Astrocyte calcium signaling under oxidative stress in acute and cultured brain tissue slices

During evolution calcium (Ca2+) has evolved into an essential signaling molecule, as it is involved in neurotransmission, muscle contraction and various metabolic processes of eukaryotic cells. Its function is highly dependant on preservation of cytosolic Ca2+ baseline levels in the nanomolar range. When oxidative stress arises after antioxidant mechanisms of the cell fail to fully reduce oxygen radicals, e.g. during aging, Ca2+ homeostasis becomes disturbed. To analyze in which way metabotropic Ca2+ signaling is affected by this, widefield Ca2+ imaging was performed in acute tissue slices of mouse hippocampus. Organotypic tissue cultures were used for analysis of medium term effects. Analysis in both was focused on astrocytes because of their variety in function within the tissue of the central nervous system (CNS). For identification of astrocytes, two methods were compared, a pharmacological approach (Dallwig et al., 2000) and use of the fluorescent dye Sulforhodamine 101 (Kafitz et al., 2008). The latter one was found to be best for the performed experiments. Next, an appropriate H2O2 concentration for induction of oxidative stress was determined. Treatment with 200 µM H2O2 for 45 minutes caused a slight increase in intracellular Ca2+ and was subsequently used to analyze the effects of acute oxidative stress. Besides an increase in intracellular Ca2+ baseline levels, metabotropic Ca2+ response was found to be significantly reduced in these experiments. To narrow down the point of interference by oxidative stress in the metabotropic signaling cascade, filling level of internal Ca2+ stores was measured and testing for hints on adenosine triphosphate (ATP) shortage was performed. Neither a decrease of Ca2+ in internal stores was found after treatment, nor were hints on ATP shortage detected. Immunohistochemical analysis of tissue cultures provided evidence for reactive astrogliosis three days after treatment. In summary, these results show that acute oxidative stress disturbs Ca2+ homeostasis and drastically alters metabotropic Ca2+ signaling in astrocytes. Although it remains to be elucidated if alterations originated from receptor protein oxidation in the membrane or from impairment of the intracellular signaling cascade, it appears reasonable to assume that the precise spatially and temporal coding of Ca2+ signals necessary for the function of Ca2+ as second messenger is affected. In addition to experiments that dealt with oxidative stress, unrestricted somatic stem cells (USSC) from human cord blood were analyzed on their functionality as a subproject of the FOR717. USSCs have been found before to support axonal regrowth and to increase locomotor recovery of rats after spinal cord injury (Schira et al., 2012). In widefield Ca2+ imaging experiments of our study, different neurotransmitters were applied on USSCs that have previously been treated to induce development into neurons. Except for ATP no substance was found to induce an intracellular Ca2+ response. To test the ability of untreated USSCs to integrate into the brain, cells were transplanted into hippocampal tissue cultures of mice directly after injury. Immunohistochemical staining detected USSCs to be still present in 6 out of 44 slices after seven days. Taken together, differentiated cells were able to show Ca2+ response which confirms their ability to respond to extracellular stimuli in principle. Yet, integration of USSCs into CNS tissue requires further analysis to make them usable for medical treatment of human beings.