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National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen, 518071, China
National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen, 518071, China
National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen, 518071, China
National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen, 518071, China
National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen, 518071, China
National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen, 518071, China
Corresponding author.Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, No. 8, Longyuan Road, Nanshan District, Shenzhen, 518055, China.
Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Medical Key Discipline of Health Toxicology (2020-2024), Shenzhen Center for Disease Control and Prevention, Shenzhen, 518055, China
National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen, 518071, China
Ultrasound enhanced delivery of Edavarone to the motor cortex in an ALS mouse model.
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The proposed approach further improved neuromuscular functions of the ALS model.
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The treatments conferred protection to both upper and lower motor neurons.
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The treatments alleviated neuroinflammation and reduced misfolded SOD1 protein.
Abstract
Background
Although amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease and unfortunately incurable yet, incremental attention has been drawn to targeting the health of corticospinal motor neurons. Focused ultrasound combined with systemically circulating microbubbles (FUS/MB) is an emerging modality capable of site-specific molecular delivery temporarily and noninvasively within a range of appropriate parameters.
Objective
To investigate the effect of FUS/MB–enhanced delivery of therapeutics to the motor cortex on the disease progression by using a transgenic mouse model of ALS.
Methods
Multiple FUS/MB–enhanced deliveries of Edaravone (Eda) to the motor cortex were performed on the SOD1G93A mouse model of ALS. The motor function of the animals was evaluated by gait analysis, grip strength and wire hanging tests. Corticospinal and spinal motor neuronal health, misfolded SOD1 protein and neuroinflammation after treatments were evaluated by histological examination.
Results
Ultrasound–enhanced delivery of Eda in the targeted motor cortex was achieved by a two-fold increase without gross tissue damage. Compared with the ALS mice administered Eda treatments only, the animals given additionally FUS/MB–enhanced brain delivery of Eda (FUS/MB + Eda) exhibited further improvements in neuromuscular functions characterized by gait patterns, muscular strength, and motor coordination along with rescued muscle atrophy. FUS/MB + Eda treatments conferred remarkable neuroprotection to both upper and lower motor neurons revealed by normalized neuronal morphology with increasing cell body size and profoundly alleviated neuroinflammation and misfolded SOD1 protein in the brains and lumbar spinal cords.
Conclusion
We report a pilot study that non-invasive ultrasound–enhanced brain delivery of Eda provides additive amelioration on disease progression of ALS and suggest that broadening the target from spinal to cortical network functions using the FUS/MB–enhanced delivery can be a rational therapeutic strategy of this debilitating disorder.
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive degeneration of upper (corticospinal motor neurons, CSMN) and lower (spinal motor neurons, SMN) components of motor neuron circuitry, leading to paralysis, respiratory failure, and death eventually. With poorly understood etiology, ALS is unfortunately incurable yet and current medical interventions could only mildly slow disease progression so far, leading to an urgent call for effective therapy strategies [
Although the site of origin of ALS is unraveled, increasing evidence from studies underscores the importance of cortical dysfunction, supporting the central premise for the “dying forward” hypothesis in ALS [
]. Promising results from animal studies have been reported that injection of therapeutic agents, including adeno-associated virus encoding short hairpin RNA [
Delayed disease onset and extended survival in the SOD1G93A rat model of amyotrophic lateral sclerosis after suppression of mutant SOD1 in the motor cortex.
Transplantation of neural progenitor cells expressing glial cell line-derived neurotrophic factor into the motor cortex as a strategy to treat amyotrophic lateral sclerosis.
], into the brain motor cortex contributed to delaying disease onset and extending survival of the animals. In a more recent seminal study, treating two nonoverlapping transgenic ALS mouse models by NU-9, a class of brain-penetrating molecules, eliminated the degeneration of CSMN with improved motor behavior of the animals [
However, delivering the therapeutic molecules to targeted regions of brain at a certain level of concentration to maximize curative outcome remains challenging mainly due to the stringent restrict of the blood-brain barrier (BBB). Consisting of basement membrane, pericytes, end-feet of astrocytes and endothelial cells that are connected by tight junctions, the BBB separates the brain parenchyma from the cerebral vasculature and limits the molecular trafficking. As a highly selective screener, while the BBB allows the passage of some small molecules, ions and metabolic substances, it blocks the entry of neurotoxins and macromolecules, thus confines drugs and antibodies to cerebral blood vessels, rendering them ineffective in the treatment of central nervous system disorders.
Aside from various brain delivery strategies to circumvent the BBB, such as intracerebral injection or grafts, intrathecal delivery, receptor- or carrier-mediated transcytosis and nanoparticulate systems, focused ultrasound combined with systemically circulating microbubbles (FUS/MB) is a physical modality capable of site-specific BBB opening temporarily, noninvasively, and reversibly within a range of appropriate parameters ensuring the safety [
]. These alluring features lead to a surge of interest in exploiting FUS/MB as a promising approach for drug delivery in the treatment of intractable central nervous system disorders. Myriad of preclinical studies proved that a wide range of biomolecules from small molecules to antibodies, nanoparticles and even cells are amenable to the brain delivery via FUS/MB-mediated BBB opening [
]. The pioneering safety trial, in which transcranial magnetic resonance (MR)-guided FUS/MB was performed on the primary motor cortex of four ALS patients, demonstrated safe and reversible BBB opening with the procedure well-tolerated [
]. Accumulating studies evidenced that the activation of microglia and reactive astrocytes, as well as the infiltration of peripheral immune cells, are prominent in the pathology of ALS [
]. Thus, targeting neuroinflammation has been proposed as a promising therapy strategy. As one of the only two medicines approved available for ALS patients in the clinic, the free radical scavenger edaravone (Eda) has been proven to protect neuronal cells from damaging by reactive oxygen species [
]. Previous studies have demonstrated that Eda successfully attenuated the degeneration of both motor neurons and skeletal muscles of various types of ALS rodent animal models [
], indicating its neuroprotective effects and antioxidant capacity. To date, to the best of our knowledge, there are no studies reporting the effect induced by FUS/MB–mediated drug delivery to the brain parenchyma on ALS.
