Neuronal nuclear antigen (NeuN): A useful marker of neuronal immaturity in sudden unexplained perinatal death

Abstract

Introduction

In the developing brain neuronal differentiation is associated with permanent exit from the mitotic cycle. Neuronal nuclear antigen (NeuN) is a nuclear protein widely expressed in the mature postmitotic neurons.

Methods

We applied NeuN immunocytochemistry in 65 cases of perinatal death (16 victims of sudden intrauterine unexplained death syndrome/SIUDS, 19 of sudden infant death syndrome/SIDS and 30 controls) to test the physiological status of the brain neurons. In addition we applied both TUNEL and Caspase 3 immunohistochemical methods in order to highlight a possible relation between decreased NeuN expression and apoptotic outcome. We also attempted to see whether or not NeuN pathological changes can be related to cigarette smoke absorption in pregnancy.

Results

NeuN staining was considerably reduced or lost in SIUDS/SIDS compared to controls. However neurons with decreased NeuN-labeling showed no sign of apoptosis. A significant association was found between NeuN depletion and maternal smoking.

Conclusion

Altered NeuN expression can be a marker of immature and/or suffering neurons. The exclusive presence of this pattern of expression in SIUDS/SIDS victims, leads us to recommend the NeuN immunohistochemistry as a routine method in neuropathological protocols to convalidate a diagnosis of sudden perinatal death.

Introduction

The neuronal nuclear antigen (NeuN) is a specific protein expressed in post-mitotic neurons. The corresponding antibody, developed by Mullen in 1992 [1], primarily stains the neuronal nucleus, but the cytoplasm and dendrites are also immunoreactive, though to a lesser extent. Noteworthy it does not stain the immature nerve cells until they exit from the cell cycle and achieve a stage of development that at least approaches mature function. This is coincident with the migration of the neuroblasts from their birthplace in the embryonic neural tube to their final position, outgrowth of axons and generation of synapses [2], [3], [4], [5].

With the inexplicable exception of several neuronal cell types, such as the Purkinje cells and the dentate nucleus neurons in the cerebellum, the neurons of the inferior olivary nucleus in the medulla oblongata and the glial cells, that generally are not recognized by the NeuN antibody, the vast majority of neurons is strongly NeuN positive already in fetal life [1], [2].

In addition to representing a marker of maturing neurons, the NeuN immunohistochemistry can be applied in neuropathologic studies to highlight their physiological status. Precisely, while intense NeuN expression is shown by healthy neurons, a decreased NeuN positivity in postembryonic life can be indicative of degeneration of differentiated neurons. In particular, immunoreactivity is significantly weakened after a severe injury, such as cerebral hypoxia/ischemia [6], [7], [8]. Infants who have suffered fetal distress or perinatal asphyxia may show less brain NeuN immunostaining than infants who have not experienced such insults [2].

Stressors, such as hypoxia, hypercarbia and asphyxia are known as pathogenetic factors in SIDS [9], [10], [11], [12], resulting in functional and/or morphological developmental alterations of brain neurons. Nevertheless, among the countless existing works in the literature on this field, our previous contributions included, there are no reports taking into consideration the NeuN expression as index of neuronal distress in SIDS.

Insofar, we aimed to evaluate the immunoexpression of NeuN in a group of victims already object of our prior studies but not formerly investigated in this regard. We reconsidered a total of 65 subjects aged from 17 gestational weeks to 10 postnatal months, who had died of known or unknown causes. Our aims were firstly to obtain basic information about the manifestation of NeuN in the study groups and to evaluate a possible wrong expression in sudden perinatal and infant death, in addition to specific morpho-functional alterations of the autonomic nervous system we had already reported [13], [14], [15], [16], [17], [18], [19]. Then, in order to evaluate if the NeuN depletion can be indicative of neuronal degeneration, we applied the TUNEL method as hallmark of apoptosis [20], [21] and Caspase3 immunohistochemistry as a signal of dying cells [22].

Finally, we considered a possible correlation between NeuN pathological changes and hypoxic injuries related to cigarette smoke absorption in pregnancy.