Potential value of changes in cell markers in organotypic hippocampal cultures associated with chronic EtOH exposure and withdrawal: comparison with NMDA-induced changes.
BACKGROUND
Previous studies have shown that withdrawal from ethanol (EtOH) exposure induces neuronal damage, as indicated by propidium iodide (PI) uptake, in organotypic hippocampal slice cultures. This is prevented by MK801, suggesting that damage is "excitotoxic," resulting from activation of N-methyl-d-aspartate (NMDA) receptors by endogenous glutamate. To avoid reliance on a single indicator, and to enable assessment of recovery from the EtOH withdrawal (EWD) insult, we assessed changes in cell markers for neurons and glia, as well as cell division, following either EWD or NMDA challenge (as a positive control).


METHODS
Organotypic cultures from postnatal day (PND) 8 rats were cultured for 5 days before exposure to EtOH (mean concentration approximately 65 mM) for 10 days before EWD. Cultures of the same "days in vitro" age (DIV16) were exposed to NMDA (200 microM) for 1 hour. Neuronal injury was visualized using PI and indices of neurons, glia, or cell division were measured at intervals up to 10 days following the neurotoxic insults. Each time point and measurement used separate slice cultures, and these were treated as separate experiments with paired controls. Regional neuronal content was assessed by neuronal nuclear protein (NeuN) and calbindin D28k (Calb), glial content by glial fibrillary acidic protein (GFAP), and cell division by bromodeoxyuridine (BrdU) incorporation, all measured immunohistochemically.


RESULTS
Chronic exposure to EtOH was associated with a dramatic reduction in BrdU incorporation in all regions of cultures. Propidium iodide fluorescence in the CA1 region was elevated significantly after EWD and more so after NMDA challenge. Reduced immunoreactivity (IR) of NeuN and Calb suggested that loss of neurons resulted from the EWD insult. Bromodeoxyuridine incorporation was initially depressed even further by EWD, but had returned to control levels after 3 days. In contrast, following NMDA insult, BrdU incorporation was significantly and persistently elevated above control levels after 3 days. Glial fibrillary acidic protein was reduced immediately after both EWD and NMDA challenge. Several days after EWD, expression of neuronal and glial markers, although variable, had generally returned to control levels. In contrast, NeuN IR remained significantly reduced after NMDA challenge.


CONCLUSIONS
In general, the use of additional markers supports data obtained with PI uptake alone and suggests that neurons (and glia) are lost from the culture following EWD or NMDA challenge. These cell markers recover several days after EWD, but it is unclear whether functional recovery accompanies these changes. If the dramatic effect of EtOH exposure and EWD on BrdU incorporation reflects reduced neuro- and gliogenesis, it is likely that this adversely affects long-term recovery from EWD. Finally, some markers showed significant and consistent changes after EWD, whereas others did not. This information may facilitate the use of this model in evaluation of potential medications that protect against and/or promote recovery from neurotoxicity.
Wilkins
Lincoln H
LH
Department of Behavioral Sciences, University of Kentucky, Lexington, Kentucky 40536-0298, USA.
Prendergast
Mark A
MA
Blanchard
John
J
Holley
Robert C
RC
Chambers
Elton R
ER
Littleton
John M
JM
D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D064092:Calbindin 1; D064026:Calbindins; D002455:Cell Division / Q000187:drug effects; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D004195:Disease Models, Animal; D000431:Ethanol / Q000494:pharmacology*; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D005260:Female; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D016202:N-Methylaspartate / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009924:Organ Culture Techniques; D011419:Propidium / Q000493:pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D013375:Substance Withdrawal Syndrome / Q000378:metabolism*
D034961:Antigens, Nuclear; D015415:Biomarkers; C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D002492:Central Nervous System Depressants; D018690:Excitatory Amino Acid Agonists; D005904:Glial Fibrillary Acidic Protein; D009419:Nerve Tissue Proteins; D064030:S100 Calcium Binding Protein G; D011419:Propidium; D000431:Ethanol; D016202:N-Methylaspartate