TARGETING MACROPHAGES TO LIMIT MUSCLE DAMAGE

Overview
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Table of Contents

Acknowledgements .................................................................................................................. II

Abstract ................................................................................................................................... III

List of tables ........................................................................................................................... VI

List of figures ........................................................................................................................ VII

List of abbreviations ............................................................................................................ VIII

1.Introduction .................................................................................................................. 1

1.1. Muscle damage ................................................................................................... 2

1.2. Role of Macrophages ......................................................................................... 4

1.3. Macrophage Ablation ......................................................................................... 6

1.4. Macrophage depletion in mouse models ............................................................ 7

1.5. Mouse injury models ........................................................................................... 9

1.6. Rationale ............................................................................................................. 9

1.6.1Hypothesis ..................................................................................... 9

1.6.2Objectives...................................................................................... 9

2.Materials and Methods ............................................................................................ 11

2.1 Animal maintenance and treatments ............................................................... 12

2.1.1 Ethical Approval .......................................................................... 12

2.1.2 Treatment of mice with doxycycline ............................................ 12

2.1.3 Muscle injury and tissue harvest .................................................. 12

2.2 Processing tissue samples ............................................................................... 13

2.4. Histochemical and immunochemistry antibody stains ................................... 14

2.4.1 Haematoxylin and eosin staining ................................................. 14

2.4.2 F480 stain ..................................................................................... 14

2.4.3 CD68 antibody stain ..................................................................... 16

2.4.4 IgG stain ....................................................................................... 17

2.4.5 Laminin stain ................................................................................ 17

2.5 Statistical analysis ........................................................................................... 17

2.5.1 Morphometric analysis ................................................................. 18

2.5.2 Quantification of macrophages .................................................... 18

2.5.3 Calculation of fibre size diameter and central nucleation ............ 18

2.5.4 Percentage of tissue damage ........................................................ 18

3. Results ........................................................................................................................ 21

3.1. Overview ........................................................................................................ 22

3.2. Inducing macrophage depletion ..................................................................... 22

3.3. Assessing muscle injury ................................................................................. 24

3.4. Assessing tissue damage ................................................................................ 26

3.5. Determining the effects of CD68

cell depletion on regeneration ................. 28

4. Discussion ................................................................................................................... 31

4.1. Macrophage depletion .................................................................................... 32

4.2. Confirmation of muscle injury ....................................................................... 33

4.3. The effects of CD68

cell depletion on tissue damage and repair ................. 33

4.4. Limitations and Future work .......................................................................... 34

4.5. Conclusion ..................................................................................................... 36

References ............................................................................................................................... 37

Appendix A. Histochemical images....................................................................................... 42

A.1. F480 images .................................................................................................................... 43

A.2. CD68 images ................................................................................................................... 44

A.3. Haematoxylin and Eosin images ..................................................................................... 45

A.4. IgG images ...................................................................................................................... 46

A.5. Laminin images ............................................................................................................... 47

Appendix B Muscle morphometry plugin images ............................................................... 48

Appendix D Literature review ............................................................................................. 50

List of tables

Table 1.1 M1 and M2 macrophage subset phenotype .............................................................. 5

Table 2.6 Components of histochemical and immunochemistry antibody stains ................... 19

Table 2.7 Components of reagents, manufacturers and location ............................................ 20

vii

List of figures

Fig 1.1 Cyclic cascade of muscle damage and repair ............................................................... 3

Fig 1.2 Generation of double transgenic mouse model to induce macrophage depletion......... 5

Fig 2.1 Sectioned slides .......................................................................................................... 13

Fig 2.2Distribution of the CD68 antibody and negative control ............................................. 16

Fig 3.1 Macrophage depletion ................................................................................................ 23

Fig 3.2 Muscle injury via cardiotoxin ..................................................................................... 25

Fig 3.3 Tissue damage ............................................................................................................ 27

Fig 3.4 The effect of CD68

cells depletion on muscle regeneration ..................................... 29

Fig A.1 F480 images ............................................................................................................... 43

Fig A.2 CD68 images ............................................................................................................ 44

Fig A.3 Haematoxylin and eosin images ................................................................................ 45

Fig A.4 IgG images ................................................................................................................. 46

Fig A.5 Laminin images ........................................................................................................ 47

Fig B.1 Plugin images ............................................................................................................. 49

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TARGETING MACROPHAGES TO LIMIT MUSCLE DAMAGE

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