Blast waves from detonated military explosive reduce GluR1 and synaptophysin levels in hippocampal slice cultures

Abstract

Explosives create shockwaves that cause blast-induced neurotrauma, one of the most common types of traumatic brain injury (TBI) linked to military service. Blast-induced TBIs are often associated with reduced cognitive and behavioral functions due to a variety of factors. To study the direct effects of military explosive blasts on brain tissue, we removed systemic factors by utilizing rat hippocampal slice cultures. The long-term slice cultures were briefly sealed air-tight in serum-free medium, lowered into a 37°C water-filled tank, and small 1.7-gram assemblies of cyclotrimethylene trinitramine (RDX) were detonated 15cm outside the tank, creating a distinct shockwave recorded at the culture plate position. Compared to control mock-treated groups of slices that received equal submerge time, 1-3 blast impacts caused a dose-dependent reduction in the AMPA receptor subunit GluR1. While only a small reduction was found in hippocampal slices exposed to a single RDX blast and harvested 1-2days later, slices that received two consecutive RDX blasts 4min apart exhibited a 26-40% reduction in GluR1, and the receptor subunit was further reduced by 64-72% after three consecutive blasts. Such loss correlated with increased levels of HDAC2, a histone deacetylase implicated in stress-induced reduction of glutamatergic transmission. No evidence of synaptic marker recovery was found at 72h post-blast. The presynaptic marker synaptophysin was found to have similar susceptibility as GluR1 to the multiple explosive detonations. In contrast to the synaptic protein reductions, actin levels were unchanged, spectrin breakdown was not detected, and Fluoro-Jade B staining found no indication of degenerating neurons in slices exposed to three RDX blasts, suggesting that small, sub-lethal explosives are capable of producing selective alterations to synaptic integrity. Together, these results indicate that blast waves from military explosive cause signs of synaptic compromise without producing severe neurodegeneration, perhaps explaining the cognitive and behavioral changes in those blast-induced TBI sufferers that have no detectable neuropathology.

Keywords: Blast-induced injury; GluR1; Military explosive; RDX; Shockwave; Synaptic decline; Synaptophysin; TBI; Traumatic brain injury.

Fig. 1

Hippocampal slice cultures were used to study the direct effects of blast waves from military explosive. Slices at culture day 4 are shown distributed on a 3-cm culture insert membrane (A). A hippocampal slice at culture day 28 displays features of the adult brain including intact hippocampal subfields visualized by H and E staining (B; view-field width: 3.5 mm). Slice cultures in a six-well plate were sealed in serum-free media then positioned within a warmed, water-filled tank (C). Three piezoelectric, high frequency pressure sensors are located directly above the clamped culture plate. A spherical assembly of RDX explosive (D) was detonated outside the water tank and the generated blast wave traveled through the wall of the tank, into the water medium, and was measured as a pressure history profile (E). DG, dentate gyrus.

Fig. 2

Military explosive blasts from 1.7-gram RDX assemblies cause a dose-dependent and time-dependent reduction in the AMPA receptor subunit GluR1. Hippocampal slice cultures exposed to 1–3 RDX blasts (1×–3×) were assessed together with mock submerge control slices (SC) by anti-GluR1 immunoblotting (A). Each blot was subsequently stained for actin for a protein load control, and positions for molecular weight standards are shown for 38–104 kDa.GluR1 immunoreactivities (means±SEM) are shown as percent to levels found in submerge control groups of slices (B) (ANOVA: p < 0.0001; compared to submerge control slices: ^#p = 0.011, ***p < 0.0001). Other slice cultures were subjected to RDX detonations or mock treatment, then harvested to assess GluR1 levels across post-blast times (C) (two-way ANOVA: p < 0.0001; comparison between control and RDX blast groups at each time point: *p < 0.05; **p < 0.01). The blast samples were also assessed for actin levels (D).

Fig. 3

RDX blast-induced loss of GluR1 immunoreactivity in brain tissue samples corresponds with increased levels of the HDAC2 protein. Hippocampal slice cultures were subjected to three consecutive detonations of the military explosive then harvested over time in order to assess GluR1 and HDAC2 in the same immunoblot lanes (top). The immunoreactivity levels of the synaptic marker and HDAC2 across individual slice samples were plotted again each other and linear regression resulted in a significant correlation (R² = 0.373; two-tailed, one-sample t-test on resulting slope: p = 0.004).

Fig. 4

Pre- and postsynaptic markers exhibit similar susceptibility to military RDX blasts. Hippocampal slice cultures were subjected to 3 consecutive RDX detonations then harvested over time in order to assess synaptophysin and actin levels by immunoblot (A). Slices from single (1×) and 3 consecutive RDX blasts (3×) were assessed for levels of the two proteins, as well as for GluR1 for comparison, which were normalized within-blot to respective measures from submerge control slices (SC) and the data sets shown (B) (means ± SEM; unpaired t tests compared to controls: **p < 0.01, ***p < 0.0001). At 24 h post-blast, slice cultures treated with 2 RDX blasts and control slices were infused with 100 μM NMDA for 5 min and harvested in buffer containing phosphatase inhibitors. The identical samples on two nitrocellulose strips were incubated with or without alkaline phosphatase (AP) for 2 h at 37 °C before phospho-cofilin immunostaining (C). Each blot was subsequently stained for actin. Another set of double-blast slices were subjected to the NMDA treatment to assess pCofilin and pERK2 in the same slice samples (D).

Fig. 5

Military explosive detonations of 1.7-gram RDX assemblies produced synaptic marker decline but no evidence of cellular damage. Hippocampal slice cultures from a control mock treatment group and a triple-blast group were fixed 45 h after the detonation events and assessed for anti-synaptophysin immunostaining and DAPI counterstaining (A), and images from the same CA1 area are shown (view-field width: 180 μm). The same slices were also subjected to Fluoro-Jade B staining and a larger area imaged due to no cellular staining evident (A; view-field width: 400 μm). A positive control for the Fluoro-Jade stain consisted of slice cultures treated with 100 μM AMPA for 15 min, the excitotoxic insult then quenched with washes containing CNQX and MK-801, and the tissue fixed 48 h post-insult for CA1 imaging (B). In the same area of a slice subjected to a single RDX blast, Fluoro-Jade B did not stain any degenerating pyramidal neurons (view-field width: 450 μm). sp, stratum pyramidale; sr, stratum radiatum. Spectrin breakdown product (BDP) was assessed by immunoblot in a positive control sample of calpain-mediated BDP (pos) and in hippocampal slices from the submerge control group (SC), a triple-blast group (3×), untreated control group (con), and AMPA-insult group (D). The same blots were assessed for GluR1 immunostaining.

Fig. 6

The blast-induced synaptic decline profile was compared to synaptic decline profiles related to excitotoxicity (small gray zone) and protein accumulation stress (large gray zone) summarized from previous hippocampal slice studies. Synaptic marker results normalized to control slices were compiled from reports on excitotoxic over-activation of glutamate receptors (Bahr et al., 2002; Karanian et al., 2005; Naidoo et al, 2012) and protein accumulation stress (Bendiske and Bahr, 2003; Butler et al., 2007; Wisniewski et al., 2011) to generate the most common “zone” for the two types of synaptic decline, the disparate insults being initiated at the typical time of culture day 25. Control slices from the current study as well as from several previous studies together showed that synaptic marker levels were stable across the period of 10–40 days in culture (hatched area). Triple RDX blast-induced synaptic marker loss is shown with the solid black line.