Pressure-dependent effect of shock waves on rat brain: induction of neuronal apoptosis mediated by a caspase-dependent pathway

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Shock waves have been experimentally applied to various neurosurgical treatments including fragmentation of cerebral emboli, perforation of cyst walls or tissue, and delivery of drugs into cells. Nevertheless, the application of shock waves to clinical neurosurgery remains challenging because the threshold for shock wave–induced brain injury has not been determined. The authors investigated the pressure-dependent effect of shock waves on histological changes of rat brain, focusing especially on apoptosis.

Methods

Adult male rats were exposed to a single shot of shock waves (produced by silver azide explosion) at over-pressures of 1 or 10 MPa after craniotomy. Histological changes were evaluated sequentially by H & E staining and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL). The expression of active caspase-3 and the effect of the nonselective caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) were examined to evaluate the contribution of a caspase-dependent pathway to shock wave–induced brain injury.

High-overpressure (> 10 MPa) shock wave exposure resulted in contusional hemorrhage associated with a significant increase in TUNEL-positive neurons exhibiting chromatin condensation, nuclear segmentation, and apoptotic bodies. The maximum increase was seen at 24 hours after shock wave application. Low-overpressure (1 MPa) shock wave exposure resulted in spindle-shaped changes in neurons and elongation of nuclei without marked neuronal injury. The administration of Z-VAD-FMK significantly reduced the number of TUNEL-positive cells observed 24 hours after high-overpressure shock wave exposure (p < 0.01). A significant increase in the cytosolic expression of active caspase-3 was evident 24 hours after high-overpressure shock wave application; this increase was prevented by Z-VAD-FMK administration. Double immunofluorescence staining showed that TUNEL-positive cells were exclusively neurons.

Conclusions

The threshold for shock wave–induced brain injury is speculated to be under 1 MPa, a level that is lower than the threshold for other organs. High-overpressure shock wave exposure results in brain injury, including neuronal apoptosis mediated by a caspase-dependent pathway. This is the first report in which the pressure-dependent effect of shock wave on the histological characteristics of brain tissue is demonstrated.

Abbreviations used in this paper:CNPase = 2,3-cyclic nucleotide 3-phosphohydrolase; DMSO = dimethyl sulfoxide; GFAP = glial fibrillary acidic protein; Nd:YAG = neodymium:yttrium-aluminum-garnet; NeuN = anti–neuron-specific nuclear protein; PBS = phosphate-buffered saline; TdT = terminal deoxynucleotidyl transferase; TUNEL = terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; Z-VAD-FMK = N-benzyl-oxycarbonyl-Val-Ala-Asp-fluoromethylketone.

Article Information

Address reprint requests to: Miki Fujimura, M.D., Ph.D., Department of Neurosurgery, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. email: fujimur@nsg.med.tohoku.ac.jp.

© AANS, except where prohibited by US copyright law.

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Figures

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    Upper: Schematic diagram of shock wave generator using silver azide (AgN3) microexplosive. Lower: Schematic diagram of experimental setup for evaluation of shock wave–induced brain damage. PVDF = polyvinylidene difluoride; YAG = yttrium aluminum garnet.

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    Photomicrographs showing sections obtained from animals in Group 2 (high-overpressure group) 4 (A and D), 24 (B and E), and 72 (C and F) hours after application of high-overpressure shock waves. Intracerebral hemorrhage was observed in both cortical and subcortical regions from 4 hours after shock wave exposure (A and D). Contusional brain injury was evident around the hemorrhage as early as 24 hours after shock wave exposure (B and E), and was further increased at 72 hours (C and F). The arrows indicate the direction of shock wave application. H & E. Original magnifications × 100 (A–C) and × 400 (D–F).

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    Photomicrographs of sections obtained from animals in Group 3 (low-overpressure group) 4 (A and D), 24 (B and E), and 72 (C and F) hours after shock wave application. Although no hemorrhage was seen, mild morphological changes such as spindle-shaped changes of neurons and elongation of nuclei in the direction of shock wave exposure were detected about 24 hours after application of shock waves. The arrows indicate the direction of shock wave application. H & E. Original magnifications × 100 (A–C) and × 400 (D–F).

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    Photomicrographs of sections obtained 24 hours after high-overpressure shock wave application (Group 2). The arrows indicate the direction of shock wave application. Two different patterns of staining may be observed. Significant quantitities of cells around the contusional lesion created by high-overpressure shock waves show strong TUNEL positivity in their nuclei with small particles around the nuclei that resemble apoptotic bodies. Other cells demonstrate slight TUNEL positivity with diffuse nuclear and cytoplasmic staining, a pattern consistent with necrosis. H & E (A and C) and TUNEL staining (B and D). Original magnifications × 100 (A and B) and × 400 (C and D).

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    Photomicrographs demonstrating results of TUNEL assay performed on sections from animals exposed to high-overpressure (A–C) and low-overpressure (D–F) shock waves at 4 (A and D), 24 (B and E), and 72 hours (C and F) after exposure. At 24 and 72 hours, TUNEL-positive cells were observed dispersed throughout the area around the contusional lesion created by high-overpressure shock wave application (B and C). In contrast, there was no significant increase in the number of TUNEL-positive cells in the low-overpressure group (D–F). Original magnification × 400.

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    Bar graph illustrating the relationship between time after shock wave application and the number of DNA-fragmented cells (expressed as cells/mm2). Light bars = high-overpressure group; dark bars = low-overpressure group. *p < 0.01; **p < 0.05.

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    Photomicrographs showing results of immunofluorescence analysis at 24 hours after high-overpressure shock wave application. Single immunofluorescence after performing TUNEL alone (A, D, and G) and after staining for NeuN (B), GFAP (E), and CNPase (H). Double immunofluorescence (merged images) show NeuN and TUNEL positivity (C), GFAP and TUNEL positivity (F), and CNPase and TUNEL positivity (I). The TUNEL-positive cells were double-labeled with staining for NeuN, but not with staining for GFAP, indicating the predominant involvement of neuronal apoptosis after high-overpressure shock wave exposure. Original magnification × 400.

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    Bar graph showing the relationship between intraventricular administration of Z-VAD-FMK and numbers of TUNEL-positive cells after application of high-overpressure shock waves. Group A animals received vehicle only, Group B animals received a single low dose of Z-VAD-FMK, Group C animals received a single high dose of Z-VAD-FMK, and Group D animals received high doses of Z-VAD-FMK repeatedly.

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    Photomicrographs showing the results of TUNEL in sections from animals exposed to high-overpressure shock waves and treated with vehicle (A and C) or repeated intraventricular administration of high doses of Z-VAD-FMK (B and D). Original magnifications × 100 (A and B) and × 400 (C and D).

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    Photomicrographs demonstrating results of immunohistochemical assay for active caspase-3 in sections from animals exposed to high-overpressure shock waves and treated with vehicle (A and B) or the repeated administration of a high dose of Z-VAD-FMK (C and D). Cytosolic expression of active caspase-3 was evident in the ipsilateral hemisphere after shock wave application (A) and was partially prevented by Z-VAD-FMK administration (C), whereas it was barely detectable in the contralateral cortex (B and D). Original magnification × 200.

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