Low-intensity pulsed ultrasound improves behavioral functions and alleviates neuroinflammation in a rat model of Parkinson’s disease

Background: Parkinson’s disease (PD) is characterized by a reduction of dopamine level in the substantia nigra pars compacta (SNpc) and striatum of the brain. Low-intensity pulsed ultrasound (LIPUS) has been demonstrated recently as a non-invasive neuromodulation tool in a number of fields. LIPUS has also been reported to improve behavioral functions in PD animal models; however, the effect of LIPUS stimulation on the neurotrophic factors and neuroinflammation has not yet been addressed. Methods: PD rat model was built by injection of 6-hydroxydopamine (6-OHDA) in two sites in the right striatum. The levels of neurotrophic factors and lipocalin-2 (LCN2)-induced neuroinflammation were quantified using a western blot. Rotational test and cylinder test were conducted biweekly for eight weeks. The safety of LIPUS was assessed using Hematoxylin and Esosin (H&E) staining and Nissl staining. Results: When the 6-OHDA+LIPUS and 6-OHDA groups were compared, the locomotor function of the 6-OHDA+LIPUS rats was significantly improved. After LIPUS stimulation, the number of neurons was remarkably increased in the striatum and SNpc of lesioned rats. Unilateral LIPUS stimulation did not increase brain-derived neurotrophic factor (BDNF) in the striatum and SNpc of lesioned rats. In contrast, unilateral LIPUS stimulation increased glial cell line-derived neurotrophic LCN2-induced neuroinflammation can be attenuated following LIPUS stimulation. Conclusions: Our data indicated that LIPUS stimulation increased GDNF and dopaminergic (DA) neuron density in the 6-OHDA-induced rat model of PD. Moreover, this technology attenuated proinflammatory mediators and reversed behavioral indicators of PD-associated motor dysfunction with no evidence of brain tissue injury. These results show that LIPUS stimulation may be a potential therapeutic tool against PD via enhancement of GDNF level and inhibition of inflammatory responses in the SNpc of the brain.


