Calvarial nerves,along with vasculature,influence skull formation during development and following injury,but it remains unclear how calvarial nerves are spatially distributed during postnatal growth and aging.Studyin...Calvarial nerves,along with vasculature,influence skull formation during development and following injury,but it remains unclear how calvarial nerves are spatially distributed during postnatal growth and aging.Studying the spatial distribution of nerves in the skull remains a challenge due to a lack of methods to quantify 3D structures in intact bone.To visualize calvarial 3D neurovascular architecture,we imaged nerves and endothelial cells with lightsheet microscopy.展开更多
Dear Editor,The mammalian brain exhibits cross-scale complexity in neuronal morphology and connectivity,the study of which demands high-resolution morphological reconstruction of individual neurons across the entire b...Dear Editor,The mammalian brain exhibits cross-scale complexity in neuronal morphology and connectivity,the study of which demands high-resolution morphological reconstruction of individual neurons across the entire brain[1-4].Current commonly used approaches for such mesoscale brain mapping include two main types of three-dimensional fluorescence microscopy:the block-face methods,and the lightsheet-based methods[5,6].In general,the high imaging speed and light efficiency of light-sheet microscopy make it a suitable tool for high-throughput volumetric imaging,especially when combined with tissue-clearing techniques.However,large brain samples pose major challenges to this approach.展开更多
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 o...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.展开更多
基金supported by funding from NIDCR(1R01DE027957)Maryland Stem Cell Research Fund(2022-MSCRFV-5782)the NSF GRFP and NCI(5R01CA237597-05,2R01CA196701-06A1).
文摘Calvarial nerves,along with vasculature,influence skull formation during development and following injury,but it remains unclear how calvarial nerves are spatially distributed during postnatal growth and aging.Studying the spatial distribution of nerves in the skull remains a challenge due to a lack of methods to quantify 3D structures in intact bone.To visualize calvarial 3D neurovascular architecture,we imaged nerves and endothelial cells with lightsheet microscopy.
基金supported by the STI 2030-Major Project(2021ZD0204400,2022ZD0205203,2021ZD0200104,2022ZD0211900)the Shenzhen Science and Technology Program(RCYX20210706092100003,RCBS20221008093311027)+3 种基金the Shenzhen Medical Research Funds(A2303005)the Youth Innovation Promotion Association CAS(2022367)the National Natural Science Foundation of China(32100896)NSFC-Guangdong Joint Fund(U20A6005).
文摘Dear Editor,The mammalian brain exhibits cross-scale complexity in neuronal morphology and connectivity,the study of which demands high-resolution morphological reconstruction of individual neurons across the entire brain[1-4].Current commonly used approaches for such mesoscale brain mapping include two main types of three-dimensional fluorescence microscopy:the block-face methods,and the lightsheet-based methods[5,6].In general,the high imaging speed and light efficiency of light-sheet microscopy make it a suitable tool for high-throughput volumetric imaging,especially when combined with tissue-clearing techniques.However,large brain samples pose major challenges to this approach.
基金This work was supported by a Veteran Affairs Career Development Award(IK2-BX003845).
文摘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.
