The present study has established for the first time the early damage indices of cerebellum, as revealed by histological and immunohistochemical techniques (see ), in adult mice subjected to a sublethal dose of TBI. Loss of Purkinje cells occur 3.5 days post-TBI and deformation of neuronal projections, the axons and dendritic arbor, appears as early as 1 day following onset of TBI. Immunohistochemical indices of early cerebellar damage include reduced expression of CB-28, and increased expression of VGluT2, MDA and 8-OHdG. In addition, expressions in VEGFR2, CD45 and p53 are minimal in the cerebellums of the control animals, but are readily detectable in the cerebellums after TBI. These findings have substantial translational implications in that they suggest a significant performance decrement in radiation exposed individuals after a radiation accident or in the nuclear/radiological terrorism situation, as well as the potential for long term neurological deficits in patients who have undergone radiation therapy of cancer.
Sublethal TBI-elicited histological and immunofluorescent changes in the cerebellum
Mechanistically, irradiation induces radiolysis of water and generates reactive radicals, which initiate oxidative damage of intracellular target molecules including RNA, DNA and membrane lipid [19
]. As shown in , total arbitrative intensities of MDA and 8-OHdG immunofluorescence increased sharply, indicating lipid peroxidation and RNA and/or DNA oxidation in acute phase (1 – 3.5 days) of TBI-induced cerebellar damage. While the method applied in the present study is different from the previous study in determining oxidative levels, the acute increases in MDA and 8-OHdG following irradiation appears much more dramatic as compared to chronic, aging-associated MDA and 8-OHdG changes (increase of 1 fold or less) in the cerebellum [31
]. It is plausible that aging process is “physiological” and/or compensative mechanisms at least partly inhibit aging-associated increases of the oxidative products, while TBI at sublethal dose of irradiation compromises the existing anti-oxidative mechanisms in the cerebellum. It is worthy of mentioning that 8-OHdG is primarily localized in the cellular nuclei, suggesting DNA oxidation [31
]. Similar findings are reported recently in irradiation-induced chronic neuronal damage: nuclear deposit of 8-OHdG in hippocampal granular cells [9
]. Interestingly, the present study demonstrates deposit of 8-OHdG as well as MDA in microvessel-like structures. The discrepancy may be attributable to the fact that MDA is anchored to the cellular membrane while 8- OHdG, degraded RNA and DNA sections, were permeable to the cellular membrane and were drained to the micro-vessels. Increase in MDA may increase cellular membrane permeability, facilitating damaged RNA and DNA leakage. The present study applied a protocol, which did not include the perfusion of animals and therefore 8-OHdG was retained in the serum of the microvessels. 8-OHdG is detectable in serum of animals and it increases after exposure to cadmium [32
] or to irradiation [8
], arguing for this assumption.
The neurovascular syndrome that develops after exposure to high doses of ionizing radiation is well known. The syndrome is attributable to occurrence of brain-blood-barrier (BBB) damage and inflammation. Using the VEGF-R2, endothelial DNA damage of the microvessels was identified by the co-immunostaining of the VEGF-R2 with the 8- OHdG after onset of the TBI (). VEGF-R2 is a transmembrane kinase receptor that regulates multiple vas-cular endothelial functions including proliferation, migration, survival and permeability [33
]. Our results concerning VEGF-R2 expression are consistent with others, demonstrating that VEGF-R2 expression is minimal in normal brain tissue [34
]. Up-regulation of VEGF-R2 has been shown in many pathologic conditions including cancer [35
] and stroke [34
] in which active angiogenesis and increased vascular permeability occur. The mechanisms of how radiation up-regulates VEGF-R2 remain unclear, nevertheless, radiation indeed results in increased VEGF-R2 expression in tumor [36
]. The present study indicates that oxidative stress/DNA damage may trigger VEGF-R2 up-regulation because VEGF-R2 is co-immunostained with 8-OHdG.
Inflammatory responses occur after exposure to ionizing irradiation, displaying increased microphage activation [37
]. p53 induction has been proposed as an in vivo marker for irradiation-induced genomic instability [38
]. Ionizing irradiation induced cell death in the developing central nervous system requires p53 [39
]. After TBI, p53 is activated in the cerebellum and p53 may induce apoptotic neuron death via the caspase-dependent or the caspase-independent pathway [40
]. The present study is consistent with the above findings that p53 induction occurs in the cerebellar granular layer after TBI. Noticeably, many cells in the granular layer expressed CD45 without co-immunostained with p53 1 day after TBI, indicating that microphages might respond to granular neuron damage/death. Because the Purkinje cell counts did not decrease 1 day but did decrease 3.5 days after the sublethal TBI, multiple granular cell deaths 1 day after TBI might accelerate degeneration of the Purkinje cells that had been damaged by exposure of the TBI. Interestingly, the p53 expression after TBI was also found in the microvessels, noticeably 3.5 days after TBI. The co-immunostaining of p53 with CD45 () indicates that the TBI may synchronize cell death of the hematopoietic nucleated cells. Nevertheless, because no p53 immunoreactivities were detected in the Purkinje cell layer, the significant Purkinje cell loss 3.5 days after TBI might not result from the p53 mediated pathogenic mechanism. On the other hand, the mechanisms by which the Purkinje cells die of the sublethal TBI warrant further investigations.
