A single low-energy shockwave pulse opens blood-cerebrospinal fluid barriers and facilitates gastrodin delivery to alleviate epilepsy.

The blood-cerebrospinal fluid barrier (BCSFB) is another gatekeeper between systemic circulation and the central nervous system (CNS), mainly present at the boundary between choroid plexuses and the ventricular system. This study demonstrates BCSFB opening in rats by single pulse of low-energy focused shockwave (FSW, energy flux density 0.03 mJ/mm2, 2 × 106 microbubbles/kg) treatment at lateral ventricle, resulting in significantly elevated cerebrospinal fluid (CSF) concentrations of systemically-administered gastrodin (GTD) (4 times vs. control within 3 hrs) that remained detectable for 24 hrs. The FSW-GTD group had significantly lower Racine's scale (<4) and zero mortality (n = 30) after lithium-pilocarpine-induced epilepsy. Electrophysiological recordings showed decreased epileptiform discharges, and brain section histology revealed reduced inflammation, oxidative stress and apoptosis, when compared with groups without FSW (Racine's scale: 4 ∼ 5; mortality: 26.67 ∼ 36.67%). FSW-mediated BCSFB opening provides a promising alternative for controlled-delivery of therapeutics into the CNS, offering rapid and widespread medication distribution. The technique could by applied in the development of novel therapies for various CNS diseases.

Introduction

Focused ultrasound (FUS) has been applied in blood–brain-barrier (BBB) opening in both animal models and human trials [1], [2], [3], with promising results reported for targeted drug delivery and gene therapy in rodent disease models including Parkinson’s and Huntington’s disease [4], [5], [6]. Focused shockwave (FSW) is a special form of acoustic wave that also offers excellent spatial resolution at a millimeter scale, as does FUS. Our group has developed FSW-mediated BBB opening techniques, achieving controllable and reversible BBB opening and successful brain transfection in a rat model [7], [8]. However, the drug delivery area of FUS/FSW-mediated BBB opening is limited by its small focal zone, which may limit application in diffuse or disseminated central nervous system (CNS) diseases when universal spreading of medication is desired.

The blood-cerebrospinal fluid barrier (BCSFB) is another gatekeeper between systemic circulation and the CNS, mainly present at the boundary between choroid plexuses and the ventricular system. The BCSFB differs from the BBB in that the choroidal vessels are highly permeable, and are not bounded by the foot processes of pericytes and astrocyte [9], [10], [11]. Once the BCSFB is disrupted, systemic medication immediately enters the CSF circulation, encompassing the whole brain and spinal cord, potentially offering a rapid and widespread drug delivery into the CNS.

Following successful FSW-mediated BBB opening, we further demonstrate the efficacy and safety of FSW-induced reversible BCSFB opening at the choroid plexus of the lateral ventricles. Single-shot, low-intensity FSW can significantly elevate concentrations of several BBB/BCSFB impermeable fluorescent indicators and medications (unpublished results), indicating the possibility of applying this technique in the treatment of CNS diseases.

Gastrodin (GTD) is the main bioactive compound of the herbal medicine Gastrodia elata. It is a phenolic glycoside that has potent CNS effects, including anti-epileptic, sedative- hypnotic and cell protective [12], [13]. These effects are mainly attributed to the inhibition of the degradative enzyme activities of gamma-aminobutyric acid (GABA), including GABA transaminase and succinic semialdehyde reductase [14], [15]. Animal models have demonstrated its therapeutic effect in diseases including epilepsy [16], Alzheimer's disease [17] and Parkinson's disease [18], [19].

Despite its potent CNS effects, the brain-to-blood distribution ratio of gastrodin is extremely low (only 0.007 at the dose of 100 mg/kg (i.v.) in rats) [20], owing to its limited permeability across the BBB and rapid metabolism in systemic circulation [20], [21]. Therefore, in pre-clinical studies, repetitive high dosing of systemically-administered GTD has often been required to maintain its therapeutic effect [12]. For potential clinical applications, the efficiency of CNS delivery of GTD after systemic administration needs to be enhanced.

