Elsevier

Brain Research

Volume 1015, Issues 1–2, 23 July 2004, Pages 169-174
Brain Research

Research report
Loss of NeuN immunoreactivity after cerebral ischemia does not indicate neuronal cell loss: a cautionary note

https://doi.org/10.1016/j.brainres.2004.04.032Get rights and content

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.

Introduction

In order to differentiate neurons from glial cells in brain sections, specific neuronal markers, such as neuron-specific enolase, neurofilament proteins and calbindin, can be used in immunohistochemical procedures [17]. However, most of these proteins are present in distinct subpopulations of neurons, limiting their application for histological identification of these cells. In order to overcome this obstacle, Mullen et al. [15] developed a monoclonal antibody that recognizes a neuron specific nuclear protein. NeuN, which stands for neuron-specific nuclear protein, is expressed in nucleus and cell body of most neuronal cell types in rodents, chicks and humans [15]. It is not expressed in glial cells; neither oligodendrocytes nor astrocytes nor do microglial cells express NeuN. Since its development, NeuN has been successfully used as a neuronal marker in a wide variety of studies in cell cultures as well as in diagnostic histopathology. Thus, NeuN has been established as a universal marker for neurons although a few neuronal cell types such as cerebellar Purkinje cells, retinal photoreceptor cells, mitral cells of the olfactory bulbs, and sympathetic chain ganglion cells are devoid of staining [15].

NeuN primarily stains the nucleus but the cytoplasm is also immunoreactive, though to a lesser extent. For the most part, the cytoplasmic staining is concentrated in the soma although it extends a short distance into some of the processes (mostly dendritic). NeuN is likely to be a transcription factor that is expressed in the nucleus and cytoplasm of mature neurons [15]. NeuN does not stain the nuclei of immature nerve cells until they complete their development [18].

Although healthy neurons normally express an intense NeuN signal, NeuN immunoreactivity has been reported to decrease under several pathological conditions that adversely affect neuronal viability such as cerebral ischemia, hypoxia and trauma [2], [9], [19]. Interestingly, this reduction in immunostainability has been considered as an evidence of neuronal loss in several studies [2], [9], [19], [20], [21]. However, loss of NeuN immunoreactivity may not necessarily indicate disintegration and disappearance of neurons, but may also be due to the depletion of NeuN protein or loss of its antigenicity.

In this study, we investigated whether or not the neurons that lost their NeuN immunoreactivity were indeed dissipated and lost their morphological integrity after mild cerebral ischemia. We preferred a mild ischemia model because it induces less tissue destruction and less neuronal cell loss.

Section snippets

MCA occlusion

Twenty-four Swiss albino mice weighing 35–40 g were housed under diurnal lighting conditions and fasted overnight but allowed free access to water before the experiment. Animal housing, care and application of experimental procedures were all done in accordance with institutional guidelines and all efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data. Mice were intraperitoneally anesthetized with chloral hydrate 300

Results

The number of NeuN-labeled cells was significantly (p<0.05) decreased by 27% in the penumbra and by 62% in the ischemic core 6 h after brief focal ischemia (Fig. 1; for absolute values of cell counts, see Fig. 2). Despite a reduction in the number of NeuN-positive neurons, H&E staining of adjacent sections did not disclose advanced degenerative changes such as ghost cells and cellular disintegration in the penumbra region, but instead showed that presumable neurons (by H&E criteria) had

Discussion

Velier et al. [20] examined the temporal kinetics of neuronal cell loss and of cellular DNA damage after permanent MCA occlusion by using the neuron-specific marker NeuN and TUNEL. They concluded that there was an early loss of neurons (46% of the entire population) within the first 24 h that did not involve DNA damage and was presumably a form of necrotic cell loss. This conclusion was based on loss of NeuN immunoreactivity. Apparently, the authors considered loss of NeuN staining as a marker

Acknowledgments

Dr. T. Dalkara's work is supported by the Turkish Academy of Sciences.

References (22)

  • K Fink et al.

    Prolonged therapeutic window for ischemic brain damage caused by delayed caspase activation

    J. Cereb. Blood Flow Metab.

    (1998)
  • Cited by (225)

    View all citing articles on Scopus
    View full text