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Transcranial ultrasound stimulation promotes brain-derived neurotrophic factor and reduces apoptosis in a mouse model of traumatic brain injury
Accepted Manuscript Transcranial ultrasound stimulation promotes brain-derived neurotrophic factor and reduces apoptosis in a mouse model of traumatic brain injury Wei-Shen Su, Chun-Hu Wu, Szu-Fu Chen, Feng-Yi Yang PII:
S1935-861X(17)30895-1
DOI:
10.1016/j.brs.2017.09.003
Reference:
BRS 1103
To appear in:
Brain Stimulation
Received Date: 31 March 2017 Revised Date:
31 July 2017
Accepted Date: 2 September 2017
Please cite this article as: Su W-S, Wu C-H, Chen S-F, Yang F-Y, Transcranial ultrasound stimulation promotes brain-derived neurotrophic factor and reduces apoptosis in a mouse model of traumatic brain injury, Brain Stimulation (2017), doi: 10.1016/j.brs.2017.09.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical Abstract
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LIPUS stimulation increased the protein levels of BDNF through TrkB/Akt-CREB signaling in a mouse model of traumatic brain injury.
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Transcranial
Ultrasound
Stimulation
Promotes
Brain-derived
Neurotrophic Factor and Reduces Apoptosis in a Mouse Model of Traumatic Brain Injury
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Wei-Shen Su1#, Chun-Hu Wu2#, Szu-Fu Chen3,4*, Feng-Yi Yang1,5*
1
Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan 2
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Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan 3 Departments of Physiology and Biophysics, National Defense Medical Center, Taipei, Taiwan 4 Department of Physical Medicine and Rehabilitation, Cheng Hsin General Hospital, Taipei, Taiwan 5 Biophotonics and Molecular Imaging Research Center, National Yang-Ming University, Taipei, Taiwan These authors contributed equally to this work.
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*Correspondence to: Feng-Yi Yang, Ph.D. Professor, Department of Biomedical Imaging and Radiological Sciences, School of Biomedical Science and Engineering, National Yang-Ming University, Taipei, Taiwan No. 155, Sec. 2, Li-Nong St., Taipei 11221, Taiwan
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Tel: 886-2-2826-7281, Fax: 886-2-2820-1095 E-mail: fyyang@ ym.edu.tw *Co-correspondence to : Szu-Fu Chen, MD & Ph.D. Associate Professor, Department of Physical Medicine and Rehabilitation, Cheng Hsin General Hospital, Taipei, Taiwan No. 45, Cheng Hsin St., Taipei 11221, Taiwan Tel: 886-2-2826-4400, Fax: 886-2-2826-7400 E-mail: [email protected] Running Title: Protective effects of ultrasound on TBI 1
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ABSTRACT Background: The protein expressions of brain-derived neurotrophic factor (BDNF)
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can be elevated by transcranial ultrasound stimulation in the rat brain. Objective: The purpose of this study was to investigate the effects and underlying mechanisms of BDNF enhancement by low-intensity pulsed ultrasound (LIPUS) on
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traumatic brain injury (TBI).
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Methods: Mice subjected to controlled cortical impact injury were treated with LIPUS in the injured region daily for a period of 4 days. Western blot analysis and immunohistochemistry were performed to assess the effects of LIPUS. Results: The results showed that the LIPUS treatment significantly promoted the
after
TBI.
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neurotrophic factors BDNF and vascular endothelial growth factor (VEGF) at day 4 Meanwhile,
LIPUS
also
enhanced
the
phosphorylation
of
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Tropomyosin-related kinase B (TrkB), Akt, and cAMP-response element binding
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protein (CREB). Furthermore, treatment with LIPUS significantly decreased the level of cleaved caspase-3. The reduction of apoptotic process was inhibited by the anti-BDNF antibody.
Conclusions: In short, post-injury LIPUS treatment increased BDNF protein levels and inhibited the progression of apoptosis following TBI. The neuroprotective effects of LIPUS may be associated with enhancements of the protein levels of neurotrophic
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ACCEPTED MANUSCRIPT factors, at least partially via the TrkB/Akt-CREB signaling pathway.
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Keywords: Ultrasound; Traumatic brain injury; Neurotrophic factor; BDNF;
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Apoptosis
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Introduction Traumatic brain injury (TBI) triggers a complex cascade of apoptotic events that
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cause delayed secondary injury processes [1]. Clinically, cleavage of caspase-1, caspase-3, and caspase-8 were observed in human brain after TBI, suggesting activation of caspase-dependent apoptosis [2, 3]. It has been shown that caspase were
protective
against
TBI
[4].
Furthermore,
the
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inhibitors
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phosphoinositide-3-kinase (PI3K)/Akt signaling pathway plays a crucial role in regulating cell survival. Activation of Akt involves phosphorylation on both Thr308 and Ser473, and then p-Akt functions through its kinase activity. Activated Akt phosphorylates several downstream proteins to prevent apoptosis. Thus, the activation
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of Akt signaling may be a useful strategy for protection of the injured brain [5]. Neuroprotection is a potential approach for the treatment of brain injuries, but no
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therapeutic agents have been shown to be effective for TBI in clinical trials [6]. At the
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same time, a lack of sufficient pharmacokinetic analysis to determine the optimal doses and therapeutic windows for therapeutic agents may limit potential proof of their clinical efficacy [7]. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, which plays an important role in the survival of existing neurons, the differentiation of new neurons, and synaptic plasticity [8, 9]. BDNF is involved in neuroprotection, neuronal repair, and functional recovery after TBI [10,
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ACCEPTED MANUSCRIPT 11]. Glial cell line-derived neurotrophic factor (GDNF) is a small protein that has been found to improve survival, promote neuronal differentiation, and reduce
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apoptotic cells following TBI [12]. Vascular endothelial growth factor (VEGF) significantly augments neurogenesis and angiogenesis after TBI [13]. However, exogenous BDNF administration following TBI does not protect against behavioral or
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the injured regions of the brain has been problematic.
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histological deficits [14]. Moreover, assuring the delivery of neurotrophic factors to
The injured brain activates self-protective mechanisms to counteract cerebral damage and promote neuronal survival [15]. Tropomyosin-related kinase B (TrkB) signaling is activated by binding to BDNF, which is one of the important protective
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mechanisms induced by brain damage and a key regulator of neuronal survival [16]. PI3K/Akt and Erk signaling pathways are the major TrkB-mediated survival pathways
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that promote neuronal survival and protect against apoptosis [17]. BDNF/TrkB
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signaling can enhance further BDNF induction through cAMP-response element binding protein (CREB), a key transcription factor for BDNF production via PI3K/Akt or Erk signaling [18, 19]. These data suggest the modulation of BDNF/TrkB signaling have a therapeutic role in brain injury. Ultrasound (US) can induce bioeffects by acting as high intensity or low intensity energy as it propagates through tissues in pulsed or continuous waves [20,
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ACCEPTED MANUSCRIPT 21]. It has been demonstrated that low-intensity pulsed US (LIPUS) could be a powerful neuromodulation tool [22, 23]. Experimental studies indicated that LIPUS
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has neuroprotective effects against cerebral damages in terms of myelin loss and apoptosis induced by AlCl3 through enhancement of neurotrophic factors [24, 25]. Evidence suggests that elevated levels of BDNF in the brain have protective effects
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against TBI [26]. Meanwhile, an increase in neurotrophic factors such as VEGF can
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improve functional outcomes and reduce lesion volume in TBI [13]. Therefore, the goal of the present study was to investigate whether LIPUS stimulation could promote the TrkB downstream PI3K/Akt or Erk pathways and increase endogenous BDNF
model of TBI.