Background
Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and a corresponding decrease in DA innervation of the striatum, which is associated with clinical symptoms such as bradykinesia, resting tremor, postural instability, and limb rigidity [1,2]. Currently, pharmacological dopamine replacement is established as the gold standard of treatment for PD, however, its long-term administration causes abnormal, uncontrollable, and involuntary movements [3]. An alternative invasive therapy of PD is deep-brain stimulation of the subthalamic nucleus (STN DBS), which was to be considered after adequate control of symptoms can no longer be achieved through pharmacotherapy [4]. Current therapies are not neuroprotective and continued neuronal degeneration ultimately results in recurrence of symptoms [5]. Furthermore, therapies that can attenuate the disease progression have remained elusive [6].
Gene therapy has been developed as a feasible approach for PD for many years. Glial cell-line derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) are known to be potent neurotrophic factors that are crucial for the survival of the DA neurons in the substantia nigra (SN). GDNF is a potent agent for PD therapy due to its effect to protect neurons from degeneration and 5 promote dopamine production from the remaining neurons [7,8]. Although the potential treatment of GDNF for PD, systemic administration is prevented by the blood-brain barrier (BBB) blocking most larger molecules from entering the brain [9].
While direct injection methods may improve outcomes, such invasive operations pose a risk for complications. Another approach is the use of gene therapy with different strains of modified viruses carrying the GDNF gene [10,11]. Focused ultrasound in conjunction with microbubbles has been shown to locally open the BBB and successfully deliver GDNF into the brain. Nevertheless, FUS-induced BBB leads to a transient inflammatory response in the sonicated brain [12,13]. A novel approach for noninvasion and minimal side effects that can be used for PD patients is critically required.
Increasing evidence suggests that neuroinflammatory responses are assumed to contribute to the progression of PD [14,15]. Observations of brain tissues from PD patients have found a significant increase in the number of reactive microglia in the SNpc [16,17]. Meanwhile, there is an increase in the expression of proinflammatory mediators, especially interleukin-1 (IL-1) and tumor necrosis factor- (TNF-) [18,19]. Lipocalin-2 (LCN2), a member of the highly heterogeneous secretory protein family of lipocalins, may cause neuroinflammation and neurodegeneration in the brain [20]. It has been reported that LCN2 is upregulated in the SN of patients with 6 PD and 6-hydroxydopamine (6-OHDA)-treated animal model of PD [21]. LCN2 upregulation in the SN of patients with PD may be a key trigger of neuroinflammation, leading to further disruption of the nigrostriatal DA system. Therefore, the control of neuroinflammation may be useful in the prevention and treatment of PD.
In addition to treatment with DA medications and DBS, noninvasive brain stimulation such as transcranial direct current stimulation (tDCS) and repetitive transcranial magnetic stimulation (rTMS) have the potential to fulfill the treatment of some neurological disorders, for example, PD [22]. These noninvasive neuromodulatory methods are generally safe and avoid both the side effects of systemic medications and the complications associated with DBS surgery. Several clinical trials have shown that rTMS provides a beneficial effect on functional recovery in PD [23,24]. However, the application of rTMS is limited due to poor spatial resolution and magnetic contraindications. Compared with rTMS or tDCS, transcranial ultrasound stimulation (TUS) has a deeper penetration ability in the brain with a higher spatial resolution [25]. Studies have suggested that low-intensity pulsed ultrasound (LIPUS) may hold a great promise for the modulation of brain function and reversal of neurological dysfunction [26][27][28]. To clarify the neuroprotective and anti-inflammatory effect of LIPUS on SNpc DA neurons after LIPUS treatment, this study investigated motor functions and tyrosine hydroxylase (TH)-immunoreactive 7 DA neurons in SNpc and striatum. Additionally, the expression of proinflammatory mediators was examined after LIPUS application in an animal model of PD.

PD animal model
Female Sprague-Dawley (SD) rats weighing from 180g to 200g were purchased from LASCO, housed on 12/12h light/dark cycle, and given food ad libitum. All procedures involving animals were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals. This study protocol was approved by the Animal Care and Use Committee of National Yang Ming University. Partial striatal lesioning with 6-OHDA was performed to establish the PD animal model. Prior to surgery, each animal was anesthetized in the prone position by inhalation of 2% isoflurane in 2 l/min oxygen, and the body temperature was maintained at 37°C using a heating pad.
The rat heads were mounted on a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA), and the top of the cranium was shaved. A midline scalp incision was made, and the skull was exposed. A total of 12 g 6-OHDA (Sigma-Aldrich, St. Louis, MO) dissolved in 2 l 0.02% ascorbic acid/0.9% saline was injected in two sites in the right striatum at the coordinates: AP: +0.5, ML: +2.1, DV: -5 and AP: -0.5, ML: +3.8, DV: -5 mm at 0.5 l/min. Two weeks after the injections, animals that showed inadequate 8 (<5 rotations/min) or severe (>9 rotations/min) apomorphine-induced rotation were excluded.

Pulsed ultrasound equipment and treatment protocols
The LIPUS setup was similar to that used in our previous study [29]. LIPUS was generated by a 1-MHz focused piezoelectric transducer (A392S; Panametrics, Waltham, MA, USA) with 50 ms burst lengths at a 5% duty cycle and a repetition frequency of 1 Hz. The spatial-peak, temporal-average intensity (I SPTA ) over the focused transducer head was 528 mW/cm 2 and was measured with a radiation force balance (RFB, Precision Acoustics, Dorset, UK) in degassed water. The focused transducer was mounted on a removable cone filled with deionized and degassed water, the tip of which was capped by a polyurethane membrane, with the center of the focal zone placed about 5.7 mm away from the cone tip. The sonication was precisely targeted using a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA) ( Figure 1A). The acoustic wave was delivered to the targeted region in the lesions of the brain. Each rat's right hemisphere was treated by LIPUS with triple sonications.
The duration of each sonication was 5 min and there was an interval of 5 min between each sonication. The parameters of the LIPUS exposures were selected based on data from our previous studies [26,30]. Figure 1B describes the experimental procedure 9 from week 1 to week 8. Rats were assessed with rotation test and divided into 6-OHDA or 6-OHDA+LIPUS groups two weeks after the 6-OHDA injections. After anesthetic induction with isoflurane mixed with oxygen, LIPUS was applied consecutively to the rats in the 6-OHDA+LIPUS group five days per week for a period of six weeks. The rats in the 6-OHDA group were also anesthetized with isoflurane mixed with oxygen during this period.

