HistoPathology of MultiScale Biology PDF

HistoPathology of MultiScale Biology: Molecules, Cells, Tissues, Organs Robert L Sah, MD, ScD  Goals Introduce terminology – anatomy – morphology – histo(patho)logy Provide framework for interpreting tissue HistoPathology  I. Biolog...

HistoPathology of MultiScale Biology: Molecules, Cells, Tissues, Organs Robert L Sah, MD, ScD  Goals Introduce terminology – anatomy – morphology – histo(patho)logy Provide framework for interpreting tissue HistoPathology  I. Biological Hierarchy A. Anatomy vs Physiology the study of structure function “a cutting up” “relationship to nature” structure dictates function Hole’s Anatomy & Physiology, McGraw-Hill  I.B. Levels of Organization & Study subatomic particles atom organ system anatomy, molecule pathology (eye) organism macromolecule organelle organ cell tissue histology, histopathology ultrastructure (light microscopy) (EM, SRM) Hole’s Anatomy & Physiology, McGraw-Hill  I.C. Tissue vs Organ “tissue” – an aggregation of similar cells and their associated extracellular matrix (ECM) – acting together to perform one or more specific functions in the body “organ” – a structure made of 2 or more tissues I.D. Hierarchy of Biological Organization I.D. Biology Hierarchy: Building Up I.D. Hierarchy of Biological Organization I.D. Hierarchy of Biological Organization  II. Histology (Histopathology) A. Definition & Processing “tissue” + “science” - the study of the microscpoic anatomy of (ab)normal cells and tissues of plants and animals workflow – isolate sample – fix – dehydrate & paraffin embed vs “OCT” & freeze – section – stain – microscopy (light, EM)  Royal College of Pathologists, 2018.11.05 What is a Histopathologist ? (5m1s, 16Kviews) https://www.youtube.com/watch?v=jBDsiUw0YQg Histopathologists are … responsible for diagnosing and studying disease in tissues and organs by examining samples of tissue. “These [glass slides] are our patients. We care as much about them as members of … the ITU care about the ITU patients.”  MedScienceAp, SOM, U Adelaide, 2016.02.18 From Biopsy to Microscopy - Tissue processing for light microscopy (6m49s, 88Kviews) https://www.youtube.com/watch?v=__D-DE0X0zk Summary of rationale, concepts, & methods of histopathology: Fixation. Processing. Sectioning. Staining.  Labs for Life Project, Ministry of Health & Family Welfare & U.S. Centers for Disease Control, 2018.06.07 Histopathology (23m21s, 74Kviews) optional (long) video https://www.youtube.com/watch?v=qAoa94WBaIc Concepts & Pointers on: sample collection, transport, accessioning; grossing; tissue processing; automated tissue processor; trimming; sectioning; labeling; hematoxylin and eosin staining.  HistoPathology Workflow http://stmichaelshospitalresearch.ca/wp-content/uploads/2017/01/Workflow-Histology-Diagram.png  II. Histology (HistoPathology) B. Staining Hematoxylin and Eosin (H&E) – primarily to display structural features – hematoxylin: colors basophilic structures with a blue-purple hue » nuclei acids – eosin: colors eosinophiic structures bright pink » cytoplasm other dyes are useful to enhance certain types of molecules (e.g., elastin fibers, collagen)  Leica Biosystems, 2019.08.09 Introducing Aperio GT 450 (4m, 9Kviews) https://www.youtube.com/watch?v=YmBrGzK_msw This is a manufacturers video advertising their whole slide scanner. See also https://www.leicabiosystems.com/digital-pathology.  