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levels and to determine whether LIPUS is protective against apoptosis in a mouse
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Materials and methods
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Animals and Surgical Procedures All procedures were approved according to guidelines stipulated by the Animal
Care and Use Committee of National Yang Ming University. The TBI model was induced by controlled cortical impact (CCI) injury in mice as described previously [27]. Male C57BL/6J mice (8 weeks old, about 22-25 g in weight) were intraperitoneally anesthetized with sodium pentobarbital (65 mg/kg; Rhone Merieux,
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ACCEPTED MANUSCRIPT Harlow, UK) and placed in a stereotaxic frame. A 5 mm craniotomy was performed over the right parietal cortex, centered on the coronal suture and 0.1 mm lateral to the
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sagittal suture, and injury to the dura was avoided. Injury was produced by a pneumatic piston with a rounded metal tip (2.5 mm in diameter) that was angled at 22.5° to the vertical so that the tip was perpendicular with the brain surface at the
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center of the craniotomy. A velocity of 4 m/s and a deformation depth of 2 mm below
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the dura were applied. The bone flap was immediately replaced and sealed, and the scalp was sutured closed. Mice were placed in a heated cage to maintain body temperature while recovering from anesthesia. Sham-operated mice received craniotomy as described before, but without CCI; the impact tip was placed lightly on
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the dura before sealing the wound. After the trauma or sham surgery, animals were
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housed under the conditions mentioned above.
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Pulsed Ultrasound Apparatus
The pulsed ultrasound setup was similar to that used in our previous study [28].
LIPUS exposures were generated by a 1.0-MHz, single-element focused transducer (A392S, Panametrics, Waltham, MA, USA) with a diameter of 38 mm and a radius of curvature of 63.5 mm. The half-maximum of the pressure amplitude of the focal zone had a diameter and length of 3 mm and 26 mm, respectively. The transducer was
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ACCEPTED MANUSCRIPT applied with a duty cycle of 5% and a repetition frequency of 1 Hz. The transducer was mounted on a removable cone filled with deionized and degassed water whose tip
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was capped by a polyurethane membrane, and the center of the focal zone was about 2.0 mm away from the cone tip. The mice were anesthetized with isoflurane mixed with oxygen during the sonication procedure. The sonication was precisely targeted
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using a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). The acoustic wave
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was delivered to the targeted region in the injured cortical areas. LIPUS was applied for a sonication time of 5 min at an acoustic power of 0.51 W (corresponding to a spatial-peak temporal-average intensity (ISPTA) of 528 mW/cm2) 5 mins after TBI and subsequently daily for a period of 3 days. Mice were sacrificed for analysis at 1 or 4
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days. The intensity of the LIPUS exposures was selected based on data from our
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previous studies [25, 29].
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Histological Evaluation
Four days following TBI, mice were sacrificed by transcardial perfusion with
phosphate-buffered saline (PBS), and then the tissues were fixed with 4% paraformaldehyde. Brains were collected and post-fixed in 4% paraformaldehyde overnight and transferred to PBS containing 30% sucrose for cryoprotection. Coronal sections were cut in a cryostat at 10 µm from the level of the olfactory bulbs to the
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ACCEPTED MANUSCRIPT visual cortex and used for immunohistochemistry. Double immunofluorescence was performed by simultaneous incubation of either anti-BDNF (1:100; Sanata Cruz) or
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anti-phospho-TrkB (1:200; Cell Signaling, MA, USA) with anti-neuronal nuclei antigen (NeuN, neuronal marker; 1:100; Millipore, Billerica, MA, USA) overnight at 4°C. Sections were then washed, incubated with Alexa Fluor 488- or Alexa Fluor
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594-conjugated secondary antibodies (1:400; Molecular Probes, Eugene, OR, USA)
Quantification of double staining
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for 2 h, observed under a fluorescence microscope, and photographed.
Double immunofluoresence labeling of neurons (NeuN) and BDNF was
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quantified on three consecutive sections from the injury core at the level of 0.74 mm from the bregma. The number of positive cells was counted in an area of 920 × 860
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µm2 in 8-10 non-overlapping fields immediately adjacent to the cortical contusion
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margin using a magnification of ×200 as previously described [30]. The total number of NeuN-positive or NeuN-BDNF-double-label cells was expressed as the mean number per field of view. Analysis was performed by two experimenters who were blinded to all animal groups. Inter-rater reliability was within 10 %.
Western blotting analysis
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ACCEPTED MANUSCRIPT One and 4 days after TBI, a 4-mm coronal section was taken from the injured area over the parietal cortex and then homogenized by T-Per extraction reagent
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supplemented with the Halt Protease Inhibitor Cocktail (Pierce Biotechnology, Inc.). Lysates were centrifuged and the supernatants were harvested, and protein concentrations were assayed with Protein Assay Reagent (Bio-Rad, CA, USA).
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Samples containing 30 µg protein were resolved on 12% sodium dodecyl sulfate
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polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immun-Blot® polyvinyldifluoride (PVDF) membranes (Bio-Rad, CA, USA). After blotting, the membranes were blocked for at least 1 h in blocking buffer (Hycell, Taipei, Taiwan), and then the blots were incubated overnight at 4°C in a solution with antibodies
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against BDNF (1:250, sc-546), GDNF (1:250, sc-328), VEGF (1:250, sc-152), and t-TrkB (1:2000, sc-8316) from Santa Cruz; cleaved caspase-3 (cCP3, #9661),
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p-Akt473 (#9271), p-Akt308 (#4056), t-Akt (#9272), p-Erk44/42 (#9101), t-Erk44/42
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(#9102), p-CREB (#9198), and t-CREB (#9197), all at 1:1000 dilution, from Cell Signaling; and p-TrkB (1:1000, ab52191) from Abcam (MA, USA). After being washed with PBST buffer, the membrane was incubated with the secondary antibodies for 1h at room temperature. After being washed with PBST buffer, signals were developed using a Western Lightning ECL reagent Pro (Bio-Rad, California, USA). The gel image was captured using an ImageQuant™ LAS 4000 biomolecular imager
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Intracerebroventricular injection of anti-BDNF antibody
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system (ImageJ) to estimate the integral optical density of the protein bands.
In this study, the rabbit anti-BDNF antibody (100 µg/ml; sc-546, Santa Cruz)
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was used to block the BDNF-TrkB system. The 2 µl of neutralizing BDNF antibody
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or an equal volume of control IgG (100 µg/ml; I5006, Sigma-Aldrich, MO, USA) was intracerebroventricularly (icv) injected at 30 min before CCI as previously described [31]. Briefly, a 30-gauge needle of a Hamilton syringe was inserted into the lateral ventricle (0.5 mm posterior to the bregma, 1 mm right lateral to the midline, and 2
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mm in depth). Then anti-BDNF antibody or control IgG was infused with an infusion pump at a rate of 0.2 µl/min for 10 min. The needle was removed 20 min after the
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thereafter.
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infusion to prevent reflux, and the CCI procedure was performed immediately
Statistics
All data are shown as means ± standard error of the mean (SEM). For comparisons among multiple groups, one-way analysis of variance (ANOVA), followed by Tukey’s test, was used to determine significant differences. Differences
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ACCEPTED MANUSCRIPT between two groups were performed using Student’s t test. The level of statistical
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significance was set at p value ≤ 0.05.
Results
Ultrasound Stimulation Increases Neurotrophic Factor Expression after TBI
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To determine whether LIPUS would trigger neurotrophic factor production
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following TBI, the protein levels of BDNF, VEGF, and GDNF were measured in the brain (Fig. 1). The protein levels of BDNF and VEGF were significantly decreased in the ipsilateral cortex at day 1 after TBI compared with the sham group, but LIPUS significantly increased VEGF to 149% of the TBI-level in the ipsilateral cortex
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(p<0.001; Fig. 1B). In addition, the protein levels of BDNF, VEGF, and GDNF were all significantly decreased in the ipsilateral cortex at day 4 after TBI compared with
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the sham group. LIPUS caused significant increases of 256% and 60% in the protein
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levels of BDNF and VEGF, respectively, at day 4 after TBI compared with the non-treated group (both p<0.001; Figs. 1A and B). However, there was no difference in GDNF protein level at both 1 and 4 days post-TBI between the non-treated and LIPUS-treated groups (Fig. 1C).
Ultrasound Stimulation Increases BDNF Expression in Neurons after TBI
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ACCEPTED MANUSCRIPT We used double immunofluorescence to further analyze whether LIPUS affected BDNF expression in neurons (Fig. 2A). LIPUS treatment significantly increased the
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total number of neurons and BDNF-positive neurons (Fig. 2B). The percentage of BDNF-positive neurons was also increased following LIPUS treatment. These data suggest that enhanced levels of neuronal BDNF protein contributes to the protective
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effects of LIPUS treatment.
