The Study On Compatibility Of Neural Tracers With Tissue Optical Clearing Techniques

Background and objectives:One obvious drawback of traditional tissue sectioning techniques is the potential loss of valuable tissue information and the disruption of tissue integrity between slices.To counter this shortcoming,a new histological technology,known as tissue optical clearing techniques,was developed and has quickly attracted widespread attention.Tissue optical clearing technique is a sectioning technique that uses a reagent,or a mixture of several reagents,to provide transparency or clarity to large or complete organs that are being studied under optical instruments after soaking,electrophoresis or perfusion treatment.Compared to traditional histology technology,tissue optical clearing technique offers the distinct advantage of being able to observe the internal structure of organs intact.The technique is especially suitable for the study of nervous systems,because of its 'long-distance communication' feature,which can depict a complete nerve conduction pathway along with neural tract tracing technology.Although researchers have used tissue clearing in combination with individual tracers to study a variety of scientific problems,there is,as yet,no literature that systematically explains the compatibility of different tracers and different tissue optical clearing techniques.Method:This study was divided into two parts.First,we selected two different types of representative tracers,namely fluorescent dye tracers(Fluoro-Gold and Fluoro-Ruby)and viral vectors with fluorescent protein(rAAV9-hSyn-mCherry-WPRE-pA and rAAV9-hSyn-EGFP-WPRE-pA).We then traced the motor conduction bundles in laboratory rats,after microinjection with these tracers,and compared the signal changes evident after using three representative clearing methods(FRUIT,3DISCO,and uDISCO).The second part of this experiment was subsequently initiated on the basis of these results.Fluoro-Gold was microinjected into one side of each rat's spinal cord,as a retrograde tracer on the motor conduction bundle to the contralateral primary motor cortex.Fluoro-Ruby was simultaneously microinjected to the rat's unilateral primary motor cortex as an anterograde tracer on the pyramidal tract to the spinal cord.After two weeks,the animals were sacrificed and perfused.Whole brains and spinal cords were removed for tissue clearing with 3DSCO.Three-dimensional imaging was performed by means of optical microscopy imaging equipment.Result:Part I:(1)The Fluoro-Gold retrograde tracer provide good compatibility with the three clearing methods;(2)The Fluoro-Ruby anterograde tracer also showed good compatibility with the three tissue clearing methods;(3)rAAV9-hSyn-mCherry-WPRE-pA and rAAV9-hSyn-EGFP-WPRE-pA were clearly compatible with the FRUIT and the fluorescence signal was well preserved;(4)In experimentation with the rats,clearing with 3DISCO and uDISCO caused the quenching of GFP and Cherry protein;(5)Under the same experimental conditions,the quenching occurred earlier with the 3DISCO,and the GFP quenched earlier and more severely than the Cherry protein.Part II:Both tracers were found to be capable of performing 3D imaging of the conduction bundle when the tissue was cleared,however,the Fluoro-Gold marker proved difficult when showing axonal morphology continuously,while the Fluoro-Ruby marker was able to show long axis axons.In the selection of microscopy imaging devices,it was found that ordinary fluorescence microscopy could only be used for rough observations,while Light sheet microscopy and two-photon microscopy were able to observe the tracer signal inside the transparent tissue more accurately.Conclusions:1.Fluoro-Gold and Fluoro-Ruby provide better compatibility with tissue clearing.2.Except for mice,the tissue clearing technology,whose key techniques are'dehydration and degreasing' could easily lead to the quenching of fluorescent proteins and shows poor compatibility with virus tracers.3.Along with tissue clearing and optical microscopy,the anterograde tracer Fluoro-Ruby stereoscopically demonstrates the complete pathway of nerve conduction.4.Light sheet microscopy and two-photon microscopy are more suitable for stereo imaging in cleared tissue.