Focused ultrasound enhances targeted curcumin delivery and alleviates behavioral and neuropathological deficits in a Parkinson's disease mouse model

Abstract

Introduction: Curcumin exhibits potent neuroprotective properties but is limited by poor blood-brain barrier (BBB) permeability. This study aimed to evaluate whether focused ultrasound (FUS)-mediated BBB opening enhances curcumin delivery and therapeutic efficacy in a 6-hydroxydopamine (6-OHDA) mouse model of Parkinson's disease (PD).

Methods: Male C57BL/6 mice with unilateral 6-OHDA lesions received intravenous curcumin with or without FUS targeted to the lesioned striatum for 4 weeks. Behavioral performance was assessed using rotarod and open-field tests. Postmortem analyses included tyrosine hydroxylase (TH) immunostaining in the striatum and substantia nigra pars compacta (SNpc), glial fibrillary acidic protein expression, and fluorescence quantification of curcumin accumulation. Microglial activation and pro-inflammatory cytokine expression were evaluated using ionized calcium-binding adaptor molecule 1 (Iba1) and interleukin-6 (IL-6) staining. Safety was evaluated by histological examination for hemorrhage or necrosis.

Results: Significant improvements in motor coordination and exploratory activity were observed in the FUS + curcumin group, particularly during early stages of degeneration. Histologically, combined treatment was associated with greater preservation of TH-positive dopaminergic neurons in the SNpc and reduced astrocytic activation in the striatum compared with curcumin alone, whereas striatal TH fiber density did not differ between curcumin-treated groups. No significant differences were observed in striatal Iba1 or IL-6 expression across groups at the 4-week time point. Enhanced curcumin accumulation in the brain was observed following FUS, as demonstrated by fluorescence quantification. No tissue damage or adverse effects were observed after repeated sonications.

Discussion: This is the first study to demonstrate the efficacy of unmodified curcumin combined with FUS in a 6-OHDA PD model. The findings support FUS as a safe and effective strategy to transiently disrupt the BBB and enhance the central nervous system delivery of small-molecule therapeutic agents, offering translational potential for regionally targeted, non-invasive treatment of PD.

Keywords: Parkinson’s disease; anti-inflammatory; blood–brain barrier; curcumin; drug delivery; focused ultrasound; neuroprotection.

FIGURE 1

Schematic overview of the experimental design and treatment timeline. (A) Schematic representation of the experimental procedure involving focused ultrasound (FUS). Mice received intravenous injection of microbubbles followed by FUS targeting the left striatum to transiently open the blood–brain barrier. Curcumin was administered immediately after each sonication session to enhance brain delivery. (B) Timeline of the experimental procedures. The study included four groups: the Sham group, the 6-OHDA group, the 6-OHDA + FUS group, the 6-OHDA + Curcumin group, and the 6-OHDA + FUS + Curcumin group. Treatments began 1 week after lesion induction and were administered once per week for four consecutive weeks. Rotarod testing was conducted weekly to assess motor coordination, and the open field test was performed in the fourth week. Body weight was recorded weekly. Mice were sacrificed 24 h after the final behavioral test for brain tissue collection and subsequent histological and immunofluorescence analyses (blue arrows indicate weekly FUS application and/or curcumin administration; orange arrows indicate weekly rotarod testing; green arrow indicates the open field test).

