Junqueira's Basic Histology practicing flashcards

HISTOLOGY: FUNDAMENTAL PRINCIPLES AND METHODS OF STUDY

• Histology is the study of body tissues and their arrangement into organs, derived from the Greek root histo, meaning "tissue" or "web."

• Tissues consist of two interacting components: cells and the extracellular matrix (ECM). The ECM is a complex of organized molecules, such as collagen fibrils and basement membranes, that provides mechanical support, transports nutrients, and carries away catabolites.

• Modern histology recognizes an intense interaction between cells and the matrix, where matrix molecules connect to intracellular cytoplasm via cell-surface receptors.

PREPARATION OF TISSUES FOR MICROSCOPY

• The most common procedure is the preparation of histological sections or thin slices for light microscopy, allowing light to pass through the specimen attached to glass slides.

• Fixation: This process uses chemical or physical methods to preserve tissue structure and molecular composition, preventing autolysis and bacterial digestion.

  • Chemical Fixatives: Stabilize or cross-link proteins. Formalin (37% formaldehyde in buffered isotonic solution) is standard for light microscopy.

  • Double Fixation: Glutaraldehyde (to cross-link proteins) followed by buffered osmium tetroxide (to preserve and stain lipids and proteins) is used for electron microscopy.

• Embedding & Sectioning: Tissues are infiltrated with hard substances like paraffin (for light microscopy) or epoxy resins (for electron microscopy) to impart rigidity.

  • Dehydration: Water is extracted using a graded series of ethanol (70% to 100%).

  • Clearing: Ethanol is replaced with a solvent (e.g., xylene) miscible with both alcohol and the embedding medium.

  • Sectioning: A microtome slices paraffin blocks into sections of $1$ to $10\,\mu m$ thick. For transmission electron microscopy (TEM), an ultramicrotome with a glass or diamond knife creates sections less than $1\,\mu m$ ($40-90\,nm$).

• Cryostat / Freezing: Tissues are fixed by rapid freezing (physical fixation), which is faster than embedding and preserves enzyme activity and lipids.

STAINING AND LIGHT MICROSCOPY

• Most tissues are colorless and must be stained selectively. Dyes typically behave as acidic or basic compounds.

• Basophilia: Components with a net negative charge (anionic), such as nucleic acids and glycosaminoglycans, stain with basic dyes like toluidine blue, alcian blue, and methylene blue. Hematoxylin also behaves like a basic dye.

• Acidophilia: Components with a net positive charge (cationic), such as proteins, mitochondria, and collagen, stain with acidic dyes like eosin, orange G, and acid fuchsin.

• Hematoxylin and Eosin (H&E): The most widely used combination. Hematoxylin stains acidic structures (DNA/RNA) blue/purple; eosin stains basic structures (collagen/cytoplasmic proteins) pink.

• Periodic acid-Schiff (PAS): Based on the transformation of 1,2-glycol groups in sugars into aldehyde residues, producing a magenta color; used for hexose sugars, glycogen, and glycoproteins.

• Lipid Staining: Best revealed with lipid-soluble dyes like Sudan black on frozen sections.

• Bright-Field Microscopy: Uses three lens systems: condenser (focuses light), objective (enlarges image), and eyepiece/ocular (further magnifies). Total magnification = objective power $\times$ ocular power. Resolving power is the smallest distance between two particles seen as separate ($\approx 0.2\,\mu m$).

• Fluorescence Microscopy: Substances are irradiated with UV light and emit visible light. Acridine orange binds DNA and RNA. DAPI and Hoechst bind DNA to show nuclei.

• Phase-Contrast & Differential Interference Microscopy: Allows observation of unstained/living cells by using refractive index differences to create contrast.

• Confocal Microscopy: Uses a laser point light source and a pinhole aperture to eliminate stray light, allowing for sharp "optical sections" and 3D reconstruction.

• Polarizing Microscopy: Identifies repetitive structures like collagen or cellulose based on their ability to rotate polarized light (birefringence).

ELECTRON MICROSCOPY AND SPECIAL TECHNIQUES

• Transmission Electron Microscopy (TEM): Works like an upside-down light microscope with an electron beam. Wavelengths are shorter than light, allowing resolution of $\approx 3\,nm$ and magnifications up to $400,000\times$. Areas where electrons pass easily are "electron-lucent" (bright); dense areas are "electron-dense" (dark).

• Scanning Electron Microscopy (SEM): Provides 3D surface views. The specimen is coated with metal atoms, and a narrow electron beam scans the surface to produce reflected/secondary electrons.

• Autoradiography: Localizes newly synthesized macromolecules by incorporating radioactively labeled metabolites (e.g., $^3H$-thymidine for DNA). Radioactive decays reduce silver bromide crystals in overcoated photographic emulsion to black metallic silver grains.

• Cell Culture: Primary cell cultures are isolated directly from tissue; permanent cell lines are immortalized/transformed cells. All must be handled in sterile environments.

• Histochemistry/Cytochemistry: Localizes enzyme activity (e.g., phosphatases, dehydrogenases, peroxidase) by providing substrates that produce insoluble, colored, or electron-dense precipitates.