Therefore, the present study aims to evaluate the feasibility and efficacy of CNS delivery of gastrodin by FSW-induced BCSFB opening, as well as its therapeutic effect in a rat model of lithium-pilocarpine-induced epilepsy. After intravenous GTD, a single shot, low-intensity FSW was targeted at the lateral ventricle where the choroidal plexus is most abundant. CSF concentration of GTD was sampled, and outcomes were evaluated by behavior analysis, electrophysiological recording using implanted electrodes, and immunohistochemistry stain of neuroinflammation, oxidative stress and apoptosis in brain section of the rat epilepsy model.

Materials & methods

Materials & instruments

This study was approved by the ethics committee of the Laboratory Animal Center at the National Taiwan University College of Medicine (approval No. 20,200,235 for the use of rats), and adhered to the experimental animal care guidelines. All rats (adult Sprague Dawley rats between 8 and 10 weeks of age) were obtained from the National Laboratory Animal Center (Taipei, Taiwan), fed with a standard diet and housed in a temperature- and humidity-controlled room (19 – 23 °C; 40 – 70 %, respectively) under a 12-hour light–dark cycle.

GTD, chloral hydrate, and Evans blue were purchased from Sigma-Aldrich, Inc. (Missouri, USA). GTD ELISA kit (GTD; MBS2700696) was purchased from MyBioSource Inc. (SD, US). Glutathione (K261), total antioxidant capacity (TAC; K274), catalase activity (K773), Malondialdehyde (E4601), and superoxide dismutase (SOD; K335) assay kits were purchased from BioVision Inc. (CA, US). Rat IL-1β (BSKR1006) assay kit was purchased from Bioss Inc. (MA, US). Isotonic sodium chloride solution (0.9 %) was provided by Taiwan Biotech Co., LTD. (Taoyuan, Taiwan) and sterile-filtered by 0.22 μm PES membrane (Millipore syringe filter) from Polyplus-transfection (Illkirch, France). Ultrasound coupling gel (CG955, sonic resistance: 1.55 ± 0.05 Mrayl, pH 7.0 ± 0.05) was obtained from Ceyotek (Chiayi City, Taiwan). Peroxidase in situ apoptosis detection kits (TUNEL S7100), Glial fibrillary acidic protein (GFAP), hematoxylin and eosin (H&E), caspase-3, and NeuN stain kits were purchased from Merck Millipore (Darmstadt, Germany). Ventana Benchmark XT tissue diagnostic system, EZ-prep, ultraview DAB and ultraview universal alkaline phosphatase red detection kits were purchased from Ventana Medical Systems, Inc. (AZ, US).

The FSW device (PiezoWave) and gel pad (F10 G4) was purchased from Richard Wolf (Knittlingen, Germany). SonoVue (Microbubbles, UCA) was acquired from Diagnostics (Milan, Italy). The slide scanner (Ventana Dp200) and its software (Ventana Image Viewer v3.2) were obtained from F. Hoffmann-La Roche (Basel, Switzerland). A microplate reader (Infinite 2000 Pro) and its software (i-control) were procured from Tecan Austria (Grodig, Austria). A neurodiagnostic system (Nicolet VikingQuest), TECA elite disposable concentric needle electrodes (S53153), and needle holders (X21001) were purchased from Natus Medical Inc. (CA, US).

FSW-UCA induced BCSFB opening via choroid plexus on lateral ventricles

The FSW probe was placed on the left lateral side of the rat head while the neurodiagnostic electrodes were placed top of the rat head (Fig. 1). The setup conditions of the commercial shockwave device and the FSW probe positioning platform were modified based on Kung [7], [8]. Microbubbles (SonoVue) are injected after implementing single low-energy extracorporeal focused shockwave pulse in order to minimize brain damage in a safe operating range to open the BBB and BCSFB according to shockwave pulse technique [11].

