HOMEOSTASIS
• 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.
• 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
Key events during tissue repair
1. Sealing: to limit the damage2. 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 populations5. Cell differentiation: to generate specialised cells6. Maturation and re-innervation: to restore full function
Damage to the skin and repair
1. Sealing2. Inflammation
3. Angiogenesis4. Cell proliferation
5. Differentiation6. Maturation
Examples of cellular eventsafter damage to skeletal muscle:
&techniques to investigate
Sarcolemnal damage‘Delta lesions’
*
*
Reseal the sarcolemma
10µm Vesicle-vesicle fusion
Exocytotic addition patchVesicle transport/cortexdissolution
Vesicles ‘patch’ the membrane lesion
McNeil PL. Kirchhausen T (2005) An emergency response team for membrane repair.
Nat Rev Mol Cell Biol. 2005 6:499-505.
A C
B D
Necrosis of the myofibre
There is much necrosisin DMD and the
mdx mouse model of DMD
Hypercontraction of damaged segment of myofibre: at 3 hours
* *
3 HOURS
6 HOURS
* *damaged segment
PMLs
inflammatory cells&
sealing the damaged zone
Neutrophils within the basal lamina (arrow) of a myofibre.
Sarcomeres of myofibre
T.Robertson
PMLs
Resealed segment of damaged myofibre
Necrotic tissue and inflammatory cells
**
*
9 hours
*
*
**Necrotic tissue
Resealed myofibre
Convoluted new demarcation membrane * to re-seal end of damaged myofibre: 12 hours
12 HOURS
24 HOURS
damaged segment
PMLs
macrophages
Roles of inflammatory cellsin muscle necrosis and subsequent repair
Damage1. ▲ susceptibility to necrosis (directly cause?)
Repair1. Phagocytosis/removal of necrotic tissue2. Produce many cytokines/enzymes for:
– Remodelling of ECM– Chemotaxis– Cell (myoblast) proliferation, – Cell (myoblast) differentiation – Myoblast fusion & myotube maturation
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. 207:321-331
Grounds MD, Davies MJ (1996) Chemotaxis in myogenesis. Basic and Applied Myology 6(6): 469-483.
MODIFIED BOYDEN CHAMBERS
Chemotactic Responseof leukocytes (PMLs and macrophages)
to uninjured and damaged muscle
5.0µm
0.8µm
Muscle (undamaged, injured, WBI)
macrophages….leukocytes
Exudate macrophage responding to a chemotactic signal:emerging onto the underside of the membrane in a Boyden chamber
Pore in membrane
MODIFIED BOYDEN CHAMBERS
Chemotactic Response
of myoblasts to macrophages
of myoblasts to growth factors
0.8µm12.0µm
C2C12myoblasts
macrophages
GF
….of myoblasts to cytokines produced by macrophages
Chemotactic index of myoblasts in response to leukocytes or growth factors
Activated macrophages
PDGF-ABLIF
Our studies (1993, 1996):Activated m/phages, LIF, PDGF-AB
> PDGF-BB/AA>TGF-ßPrevented by IRRADIATIONRequires intact VASCULAR system
Bischoff (1997): TGF-ß (platelets), HGF
PDGF-AB/BB/AA, FGF-2, EGFEffect was very DOSE DEPENDENTComplex gradients of many factors
Torrente et al (2003)TNF-α : 2 fold (also in vivo)
Also acts indirectly via MMPs and ECM breakdownLeukocytes Growth Factors
Grounds MD, Davies MJ (1996) Chemotaxis in myogenesis. Basic and Applied Myology 6(6): 469-483.
Chemotactic pathways after muscle damage
PMLs from vasculature macrophages myoblasts
DAMAGED SKELETAL MUSCLE
Key events during tissue repair
1. Sealing: to limit the damage2. 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 populations5. Cell differentiation: to generate specialised cells6. Maturation and re-innervation: to restore full function
Cell proliferation
Differentiation (and fusion)