Therefore, we aimed to evaluate whether FUS/MB–enhanced delivery of Eda to the motor cortex can generate positive effect on the disease progression to this treatment modality by using the SOD1G93A mouse model of ALS. FUS/MB treatments were performed on four non-overlapping sites in the motor cortex of the brain and the BBB opening was validated by contrast-enhanced T1-weighted magnetic resonance (MR) imaging. The outcome of the delivery was then compared with which of the cohorts that were injected with Eda only. The motor function of the mice was evaluated by gait analysis, grip strength and wire hanging tests, which were performed to assess gait patterns, muscular strength, and motor coordination. Additionally, CSMN and SMN health, as well as misfolded SOD1 protein and neuroinflammation after treatments were evaluated by histological examination.
2. Materials and methods
2.1 Animals
Transgenic female mice expressing a high copy number of the human SOD1 gene with a G93A mutation (B6.Cg-Tg (SOD1G93A)1Gur/J, the Jackson Laboratory) were used in this study. Animals were housed in a 12-h light-dark cycle room with relatively constant temperature (23°C–25 °C) and humidity (55 ± 5%). Animal care and experiments were approved by the Animal Care and Use from the Committee of the Experimental Animal Center at Shenzhen University.
2.2 Sonication setup of the FUS/MB treatment
Focused ultrasound beam was generated by a single-element spherical transducer (Center frequency: 1.1 MHz, F-number: 0.98, H-102, Sonic Concepts, U.S.A.), which was immersed in a cone filled with degassed water (Fig. 1A) and mounted on an automated stereotaxic apparatus (71000, RWD life science, China) for positioning to the target brain region (Fig. 1A). The tip of the cone was capped with a thin polyurethane membrane providing an acoustic window which allows ultrasound beam to pass through. The pressure amplitudes and beam dimensions of the transducer were measured using a needle hydrophone (HNR-0500; Onda, U.S.A.). The lateral and axial full-width at half maximum intensity of the beam is 1.8 mm and 13.5 mm.
Fig. 1Schematic of the study design. (A) Illustration of the sonication setup of focused ultrasound combined with microbubbles (FUS/MB) treatment. (B) Representative photomicrograph of MBs with a lipid shell and perfluoropropane core, and the size distribution. MBs were polydisperse with diameters ranging from 0.4 μm to 8 μm. (C) Contrast-enhanced B-mode images of a mouse brain with craniotomy before and after MBs injection and the corresponding time-intensity curve. (D) The timeline of a single FUS/MB treatment procedure. Before sonication, a bolus of MBs (0.2 μL/g) was injected through the tail vein of the mouse. Four intermittent non-overlapping FUS sonication with a 15-min interval between each other (peak-rarefactional pressure: 0.52 MPa; burst length: 9.09 msec; pulse repetition frequency: 1 Hz; duration: 60 sec) was then applied to the brain. The BBB opening was validated by T1-weighted MR imaging. (E) Scheme of the treatments and motor function tests. The treatments started at the age of 13 weeks and lasted for six weeks. For the Eda only and the FUS/MB + Eda group, Eda solutions were administered (15 mg/kg) through the tail vein injection (i. v.) and intraperitoneal injection (i. p.) alternately every two days due to the difficulty of tail vein injection on mice for such a long period. For FUS/MB + Eda group, FUS/MB treatments were conducted additionally at the same time of Eda administration through i. v. injection every four days.
Microbubbles with lipid shell and perfluoropropane gas core were prepared. The molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2000) (Lipoid) was 9:1. After preparation, the concentration and particle size distribution were measured by a Coulter Counter (Multisizer IV, Beckman Coulter, U.S.A.) after dilution with Isoton II solution. The freshly prepared MBs were polydisperse with the mean diameter of 1.8 μm and the concentration was 5.5 × 109/mL (Fig. 1B). To investigate the lifetime of MBs in the brain, contrast-enhanced ultrasound imaging was performed on mice with craniotomy using a small animal acoustic imaging system (Vevo® LAZR; VisualSonics, U.S.A.) with a 40-MHz transducer. The signal intensity of the images elevated immediately after MBs administration and then gradually dropped to the half level at about 10 mins later (Fig. 1C).
2.4 The procedure of FUS/MB-mediated BBB opening
Animals were placed in the prone position and anesthetized with 1.5% isoflurane (RWD Life Science, China). The body temperature of each mouse was maintained through a heating pad. Through the 3-D positioning system of the stereotaxic apparatus, a metal pointer was used to locate the transducer to the lambda on the skull as the reference. Before sonication, a bolus of MBs (0.2 μL/g) was injected through a catheter inserted in the tail vein of the mouse followed by saline flush. Aiming to achieve favorable delivery outcomes without tissue damaging, the parameters were chosen based on our previous study: 0.52-MPa peak-rarefactional pressure, 9.09-msec burst length, 1-Hz pulse repetition frequency and 60-sec duration for single sonication procedure [
]. Four intermittent non-overlapping sonication with a 15-min interval between each other was then applied to the brain with the centers spatially distributing in the corners of a 2-mm square, which centered 0.5 mm anterior to the lambda. After FUS/MB treatment was completed, a bolus of gadopentatate dimeglumine MR contrast agent (Gd-DTPA; Magnevist; Berlex Laboratories; 0.2 mL/kg) was infused into the tail vein catheter and T1-weighted fast spin-echo images of the brain were acquired in coronal and transverse planes to confirm the opening of the BBB (3T, uPMR790, Shanghai United Imaging Healthcare; repetition/echo time: 450/15 msec; resolution: 0.25×0.25×1.0 mm3; slice thickness: 1.0 mm).
2.5 The quantification of Eda delivered to the brain by FUS/MB
The plasma concentrations of Eda after intravenous administration were first measured. Eda (MB144, Meilun Bio, Shanghai, China) solution were injected through the tail vein of the mice (5 mg/kg of body weight). Blood samples (n = 3 for each time point) were obtained at 3 min, 10 min, 30 min, 60 min and 120 min post injection and further processed into plasma. Another two cohorts of normal mice (n = 5 for Eda and n = 5 for FUS/MB + Eda) was used to quantify the Eda delivered to the brain by FUS/MB. Cardiac perfusions with saline were performed 3 mins after Eda injection. Then the motor cortices were harvested, weighed, and homogenized on ice quickly. Eda concentrations in plasma and motor cortex were determined by liquid chromatography-tandem mass spectrometry method (LC-MS/MS, Wuhan Servicebio Technology, China).