Ultrasound Stimulation Enhances Activation of TrkB and Downstream Akt Signaling, but Does Not Affect Erk Signaling after TBI
We examined the phosphorylation of TrkB and the activation of its major
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downstream survival signaling pathways (the Akt and Erk pathways). CCI induced a significant decrease in the TrkB phosphorylation level at both day 1 and day 4
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compared with the sham group (both p<0.01). LIPUS significantly increased TrkB
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phosphorylation to 136% of the non-treated TBI-level at day 4 (p=0.016; Fig. 3A). Likewise, levels of Akt Thr308 and Akt Ser473 phosphorylation were significantly diminished following TBI at either day 1 or day 4 (both p<0.05; Fig. 3B). The levels of both Akt phosphorylation forms were significantly higher in the LIPUS-treated TBI group than in the non-treated TBI group at day 4 (phospho-Akt Thr308: 172% of non-treated TBI, p=0.014; phospho-Akt Ser473: 185% of non-treated TBI, p=0.003;
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following LIPUS at either time point (all P > 0.05; Fig. 3C).
Ultrasound Stimulation Enhances Activation of CREB and TrkB Phosphorylation in Neurons after TBI
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We further evaluated the phosphorylation levels of CREB, which are
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downstream factors of Akt. Similar to the pattern observed for Akt phosphorylation, the CREB phosphorylation levels significantly increased following LIPUS (179% and 361% of non-treated TBI, p=0.032 and 0.02; Fig. 4A) at 4 days post-injury. To further investigate whether the TrkB activation induced by LIPUS directly occurred in
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neurons, we examined the localization of the phospho-TrkB following CCI. TrkB phosphorylation was found in neurons around the impact site, and increased following
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LIPUS treatment at 4 days (Fig. 4B).
Ultrasound Stimulation Reduces Apoptotic process after TBI We next assessed whether LIPUS reduced post-traumatic apoptosis. Cleaved
caspase-3 protein level was low in the sham-operated brains but it was induced significantly by CCI at 1 and 4 days post-injury. LIPUS significantly decreased the level of cleaved caspase-3, a final effector of apoptotic death, by 40% at day 1
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TBI.
Neutralization of BDNF Attenuates LIPUS-induced Neuroprotection after TBI
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To further confirm that BDNF/TrkB signaling contributes to the beneficial effect
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of LIPUS, a neutralizing antibody against BDNF was administered to LIPUS-treated CCI mice. The ability of the anti-BDNF antibody to block the action of BDNF was validated with Western blot analysis, in which BDNF-mediated TrkB phosphorylation was prevented by the anti-BDNF antibody but not by control IgG (Fig. 6B).
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Treatment with the neutralizing antibody significantly reduced LIPUS-induced TrkB phosphorylation at 4 days post-TBI (Fig. 6C). However, mice treated with the
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neutralizing antibody alone in the absence of LIPUS stimulation showed no difference
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in TrkB phosphorylation compared with mice treated with antibody and LIPUS. The total TrkB levels in brains treated with anti-BDNF antibody were reduced compared with those in control IgG-treated brains (Fig. 6C). One potential reason for the reduction in TrkB levels is the cellular response to the lack of ligand (BDNF) in neurons treated with the BDNF neutralizing antibody, a finding that was previously reported [32]. The protective effects of LIPUS against trauma-induced apoptosis were
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ACCEPTED MANUSCRIPT inhibited by the anti-BDNF antibody. There was no difference in the cleaved caspase-3 level between the BDNF Ab group and BDNF Ab+LIPUS group at 4 days
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post-TBI (Fig. 6D).
Discussion
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In this study, we demonstrated that transcranial LIPUS stimulation elevated the
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protein levels of neurotrophic factors and inhibited the progression of apoptosis in mice subjected to TBI. LIPUS increased BDNF and VEGF protein expression and enhanced
the
activation
of
the
TrkB/Akt-CREB
pathway.
Furthermore,
co-administration of a neutralizing anti-BDNF antibody attenuated the reduction of
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apoptosis by LIPUS treatment, indicating that its therapeutic action is at least partly mediated by the BDNF signaling pathway (Fig. 7). Our results suggest that LIPUS
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stimulation may be a promising new technique for treating TBI.
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US has been widely used for both diagnostic and therapeutic purposes due to its non-ionizing and non-invasive nature. LIPUS is mainly used clinically in the treatment of bone fractures to accelerate the proliferation and differentiation of osteoblasts. Experimental studies have also demonstrated that LIPUS can promote peripheral nerve regeneration. Recently, our studies demonstrated that the brain damage and memory impairments can be improved by LIPUS stimulation in animal
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ACCEPTED MANUSCRIPT models of neurodegenerative diseases [25, 33]. These beneficial effects of LIPUS may be attributed partially to its enhancement of BDNF expression.
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We showed that TBI induced a decrease of the cerebral BDNF protein level and that subsequent LIPUS stimulation for 4 days non-invasively promoted the BDNF protein level and increased the number of BDNF-positive neurons. The observation of
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LIPUS stimulation increased the BDNF protein expression in the injured brain
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extended our previous finding that LIPUS promotes cerebral BDNF protein level in the normal brain [25, 33]. The importance of BDNF to functional recovery following TBI has been documented in a previous clinical study showing that the polymorphisms of human BDNF affect the recovery of cognitive intelligence in
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patients with penetrating TBI [34]. Accumulating experimental evidence also indicates the critical role of BDNF signaling in promoting neuronal survival. BDNF
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mediates its effect through its high affinity for the TrkB receptor. The activation of
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TrkB triggers the downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which is the major TrkB-mediated survival pathway against TBI [27, 31]. We observed that 4 days of LIPUS stimulation increased TrkB and Akt phosphorylation, along with increasing the phosphorylation of CREB, the Akt downstream targets. In addition, LIPUS reduced the cleaved caspase-3 level in the injured brain. Neutralizing BDNF by using the BDNF neutralizing antibody
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signaling. Given that the therapeutic potential of recombinant BDNF is limited due to its short half-life and poor BBB penetration [35], the enhancement of endogenous BDNF via LIPUS could be a novel therapeutic strategy for TBI and various
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neurological diseases related to the dysregulation of BDNF.
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Instead of the thermal contribution, changes in transmembrane capacitance and subsequent excitation of neural cells due to the exposure to the acoustic pressure waves may serve as a probable contributing factor for enhancement of BDNF secretion
[36].
LIPUS-mediated
mechanical
activation
of
glial
cells
via
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mechanoreceptors may be another potential mechanism. The mechanisms involved in LIPUS-stimulated BDNF protein expression in damaged brains have not been
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elucidated yet; however, previous studies have demonstrated that LIPUS can enhance
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the BDNF protein level in cultured astrocytes through the activation of integrin receptors and NFκB signaling [24, 37]. Integrins, which are transmembrane receptors that connect the cytoskeleton to the extracellular matrix, are involved in the transduction of mechanical stimuli to biochemical signals [38]. US stimuli are transmitted to adherent cells via their contact with surrounding extracellular matrices, and integrins may transmit US signals through the cytoskeleton and activate several
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ACCEPTED MANUSCRIPT downstream protein kinases [38, 39]. In this regard, LIPUS stimulation has been demonstrated to increase the expression of integrins in cultured chondrocytes [39],
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while an integrin inhibitor was shown to attenuate LIPUS-induced BDNF expression in cultured astrocytes [24]. The activation of NF-κB has also been demonstrated to contribute to LIPUS-induced BDNF expression in astrocytes [37]. Since
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astrocyte-derived BDNF is a source of trophic support that can be used to reduce
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damage induced by brain injury [40], it is possible that LIPUS promotes neuronal survival by inducing astrocyte-derived BDNF expression in the damaged brain. The mechanisms underlying the interaction between astrocytes and neurons following LIPUS stimulation in the damaged brain will be the subject of further investigation.
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We also observed that LIPUS enhanced the protein level of VEGF in injured brains. In this regard, we have recently reported that LIPUS promoted VEGF protein
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levels in normal brains [25] and cultured astrocytes [24]. Previous studies in animal
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models of myocardial ischemia also demonstrated that LIPUS upregulated the protein expression of VEGF and eNOS and induced angiogenesis [41]. Since VEGF has been reported to regulate neuronal survival, neurogenesis, and angiogenesis [42], it is possible that LIPUS stimulation contributes to neurovascular remodeling, leading to improved neurobehavioral outcomes following TBI. This point remains to be examined in future studies.
