Neurodegeneration, the progressive loss of structure or function of neurons, results in many neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and Huntington’s [1,2,3,4]. The hippocampus, where neuronal circuitry ranging from molecular to systemic exists, has been recognized as a key region related to many neurological diseases [5,6,7]. Doublecortin (DCX) as a progenitor neural cell marker, a microtubule-associated phospho-protein, has been utilized for analyzing alterations in neurogenesis in the adult dentate gyrus (DG) because it is believed to be specific to neuronal antigens expressed by newly born neurons [8]. The rate of hippocampal neurodegeneration is known to be affected by age, hormonal status, growth factor, chemicals, physiologic stimuli and environmental effects. For many reasons, there is an increasing interest in models used to understand the mechanism of neurodegeneration [9,10,11,12].

Ionizing radiation has been hazardous to human health due to overexposure from natural and artificial sources, such as diagnostic and therapeutic medical usage [13,14]. Overexposure to radiation causes damage to living tissue, and can result in mutations, radiation sickness, cancer, and death [15,16]. Even though the risk to radiation exposure in the brain is a big issue, the changes in the hippocampal neurodegeneration are poorly understood. However, recently, the risk of short-term neurodegeneration caused by radiation exposure has been reported [17,18,19,20,21,22]. Kim’s group suggested that transient impairment of the functioning of the hippocampus is linked to inhibition of hippocampal neurogenesis after acute γ-irradiation [9]. The Abdallah group reported that irradiation can reversibly alter proliferation, neurogenesis, and cell death in the dentate gyrus of adult mice. The early cognitive deficits may be related to the effects of a short-term reduction of neurogenesis [17]. The Acevedo group reported that older female mice deficient in apoE (Apoe^−/−) are more susceptible to effects of gamma irradiation on novel location recognition [23].

In this study, we addressed the long-term effect of γ-irradiation exposure on neurogenesis in a C57BL/6 female model. We were interested in determining if there is a dose response relationship in terms of radiation-induced long-term impairments in neurogenesis. We elucidated the number of DCX-positive cells on the hippocampus with altered radiation dose in immunohistochemistry. We hypothesized that the long-term changes in DCX-positive cells caused by γ-irradiation would continuously increase neurodegeneration resulting in damage to the hippocampal function as opposed to the recovering tendency of neurogenesis impairment in the short term. Understanding how irradiation affects neurogenesis may provide useful insight into potential approaches to reduce brain-related diseases.

Figure 1 shows the scheme for the procedure of radiation exposure in an animal model. The mice were exposed to whole-body radiation at acute dose (5 Gy) or fractional doses (1 Gy × 5 times or 0.5 Gy × 10 times), and further sustained for 6 months.

To investigate the long-term effect of radiation exposure on hippocampal neurogenesis in an adult mouse DG, the number of DCX-positive cells in the subgranular zone (SGZ) of the hippocampal DG was analyzed by using immunohistochemistry (Figure 2). Six months after radiation exposure, the number of DCX-positive cells in the hippocampal DG of the control group was 10.75 ± 2.25 cells/DG (Figure 2A, a and e). In the fractional (0.5 Gy × 10 times and 1 Gy × 5 times) and acute irradiation (5 Gy × 1 times) groups, the numbers of DCX-positive cells were 5.5 ± 1.5 cells/DG (Figure 2A, b and f), 5.25 ± 0.75 cells/DG (Figure 2A, c and g) and 1.5 ± 0.5 cells/DG (Figure 2A, d and h), respectively. The groups of fractionated irradiation (0.5 Gy × 10 times and 1 Gy × 5 times) showed approximately 49% and 51% reductions of DCX-positive cells in the hippocampal DG compared to the control group. Importantly, the group with acute irradiation (5 Gy × 1 times) resulted in an 86% reduction of DCX-positive cells.

DCX is a reliable marker of newly generated neurons because neuronal precursor cells begin to express DCX while actively dividing [8,24]. DCX immunoreactivity, which quantifies both the absolute number and dendritic growth of new neurons, can be useful for analyzing the modulation of hippocampal neurogenesis as a function of neurodegenerative diseases. In this study, it was shown that the number of DCX-positive cells by acute or fractionated irradiation groups tended to be significantly reduced (Figure 2B). Thus, the results of DCX immunoreactivity suggests that the long-term effect of γ-irradiation exposure can suppress hippocampal neurogenesis resulting in damage to the hippocampal function unlike the tendency to fully restore transient impairments through the short-term effect of irradiation in previous studies [17,18].

Radiation therapy plays a major role in the treatment of primary brain tumors and cancers. Unfortunately, however, radiation exposure can cause severe side effects including cognitive deficits. According to recent studies, irradiation effects in normal brain tissue have been principally limited to studies of acute dose irradiation [25]. Our goal in this study was to characterize the long-term effects by acute and fractionated irradiation on hippocampal neurogenesis. DCX immunoreactivity was relatively lower in the acute irradiation group than in the fractionated irradiation groups, but there was no significant difference in the fractionated irradiation (Figure 2B). These results showed that hippocampal neurogenesis was more vulnerable to acute than fractionated irradiation.

In conclusion, this is the first report to elucidate the long-term effects of DCX-positive cells as an immature progenitor cell marker by acute and fractionated irradiation on hippocampal neurogenesis in vivo. To the best of our knowledge, no data exist with respect to the radiation response of acute and fractionated irradiation on hippocampal neurogenesis in animal model. This is important because the effect of brain treatment in acute irradiation is higher than that seen in fractionated irradiation. The long-term effects of irradiation are important in clinic because of chronic progressive nature and significant long-term morbidity.