II.C. Histology: Tissue Sections transverse oblique longitudinal Hole’s Anatomy & Physiology, McGraw-Hill III. HistoPathology of Tissue Types http://www.path.uiowa.edu/virtualslidebox/ http://www.youtube.com/watch?v=ux9rvC5NvQ8 “Help with Histology” The 4 Tissue Types (Embryology+) are Epithelial, Muscle, Nervous, Connective  What type of tissue is it ? http://www.youtube.com/watch?v=ux9rvC5NvQ8 What type of tissue is it ? http://www.youtube.com/watch?v=ux9rvC5NvQ8  (1) Connective Tissue tissue that connects (including blood) characterized by its extracellular matrix ECM loose connective tissue – loosely arranged collagen fibers and many cells – suspends, supports, protects - vessels, nerves and epithelia – fills potential space between other tissues dense connective tissue – predominantly collagen fibers – regular – collagen packed in parallel bundles (e.g. tendons, ligaments) – irregular – collagen packed in bundles that are woven into a pattern that resists multidirectional mechanical stresses (e.g. organ capsules) Some Specialized Connective Tissues adipose bone cartilage tendon ligament intervertebral disc labrum meniscus periosteum perichondrium Intervertebral disk Bone  (2) Epithelial Tissue sheet-like layer of tightly packed cells little intercellular matrix sits on basement membrane at free surface principle functions: – covering and lining surfaces (skin, blood vessels) – absorption (intestine, kidney) – secretion (glands, kidney) – sensation (neuroepithelium) – contraction (myoepithelium) ` Stomach  Skin Acts as a barrier Regulates body temperature and water loss Conveys sensory information Endocrine functions by secreting hormones Exocrine secretion of sweat, sebaceous, and apocrine glands Blood Vessels Lung  Pancreas Exocrine: synthesizes and secretes ezymes into duodenum that are essential for digestion in instestine Endocrine: synthesizes and secretes the hormones insulin and glucagaon into the blood  (3) Muscle, Muscular Tissue characterized by ability to contract striated – skeletal: attached to bone and responsible for voluntary movement – visceral: voluntary and identical skeletal muscle but found in soft tissues (e.g. tongue, diaphragm) – cardiac smooth – involuntary movement of blood vessels and hollow organs Skeletal Muscle Heart  (4) Nervous Tissue consists of nerve cells (neurons) and associated supporting cells Spinal Cord  Summary I. Biological Hierarchy.A. Anatomy v Physiology; Structure v Function.B. Levels of Organization: molecules-organism, ultrastructure, histology/pathology, anatomy/pathology II. Histo(Patho)logy.A. Definition, Process.B. Stains III. HistoPathology of Tissues (1) connective tissue (2) epithelial (3) muscle (4) nerve Digital Image Characteristics  Continuous images are represented as arrays of digital elements, x called pixels in 2-D and voxels in 3-D. Cross-stitch samplers: physical forms of pixelation. Viewer interprets image as continuous, filling in gaps subconsciously. few “rules” on pixel dimension and organization square pixels arranged in a Cartesian grid allow for convenient data processing. y Adapted from Fig. 1.26 Images are represented as a matrix, where each element at a position (row and column) is a pixel. 1 Russ JC, Neal FB. The image processing handbook. In 7th ed. Boca Raton, CRC Press, 1053pp, 2016. 2 https://www.geeksforgeeks.org/digital-image-processing-basics 14  Image processing has several purposes. 1. Improve visual appearance of images (print & transmitted) for human observer. 2. Prepare images for measurement and analysis of revealed features & structures. The pinwheel galaxy Latent fingerprint fluorescing Fig. 1.1 Fig. 1.3 Russ JC, Neal FB. The image processing handbook. In 7th ed. Boca Raton, CRC Press, 1053pp, 2016. 15  Resolution varies with pixel density & gray levels. high spatial resolution low pixels 256 x 256 128 x 128 64 x 64 32 x 32 gray levels 32 16 8 4 high tonal resolution low Russ JC, Neal FB. The image processing handbook. In 7th ed. Boca Raton, CRC Press, 1053pp, 2016.  