Investigation of Macrophages Types Present within NF2 knock-out Peripheral Nerves Post-Injury
Paul Cookson, Peninsula College of Medicine and Dentistry
Introduction
Neurofibromatosis type 2 is a condition in humans linked with a mutation of the NF2 gene. Classically,
the disease in humans is associated with bilateral vestibular schwannomas as well as other Schwann
cell tumours. In previous work, Schwann cell selective NF2 knock-out has been achieved in mice,
suggesting the possibility of using this line of mice as an animal model for the disease in humans. In my
study, I aimed to investigate the inflammatory properties of the Schwann cell derived neoplasia that
occurs following nerve injury using the above mentioned animal model. Macrophages can be
described as either type M1 (pro-inflammatory) or type M2 (pro-proliferative). Using M1 and M2
specific genes, we hope to identify a difference in the populations of macrophages after peripheral
nerve injury in the knock-out mice compared to the control wild-type mice.
Aims:
To use semi-quantitative RT-PCR to identify a difference between the populations of macrophages
found in NF2 knockout mice compared with wild type mice.
Methods:
We used Schwann cell-selective NF2 knock out mice and wild type mice in this experiment. Both
groups of mice were treated with a sciatic nerve crush in one hind leg and 21 days post-injury, the
sciatic nerve tissue from both groups were harvested and frozen. We used a standard protocol to
isolate the mRNA from mouse sciatic nerve samples. The RNA was then run in a RT-PCR protocol in
order to produce a cDNA fragments. The cDNA from each sample was then run in PCR with primers
specific to either M1 or M2 macrophages as well as general indicators of inflammation and
macrophage markers. Each PCR was run in triplicate and the results were isolated and visualised using
electrophoresis. The gels were photographed and the density of the bands was recorded, averaged
and compared.
In a separate experiment using immunofluorescence, proliferating cells were found by overlaying
Hoechst counterstain in blue (a dye that stains DNA) with Ki-67 (a protein that is expressed only during
cell division) in red. The cells where these stains overlapped were identified and counted.
Results
The data gathered is summarised in the table below. The most significant differences could be seen
between the uncrushed intact and the crushed nerves, regardless of genotype. We found no
significant evidence for M1 macrophages in any of the samples. However we found decreases in the
concentrations of generic and M2 macrophage markers in the knock-out mice compared to the wildtype mice.
No significant difference was observed between the knock-out and wild type mice in the
immunofluorescence experiment.
Average Results by Genetype and Crush Status
10000
8000
6000
4000
2000
0
Intact
Crushed
Intact
Crushed
WT
WT
NF2KO
NF2KO
18S
F4/80
MCP-1
Arg-1
CTGF
Trem2
iNOS
Discussion and conclusions
These results contrasted with previous research as we found decreased amounts of mRNA coding for
M2 macrophage markers in the post crush samples and of the generic macrophage markers, only the
F4/80 in the wild type mice increased after the crush. Increases in connective tissue growth factor
(CTGF) in the crushed nerve is consistent with laying down of new collagen in the repairing nerve. The
findings contrast with previous research which indicated increased M2 macrophages to be present.
This may be due to the timing that the samples were taken (in this case 21 days) since previous
research was done 7 days after injury. Further research with earlier samples may be warranted.
Acknowledgements
I would like to thank the Wellcome Trust and the Academy of Medical Sciences for the Inspire
Studentship that made this research possible. I also want to thank the Peninsula Schools of Medicine
and Dentistry Centre for Biomedical Research, particularly Professor David Parkinson and Dr Thomas
Mindos for their encouragement and guidance in this research. I found the experience of working in
the laboratory extremely enjoyable, confidence-building and enriching. I believe I have developed
useful and transferable skills at each stage of the project: reviewing the previous research, writing the
grant proposal, conducting the experiments, investigating the results and reporting my findings.
Having found the experience to be extremely rewarding, I have no doubt that I will be aggressively
pursuing research opportunities in the future.