Behavioral test
Behavioral analysis was performed one to three days before and two, four, six, and eight weeks after injection of 6-OHDA. Rotational tests and cylinder tests were performed similar to those previous studies [31,32]. For Apomorphine-induced rotation, the rats were intraperitoneally injected with apomorphine (0.4mg/kg, Sigma-Aldrich, St. Louis, MO) and placed in a hemispheric plastic bowl. Left and right rotations were counted for 40 min. For cylinder test, the rats were placed in a 20-cm diameter glass cylinder and their activity was recorded. Contralateral and ipsilateral weight-bearing forepaw contacts with walls were counted and a minimum of 20 contacts were required to complete the test. Data are presented as percentage of contacts with the contralateral forepaw.

Western blotting analysis
Twelve hours after the last sonication, rats were sacrificed for analysis of LCN2 protein expression ( Figure 5A). The other protein expressions were measured at 48 h after the last sonication in the rats for locomotor tests ( Figure 1B). The 2-mm coronal section was taken from the sonicated area over the striatum and SNpc, and then homogenized by a T-Per extraction reagent 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). Samples containing 30 μg protein were resolved on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immun-Blot ® polyvinyl difluoride (PVDF) membranes (Bio-Rad, CA, USA). After blotting, the membranes were blocked for at least 1 h in a blocking buffer (Hycell, Taipei, Taiwan), and then the blots were incubated overnight at 4°C in a solution with primary antibodies raised in rabbits against tyrosine hydroxylase (TH), GDNF, BDNF, LCN2, and IL-1β. After being washed with PBST buffer, each membrane was incubated with the secondary antibodies for 1 h at room temperature.
After the given membrane was 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 (GE 11 Healthcare Life Sciences, Pennsylvania, USA) and analyzed using a gel image system (ImageJ) to estimate the integral optical density of the protein bands. The protein expressions of TH were normalized to the contralateral site of the brain region. The other protein expressions were normalized to the same region of the untreated 6-OHDA control group.

Histological assessment
Four hours after the last sonication, 6-OHDA-induced rats 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. The first coronal section containing the striatum was prepared by cutting 3 mm and 6 mm anterior to the front of the brain. The second coronal section containing the SNpc was prepared by cutting 9 mm and 12 mm anterior to the front of the brain. The primary antibodies were rabbit anti-tyrosine hydroxylase ( Considering that 6-OHDA would cause tissue damage in the brain, the healthy rats were selected for safety evaluation. Hematoxylin and eosin (H&E) and Nissl staining were performed to observe any morphological changes caused by LIPUS treatment with five days for one week.
Statistical analysis 13 All data are shown as means ± standard error of the mean (SEM). Differences between two groups were analyzed using Student's t test. The level of statistical significance was set at p value  0.05.

