A robust vessel-labeling pipeline with high tissue clearing compatibility for 3D mapping of vascular networks.

The combination of vessel-labeling, tissue-clearing, and light-sheet imaging techniques provides a potent tool for accurately mapping vascular networks, enabling the assessment of vascular remodeling in vascular-related disorders. However, most vascular labeling methods face challenges such as inadequate labeling efficiency or poor compatibility with current tissue clearing technology, which significantly undermines the image quality. To address this limitation, we introduce a vessel-labeling pipeline, termed Ultralabel, which relies on a specially designed dye hydrogel containing lysine-fixable dextran and gelatins for double enhancement. Ultralabel demonstrates not only excellent vessel-labeling capability but also strong compatibility with all tissue clearing methods tested, which outperforms other vessel-labeling methods. Consequently, Ultralabel enables fine mapping of vascular networks in diverse organs, as well as multi-color labeling alongside other labeling techniques. Ultralabel should provide a robust and user-friendly method for obtaining 3D vascular networks in different biomedical applications.

Dual-Channel in Spatial-Frequency Domain CycleGAN for perceptual enhancement of transcranial cortical vascular structure and function.

Observing cortical vascular structures and functions using laser speckle contrast imaging (LSCI) at high resolution plays a crucial role in understanding cerebral pathologies. Usually, open-skull window techniques have been applied to reduce scattering of skull and enhance image quality. However, craniotomy surgeries inevitably induce inflammation, which may obstruct observations in certain scenarios. In contrast, image enhancement algorithms provide popular tools for improving the signal-to-noise ratio (SNR) of LSCI. The current methods were less than satisfactory through intact skulls because the transcranial cortical images were of poor quality. Moreover, existing algorithms do not guarantee the accuracy of dynamic blood flow mappings. In this study, we develop an unsupervised deep learning method, named Dual-Channel in Spatial-Frequency Domain CycleGAN (SF-CycleGAN), to enhance the perceptual quality of cortical blood flow imaging by LSCI. SF-CycleGAN enabled convenient, non-invasive, and effective cortical vascular structure observation and accurate dynamic blood flow mappings without craniotomy surgeries to visualize biodynamics in an undisturbed biological environment. Our experimental results showed that SF-CycleGAN achieved a SNR at least 4.13 dB higher than that of other unsupervised methods, imaged the complete vascular morphology, and enabled the functional observation of small cortical vessels. Additionally, the proposed method showed remarkable robustness and could be generalized to various imaging configurations and image modalities, including fluorescence images, without retraining.

Tau induces inflammasome activation and microgliosis through acetylating NLRP3.

BACKGROUND: Alzheimer's disease (AD) and related Tauopathies are characterised by the pathologically hyperphosphorylated and aggregated microtubule-associated protein Tau, which is accompanied by neuroinflammation mediated by activated microglia. However, the role of Tau pathology in microglia activation or their causal relationship remains largely elusive. METHODS: The levels of nucleotide-binding oligomerisation domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) acetylation and inflammasome activation in multiple cell models with Tau proteins treatment, transgenic mice with Tauopathy, and AD patients were measured by Western blotting and enzyme-linked immunosorbent assay. In addition, the acetyltransferase activity of Tau and NLRP3 acetylation sites were confirmed using the test-tube acetylation assay, co-immunoprecipitation, immunofluorescence (IF) staining, mass spectrometry and molecular docking. The Tau-overexpressing mouse model was established by overexpression of human Tau proteins in mouse hippocampal CA1 neurons through the adeno-associated virus injection. The cognitive functions of Tau-overexpressing mice were assessed in various behavioural tests, and microglia activation was analysed by Iba-1 IF staining and [18F]-DPA-714 positron emission tomography/computed tomography imaging. A peptide that blocks the interaction between Tau and NLRP3 was synthesised to determine the in vitro and in vivo effects of Tau-NLRP3 interaction blockade on NLRP3 acetylation, inflammasome activation, microglia activation and cognitive function. RESULTS: Excessively elevated NLRP3 acetylation and inflammasome activation were observed in 3xTg-AD mice, microtubule-associated protein Tau P301S (PS19) mice and AD patients. It was further confirmed that mimics of 'early' phosphorylated-Tau proteins which increase at the initial stage of diseases with Tauopathy, including TauT181E, TauS199E, TauT217E and TauS262E, significantly promoted Tau-K18 domain acetyltransferase activity-dependent NLRP3 acetylation and inflammasome activation in HEK293T and BV-2 microglial cells. In addition, Tau protein could directly acetylate NLRP3 at the K21, K22 and K24 sites at its PYD domain and thereby induce inflammasome activation in vitro. Overexpression of human Tau proteins in mouse hippocampal CA1 neurons resulted in impaired cognitive function, Tau transmission to microglia and microgliosis with NLRP3 acetylation and inflammasome activation. As a targeted intervention, competitive binding of a designed Tau-NLRP3-binding blocking (TNB) peptide to block the interaction of Tau protein with NLRP3 inhibited the NLRP3 acetylation and downstream inflammasome activation in microglia, thereby alleviating microglia activation and cognitive impairment in mice. CONCLUSIONS: In conclusion, our findings provide evidence for a novel role of Tau in the regulation of microglia activation through acetylating NLRP3, which has potential implications for early intervention and personalised treatment of AD and related Tauopathies.

