Characterization of Blood-Brain Barrier Opening Induced by Transcranial Histotripsy in Murine Brains.

OBJECTIVE: Transcranial histotripsy has shown promise as a non-invasive neurosurgical tool, as it has the ability to treat a wide range of locations in the brain without overheating the skull. One important effect of histotripsy in the brain is the blood-brain barrier (BBB) opening (BBBO) at the ablation site, but there is a knowledge gap concerning the extent of histotripsy-induced BBBO. Here we describe induction of BBBO by transcranial histotripsy and use of magnetic resonance imaging (MRI) and histology to quantify changes in BBBO at the periphery of the histotripsy ablation zone over time in the healthy mouse brain. METHODS: An eight-element, 1 MHz histotripsy transducer with a focal distance of 32.5 mm was used to treat the brains of 23 healthy female BL6 mice. T1-gadolinium (T1-Gd) MR images were acquired immediately following histotripsy treatment and during each of the subsequent 4 wk to quantify the size and intensity of BBB leakage. RESULTS: The T1-Gd MRI results revealed that the hyperintense BBBO volume increased over the first week and subsided gradually over the following 3 wk. Histology revealed complete loss of tight junction proteins and blood vessels in the center of the ablation region immediately after histotripsy, partial recovery in the periphery of the ablation zone 1 wk following histotripsy and near-complete recovery of tight junction complex after 4 wk. CONCLUSION: These results provide the first evidence of transcranial histotripsy-induced BBBO and repair at the periphery of the ablation zone.

Transcranial histotripsy parameter study in primary and metastatic murine brain tumor models.

OBJECTIVE: This study investigated the effect of various histotripsy dosages on tumor cell kill and associated bleeding in two murine brain tumor models (glioma [Gl261] and lung metastasis [LL/2-Luc2]). METHODS AND MATERIALS: GL261 or LL/2-Luc2 cells were cultured and implanted into the brains of C57BL/6 mice. Histotripsy (1-cycle pulses, 5 Hz PRF, 30 MPa-P) was performed using a 1 MHz transducer for five different dosages for each cell line: 5, 20 or 200 pulses per location (PPL) at a single treatment point, or 5 or 10-20 PPL at multiple treatment points. MRI, bioluminescence imaging and histology were used to assess tumor ablation and treatment effects within 4-6 h post-treatment. RESULTS: All treatment groups resulted in a reduction of BLI intensity for the LL/2-Luc2 tumors, with significant signal reductions for the multi-point groups. The average pre-/post-treatment BLI flux (photons/s, ×108) for the different treatment groups were: 4.39/2.19 (5 PPL single-point), 5.49/1.80 (20 PPL single-point), 3.86/1.73 (200 PPL single-point), 2.44/1.11 (5 PPL multi-point) and 5.85/0.80 (10 PPL multi-point). MRI and H&E staining showed increased tumor damage and hemorrhagic effects with increasing histotripsy dose for both GL261 and LL/2-Luc2 tumors, but the increase in tumor damage was diminished beyond 10-20 PPL for single-point treatments and outweighed by increased hemorrhage. In general, hemorrhage was confined to be within 1 mm of the treatment boundary for all groups. CONCLUSIONS: Our results suggest that a lower number of histotripsy pulses at fewer focal locations can achieve substantial tumor kill while minimizing hemorrhage.

Histotripsy Treatment of Murine Brain and Glioma: Temporal Profile of Magnetic Resonance Imaging and Histological Characteristics Post-treatment.

OBJECTIVE: Currently, there is a knowledge gap in our understanding of the magnetic resonance imaging (MRI) characteristics of brain tumors treated with histotripsy to evaluate treatment response as well as treatment-related injuries. Our aim was to bridge this gap by investigating and correlating MRI with histological analysis after histotripsy treatment of mouse brain with and without brain tumors and evaluating the evolution of the histotripsy ablation zone on MRI over time. METHODS: An eight-element, 1 MHz histotripsy transducer with a focal distance of 32.5 mm was used to treat orthotopic glioma-bearing mice and normal mice. The tumor burden at the time of treatment was ∼5 mm3. T2, T2*, T1 and T1-gadolinium (Gd) MR images and histology of the brain were acquired on days 0, 2 and 7 for tumor-bearing mice and days 0, 2, 7, 14, 21 and 28 post-histotripsy for normal mice. RESULTS: T2 and T2* sequences most accurately correlated with histotripsy treatment zone. The treatment-induced blood products, T1 along with T2, revealed blood product evolution from oxygenated, de-oxygenated blood and methemoglobin to hemosiderin. And T1-Gd revealed the state of the blood-brain barrier arising from the tumor or histotripsy ablation. Histotripsy leads to minor localized bleeding, which resolves within the first 7 d as evident on hematoxylin and eosin staining. By day 14, the ablation zone could be distinguished only by the macrophage-laden hemosiderin, which resides around the ablation zone, rendering the treated zone hypo-intense on all MR sequences. CONCLUSION: These results provide a library of radiological features on MRI sequences correlated to histology, thus allowing for non-invasive evaluation of histotripsy treatment effects in in vivo experiments.

Stereotactic Transcranial Focused Ultrasound Targeting System for Murine Brain Models.