Fig. 1

(a) Top view of the experimental implementation of shockwave probe; (b) lateral view. The brown circle indicates the bregma; the red dot is the focal point (AP 0, L 4, V −5) with the target region at the choroid plexus on the lateral ventricles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The concave shockwave probe (46 mm in radius, with a curvature radius of 62.9 mm) was coupled with a gel pad to ensure the focus of the FSW probe was 5 mm from the bottom of the gel pad. The shockwave probe, together with the gel pad, was always focused at the same location on the left lateral ventricle (at the point: antero-posterior (AP) 0, mediolateral (ML) 4, Dorsoventral (DV) −5 according to the atlas of Praxinos and Watson. Ultrasound gel was spread at the interfaces between the shockwave probe and the gel pad, and also between the bottom of the gel pad and the rat scalp (Fig. 1).

0.5 ml of 3 % Evans blue was used as an indicator to assess the macromolecule delivery rate (CSF/blood) into CSF circulation by applying FSW on the left lateral side of the rat head. Three hours after FSW application (single pulse, intensity level 0.1, peak negative pressure −4.2 MPa; energy flux density 0.03 mJ/mm2) with microbubbles (2 × 106 MBs/kg, SonoVue) to open the BCSFB, CSF was sampled from the cisterna magna.

Time responses of GTD in CSF circulation

In this study, the sampling process of GTD was based on that described in section 2.2. Time 0 is the time point at which FSW-UCA is applied, with sampling time points at 0.5 hr, 1 hr, 2 hrs, 3 hrs, and 1 day, 2 days, 4 days following FSW-UCA application (5 rats at each time point). The drug concentration ratios (CSF/blood; cotex/blood) of GTD were analyzed using the Gastrodin ELISA kits (MBS2700696, MyBioSource).

Lithium-Pilocarpine-induced epilepsy model and FSW-GTD therapy

To induce epilepsy, Lithium chloride (27 mg/kg) was administered intraperitoneally to rats 18 hrs before the subcutaneous injection of pilocarpine (30 mg/kg) [22]. Then, 3 hrs after injecting pilocarpine, Chloral hydrate (300 mg/kg; i.p.) was applied to stop the seizure (as Fig. 2 shown).

Fig. 2

The experimental protocol. The rats were sacrificed 3 hrs (T 1), 24 hrs (T 2), and 72 hrs (T3) after seizure induction to evaluate Li-Pilo-epilepsy induced hippocampal neuronal damage (CA1-CA3-DG area) and FSW-GTD treatment efficacy.

The time points of the FSW-GTD and GTD (GTD only as Sham group) (60 mg/kg; i.v.) treatments were set 18 hrs before the subcutaneous injection of pilocarpine. The experimental set up of FSW-GTD treatment follows that in section 2.2.

Electrophysiology

Electrocorticography data is wildly used to estimate brain activity patterns on the epilepsy patients [23]. 7 days before processing the Lithium-Pilocarpine-induced epilepsy model, rats were surgically implanted with electrodes (directly cut to a length of 3 mm) under stereotaxic guidance. The rats were fully anesthetized with chloral hydrate (0.35 g/kg, i.p). The concentric needle electrode was implanted into the right hippocampus. Stereotaxic coordinates in millimeters relative to the bregma according to the atlas of Paxinos and Watson were as follows: AP-3.9, L-1.7, V-2.9 [24]. The electrodes were secured to the skull by acrylic dental cement. The rats were allowed 7 days to recover from the surgical procedure before start of the study.

The electrophysiological activity was simultaneously acquired in freely moving rats using a neurodiagnostic system (Nicolet VikingQuest). Each animal was recorded 3 hrs after lithium injection.

Behaviors

During the 3 hrs between injecting pilocarpine and injecting chloral hydrate, the behaviors of epilepsy rat were evaluated by seizure latency and seizure score. Seizure level was classified using Racine’s scale as follows: stage 1, immobility, eyes closed, and facial clonus; stage 2, head nodding and more severe facial clonus; stage 3, clonus of one forelimb; stage 4, rearing with bilateral forelimb clonus; and stage 5, generalized tonic-clonic seizures.

