Cavitation-induced traumatic cerebral contusion and intracerebral hemorrhage in the rat brain by using an off-the-shelf clinical shockwave device

Abstract

Traumatic cerebral contusion and intracerebral hemorrhages (ICH) commonly result from traumatic brain injury and are associated with high morbidity and mortality rates. Current animal models require craniotomy and provide less control over injury severity. This study proposes a highly reproducible and controllable traumatic contusion and ICH model using non-invasive extracorporeal shockwaves (ESWs). Rat heads were exposed to ESWs generated by an off-the-shelf clinical device plus intravenous injection of microbubbles to enhance the cavitation effect for non-invasive induction of injury. Results indicate that injury severity can be effectively adjusted by using different ESW parameters. Moreover, the location or depth of injury can be purposefully determined by changing the focus of the concave ESW probe. Traumatic contusion and ICH were confirmed by H&E staining. Interestingly, the numbers of TUNEL-positive cells (apoptotic cell death) peaked one day after ESW exposure, while Iba1-positive cells (reactive microglia) and GFAP-positive cells (astrogliosis) respectively peaked seven and fourteen days after exposure. Cytokine assay showed significantly increased expressions of IL-1β, IL-6, and TNF-α. The extent of brain edema was characterized with magnetic resonance imaging. Conclusively, the proposed non-invasive and highly reproducible preclinical model effectively simulates the mechanism of closed head injury and provides focused traumatic contusion and ICH.

Figure 1

ESW application setup. (A) Device setup. The ESW probe was fixed on a turned U-shaped positioning platform (white arrow) and positioned over the shaved rat head fixed on a sterotaxic frame. The rat was anesthetized with 2% Forane delivered via a hose (black arrow). (B) The red spot labeled on the scalp indicates the target of the concave ESW probe. (C) A close-up photo showing the bud (arrow; i.e., probe center) on the bottom of the gel pad positioned over the labeled spot (i.e., target). (D) Depiction of the exact position of the ESW focal point in the rat brain, i.e., 5 mm below the gel bud.

Figure 2

Gross observation of rat brains following ESW exposure. The blue and dark brown signals indicate EB dye leaked from the disrupted BBB and hemorrhaged on the brain surface. These gross findings are identical to the surgical findings in patients with traumatic contusion and ICH.

Figure 3

Histopathological changes in rat brains two weeks after ESW-induced focal injury. (A) H&E-stained sections of brain tissues, chosen from six candidates at each time point. Upper panel, mildly injured brain sections; lower panel, severely injured brain sections; arrows, areas enlarged and shown in (B). (B) Enlarged views (scale bar = 100 µm). Arrowheads, intact small vessels in the injured tissues.

Figure 4

TUNEL assay of apoptotic cell death in brain tissues after ESW exposure. (A) TUNEL-stained sections of brain tissues, chosen from six candidates at each time point. Arrows, the areas with obvious TUNEL signals. (B) Enlarged detailed section views (scale bar = 100 µm).

Figure 5

Iba1 immunostaining of microglial changes in brain tissue after ESW exposure. (A) Iba1-stained sections of brain tissues, chosen from six candidates at each time point. Arrows, the areas with obvious Iba1 signals. (B) Enlarged detailed section views (scale bar = 100 µm).

Figure 6

GFAP immunostaining of astrogliosis in brain tissue after ESW exposure. (A) GFAP-stained sections of brain tissues, chosen from six candidates at each time point. (B) Enlarged detailed section views (scale bar = 100 µm). The typical cells were further enlarged in the black squares (scale bar = 40 µm).

Figure 7

Time course of TUNEL-, Iba1-, and GFAP-positive cells in ESW-induced focal injury. (A) TUNEL-positive apoptotic cells. (B) Iba1-postive microglial cells. (C) GFAP-positive astrocytes. Six rats were sampled at each time point. Severe injury group (solid circle) vs. mild injury group (open circle), *p < 0.05, **p < 0.01.

Figure 8

Expression of inflammatory cytokines after ESW exposure at severe condition. (A) IL-1β. (B) IL-6. (C) TNF-α. Six rats were sampled on Day 3 for each condition. ESW group (solid bar) vs. control group (open bar), *p < 0.05, **p < 0.01.

Figure 9

T2WI showing the different patterns of brain edema, ICH, and enlarged lateral ventricle in the mildly and severely injured brains. Images were acquired at the level of Bregma 0.5 mm (upper) or −3.5 mm (lower). White arrow, hyperintensity related to brain edema; white arrowhead, hypointensity related to ICH; red arrow, enlarged lateral ventricle; red arrowhead, hyperintensity related to the further enlargement of lateral ventricle.

Figure 10

The biophysics behind our model. (A) Generation and collapse of the microbubbles under acoustic pressure. P+, positive pressure; P−, negative pressure. (B) Microject (a mechanical stress) generation and surrounding tissue damage.