Spectrophotometric Assay of Hemoglobin Volume

At 24 h after SBI, the animals were placed under deep anesthesia and transcardially perfused with 0.1 M PBS until the outflowing fluid from the right atrium became colorless. The brain was removed and dissected into left and right hemispheres. Hematoma volume was quantified by spectrophotometric assay of brain hemoglobin content as described previously⁴⁸. A standard curve was obtained by adding incremental volumes of homologous blood to perfused brain tissue of naïve animals. The hemispheric samples were then homogenized and sonicated in distilled water followed by a 30-min centrifugation (13,000 g at 4 °C); Drabkin reagent (1.6 mL; Sigma) was added to 0.4 mL supernatant aliquots and optical density was measured at 540 nm via a spectrophotometer (Spectronix 3000; Milton-Roy). Hemoglobin measurements were compared with the standard curve to obtain hemorrhage volume expressed as μL of blood per ipsilateral hemisphere.

Intraoperative hemorrhage volume was collected by suction throughout surgery and was added to packing material used for hemostasis during the procedure; distilled water was added bringing the volume to 50 mL for each collected hemorrhage sample. A standard curve was obtained by adding incremental volumes of homologous blood to distilled water to create solutions of 50 mL total volume. Samples were homogenized, sonicated, and prepared for spectrophotometric assay as described above. Testing was done in duplicate.