Question: 

Ca²⁺ is a cofactor of many cellular processes including apoptosis and necrosis. This study's hypothesis was that bloody cerebrospinal fluid (CSF) from patients after intraventricular hemorrhage may cause a Ca²⁺-rise which induces apoptosis or necrosis in a in-vitro model of human cerebral astrocytes.

Method: 

Human astrocytes were incubated with CSF from patients with intraventricular hemorrhage. In control experiments native CSF was used. Single cell cytosolic Ca²⁺-concentration was measured by fura-2 microfluometry. Three blockers were used: Nimodipine (10mM), XestosponginD (6mM), suramine (100mM), 8-phenyltheophilline (8-PT; 10mM) and D(-)-2-Amino-5-Phosphopentanoic Acid (2A5P; 50mM), that blocks the L-type Ca²⁺-channels, the endoplasmic reticulum Ca²⁺ release channels, purinergic P2 and P1 receptors and NDMA receptors, respectively. Apoptosis and necrosis were evaluated by staining with Hoechst-3342 and propidium iodide.

Results: 

Incubation of astrocytes with bloody-CSF provoked a cytosolic Ca²⁺ rise in a characteristic pattern (straight line; see figure): after an initial peak, Ca²⁺ concentration almost returned to baseline but then increased slowly but long lasting over the observation period of 60 min. The Ca²⁺ peak was nearly completely blocked by XestosponginD and suramin (p<0.05). Nimodipine, 8-PT and 2A5P had no significant effect. Incubation of the astrocytes with bloody-CSF induced necrosis but not apoptosis. The blockade of the Ca²⁺-rise by XestosponginD or Suramin reduced necrosis significantly (p<0.05). Nimodipine, 8-PT and 2A5P did not prevent astrocytic necrosis.

Conclusion: 

Bloody CSF induces an Ca²⁺ peak leading to necrosis but not apoptosis in human astrocytes. The initial cytosolic Ca²⁺ peak and cellular necrosis depends on the activation of the ATP sensitive P2 receptors and subsequent IP₃ dependent calcium release. L-type Ca²⁺-channels, purinergic P1 receptors and NDMA receptors are not significantly involved in bCSF induced astrocytic calcium overload and necrosis.

Figure 1