2.6 Experimental groups
There were totally fifty female ALS mice randomly assigned to five groups (n = 10 per group). The mice in the ALS group were administered saline through the tail vein injection every four days. The FUS group was treated by FUS sonication alone without MB, while the FUS/MB group received FUS sonication with MB but without Eda every four days. For the Eda only and the FUS/MB + Eda group, Eda solutions were administered (15 mg/kg) through the tail vein injection (i. v.) and intraperitoneal injection (i. p.) alternately every two days due to the difficulty of tail vein injection every day on mice for such a long period and alleviating the pressure of the injections. For FUS/MB + Eda group, FUS/MB treatments were conducted additionally and immediately followed by Eda administration through i. v. injection every four days lasting from the age of 93 days–133 days (Fig. 1E). Our previous study revealed that the BBB had recovered within 24 h after single FUS/MB treatment [
Ultrasound with microbubbles improves memory, ameliorates pathology and modulates hippocampal proteomic changes in a triple transgenic mouse model of Alzheimer.
], thus this intermittent period is acceptable. Non-transgenic mice of gender-matched littermates (WT, n = 10) were treated with saline i. v. every four days. In this study, we chose the disease onset as the initiation of the treatment to make close to the clinical situation. The behavioral tests showed that the animals started to show abnormalities in the gait pattern and reduction in grip strength at the age of 13 weeks. The treatments started at the age of 13 weeks and lasted for six weeks (Fig. 1E). Due to the reason that the mice in the ALS, FUS, and FUS/MB group were weak at 19-week age, we decided to terminate all the treatments at that time. During the experiment, the intravenous injections were performed by a skilled technician to reduce the stress as much as possible.
2.7 Gait analysis
Experimenters were blind in all instances to both genotype and treatment regimen. The gait of voluntary movement was assessed by a gait analysis system (BT60601, Stones Scientific Instruments, Shenzhen, China), which allows recording the walking of animals from a ventral view by a high-speed digital video camera (150 frames per second) and analyzes automatically various gait parameters. All the tests were performed in the dark. Once the animal entered the walkway, 3–5 complete strides were recorded and the software of the gait system could automatically classify compliant run. After each animal test, the walkway was carefully cleaned with ethanol 70% in order to avoid ordor interference from previous animal. Stride length, average speed, stance time of hindlimbs and time of diagonal support were used to describe gait characteristics.
2.8 Grip strength test
Mice were placed on a wire grid with all the four limbs gripping the grid connected to a grip strength meter (ZS-ZL, Stones Scientific Instruments, Shenzhen, China). When pulling the mice backwards by the tail, the peak force at the time of release was recorded in gram-force (gf) and normalized to body weight (g). This was repeated three times with 5-min intervals between measurements and the maximum value was taken.
2.9 Wire hanging test
Mice were placed in the center of a wire grid (21 cm × 21 cm; line width, 0.1 cm) first and the grid was then slowly inverted. The latency to fall was recorded. This test was performed three times with a 5-min interval between each and the longest endurance was taken with 120 s as the cut-off value.
2.10 Histological examination and immunofluorescence analysis
Mice were deeply anesthetized and followed by intracardiac perfusion with 4% paraformaldehyde (PFA). To assess the safety of FUS/MB treatment, brains were postfixed in PFA overnight and then embedded in paraffin. Their coronal sections (7 μm) were stained with hematoxylin and eosin (H&E) and toluidine blue (Nissl) in order to examine microvascular injury and neuronal damage. After sacrifice, gastrocnemius muscles were dissected from hindlimbs after hair, skin, and surrounding fascia removal. They were fixed in 4% PFA for 48 h and then processed for H&E staining. The sections were imaged by a microscope (BX53; Olympus Corporation, Japan) using a 20× objective.
For immunofluorescence analysis, four mice in each group were used and serial sections of brains and lumbar spinal cords (L3 - L5) were cut into 30-μm slices using a vibratome (Leica VT1000S, Leica Inc., Germany) and free-floating immunohistochemistry was performed. To identify CSMN in motor cortex, the nuclei of motor neurons in layer V were immunohistochemically stained using primary antibodies against COUP-TF-interacting protein 2 (CTIP2; rabbit; 1:500; Abcam, ab240636) and neuronal bodies were stained by Nissl staining (Neurotrace; 1:300; Invitrogen, N21482). For SMN, spinal cord sections were stained using primary antibodies against choline acetyltransferase (ChAT; goat; 1:200; Millipore, U.S.A.). To assess the neuroinflammation, glial fibrillary acidic protein (GFAP; mouse; 1:400; Millipore, U.S.A.) for astrocytes and ionized calcium binding adaptor molecule 1 (Iba1; rabbit, 1:600; Wako, Japan) for microglia were used. Anti-misfolded SOD1 mouse monoclonal antibodies B8H10 (mouse; 1:400; Medimabs, Canada, MM-0070-P) were used for immunofluorescence. The secondary antibodies used were donkey Alexa 568-conjungated anti-rabbit IgG (1:1000, A10042, Invitrogen), donkey Alexa 488-conjungated anti-mouse IgG (1:500, A21202, Invitrogen) and donkey Alexa 488-conjungated anti-rabbit IgG (1:500, A21206, Invitrogen). Sections were coverslipped with DAPI (4’,6-diamidino-2-phenylindole) fluorescence mounting medium (Dako) to counterstain the nuclei. Fluorescence images were acquired with a confocal laser scanning microscope (LSM 880, Carl ZEISS, Germany) using a 63× oil objective or microscope (Nikon Eclipse Ti2, Japan) using a 20× objective.
For quantification, three lumbar (L4-L5) spinal cord sections and brain sections (∼1–2 mm anterior to the bregma, 400 μm apart) were used in each animal (n = 4). To analyze CSMN in motor cortex, CTIP2+ cells in layer V were counted in three adjacent fields defined by the 20× objective and the neuron body size was measured based on Nissl staining using ImageJ software (NIH, Bethesda, MD, USA). To analyze SMN, ChAT positive (ChAT+) motor neurons were counted per section and the cell body size was measured. For the assessment of microglial morphologies, skeleton analysis was carried out on fluorescence images of brain or spinal cord sections stained by anti-Iba1 antibodies as described previously [
]. All counts were performed blinded to the genotype of the sample by two independent observers.
2.11 Statistical analysis
Statistical analysis was performed by using GraphPad Prism (Version 8.3; GraphPad). All data were expressed as the mean ± standard deviation (SD). Statistical differences were evaluated by one-way analysis of variance (ANOVA) among the groups. The post-hoc analysis was used by Tukey's multiple comparison test. Statistically significant differences were taken at p < 0.05.