Biomarkers

To evaluate the therapeutic effect between the FSW-GTD group and the Sham group, 3, 24, 72 hrs after lithium-pilocarpine induced seizures, the brains of the rats were removed. In addition, 5 normal rat brains without pilocarpine-induced seizures were used as a blank group to evaluate the difference of related biomarker expression in SOD, CAT, GSH, T-AOC, IL-1β, MDA. The dissected hippocampus was at 4 °C. Subsequently, the homogenized brain tissues were analyzed as described in the kit protocol.

Histopathologic sections and immunohistochemistry

To evaluate the therapeutic effect between the FSW-GTD group and the Sham group, 3, 24, 72 hrs after lithium-pilocarpine induced seizures, the brains of the rats were removed. Brain fillets were then immersed in 10 % formaldehyde solution for 24 hrs. Subsequently, the specimens were embedded in paraffin and subjected to the following analysis.

For GFAP detection, after serial 4 µm paraffin sections were deparaffinized by EZ prep, the slides were incubated with anti-Glial Fibrillary Acidic Protein in 1:1500 titration for 32 min using the automated Ventana Benchmark XT. Labeling was detected with the ultraview DAB detection kit. All sections were counterstained with hematoxylin in Ventana reagent.

For TUNEL analysis, 4 µm paraffin sections were deparaffinized by xylene and rehydrated in a series of graded ethanol similar to tissues stained for immunohistochemistry. Protein digestion was done with 0.5 units/ml proteinase1 for 8 min at room temperature. Tissues sections were rinsed in distilled water for 2 min followed by quenching of endogenous peroxidase with 3% H2O2 in 1 × PBS for 30 min at room temperature. Samples were rinsed in 1 × PBS, excess liquid was removed by blotting, and a 1 × equilibrium buffer was applied for 10–15 s at room temperature. Residues of digoxigenin–nucleotide (digoxigenin-11-dUTP and dATP) were added to DNA using TdT enzyme in a reaction buffer, with the samples incubated in a humidified chamber at 37 °C for 1 hr. The tissue sections were then placed in a prewarmed stop/wash buffer for 30 min at 37 °C. Next, the samples were incubated with anti-digoxigenin conjugated to peroxidase and visualized using DAB, followed by counterstaining with Mayer’s hematoxylin and dehydration with a series of graded ethanol.

For Caspase3-NeuN double staining, serial 4 µm paraffin sections were deparaffinized by EZ prep. The slides were incubated with anti-Caspase3 in 1:200 titration, and anti-NeuN in 1:150 titration for 32 min using the automated Ventana Benchmark XT. Labeling was detected with the ultraview DAB and ultraview universal alkaline phosphatase red detection kits. All sections were counterstained with hematoxylin in Ventana reagent.

Statistics

Statistical analysis was performed using the statistical analysis software SPSS version 26. All data were expressed as mean ± standard deviation (SD) of at least five independent samples (n). In group comparisons, the statistical evaluations were carried out with one-way ANOVA and post-hoc analysis (Tukey). A p-value of less than 0.05 was considered significant.

Results

At the beginning of the study, Evans blue was used as an indicator to test the efficacy of the FSW-BCSFB. After the FSW-BCSFB procedure, the concentration of Evans blue in CSF circulation can reach 0.52 ± 0.11% (EB in CSF vs. in blood) (N = 5). The content of Evans blue without FSW-BCSFB is only 0.04 ± 0.04% (EB in CSF vs. in blood) (n = 5). This proves that the FSW-BCSFB technique can significantly enhance delivery of drugs into CSF circulation.

In terms of the target drug GTD of this study, Fig. 3 shows the response to CSF circulation following FSW-BCSFB application. Fig. 3 (a) shows that the half-life of GTD in the blood is about 0.5 hrs. The concentration of GTD drops rapidly in the first 1 hr, and was almost completely metabolized in blood circulation after 24 hrs. Fig. 3 (b) and (c) show that after FSW-BCSFB procedure, the GTD concentration in CSF circulation was elevated more than 3 times that in the control group through the first 24 hrs. The ratio of GTD in CSF and in blood circulation was enhanced even more prominently. Therefore, the FSW-BCSFB technique can increase and prolong the circulation of drugs in the CSF.

