A fluorescent method to quantify neuronal injury after intracerebral hemorrhage

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Intracerebral hemorrhage (ICH) is the primary event in 10-15% of strokes. Although injury to tissue surrounding a hematoma was initially attributed to its mass effect, experimental evidence suggests that the hematoma per se may be toxic to neighboring cells. One likely neurotoxin is hemoglobin (Hb), which is present in millimolar concentrations in the clot and is released from erythrocytes in the days after hemorrhage. Breakdown of heme moieties by the heme oxygenase (HO) enzymes may then result in an iron-dependent oxidative injury. However, conflicting results in the collagenase and blood injection ICH models suggest that more detailed investigation of the consequences of heme breakdown is warranted. Neuronal injury in tissue surrounding an experimental ICH is usually quantified by cell counts. Specific histological methods vary considerably from laboratory to laboratory, but do share common features of being laborious, operator- dependent, and quite expensive. The latter characteristic may account for the limited scope of many studies, which typically test only one hemorrhage volume and one or two drug doses. Since the efficacy of any therapeutic intervention will likely be a function of clot size and drug dose, a more efficient method to quantify cell injury is urgently needed. Fluorescent protein expression has been successfully used as a marker of cell viability in vitro and in retinal studies in vivo, but not in any ICH model to date. In order to evaluate the potential of this approach, a colony of transgenic mice that constitutively express the red fluorescent protein variant dTomato in neurons has been established by the applicant. Preliminary studies indicate that: 1) dTomato expression is nontoxic per se, as detected by behavioral phenotyping and histological analysis; 2) Striatal fluorescence correlates very well with the MTT cell viability assay after experimental ICH. The goal of this proposed project is to validate striatal fluorescence as a rapid and efficient marker of neuronal loss after experimental ICH. The transgene is currently being incorporated into mice lacking HO-2 for further evaluation of the effect of this enzyme on hemorrhagic CNS injury. Specific aims are as follows: 1) Induce striatal hemorrhage in HO-2 knockout-dTomato and HO-2 wild-type-dTomato mice by stereotactic injection of autologous blood (5, 10, or 20 5l) or collagenase (0.01, 0.03 or 0.075 units). At 3 and 8 days, compare neuronal injury as quantified by: a) MTT assay after striatal cell dissociation, normalizing formazan production to that in the contralateral striatum; b) Striatal cell lysis, followed by measurement of lysate fluorescence, also normalized to that in the contralateral striatum; c) Cell counts, guided by a design-based stereology program. [Compare neuronal loss with perihematomal astrogliosis and inflammatory cell infiltration. 2) Correlate striatal neuronal injury with behavioral outcome, quantified with corner turn, adhesive removal, and elevated body swing tests, and digital analysis of home cage video recordings.] It is hoped that the proposed studies will establish the validity of this rapid fluorescent method for quantifying neuronal injury after experimental ICH.
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