Transcranial photobiomodulation for brain diseases: review of animal and human studies including mechanisms and emerging trends.

The brain diseases account for 30% of all known diseases. Pharmacological treatment is hampered by the blood-brain barrier, limiting drug delivery to the central nervous system (CNS). Transcranial photobiomodulation (tPBM) is a promising technology for treating brain diseases, due to its effectiveness, non-invasiveness, and affordability. tPBM has been widely used in pre-clinical experiments and clinical trials for treating brain diseases, such as stroke and Alzheimer's disease. This review provides a comprehensive overview of tPBM. We summarize emerging trends and new discoveries in tPBM based on over one hundred references published in the past 20 years. We discuss the advantages and disadvantages of tPBM and highlight successful experimental and clinical protocols for treating various brain diseases. A better understanding of tPBM mechanisms, the development of guidelines for clinical practice, and the study of dose-dependent and personal effects hold great promise for progress in treating brain diseases.

Photostimulation of lymphatic clearance of ß-amyloid from mouse brain: a new strategy for the therapy of Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder that poses a significant burden on socio-economic and healthcare systems worldwide. However, the currently available therapy of AD is limited, and new strategies are needed to enhance the clearance of ß-amyloid (Aß) protein and improve cognitive function. Photobiomodulation (PBM) is a non-invasive and effective therapeutic method that has shown promise in treating various brain diseases. Here, we demonstrate that 1267-nm PBM significantly alleviates cognitive decline in the 5xFAD mouse model of AD and is safe as it does not induce a significant increase in cortical temperature. Moreover, with the combination of 3D tissue optical clearing imaging and automatic brain region segmentation, we show that PBM-mediated reductions of Aß plaques in different subregions of prefrontal cortex and the hippocampus are different. The PBM-induced lymphatic clearance of Aß from the brain is associated with improvement of memory and cognitive functions in 5xFAD mice. Our results suggest that the modulation of meningeal lymphatic vessels (MLVs) should play an important role in promoting Aß clearance. Collectively, this pilot study demonstrates that PBM can safely accelerate lymphatic clearance of Aß from the brain of 5xFAD mice, promoting improvement of neurocognitive status of AD animals suggesting that PBM can be an effective and bedside therapy for AD.

Transcranial photobiomodulation improves insulin therapy in diabetic microglial reactivity and the brain drainage system.

The dysfunction of microglia in the development of diabetes is associated with various diabetic complications, while traditional insulin therapy is insufficient to rapidly restore the function of microglia. Therefore, the search for new alternative methods of treating diabetes-related dysfunction of microglia is urgently needed. Here, we evaluate the effects of transcranial photobiomodulation (tPBM) on microglial function in diabetic mice and investigate its mechanism. We find tPBM treatment effectively improves insulin therapy on microglial morphology and reactivity. We also show that tPBM stimulates brain drainage system through activation of meningeal lymphatics, which contributes to the removal of inflammatory factor, and increase of microglial purinergic receptor P2RY12. Besides, the energy expenditure and locomotor activity of diabetic mice are also improved by tPBM. Our results demonstrate that tPBM can be an efficient, non-invasive method for the treatment of microglial dysfunction caused by diabetes, and also has the potential to prevent diabetic physiological disorders.

Protocol for fine casting, imaging, and analysis of murine vascular networks with VALID.