Fig. 3

Time responses of GTD in blood and in CSF circulation, treated with FSW (FSW) and without FSW (Control). (a) time responses of GTD in blood; (b) time responses of GTD in CSF with and without FSW treatment; (c) the ratio of GTD in CSF vs. in blood with and without FSW treatment, where **p < 0.01 vs. control group in one-way ANOVA with the Tukey post hoc test. (n = 5).

In our study, the same sampling was also carried out over 2 and 4 days. Unfortunately, at this time, the concentrations of GTD in blood and CSF are lower than the detection limit of the ELISA Kits. Therefore, these results are not included in Fig. 3.

Behaviors and its electrophysiologies

After determining the performance of the FSW-BCSFB technique to deliver GTD into CSF circulation, this method (FSW-GTD) is applied in the treatment of Lithium-Pilocarpine-induced epilepsy (Li-Pilo-epilepsy)

In terms of behavior, FSW-GTD treatment produced statistically significant improvements in seizure latency and seizure score on day 3 as compared with the epilepsy and GTD treatment groups (Table 1). Of note, there was zero mortality in the FSW-GTD group, as opposed to mortality rates of 36.67% and 26.67%, respectively, in epilepsy and GTD treatment groups. Representative video recordings of the behavior are included in the supporting material. In particular, the seizure score of the FSW-GTD treatment group can be controlled at stage 3 to 4 of Racine’s scale. The GTD only treatment group is at stage 4 to 5, while all of the epilepsy group subjects fall into stage 5.

Table 1

Behavior comparison of different conditions on epilepsy.

Condition			Epilepsy
Epilepsy	GTD	FSW	Seizure latency (min)	Seizure score	Mortality rate on day 3 (%)
V			67.86 ± 9.33	5.00 ± 0.00	36.67
V	V		79.59 ± 13.76*	4.63 ± 0.78*	26.67
V	V	V	112.64 ± 16.41**,#	3.97 ± 0.75**,#	0.00*,#

* and ** were p < 0.05 and p < 0.01 vs. epilepsy in in one-way ANOVA with the Tukey post hoc test. (n = 30)

were p < 0.05 vs. epilepsy-GTD in one-way ANOVA with the Tukey post hoc test. (n = 30)

Furthermore, electrophysiology is another indicator of epilepsy severity. It can be seen from Table 2 and Fig. 4 that, after the FSW-GTD treatment, the epileptiform discharges were significantly improved as compared to the epilepsy and GTD treatment groups.

Table 2

Electrophysiological comparison of different conditions on epilepsy.

Epilepsy	GTD	FSW		Electrophysiological amplitude
				Max	Min	Mean
				5.27 ± 1.84	−2.63 ± 0.93	0.78 ± 2.26
V				51.34 ± 17.35	−36.53 ± 13.22	23.68 ± 8.57
V	V			34.06 ± 7.41	−19.35 ± 8.57	17.62 ± 7.54
V	V	V		19.98 ± 6.54	−10.92 ± 6.30*,#	8.38 ± 3.67*,#

* and # were p < 0.05 vs. epilepsy and vs. epilepsy-GTD in one-way ANOVA with the Tukey post hoc test. (n = 5)

Unit: mV

Fig. 4

Electrophysiological waveform under different conditions: Control - without any treatment; Epilepsy - epilepsy group; Epilepsy - epilepsy rats treated with GTD; Epilepsy - epilepsy rats treated with GTD and FSW.

Epilepsy related biomarkers

Behavior and electrophysiology evaluations were followed by brain tissue analysis and tracking. Overall, FSW-GTD treatment produced a statistically significant improvement in IL-1β, SOD, CAT, GSH, T-AOC, and MDA expression as compared with the epilepsy and GTD treatment groups at all sampling points 3 hr, 24 hr, and 72 hr after Li-Pilo-epilepsy (Fig. 5).

Fig. 5

Comparisons of time responses of epilepsy related biomarks. (a) IL-1β expression; (b) SOD expression; (c) CAT expression; (d) GSH expression; (e) T-AOC expression; (f) MDA expression, in which, *p, #p, ξp < 0.05 vs. control, epilepsy + GTD, and epilepsy groups, respectively; and **p, ##p, ξξp < 0.01 vs. control, epilepsy + GTD, and epilepsy groups, respectively, in one-way ANOVA with the Tukey post hoc test. (n = 5).

