Nitric oxide synthase mediates cerebellar dysfunction in mice exposed to repetitive blast-induced mild traumatic brain injury

Abstract

We investigated the role of nitric oxide synthase (NOS) in mediating blood-brain barrier (BBB) disruption and peripheral immune cell infiltration in the cerebellum following blast exposure. Repetitive, but not single blast exposure, induced delayed-onset BBB disruption (72 hours post-blast) in cerebellum. The NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) administered after blast blocked BBB disruption and prevented CD4⁺ T-cell infiltration into cerebellum. L-NAME also blocked blast-induced increases in intercellular adhesion molecule-1 (ICAM-1), a molecule that plays a critical role in regulating blood-to-brain immune cell trafficking. Blocking NOS-mediated BBB dysfunction during this acute/subacute post-blast interval (24-71 hours after the last blast) also prevented sensorimotor impairment on a rotarod task 30 days later, long after L-NAME cleared the body. In postmortem brains from Veterans/military Servicemembers with blast-related TBI, we found marked Purkinje cell dendritic arbor structural abnormalities, which were comparable to neuropathologic findings in the blast-exposed mice. Taken collectively, these results indicate that blast provokes delayed-onset of NOS-dependent pathogenic cascades that can later emerge as behavioral dysfunction. These results also further implicate the cerebellum as a brain region vulnerable to blast-induced mTBI.

Figure 1

Blast overpressures generated with a well-established pneumatic shock tube simulate a Friedlander waveform expected from high explosives. Red trace denotes the mean waveform of 24 blasts sampled throughout the experiments comprising this report. Black trace shows an estimated Friedlander waveform expected from detonation of approximately 21 kg TNT detonated at a distance of 8 m in the open field. Error bars denote SEM.

Figure 2

Repetitive blast exposure increases BBB permeability to radiolabeled albumin. A significant increase (**p ≤ 0.01) in brain/serum (uμl/g) ratios of ^99mTc-albumin was observed in whole brain at 72 h after repetitive mTBI (3X blast; n = 12) compared to 1X blast (n = 12), and sham control mice (n = 12). One-way ANOVA post-hoc Newman-Keuls. Values represent means ± SEM and are expressed as microliters per gram of brain tissue. Brain/serum ratios were calculated by dividing the cpm per brain by the cpm per microliter in the corresponding serum and then by the weight of the brain.

Figure 3

Nitric oxide synthase inhibition blocks blast-induced delayed-onset BBB disruption in cerebellum. (a) 3X blast significantly increased brain/serum ratios of ^99mTc-albumin in the cerebellum (*p ≤ 0.05), which was significantly attenuated by L-NAME administration (10 mg/kg; ip at 48, 54, and 71 h after the last blast) (*p ≤ 0.05). (b) No differences were observed in brain/serum ratios of ^99mTc-albumin in the forebrain after repetitive mTBI (p > 0.05). One-way ANOVA post-hoc Newman-Keuls. Values represent means ± SEM. Timeline portrays the mTBI exposure, L-NAME treatment paradigm, and measurement of BBB permeability.

Figure 4

Nitric oxide inhibition attenuates CD4⁺ T-cell infiltration in the cerebellum following repetitive blast. (a) Flow cytometry revealed that repetitive TBI significantly increased CD4⁺ counts in the cerebellum (**p ≤ 0.01), which was significantly attenuated by L-NAME administration (*p ≤ 0.05). (b) No differences were measured in cerebellar CD8⁺ counts after repetitive TBI (p > 0.05). (c) Scatter plots for cerebellum of shams, (d) blast vehicle-treated, and (e) blast L-NAME-treated mice. (f) No differences in CD4⁺ counts (p > 0.05), or (g) CD8⁺ counts were measured in forebrain after repetitive mTBI (p > 0.05). (h) Scatter plots for forebrain of shams, (i) blast + vehicle-treated, and (j) blast + L-NAME-treated mice. One-way ANOVA post-hoc Newman-Keuls. Values represent means ± SEM.

Figure 5

Repetitive blast significantly increased ICAM-1, but not VCAM-1 expression in cerebellum. (a) Western blot analysis revealed that repetitive mTBI significantly increased ICAM-1 expression in cerebellum at 72 h (**p ≤ 0.01), and that L-NAME significantly attenuated this effect (*p ≤ 0.05). (b) No significant differences were observed in VCAM-1 expression in cerebellum at 72 h after repetitive mTBI (p > 0.05). One-way ANOVA post-hoc Newman-Keuls. Values represent means ± SEM.

Figure 6

L-NAME improves sensorimotor impairment in mice exposed to repetitive blast. 3X blast and sham mice received L-NAME mice. In shams, L-NAME treatment had no significant effect on performance and were thus pooled (sham-vehicle n = 10, and sham L-NAME-treated n = 11). There was a significant overall difference (p ≤ 0.045) between shams, blast vehicle-treated (n = 14), and blast L-NAME-treated (n = 14) mice. Mice exposed to repetitive mTBI one month prior exhibited impaired sensorimotor performance on a rotarod task (p ≤ 0.05) compared to sham controls (pooled mice, mice administered L-NAME). Post-hoc analysis revealed blast L-NAME-treated mice performed significantly better than blast vehicle-treated mice (p ≤ 0.02). Two-way ANOVA Helmert’s test. Values represent means ± SEM.

Figure 7

Reduced EAAT4 expression on cerebellar Purkinje cells was observed in mice exposed to repetitive blast injury. (a) Shows representative images of secondary only controls (no primary antibodies) for Alexa 488, (b) Alexa Cy3, (c) merged, and (d) zoomed (40×). (e) Shows representative immunofluorescent images of EAAT4 (green), (f) calbindin (red), (g) merged, and (h) zoomed images in lobule IX of the cerebellum in control mice at one month after 3X sham and L-NAME administration. (i) Shows representative images of EAAT4, (j) calbindin, (k) merged, and (l) zoomed images at one month after repetitive mTBI. (m) Shows representative images of EAAT4, (n) calbindin, (o) merged, and (p) zoomed images at one month after repetitive mTBI with L-NAME. A significant decrease in (q) EAAT4 (*p ≤ 0.05), and (r) calbindin (*p ≤ 0.05) immunofluorescence was observed in lobule IX of the cerebellum of mice exposed to both repetitive mTBI plus vehicle (*p ≤ 0.05), and repetitive mTBI plus L-NAME (*p ≤ 0.05). One-way ANOVA post hoc Newman-Keuls. Values represent means ± SEM. Arrowheads highlight EAAT4+/calbindin+ Purkinje cell bodies. Scale bars = 30 µm, 20 µm (zoomed).

Figure 8

Dystrophic EAAT4 expression in cerebellar Purkinje cells in human with blast-related mTBI. Confocal microscopy shows representative immunofluorescent images of EAAT4 (green), and calbindin (red) in the cerebella of Veteran/military Servicemember controls with no blast TBI (a–c and d–f, Controls₁–₂, respectively described in Results), and in the cerebellum of Veteran/military Servicemembers with blast-related mTBI (g–i, j–l, m–o, and p–r, TBIs_1–4, respectively as described in Results) Scale bars = 50 µm.