Focused ultrasound-induced blood-brain barrier opening improves adult hippocampal neurogenesis and cognitive function in a cholinergic degeneration dementia rat model

All animal experimental procedures were conducted in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee (IACUC; 2016-0339) of Yonsei University. Animals were housed in groups of three in laboratory cages with food and water available ad libitum in a 12-h light/dark (lights on at 07:00) cycle in a room with controlled temperature (22 ± 2 °C) and humidity (55 ± 5%).

Previous studies have modeled cholinergic degeneration and cognitive function-impaired dementia in rats by intraventricularly administering the selective immunotoxin 192 IgG-saporin (SAP) to induce lesions in BFC neurons [32‐35]. To investigate the effect of FUS on AHN in a cholinergic degeneration rat model of dementia, adult male Sprague Dawley rats (n = 48; 200–250 g) were divided into control (phosphate-buffered saline [PBS] injection), SAP, and SAP+FUS groups. The dementia rat model (SAP, n = 16; SAP+FUS, n = 16) was generated by injecting SAP (Chemicon, Temecula, CA, USA), and the control group (n = 16) received a bilateral ventricular infusion of 1× PBS (PH 7.4) into the brain. All 48 rats were anesthetized with a mixture of ketamine (75 mg/kg), xylazine (Rompun™; 4 mg/kg), and acepromazine (0.75 mg/kg) and were fixed in a stereotaxic frame. As previously described, scalp skin was incised, and two holes were drilled into the skull at the following coordinates: from the bregma anterior-posterior, − 0.8 mm; medial-lateral, ± 1.2 mm; and dorsal-ventral, − 3.4 mm [36]. Thereafter, 4 μl of SAP (0.63 μg/μl) was bilaterally injected at a rate of 1 μl/min into the lateral ventricle of the rats in the SAP and SAP+FUS groups using a syringe pump (Legato 130, 788130, KD Scientific, Holliston, MA, USA). As shown in Fig. 1a, rats were sacrificed at different time points, i.e., 24 h, 5 days, and 18 days after FUS. To detect changes in AChE and BDNF expression levels, observe proliferation and neuroblast production, and observe neuronal differentiation of BrdU-positive cells and long-term effects of AChE and BDNF, the rats were sacrificed 24 h, 15 days, and 18 days after FUS, respectively.

The pulsed ultrasound was generated using a 0.5-MHz single-element spherically focused transducer (H-107MR, Sonic Concept Inc., Bothell, WA, USA) with a diameter of 51.7 mm and radius of curvature of 63.2 mm. A waveform generator (33220A, Agilent, Palo Alto, CA, USA) was connected to a 50-dB Radio Frequency Power Amplifier (240 L, ENI Inc., Rochester, NY, USA) to drive the FUS transducer, and a power meter (E4419B, Agilent) was used to measure the input electrical power. The transducer electrical impedance was matched to the output impedance of the amplifier (50 Ω) with an external matching network (Sonic Concept Inc., Bothell, WA, USA). A cone filled with distilled, degassed water was mounted onto the transducer assembly (Additional file 1: Figure S1). A needle-type hydrophone (HNA-0400, Onda, Sunnyvale, CA, USA) was used for the transducer calibration, which measured the acoustic beam profile in the tank filled with degassed water. The transducer was mounted on the cone filled with degassed water, and the end of its tip was wrapped in a polyurethane membrane.

The experimental procedure is shown in Fig. 1. Briefly, rats were anesthetized with a mixture of ketamine (75 mg/kg) and xylazine (4 mg/kg), and their heads mounted on a stereotaxic frame (Narishige, Tokyo, Japan) with ear and nose bars. Ultrasound transmission gel (ProGel-Dayo Medical Co., Seoul, South Korea) was used to cover the area between the animal’s skull and the cone tip to maximize the transmission efficiency of the ultrasound. FUS was targeted bilaterally to the region containing the hippocampus according to the 3D positioning system. DEFINITY® microbubble contrast agents (mean diameter range, 1.1–3.3 μm; Lantheus Medical Imaging, North Billerica, MA, USA) were diluted in saline and injected intravenously into the tail vein 10 s before sonication. Sonication parameters were set as follows: burst duration, 10 ms; pulse repetition frequency, 1 Hz; total duration, 120 s; and average peak-negative pressure, 0.25 MPa.