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Conclusion
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In conclusion, we demonstrated that post-injury LIPUS treatment enhanced the protein levels of neurotrophic factors and attenuated apoptotic process in mouse TBI. The enhancement of BDNF with LIPUS may be related to modulation of the
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TrkB/Akt-CREB signaling pathway. Thus, LIPUS stimulation may play a crucial and
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beneficial role in TBI patients.
Acknowledgments
This study was supported by grants from the Ministry of Science and Technology of
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Taiwan (no. MOST 105-2221-E-010-003, MOST 104-2314-B-010-003-MY3, and 101-2314-B-350-001-MY3), the Veterans General Hospitals University System of
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Taiwan Joint Research Program (#VGHUST106-G7-6-1), the Cheng Hsin General
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Hospital Foundation (no. CY10622, CY10418 and CHGH103-34), and the Taiwan Ministry of Education’s Aim for the Top University Plan.
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BDNF production by activation of MAPK-CREB pathway in rat primary cortical neuronal culture. Neurosci Res 2011;69:214-22. [20] Dalecki D. Mechanical bioeffects of ultrasound. Annu Rev Biomed Eng 2004;6:229-48. [21] ter Haar G. Therapeutic applications of ultrasound. Prog Biophys Mol Biol 2007;93:111-29.
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ACCEPTED MANUSCRIPT [22] Fry WJ. Electrical stimulation of brain localized without probes--theoretical analysis of a proposed method. J Acoust Soc Am 1968;44:919-31.
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[23] Gavrilov LR, Tsirulnikov EM, and Davies IA. Application of focused ultrasound for the stimulation of neural structures. Ultrasound Med Biol 1996;22:179-92. [24] Yang FY, Lu WW, Lin WT, Chang CW, and Huang SL. Enhancement of
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Using Low-intensity Pulsed Ultrasound Stimulation. Brain Stimul 2015;8:465-73. [25] Lin WT, Chen RC, Lu WW, Liu SH, and Yang FY. Protective effects of low-intensity pulsed ultrasound on aluminum-induced cerebral damage in Alzheimer's disease rat model. Sci Rep 2015;5:9671.
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[27] Chen SF, Tsai HJ, Hung TH, Chen CC, Lee CY, Wu CH, et al. Salidroside improves behavioral and histological outcomes and reduces apoptosis via PI3K/Akt signaling after experimental traumatic brain injury. PloS One 2012;7:e45763. [28] Yang FY, Chang WY, Chen JC, Lee LC, and Hung YS. Quantitative assessment of cerebral glucose metabolic rates after blood-brain barrier disruption induced by focused ultrasound using FDG-MicroPET. Neuroimage 2014;90:93-8.
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ACCEPTED MANUSCRIPT [29] Su WS, Tsai ML, Huang SL, Liu SH, and Yang FY. Controllable permeability of blood-brain barrier and reduced brain injury through low-intensity pulsed ultrasound
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stimulation. Oncotarget 2015;6:42290-9. [30] Chen CC, Hung TH, Lee CY, Wang LF, Wu CH, Ke CH, et al. Berberine protects against neuronal damage via suppression of glia-mediated inflammation in traumatic
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[31] Wu CH, Hung TH, Chen CC, Ke CH, Lee CY, Wang PY, et al. Post-injury treatment with 7,8-dihydroxyflavone, a TrkB receptor agonist, protects against experimental traumatic brain injury via PI3K/Akt signaling. PloS One 2014;9:e113397.
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[32] Pandya CD and Pillai A. TrkB interacts with ErbB4 and regulates NRG1-induced NR2B phosphorylation in cortical neurons before synaptogenesis. Cell Commun
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[33] Huang SL, Chang CW, Lee YH, and Yang FY. Protective Effect of Low-Intensity Pulsed Ultrasound on Memory Impairment and Brain Damage in a Rat Model of Vascular Dementia. Radiology 2017;282:113-22. [34] Rostami E, Krueger F, Zoubak S, Dal Monte O, Raymont V, Pardini M, et al. BDNF polymorphism predicts general intelligence after penetrating traumatic brain injury. PloS One 2011;6:e27389.
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ACCEPTED MANUSCRIPT [35] Poduslo JF and Curran GL. Permeability at the blood-brain and blood-nerve barriers of the neurotrophic factors: NGF, CNTF, NT-3, BDNF. Brain Res Mol Brain
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Res 1996;36:280-6. [36] Krasovitski B, Frenkel V, Shoham S, and Kimmel E. Intramembrane cavitation
as a unifying mechanism for ultrasound-induced bioeffects. Proc Natl Acad Sci U S A
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2011;108:3258-63.
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[37] Liu SH, Lai YL, Chen BL, and Yang FY. Ultrasound Enhances the Expression of Brain-Derived Neurotrophic Factor in Astrocyte Through Activation of TrkB-Akt and Calcium-CaMK Signaling Pathways. Cereb Cortex 2016.
[38] Humphries MJ. Integrin structure. Biochem Soc Trans 2000;28:311-39.
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[39] Xia P, Ren S, Lin Q, Cheng K, Shen S, Gao M, et al. Low-Intensity Pulsed Ultrasound Affects Chondrocyte Extracellular Matrix Production via an
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Integrin-Mediated p38 MAPK Signaling Pathway. Ultrasound Med Biol
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[40] Fulmer CG, VonDran MW, Stillman AA, Huang Y, Hempstead BL, and Dreyfus CF. Astrocyte-derived BDNF supports myelin protein synthesis after cuprizone-induced demyelination. J Neurosci 2014;34:8186-96. [41] Hanawa K, Ito K, Aizawa K, Shindo T, Nishimiya K, Hasebe Y, et al. Low-intensity pulsed ultrasound induces angiogenesis and ameliorates left ventricular
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[42] Greenberg DA and Jin K. From angiogenesis to neuropathology. Nature
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2005;438:954-9.
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Figure captions Figure 1. Effects of LIPUS treatment on neurotrophic factor expression in TBI mice. Representative western blots and optical densitometric quantification of (A) BDNF,
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(B) VEGF, and (C) GDNF in the ipsilateral hemisphere of sham-injured, LIPUS-treated sham, non-treated TBI, and LIPUS-treated TBI mice at 1 and 4 days
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post-injury. The protein expressions of BDNF and VEGF were enhanced by LIPUS treatment in the traumatized brain. * and # denote significantly different from sham
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and non-treated TBI group, respectively (***,###, p<0.001, n = 6-7).
Figure 2. Identification of BDNF-positive neurons at 4 days post-injury in the
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peri-contusional margin by double immunofluorescence staining. (A) BDNF is shown in red, and NeuN (neurons) is shown in green. Co-localization of BDNF with NeuN is
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shown by yellow labeling. The representative sections were taken from the injury core
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at the level of 0.74 mm from the bregma. Sections were stained with DAPI (blue) to show all nuclei. (B) LIPUS significantly increased the total number of neurons and BDNF-positive neurons, as well as the percentage of BDNF-positive neurons. The number of NeuN-positive cells and BDNF-positive neurons is expressed as the mean number per field of view (0.8 mm2 ). # denote significantly different from non-treated TBI group (##, p<0.01; ###, p<0.001, n = 6-7).
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ACCEPTED MANUSCRIPT Figure 3. Effects of LIPUS treatment on TrkB and downstream Akt and Erk signaling in TBI mice. Representative western blots and optical densitometric quantification of
hemisphere
of
sham-injured,
LIPUS-treated
sham,
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(A) phospho-TrkB, (B) phospho-Akt, and (C) phospho-Erk 1/2 in the ipsilateral non-treated
TBI,
and
LIPUS-treated TBI mice at 1 and 4 days post-injury. LIPUS significantly enhanced
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phosphorylation of TrkB, Akt at Thr308, and Akt at Ser473 at 4 days. Erk 1/2
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phosphorylation was not affected by LIPUS at either time point. * and # denote significantly different from sham and non-treated TBI group, respectively (*,#, p p<0.01; ***,
p<0.001, n = 6-7).