The majority of fluorescent vessel labeling techniques currently available are limited by their expense, incomplete labeling, or complexity. Here, we present VALID (vessel labeling via gelatin-based lipophilic dye solution)-a protocol for complete labeling of different vascular networks. We describe steps for preparing different dye hydrogels, murine vascular casting and tissue harvesting, immunolabeling, tissue clearing, and imaging, as well as detailed analysis of the vascular networks. This protocol is helpful for evaluating vascular lesions in studying different vessel-associated diseases. For complete details on the use and execution of this protocol, please refer to Zhu et al.1.

In vivo skin optical clearing for improving imaging and light-induced therapy: a review.

Significance: Skin is the largest organ and also the first barrier of body. Skin diseases are common, and cutaneous microcirculation is relative to various diseases. Researchers attempt to develop novel imaging techniques to obtain the complex structure, components, and functions of skin. Modern optical techniques provide a powerful tool with non-invasiveness, but the imaging performance suffers from the turbid character of skin. In vivo skin optical clearing technique has been proposed to reduce tissue scattering and enhance penetration depth of light and became a hot topic of research. Aim: The aim of this review is to provide a comprehensive overview of recent development of in vivo skin optical clearing methods, how in vivo skin optical clearing enhances imaging performance, and its applications in study and light therapy of various diseases. Approach: Based on the references published over the last decade, the important milestones on the mechanism, methods, and its fundamental and clinical applications of in vivo skin optical clearing technique are provided. Results: With the deepening understanding of skin optical clearing mechanisms, efficient in vivo skin optical clearing methods were constantly screened out. These methods have been combined with various optical imaging techniques to improve imaging performances and acquire deeper and finer skin-related information. In addition, in vivo skin optical clearing technique has been widely applied in assisting study of diseases as well as achieving safe, high-efficiency light-induced therapy. Conclusions: In the last decade, in vivo skin optical clearing technique has developed rapidly and played an important role in skin-related studies.

A versatile vessel casting method for fine mapping of vascular networks using a hydrogel-based lipophilic dye solution.

Efficient labeling of the vasculature is important for understanding the organization of vascular networks. Here, we propose VALID, a vessel-labeling method that enables visualization of vascular networks with tissue clearing and light-sheet microscopy. VALID transforms traditional lipophilic dye solution into hydrogel by introducing gelatin and restrains the dye aggregation, resulting in complete and uniform vessel-labeling patterns with high signal-to-background ratios. VALID also enhances the compatibility of lipophilic dyes with solvent-based tissue-clearing protocols, which was hard to achieve previously. Using VALID, we combined lipophilic dyes with solvent-based tissue-clearing techniques to perform 3D reconstructions of vasculature within mouse brain and spinal cord. We also employed VALID for 3D visualization and quantification of microvascular damage in a middle cerebral artery occlusion mouse model. VALID should provide a simple, cost-effective vessel-labeling protocol that would significantly widen the applications of lipophilic dyes in research on cerebrovascular complications.

A labeling strategy for the three-dimensional recognition and analysis of microvascular obstruction in ischemic stroke.

Rationale: Large vessel recanalization in ischemic stroke does not always go along with tissue reperfusion, a phenomenon called "no-reflow". However, knowledge of the mechanism of no-reflow is limited because identifying microvascular obstruction across the cortex and subcortex both in clinical and experimental models is challenging. In this study, we developed a smart three-dimensional recognition pipeline for microvascular obstruction during post-ischemia reperfusion to examine the underlying mechanism of no-reflow. Methods: Transient (60 min) occlusion of the middle cerebral artery (tMCAo) in mice was induced using a filament. Two different fluorophore-conjugated tomato lectins were injected into mice via the tail vein before and after ischemia/reperfusion (I/R), respectively, one to label all blood vessels and the other to label functional blood vessels. Post-I/R microvascular obstruction was visualized using combined iDISCO+-based tissue clearing and optical imaging. Arterioles and capillaries were distinguished using whole-mount immunolabeling with an anti-αSMA antibody. Circulating neutrophils were depleted utilizing an anti-Ly6G antibody. Brain slices were immunostained with the anti-Ly6G antibody to identify co-localized blockage points and neutrophils. MATLAB software was used to quantify the capillary diameters in the ipsilateral brain from the normal and tMCAo mice. Results: Microcirculatory reperfusion deficit worsened over time after I/R. Microvascular obstruction occurred not only in arterioles but also in capillaries, with capillary obstruction associated with local capillary lumen narrowing. In addition, the depletion of circulating neutrophils mitigated reperfusion deficit to a large extent after I/R. The co-localization of blockage points and neutrophils revealed that some neutrophils plugged capillaries with coexisting capillary lumen narrowing and that no neutrophil was trapped in heaps of blockage points. Quantification of the capillary diameter showed that capillary lumen shrunk after I/R but returned to typical measurements when intravascular neutrophils were depleted. Conclusions: According to our findings, both vascular lumen narrowing and neutrophil trapping in cerebral microcirculation are the key causes of microvascular obstruction after I/R. Also, the primary contribution by neutrophils to microvascular obstruction does not occur through microemboli plugging but rather via the exacerbation of capillary lumen narrowing. Our proposed method will help monitor microcirculatory reperfusion deficit, explore the mechanism of no-reflow, and evaluate the curative effect of drugs targeting no-reflow.