3. Results
3.1 FUS/MB enhanced delivery efficiency of Eda across the BBB to the motor cortex
BBB opening by FUS/MB was evaluated by contrast-enhanced T1-weighted MR imaging. Significant gadolinium enhancement can be seen in the targeted region on T1-weighted images (Fig. 2A), indicating successful opening of the BBB. Moreover, H&E and Nissl staining of the brain slices revealed that there was no hemorrhage and neuron damage in the sonicated region compared to the control ones (Fig. 2B). The performance of mice in the grip strength and wire hanging tests showed that there were no differences before and after FUS/MB treatment (Fig. 2C).
Fig. 2FUS/MB–enhanced brain delivery outcome of edaravone (Eda). (A) Representative Gd-enhanced T1-weighted MR images of horizontal (top panel) and coronal (bottom panel) slices of the mice brains. Four non-overlapping sonications were applied to the motor cortex with the centers spatially distributing in the corners of a 2-mm square, which centered 0.5 mm anterior to the lambda. The yellow line outlines the contrast enhancement area. Scale bar: 5 mm. (B) H&E and Nissl staining images of non-treated and FUS/MB-treated motor cortices. Bottom panel: magnified images from the black boxes in the top panel. Top scale bar: 1 mm. Bottom scale bar: 100 μm. Scale bar in the white inserts: 25 μm. (C) The grip strength and wire hanging tests of the mice before and 24 hr after single FUS/MB treatments. ns: no significant difference. (D) Eda concentration in plasma at different times after intravenous administration (5 mg/kg, n = 3 for each time point). (E) Eda concentrations in motor cortices of the mice treated by Eda only and FUS/MB + Eda measured by LC-MS/MS method. All data are shown as means ± SD.
Next, FUS/MB-mediated delivery efficiency of Eda to the motor cortex was quantified by LC-MS/MS method. We first examined the Eda concentration in plasma at different time points after intravenous injection and found that the Eda concentration decreased rapidly in plasma (Fig. 2D), indicating fast distribution into the tissues. As shown in Fig. 2E, the LC-MS/MS results showed that the concentration of Eda extracted from the homogenate of motor cortices in the FUS/MB + Eda group was a two-fold increase compared to the Eda group (117.7 ± 27.3 ng/g vs. 52.6 ± 16.5 ng/g, p = 0.003). Thus, enhanced delivery of Eda to the motor cortex could be achieved safely by using FUS/MB with the parameters used in this study.
3.2 FUS/MB + Eda treatments provided additive amelioration on motor function impairment of SOD1G93A mice
In this study, the motor function of the mice was evaluated by gait analysis, grip strength and wire hanging tests, which were performed to assess gait patterns, muscular strength, and motor coordination. There were no differences among the five groups of the ALS mice at the age of 13 weeks (Fig. S1). However, the animals started to show abnormalities in the gait and reduction in grip strength. Fig. 3A and Supplemental video showed representative footprint records of the six groups at the age of 19 weeks, which were quantified by stride length, average speed, and stance time of hindlimbs, and time of diagonal support (Fig. 3B). Compared with WT, ALS mice showed significant gait abnormalities characterized by reduced stride length (5.7 ± 0.4 cm in WT vs. 2.3 ± 0.7 cm in ALS, p < 0.001), increased stance time (0.15 ± 0.02 s in WT vs 0.7 ± 0.2 s in ALS, p < 0.001) and decreased average speed (18.4 ± 1.9 cm/s in WT vs. 3.8 ± 1.6 cm/s in ALS, p < 0.001). Disturbed gait pattern in ALS mice was also evident with less time supporting themselves with diagonal limbs (71.1% ± 7.0% in WT vs. 34.9% ± 11.7% in ALS, p < 0.001), reflecting a reduced stability of gait. There were no significant differences between FUS or FUS/MB group vs. the ALS group. In contrast, gaits of the animals in both the Eda alone group and FUS/MB + Eda group benefit from 6-week treatments, evidenced by tangible improvements in the four gait parameters (p < 0.001). Conspicuously, compared with the Eda alone treatments, the FUS/MB + Eda treatments provided effective therapeutic effects additively on the ALS mice gaits, supported by further improvements in gait parameters. The hindlimb stride length, hindlimb stance and the average speed of the FUS/MB + Eda group increased by 19.5% (4.9 ± 0.7 cm in FUS/MB + Eda vs. 4.1 ± 0.9 cm in Eda, p = 0.019), and by 20.7% (13.3 ± 2.7 cm/s in FUS/MB + Eda vs. 11.0 ± 2.0 cm/s in Eda, p = 0.002), accompanied with a 29.0% reduction in the stance time (0.2 ± 0.06 s in FUS/MB + Eda vs. 0.3 ± 0.09 s in Eda, p = 0.021). The percentage of time spent on diagonal paws also increased after FUS/MB + Eda treatments by a further 15.6% increment (66.7% ± 6.4% in FUS/MB + Eda vs. 56.3% ± 5.4% in Eda, p = 0.020), indicating much more improvement of unsteady gait than the Eda only treatments.
Fig. 3Behavioral assessment results of gait analysis, grip strength and wire hanging tests after 6-week treatments. (A) Representative footprint records of the mice in the six groups after 6-week treatments. WT: Non-transgenic mice of gender-matched littermates. ALS: SOD1G93A mice administered saline through the tail vein injection (i. v.). FUS or FUS/MB: SOD1G93A mice treated by FUS sonication alone without or with MB every four days. Eda: SOD1G93A mice administered Eda (15 mg/kg) through i. v. and intraperitoneal injection (i. p.) alternately every two days. FUS/MB + Eda: Besides Eda administration, FUS/MB treatments were conducted additionally at the same time of Eda administration (i. v.) every four days. (B) Four gait parameters including stride length, average speed, stance time of hindlimbs and time of diagonal support among the six groups (n = 10 per group). Compared with WT, ALS mice showed significant gait abnormalities with disturbed patterns. There were no significant differences between FUS or FUS/MB group vs. the ALS group. Gaits of the Eda alone group showed tangible improvements in the four gait parameters (p < 0.001). Conspicuously, compared with the Eda alone treatments, the FUS/MB + Eda treatments provided effective therapeutic effects additively on the ALS mice gaits, supported by further improvements in gait parameters. (C) Grip strength and (D) the latency to fall in the wire hanging tests of the six groups. After 6-week Eda treatments, the mice exhibited slower decline of the muscle strength compared to the ALS group (p < 0.001). Notably, the latency to fall in wire hanging test of the FUS/MB + Eda group extended significantly by a 20.3% increase (73.6 ± 8.4 s in FUS/MB + Eda vs. 61.2 ± 8.4 s in Eda, p = 0.017). #: vs. WT, p < 0.01. All data are shown as means ± SD.