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<0.05; **,##,
Figure 4. Effects of LIPUS treatment on phosphorylation of CREB and TrkB in TBI mice. Representative western blots and optical densitometric quantification of (A)
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phospho-CREB in the ipsilateral hemisphere of sham-injured, LIPUS-treated sham,
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non-treated TBI, and LIPUS-treated TBI mice at 1 and 4 days post-injury. LIPUS significantly enhanced phosphorylation of CREB at 4 days. * and # denote significantly different from sham and non-treated TBI group, respectively (#, p <0.05; **, p<0.01; n = 6-7). (B) Cellular localization of phospho-TrkB in the peri-contusional margin at 4 days post-TBI observed by immunofluorescence labeling. Phospho-TrkB is shown in red, and NeuN (neurons) is shown in green. Colocalization
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Sections were stained with DAPI (blue) to show all nuclei.
Figure 5. Effects of LIPUS treatment on apoptosis expression in TBI mice.
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Representative western blots and optical densitometric quantification of cleaved
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caspase-3 in the ipsilateral hemisphere of sham-injured, LIPUS-treated sham, non-treated TBI, and LIPUS-treated TBI mice at 1 and 4 days post-injury. LIPUS significantly decreased the cleaved caspase-3 level at both 1 and 4 days post-injury. * and # denote significantly different from sham and non-treated TBI group, p<0.01; ***,###, p<0.001, n = 6-7).
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respectively (##,
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Figure 6. Neutralization of BDNF attenuates LIPUS-induced neuroprotection in TBI
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mice. (A) Experimental design. icv: intracerebroventricularly; WB: western blots. (B) Representative western blots of phospho-TrkB in the ipsilateral hemisphere of a non-treated TBI, a control IgG-treated TBI, and a BDNF Ab-treated TBI mouse at 4 days post-injury. TrkB phosphorylation was attenuated by the anti-BDNF antibody but not by the control IgG. Representative western blots and optical densitometric quantification of (C) phospho-TrkB and (D) cleaved caspase-3 in the ipsilateral
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ACCEPTED MANUSCRIPT hemisphere of LIPUS+IgG, BDNF Ab, and LIPUS+BDNF Ab mice at 4 days post-injury. Treatment with anti-BDNF Ab significantly reduced LIPUS-induced TrkB
LIPUS+IgG group at day 4 post-TBI.
denotes significantly different from
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LIPUS+IgG TBI group (§, p<0.05; n = 6-7).
§
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phosphorylation and increased cleaved caspase-3 level compared with the
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Figure 7. Schematic diagram of the mechanisms involved in LIPUS-induced protection in a TBI mouse. LIPUS stimulation increased the protein levels of BDNF and VEGF, in addition to enhancing the phosphorylation of TrkB, Akt, and CREB. Treatment with BDNF neutralizing antibody inhibited the protective effects of LIPUS
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against trauma-induced apoptosis. Taken together, post-traumatic LIPUS stimulation
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promotes BDNF production and reduces apoptosis following TBI.
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ACCEPTED MANUSCRIPT Highlights: Low-intensity pulsed ultrasound (LIPUS) stimulation increased the protein levels of BDNFand VEGF in a mouse model of traumatic brain injury (TBI).
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LIPUS stimulation inhibited the progression of apoptosis following TBI.
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The neuroprotective effects of LIPUS may be associated with enhancements of the protein levels of BDNF, at least partially via the TrkB/Akt-CREB signaling pathway.
S1935-861X(17)30895-1
DOI:
10.1016/j.brs.2017.09.003
Reference:
BRS 1103
To appear in:
Brain Stimulation
Received Date: 31 March 2017 Revised Date:
31 July 2017
Accepted Date: 2 September 2017
Please cite this article as: Su W-S, Wu C-H, Chen S-F, Yang F-Y, Transcranial ultrasound stimulation promotes brain-derived neurotrophic factor and reduces apoptosis in a mouse model of traumatic brain injury, Brain Stimulation (2017), doi: 10.1016/j.brs.2017.09.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical Abstract
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LIPUS stimulation increased the protein levels of BDNF through TrkB/Akt-CREB signaling in a mouse model of traumatic brain injury.
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Transcranial
Ultrasound
Stimulation
Promotes
Brain-derived
Neurotrophic Factor and Reduces Apoptosis in a Mouse Model of Traumatic Brain Injury
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Wei-Shen Su1#, Chun-Hu Wu2#, Szu-Fu Chen3,4*, Feng-Yi Yang1,5*
1
Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan 2
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Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan 3 Departments of Physiology and Biophysics, National Defense Medical Center, Taipei, Taiwan 4 Department of Physical Medicine and Rehabilitation, Cheng Hsin General Hospital, Taipei, Taiwan 5 Biophotonics and Molecular Imaging Research Center, National Yang-Ming University, Taipei, Taiwan These authors contributed equally to this work.
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#
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*Correspondence to: Feng-Yi Yang, Ph.D. Professor, Department of Biomedical Imaging and Radiological Sciences, School of Biomedical Science and Engineering, National Yang-Ming University, Taipei, Taiwan No. 155, Sec. 2, Li-Nong St., Taipei 11221, Taiwan
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Tel: 886-2-2826-7281, Fax: 886-2-2820-1095 E-mail: fyyang@ ym.edu.tw *Co-correspondence to : Szu-Fu Chen, MD & Ph.D. Associate Professor, Department of Physical Medicine and Rehabilitation, Cheng Hsin General Hospital, Taipei, Taiwan No. 45, Cheng Hsin St., Taipei 11221, Taiwan Tel: 886-2-2826-4400, Fax: 886-2-2826-7400 E-mail: [email protected] Running Title: Protective effects of ultrasound on TBI 1
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ABSTRACT Background: The protein expressions of brain-derived neurotrophic factor (BDNF)
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can be elevated by transcranial ultrasound stimulation in the rat brain. Objective: The purpose of this study was to investigate the effects and underlying mechanisms of BDNF enhancement by low-intensity pulsed ultrasound (LIPUS) on
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traumatic brain injury (TBI).
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Methods: Mice subjected to controlled cortical impact injury were treated with LIPUS in the injured region daily for a period of 4 days. Western blot analysis and immunohistochemistry were performed to assess the effects of LIPUS. Results: The results showed that the LIPUS treatment significantly promoted the
after
TBI.
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neurotrophic factors BDNF and vascular endothelial growth factor (VEGF) at day 4 Meanwhile,
LIPUS
also
enhanced
the
phosphorylation
of
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Tropomyosin-related kinase B (TrkB), Akt, and cAMP-response element binding
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protein (CREB). Furthermore, treatment with LIPUS significantly decreased the level of cleaved caspase-3. The reduction of apoptotic process was inhibited by the anti-BDNF antibody.
Conclusions: In short, post-injury LIPUS treatment increased BDNF protein levels and inhibited the progression of apoptosis following TBI. The neuroprotective effects of LIPUS may be associated with enhancements of the protein levels of neurotrophic
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Keywords: Ultrasound; Traumatic brain injury; Neurotrophic factor; BDNF;
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Apoptosis
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Introduction Traumatic brain injury (TBI) triggers a complex cascade of apoptotic events that
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cause delayed secondary injury processes [1]. Clinically, cleavage of caspase-1, caspase-3, and caspase-8 were observed in human brain after TBI, suggesting activation of caspase-dependent apoptosis [2, 3]. It has been shown that caspase were
protective
against
TBI
[4].
Furthermore,
the
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inhibitors
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phosphoinositide-3-kinase (PI3K)/Akt signaling pathway plays a crucial role in regulating cell survival. Activation of Akt involves phosphorylation on both Thr308 and Ser473, and then p-Akt functions through its kinase activity. Activated Akt phosphorylates several downstream proteins to prevent apoptosis. Thus, the activation
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of Akt signaling may be a useful strategy for protection of the injured brain [5]. Neuroprotection is a potential approach for the treatment of brain injuries, but no
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therapeutic agents have been shown to be effective for TBI in clinical trials [6]. At the
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same time, a lack of sufficient pharmacokinetic analysis to determine the optimal doses and therapeutic windows for therapeutic agents may limit potential proof of their clinical efficacy [7]. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, which plays an important role in the survival of existing neurons, the differentiation of new neurons, and synaptic plasticity [8, 9]. BDNF is involved in neuroprotection, neuronal repair, and functional recovery after TBI [10,
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ACCEPTED MANUSCRIPT 11]. Glial cell line-derived neurotrophic factor (GDNF) is a small protein that has been found to improve survival, promote neuronal differentiation, and reduce
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apoptotic cells following TBI [12]. Vascular endothelial growth factor (VEGF) significantly augments neurogenesis and angiogenesis after TBI [13]. However, exogenous BDNF administration following TBI does not protect against behavioral or
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the injured regions of the brain has been problematic.