Pixels (voxels) exhibit “partial volume” effects. high resolution 32 x 32 pixels average gradient is gray light to dark 17 Russ JC, Neal FB. The image processing handbook. In 7th ed. Boca Raton, CRC Press, 1053pp, 2016.  Brightness values are often expressed as 0-255 (8-bit grayscale, where 256=28) Histogram A # of pixels E F A B C E F B 0 255 C A brightness histogram shows the frequency of particular brightness values. Boundaries exhibit partial volume effects with intermediate grayscale values (blue ovals). Fig. 1.31 Russ JC, Neal FB. The image processing handbook. In 7th ed. Boca Raton, CRC Press, 1053pp, 2016.  “Noise” causes characteristic peaks to be wide and can blur the distinction between different features. Fig. 1.34 Russ JC, Neal FB. The image processing handbook. In 7th ed. Boca Raton, CRC Press, 1053pp, 2016.  Images can be stored in many ways (different file types). Russ JC, Neal FB. The image processing handbook. In 7th ed. Boca Raton, CRC Press, 1053pp, 2016. Light Microscopy for Cell & Tissue Engineering Stereo vs Compound Microscope Upright vs Inverted Microscope Transmitted, Phase, Fluorescence Optics –  Compound vs Stereo Microscope 2+ lenses 1 – https://www2.mrc-lmb.cam.ac.uk/microscopes4schools/microscopes1.php  Upright vs Inverted Microscope – slides with cover-slip, flasks, object below histology sections (no liquid) cell cultures (w/ liquid)  Brightfield vs Phase Contrast Optics Brightfield: contrast is caused by absorbance of transmitted light Phase Contrast: converts phase – shifts in light to brightness changes (contrast) Fluorescence Microscopy Fluorophores absorb and emit light at different wavelengths Excitation filters select wavelengths (from the lamp) which the fluorophore will absorb Dichroic mirrors reflect light in the excitation band (back to the sample) and transmit light in the emission band (to the emission filter) Emission filters select wavelengths that have been emitted from the sample –  The ExtraCellular Matrix Insoluble macromolecular networks Determines the shape of animals and maintains positional homeostasis or organs Structure and chemical makeup varies with organ Not static; changes during – development/growth – injury – disease  Major ECM Molecules Collagens Elastin Proteoglycans and glycosaminoglycans (GAGs) Cell adhesion molecules – fibronectin, laminin,... (ECM is hydrated ~ 65% water) ECM Life Cycle: Secretion, Activation, Assembly, Binding, & Degradation  Proteoglycans (PGs) and Glycosaminoglycans (GAGs) GAG = long repeating disaccharides, (unbranched polysaccharides) e.g., hyaluronan, keratan sulfate, chondroitin sulfate, - negatively charged - binds growth factors - provides compressive stiffness can be linked to a protein to form proteoglycans (PG) Glycosaminoglycans (GAGs) Laminin Major component of basement membranes Epithelial cells Epithelial cells Basal lamina Collagen fibrils SEM of a basal lamina in the cornea of a chick embryo  Collagens: diversity, structure, biosynthesis and cross-linking David M. Hudson Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, WA, USA  What makes a protein a Collagen? If the protein consists largely of a triple-chain helix 1o 2o 3o Collagens have a unique primary, secondary and tertiary structure: 1o: Gly-X-Y repeat 2o: left-handed alpha helix 3o: right-handed triple helix Structure of Collagen The backbone of the collagen triple helix Gly Pro Hypro 1. Three polypeptide chains (alpha chains) with repeating Gly-X-Y amino acid sequence staggered by one residue relative to each other 2. Individual chains are left-handed helices (~ 1000aa) 3. Three chains are wrapped around each other in a right-handed supercoiled helix  COLLAGEN STRUCTURE Every third amino acid is a glycine. Molecular packing Only glycine is small enough to fit in tight junction where chains are in contact. Inside of the 3-chain structure (triple helix) is lined with glycine residues. The side chains in X and Y position (commonly proline and hydroxyproline) are directed outwards and participate in inter- and intra molecular interactions. Nelson DL and Cox MM, 2003. Lehninger Principles of Biochemistry, 3rd ed. Structure of Collagen Helix Stabilization Gly Pro Hypro Collagen triple helix is stabilized by: 1. Interchain Hydrogen bonds between the peptide –NH of glycine and the C=O of the second residue in the triplet of a neighboring chain. 