• Immunohistochemistry: Uses the highly specific interaction between antigens and antibodies.

  • Direct Method: The specific antibody is tagged with a label (fluorescent, peroxidase, or gold).

  • Indirect Method: A primary antibody binds the target; a labeled secondary antibody then binds the primary, amplifying the signal.

• In Situ Hybridization (ISH): Uses a complementary nucleotide "probe" to detect specific DNA or RNA sequences in cells or tissues.

THE CYTOPLASM AND ORGANELLES

• Animal cells are eukaryotic, possessing membrane-limited nuclei and organelles ($Gr.\,eu$, good + $karyon$, nucleus).

• Plasma Membrane: A lipid bilayer ($7.5-10\,nm$ thick) composed of phospholipids, cholesterol, and proteins. Hydrophilic polar heads face outward; hydrophobic nonpolar fatty acid chains face inward.

  • Proteins: Integral proteins are embedded in the bilayer; peripheral proteins are loosely associated with surfaces. Transmembrane proteins span the bilayer.

  • Glycocalyx: A carbohydrate-rich cell coat involved in cell recognition and attachment.

  • Endocytosis: Includes phagocytosis ("cell eating"), pinocytosis ("cell drinking"), and receptor-mediated endocytosis (utilizing clathrin-coated pits).

  • Signal Transduction: Extracellular signals (ligands) bind receptors, often activating G proteins to generate second messengers ($cAMP$, $DAG$, $IP_3$).

• Mitochondria: Double-membrane organelles specialized for aerobic respiration and ATP production. The inner membrane forms cristae to increase surface area and contains the respiratory chain. The matrix contains DNA and ribosomes. Oxygen debt during anaerobic exercise produces lactate in muscle.

• Ribosomes: Small granules ($20 \times 30\,nm$) of rRNA and protein. Free polyribosomes synthesize proteins for cytosol; membrane-bound polysomes on RER synthesize proteins for secretion or membranes.

• Endoplasmic Reticulum (ER):

  • Rough ER (RER): Continuous with the nuclear envelope; covered in polyribosomes. Functions in protein segregation, glycosylation, and phospholipid synthesis.

  • Smooth ER (SER): Lacks ribosomes; continuous with RER. Involved in lipid synthesis, steroid production, detoxification (cytochrome P-450), and calcium sequestration (sarcoplasmic reticulum).

• Golgi Apparatus: System of flattened saccules (cisternae). The cis face receives transport vesicles from RER; the trans face matures and sorts proteins into lysosomes, secretory granules, or plasma membrane vesicles.

• Lysosomes: Membrane-limited vesicles filled with $\approx 40$ hydrolytic enzymes active at $pH \approx 5.0$. They manage intracellular digestion, turnover of organelles (autophagy), and removal of materials from endocytosis.

• Proteasomes: Non-membranous protein complexes that degrade ubiquitinated, denatured, or nonfunctional polypeptides.

• Peroxisomes: Spherical organelles containing oxidases and catalase. They breakdown fatty acids and detoxify substances (e.g., ethanol to acetic aldehyde).

THE CYTOSKELETON AND NUCLEUS

• Microtubules: Hollow tubes ($24\,nm$ diameter) of $\alpha$ and $\beta$ tubulin dimers. They maintain cell shape, form mitotic spindles, and serve as tracks for motor proteins (kinesins and dyneins). Found in cilia and flagella ($9 + 2$ axoneme arrangement).

• Microfilaments (Actin): Thin ($5-7\,nm$) polarized filaments. Involved in cell motility, endocytosis, and muscle contraction (interacting with myosin).

• Intermediate Filaments: Size ($10-12\,nm$) between actin and microtubules. Includes keratins (epithelial), vimentin (mesenchymal), desmin (muscle), neurofilaments (neurons), and lamins (nucleus).

• Nucleus: Contains chromosomal DNA. Surrounded by the nuclear envelope (two membranes separated by a $30-50\,nm$ perinuclear space). Nuclear pore complexes (NPCs) regulate transport via importins/exportins.

• Chromatin: DNA bound to histones. Heterochromatin is condensed/inactive; Euchromatin is uncoiled/active. Basic unit is the nucleosome ($8$ histones with $\approx 150$ base pairs of DNA).

• Cell Cycle: Includes Interphase ($G_1$, $S$ for DNA replication, $G_2$) and Mitosis ($M$). Control involves cyclins and cyclin-dependent kinases (CDKs).

EPITHELIAL TISSUE

• Epithelia cover bodies, line cavities, and form glands. Features include high cellularity, minimal ECM, and strong adhesion.

• Junctions:

  • Tight (Zonula Occludens): Apical seal, paracellular barrier (transmembrane protein claudin).

  • Adherent (Zonula Adherens): Continuous belt, anchors actin filaments (cadherins and catenin).

  • Desmosome (Macula Adherens): Spot-weld, anchors intermediate filaments.

  • Gap (Communicating): Connexons ($6$ connexins) forming $1.5\,nm$ pores for ionic coupling.

  • Hemidesmosome: Binds basal pole to the basal lamina (integrins).

