Use of a tissue clearing technique combined with retrograde trans-synaptic viral tracing to evaluate changes in mouse retinorecipient brain regions following optic nerve crush

Successful establishment of reconnection between retinal ganglion cells and retinorecipient regions in the brain is critical to optic nerve regeneration.However,morphological assessments of retinorecipient regions are limited by the opacity of brain tissue.In this study,we used an innovative tissue cleaning technique combined with retrograde trans-synaptic viral tracing to observe changes in retinorecipient regions connected to retinal ganglion cells in mice after optic nerve injury.Specifically,we performed light-sheet imaging of whole brain tissue after a clearing process.We found that pseudorabies virus 724(PRV724)mostly infected retinal ganglion cells,and that we could use it to retrogradely trace the retinorecipient regions in whole tissue-cleared brains.Unexpectedly,PRV724-traced neurons were more widely distributed compared with data from previous studies.We found that optic nerve injury could selectively modify projections from retinal ganglion cells in the hypothalamic paraventricular nucleus,intergeniculate leaflet,ventral lateral geniculate nucleus,central amygdala,basolateral amygdala,Edinger-Westphal nucleus,and oculomotor nucleus,but not the superior vestibular nucleus,red nucleus,locus coeruleus,gigantocellular reticular nucleus,or facial nerve nucleus.Our findings demonstrate that the tissue clearing technique,combined with retrograde trans-synaptic viral tracing,can be used to objectively and comprehensively evaluate changes in mouse retinorecipient regions that receive projections from retinal ganglion cells after optic nerve injury.Thus,our approach may be useful for future estimations of optic nerve injury and regeneration.

Optical tissue clearing enables rapid,precise and comprehensive assessment of three-dimensional morphology in experimental nerve regeneration research

Morphological analyses are key outcome assessments for nerve regeneration studies but are historically limited to tissue sections.Novel optical tissue clearing techniques enabling three-dimensional imaging of entire organs at a subcellular resolution have revolutionized morphological studies of the brain.To extend their applicability to experimental nerve repair studies we adapted these techniques to nerves and their motor and sensory targets in rats.The solvent-based protocols rendered harvested peripheral nerves and their target organs transparent within 24 hours while preserving tissue architecture and fluorescence.The optical clearing was compatible with conventional laboratory techniques,including retrograde labeling studies,and computational image segmentation,providing fast and precise cell quantitation.Further,optically cleared organs enabled three-dimensional morphometry at an unprecedented scale including dermatome-wide innervation studies,tracing of intramuscular nerve branches or mapping of neurovascular networks.Given their wide-ranging applicability,rapid processing times,and low costs,tissue clearing techniques are likely to be a key technology for next-generation nerve repair studies.All procedures were approved by the Hospital for Sick Children’s Laboratory Animal Services Committee(49871/9)on November 9,2019.

A"messenger zone hypothesis"based on the visual three-dimensional spatial distribution of motoneurons innervating deep limb muscles

Coordinated contraction of skeletal muscles relies on selective connections between the muscles and multiple classes of the spinal motoneuro ns.Howeve r,current research on the spatial location of the spinal motoneurons innervating differe nt muscles is limited.In this study,we investigated the spatial distribution and relative position of different motoneurons that control the deep muscles of the mouse hindlimbs,which were innervated by the obturator nerve,femoral nerve,inferior gluteal nerve,deep pe roneal nerve,and tibial nerve.Locations were visualized by combining a multiplex retrograde tracking technique compatible with three-dimensional imaging of solvent-cleared o rgans(3DISCO)and 3-D imaging technology based on lightsheet fluorescence microscopy(LSFM).Additionally,we propose the hypothesis that"messenger zones"exist as interlaced areas between the motoneuron pools that dominate the synergistic or antagonist muscle groups.We hypothesize that these interlaced neurons may participate in muscle coordination as messenger neurons.Analysis revealed the precise mutual positional relationships among the many motoneurons that innervate different deep muscles of the mouse.Not only do these findings update and supplement our knowledge regarding the overall spatial layout of spinal motoneurons that control mouse limb muscles,but they also provide insights into the mechanisms through which muscle activity is coordinated and the architecture of motor circuits.

Ex-vivo confocal Raman microspectroscopy of porcine skin with 633/785-NM laser excitation and optical clearing with glycerol/water/DMSO solution

Confocal Raman microspectroscopy(CRM)with 633-and 785-nm excitation wavelengths combined with optical clearing(OC)technique was used for ex-vivo study of porcine skin in the Raman fingerprint region.The optical clearing has been performed on the skin samples by applying a mixture of glycerol and distilled water and a mixture of glycerol,distilled water and chemical penetration enhancer dimethyl sulfoxide(DMSO)during 30 min and 60 min of treat-ment.It was shown that the combined use of the optical clearing technique and CRM at 633 nm allowed one to preserve the high probing depth,signal-to-noise ratio and spectral resolution simultaneously.Comparing the effect of different optical clearing agents on porcine skin showed that an optical clearing agent containing chemical penetration enhancer provides higher optical clearing efficiency.Also,an increase in treatment time allows to improve the optical clearing efficiency of both optical clearing agents.As a result of optical clearing,the detection of the amide-Ⅲ spectral region indicating well-distinguishable structural differences between the type-Ⅰ and type-Ⅳ collagens has been improved.