Rapid 3D isotropic imaging of whole organ with double-ring light-sheet microscopy and self-learning side-lobe elimination.

Bessel-like plane illumination forms a new type of light-sheet microscopy with ultra-long optical sectioning distance that enables rapid 3D imaging of fine cellular structures across an entire large tissue. However, the side-lobe excitation of conventional Bessel light sheets severely impairs the quality of the reconstructed 3D image. Here, we propose a self-supervised deep learning (DL) approach that can completely eliminate the residual side lobes for a double-ring-modulated non-diffraction light-sheet microscope, thereby substantially improving the axial resolution of the 3D image. This lightweight DL model utilizes the own point spread function (PSF) of the microscope as prior information without the need for external high-resolution microscopy data. After a quick training process based on a small number of datasets, the grown-up model can restore sidelobe-free 3D images with near isotropic resolution for diverse samples. Using an advanced double-ring light-sheet microscope in conjunction with this efficient restoration approach, we demonstrate 5-minute rapid imaging of an entire mouse brain with a size of ∼12 mm × 8 mm × 6 mm and achieve uniform isotropic resolution of ∼4 µm (1.6-µm voxel) capable of discerning the single neurons and vessels across the whole brain.

Optimal combinations of fluorescent vessel labeling and tissue clearing methods for three-dimensional visualization of vasculature.

Significance: Visualization of intact vasculatures is crucial to understanding the pathogeneses of different neurological and vascular diseases. Although various fluorescent vessel labeling methods have been used in combination with tissue clearing for three-dimensional (3D) visualization of different vascular networks, little has been done to quantify the labeling effect of each vessel labeling routine, as well as their applicability alongside various clearing protocols, making it difficult to select an optimal combination for finely constructing different vasculatures. Therefore, it is necessary to systematically assess the overall performance of these common vessel labeling methods combined with different tissue-clearing protocols. Aim: A comprehensive evaluation of the labeling quality of various vessel labeling routines in different organs, as well as their applicability alongside various clearing protocols, were performed to find the optimal combinations for 3D reconstruction of vascular networks with high quality. Approach: Four commonly-used vessel labeling techniques and six typical tissue optical clearing approaches were selected as candidates for the systematic evaluation. Results: The vessel labeling efficiency, vessel labeling patterns, and compatibility of each vessel labeling method with different tissue-clearing protocols were quantitatively evaluated and compared. Based on the comprehensive evaluation results, the optimal combinations were selected for 3D reconstructions of vascular networks in several organs, including mouse brain, liver, and kidney. Conclusions: This study provides valuable insight on selecting the proper pipelines for 3D visualization of vascular networks, which may facilitate understanding of the underlying mechanisms of various neurovascular diseases.

Three-dimensional mapping reveals heterochronic development of the neuromuscular system in postnatal mouse skeletal muscles.

The development of the neuromuscular system, including muscle growth and intramuscular neural development, in addition to central nervous system maturation, determines motor ability improvement. Motor development occurs asynchronously from cephalic to caudal. However, whether the structural development of different muscles is heterochronic is unclear. Here, based on the characteristics of motor behavior in postnatal mice, we examined the 3D structural features of the neuromuscular system in different muscles by combining tissue clearing with optical imaging techniques. Quantitative analyses of the structural data and related mRNA expression revealed that there was continued myofiber hyperplasia of the forelimb and hindlimb muscles until around postnatal day 3 (P3) and P6, respectively, as well as continued axonal arborization and neuromuscular junction formation until around P3 and P9, respectively; feature alterations of the cervical muscle ended at birth. Such structural heterochrony of muscles in different body parts corresponds to their motor function. Structural data on the neuromuscular system of neonatal muscles provide a 3D perspective in the understanding of the structural status during motor development.