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histological deficits [14]. Moreover, assuring the delivery of neurotrophic factors to
The injured brain activates self-protective mechanisms to counteract cerebral damage and promote neuronal survival [15]. Tropomyosin-related kinase B (TrkB) signaling is activated by binding to BDNF, which is one of the important protective
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mechanisms induced by brain damage and a key regulator of neuronal survival [16]. PI3K/Akt and Erk signaling pathways are the major TrkB-mediated survival pathways
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that promote neuronal survival and protect against apoptosis [17]. BDNF/TrkB
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signaling can enhance further BDNF induction through cAMP-response element binding protein (CREB), a key transcription factor for BDNF production via PI3K/Akt or Erk signaling [18, 19]. These data suggest the modulation of BDNF/TrkB signaling have a therapeutic role in brain injury. Ultrasound (US) can induce bioeffects by acting as high intensity or low intensity energy as it propagates through tissues in pulsed or continuous waves [20,
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has neuroprotective effects against cerebral damages in terms of myelin loss and apoptosis induced by AlCl3 through enhancement of neurotrophic factors [24, 25]. Evidence suggests that elevated levels of BDNF in the brain have protective effects
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against TBI [26]. Meanwhile, an increase in neurotrophic factors such as VEGF can
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improve functional outcomes and reduce lesion volume in TBI [13]. Therefore, the goal of the present study was to investigate whether LIPUS stimulation could promote the TrkB downstream PI3K/Akt or Erk pathways and increase endogenous BDNF
model of TBI.
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levels and to determine whether LIPUS is protective against apoptosis in a mouse
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Materials and methods
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Animals and Surgical Procedures All procedures were approved according to guidelines stipulated by the Animal
Care and Use Committee of National Yang Ming University. The TBI model was induced by controlled cortical impact (CCI) injury in mice as described previously [27]. Male C57BL/6J mice (8 weeks old, about 22-25 g in weight) were intraperitoneally anesthetized with sodium pentobarbital (65 mg/kg; Rhone Merieux,
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ACCEPTED MANUSCRIPT Harlow, UK) and placed in a stereotaxic frame. A 5 mm craniotomy was performed over the right parietal cortex, centered on the coronal suture and 0.1 mm lateral to the
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sagittal suture, and injury to the dura was avoided. Injury was produced by a pneumatic piston with a rounded metal tip (2.5 mm in diameter) that was angled at 22.5° to the vertical so that the tip was perpendicular with the brain surface at the
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center of the craniotomy. A velocity of 4 m/s and a deformation depth of 2 mm below
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the dura were applied. The bone flap was immediately replaced and sealed, and the scalp was sutured closed. Mice were placed in a heated cage to maintain body temperature while recovering from anesthesia. Sham-operated mice received craniotomy as described before, but without CCI; the impact tip was placed lightly on
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the dura before sealing the wound. After the trauma or sham surgery, animals were
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housed under the conditions mentioned above.
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Pulsed Ultrasound Apparatus
The pulsed ultrasound setup was similar to that used in our previous study [28].
LIPUS exposures were generated by a 1.0-MHz, single-element focused transducer (A392S, Panametrics, Waltham, MA, USA) with a diameter of 38 mm and a radius of curvature of 63.5 mm. The half-maximum of the pressure amplitude of the focal zone had a diameter and length of 3 mm and 26 mm, respectively. The transducer was
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was capped by a polyurethane membrane, and the center of the focal zone was about 2.0 mm away from the cone tip. The mice were anesthetized with isoflurane mixed with oxygen during the sonication procedure. The sonication was precisely targeted
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using a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). The acoustic wave
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was delivered to the targeted region in the injured cortical areas. LIPUS was applied for a sonication time of 5 min at an acoustic power of 0.51 W (corresponding to a spatial-peak temporal-average intensity (ISPTA) of 528 mW/cm2) 5 mins after TBI and subsequently daily for a period of 3 days. Mice were sacrificed for analysis at 1 or 4
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previous studies [25, 29].
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Histological Evaluation
Four days following TBI, mice were sacrificed by transcardial perfusion with
phosphate-buffered saline (PBS), and then the tissues were fixed with 4% paraformaldehyde. Brains were collected and post-fixed in 4% paraformaldehyde overnight and transferred to PBS containing 30% sucrose for cryoprotection. Coronal sections were cut in a cryostat at 10 µm from the level of the olfactory bulbs to the
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anti-phospho-TrkB (1:200; Cell Signaling, MA, USA) with anti-neuronal nuclei antigen (NeuN, neuronal marker; 1:100; Millipore, Billerica, MA, USA) overnight at 4°C. Sections were then washed, incubated with Alexa Fluor 488- or Alexa Fluor
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594-conjugated secondary antibodies (1:400; Molecular Probes, Eugene, OR, USA)
Quantification of double staining
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for 2 h, observed under a fluorescence microscope, and photographed.
Double immunofluoresence labeling of neurons (NeuN) and BDNF was
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quantified on three consecutive sections from the injury core at the level of 0.74 mm from the bregma. The number of positive cells was counted in an area of 920 × 860
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µm2 in 8-10 non-overlapping fields immediately adjacent to the cortical contusion
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margin using a magnification of ×200 as previously described [30]. The total number of NeuN-positive or NeuN-BDNF-double-label cells was expressed as the mean number per field of view. Analysis was performed by two experimenters who were blinded to all animal groups. Inter-rater reliability was within 10 %.
Western blotting analysis
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ACCEPTED MANUSCRIPT One and 4 days after TBI, a 4-mm coronal section was taken from the injured area over the parietal cortex and then homogenized by T-Per extraction reagent
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supplemented with the Halt Protease Inhibitor Cocktail (Pierce Biotechnology, Inc.). Lysates were centrifuged and the supernatants were harvested, and protein concentrations were assayed with Protein Assay Reagent (Bio-Rad, CA, USA).
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Samples containing 30 µg protein were resolved on 12% sodium dodecyl sulfate
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polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immun-Blot® polyvinyldifluoride (PVDF) membranes (Bio-Rad, CA, USA). After blotting, the membranes were blocked for at least 1 h in blocking buffer (Hycell, Taipei, Taiwan), and then the blots were incubated overnight at 4°C in a solution with antibodies
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against BDNF (1:250, sc-546), GDNF (1:250, sc-328), VEGF (1:250, sc-152), and t-TrkB (1:2000, sc-8316) from Santa Cruz; cleaved caspase-3 (cCP3, #9661),
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p-Akt473 (#9271), p-Akt308 (#4056), t-Akt (#9272), p-Erk44/42 (#9101), t-Erk44/42
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(#9102), p-CREB (#9198), and t-CREB (#9197), all at 1:1000 dilution, from Cell Signaling; and p-TrkB (1:1000, ab52191) from Abcam (MA, USA). After being washed with PBST buffer, the membrane was incubated with the secondary antibodies for 1h at room temperature. After being washed with PBST buffer, signals were developed using a Western Lightning ECL reagent Pro (Bio-Rad, California, USA). The gel image was captured using an ImageQuant™ LAS 4000 biomolecular imager
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Intracerebroventricular injection of anti-BDNF antibody
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system (ImageJ) to estimate the integral optical density of the protein bands.
In this study, the rabbit anti-BDNF antibody (100 µg/ml; sc-546, Santa Cruz)
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was used to block the BDNF-TrkB system. The 2 µl of neutralizing BDNF antibody
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or an equal volume of control IgG (100 µg/ml; I5006, Sigma-Aldrich, MO, USA) was intracerebroventricularly (icv) injected at 30 min before CCI as previously described [31]. Briefly, a 30-gauge needle of a Hamilton syringe was inserted into the lateral ventricle (0.5 mm posterior to the bregma, 1 mm right lateral to the midline, and 2
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mm in depth). Then anti-BDNF antibody or control IgG was infused with an infusion pump at a rate of 0.2 µl/min for 10 min. The needle was removed 20 min after the
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infusion to prevent reflux, and the CCI procedure was performed immediately
Statistics
All data are shown as means ± standard error of the mean (SEM). For comparisons among multiple groups, one-way analysis of variance (ANOVA), followed by Tukey’s test, was used to determine significant differences. Differences
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significance was set at p value ≤ 0.05.