Varying of up-conversion nanoparticles luminescence from the muscle tissue depth during the compression

The current work is focused on the study of optical clearing of skeletal muscles under local compression.The experiments were performed on in vitro bovine skeletal muscle.The time dependence of optical clearing was studied by monitoring the luminescence intensity of NaYF_(4)∶Er,Yb upconverting particles located under tissue layers.This study shows the possibility to use upconverting nanoparticles(UCNPs)both for studying the dynamics of the optical clearing of biological tissue under compression and to detect moments of cell wall damage under excessive pressure.The advantage of using UCNPs is the presence of several bands in their luminescence spectra,located both at close wavelengths and far apart.

ALTERATIONS IN AUTOFLUORESCENCE SIGNAL FROM RAT SKIN EX VIVO UNDER OPTICAL IMMERSION CLEARING

For the first time,the changes in autofluorescence spectra of ex vivo rat skin have been experimentally investigated using the combination of fluorescence spectroscopy and optical immersion clearing.The glucose,glycerol and propylene glycol solutions were used as clearing agents.The optical clearing was performed from the dermal side of skin imitating the in vivo injection of clearing agent under the dermal layers.In this contribution,the common properties of autofluorescence variation during optical immersion clearing were determined.The tendency of autofluorescence signal to decrease with reduction of scattering in tissue was noticed and discussed in detail.However,the differences in the shape of spectral curves under application of different clearing agents showed that optical clearing affects the autofluorescence properties of tissue differently depending on the type of clearing liquid.The results obtained are useful for the understanding of tissue optical clearing mechanisms and for improving techniques such as fluorescence spectroscopy.

Tissue optical clearing imaging for structural changes of neuromuscular junctions after mice ischemic stroke[Invited]

Ischemic stroke causes long-term disability and results in motor impairments.Such impairments are associated with structural changes in the neuromuscular junction(NMJ),including detailed morphology and three-dimensional(3D)distribution.However,previous studies only explored morphological changes of individual NMJs after stroke,which limits the understanding of their role in post-stroke motor impairment.Here,we examine 3D distributions and detailed morphology of NMJs in entire mouse muscles after unilateral and bilateral strokes induced by photothrombosis.The results show that 3D distributions and numbers of NMJs do not change after stroke,and severe unilateral stroke causes similar levels of NMJ fragmentation and area enlargement to bilateral stroke.This research provides structural data,deepening the understanding of neuromuscular pathophysiology after stroke.

MACS-W:A modified optical clearing agent for imaging 3D cell cultures

Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.

Optical angiography for diabetes-induced pathological changes in microvascular structure and function:An overview

Diabetes mellitus(DM)is a kind of metabolic disorder characterized by chronic hyperglycemia and glucose intolerance due to absolute or relative lack of insulin,leading to chronic damage of vasculature within various organ systems.These detrimental e®ects on the vascular networks will result in the development of various diseases associated with microvascular injury.Modern optical imaging techniques provide essential tools for accurate evaluation of the structural and functional changes of blood vessels down to capillaries level,which can o®er valuable insight on understanding the development of DM-associated complications and design of targeted therapy.This review will brie°y introduce the DM-induced structural and functional alterations of vasculature within di®erent organs such as skin,cerebrum and kidneys,as well as how novel optical imaging techniques facilitate the studies focusing on exploration of these pathological changes of vasculature caused by DM both in-vivo and ex-vivo.

Recent advances in stem cell therapy for neurodegenerative disease: Three dimensional tracing and its emerging use

Neurodegenerative disease is a brain disorder caused by the loss of structure andfunction of neurons that lowers the quality of human life. Apart from the limitedpotential for endogenous regeneration, stem cell-based therapies hold considerablepromise for maintaining homeostatic tissue regeneration and enhancingplasticity. Despite many studies, there remains insufficient evidence for stem celltracing and its correlation with endogenous neural cells in brain tissue with threedimensionalstructures. Recent advancements in tissue optical clearing techniqueshave been developed to overcome the existing shortcomings of cross-sectionaltissue analysis in thick and complex tissues. This review focuses on recentprogress of stem cell treatments to improve neurodegenerative disease, andintroduces tissue optical clearing techniques that can implement a threedimensionalimage as a proof of concept. This review provides a more comprehensiveunderstanding of stem cell tracing that will play an important role inevaluating therapeutic efficacy and cellular interrelationship for regeneration inneurodegenerative diseases.