After sonication, magnetic resonance imaging (MRI) experiments were performed with a Bruker 9.4 T 20-cm-bore MRI system (Biospec 94/20 USR; Bruker, Ettlingen, Germany) and a rat head coil. A gadolinium-based contrast agent, gadobutrol (Gd, Gadovist; Bayer Schering Pharma AG, Berlin, Germany; 0.2 mL/kg), was injected into the tail vein, and contrast-enhanced T1-weighted images were used to confirm the BBB opening from the FUS. T1-weighted MRI was performed with and without the use of gadobutrol contrast (Fig. 1d). T2-weighted images were used to confirm edema with FUS (Fig. 1e). Sequence parameters are summarized in Table 1.

Rats underwent the Morris water maze (MWM) test at 2 weeks after receiving SAP injection. The MWM apparatus comprised a circular pool (diameter, 2 m; height, 50 cm) filled to a depth of 30 cm with dark water (23 °C). A concealed black, round platform (diameter, 15 cm) was situated 1–2 cm below the surface of the water in the center of a target quadrant. All rats were trained for four trials per day for 5 consecutive days. During training, the location of the hidden platform was fixed, and spatial cues were provided for guidance. For each training trial, the rats were placed in the water facing the wall at one of the four starting points and were given 60 s to reach the hidden platform. After finding the platform, the rats were allowed to remain on the platform for 10 s. The rats that could not find the platform within 60 s were led to the platform by the experimenter and were allowed to remain on the platform for 10 s. The rats were given a 60-s probe test without the platform 72 h after the last training trial. Swimming speed, swim path, time spent in each zone, and distance swam were recorded using the SMART video-tracking system (Harvard Apparatus, Holliston, MA, USA).

To investigate the effect of FUS on neurogenesis, animals were injected intraperitoneally with 5-bromo-2′-deoxyuridine (BrdU; Sigma-Aldrich, St. Louis, MO, USA), used for the detection of proliferating cells, twice a day for 4 consecutive days, 24 h after sonication [30, 37].

To confirm cholinergic degeneration in our model, we quantified ChAT-immunopositive cells in MS/DB of each group of rats. Five days after sonication, compared with the control group (100 ± 3.5%), both the SAP (65.5 ± 13.1%; P < 0.05) and SAP+FUS (48.6 ± 5.05; P < 0.01) groups displayed a significantly reduced number of ChAT-immunopositive neurons (Fig. 2b). Eighteen days after sonication, compared with the control group (100 ± 10), both the SAP (18.7 ± 4.3; P < 0.001) and SAP+FUS (13.89 ± 5.9; P < 0.001) groups had significantly fewer ChAT-immunopositive neurons and less neuronal damage to cholinergic neuron bodies (Fig. 2c).

These results indicate that the number of cholinergic neurons were decreased in all groups at 5 and 18 days, which provide supporting evidence that dementia model using SAP was effective.

To determine whether FUS affects cholinergic neuronal activity, we quantified AChE activity in each group. Twenty-four hours after sonication, AChE activity was significantly reduced in the SAP group in the frontal cortex (FC; 68.61 ± 3.02%; P < 0.05) and hippocampus (86.12 ± 1.43%; P < 0.05) compared with that in the control group (Fig. 3a, b).

Eighteen days after sonication, AChE activity was significantly decreased in the SAP group in the FC (74.85 ± 3.62%; P < 0.05) and hippocampus (83.70 ± 1.61%; P < 0.01) compared with that in the control group (Fig. 3c, d). However, the AChE activity of the hippocampus was significantly increased in the SAP+FUS group (94.03 ± 2.33%; P < 0.01) compared with that in the SAP group. The AChE activity of the FC was increased in the SAP+FUS group (90.79 ± 5.30%; P = 0.09) compared with that in the SAP group, but there was no significant difference between the two groups (Fig. 3c, d).

As a result, cholinergic degeneration of MS induced decreased activities of AChE in both FC and hippocampus at 24 h and 18 days. The effect of FUS treatment in AChE activities was only observed in the hippocampus at 18 days.

BDNF acts on specific neurons by promoting neurogenesis, which is crucial for long-term memory. To examine the effects of FUS on BDNF expression in the hippocampus, we performed immunoblotting analyses using brain samples from the hippocampal region obtained at 24 h and 18 days after sonication. The BDNF gene produces immature BDNF protein (17~32 kDa) and BDNF mature form (~ 13 kDa) by intracellular and extracellular proteases (Additional file 1: Figure S2) [39]. At both time points, compared with the control group, the SAP group (24 h: 80.15 ± 6.16%; 18 days: 60.79 ± 4.09%; P < 0.05) exhibited a significantly reduced expression level of mature-BDNF in the hippocampus, whereas compared with the SAP group, the SAP+FUS group (24 h: 108 ± 4.81%; P < 0.01; 18 days: 73.37 ± 10.63%; P < 0.05) showed a significantly increased level of mature-BDNF (Fig. 3e–h).