Results
Ultrasound Stimulation Increases Neurotrophic Factor Expression after TBI
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following TBI, the protein levels of BDNF, VEGF, and GDNF were measured in the brain (Fig. 1). The protein levels of BDNF and VEGF were significantly decreased in the ipsilateral cortex at day 1 after TBI compared with the sham group, but LIPUS significantly increased VEGF to 149% of the TBI-level in the ipsilateral cortex
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(p<0.001; Fig. 1B). In addition, the protein levels of BDNF, VEGF, and GDNF were all significantly decreased in the ipsilateral cortex at day 4 after TBI compared with
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the sham group. LIPUS caused significant increases of 256% and 60% in the protein
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levels of BDNF and VEGF, respectively, at day 4 after TBI compared with the non-treated group (both p<0.001; Figs. 1A and B). However, there was no difference in GDNF protein level at both 1 and 4 days post-TBI between the non-treated and LIPUS-treated groups (Fig. 1C).
Ultrasound Stimulation Increases BDNF Expression in Neurons after TBI
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total number of neurons and BDNF-positive neurons (Fig. 2B). The percentage of BDNF-positive neurons was also increased following LIPUS treatment. These data suggest that enhanced levels of neuronal BDNF protein contributes to the protective
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Ultrasound Stimulation Enhances Activation of TrkB and Downstream Akt Signaling, but Does Not Affect Erk Signaling after TBI
We examined the phosphorylation of TrkB and the activation of its major
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downstream survival signaling pathways (the Akt and Erk pathways). CCI induced a significant decrease in the TrkB phosphorylation level at both day 1 and day 4
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compared with the sham group (both p<0.01). LIPUS significantly increased TrkB
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phosphorylation to 136% of the non-treated TBI-level at day 4 (p=0.016; Fig. 3A). Likewise, levels of Akt Thr308 and Akt Ser473 phosphorylation were significantly diminished following TBI at either day 1 or day 4 (both p<0.05; Fig. 3B). The levels of both Akt phosphorylation forms were significantly higher in the LIPUS-treated TBI group than in the non-treated TBI group at day 4 (phospho-Akt Thr308: 172% of non-treated TBI, p=0.014; phospho-Akt Ser473: 185% of non-treated TBI, p=0.003;
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following LIPUS at either time point (all P > 0.05; Fig. 3C).
Ultrasound Stimulation Enhances Activation of CREB and TrkB Phosphorylation in Neurons after TBI
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We further evaluated the phosphorylation levels of CREB, which are
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downstream factors of Akt. Similar to the pattern observed for Akt phosphorylation, the CREB phosphorylation levels significantly increased following LIPUS (179% and 361% of non-treated TBI, p=0.032 and 0.02; Fig. 4A) at 4 days post-injury. To further investigate whether the TrkB activation induced by LIPUS directly occurred in
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neurons, we examined the localization of the phospho-TrkB following CCI. TrkB phosphorylation was found in neurons around the impact site, and increased following
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LIPUS treatment at 4 days (Fig. 4B).
Ultrasound Stimulation Reduces Apoptotic process after TBI We next assessed whether LIPUS reduced post-traumatic apoptosis. Cleaved
caspase-3 protein level was low in the sham-operated brains but it was induced significantly by CCI at 1 and 4 days post-injury. LIPUS significantly decreased the level of cleaved caspase-3, a final effector of apoptotic death, by 40% at day 1
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TBI.
Neutralization of BDNF Attenuates LIPUS-induced Neuroprotection after TBI
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To further confirm that BDNF/TrkB signaling contributes to the beneficial effect
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of LIPUS, a neutralizing antibody against BDNF was administered to LIPUS-treated CCI mice. The ability of the anti-BDNF antibody to block the action of BDNF was validated with Western blot analysis, in which BDNF-mediated TrkB phosphorylation was prevented by the anti-BDNF antibody but not by control IgG (Fig. 6B).
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Treatment with the neutralizing antibody significantly reduced LIPUS-induced TrkB phosphorylation at 4 days post-TBI (Fig. 6C). However, mice treated with the
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neutralizing antibody alone in the absence of LIPUS stimulation showed no difference
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in TrkB phosphorylation compared with mice treated with antibody and LIPUS. The total TrkB levels in brains treated with anti-BDNF antibody were reduced compared with those in control IgG-treated brains (Fig. 6C). One potential reason for the reduction in TrkB levels is the cellular response to the lack of ligand (BDNF) in neurons treated with the BDNF neutralizing antibody, a finding that was previously reported [32]. The protective effects of LIPUS against trauma-induced apoptosis were
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post-TBI (Fig. 6D).
Discussion
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In this study, we demonstrated that transcranial LIPUS stimulation elevated the
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protein levels of neurotrophic factors and inhibited the progression of apoptosis in mice subjected to TBI. LIPUS increased BDNF and VEGF protein expression and enhanced
the
activation
of
the
TrkB/Akt-CREB
pathway.
Furthermore,
co-administration of a neutralizing anti-BDNF antibody attenuated the reduction of
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apoptosis by LIPUS treatment, indicating that its therapeutic action is at least partly mediated by the BDNF signaling pathway (Fig. 7). Our results suggest that LIPUS
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stimulation may be a promising new technique for treating TBI.
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US has been widely used for both diagnostic and therapeutic purposes due to its non-ionizing and non-invasive nature. LIPUS is mainly used clinically in the treatment of bone fractures to accelerate the proliferation and differentiation of osteoblasts. Experimental studies have also demonstrated that LIPUS can promote peripheral nerve regeneration. Recently, our studies demonstrated that the brain damage and memory impairments can be improved by LIPUS stimulation in animal
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ACCEPTED MANUSCRIPT models of neurodegenerative diseases [25, 33]. These beneficial effects of LIPUS may be attributed partially to its enhancement of BDNF expression.
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We showed that TBI induced a decrease of the cerebral BDNF protein level and that subsequent LIPUS stimulation for 4 days non-invasively promoted the BDNF protein level and increased the number of BDNF-positive neurons. The observation of
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LIPUS stimulation increased the BDNF protein expression in the injured brain
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extended our previous finding that LIPUS promotes cerebral BDNF protein level in the normal brain [25, 33]. The importance of BDNF to functional recovery following TBI has been documented in a previous clinical study showing that the polymorphisms of human BDNF affect the recovery of cognitive intelligence in
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patients with penetrating TBI [34]. Accumulating experimental evidence also indicates the critical role of BDNF signaling in promoting neuronal survival. BDNF
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mediates its effect through its high affinity for the TrkB receptor. The activation of
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TrkB triggers the downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which is the major TrkB-mediated survival pathway against TBI [27, 31]. We observed that 4 days of LIPUS stimulation increased TrkB and Akt phosphorylation, along with increasing the phosphorylation of CREB, the Akt downstream targets. In addition, LIPUS reduced the cleaved caspase-3 level in the injured brain. Neutralizing BDNF by using the BDNF neutralizing antibody
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ACCEPTED MANUSCRIPT attenuated the LIPUS-induced protection against apoptosis, indicating that the neuroprotective mechanism of LIPUS is at least partially mediated by BDNF
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signaling. Given that the therapeutic potential of recombinant BDNF is limited due to its short half-life and poor BBB penetration [35], the enhancement of endogenous BDNF via LIPUS could be a novel therapeutic strategy for TBI and various
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neurological diseases related to the dysregulation of BDNF.
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Instead of the thermal contribution, changes in transmembrane capacitance and subsequent excitation of neural cells due to the exposure to the acoustic pressure waves may serve as a probable contributing factor for enhancement of BDNF secretion
[36].
