title: Loss of NeuN immunoreactivity after cerebral ischemia does not indicate neuronal cell loss: a cautionary note.
abstract: NeuN immunoreactivity is used as a specific marker for neurons. The number of NeuN-positive cells decreases under pathological conditions. This finding is usually considered as an evidence of neuronal loss. However, decrease in NeuN labeling may also be caused by depletion of the protein or loss of its antigenicity. Hence, we have investigated the morphological features of neurons that lost NeuN immunoreactivity and the NeuN protein levels in mouse brain after cerebral ischemia. The number of NeuN-labeled cells was decreased 6 h after a mild ischemic insult (30 min middle cerebral artery occlusion) in penumbral and core regions. Hematoxylin and eosin (H&E) staining of adjacent sections showed that neurons in the penumbra were not disintegrated but displayed early ischemic changes. The nuclear NeuN staining was dramatically reduced or lost in some neurons. However, Hoechst 33258 staining of the same sections revealed that these nuclei were preserved with an intact membrane. Labeling of neurons that had lost NeuN-positivity with antibodies against caspase-3-p20, which is constitutively not present but emerges in neurons after ischemia, disclosed that these neurons still preserved their integrity. Moreover, Western blots showed that NeuN protein levels were not decreased, suggesting that reduced NeuN antigenicity accounted for loss of immunoreactivity in this mild brain injury model. Supporting this idea, NeuN labeling was partially restored after antigenic retrieval. In conclusion, since NeuN immunoreactivity readily decreases after metabolic perturbations, reduced NeuN labeling should not be taken as an indicator of neuronal loss and, quantitative analysis based on NeuN-positivity should be used cautiously after central nervous system (CNS) injury.
authors.affiliation: Institute of Neurological Sciences and Psychiatry, and Faculty of Medicine, Department of Neurology, Hacettepe University, Sihhiye Ankara 06100, Turkey.
authors.forename: Isin
authors.initials: I
authors.lastname: Unal-Cevik
authors.forename: Münire
authors.initials: M
authors.lastname: Kilinç
authors.forename: Yasemin
authors.initials: Y
authors.lastname: Gürsoy-Ozdemir
authors.forename: Gunfer
authors.initials: G
authors.lastname: Gurer
authors.forename: Turgay
authors.initials: T
authors.lastname: Dalkara
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D015415:Biomarkers / Q000032:analysis; D001921:Brain / Q000378:metabolism / Q000473:pathology; D001923:Brain Chemistry; D002545:Brain Ischemia / Q000378:metabolism* / Q000473:pathology*; D016923:Cell Death / Q000502:physiology; D004268:DNA-Binding Proteins; D007150:Immunohistochemistry; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism* / Q000473:pathology*; D009687:Nuclear Proteins / Q000378:metabolism*
chemical_list: D034961:Antigens, Nuclear; D015415:Biomarkers; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; C492380:NeuN protein, mouse; D009687:Nuclear Proteins