An inexpensive, accurate focused ultrasound stereotactic targeting method guided by pretreatment magnetic resonance imaging (MRI) images for murine brain models is presented. An uncertainty of each sub-component of the stereotactic system was analyzed. The entire system was calibrated using clot phantoms. The targeting accuracy of the system was demonstrated with an in vivo mouse glioblastoma (GBM) model. The accuracy was quantified by the absolute distance difference between the prescribed and ablated points visible on the pre treatment and posttreatment MR images, respectively. A precalibration phantom study ( N = 6 ) resulted in an error of 0.32 ± 0.31, 0.72 ± 0.16, and 1.06 ± 0.38 mm in axial, lateral, and elevational axes, respectively. A postcalibration phantom study ( N = 8 ) demonstrated a residual error of 0.09 ± 0.01, 0.15 ± 0.09, and 0.47 ± 0.18 mm in axial, lateral, and elevational axes, respectively. The calibrated system showed significantly reduced ( ) error of 0.20 ± 0.21, 0.34 ± 0.24, and 0.28 ± 0.21 mm in axial, lateral, and elevational axes, respectively, in the in vivo GBM tumor-bearing mice ( N = 10 ).

Level-wise differences in in vivo lateral bending moment are associated with microstructural alterations in bovine caudal intervertebral discs.

Despite its common use as a laboratory model, little is known about the in vivo forces and moments applied to the bovine caudal intervertebral disc. Such aspects are crucial, as intervertebral disc tissue is known to remodel in response to repeated loading. We hypothesized that the magnitude of loading from muscle contraction during a typical lateral bending motion varies between caudal levels and is accompanied by variations in tissue microstructure. This hypothesis was tested by estimating level-wise forces and bending moments using two independent approaches: a dynamic analytical model of the motion and analysis of muscle cross-sections obtained via computed tomography. Microstructure was assessed by measuring the collagen fiber crimp period in the annulus fibrosus, and composition was assessed via quantitative histology. Both the analytical model and muscle cross-sections indicated peak bending moments of over 3 N m and peak compressive force of over 125 N at the c1c2 level, decreasing distally. There was a significant downward trend from proximal to distal in the outer annulus fibrosus collagen crimp period in the anterior and posterior regions only, suggesting remodeling in response to the highest lateral bending moments. There were no observed trends in composition. Our results suggest that although the proximal discs in the bovine tail are subjected to forces and moments from muscle contraction that are comparable (relative to disc size) to those acting on human lumbar discs, the distal discs are not. The resulting pattern of microstructural alterations suggests that level-wise differences should be considered when using bovine discs as a research model.

Mapping of Intervertebral Disk Annulus Fibrosus Compressive Properties Is Sensitive to Specimen Boundary Conditions.

Predicting the mechanical behavior of the intervertebral disk (IVD) in health and in disease requires accurate spatial mapping of its compressive mechanical properties. Previous studies confirmed that residual strains in the annulus fibrosus (AF) of the IVD, which result from nonuniform extracellular matrix deposition in response to in vivo loads, vary by anatomical regions (anterior, posterior, and lateral) and zones (inner, middle, and outer). We hypothesized that as the AF is composed of a nonlinear, anisotropic, viscoelastic material, the state of residual strain in the transverse plane would influence the apparent values of axial compressive properties. To test this hypothesis, axial creep indentation tests were performed, using a 1.6 mm spherical probe, at nine different anatomical locations on bovine caudal AFs in both the intact (residual strain present) and strain relieved states. The results showed a shift toward increased spatial homogeneity in all measured parameters, particularly instantaneous strain. This shift was not observed in control AFs, which were tested twice in the intact state. Our results confirm that time-dependent axial compressive properties of the AF are sensitive to the state of residual strain in the transverse plane, to a degree that is likely to affect whole disk behavior.

Residual strains in the intervertebral disc annulus fibrosus suggest complex tissue remodeling in response to in-vivo loading.

The annulus fibrosus (AF) of the intervertebral disc (IVD) serves the dual roles of containing hydrostatic pressure from the inner nucleus pulposus (NP) and allowing flexible connection between adjacent vertebral bodies. Previous work has indicated that in the unloaded state, the AF is under a state of residual circumferential strain that, on average, is comparable to that which is believed to reduce peak stresses in other pressure containing organs. The complex in-vivo loading of the IVD, however, led us to hypothesize that variations with anatomical region should also exist. Residual strains were measured by imaging bovine caudal IVDs at both macro and micro scales in both the intact state (under residual strain) and opened into anterior, posterior, and lateral quadrants (residual strains relieved). Calculation of macro scale residual strains using changes in lamellar arc length and thickness confirmed circumferential tension (anterior: 0.63±2.1%, lateral: 8.3±1.5%, posterior: 4.4±2.1%) and radial compression (anterior: 12.4±5.8%, lateral: 11.120±2.8%, posterior: 4.8±4.2%) around the outer zone of the AF. The inner zone, however, had residual circumferential strains ranging from 28.7±3.4% compression in the anterior region to 3.4±3% tension in the posterior region, with radial strains of 9.7±5.5% tension and 0.2±4.4% compression respectively. This pattern of residual circumferential strain was corroborated at the microscale by comparing the crimp period of collagen fiber bundles in the intact and open states. The results of this study point toward a complex pattern of residual strains in the AF, which develop in response to stresses from both NP pressurization and bending movements.