The cholinergic degeneration of MS induced decreased expression level of BDNF in the hippocampus at 24 h and 18 days. In contrast, FUS could upregulate the BDNF at the same time points.

EGR1, a transcriptional regulator, is extensively used as a marker for neuronal plasticity. To investigate whether FUS affects the transcription factor of EGR1 expression at 5 days after sonication, the number of EGR1-positive cells was visualized using immunohistochemistry. The SAP group exhibited a significantly lower number of EGR1-positive cells in CA1 (117 ± 4; P < 0.001), CA3 (67 ± 9; P < 0.01), and DG (159 ± 6; P < 0.01) of the hippocampus than the control group (CA1, 163 ± 2; CA3, 87 ± 4; DG, 229 ± 15). However, the EGR1 activity in the FUS group indicated a significant increase in CA1 (135 ± 4; P < 0.05), CA3 (77 ± 4; P < 0.05), and DG (199 ± 5; P < 0.05) compared with that in the SAP group (Fig. 4a, b).

These results indicate that cholinergic degeneration of MS caused the decreased activities of EGR1 in hippocampal region at 5 days, and FUS significantly upregulated the activities of EGR1 than SAP group.

The rats in each group were sacrificed 5 days after bilateral sonication of the hippocampal regions. Twenty-four hours after sonication, BrdU labeling was performed for 4 consecutive days for each group to observe progenitor cell proliferation in SGZ of DGs. We observed a decrease in the number of BrdU-positive cells in the SAP group (65 ± 6; P < 0.05) compared with that in the control group (117 ± 18), while this number was significantly increased in the SAP+FUS group (137 ± 10; P < 0.01) compared with that in the SAP group (Fig. 4c, d).

To investigate whether FUS affects newly generated immature neurons, the number of neuroblasts was visualized using doublecortin (DCX, a marker for neurogenesis) immunohistochemistry. Compared with the control (196 ± 21; P < 0.05) and SAP+FUS (183 ± 9; P < 0.05; Fig. 4e, f) groups, the SAP group exhibited a significantly reduced number of DCX-positive cells in DG of the hippocampus (113 ± 14).

It has been suggested that cholinergic degeneration of MS showed decreased activities of proliferation and neuroblast production in SGZ at 24 h and 18 days, which was reversed by FUS.

To determine the phenotypic characterization of BrdU-positive cells 18 days after sonication, sections were analyzed 2 weeks after the last injection of BrdU: neuronal phenotypes were identified by double-immunofluorescence labeling for NeuN and BrdU and glial phenotypes by double-immunofluorescence labeling for GFAP (astrocyte-specific marker) and BrdU (Fig. 5a–c). Compared with the control group (40 ± 2), the SAP group (25 ± 2; P < 0.01) displayed significantly reduced neurogenesis (NeuN+/BrdU+) in SGZ/GCL of DG. Compared with the SAP group, the SAP+FUS group (49 ± 1; P < 0.001) featured a significantly increased number of co-expression cells (NeuN+/BrdU+) in DG. No significant differences in gliogenesis (GFAP+/BrdU+) (Fig. 5e) and the phenotypes of BrdU-positive cells expressing NeuN or GFAP in DG (Fig. 5g) were identified among the groups.

The decreased AHN induced by cholinergic degeneration of MS was enhanced by FUS at 18 days. Interestingly, gliogenesis was not affected by FUS.

To investigate the effects of FUS on memory and cognitive function, rats (n = 8/group) underwent training for MWM for 5 consecutive days (14 days post-modeling). All groups showed a gradual decrease in escape latency to the platform from the first day to the last day of the training (Fig. 6a).

In the MWM probe test, rats in the SAP group made significantly a lower number of crossing over (0.62 ± 0.26; P < 0.01) and spent less time in the platform area (0.47 ± 0.16; P < 0.05) compared with those in the control group (crossing over, 3.37 ± 0.7; platform area, 1.44 ± 0.32). In contrast, rats in the SAP+FUS group showed a significantly higher number of crossing over (3.12 ± 0.61; P < 0.05) and time in the platform area (1.51 ± 0.21; P < 0.05) compared with those in the control group (Fig. 6b, c). However, the time spent in the target quadrant and movement speed were not significantly different among the groups (Fig. 6d, e).

These results implicate that cholinergic degradation in MS reduces the memory and cognitive function, but FUS can reverse the impairment.