2. Proline ring structure (pyrrolidine) prevents rotation about the N-Ca bond in a polypeptide chain. 3. Hydroxyproline hydroxyl group is involved in water bridged hydrogen bonding with a backbone carbonyl oxygen. 4. Side-chain interactions as charged-pair interactions, including intra inter molecular charge clustering. 5. Disulfide bonds in certain collagens (III, IV, IX etc.) Structurally related superfamily Singh et al. (2012). FEMS Microbiol. Rev. 36, 1122-1180. Collagen Type Type I collagen Chain composition Two a1(I) chains and one a2(I) chain make up the type I collagen molecule Tissue distribution Ubiquitous e.g., bone, skin, tendon, ligament  Collagen-related diseases Human gene Human disease COL1A1, A2 Osteogenesis imperfecta; osteoporosis; Ehlers-Danlos type VIIA type VIIB COL2A1 Several chondrodysplasias; osteoarthrosis COL3A1 Ehlers-Danlos type IV; arterial aneurysms COL4A3, A4 Autosomal forms of Alport syndrome COL4A5 X-linked forms of Alport syndrome COL4A5, A6 Alport syndrome with diffuse oesophageal leiomyomatosis COL5A1, A2 Ehlers-Danlos type I; Ehlers-Danlos type II COL6A1, A2, A3 Bethlem myopathy; Ullrich muscular dystrophy COL7A1 Dystrophic forms of Epidermolysis bullosa COL8A2 Two forms of corneal endothelial dystrophy COL9A1, A2, A3 Multiple epiphyseal dysplasia; osteoarthrosis; intervertebral disc disease COL10A1 Schmid metaphyseal chondrodysplasia COL11A1, A2 Several mild chondrodysplasias; non-syndromic hearing loss; osteoarthrosis COL12A1 Ullrich congenital muscular dystrophy 2; Bethlem myopathy 2 COL13A1 Congenital myasthenic syndrome type 19 COL15A1 Not identified; skeletal myopathy and cardiovascular defects in mouse COL17A1 Two forms of Epidermolysis bullosa COL18A1 Knobloch and pigment dispersion syndromes COL19A1 Not identified; abnormal muscle layer in the oesophagus in mouse COL25A1 Congenital cranial dysinnervation disorder COL27A1 Steel syndrome Mutations in collagen modifying genes can also cause disease (OI) Table 1 Non-Collagen Genes in which Mutations Cause Osteogenesis Imperfecta Variants Gene Protein Phenotype Bone Collagen Abnormalities } CRTAP CRTAP Mild to severe OI, α1(I)P986 and α2(I)P707 LEPRE1 P3H1, prolyl hydroxylase P3H1 reduced mineral under prolyl-3-hydroxylation, PPIB CYPB, cyclophilin B Complex density high HP/LP (CRTAP & LEPRE1), low HP/LP (PPIB) TMEM38B TRIC-B Bone fragility, Reduced helical Lys moderate/severe OI hydroxylation, increased telo-peptide hydroxylation FKPB10 PLOD2 FKBP65 LH2, lysyl hydroxylase 2 } Bruck Syndrome: bone fragility, joint contractures Lack of telopeptide hydroxylysines produces skin-like cross-links LEPREL2 LEPREL4 P3H3 Sc65 } Low bone mass, skin fragility Low HP/LP, reduced helical Lys hydroxylation, altered divalent cross-links MBTPS2 SP2, site-2 metalloprotease Moderate/severe Reduced helical Lys87 X-linked OI hydroxylation and glycosylation BMP1 Procollagen type I C-propeptidase High mineral density, Defective C-propeptide removal, mild to severe OI potential cross-linking defects SERPINH1 HSP47, heat shock protein 47 Moderate/severe OI, High HP/LP, and Bone fragility abnormal arrangement of cross-linking bonds IFITM5 Bril, osteoblast-specific small Normal to severe OI, Altered