TARGETING MACROPHAGES TO LIMIT MUSCLE DAMAGE

Imoh Essien 85 PAGES (15673 WORDS) Dissertation
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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
1.6.1 Hypothesis ..................................................................................... 9
1.6.2 Objectives...................................................................................... 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
v
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
vi
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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APA

Imoh, E (2019). TARGETING MACROPHAGES TO LIMIT MUSCLE DAMAGE. Afribary.com: Retrieved March 04, 2021, from https://afribary.com/works/essien-imoh

MLA 8th

Essien, Imoh. "TARGETING MACROPHAGES TO LIMIT MUSCLE DAMAGE" Afribary.com. Afribary.com, 19 Aug. 2019, https://afribary.com/works/essien-imoh . Accessed 04 Mar. 2021.

MLA7

Essien, Imoh. "TARGETING MACROPHAGES TO LIMIT MUSCLE DAMAGE". Afribary.com, Afribary.com, 19 Aug. 2019. Web. 04 Mar. 2021. < https://afribary.com/works/essien-imoh >.

Chicago

Essien, Imoh. "TARGETING MACROPHAGES TO LIMIT MUSCLE DAMAGE" Afribary.com (2019). Accessed March 04, 2021. https://afribary.com/works/essien-imoh