ABSTRACT
Organotypic hippocampal cultures have been recently used to study in vitro ischaemic neuronal death. Sub-lethal periods of ischaemia in vivo confer resistance to lethal insults and many studies have demonstrated the involvement of heat shock proteins in this phenomenon. We used organotypic hippocampal cultures to investigate the involvement of heat shock protein (HSP) 27 in preconditioning to oxygen and glucose deprivation. Neuronal damage was assessed using propidium iodide fluorescence; HSP27 phosphorylation and immunocontent were obtained using (32)Pi labelling followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. We observed that immunocontent of HSP27 was increased after lethal or sub-lethal treatment, indicating it is a response to metabolic stress. Treatments with 5 or 10 min of oxygen and glucose deprivation (OGD) or 1- microM N-methyl-D-aspartate (NMDA) induced tolerance to 40 min of OGD associated with an increase in HSP27 immunocontent and phosphorylation. These data suggest that, in vitro, phosphorylated HSP27 might be involved in preconditioning, probably acting as a modulator of actin filaments or by the blockage of neurodegenerative processes.
Subject(s)
Glucose/metabolism , Heat-Shock Proteins/metabolism , Hippocampus/metabolism , Oxygen/metabolism , Animals , Blotting, Western , Cell Death/drug effects , Excitatory Amino Acid Agonists/pharmacology , Glucose/deficiency , HSP72 Heat-Shock Proteins , Hypoxia/metabolism , Immunohistochemistry/methods , Ischemic Preconditioning/methods , N-Methylaspartate/pharmacology , Organ Culture Techniques , Phosphorylation , Rats , Rats, Wistar , Time FactorsABSTRACT
Global cerebral ischemia, with or without preconditioning, leads to an increase in heat shock protein 27 (HSP27) immunocontent and alterations in HSP27 phosphorylation in CA1 and dentate gyrus areas of the hippocampus. We studied different times of reperfusion (1, 4, 7, 14, 21 and 30 days) using 2 min, 10 min or 2+10 min of ischemia. The results showed an increase in HSP27 immunocontent of about 300% after 10 min of ischemia in CA1 and dentate gyrus. CA1, a hippocampal vulnerable area, showed an increase in HSP27 phosphorylation, parallel with immunocontent. In dentate gyrus, a resistant area, the increase in HSP phosphorylation was lower than immunocontent. After preconditioned ischemia (2+10 min), when CA1 neurons are protected to a lethal, 10 min insult, we observed an increase in HSP immunocontent and a decrease in phosphorylation in both regions of the hippocampus, suggesting that, when there is no neuronal death, HSP27 in a vulnerable area responds similarly to the resistant area.When dephosphorylated, HSP27 acts as a chaperone, protecting other proteins from denaturation. As it is markedly expressed in astrocytes, we suggest that HSP27 could be protecting hippocampal astrocytes, which could then be helping neurons to resist to the insult, maintaining tissue normal homeostasis.
Subject(s)
Brain Ischemia/metabolism , Heat-Shock Proteins , Hippocampus/metabolism , Ischemic Preconditioning , Neoplasm Proteins/metabolism , Nerve Degeneration/metabolism , Neurons/metabolism , Animals , Brain Ischemia/pathology , Brain Ischemia/physiopathology , Dentate Gyrus/metabolism , Dentate Gyrus/pathology , Dentate Gyrus/physiopathology , HSP27 Heat-Shock Proteins , Hippocampus/pathology , Hippocampus/physiopathology , Immunohistochemistry , Male , Nerve Degeneration/pathology , Nerve Degeneration/physiopathology , Neurons/pathology , Phosphorylation , Rats , Rats, Wistar , Time FactorsABSTRACT
Brain ischemia results in cellular degeneration and loss of function. Here we investigated the neuroprotective effect of lithium in an in vitro model of ischemia. Organotypic hippocampal slice cultures were exposed to oxygen and glucose deprivation. Cellular death was quantified by measuring uptake of propidium iodide (PI). Lithium chloride (0.2-1.2 mM) was added to the medium before, during and after lesion induction. A decrease in incorporation of PI was observed, indicating a neuroprotective effect in all doses tested. We also studied the effect of lithium on the phosphorylation of HSP27, a heat shock protein involved in cellular protection in its dephosphorylated state. In the lesioned hippocampus, 0.4 mM lithium chloride decreased the proportion of phosphorylated HSP27 to total HSP27. These results suggest that lithium may be useful in the treatment of brain ischemia.
Subject(s)
Antimanic Agents/pharmacology , Glucose/deficiency , Heat-Shock Proteins , Hippocampus/drug effects , Lithium Chloride/pharmacology , Neuroprotective Agents/pharmacology , Animals , Cell Death , Cell Hypoxia , Culture Media , Culture Techniques , Dizocilpine Maleate/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Fluorescence , HSP27 Heat-Shock Proteins , Hippocampus/cytology , Immunoblotting , Neoplasm Proteins/metabolism , Phosphorylation , Rats , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitorsABSTRACT
In recent years organotypic slice cultures of hippocampal tissue of rats have been widely used to study factors involved in neuronal death. Here we used 2D electrophoresis to study the phosphoprotein profile in such cultures and the effect of oxygen/glucose deprivation on this profile. Cultures were prepared from 7-day-old rats. After 14 days in culture the phosphorylation profile in the cultures, as shown by phospho-protein markers undergoing developmental change, closely resembled the profile of fresh tissue from 23-day-old rats. The results suggest that this model could be a good method to observe the development of the tissue and its response to an ischaemic lesion