FIGURE 2

Focused ultrasound (FUS)-induced blood–brain barrier (BBB) opening, histological evaluation, and enhancement of curcumin brain delivery. (A) Representative coronal brain sections from mice injected with Evans blue (EB) dye following FUS at 40, 54, or 75 m V (n = 3). No visible EB leakage was observed in the 40 mV group. In contrast, localized EB accumulation was detected in the striatum of the 54 mV group, indicating successful and focal BBB opening. The 54 mV group exhibited pronounced EB staining. While no hemorrhagic signs were noted in the 40 and 54 mV groups, visible bleeding was observed in the 75 mV group. (B) Macroscopic brain images under each FUS condition. In the 40mV group, sonication did not reach the deep brain structures. In contrast, both the 54 and 75mV groups demonstrated effective targeting of the striatal region. (C) Hematoxylin and eosin-stained coronal brain sections of the cerebral cortex and striatum under each FUS condition. Low-magnification images (4 × ) encompassing the sonicated region are shown, together with higher-magnification views (20 × ) to illustrate local tissue morphology. No histological evidence of hemorrhage, edema, or neuronal damage was found in the 40 and 54 mV groups. Mild hemorrhagic changes were observed in the 75mV group. (D) Curcumin fluorescence in brain homogenates was measured 3 h after injection. Fluorescence intensity was normalized to total protein concentration and expressed as arbitrary units per milligram of protein (A.U./mg protein). At 3 h post-injection, the FUS + Curcumin group (n = 5) showed significantly higher normalized fluorescence intensity compared to the Curcumin-only group (n = 4), indicating enhanced delivery via FUS-mediated BBB opening. Statistical significance was determined using Student’s t-test. **p < 0.01.

FIGURE 3

Body weight changes and rotarod performance during the 4-week treatment period. (A) Body weight was recorded weekly for all experimental groups throughout the 4-week treatment and observation period. Data are presented as mean ± SEM. No significant differences in body weight were observed among the groups. (B) Latency to fall on the rotarod was measured weekly over the 4-week treatment period to assess motor coordination and balance. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. Symbols indicate the following comparisons: *p and ^#p < 0.05 versus the Sham, 6-OHDA groups, respectively; corresponding double symbols (**p, ^##p) indicate p < 0.01, and triple symbols (***p, ^###p) indicate p < 0.001 for the same group comparisons, when statistically significant. Group sizes were as follows: Sham (n = 8), 6-OHDA (n = 9), 6-OHDA + FUS (n = 5), 6-OHDA + Curcumin (n = 9), and 6-OHDA + FUS + Curcumin (n = 9).

FIGURE 4

Open field test evaluating locomotor activity and exploratory behavior after 4 weeks of treatment. Open field performance was assessed by measuring (A) total distance traveled, (B) immobilization time, (C) average speed and (D) the number of zone transitions, with (E) showing representative movement paths for each group. Data are presented as mean ± SEM. The dots represent individual mice in each group (Sham group, n = 8; 6-OHDA group, n = 9; 6-OHDA + FUS group, n = 5; 6-OHDA + Curcumin group, n = 9; and 6-OHDA + FUS + Curcumin group, n = 9). Significant differences were determined using one-way ANOVA followed by Tukey’s post-hoc test and are indicated with asterisks. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

Tyrosine hydroxylase (TH) immunohistochemistry evaluating dopaminergic fiber integrity in the striatum after 4 weeks of treatment. (A) Representative TH-immunostained coronal sections of the striatum from each experimental group, obtained from anatomically matched anteroposterior levels corresponding to the central striatal region and processed simultaneously under identical staining conditions. Low-magnification images were acquired at 4 × magnification, with representative higher-magnification images (20 × ) shown to illustrate local differences in TH-positive fiber density. The 6-OHDA group showed marked loss of TH-positive fibers compared with the Sham group. Visual preservation of TH immunoreactivity was observed in the 6-OHDA + FUS + Curcumin group relative to the 6-OHDA group. (B) Quantification of striatal TH immunoreactivity, expressed as optical density of TH-positive fibers in the lesioned striatum and normalized to the contralateral side. Data are presented as mean ± SEM. The dots represent individual mice in each group (Sham group, n = 5; 6-OHDA group, n = 8; 6-OHDA + FUS group, n = 5; 6-OHDA + Curcumin group, n = 7; and 6-OHDA + FUS + Curcumin group, n = 9). Significant differences were determined using one-way ANOVA followed by Tukey’s post-hoc test and are indicated with asterisks. **p < 0.01, ***p < 0.001.

