Research reportLoss of NeuN immunoreactivity after cerebral ischemia does not indicate neuronal cell loss: a cautionary note
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)
- et al.
Immunohistochemical and biochemical assessment of caspase-3 activation and DNA fragmentation following transient focal ischemia in the rat
Neuroscience
(2002) - et al.
Relationship between metabolic dysfunctions, gene responses and delayed cell death after mild focal cerebral ischemia in mice
Neuroscience
(2001) Disturbances of cerebral protein synthesis and ischemic cell death
Prog. Brain Res.
(1993)- et al.
Regional vulnerability after traumatic brain injury: gender differences in mice that over express human copper, zinc superoxide dismutase
Exp. Neurol.
(2001 (Dec)) - et al.
Axotomy abolishes NeuN expression in facial but not rubrospinal neurons
Exp. Neurol.
(2004) - et al.
Glutamate receptor antagonists inhibit calpain-mediated cytoskeletal proteolysis in focal cerebral ischemia
Brain Res.
(1998) - et al.
Neuronal nuclear antigen (NeuN): a marker of neuronal maturation in the early human fetal nervous system
Brain Dev.
(1998) - et al.
Improvement in neuronal survival after ischemic preconditioning in hippocampal slice cultures
Brain Res.
(2002) - et al.
Cerebral ischemia/reperfusion injury in rat brain: effects of naloxane
NeuroReport
(2001 (May 8)) - et al.
Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family
J. Cereb. Blood Flow Metab.
(1998)
Prolonged therapeutic window for ischemic brain damage caused by delayed caspase activation
J. Cereb. Blood Flow Metab.
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