LIPUS-mediated
mechanical
activation
of
glial
cells
via
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mechanoreceptors may be another potential mechanism. The mechanisms involved in LIPUS-stimulated BDNF protein expression in damaged brains have not been
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elucidated yet; however, previous studies have demonstrated that LIPUS can enhance
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the BDNF protein level in cultured astrocytes through the activation of integrin receptors and NFκB signaling [24, 37]. Integrins, which are transmembrane receptors that connect the cytoskeleton to the extracellular matrix, are involved in the transduction of mechanical stimuli to biochemical signals [38]. US stimuli are transmitted to adherent cells via their contact with surrounding extracellular matrices, and integrins may transmit US signals through the cytoskeleton and activate several
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ACCEPTED MANUSCRIPT downstream protein kinases [38, 39]. In this regard, LIPUS stimulation has been demonstrated to increase the expression of integrins in cultured chondrocytes [39],
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while an integrin inhibitor was shown to attenuate LIPUS-induced BDNF expression in cultured astrocytes [24]. The activation of NF-κB has also been demonstrated to contribute to LIPUS-induced BDNF expression in astrocytes [37]. Since
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astrocyte-derived BDNF is a source of trophic support that can be used to reduce
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damage induced by brain injury [40], it is possible that LIPUS promotes neuronal survival by inducing astrocyte-derived BDNF expression in the damaged brain. The mechanisms underlying the interaction between astrocytes and neurons following LIPUS stimulation in the damaged brain will be the subject of further investigation.
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We also observed that LIPUS enhanced the protein level of VEGF in injured brains. In this regard, we have recently reported that LIPUS promoted VEGF protein
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levels in normal brains [25] and cultured astrocytes [24]. Previous studies in animal
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models of myocardial ischemia also demonstrated that LIPUS upregulated the protein expression of VEGF and eNOS and induced angiogenesis [41]. Since VEGF has been reported to regulate neuronal survival, neurogenesis, and angiogenesis [42], it is possible that LIPUS stimulation contributes to neurovascular remodeling, leading to improved neurobehavioral outcomes following TBI. This point remains to be examined in future studies.
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Conclusion
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In conclusion, we demonstrated that post-injury LIPUS treatment enhanced the protein levels of neurotrophic factors and attenuated apoptotic process in mouse TBI. The enhancement of BDNF with LIPUS may be related to modulation of the
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TrkB/Akt-CREB signaling pathway. Thus, LIPUS stimulation may play a crucial and
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beneficial role in TBI patients.
Acknowledgments
This study was supported by grants from the Ministry of Science and Technology of
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Taiwan (no. MOST 105-2221-E-010-003, MOST 104-2314-B-010-003-MY3, and 101-2314-B-350-001-MY3), the Veterans General Hospitals University System of
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Taiwan Joint Research Program (#VGHUST106-G7-6-1), the Cheng Hsin General
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Hospital Foundation (no. CY10622, CY10418 and CHGH103-34), and the Taiwan Ministry of Education’s Aim for the Top University Plan.
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Figure captions Figure 1. Effects of LIPUS treatment on neurotrophic factor expression in TBI mice. Representative western blots and optical densitometric quantification of (A) BDNF,
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(B) VEGF, and (C) GDNF in the ipsilateral hemisphere of sham-injured, LIPUS-treated sham, non-treated TBI, and LIPUS-treated TBI mice at 1 and 4 days
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post-injury. The protein expressions of BDNF and VEGF were enhanced by LIPUS treatment in the traumatized brain. * and # denote significantly different from sham
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and non-treated TBI group, respectively (***,###, p<0.001, n = 6-7).
Figure 2. Identification of BDNF-positive neurons at 4 days post-injury in the
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peri-contusional margin by double immunofluorescence staining. (A) BDNF is shown in red, and NeuN (neurons) is shown in green. Co-localization of BDNF with NeuN is
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shown by yellow labeling. The representative sections were taken from the injury core
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at the level of 0.74 mm from the bregma. Sections were stained with DAPI (blue) to show all nuclei. (B) LIPUS significantly increased the total number of neurons and BDNF-positive neurons, as well as the percentage of BDNF-positive neurons. The number of NeuN-positive cells and BDNF-positive neurons is expressed as the mean number per field of view (0.8 mm2 ). # denote significantly different from non-treated TBI group (##, p<0.01; ###, p<0.001, n = 6-7).
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ACCEPTED MANUSCRIPT Figure 3. Effects of LIPUS treatment on TrkB and downstream Akt and Erk signaling in TBI mice. Representative western blots and optical densitometric quantification of
hemisphere
of
sham-injured,
LIPUS-treated
sham,
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(A) phospho-TrkB, (B) phospho-Akt, and (C) phospho-Erk 1/2 in the ipsilateral non-treated
TBI,
and
LIPUS-treated TBI mice at 1 and 4 days post-injury. LIPUS significantly enhanced
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phosphorylation of TrkB, Akt at Thr308, and Akt at Ser473 at 4 days. Erk 1/2
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phosphorylation was not affected by LIPUS at either time point. * and # denote significantly different from sham and non-treated TBI group, respectively (*,#, p p<0.01; ***,
p<0.001, n = 6-7).
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<0.05; **,##,
Figure 4. Effects of LIPUS treatment on phosphorylation of CREB and TrkB in TBI mice. Representative western blots and optical densitometric quantification of (A)
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phospho-CREB in the ipsilateral hemisphere of sham-injured, LIPUS-treated sham,
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non-treated TBI, and LIPUS-treated TBI mice at 1 and 4 days post-injury. LIPUS significantly enhanced phosphorylation of CREB at 4 days. * and # denote significantly different from sham and non-treated TBI group, respectively (#, p <0.05; **, p<0.01; n = 6-7). (B) Cellular localization of phospho-TrkB in the peri-contusional margin at 4 days post-TBI observed by immunofluorescence labeling. Phospho-TrkB is shown in red, and NeuN (neurons) is shown in green. Colocalization
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ACCEPTED MANUSCRIPT of phospho-TrkB with neurons is shown by yellow labeling. The representative sections were taken from the injury core at the level of 0.74 mm from the bregma.
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Sections were stained with DAPI (blue) to show all nuclei.
Figure 5. Effects of LIPUS treatment on apoptosis expression in TBI mice.
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Representative western blots and optical densitometric quantification of cleaved
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caspase-3 in the ipsilateral hemisphere of sham-injured, LIPUS-treated sham, non-treated TBI, and LIPUS-treated TBI mice at 1 and 4 days post-injury. LIPUS significantly decreased the cleaved caspase-3 level at both 1 and 4 days post-injury. * and # denote significantly different from sham and non-treated TBI group, p<0.01; ***,###, p<0.001, n = 6-7).
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respectively (##,
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Figure 6. Neutralization of BDNF attenuates LIPUS-induced neuroprotection in TBI
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mice. (A) Experimental design. icv: intracerebroventricularly; WB: western blots. (B) Representative western blots of phospho-TrkB in the ipsilateral hemisphere of a non-treated TBI, a control IgG-treated TBI, and a BDNF Ab-treated TBI mouse at 4 days post-injury. TrkB phosphorylation was attenuated by the anti-BDNF antibody but not by the control IgG. Representative western blots and optical densitometric quantification of (C) phospho-TrkB and (D) cleaved caspase-3 in the ipsilateral
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ACCEPTED MANUSCRIPT hemisphere of LIPUS+IgG, BDNF Ab, and LIPUS+BDNF Ab mice at 4 days post-injury. Treatment with anti-BDNF Ab significantly reduced LIPUS-induced TrkB
LIPUS+IgG group at day 4 post-TBI.
denotes significantly different from
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LIPUS+IgG TBI group (§, p<0.05; n = 6-7).
§
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phosphorylation and increased cleaved caspase-3 level compared with the
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Figure 7. Schematic diagram of the mechanisms involved in LIPUS-induced protection in a TBI mouse. LIPUS stimulation increased the protein levels of BDNF and VEGF, in addition to enhancing the phosphorylation of TrkB, Akt, and CREB. Treatment with BDNF neutralizing antibody inhibited the protective effects of LIPUS
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against trauma-induced apoptosis. Taken together, post-traumatic LIPUS stimulation
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promotes BDNF production and reduces apoptosis following TBI.
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ACCEPTED MANUSCRIPT Highlights: Low-intensity pulsed ultrasound (LIPUS) stimulation increased the protein levels of BDNFand VEGF in a mouse model of traumatic brain injury (TBI).
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LIPUS stimulation inhibited the progression of apoptosis following TBI.
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The neuroprotective effects of LIPUS may be associated with enhancements of the protein levels of BDNF, at least partially via the TrkB/Akt-CREB signaling pathway.