As additional measures of neuromuscular function, grip strength and wire hanging tests were employed to assess muscle strength of combined forelimbs and hindlimbs (Fig. 3C and D). Compared with WT, the grip strength and hanging duration of the ALS group decreased drastically at the age of 19 weeks due to the muscle weakness (10.1 ± 1.1 gf/g in WT vs 5.0 ± 0.7 gf/g in ALS, p < 0.001, and 120.0 ± 0.0 s in WT vs 31.7 ± 9.6 s in ALS, p < 0.001). There were also no significant differences between FUS or FUS/MB group vs. the ALS group. After 6-week Eda treatments, the mice exhibited slower decline of the muscle strength compared to the ALS group (p < 0.001). Notably, the latency to fall in wire hanging test of the FUS/MB + Eda group extended significantly by a 20.3% increase (73.6 ± 8.4 s in FUS/MB + Eda vs. 61.2 ± 8.4 s in Eda, p = 0.017, Fig. 3D).
Histological analysis of gastrocnemius muscle revealed that myofiber atrophy in the ALS, FUS, and FUS/MB group became evident, characterized by groups of small and atrophic muscle fibers (Fig. 4A) and decreased cross-sectional area compared with the WT group (Fig. 4B, p < 0.001). Eda treatments alleviated pathological alterations apparently and increased the average myofiber area compared with non-treated ALS mice (788.2 ± 84.1 μm2 in Eda and 384.3 ± 43.7 μm2 in ALS, p < 0.001). However, atrophic and round muscle fibers could still be detectable in the Eda group. In line with the motor performance improvements, FUS/MB + Eda treatments exerted more profound effect in delaying the muscle atrophy with relatively ordered arrangement and larger myofibers by 13.9% compared with the Eda group (897.9 ± 104.4 μm2 in FUS/MB + Eda, p = 0.004). Along with the improvement in the muscle health, the body weight of the FUS/MB + Eda group exhibited a slightly increasing tendency until the end of the treatments (Fig. 4C).
Fig. 4Histology of the gastrocnemius muscle and weights of the animals. (A) Cross-sections of the gastrocnemius muscle fibers stained by hematoxylin and eosin. Scale bar: 50 μm. The insets: magnified images in the white dashed boxes; scale bar: 20 μm. Black arrows: myofiber atrophy. (B) Quantification of the average cross-sectional area of gastrocnemius muscle fibers in sections from mice of the six groups. Myofiber atrophy in the ALS, FUS, and FUS/MB group was evident and characterized by groups of small and atrophic muscle fibers and decreased cross-sectional area compared with the WT group (p < 0.001). Compared with the Eda group, FUS/MB + Eda treatments exerted more profound effect in delaying the muscle atrophy with relatively ordered arrangement and larger myofibers by 13.9%. (C) Body weights of the mice in the six groups during the treatment period. Along with the improvement in the muscle health, the body weight of the FUS/MB + Eda group exhibited a slightly increasing tendency until the end of the treatments. * (p < 0.05), ** (p < 0.01) and *** (p < 0.001): WT, ALS, FUS, and FUS/MB vs. FUS/MB + Eda; # (p < 0.05) and ### (p < 0.001): WT, ALS, FUS, and FUS/MB vs. Eda; %% (p < 0.01): FUS/MB + Eda vs. Eda. All data are shown as means ± SD.
]. By counterstaining CSMN with fluorescent Nissl staining, which was used to evaluate the CTIP2+ cell size, the CSMN of the WT mice showed a large pyramidal cell body with an average size of 133.9 ± 37.3 μm2 (Fig. 5A and B). In contrast with the WT group, the CSMN in the ALS group exhibited atrophic morphology with a significant size reduction by 51.1% (65.5 ± 6.8 μm2 in ALS, p < 0.001). Eda treatments improved the CSMN health with averagely larger size compared to the ALS group (95.5 ± 13.1 μm2 in Eda, p = 0.004). Conspicuously, the atrophy of CSMN in the FUS/MB + Eda group was slowed more evidently, indicated by normalized neuronal morphology with 1.84-fold cell body size of that in the ALS group (120.2 ± 18.2 μm2 in FUS/MB + Eda, p < 0.001) and 1.21-fold size of the Eda group (p < 0.001).
Fig. 5Immunostaining images and corresponding quantification of corticospinal motor neurons (CSMN) and spinal motor neurons (SMN) in the WT and SOD1G93A mice after different treatments. (A) Representative confocal fluorescence images of CTIP2 positive (CTIP2+, green nuclei) CSMN in layer V of the motor cortex. The neuronal bodies were stained by Nissl staining (Red). Scale bar: 20 μm. The insets: enlarged CSMN morphology. (B) The average cell body size of CTIP2+ CSMN in the mice of the six groups. (C) Representative confocal fluorescence images of ChAT positive (ChAT+, red cell body) SMN in the lumbar spinal cord. Scale bar: 20 μm. The nuclei were stained by DAPI (Blue). (D) The number and (E) the average cell body size of ChAT + SMN in the lumbar spinal cord sections from the mice of the six groups. # (p < 0.001): ALS, FUS, FUS/MB, and Eda vs. WT. All data are shown as means ± SD.
We also performed ChAT immunostaining in the lumbar spinal cord to evaluate the therapeutic effects on the SMN (Fig. 5C). Compared with WT, SOD1G93A mice exhibited significant degeneration of SMN with a 70.2% loss in the total number (26.2 ± 3.3 in WT vs. 7.8 ± 1.6 in ALS, p < 0.001) and a 42.9% decrease in the cell body size of ChAT + neurons (1024.5 ± 189.7 μm2 in WT vs. 584.2 ± 150.2 μm2 in ALS, p < 0.001, Fig. 5D and E). Although Eda treatments induced an increase in the number of SMN (10.7 ± 1.5, p = 0.016), there was no significant improvement in the cell body size (692.4 ± 132.3 μm2 in Eda) compared to the ALS group (p = 0.507). In contrast, SOD1G93A mice received FUS/MB + Eda treatments showed a more favorable effect on ameliorating the pathological abnormalities in SMN compared with the Eda group, with a significant 25.2% increase in the number (13.4 ± 2.2 in FUS/MB + Eda, p = 0.028) and a 28.6% improvement in the average size (890.7 ± 130.0 μm2 in FUS/MB + Eda, p = 0.025).
