.     * The grading rubric is available on Canvas.
• Assessment Scoring Breakdown:     * $10$ Block Exams (Weeks $3-12$): $12$ points each = $120$ total points.     * $1$ Block Exam (Week $2$): $6$ points.     * $1$ DOPPS Assessment: $6$ points.     * Final Exam: $80$ points.     * Subtotal Points: $212$.     * Lowest score dropped: $-12$ points.     * Total Course Points: $200$.

Course Objectives and Core Definitions

• Primary Learning Objectives:     * Achieve proficiency in the use of the light microscope.     * Identify, describe, and name normal cells, tissues, and organs of the animal body.     * List functions of cells, tissues, and organs and relate them to animal physiology.     * Outline early embryonic development and relate it to common developmental anomalies.     * Integrate microscopic and developmental anatomy with other academic classes.
• Subject Definitions:     * Microanatomy / Microscopic Anatomy (Histology): The study of the cells and tissues of the body and how these integrate to form organs.     * Embryology / Developmental Biology: The study of the development of a new individual, including the development of the embryo and fetus.

Study Strategies and Expectations

• Subject Difficulty: Microanatomy is categorized as a challenging subject.
• Self-Directed Learning: Students are expected to use digital slides and repeat lab objectives using an atlas independently.
• Support Systems: Teaching Assistants (TAs) and scheduled exam reviews are available.
• Recommendations for Success:     * Take ownership of learning and maintain a strict timetable.     * Write things down and make study notes for later in the semester.     * Draw diagrams to visualize structures.     * Categorize study sessions into "harder" and "easier."     * Schedule difficult topics for the morning and easier reviews for later.     * Avoid studying when tired; maintain a routine and do not prevaricate.
• Reading Materials:     * Required: Color Atlas of Veterinary Histology, Second Edition. Authors: William J. Bacha, Jr. & Linda M. Bacha ($2000$).     * Additional: Dellmann's Textbook of Veterinary Histology, Sixth Edition. Authors: Jo Ann Eurell, Horst-Dieter Dellmann (Editor), Brian L. Frappier (Editor) ($2006$).

Clinical Applications of Microanatomy

• Gross Anatomy vs. Microanatomy: Gross anatomy involves structures visible to the naked eye (e.g., the liver, esophagus, spleen, colon, trachea, lungs, stomach, and heart), whereas microanatomy requires a microscope to view tissues and cells.
• Cytology: The study of the structure and function of cells. An example is a vaginal smear used for estrus detection in canines.
• Cytopathology: The study of diseased cells obtained from fluids or tissues (e.g., fine needle aspiration of "lumps and bumps"/masses or normal liver cell aspirates).
• Histopathology: The study of diseased tissues (e.g., a liver biopsy or intestinal biopsy).

Principles and Modalities of Light Microscopy (LM)

• Mechanism: A light beam is transmitted through a tissue.
• Resolving Power: The resolving power of LM is limited by the wavelength of light and is measured at $0.2\,\mu m$.
• Key Modalities:     * Bright Field Microscopy: Requires staining for contrast. Digital scanners use the same objectives to produce digital images.     * Phase Contrast Microscopy: Used for observing LIVING, non-stained structures such as spermatozoa, leukocytes, and cell/tissue cultures. Dense structures possess a higher refractive index. This method requires the specimen to be alive to "Phase through walls."     * Fluorescence Microscopy: Fluorescent dyes stain specific cell components. These are visualized under ultraviolet light. Example: kidney cell cultures where blue fluorescence binds to nuclear DNA and green dye binds to actin filaments.     * Polarized Microscopy: Utilizes a polarizing filter to highlight birefringent materials (e.g., crystalline materials). In this mode, collagen fibers may appear bright red or yellow, while elastic fibers and nuclei are not detected.     * Dissecting Stereomicroscope:         * Advantages: Inexpensive, practical, versatile, provides a $3$-dimensional image, useful for microsurgery (e.g., identifying mosquito species or examining a $10\,mm$, $19$-day-old pig embryo whole mount).         * Disadvantages: Low resolving power and requires maintenance.
• General Advantages of LM: Relatively inexpensive, provides rapid diagnosis, and allows observation of living specimens.
• General Disadvantages of LM: Image is $2$-dimensional, requires maintenance, and requires expertise for quality analysis and control.

Electron Microscopy (TEM and SEM)

• Transmission Electron Microscopy (TEM):     * Based on the interaction between electrons and tissue components.     * The electron beam wavelength is shorter than light, resulting in a $1,000$-fold increase in resolution.     * Resolving Power: $0.16$ to $0.18\,nm$.     * Advantages: High resolving power; useful for rapid diagnosis of viruses, storage diseases, and microorganisms.     * Disadvantages: Image is $2$-dimensional, black and white only, specimen cannot be living, and the equipment is very expensive.     * Example: Cross-section of a sperm tail at $50,000\times$ magnification.
• Scanning Electron Microscopy (SEM):     * The electron beam scans the surface to produce a $3D$ effect.     * Visualizes only external structures.     * Resolving Power: Lower resolution than TEM.     * Example: $3D$ structure of sperm cells and the surface of uterine epithelial ciliated and secretory cells.

Tissue Retrieval, Preservation, and Processing

• Terminology:     * Biopsy: A tissue sample taken from a living animal.     * Tissue Sample/Biospecimen: A sample of tissue or a whole organ taken from a dead animal.
• Processing Requirements:     * Trim samples to $1\,cm^3$.     * Place in $10$ times the volume of $10\%$ formalin fixative.     * The specimen must be well preserved (retain structure and molecular composition), sufficiently thin for transmission, and have sufficient contrast.
• Tissue Preservation Steps:     * Fixation: $10\%$ buffered formalin (hazardous) coagulates proteins in a life-like manner.     * Dehydration: Ascending percentages of alcohol.     * Clearing: Xylene is used to remove the alcohol.     * Embedding: Liquid paraffin wax is used, which then solidifies.
• Sectioning (Microtome):     * A microtome slices embedded tissue into thin sections ranging from $1$ to $7\,\mu m$.     * Sections are floated on water for retrieval and subsequent staining.

Histological Staining Techniques

• Routine Staining (H&E):     * Hematoxylin: A basic stain that colors DNA and RNA blue. These components are termed "basophilic."     * Eosin: An acidic stain that colors proteins pink. These components are termed "eosinophilic."
• Special Stains:     * Masson's Trichrome: Stains collagen blue and nuclei red.     * Periodic Acid Schiff (PAS): Localizes glycogen, glycoproteins, and mucins. Goblet cells, which appear pale in H&E, stain magenta in PAS.     * Enzyme Histochemistry: Stains enzymes in cells. Example: Gomori’s method stains alkaline phosphatase black in the brush border of proximal convoluted tubules in the kidney.
• Immunohistochemistry (IHC):     * Highly specific method using antibodies labeled with fluorescent dyes or enzymes to bind to antigens.     * Direct Method: Labeled antibody binds directly to the antigen.     * Indirect Method: Unlabeled primary antibody binds to the antigen, and a labeled secondary antibody binds to the primary.     * Example: Using visible stains to identify Glucagon in A cells and Insulin in B cells within the pancreatic Islets of Langerhans.