The histopathologic sections and immunohistochemistry at the CA1-CA3-DG region showed little variation in terms of the configuration and cell death among the groups (Fig. 6 (a) H&E stains and (b) TUNEL stains). However, FSW-GTD treatment was followed by statistically significant differences in GFAP stains (Fig. 6 (c)), and Caspase3-NeuN stains (Fig. 6 (d)) as compared with the epilepsy and GTD treatment groups. Fig. 6 (e) shows a statistical improvement in the quantitative comparison of Caspase3-NeuN stains in the FSW-GTD treatment group, as compared with the epilepsy and GTD treatment groups.

Fig. 6

Comparisons of histopathologic and immunohistochemistrical sections by blank, epilepsy, epilepsy with GTD only (Sham), and epilepsy with FSW-GTD (FSW-GTD) groups. (a) H&E stains; (b) TUNEL stains; (c) GFAP stains; (d) Caspase3-NeuN stains, arrowed is the Caspase-3-stained cell; (e) time responses of Caspase-3 density. *p, #p, ξp < 0.05 vs. control, epilepsy + GTD, and epilepsy groups, respectively; **p < 0.01 vs. control groups, respectively, in one-way ANOVA with the Tukey post hoc test. (n = 5).

Discussions

We demonstrated that a single pulse of low-energy FSW treatment at the lateral ventricle significantly elevates the CSF concentration of systemically-administered single-dose GTD, indicating a successful opening of the BCSFB. Rats pretreated with intravenous GTD and FSW-mediated BCSFB opening scored significantly lower on Racine’s scale and experienced significantly reduced mortality after seizure induction, as compared with groups without FSW treatment. In addition, electrophysiological recordings showed decreased epileptiform discharges, and brain section histology revealed reduced inflammation, oxidative stress and apoptosis in the FSW treatment group. The results indicated that FSW-mediated BCSFB opening provides an effective alternative to controlled delivery of therapeutics into the CNS, and could be applied in the treatment of CNS diseases particularly when widespread medication is desired.

Targeting the BCSFB might be a useful surrogate drug delivery method to circumvent the barrier between systemic circulation and the CNS. The BCSFB is mainly distributed at the ventricular choroid plexuses, secreting various hormones and neurotrophic factors [25]. Differing from the BBB of the cerebral capillaries, the blood flow in the choroid plexus is much larger, and the vessel wall is more permeable with a high fluid turnover rate. Because of these features, the proposed FSW-mediated BCSFB opening for drug delivery offers the following advantages. First, it enables rapid and widespread medication delivery into the CSF, which surrounds the entire brain and spinal cord. In our unpublished study, accumulated fluorescence intensities could be observed in brain regions distant to the FSW stimulation site, the contralateral hemisphere, and even the spinal cord 3 hrs after FSW stimulation, and remained detectable for at least 120 hrs. Second, shifting of medication from the vascular to the ventricular compartment prevents rapid elimination of medication in blood attributed to the presence of plasma protein and enzymes [26]. Fig. 3 shows markedly persistent elevated CSF concentrations of GTD in the FSW-GTD group in the first 24 hrs despite rapidly declining blood concentrations. A longer half-life in the CSF than in the blood further increases the possibility of drug delivery into the parenchyma.

Despite being a potent GABAergic medication and widely used in seizure treatment both in animal and human trials [12], therapeutic applications of GTD have been limited by its short half-life in blood and poor penetration across the BBB [13], [24], [27]. Our results show that GTD concentrations in blood after systemic administration dropped by more than half in the first hour, consistent with previous in vivo rat studies [20], [24]. Several previous studies gave reported that positive outcomes using GTD as seizure therapy required prolonged and repeated medication loading to reach therapeutic effect (50–200 mg/kg, intraperitoneal, 7–28 d) [28], [29], [30]. Our study showed that, using FSW-mediated BCSFB opening, single dose GTD pretreatment could effectively suppress seizure activity induced 18 hrs after FSW-GTD.