FIGURE 6

Tyrosine hydroxylase (TH) immunohistochemistry evaluating dopaminergic neuron preservation in the substantia nigra pars compacta (SNpc) after 4 weeks of treatment. (A) Representative TH-immunostained coronal sections of the substantia nigra pars compacta (SNpc) from each experimental group, obtained from anatomically matched anteroposterior levels and processed simultaneously under identical staining conditions. Low-magnification images were acquired at 4 × magnification. Higher-magnification images (10 × ) are shown for comparison of TH-positive dopaminergic neurons. The 6-OHDA group exhibited a marked reduction in TH-positive neurons in the SNpc. Increased TH immunoreactivity was observed in the treatment groups, with the 6-OHDA + FUS + Curcumin group showing greater preservation of TH-positive neurons relative to the 6-OHDA group. (B) Quantification of TH-positive cells in the SNpc, expressed as the number of TH-immunoreactive neurons in the lesioned SNpc and normalized to the contralateral side. Data are presented as mean ± SEM. Dots represent individual animals in each group (Sham group, n = 4; 6-OHDA group, n = 6; 6-OHDA + FUS group, n = 5; 6-OHDA + Curcumin group, n = 7; 6-OHDA + FUS + Curcumin group, n = 7). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test and is indicated with asterisks. *p < 0.05, ***p < 0.001.

FIGURE 7

Glial fibrillary acidic protein (GFAP) immunofluorescence analysis of astrocytic activation in the striatum after 4 weeks of treatment. (A) Representative GFAP immunofluorescence images showing astrocytic activation in the lesioned striatum from each experimental group. Images were obtained from anatomically matched coronal sections and acquired using identical imaging parameters at 20 × magnification. Representative higher-magnification images (40 × ) are shown to illustrate regional differences in GFAP expression. The 6-OHDA-lesioned group exhibited increased GFAP immunoreactivity, indicative of reactive astrogliosis. (B) Quantification of GFAP-positive cell density within regions of interest encompassing the striatum, normalized to the contralateral side and expressed as a percentage. Data are presented as mean ± SEM. The dots represent individual mice in each group (Sham group, n = 5; 6-OHDA group, n = 9; 6-OHDA + FUS group, n = 5; 6-OHDA + Curcumin group, n = 9; and 6-OHDA + FUS + Curcumin group, n = 9). Significant differences were determined using one-way ANOVA followed by Tukey’s post-hoc test and are indicated with asterisks. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 8

Glial fibrillary acidic protein (GFAP) immunofluorescence analysis of astrocytic activation in the substantia nigra pars compacta (SNpc) after 4 weeks of treatment. (A) Representative GFAP immunofluorescence images showing astrocytic activation in the lesioned SNpc from each experimental group. Images were obtained from anatomically matched coronal sections and acquired using identical imaging parameters at 20 × magnification. Representative higher-magnification images (40 × ) are shown to illustrate regional differences in GFAP expression. The 6-OHDA-lesioned group exhibited increased GFAP immunoreactivity, consistent with astrogliosis. (B) Quantification of GFAP-positive cell density within regions of interest encompassing the SNpc, normalized to the contralateral side and expressed as a percentage. Data are presented as mean ± SEM. Dots represent individual animals in each group (Sham group, n = 5; 6-OHDA group, n = 7; 6-OHDA + FUS group, n = 5; 6-OHDA + Curcumin group, n = 7; 6-OHDA + FUS + Curcumin group, n = 7). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test and is indicated with asterisks. **p < 0.01.

FIGURE 9

Microglial activation and pro-inflammatory cytokine expression in the striatum after 4 weeks of treatment. (A) Quantification of Iba1-positive cell density in the lesioned striatum. Iba1 immunofluorescence was analyzed in anatomically matched coronal sections, and signal density within the striatal region of interest was quantified and expressed as mean ± SEM. Dots represent individual animals in each group (Sham group, n = 5; 6-OHDA group, n = 9; 6-OHDA + FUS group, n = 8; 6-OHDA + Curcumin group, n = 9; 6-OHDA + FUS + Curcumin group, n = 9). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. (B) Quantification of IL-6 immunoreactivity in the lesioned striatum. IL-6-positive cell density was quantified within the striatal region of interest, normalized to the contralateral side, and expressed as mean ± SEM. Dots represent individual animals in each group (Sham group, n = 2; 6-OHDA group, n = 5; 6-OHDA + FUS group, n = 5; 6-OHDA + Curcumin group, n = 5; 6-OHDA + FUS + Curcumin group, n = 5). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test.