Results
Ultrasound improves locomotor functions in 6-OHDA-induced rats Ultrasound prevents 6-OHDA-induced decrease in TH levels in striatum and

SNpc of rats
In order to examine the neuroprotective effects of LIPUS on the nigrostriatal pathway, 14 TH immunohistochemistry and western blot were performed to assess the section of the striatum and SNpc eight weeks after 6-OHDA administration (Figure 3). In the control 6-OHDA group, 6-OHDA administration led to 85.7% and 73.9% reductions in DA staining density in the striatum and SNpc of ipsilateral site, respectively ( Figure   3B). Compared to 6-OHDA group, there is a 2.6-fold and a 1.7-fold increase in DA density in the striatum and SNpc of 6-OHDA+LIPUS, respectively (p<0.05). We further evaluated the TH protein level in the striatum and SNpc. As shown in Figure   3C, Effects of ultrasound on the levels of GDNF and BDNF in 6-OHDA-induced rats There was no significant difference in GDNF and BDNF levels in the striatum between 6-OHDA and 6-OHDA+LIPUS groups (p>0.05; Figures 4A, B). In the SNpc  Figure 4D) compared to the 6-OHDA group.
Ultrasound decreases the levels of LCN2 and IL-1 in the SNpc of 6-OHDA-induced rats 16 To investigate the upregulation of LCN2 expression in the SNpc of 6-OHDA lesioned brains, we examined the neuroprotective effect of LIPUS against neurotoxicity in the nigrostriatal DA system. Similar to the upregulation of LCN2 after 6-OHDA administration in the previous study [21], an intrastriatal injection of 6-OHDA caused a significant increase in LCN2 expression (p<0.05) in the SNpc compared with control group ( Figure 5C). However, LIPUS treatment significantly reduced the expression of LCN2 (p<0.01) in the SNpc induced by 6-OHDA. Furthermore, Western blot analyses showed that the significant decreases in the protein levels of neurotoxic inflammatory cytokine such as IL-1 were observed in the bilateral SNpc compared to the 6-OHDA group ( Figure 5D). These results suggest that neurotoxicity in the nigrostriatal DA system induced by upregulated LCN2 levels might be a common feature of this animal model of PD and the increased LCN2 expression may have a direct neurotoxicity with the induction of proinflammatory mediators, thereby playing a central role in PD pathogenesis.

Safety Evaluation
Rats in 6-OHDA+LIPUS treatment group did not show changes in weight gain compared to the 6-OHDA group ( Figure 6A). Besides, H&E staining is widely used to observe the hemorrhage or tissue damage and Nissl staining is used to visualize 17 neurons. Here, H&E and Nissl staining were performed to assess the safety of LIPUS in healthy rats. H&E staining ( Figure 6B) did not show any tissue damage or hemorrhage associated in a five-day LIPUS effect. Nissl staining ( Figure 6C) showed that neuronal density appeared to be normal and no neuronal damage was found through the entire brains.