• Apical Specializations: Microvilli (absorption), Stereocilia (long branched microvilli), and Cilia/Flagella (motility).

• Classification:

  • Simple: Squamous, cuboidal, or columnar.

  • Stratified: Squamous (keratinized or non-keratinized), cuboidal, columnar, or transitional (urothelium).

  • Pseudostratified: Appears stratified as all cells touch basal lamina but nuclei are at different heights (e.g., respiratory tract).

• Glandular Secretion: Merocrine (exocytosis), Apocrine (apical cytoplasm loss), or Holocrine (entire cell shed).

CONNECTIVE TISSUE

• Characterized by abundant ECM consisting of fibers and ground substance.

• Cells:

  • Fibroblasts: Synthesize fibers and ground substance. Active state vs. quiescent fibrocytes.

  • Macrophages: Phagocytic defense cells derived from blood monocytes.

  • Mast Cells: Store heparin, histamine, and leukotrienes; involved in allergies (immediate hypersensitivity).

  • Plasma Cells: Derived from B lymphocytes; produce antibodies.

• Fibers:

  • Collagen: Most abundant protein in the body. Type I (bone, tendons), Type II (cartilage), Type III (reticular), Type IV (basal laminae).

  • Reticular Fibers: Thin, heavily glycosylated (argyrophilic) networks of Type III collagen.

  • Elastic Fibers: Composed of fibrillin and elastin; provide resiliency.

• Ground Substance: Viscous, hydrated mix of glycosaminoglycans (GAGs), proteoglycans, and multiadhesive glycoproteins (e.g., laminin, fibronectin).

• Varieties: Loose (areolar), Dense (regular or irregular), Reticular, and Mucous (Wharton's jelly).

ADIPOSE, CARTILAGE, AND BONE

• White Adipose: Unilocular cells specialized for long-term energy storage ($9.3\,kcal/g$) and hormone production (leptin).

• Brown Adipose: Multilocular cells; thermogenesis driven by uncoupling protein-1 (thermogenin) in mitochondria.

• Cartilage: Avascular, flexible support. Types: Hyaline (Type II collagen), Elastic (Elastin), and Fibrocartilage (Type I collagen; found in intervertebral disks).

• Bone: Calcified ECM (hydroxyapatite). Cells: Osteoblasts (secrete matrix), Osteocytes (in lacunae), and Osteoclasts (large multinucleated cells for resorption).

  • Primary (Woven): First to appear, random fiber arrangement.

  • Secondary (Lamellar): Organized into osteons (Haversian systems) with concentric lamellae around vascular canals.

NERVE AND MUSCLE TISSUE

• Nerve Tissue: Neurons (cell body, dendrites, axon) and Glia. CNS glia: Astrocytes (BBB), Oligodendrocytes (myelin), Microglia (immune), Ependymal (line ventricles). PNS glia: Schwann (myelin) and Satellite cells.

• Muscle Tissue:

  • Skeletal: Multinucleated fibers with cross-striations of sarcomeres. Sarcomere limits are Z lines.

  • Cardiac: Striated, branched fibers with intercalated disks (gap junctions/desmosomes).

  • Smooth: Fusiform cells, non-striated; utilize calmodulin and myosin light chain kinase for contraction.

THE CARDIOVASCULAR AND IMMUNE SYSTEMS

• Vessel Structure: Tunica intima (endothelium), Tunica media (smooth muscle), and Tunica adventitia (connective tissue). Capillaries consist only of endothelium.

• Spleen: Largest lymphoid accumulation. White pulp (lymphoid follicles) and Red pulp (splenic cords and venous sinusoids for blood filtration).

• Lymph Nodes: Encapsulated in-line filters for lymph. Cortex (B cells), Paracortex (T cells), Medulla (cords and sinuses).

• Thymus: Primary lymphoid organ for T cell maturation. Features include thymic (Hassall's) corpuscles in the medulla.

DIGESTIVE AND RESPIRATORY SYSTEMS

• Gastrointestinal: Layers are Mucosa, Submucosa, Muscularis, and Serosa/Adventitia. Stomach contains parietal cells ($HCl$/intrinsic factor) and chief cells (pepsinogen). Small intestine features villi and crypts of Lieberkühn.

• Respiratory: Lined mainly by ciliated pseudostratified columnar epithelium. Alveoli possess Type I cells (gas exchange) and Type II cells (surfactant secretion).

ENDOCRINE GLANDS AND SENSE ORGANS

• Adrenal: Cortex regions: Zona glomerulosa (aldosterone), Zona fasciculata (cortisol), Zona reticularis (androgens). Medulla: Chromaffin cells (epinephrine/norepinephrine).

• Thyroid: Composed of follicles containing thyroglobulin colloid. Parafollicular C cells secrete calcitonin.

• Eye: Fibrous layer (sclera/cornea), Vascular layer (uvea/ciliary body), and Retina (photoreceptors). Retina has 10 layers; rods (low light) and cones (color).

• Ear: External, Middle (ossicles), and Internal (bony/membranous labyrinth). Organ of Corti in the cochlea mediates hearing via hair cells.