These results would suggest that FUS/MB + Eda treatments to the motor cortex conferred remarkable protection to the CSMN and bolstered the beneficial effect of Eda on the SMN of the ALS mice as well.
3.4 FUS/MB + Eda treatments ameliorated microglial activation and astrocytosis in SOD1G93A mice
Neuroinflammation has known to be involved in the disease progression of ALS with microgliosis and astrocytosis in both the motor cortex and spinal cord, which were evaluated by neuroinflammatory markers Iba1 and GFAP in the present study. The WT mice showed a normal morphology of surveillant microglia with small cell bodies and highly ramified branching processes in the layer V of the motor cortex (Fig. 6A) without astrocytosis (Fig. 7A). In contrast, activated microglia with short and protruding processes and enlarged cell body size were abundant in the ALS group as well as the FUS and FUS/MB group, along with increased GFAP immunoreactive area (vs. WT, p < 0.001, Fig. 6B). The quantification of the processes length and cell body size showed that mitigation of microglial activation in the motor cortex was manifest in the FUS/MB + Eda group compared with the ALS group (p < 0.05) whilst Eda only treatments failed to reach significance (Fig. 6B). The immunoreactive GFAP area was reduced in the motor cortex both in Eda and FUS/MB + Eda group (vs. ALS, p < 0.001).
Fig. 6Immunostaining images and corresponding quantification of microglial activation immunostained using the microglial cytoplasmic marker Iba1 (Red) in the brain and spinal cord sections from the WT and SOD1G93A mice after different treatments. (A) Representative confocal fluorescence images of microglia in the motor cortex from the mice of the six groups. Scale bar: 20 μm. (B) The process length and the average cell body size of microglia in the mice of the six groups after different treatments. (C) Representative confocal fluorescence images of microglia in the ventral horn of lumbar spinal cord sections from the mice of the six groups. Scale bar: 20 μm. (D) The process length and the average cell body size of microglia in the ventral horn of lumbar spinal cord sections from the mice of the six groups after different treatments. # (p < 0.001): ALS, FUS, FUS/MB, and Eda vs. WT. All data are shown as means ± SD.
Fig. 7Immunostaining images and corresponding quantification of astrocytosis (GFAP, green) in the brain and spinal cord sections from the WT and SOD1G93A mice after different treatments. (A) Representative fluorescence images of astrocytes in the motor cortex from the mice of the six groups. Scale bar: 500 μm. The insets: enlarged area in the white boxes; scale bar: 100 μm. (B) GFAP positive area per 20× field in the motor cortex from the mice of the six groups after different treatments. (C) Representative fluorescence images of astrocytes in the lumbar spinal cord sections from the mice of the six groups. Scale bar: 500 μm. The insets: enlarged area of the white boxes in the ventral horn; scale bar: 100 μm. (D) GFAP positive area in the ventral horn of lumbar spinal cord sections from the mice of the six groups after different treatments. # (p < 0.001): ALS, FUS, FUS/MB, and Eda vs. WT. All data are shown as means ± SD.
The Iba1 and GFAP immunofluorescence examinations were also performed in lumbar spinal cord sections (Fig. 6, Fig. 7C). The abnormal morphology of microglia with retracted processes and enlarged cell bodies and astrocytosis was especially prominent in the ventral horn of the lumbar spinal cord of the ALS group, FUS group, and FUS/MB group. After 6-week Eda only treatments, the alleviation of microglial activation in the spinal cord was apparent with smaller cell body size compared with the ALS group (300.5 ± 37.9 μm2 in Eda and 480.5 ± 57.8 μm2 in ALS, p < 0.001, Fig. 6, Fig. 7D). However, there was no significant difference in process length between the two groups (269.4 ± 70.6 μm2 in Eda and 280.9 ± 68.9 μm in ALS, p = 0.986). Noticeably, the FUS/MB + Eda group showed a profoundly morphological recovery of microglia with increasing ramifications and normalized cell body, as well as exhibiting evident improvement in the astrocytosis compared with the ALS group (p < 0.001).
3.5 FUS/MB + Eda treatments profoundly reduced levels of misfolded SOD1 accumulation in SOD1G93A mice
To determine whether FUS/MB + Eda treatments attenuates the accumulation of misfolded SOD1, we detected misfolded SOD1 forms by immunofluorescence on both brain sections and spinal cord stained with the monoclonal antibody B8H10, which recognized misfolded forms of mutant human SOD1 protein (Fig. 8). There was no B8H10 signal in the brains of WT mice and misfolded SOD1 was evident in layer V of the motor cortex where bright signal was observed within the soma of CSMN in the ALS, FUS and FUS/MB group (Fig. 8A and B). Although Eda mildly alleviated the level of misfolded SOD1 in the motor cortex, the difference did not reach significance when compared to the ALS group (16.3 ± 4.6 in Eda vs. 21.7 ± 8.4 in ALS, p = 0.173, Fig. 8B). In striking contrast, the FUS/MB + Eda group markedly reduced the accumulation of misfolded SOD1 in the motor cortex to a level without significant difference with the WT ones (9.3 ± 2.9 in FUS/MB + Eda vs. 5.7 ± 1.2 in WT, p = 0.598).
Fig. 8Immunostaining images and corresponding quantification of misfolded SOD1 protein in the brain and spinal cord sections from the WT and SOD1G93A mice after different treatments. (A) Representative confocal fluorescence images of brain sections with misfolded SOD1 specific antibody B8H10 (green) and co-labeled with CTIP2 (Red) in the motor cortex from the mice of the six groups. Scale bar: 20 μm. (B) The average fluorescence intensity of B8H10 signal per field of view in the brain sections. (C) Representative confocal fluorescence images of lumbar spinal cord sections with misfolded SOD1 specific antibody B8H10 (green) and co-labeled with ChAT (Red) in the ventral horns. Scale bar: 20 μm. (D) The average fluorescence intensity of B8H10 signal per field of view in the ventral horn of lumbar spinal cord sections from the mice of the six groups after different treatments. # (p < 0.001): ALS, FUS, FUS/MB, and Eda vs. WT. All data are shown as means ± SD.