Discussion
The present experiments demonstrate that LIPUS stimulation improves parkinsonian motor behaviors and protect TH positive neurons in the 6-OHDA rat model of PD.
Specifically, LIPUS reversed the 6-OHDA lesion-induced decrease in contralateral forepaw use as evaluated in the cylinder test. The results reveal that LIPUS has neuroprotective effects against 6-OHDA neurotoxin by promoting GDNF protein levels and attenuating the LCN2 release in the SNpc of the brain, thereby suppressing neurotoxic cytokine such as IL-1. We concluded that LIPUS stimulation protects against behavioral impairments with neurotrophic activities and anti-inflammatory responses in the nigrostriatal neurons in the PD rat model induced by 6-OHDA. These findings suggest that LIPUS could be a promising treatment for antiparkinsonian action.
GDNF has shown neuroprotective effects in animal models of PD [7,33], but 18 clinical trials have generally failed to meet primary outcomes in PD patients, probably because of the limited penetration into the brain [34]. Recently, a growing body of evidence suggests that focused ultrasound-induced BBB opening may be a useful tool for delivering such a neurotrophic factor directly into the central nervous system [35,36], and an increased GDNF level may lead to a strong trophic effect on the DA system. Injection of the GDNF protein into the striatum is effective in protecting nigral DA neurons after the 6-OHDA lesioned brain [37,38]. On the other hand, exogenous GDNF could have possible side effects such as cerebellar damage [39].
The strategy to increase the local level of endogenous neurotrophic factors is crucial for PD because the passage of large molecules into the brain is limited by the BBB. In the present experiment, a significant rescue of DA neurons was obtained in the striatum and SNpc of 6-OHDA lesioned brain after LIPUS stimulation. Although Western blot analysis showed that LIPUS only increased endogenous GDNF protein in the SNpc (Figure 4A), the increased expressions of both endogenous GDNF and BDNF were apparently observed in the TH-immunopositive neurons ( Figures 4C, D), indicating the induction of neurotrophic factors by LIPUS in DA neurons. However, LIPUS did not increase BDNF protein levels in both structures examined, including the striatum and SNpc ( Figure 4B). Besides, the loss of DA neurons in SNpc leads to PD-like behavioral symptoms [40]. Figure 4D indicated that LIPUS significantly 19 increased the number of DA neurons in the SNpc of 6-OHDA lesioned rats.
The root cause of PD remains poorly understood, but it is well known that neuroinflammation and immune activation play major roles in the pathophysiology of PD. Therefore, the characterization of the action mechanisms of reactive-glia-derived molecules involved in neurotoxicity and neuroinflammation may be indispensable for finding a novel therapeutic strategy for the protection of the nigrostriatal DA projection in the PD brain. Furthermore, previous results suggest that LCN2 may be an important pathogenic biomolecule involved in PD [21]. LIPUS has been shown to inhibit the lipopolysaccharide (LPS)-induced activation of TLR4/NF-B inflammatory signaling and affected the proinflammatory responses of microglia [28,41]. In addition to demonstrating LCN2 upregulation in the 6-OHDA-treated rats of PD, we investigated whether LIPUS stimulation in the 6-OHDA-treated rats of PD prevented the ensuing production of neurotoxic cytokine such as IL-1 ( Figure 5).
Western blot analyses showed that the level of neurotoxic inflammatory cytokine such as IL-1 was inhibited in SNpc after 6-OHDA microinjection ( Figure 5D).
The mechanisms underlying ultrasound neuromodulation in PD are still unknown. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-OHDA are commonly used to induce PD-like symptoms in animals [42,43]. The MPTP animal model is recommended for studying mitochondrial dysfunction in PD [44]. Several in 20 vivo studies have shown that LIPUS is able to alleviate motor impairments through antiapoptotic effects and antioxidant activity in MPTP mice [45,46]. Additionally, the 6-OHDA striatal model is mainly used to test neuroprotective strategies for PD [47,48]. In the present study, the results supported and extended the suggestion that neurotrophic and anti-inflammatory responses can be another important beneficial effect of LIPUS on parkinsonian motor deficits in rats administrated with 6-OHDA.
Herein, we chose a 1-MHz ultrasound transducer with a set of parameters based on our previous studies [26,28]. A wide range of bio-effects can be produced by LIPUS stimulation, depending on multiple parameters, including frequency, intensity, burst duration, pulse repetition frequency, etc. After a five-day LIPUS stimulation, no tissue damage was observed in H&E and Nissl staining. The intensity of I SPTA (528 mW/cm 2 ) was below the threshold value (720 mW/cm 2 ) of the U.S. FDA's clinical diagnostic ultrasound, ensuring the safety of the LIPUS stimulation used here. Further investigation is needed to explore the impact of ultrasound parameters on the neuromodulation effects.

Conclusions
In short, LIPUS stimulation has neuroprotective effects against the 6-OHDA-induced parkinsonian rat model, indicating that this may be a new potential 21 tool for PD treatment. These neuroprotective effects are attributed to reserving neurotrophic factor levels and an inhibitory role on different key events involved in neuroinflammation, as well as rescuing TH positive neurons in striatum and SNpc, which consequently improved behavioral impairments.