Using the proposed technique, the systemic dosage of GTD could possibly be further reduced. By FSW-mediated BCSFB opening, the concentration of GTD in CSF increased nearly 3 times compared with that without FSW by as early as 0.5 hr post-FSW, reaching a plateau at 3 hrs and remaining blood concentrations similar to those with systemic GTD without FSW at 24 hrs. The CSF/ plasma concentration was 40–60% in the first 3 hrs after FSW, and remained above 50% for 24 hrs, in contrast to the reported 4.8+/-2.4 % in a study of rats treated with intravenous high dose GTD (200 mg/kg) [27]. The successful CSF delivery of GTD possibly contributed to the significant therapeutic efficacy in the current model. Furthermore, the proximity of the lateral ventricle and hippocampus, the main focus of lithium-pilocarpine-induced epilepsy, made CSF delivery of therapeutics even more promising.

By targeting the ventricular system, the proposed method could potentially be applied in the treatment of diseases with diffuse CNS involvement, particularly the periventricular brain regions including basal ganglia, mesial temporal lobes, and brain stem. In addition, in certain brain regions such as the brain stem, where FUS or FSW-mediated BBB opening is considered hazardous, FSW-mediated BCSFB opening could provide an effective and safe alternative treatment option.

With regard to safety and efficacy, the proposed single-shot FSW method not only successfully reduces the required total acoustic energy and pulses, but also provides a more reliable method to open both the BBB and BCSFB when compared with the FUS-mediated BBB opening technique. FSW is a special form of acoustic wave composed of a unique single pulse wave, a short-duration but high positive acoustic pressure followed by a long negative wave pattern. The significantly longer negative wave enables FSW to more effectively produce a cavitation effect in the tissue. Our previous research reported a 100% successful BBB opening rate by a single shot of FSW using 20% of clinical dosage of microbubbles [7]. Another advantage of FSW is the lower total acoustic energy. Fig. 7 shows the physical characteristics of the single FSW pulse (Fig. 7 above), the device used in the current study, and the high-intensity focused ultrasound (HIFU) device, a preferred device to induce BBB opening (Fig. 7 bottom). Using the single FSW pulse, the total energy required for BBB opening under the conditions shown in Fig. 7 is about 1/60,000 times that of by using HIFU. Furthermore, as FSW generates negligible heat, its application, exploiting acoustic related biological effects, would not be limited by tissue heating or related adverse effects [9], [10], [30].

Fig. 7

Acoustic profile comparisons for a single FSW pulse (the method used in this study), and HIFU (the preferred method to induce BBB opening).

This study presents several limitations. First, optimal therapeutic dosage is not determined. The chosen intravenous dosage (60 mg/kg) is similar to that used in previous rodent epilepsy models (50–200 mg/kg, intraperitoneal) [27], [28], [29]. As mentioned above, with FSW-mediated BCSFB opening, even single dose of GTD could achieve high and sustained CSF concentrations. Further study is needed to determine the optimal dosage of systemic GTD that can effectively suppress or terminate epilepsy. Second, the exact intensity threshold for FSW-BCSFB opening has yet to be determined. The intensity used in the present study is the same as that used for FSW-induced BBB opening in our previous study, and is the lowest possible setting of the commercial FSW. Considering the secretory nature and fenestrated vessel structure, it is possible that there is a lower intensity threshold for BCSFB opening than for BBB opening. A lower FSW intensity will further ameliorate concerns of adverse effects such as hemorrhage and neuronal injury.

Conclusions

BCSFB opening by single pulse of low-energy FSW treatment at the lateral ventricle facilitated CSF delivery of systemically-administered GTD. Seizure activity could be effectively suppressed after pretreatment in the lithium-pilocarpine-induced epilepsy model, as evidenced by behavioral, electrophysiological and immunohistochemistry analysis. FSW-mediated BCSFB opening provides a promising alternative for controlled delivery of therapeutics into the CNS, offering rapid and widespread medication distribution. The technique could by applied in developing novel therapies for various CNS diseases.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.