In the ALS, FUS and FUS/MB group, abundant misfolded SOD1 proteins were co-localized with diseased SMN accompanied by profuse puncta throughout the neuropil, while absent in the WT mice (Fig. 8C). Notably, Eda only treatments reduced levels of mutant SOD1 compared with the ALS group (10.2 ± 2.4 in Eda vs. 17.0 ± 5.8 in ALS, p = 0.003, Fig. 8D), but not as profoundly as the FUS/MB + Eda group in which the B8H10 signal in the spinal cord nearly recovered to a comparable level of the WT ones (4.8 ± 1.5 in FUS/MB + Eda vs. 3.9 ± 2.6 in WT, p = 0.997).
Collectively, the results indicated that FUS/MB-enhanced Eda accumulation in the motor cortex elicited more tangible improvements in reducing misfolded SOD1 protein in SODG93A mice.
4. Discussion
Recent clinical safety trials in patients have demonstrated the great potential of FUS/MB in the treatment of neurodegenerative disorders [
]. However, the effect of FUS/MB-enhanced drug delivery on ALS models has not been reported yet. Here, we provided a pilot study to reveal that FUS/MB-enhanced delivery of Eda to the brain improved the therapeutic effect of Eda on SOD1G93A transgenic mice assessed by motor function behavioral tests and pathological examinations.
With a short half-life in the circulation, a relatively high dose (60 mg/day) of Eda is used in clinic with long-time intravenous administration and complications, such as injection-site contusion and renal function disorder, have been reported [
]. In order to avoid possible side effects, a 14-day drug-free period is arranged in the first treatment cycle. It has been reported that Eda concentration in the male rat brain tissue at 5 min after a single intravenous administration of Eda (2 mg/kg) was 590 ng/g, in a range of 1/16–1/18 of the plasma concentration, which decreased rapidly in the distribution phase with a half-life of only 5.4 min [
Pharmacokinetic studies of 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186) in rats (1): blood and plasma levels, distribution, metabolism and excretion after a single intravenous administration.
]. We also found quick decline of the Eda plasma concentration, but the one in motor cortex was much lower, nearly by one magnitude, than the value measured in the brain by Komatsu et al. Moreover, they found that the radioactivity of 14C labeled Eda in tissues disappeared quickly after a single intravenous injection indicating fast elimination effect in the body [
Pharmacokinetic studies of 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186) in rats (1): blood and plasma levels, distribution, metabolism and excretion after a single intravenous administration.
]. Thus, the effective delivery and tissue targeting method are critical for maximizing the potential for the therapeutic benefit of Eda. In the present study, the results of Gd-DTPA contrast MR imaging together with the quantification of Eda concentration demonstrated FUS/MB as a noninvasive method for enhancing local local delivery of Eda through the BBB in the motor cortex, such that the drug level shown resulted in more favorable effects on motor function behavior and motor neuron health in the FUS/MB + Eda group than in the Eda only group.
Multiple behavioral tests were used in this study to evaluate a range of motor functions, including gait patterns, motor coordination and muscular strength. Gait analysis is sensitive to motor functional deficits in SOD1G93A mice [
Locomotor analysis identifies early compensatory changes during disease progression and subgroup classification in a mouse model of amyotrophic lateral sclerosis.
]. Compared with the WT group, the ALS, FUS and FUS/MB group showed significant gait abnormalities revealed by strikingly decreased stride length and average speed along with the increased stance time during a recorded run, reflecting marked muscle atrophy. In addition, the WT mice showed predominant diagonal support in a step cycle, while the SOD1G93A mice without Eda treatments presented unsteadily wobble gait patterns due to severely deteriorated motor coordination and aberrant muscular strength of the four limbs assessed by the grip strength and wire hanging tests. Ito et al. found that a higher Eda dose of 15 mg/kg (i. p.) every day induced significantly beneficial outcomes on motor performance of SOD1G93A mice while a lower dose of 5 mg/kg did not [
]. Thus, we chose 15 mg/kg as the Eda administration dosage in the present study. Although Eda was given every two days, which was different from the dosing interval in the above study, the SOD1G93A mice in the Eda group showed consistent results with ameliorated gait deficits and improved muscular strength. Conspicuously, the disease modifying effect of FUS/MB-enhanced delivery of Eda targeting to the motor cortex of the SOD1G93A mice was more effective on the motor functions compared with the ones undergone only Eda treatments. Although it has been reported that effective Eda concentration in cerebral spinal fluid was maintained after intravenous infusion to dogs [
], we found low Eda concentration in motor cortex of the animals in the present study. Using intravitally two-photon imaging method, the dynamic process of macromolecular delivery across the BBB during FUS sonication with circulating MB was observed in vivo followed by deep penetration into the brain interstitium, thus directly raising the local accumulating concentration of drugs [
]. It was speculated that this immediate effect was closely related to the neuroprotective effect of Eda on CSMN to reinforce its therapeutic efficacy.
CSMN are an indispensable component of the motor neuron circuitry. They are essential to voluntary movement by collecting information from various neurons in the brain and conveying the cortical input to spinal cord targets. Degeneration of CSMN has been observed even in presymptomatic stage of ALS in SOD1G93A mice [
]. With the disease progression, the degeneration of CSMN was progressive, manifested by gradual decrease in CSMN soma size and neuron number. Although the origin of this disease remains controversial, recent studies have built evidence that it has significant clinical implications to improve the health of CSMN for the ALS therapy [
Delayed disease onset and extended survival in the SOD1G93A rat model of amyotrophic lateral sclerosis after suppression of mutant SOD1 in the motor cortex.
Transplantation of neural progenitor cells expressing glial cell line-derived neurotrophic factor into the motor cortex as a strategy to treat amyotrophic lateral sclerosis.
]. By counterstaining the brain sections with antibodies against CTIP2, a CSMN-specific transcription factor in layer V of the motor cortex, and fluorescent Nissl stain, the distinctions of CSMN between the WT and the ALS group could be visualized clearly in this study. Compared with the SOD1G93A mice which were given only Eda administration every two days, immunostaining results revealed that the SOD1G93A mice, which were given additionally repeated FUS/MB treatments every four days initiating at disease onset, displayed more healthier CSMN and SMN concomitantly. The delayed progression in the disease pathology of motor neurons could be a critical factor contributing to the improved motor function performance of SOD1G93A mice, as CSMN and SMN are known to directly correlate with the initiation, modulation, and execution of voluntary movement.