signaling is essential for neuronal function. Ca2+
signaling is thought to be mediated by cytosolic Ca2+
- binding proteins [41
]. CD-28 is one of the best characterized high-affinity Ca2+
-binding proteins [42
]. In general, the physiological function of CB-28 remains not fully clarified [43
], while it is thought to act either as an intracellular calcium buffer, or as a vehicle for calcium-intramembranous transport. CB-28 has also been indicated as an excellent neuroanatomic marker for neuronal subpopulations such as for one of subtypes of Purkinje cells [15
]. However, in randomly-selected intact cerebellar tissue slices, some of Purkinje cells (cell body) are CB-28 immunoreactivity-negative, while the majority of Purkinje cells, even along the same Purkinje cell layer, express CB-28. Our finding of the Purkinje cell CB-28 immunoreactivity in intact animals is consistent with those reported before [15
]. Noticeably, the present study demonstrates that CB-28 immunoreactivity can be applied to address pathophysiological status: early changes in irradiation-induced neuronal/Purkinje cell damage.
CB-28 immunoreactivity-based pathophysiological features can be classified in 2 categories: reduction in CB-28 expression and deformation of CB-28-delineated cell structures, evidently Purkinje cell bodies and Purkinje cell dendrite arbors. While application of other methods such as Western blot analysis can be used to confirm reduced CB-28 expression other than CB-28 redistribution following TBI, degradation/consumption and inhibition of synthesis of CB-28 may also explain the reduction of CB-28 expression. Reduction in CB-28 expression has been reported in other pathophysiological conditions including aging [44
] and epilepsy [45
]. The deformed Purkinje cells outlined by the CB-28 immunoreactivity may be partially derived as a result of CB-28 reduction. These deformations include reduced Purkinje cell body size, changed cell body shape, shortening in height of the Purkinje cell dendrite arbors, and reduction in size of the dendrite arbor trunk and number of the arbor branches. Although we observed a reduction in the length of Purkinje arbors, CB-28 reduction does not seem to lead distortion of the Purkinje cell dendrite arbors (see ). Thus CB-28 immunoreactivity provides a morphological parameter to define pathological Purkinje cell changes. Interestingly, these findings are consistent with the report in which rats subjected to postnatal X-ray irradiation had shown 3 weeks later reduced CB-28 immunoreactivity accompanied by thin, shortened, and disoriented Purkinje cell dendrite arbors [30
]. Similarly, a morphological study using Golgi-staining method demonstrated multiple primary dendrites, angulation of the primary dendrites, long segments of primary dendrites without branches and significantly reduced dendritic volume in postnatal rats subjected to in utero exposure to continuous irradiation [46
VGLUT2 mediates the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. VGLUT2 may also mediate the transport of inorganic phosphate. Major subcellular locations of VGLUT2 are cytoplasmic vesicle, secretory vesicle and synaptic vesicle membrane. Recent molecular biology studies have identified three subtypes of the vesicular glutamate transporter: VGluT1; VGluT2; and VGluT3 [47
]. VGLUT2 (NM_ 020346.1) is a sodium dependent inorganic phosphate co-transporter that is involved in the calcium dependent glutamate release from astrocytes [48
]. The present study demonstrates that TBI induces up-regulation of VGLUT2 along the Purkinje cell dendrite arbors (). The locations of VGLUT2 immunoreactivity indicate a pre-synaptic accumulation at the climbing fiber endings. It is likely that VGLUT2 was transferred to the locations relevant to local glutamate transport and calcium-dependent events. It is of interest whether or not VGLUT2 plays a crucial role in irradiation-induced neuronal damage. VGLUT2 may be involved in an excitotoxicity pathway: irradiation—oxidative stress
--glutamate release- Ca2+ influx into cells -CB-28 consumption
, and finally leads to neuron death. Future studies are warranted to determine the possibilities of such a pathway.
In conclusion sublethal TBI results in significant damage to the cerebellum of adult mice. Oxidative damage was detectable in Purkinje cells, cell bodies and their dendrite arbors, 1 day after radiation; however, neuronal death was not evident until 3.5 days, indicating partial delayed death of Purkinje neurons. Oxidative stress, inflammatory response and calcium neurotoxicity-associated mechanisms are involved in radiation-induced neuronal damage.
These findings have significant implications to radiation exposure of civilians or military personnel in nuclear/radio-logical accidents, warfare, or terrorism scenarios, as well as to the use of ionizing radiation as a cancer treatment modality.