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1 HOMEOSTASIS Normal maintenance and renewal of differentiated cells in many tissues This does NOT involve leukocytes. Leukocytes and inflammation occurs in response to damage NEED FOR REPAIR When tissue is damaged by mechanical injury, disease of infection, the damaged tissue must be removed and replaced by new cells This may involve a large area of tissues and is associated with inflammation Cells in some tissues of higher vertebrates cannot replicate and therefore the specialised cells are not replaced e.g CNS and heart In other instances, repair may fail after repeated injury and extensive damage e.g cirrhosis of the liver, muscular dystrophy In these instances the tissue is replaced by fibrous and fatty connective tissue scar tissue Injury & inflammation (and ageing) increased fibrous connective tissue and alters the ECM environment: this can affect the fate of precursor cells e.g in dystrophic muscle, satellite cells may become fibroblasts instead of myoblasts.
2 Key events during tissue repair 1. Sealing: to limit the damage 2. Inflammation: removes damaged tissue (phagocytosis), proteases (modify the ECM), chemokines have many effects (chemotaxis, mitogens). Involves evascularisation of leukocytes (see later lecture); neutrophils followed by macrophages 3. Angiogenesis: where blood vessels are damaged they must be replaced rapidly to allow oxygen and nutrients to the new tissue (later lecture) otherwise scar tissue results. 4. Cell proliferation: to expand cell populations 5. Cell differentiation: to generate specialised cells 6. Maturation and re-innervation: to restore full function
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4 Damage to the skin and repair 1. Sealing 2. Inflammation
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6 3. Angiogenesis 4. Cell proliferation
7 5. Differentiation 6. Maturation
8 Examples of cellular events after damage to skeletal muscle: & techniques to investigate
9 Sarcolemnal damage Delta lesions * *
10 Reseal the sarcolemma
11 McNeil PL. Kirchhausen T (2005) An emergency response team for membrane repair. Nat Rev Mol Cell Biol : A 10µm Vesicle-vesicle fusion C B Vesicle transport/cortex dissolution D Exocytotic addition patch Vesicles patch the membrane lesion
12 Necrosis of the myofibre There is much necrosis in DMD and the mdx mouse model of DMD
13 Hypercontraction of damaged segment of myofibre: at 3 hours * *
14 3 HOURS * damaged segment * PMLs 6 HOURS
15 inflammatory cells & sealing the damaged zone
16 Neutrophils within the basal lamina (arrow) of a myofibre. PMLs Sarcomeres of myofibre T.Robertson
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18 9 hours Necrotic tissue and inflammatory cells * * * Resealed segment of damaged myofibre
19 * Convoluted new demarcation membrane to re-seal end of damaged myofibre: 12 hours * Necrotic tissue * * * Resealed myofibre
20 12 HOURS damaged segment PMLs 24 HOURS macrophages
21 Roles of inflammatory cells in muscle necrosis and subsequent repair Damage 1. susceptibility to necrosis (directly cause?) Repair 1. Phagocytosis/removal of necrotic tissue 2. Produce many cytokines/enzymes for: Remodelling of ECM Chemotaxis Cell (myoblast) proliferation, Cell (myoblast) differentiation Myoblast fusion & myotube maturation
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24 Inflammatory cells and cytokines in CHEMOTAXIS How relevant are in vitro studies to repair in vivo? Robertson TA et al (1993) the role of macrophages in skeletal muscle regeneration with particular reference to chemotaxis. Exp. Cell Res. 207: Grounds MD, Davies MJ (1996) Chemotaxis in myogenesis. Basic and Applied Myology 6(6):
25 Chemotactic Response of leukocytes (PMLs and macrophages) to uninjured and damaged muscle.leukocytes macrophages 5.0µm 0.8µm Muscle (undamaged, injured, WBI) MODIFIED BOYDEN CHAMBERS
26 Exudate macrophage responding to a chemotactic signal: emerging onto the underside of the membrane in a Boyden chamber Pore in membrane
27 Chemotactic Response of myoblasts to macrophages of myoblasts to growth factors.of myoblasts to cytokines produced by macrophages C2C12 myoblasts 12.0µm 0.8µm GF macrophages MODIFIED BOYDEN CHAMBERS
28 Chemotactic index of myoblasts in response to leukocytes or growth factors Activated macrophages PDGF-AB LIF Our studies (1993, 1996): Activated m/phages, LIF, PDGF-AB > PDGF-BB/AA>TGF-ß Prevented by IRRADIATION Requires intact VASCULAR system Bischoff (1997): TGF-ß (platelets), HGF PDGF-AB/BB/AA, FGF-2, EGF Effect was very DOSE DEPENDENT Complex gradients of many factors Leukocytes Growth Factors Torrente et al (2003) TNF-α : 2 fold (also in vivo) Also acts indirectly via MMPs and ECM breakdown
29 Chemotactic pathways after muscle damage PMLs from vasculature macrophages myoblasts DAMAGED SKELETAL MUSCLE Grounds MD, Davies MJ (1996) Chemotaxis in myogenesis. Basic and Applied Myology 6(6):
30 Key events during tissue repair 1. Sealing: to limit the damage 2. Inflammation: removes damaged tissue (phagocytosis), proteases (modify the ECM), chemokines have many effects (chemotaxis, mitogens). Involves evascularisation of leukocytes (see later lecture); neutrophils followed by macrophages 3. Angiogenesis: where blood vessels are damaged they must be replaced rapidly to allow oxygen and nutrients to the new tissue otherwise scar tissue results. 4. Cell proliferation: to expand cell populations 5. Cell differentiation: to generate specialised cells 6. Maturation and re-innervation: to restore full function
31 Cell proliferation Differentiation (and fusion)
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