There is a consensus that misfolded SOD1 accumulation, motor neuron degeneration and microgliosis are pathological hallmarks in SOD1G93A mice [
]. SOD1 mutations initiate a cascade of events, including glutamate excitotoxicity, oxidative stress, and mitochondrial dysfunction, leading to motor neuron degeneration eventually. Reducing or elimination of SOD1 protein levels, which is a strategy of targeting the disease at the source, may potentially provide a therapeutic benefit to patients with SOD1-related ALS. In line with previous studies [
], we also found alleviation of misfolded SOD1 in spinal cord of the SOD1G93A mice which were given Eda therapy. However, no significant reduction of misfolded SOD1 was found in the motor cortex of the Eda group, probably associating with the limited level of Eda accumulated in the brain tissue after administration. This inadequate effect on the brain was breached by FUS/MB, as demonstrated by remarkable reductions of misfolded SOD1 and much healthier CSMN in the motor cortex compared with the Eda only group. Admittedly, the increasing or decreasing magnitudes of all the quantitative measures of the behavioral results in the FUS/MB + Eda group were no more than 30% when comparing with the ones in the Eda only group, indicating that the additive effect was mild. These results were comparable to the changing scales of indices of the gastrocnemius muscle, motor neurons in spinal cord, microglia, and astrocytes. Conspicuously, the SOD1 level in both the motor cortex and spinal cord in the FUS/MB + Eda group decreased by more profound degrees, more than 40%, suggesting other different mechanisms contributing to the ALS motor functional deficits besides aberrant SOD1 aggregation.
As Eda is a free radical scavenger with antioxidant properties, we then investigated the distinctions of neuroinflammation among the SOD1G93A mice undergone different treatments. Consistent with other studies, we found that Eda could attenuate astrocyte proliferation and microgliosis in the spinal cord [
]. Importantly, ultrasound-enhanced delivery of Eda to the motor cortex also induced an anti-inflammatory response of cortical microglia or astrocytes. Symptomatic treatment of SOD1G93A mice with FUS/MB + Eda improved motor performance additively. Whether this beneficial effect can be attributed to additional improvement due to broadly targeting both the cortical and spinal network functions remain to be determined, but this pilot study strongly highlights the brain-targeted therapy as a rational therapeutic strategy of this debilitating disorder.
There are several limitations to this study. First, Eda was administered every two days with alternate i. p. and i. v. injections because we would like to reduce injection burdens on ALS mice and ensure the success rates of sequential i.v. injections during the long-time treatment period as much as possible. Although this procedure was different from the clinic practice, the improved performance of the Eda group compared with the ALS group demonstrated the feasibility of the administration timing in this study. Second, we used only one dosing of Eda due to limited available number of transgenic mice. Future study is warranted to investigate the dose–response correlation and specifically whether FUS/MB could reinforce the therapeutic effect of Eda when using a lower dosage. Third, the present study used only one type of ALS transgenic mouse model with SOD1 gene mutation, to which only ∼20% of familial ALS cases are linked. As most ALS cases are sporadic, future studies are needed to include other ALS models, such as ones with TDP43 pathology with cortical circuit dysfunction as well [
], to recapitulate more aspects of ALS disease pathology. Fourth, the grip strength of all the four limbs were used without differentiating hindlimbs from forelimbs, thus possibly being causative of the unsignificant difference between the Eda and FUS/MB + Eda groups although there was a slightly increasing trend. Lastly, survival analysis was not included in the present study due to limited number of animals. Although it has not established whether Eda prolongs the lifespan of ALS patients [
], a larger cohort of ALS animals should be included in future preclinical studies to investigate whether FUS/MB–enhanced brain delivery of the drug can substantially extend the life expectancy of the treated animals.
5. Conclusions
In conclusion, this study showed successful ultrasound–enhanced delivery outcome of Eda in the targeted motor cortex without gross tissue damage. Compared with the ALS transgenic mice given by Eda treatments only, the animals given additionally FUS/MB–enhanced brain delivery of Eda (FUS/MB + Eda) exhibited further improvements in neuromuscular functions characterized by gait patterns, muscular strength, and motor coordination along with rescued muscle atrophy. FUS/MB + Eda treatments conferred remarkable neuroprotection to both CSMN and SMN revealed by normalized neuronal morphology with increasing cell body size. Additionally, neuroinflammation and misfolded SOD1 protein was also profoundly alleviated in the brains and lumbar spinal cords of the FUS/MB + Eda group. We provided the initial step in broadening the target from spinal to cortical network functions using the FUS/MB–enhanced delivery technology coupled with promising therapeutics in ALS.
Credit author statement
Yuanyuan Shen: Writing - Original Draft Preparation, Conceptualization, Investigation, Visualization, Funding acquisition; Ji Zhang: Investigation, Data curation, Methodology, Formal analysis; Yiluo Xu: Visualization, Investigation. Shuneng Sun: Methodology; Visualization; Kaili Chen: Validation; Siping Chen: Resources; Xifei Yang: Conceptualization, Resources; Xin Chen: Conceptualization, Funding acquisition Writing- Reviewing and Editing and Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work is supported by grants from National Key Research and Development Program of China (2020YFA0908800), National Natural Science Foundation of China (12074269, 81871429), Shenzhen Basic Science Research (20220808185138001, JCYJ20200109105622824, JSGG20210802153811034, JCYJ20210324093006017, JCYJ20200109150717745), Guangdong Natural Science Foundation (2021A1515011202), and Shenzhen-Hong Kong Institute of Brain Science- Shenzhen Fundamental Research Institutions (NYKFKT20190019). We would like to thank the Instrumental Analysis Center of Shenzhen University (Lihu Campus) for the help of fluorescence imaging.
Appendix A. Supplementary data
The following are the Supplementary data to this article.
Delayed disease onset and extended survival in the SOD1G93A rat model of amyotrophic lateral sclerosis after suppression of mutant SOD1 in the motor cortex.
Transplantation of neural progenitor cells expressing glial cell line-derived neurotrophic factor into the motor cortex as a strategy to treat amyotrophic lateral sclerosis.
Ultrasound with microbubbles improves memory, ameliorates pathology and modulates hippocampal proteomic changes in a triple transgenic mouse model of Alzheimer.
Pharmacokinetic studies of 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186) in rats (1): blood and plasma levels, distribution, metabolism and excretion after a single intravenous administration.
Locomotor analysis identifies early compensatory changes during disease progression and subgroup classification in a mouse model of amyotrophic lateral sclerosis.
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