Chemical blocking of zinc ions in CNS increases neuronal damage following traumatic brain injury (TBI) in mice

Abstract

Background: Traumatic brain injury (TBI) is one of the leading causes of disability and death among young people. Although much is already known about secondary brain damage the full range of brain tissue responses to TBI remains to be elucidated. A population of neurons located in cerebral areas associated with higher cognitive functions harbours a vesicular zinc pool co-localized with glutamate. This zinc enriched pool of synaptic vesicles has been hypothesized to take part in the injurious signalling cascade that follows pathological conditions such as seizures, ischemia and traumatic brain injury. Pathological release of excess zinc ions from pre-synaptic vesicles has been suggested to mediate cell damage/death to postsynaptic neurons.

Methodology/principal findings: In order to substantiate the influence of vesicular zinc ions on TBI, we designed a study in which damage and zinc movements were analysed in several different ways. Twenty-four hours after TBI ZnT3-KO mice (mice without vesicular zinc) were compared to littermate Wild Type (WT) mice (mice with vesicular zinc) with regard to histopathology. Furthermore, in order to evaluate a possible neuro-protective dimension of chemical blocking of vesicular zinc, we treated lesioned mice with either DEDTC or selenite. Our study revealed that chemical blocking of vesicular zinc ions, either by chelation with DEDTC or accumulation in zinc-selenium nanocrystals, worsened the effects on the aftermath of TBI in the WT mice by increasing the number of necrotic and apoptotic cells within the first 24 hours after TBI, when compared to those of chemically untreated WT mice.

Conclusion/significance: ZnT3-KO mice revealed more damage after TBI compared to WT controls. Following treatment with DEDTC or selenium an increase in the number of both dead and apoptotic cells were seen in the controls within the first 24 hours after TBI while the degree of damage in the ZnT3-KO mice remained largely unchanged. Further analyses revealed that the damage development in the two mouse strains was almost identical after either zinc chelation or zinc complexion therapy.

Figure 1. Tracing of zinc using the ZnSe^AMG method.

(A) Cryo-section: Wild Type mouse 24 hours after TBI. Asterisks denote lesion tract; a distinct layering of the neocortex is noticeable. Closest to the lesion tract no AMG development is possible because all cells are severely damaged. This is shown as a little tint of white on both sides of the lesion tract. Scale bar: 1 mm. (B) Cryo-section: ZnT3-KO mouse 24 hours after TBI. Asterisks denote lesion. In the periphery of the lesion a number of ZnSe nanocrystals containing neurons are just visible. These somata marked neurons border the area between morphologically damaged tissue and morphologically intact tissue. (C) Semi-thin section: Wild Type mouse, a close-up of what is seen in A. There is a distinct neuropil stain; the tissue is oedematous with bleeding. The neurons are distorted, with vacuolation of their cytoplasm and some with eccentrically placed, pycnotic nuclei; none of the neuronal cell-bodies contain any ZnSe nanocrystals. Scale bar: 30 µm. (D_1–2) Semi-thin sections: ZnT3-KO mouse. In the periphery of the lesion some neurons containing ZnSe nanocrystals in their somata can be seen. Going towards the lesion tract most cells are heavily distorted with condensed, eccentrically placed nuclei. The tissue is severely damaged with massive oedema. Scale bar: 30 µm. Tracing of dead and dying neurons with Fluorojade B (FJB). (E) Cryo-section: Wild Type mouse stained with FJB; fluorescent neurons border the lesion tract. Scale bar: 300 µm. (F) Cryo-section: ZnT3-KO mouse stained with FJB; numerous neurons line the lesion tract. Scale bar: 300 µm.

Figure 2. Evaluation of apoptosis and reactive gliosis.

The rows illustrate TUNEL stains in (A-F), GFAP stains in (G-L) and Caspase 3 stains in (M-R). All sections are paraffin sections 24 hours after TBI: (A) Wild Type mouse, not chelator treated. (B) Wild Type mouse treated with DEDTC 1 hour before TBI. An increased number of apoptotic neurons are seen. (C) Wild Type mouse treated with selenite 1 hour before TBI. Even more apoptotic neurons are lining the lesion tract. (D) ZnT3-KO mouse not chelator treated. Numerous apoptotic neurons are seen. (E) ZnT3-KO treated with DEDTC 1 hour before TBI. Still a number of apoptotic neurons are seen. (F) ZnT3-KO mouse treated with selenite prior to TBI. No major changes compared to the other ZnT3-KO sections are observed. (G) Wild-type mouse not chelator treated; some reactive cells are seen in the area. (H) Wild Type mouse treated with DEDTC 1 hour before TBI. No major changes are observed. (I) Wild Type mouse treated with selenite 1 hour before TBI. No major changes are observed. (J) ZnT3-KO mouse not chelator treated; some activated glia are seen. (K) ZnT3-KO mouse DEDTC treated 1 hour before TBI. The gliosis remains unchanged. (L) ZnT3-KO mouse selenite treated 1 hour before TBI. A massive increase in the reactive gliosis is seen with big amoeboid cells and increased staining intensity. (M) Wild Type mouse. Sparse Caspase 3 positive cells are seen near the lesion tract. (N) Wild Type mouse treated with DEDTC before TBI. More caspase 3 positive neurons are seen in the proximity of the lesion tract. (O) Wild Type mouse treated with selenite before TBI; a clear increase in the number of caspase 3 positive neurons is seen near the lesion. (P) ZnT3-KO mouse. Sparse Caspase 3 positive cells are seen near the lesion tract. (Q) ZnT3-KO mouse, treated with DEDTC before TBI. No significant increase in the number of Caspase 3 positive cells is observed. (R) ZnT3-KO mouse treated with selenite before TBI. A major increase in the number of positive cells is observed in the entire lesioned area. Scale bar: 300 µm.

Figure 3. Control sections.

Sections are toluidine stained in (A-H). Both the WT mice and The ZnT3-KO mice display a normal morphology 24 hours after respectively DEDTC and selenite treatment. The tissue is without oedema and the cells are all intact with normal configuration. A, B, E, F are Cryo-sections 30 µm thick. C, D, G, H are semi-thin sections 3 µm thick. No major differences between the ZnT3-KO and the WT mice are noticeable. Scale bar A,B: 100 µm. Scale bar E: 200 µm. Scale bar F: 300 µm. Scale bar C, D, G, H: 30 µm. Control sections, FJB stained (I-L). Sections I, J are pretreated with DEDTC and sections K, L are pretreated with selenite. All sections are FJB negative. Control sections, TUNEL stained (M-P). Sections M, N are pretreated with DEDTC and sections O, P are pretreated with selenite. All sections are TUNEL negative. M, P depict part of the hippocampus formation and N, O depict all the six neocortical layers. Scale bar I-P: 200 µm.