Establishing sheep as an experimental species to validate ultrasound-mediated blood-brain barrier opening for potential therapeutic interventions

Abstract

Rationale: Treating diseases of the brain such as Alzheimer's disease (AD) is challenging as the blood-brain barrier (BBB) effectively restricts access of a large number of potentially useful drugs. A potential solution to this problem is presented by therapeutic ultrasound, a novel treatment modality that can achieve transient BBB opening in species including rodents, facilitated by biologically inert microbubbles that are routinely used in a clinical setting for contrast enhancement. However, in translating rodent studies to the human brain, the presence of a thick cancellous skull that both absorbs and distorts ultrasound presents a challenge. A larger animal model that is more similar to humans is therefore required in order to establish a suitable protocol and to test devices. Here we investigated whether sheep provide such a model.Methods: In a stepwise manner, we used a total of 12 sheep to establish a sonication protocol using a spherically focused transducer. This was assisted by ex vivo simulations based on CT scans to establish suitable sonication parameters. BBB opening was assessed by Evans blue staining and a range of histological tests.Results: Here we demonstrate noninvasive microbubble-mediated BBB opening through the intact sheep skull. Our non-recovery protocol allowed for BBB opening at the base of the brain, and in areas relevant for AD, including the cortex and hippocampus. Linear time-shift invariant analysis and finite element analysis simulations were used to optimize the position of the transducer and to predict the acoustic pressure and location of the focus.Conclusion: Our study establishes sheep as a novel animal model for ultrasound-mediated BBB opening and highlights opportunities and challenges in using this model. Moreover, as sheep develop an AD-like pathology with aging, they represent a large animal model that could potentially complement the use of non-human primates.

Understanding ultrasound-mediated blood-brain barrier opening as a potential non-invasive therapeutic approach for Alzheimer’s disease

“…The exclusion of 2,000 kDa (hydrodynamic diameter 54.4nm) dextran suggests a size threshold for the molecules delivered across the BBB with the sonication parameters used in this study, which is in line with a previous report [261]. This threshold suggests that red blood cells (6-8 µm diameter) would be excluded from the brain parenchyma under these conditions, and as such any extravasation of blood cells could be used as a hematological marker for damage [172]. In addition, the result also indicates that larger therapeutic agents such as recombinant adeno-associated viruses for gene delivery (~4,000 kDa) would not be delivered at these parameters and that the size of the molecules to be delivered would dictate the ultrasound parameters.…”

“…A multimodal analysis of long-term functional cognitive read-outs, neuronal morphology and memory in aged mice reported similar positive outcomes [171]. Importantly, these studies have also been extended to larger animals, such as sheep, macaques and aged beagles demonstrating opening of the BBB without histological evidence of microhemorrhages or cell damage [172][173][174][175]. In addition, there were no deficits observed on various behavioral tasks in these studies, suggesting that ultrasound-mediated BBB opening does not lead to functional impairment.…”

The toxic effect of mobile phone radiation on rabbit organs

“…It is conceivable that the absorption of radiation would be affected by the thickness of the skulls and therefore larger animals such as rabbits would be more appropriate to examine the potential damage of the radiation to humans. In fact, large animals have been recently used to test potential effects of ultrasound on humans (Pelekanos et al 2018). Therefore, we believe that our results using New Zealand rabbits as a model to study the effect of mobile phone radiation on brain tissues would be more reliable than rodent models.…”

Multimodal analysis of aged wild-type mice exposed to repeated scanning ultrasound treatments demonstrates long-term safety

“…Safety was demonstrated by acquiring T2- and T2*- weighted images post-treatment, running a neurological test battery, and determining that there were no changes in the number of microhemorrhages in brain tissue taken after sacrifice 25. In another study, three- to eight-year-old sheep also underwent sonications, assessing multiple single treatments with a range of pressures 26. BBB opening was monitored post-treatment via Evans blue extravasation and endogenous IgG uptake, and staining with either hematoxylin & eosin (H&E) or vanadium acid fuchsin stain was used to assess cell damage and red blood cell infiltration 26.…”

“…In another study, three- to eight-year-old sheep also underwent sonications, assessing multiple single treatments with a range of pressures 26. BBB opening was monitored post-treatment via Evans blue extravasation and endogenous IgG uptake, and staining with either hematoxylin & eosin (H&E) or vanadium acid fuchsin stain was used to assess cell damage and red blood cell infiltration 26. In a study in macaques of unspecified age, the commercial ExAblate 4000 low-frequency TcMRgFUS system (InSightec) was used and was integrated with a clinical 3 T MRI unit 27.…”