mineralization, no transmembrane protein bone fragility other collagen abnormalities SERPINF1 PEDF Moderate/severe OI, Failed mineralization, no other pigment epithelium-derived factor low mineral density, collagen abnormalities osteoid seams  Collagen biosynthesis HSP47 Prolyl Hydroxylases PPIase Lysyl Hydroxylases Glycosyl Transferases PNP PCP Lysyl Oxidase Yamauchi and Sricholpelch 2012. Fibrillar collagen structure Tropocollagen ~ 300nm Fibrils ~ 1µm Fibers~ 10µm Weis et al. (2010). J. Biol. Chem. 285, 2580-2590. Hierarchical structure of tendon Richardson et al. (2007). Trends Biotechnol. 25, 409-416.  ExtraCellular Matrix 1. What is ECM ? Composition, Structure. 2. What are the functions of ECM ? 3. How is ECM characterized (at a particular “tissue state”) ? 4. What determines steady-state levels of ECM components? 5. What is an example of ECM assessment in TE-RM ? Extracellular Matrix and Interstitial Fluid. 2017.10.10, 5m44s [732Kviews, 2022.09.20]  The ExtraCellular Matrix Insoluble macromolecular networks Determines the shape of animals and maintains positional homeostasis or organs Structure and chemical makeup varies with organ Not static; changes during – development/growth – injury – disease  Major ECM Molecules Collagens Elastin Proteoglycans and glycosaminoglycans (GAGs) Cell adhesion molecules – fibronectin, laminin,... (ECM is hydrated ~ 65% water)  Collagens: diversity, structure, biosynthesis and cross-linking David M. Hudson Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, WA, USA  What makes a protein a Collagen? If the protein consists largely of a triple-chain helix 1o 2o 3o Collagens have a unique primary, secondary and tertiary structure: 1o: Gly-X-Y repeat 2o: left-handed alpha helix 3o: right-handed triple helix Structurally related superfamily Epithelial cells Epithelial cells Basal lamina Collagen fibrils SEM of a basal lamina in the cornea of a chick embryo  Collagen biosynthesis HSP47 Prolyl Hydroxylases PPIase Lysyl Hydroxylases Glycosyl Transferases PNP PCP Lysyl Oxidase Yamauchi and Sricholpelch 2012. Fibrillar collagen structure Tropocollagen ~ 300nm Fibrils ~ 1µm Fibers~ 10µm Weis et al. (2010). J. Biol. Chem. 285, 2580-2590. Hierarchical structure of tendon Richardson et al. (2007). Trends Biotechnol. 25, 409-416.  Proteoglycans (PGs) and Glycosaminoglycans (GAGs) GAG = long repeating disaccharides, (unbranched polysaccharides) e.g., hyaluronan, keratan sulfate, chondroitin sulfate, - negatively charged - binds growth factors - provides compressive stiffness can be linked to a protein to form proteoglycans (PG) Articular Cartilage in Synovial Joints Human Knee Joint Gray’s Anatomy 40th Ed, 2008 pain-free low-friction load-bearing wear-resistant Glycosaminoglycans (GAGs) Laminin Major component of basement membranes  Space-filling hydrophilic proteoglycans are pre-loaded & restrained by collagen network. courtesy of Neil Broom Hardingham TE, http://glycoforum.gr.jp http://www.geekwire.com/2015/deflategate-heres- underinflated-footballs-easier-throw-catch/  Articular cartilage load-bearing properties depend on proteoglycan-collagen interactions. courtesy of Neil Broom  Confined Compression Testing: Aggregate Modulus Stress,  0 1 0 1... HA = aggregate FLUID modulus Strain,  CARTILAGE  Articular cartilage load-bearing properties depend on proteoglycan & collagen content. Fetal Post-natal Adult (3rd trimester) (1-3 wk) (1.5-2 yr) Lateral Medial 2 cm Compr. Modulus [ MPa ] 0.4 30 GAG [ mg/ml ] 150 COL [ mg/ml ] 20 100 condyle 0.2 groove 10 50 0 0 0 fetal calf adult fetal calf adult fetal calf adult Williamson+, J Orthop Res, 2002; Han+, Biophys J, 2011 Compressive Modulus correlates with both s-GAG and COL content. Compressive Modulus [MPa] 1 R2 = 0.24 R2 = 0.36 p