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.