Three-dimensional histology:new visual approaches to morphological changes during neural regeneration

Three-dimensional（3D） histology utilizes tissue clearing techniques to turn intact tissues transparent,allowing rapid interrogation of tissue architecture in three dimensions.In this article,we summarized the available tissue clearing methods and classified them according to their physicochemical principles of operation,which provided a framework for one to choose the best techniques for various research settings.Recent attempts in addressing various questions regarding the degenerating and regenerating nervous system have been promising with the use of 3D histological techniques.

Effects of delayed repair of peripheral nerve injury on the spatial distribution of motor endplates in target muscle

Motor endplates(MEPs) are important sites of information exchange between motor neurons and skeletal muscle, and are distributed in an organized pattern of lamellae in the muscle. Delayed repair of peripheral nerve injury typically results in unsatisfactory functional recovery because of MEP degeneration. In this study, the mouse tibial nerve was transected and repaired with a biodegradable chitin conduit, immediately following or 1 or 3 months after the injury. Fluorescent α-bungarotoxin was injected to label MEPs. Tissue optical clearing combined with light-sheet microscopy revealed that MEPs were distributed in an organized pattern of lamellae in skeletal muscle after delayed repair for 1 and 3 months. However, the total number of MEPs, the number of MEPs per lamellar cluster, and the maturation of single MEPs in gastrocnemius muscle gradually decreased with increasing denervation time. These findings suggest that delayed repair can restore the spatial distribution of MEPs, but it has an adverse effect on the homogeneity of MEPs in the lamellar clusters and the total number of MEPs in the target muscle. The study procedures were approved by the Animal Ethics Committee of the Peking University People's Hospital(approval No. 2019 PHC015) on April 8, 2019.

OPTICAL MEA SUREMENTS OF RAT MUSCLE SAMPLES UND ER TREATMENT WITH ETHYLENE GLYCOL AND GLUCOSE

With the objective to study the variation of optical properties of rat muscle during optical clearing,we have performed a set of optical measurements from that kind of tissue.The.measurements performed were total tr ansmittance,ollimated transmit tance,specular reflec-tance and total reflectance.This set of measurements is suficient to determine diffuse reflectance and absorbance of the sample,also necessary to est imate the optical properties.All the per formed measurements and calculated quantities will be used later in inverse Monte Carlo(IMC)simu-lations to determine the evolution of the optical properties of muscle during treatments with ethylene glycol and glucose.The results obt ained with the measurements already provide some information about the optical c learing treatments applied to the muscle and translate the mechanisms of turning the tissue more transparent and sequence of regimes of optical clearing.

Optical Brain Imaging： A Powerful Tool for Neuroscience

As the control center of organisms, the brain remains little understood due to its complexity. Taking advantage of imaging methods, scientists have found an accessible approach to unraveling the mystery of neuroscience. Among these methods, optical imaging techniques are widely used due to their high molecular specificity and single-molecule sensitivity. Here, we overview several optical imaging techniques in neuroscience of recent years, including brain clearing, the micro-optical sectioning tomography system, and deep tissue imaging.

AAV6 as an effective gene delivery vector for prolonged transgene expression in intervertebral disc cells in vivo

Intervertebral disc degeneration is the main contributor to low back pain,now the leading cause of disability worldwide.Gene transfer,either in a therapeutic attempt or in basic research to understand the mechanisms of disc degeneration,is a fascinating and promising tool to manipulate the complex physiology of the disc.Viral vectors based on the adeno-associated virus(AAV)have emerged as powerful transgene delivery vehicles yet a systematic investigation into their respective tropism,transduction efficiency,and relative toxicity have not yet been performed in the disc in vivo.Herein,we used in vivo bioluminescence imaging to systematically compare multiple AAV serotypes,injection volumes,titers,promoters,and luciferase reporters to determine which result in high transduction efficiency of murine nucleus pulposus(NP)cells in vivo.We find that AAV6 using a CAG promoter to drive transgene expression,delivered into the NP of murine caudal discs at a titer of 1011 GC/mL,provides excellent transduction efficiency/kinetics and low toxicity in vivo.We also show,for the first time,that the transduction of NP cells can be significantly boosted in vivo by the use of small cell permeabilization peptides.Finally,to our knowledge,we are the first to demonstrate the use of optical tissue clearing and three-dimensional lightsheet microscopy in the disc,which was used to visualize fine details of tissue and cell architecture in whole intact discs following AAV6 delivery.Taken together,these data will contribute to the success of using AAV-mediated gene delivery for basic and translational studies of the IVD.