DARPP-32 to quantify intracerebral hemorrhage-induced neuronal death in basal ganglia.

Quantification of acute brain injury in basal ganglia is essential for mechanistic and therapeutic studies in experimental intracerebral hemorrhage (ICH). Using conventional counting of degenerating cells based on morphological or immunohistochemical criteria, it is hard to define the boundary of the whole lesion area. Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32) is a cytosolic protein highly enriched in medium-sized spiny neurons of the striatum. We developed new methods for quantifying lesion area by detecting the difference of the DARPP-32 negative area and the hematoma clot, and by measuring DARPP-32 protein level for semi-qualification in rat model of ICH. We found that DARPP-32 negative area around hematoma was present at day-1, peaked at day-3, and decreased at day-14 after ICH, a time course paralleled by DARPP-32 Western blots. The DARPP-32 negative area matched well with the necrotic area determined using propidium iodide. Treatment with an iron chelator, deferoxamine, attenuated the ICH-induced reduction in DARPP-32 protein levels. These results suggest that DARPP-32 is a simple and quantifiable indicator of ICH-induced neuronal death in basal ganglia.