 Proceedings of Engineering and Technology Innovation , vol. 3, 2016, pp. 13 - 15 13 Behavioural Assessment of Blood-Brain Barrier Opening Induced by Various Ultrasound Parameters Feng-Yi Yang * , Sheng-Fang Huang Department of Biomedical Imaging and Radiological Sciences , National Yang-Ming University, Taipei, Taiwan. Received 23 March 2016; received in revised form 15 April 2016; accept ed 02 May 2016 Abstract Focused ultrasound (FUS) -induced opening of the blood-brain barrier (BBB) in the presence of microbubbles is prospective for a targeted drug delivery to the bra in lesion. Ho wever, the safety of this technology has been addressed recently. The purpose of this study was to assess the behavioural changes follo wing the FUS induced-BBB disruption (BBBD). The behav- ioural assessment of rats were tested using the open field and hole-board fro m day 1 through day 32 a fter undergoing BBBD induced by FUS with either a mild or heavy parameter. We found that heavy BBBD induced behavioral changes, including significantly increased locomotor activity and a longer latency to nose poke for baits . Additionally, mild BBBD resulted in sig- nificant decrease of central activity. Therefore, the behavioural changes after FUS induced BBBD should be considered before clinica l application. Ke ywor ds : behavioural evaluation, focused ultrasound, blood-brain barrier, memory 1. Introduction Focused ultrasound (FUS) with mic robu b- bles has offered the potential to produce BBB disruption (BBBD) noninvasively in specific regions of the brain [1, 2]. However, the greatest limitat ion on the use of FUS-induced BBBD in clin ical practice consists of safety concerns relating to cavitation in the bra in. Mechanica l effects may be responsible for FUS-induced BBBD, but inert ial cavitation could usually cause hemorrhaging or apoptosis in the brain tissue from the neighboring vessel [3]. A lthough no significant negative effects resulting fro m histological e xa mination, further evaluation of brain functions follo wing FUS-induced BBBD were still needed. Such research will increase our knowledge of the behaviora l a lterations that occur following BBBD and allow for a better assessment of the safety of this technique in terms of its effects on bra in functions. Therefore , the purpose of this study was to exa mine the impact of BBBD in terms of behavioral altera - tions following FUS e xposure in the presence of microbubbles. 2. Method All procedures involving anima ls were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals. Th e study protocol was approved by the Anima l Ca re and Use Committee of National Yang Ming University. Male Sprague-Dawley (SD) rats weighing fro m 300 to 350 g we re used in this study. 2.1. Ultrasound Setup and Behavioural As- sessment The ultrasound system and equipment setup were the same as used in our previous study [4]. The BBB disruption (BBBD) can be quantified based on the extravasation of Evans blue (EB), which binds to albumin. Activ ity in the open fie ld was tested with the automated Fle x-Fie ld/Open field Photobeam Activity Sy s- tem on post-sonication days 1, 9, 18, and 32. Spatial lea rning ability was studied by means of a hole-board apparatus in which food rewa rds were used as positive motivation. 2.2. Number Synthesis Three rats fro m each group we re prepared for histological observation. The rats were per- fused with saline and 10% neutral buffered * Corresponding aut hor. Email: fyyang@ym.edu.t w Proceedings of Engineering and Technology Innovation , vol. 3, 2016, pp. 13 - 15 14 Copyright © TAETI forma lin on days 1 and 9 a fter the FUS soni- cation. The brain slices were stained by TUNEL staining (DeadEnd Co lorimetric T UNEL system, G7130, Pro mega, Mad ison, WI, USA) in order to detect DNA frag mentation and apoptotic bodies within the cells . All values are shown as means ± SEM. The behavioral assessment data were analy zed using the Mann-Whitney U test. Other data were an- alyzed using the unpaired Student’s t test. Sta- tistical significance was defined as a p value  0.05. 