Optical clearing imaging assisted evaluation of urokinase thrombolytic therapy on cerebral vessels with different sizes.

Ischemic stroke is caused by occlusion of the blood vessels in the brain, where intravenous thrombolytic therapy is the most effective treatment. Urokinase is a commonly used drug for intravenous thrombolytic therapy, while the effect of vessel size has not been thoroughly studied on urokinase. In this work, using the thrombin-combined photothrombosis model and craniotomy-free skull optical clearing window, we studied the recanalization of different cortical vessels after urokinase treatment. The results demonstrated that, compared to small vessels in distal middle cerebral artery (MCA) and large MCA, urokinase has the best therapeutic effect on secondary branches of MCA. This study holds potential to provide references for the clinical applications of urokinase.

In vivo tissue optical clearing assisted through-skull targeted photothrombotic ischemic stroke model in mice.

SIGNIFICANCE: Photothrombotic stroke is an important and widely used model for ischemic stroke research. However, the significant scattering of the skull during the procedure limits the light's ability to penetrate and focus on its target. Targeted photothrombosis uses surgery-based skull windows to obtain optical access to the brain, but it renders the brain's environment unnatural even before a stroke is established. AIM: To establish a targeted, controllable ischemic stroke model in mice through an intact skull. APPROACH: The in vivo skull optical clearing technique provides a craniotomy-free "optical window" that allows light to penetrate. Alongside the local photodynamic effect, we have established targeted photothrombosis without skull removal, effectively controlling the degree of thrombotic occlusion by changing the light dose. RESULTS: Ex vivo and in vivo results demonstrated that skull optical clearing treatment significantly enhanced light's ability to penetrate the skull and focus on its target, contributing to thrombotic occlusion. The skull optical clearing window was also used for continuous blood flow mapping, and the relationship between light dose and injury degree was evaluated over 14 days of monitoring. Per our findings, increasing the light dose was accompanied by more severe infarction, indicating that the model was easily controllable. CONCLUSIONS: Herein, a targeted, controllable ischemic stroke model was established by combinedly running an in vivo skull optical clearing technique and a photothrombotic procedure, avoiding unnecessary damage or environmental changes to the brain caused by surgery on the skull. Our established model should offer significant value to research on ischemic stroke.

Transmissive-detected laser speckle contrast imaging for blood flow monitoring in thick tissue: from Monte Carlo simulation to experimental demonstration.

Laser speckle contrast imaging (LSCI) is a powerful tool to monitor blood flow distribution and has been widely used in studies of microcirculation, both for animal and clinical applications. Conventionally, LSCI usually works on reflective-detected mode. However, it could provide promising temporal and spatial resolution for in vivo applications only with the assistance of various tissue windows, otherwise, the overlarge superficial static speckle would extremely limit its contrast and resolution. Here, we systematically investigated the capability of transmissive-detected LSCI (TR-LSCI) for blood flow monitoring in thick tissue. Using Monte Carlo simulation, we theoretically compared the performance of transmissive and reflective detection. It was found that the reflective-detected mode was better when the target layer was at the very surface, but the imaging quality would rapidly decrease with imaging depth, while the transmissive-detected mode could obtain a much stronger signal-to-background ratio (SBR) for thick tissue. We further proved by tissue phantom, animal, and human experiments that in a certain thickness of tissue, TR-LSCI showed remarkably better performance for thick-tissue imaging, and the imaging quality would be further improved if the use of longer wavelengths of near-infrared light. Therefore, both theoretical and experimental results demonstrate that TR-LSCI is capable of obtaining thick-tissue blood flow information and holds great potential in the field of microcirculation research.

Dec-DISCO: decolorization DISCO clearing for seeing through the biological architectures of heme-rich organs.

