Spectronix 3000

Hemorrhagic volume was quantified with spectrophotometric assay of brain hemoglobin content at 24 hours after MCAO. Cerebral hemorrhage was quantified using a previously described spectrophotometric assay (Hu et al., 2011 (link)). A standard curve was obtained using a “virtual” model of hemorrhage. Incremental volumes of homologous blood (0, 2, 4, 8, 16, 32 μl) were added to the perfused brain tissue. The hemispheric brain was then homogenized in distilled water followed by 30-minute centrifugation (13,000 g). Drabkin reagent (1.6 ml; Sigma) was added to 0.4 ml supernatant aliquots and optical density was measured at 540 nm via spectrophotometer (Spectronix 3000; Milton-Roy). Hemoglobin measurements were performed and compared with the standard curve to obtain data in terms of hemorrhage volume. The total hemispheric hemoglobin content was expressed as μl of blood per hemisphere.

Intra-operative hemorrhage volume was quantified using spectrophotometric hemoglobin assay as previously described (Auriat et al., 2005 (link); MacLellan et al., 2004 (link)). Hemoglobin assay was used to quantify the amount of intra-operative bleeding that was encountered during partial frontal lobe resection procedure. The intra-operative blood loss that occurred during resection procedure was collected by suction. The suctioned blood sample was subjected to hemoglobin assay to quantify the amount of intra-operative bleeding volume in each rat. Briefly, blood loss during frontal lobe resection procedure was collected, distilled water was added to the suctioned blood sample to reach a total volume of 50 mL. The sample was sonicated for 60 seconds on ice followed by centrifugation at 13,000 rpm for 20 min. The supernatant (0.4 mL) was added to Drabkin’s reagent (1.6 mL) (Sigma Aldrich, St. Louis, MO) and allowed to react for 15 min at room temperature. Absorbance of hemoglobin was measured at 540 nm using a spectrophotometer (Spectronix 3000, Milton-Roy, Rochester, NY). A standard curve was established in the spectrophotometer using incremental volumes of rat blood. According to the linear relationship between optical density and blood volume on the standard curve, the bleeding volume of each sample was obtained.