gms | German Medical Science

Objective: Ca²⁺ is a cofactor of many intracellular processes including apoptosis and necrosis. This study’s hypothesis was that bloody CSF from patients after intraventricular hemorrhage (IVH) may cause a Ca²⁺-rise which induces a mitochondrial dysfunction that results in necrosis in a in-vitro model of human cerebral astrocytes.

Methods: Human astrocytes were incubated with CSF from patients with IVH. In control experiments native CSF was used. Single cell cytosolic Ca²⁺-concentrations were measured by fura-2 microfluometry. Three blockers were used: Cyclosporin A, APB and suramine that block mitochondrial permeability transition pores (MPTP), endoplasmic reticulum IP3 sensitive Ca²⁺-channels and ATP-sensitive PY2-rezeptor, respectively. Apopotois and necrosis were evaluated by staining with Hoechst-3342 and propidium iodide, respectively.

Results: Incubation of astrocytes with bloody-CSF provoked an initial Ca²⁺ peak that was followed by a slow but long lasting Ca²⁺-rise over the observation period of 60 min. This was also true, when the extracellular Ca²⁺ was bounded by EDTA. Incubation of the astrocytes with bloody-CSF induced necrosis but not apoptosis. APB and suramin reduced both significantly the Ca²⁺-rise and the rate of necrosis. Cyclosporin A did not influence the Ca²⁺-rise but reduced significantly the rate of necrosis.

Conclusions: Bloody CSF induces a Ca²⁺-overload that leads to necrosis but not apoptosis in human astrocytes via activation of the ATP-sensitive PY2-receptor and Ca²⁺ release from IP3-dependent intracellular Ca²⁺-stores. The necrosis is due to Ca²⁺-dependent opening of MPTP resulting in breakdown of mitochondrial homoeostasis.