Fig. 9.

EPO and CEPO reduce microglial PARP-1 activation in cerebral white matter injury. We performed immunohistochemistry with an antibody for PARP-1 and examined PARP-1 expression in the white matter after H/I or H/I/L. (A–C) We noticed PARP-1 immunoreactivity present in the white matter after H/I and this appeared to decrease with EPO or CEPO treatment. (D–F) We observed strong PARP-1 immunoreactivity in the white matter after H/I/L, which appeared increased compared with H/I alone (D vs. F). Mice treated with EPO or CEPO after H/I/L displayed decreased PARP-1 immunoreactivity (E, F) compared with vehicle-treated mice (D). We performed cell counting to confirm these findings. We found a significant decline in the number of PARP-1+ cells in EPO and CEPO treated mice exposed to H/I (G, *p<0.05). Similarly, the number of PARP-1+ cells significantly decreased in H/I/L mice treated with EPO or CEPO (H, *p<0.05). (I, J) Immunostaining to identify the PARP-1 enzymatic product, PAR, was performed to show PARP-1 activity. Accumulation of PAR mainly co-localized with the macrophage lineage marker CD68 in the white matter area after H/I or H/I/L. (K, L) We examined whether EPO or CEPO treatment altered white matter injury in PARP-1^−/− mice by performing the same H/I or H/I/L procedure and treatment on P6. Lesion severity was assessed using MBP immunostaining and the same 0–5 scoring scale as in above experiments. (K) We found no difference in lesion severity between wild-type vehicle treated, PARP-1^−/− vehicle treated, PARP-1^−/− EPO treated or PARP-1^−/− CEPO treated H/I mice. (L) After H/I/L, PARP-1 deficient mice showed relatively minor injuries compared with wild-type mice (*, p<0.05). However, EPO or CEPO treatment to PARP-1^−/− mice exposed to H/I/L did not alter lesion severity. Scale bar in A (for A–F), 20 µm. Scale bar in I (for I–J), 10 µm.