The tissue optical clearing technique plays an important role in three-dimensional (3D) visualization of large tissues. As a typical solvent-based clearing method, 3DISCO can achieve the highest level of tissue transparency with favorable clearing speed. However, 3DISCO cannot deal with the residual blood within tissues, leading to tissue brownness or redness after clearing, thus greatly influencing the tissue transparency and image quality due to the strong absorption of residual blood. To address this problem, we proposed an optimized clearing method by introducing CUBIC-L solution combined with 3DISCO for effective decolorization, termed Dec-DISCO (Decolorization DISCO). Dec-DISCO achieves better transparency than 3DISCO for various heme-rich tissues and performs enhanced fluorescence preservation capability. Dec-DISCO allows high-quality 3D imaging of fluorescently labeled heme-rich organs, as well as pathological tissue with severe hemorrhage. Dec-DISCO is expected to provide a powerful tool for histological analysis of kinds of heme-rich tissues in various medical conditions.

FDISCO+: a clearing method for robust fluorescence preservation of cleared samples.

Significance: The recently reported solvent-based optical clearing method FDISCO can preserve various fluorescent signals very well. However, the strict low-temperature storage condition of FDISCO is not conducive to long-time or repetitive imaging usually conducted at room temperature. Therefore, it is important to solve the contradiction between fluorescence preservation and imaging condition. Aim: We develop a modified FDISCO clearing method, termed FDISCO+, to change the preservation condition from low temperature to room temperature. Approach: Two alternative antioxidants were screened out to effectively inhibit the peroxide generation in the clearing agent at room temperature, enabling robust fluorescence preservation of cleared samples. Results: FDISCO+ achieves comparable fluorescence preservation with the original FDISCO protocol and allows long-time storage at room temperature, making it easier for researchers to image and preserve the samples. Conclusions: FDISCO+ is expected to be widely used due to its loose operation requirements.

Tissue optical clearing for 3D visualization of vascular networks: A review.

Reconstruction of the vasculature of intact tissues/organs down to the capillary level is essential for understanding the development and remodeling of vascular networks under physiological and pathological conditions. Optical imaging techniques can provide sufficient resolution to distinguish small vessels with several microns, but the imaging depth is somewhat limited due to the high light scattering of opaque tissue. Recently, various tissue optical clearing methods have been developed to overcome light attenuation and improve the imaging depth both for ex-vivo and in-vivo visualizations. Tissue clearing combined with vessel labeling techniques and advanced optical tomography enables successful mapping of the vasculature of different tissues/organs, as well as dynamically monitoring vessel function under normal and pathological conditions. Here, we briefly introduce the commonly-used labeling strategies for entire vascular networks, the current tissue optical clearing techniques available for various tissues, as well as the advanced optical imaging techniques for fast, high-resolution structural and functional imaging for blood vessels. We also discuss the applications of these techniques in the 3D visualization of vascular networks in normal tissues, and the vascular remodeling in several typical pathological models in clinical research. This review is expected to provide valuable insights for researchers to study the potential mechanisms of various vessel-associated diseases using tissue optical clearing pipeline.

An Approach to Maximize Retrograde Transport Based on the Spatial Distribution of Motor Endplates in Mouse Hindlimb Muscles.

Knowledge regarding the relationship between muscles and the corresponding motor neurons would allow therapeutic genes to transport into specific spinal cord segments. Retrograde tracing technique by targeting the motor endplate (MEP), a highly specialized structure that offers direct access to the spinal motor neurons, has been used to elucidate the connectivity between skeletal muscles and the innervating motor neuron pools. However, current injection strategies mainly based on blind injection or the local MEP region might lead to an underestimation of the motor neuron number due to the uneven distribution of MEP in skeletal muscles. In this work, we proposed a novel intramuscular injection strategy based on the 3D distribution of the MEPs in skeletal muscles, applied the 3D intramuscular injection to the gastrocnemius and tibialis anterior for retrograde tracing of the corresponding motor neurons, and compared this with the existing injection strategy. The intramuscular diffusion of the tracer demonstrated that 3D injection could maximize the retrograde transport by ensuring a greater uptake of the tracer by the MEP region. In combination with optical clearing and imaging, we performed 3D mapping and quantification of the labeled motor neurons and confirmed that 3D injection could label more motor neurons than the current injection method. It is expected that 3D intramuscular injection strategy will help elucidate the connective relationship between muscles and motor neurons faithfully and becomes a promising tool in the development of gene therapy strategies for motor neuron diseases.