3. Results and Discussion Fig. 1 Effects of rats with FUS -induced BBBD on the open field activ ity. (A) The nu m- ber of center entries was significantly increased in the BBBD-heavy group on days 1 and 9 follo wing sonication. (B) But the number of center entries was significantly decreased in the BBBD-mild group on day 18 following sonication. * denotes significant differ- ences compared to the sham group at the same time points. (*, p<0.05; ***, p<0.005, n = 8) BBBD-heavy e xposed rats entered the center of the open field significantly more frequently on days 1 and 9 post-sonication compared with sham group (Fig. 1(A)). BBBD-mild e xposure significantly reduced the number of center en- tries on day 18 post-sonication compared with sham group (Fig. 1(B)). The BBBD-heavy group e xhibited a signif- icantly longer latency and latency to first baited hole compared with the sham group (Fig. 2(A)). No s ignificant diffe rences in latency and latency to first baited hole were found between the BBBD-mild group and sham group (Fig. 2(B)). Fig. 2 Ho le-board testing was performed on day 12 fo llo wing sonication. The latency and latency to the first baited hole were rec- orded in (A) the BBBD-heavy group and (B) the BBBD-mild group. * denotes a significant diffe rence relative to sham group. (*, p<0.05; **, p<0.01, n = 8) To our knowledge, the current study is the first to exp lore the e ffect of BBBD induced by FUS on behavioral alterations in an anima l model. The findings fro m this study indicate that BBBD-heavy rats produced hyperactivity rela - tive to sham animals under open field test. Proceedings of Engineering and Technology Innovation , vol. 3, 2016, pp. 13 - 15 15 Copyright © TAETI Moreover, the BBBD-heavy rats revea led spatial me mo ry impa irment in the hole-board test compared to sham group. In contrast, BBBD-mild rats only e xhibited an xiety-re lated behaviors in the open field test. 4. Conclusions In this paper, FUS-induced BBBD repre - sents a major advance in the targeted drug de- livery of the bra in. The current study suggests the possibility that manipulation of FUS-induced BBBD might produce a variety of behavioral changes after enhanced drug delivery. Further investigations of behavioral alterations in ani- ma ls fo llowing FUS -induced BBBD are needed in order to avoid abnorma l bra in function ing in humans following future clinica l uses of FUS-induced BBBD. Acknowledgement This study was supported by grants from the Ministry of Science and Technology of Taiwan (no. MOST 104-2314-B-010-003-M Y3). References [1] F. Y. Yang, T . T. Wong, M. C. Teng, R. S. Liu, M. Lu, and H. F. Liang, et a l., “Focused ultrasound and interleukin-4 receptor- targeted liposomal do xorubic in for en- hanced targeted drug delivery and antitu mor effect in glioblastoma multiforme ,” Journal of Controlled Release, vol. 160, no. 3, pp. 652-658, June 2012. [2] F. Y. Yang, W. Y. Chang, J. J. Li, H. E. Wang, J. C. Chen, and C. W. Chang, “Pharmacokinetic analysis and uptake of 18F-FBPA-Fr sfter ultrasound-induced blood-brain barrie r disruption for potential enhancement of boron delivery for neutron capture therapy,” Journal of Nuclear Medi- cine, vol. 55, pp. 616-621, April 2014. [3] F. Yang, W. Fu, W. Chen, W. Yeh, and W. Lin, “Quantitative evaluation of the use of microbubbles with transcranial focused u l- trasound on blood¡Vbra in-barrie r disrup- tion,” Ultrasonics Sonochemistry, vol. 15, no. 4, pp. 636-643, April 2008. [4] F. Y. Yang, W. Y. Chang, J. C. Chen, L. C. Lee, and Y. S. Hung, “Quantitative as- sessment of ce rebral glucose metabolic rates after blood-brain barrie r disruption induced by focused ultrasound using FDG-M icroPET ,” Neurolmage, vol. 90, pp. 93-98, April 2014.