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)