Three-Dimensional Reconstruction Of Microvasculature Based On Tissue Sections In Rat Optic Nerve

Objective Artificial vision brings hope through biological and engineering technology for the blind patients,and got encouraging progress.China funded this research in 2005 through the "973" project,and hope to make the blind patients' sight go to return by optic nerve interface.At an early time,we have got the 3-D reconstruction of optic nerve successful by computer technology.To make a good understanding of the distribution of microvasculature of optic nerve,we researched the 3-D reconstruction of microvasculature of optic nerve by computer assisted technology based on tissue sections.Methods Adult SD rats were used as experimental animals.After transcardiac perfusion with 10%gelatin-ink mixture,rat optic nerve was removed.To make sure of alignment of images of successive serial sections,there was one hair on nasal side of optic nerve and two hairs on temple side of optic nerve,and then embeded in OCT to perform a series of 20μm thick sections.The micrographs were obtained by Olympus Image Processing System,The micrographs were processed by Photoshop 9.0cs, Amira 3.1.1 software transformed these processed two-dimensional images into three-dimensional(3-D) visualization images in the SunGraph 6000A professional workstation,to analyze parameters such as number,area,circularity of microvasculature of optic nerve through the software of Image-J and Lecia Qwin for the features of construction and distribution.Results Perfusion with 10%gelatin-ink mixture until limbs,lip,all skin of body became black,can display microvasculature of optic nerve successfully.Using three hairs as position can meet the alignment of successive serial sections.To transform these processed two-dimensional images into three-dimensional (3-D) visualization images using Amira 3.1.1 software.From anterior view of 3-D reconstruction of three segments,microvasculature distributed around the optic nerve bundles.There are much more vacant optic nerve tracts in midpiece segment and posterior segment than anterior segment because there are more branches of microvasculature in anterior segment.From lateral view of 3-D reconstruction of microvasculature of optic nerve,there are much more laterigrade microvasculature in anterior segment and to interlace into network,there are longitudinal microvasculature until in midpiece segment and much more longitudinal microvasculature in posterior segment.There is one large vascular in nasal side both in anterior segment and midpiece segment,and then it turns the upper surfaces of the nerve in posterior segment.The diameter of rat optic nerve is diminished gradually from eyeball to optic chiasm.The diameter behind eyeball is about 0.5mm,but the diameter is about 0.46mm close to chiasm,there is no significant difference between midpiece segment and posterior segment.Under light microscope,proximal to globe their alignment are curved and gradually become straight along the optic nerve axis towards optic chiasm.According to the morphology of microvasculature of cross sections,optic nerve of rat was divided into three segments:anterior segment,midpiece segment and posterior segment.Anterior segment is 0～5mm behind eyeball,midpiece segment is 5～8mm behind eyeball,posterior segment is beyond 8 mm behind eyeball.The diameter of rat optic nerve is diminished gradually from eyeball to optic chiasm.The diameter behind eyeball is about 0.5mm,but the diameter is about 0.46mm close to chiasm,there is no significant difference between midpiece segment and posterior segment.The number of peripheral microvasculature of three segments is 21.9±3.9,17.4±3.7,20.3±3.1 respectively,compared with each segment,there is significant difference between anterior segment and midpiece segment(P＜0.01) and between midpiece segment and posterior segment(P＜0.05) and no significant difference between anterior segment and posterior segment.The number of inner microvasculature of three segments is 30.0±3.2,27.5±4.0,32.9±7.4,compared with each segment,there is significant difference between midpiece segment and posterior segment(P＜0.01) and no significant difference between other segments.The numerical density of microvasculature of three segments is 14.0±0.8/1×105μm2,16.3±0.8/1×105μm2,20.5±1.5/1×105μm2,compared with each segment,there is significant difference between anterior segment and posterior segment(P＜0.01) and between midpiece segment and posterior segment(P＜0.05) and no significant difference between anterior segment and midpiece segment.To calculate the area of microvasculature of each segment by Lecia Qwin software,the areas of microvasculature of each segment is 11616.36±1858.65μm2, 6286.10±736.32μm2,4590.50±1137.67μm2 respectively.The area density of microvasculature of each segment is 5.85%±0.92%,3.67%±0.42%,2.7%±0.50 %respectively,compared with each segment,there is significant difference between each segment(P＜0.01).To calculate the circularity of microvasculature by Image-J software,the average circularity of microvasculature of each segment is 0.52±0.03,0.58±0.04,0.68±0.05 respectively,compared with each segment,there is significant difference between each segment(P＜0.01).Conclusion The number of microvasculature of optic nerve increases from eyeball to optic chiasm gradually,the change is unobvious between anterior segment and midpiece segment but the number of posterior segment increases obviously. Numerical density increase from eyeball to optic chiasm gradually.But the areas of microvasculature and area density diminish from eyeball to optic chiasm gradually. All of light microscope,Image-J software and 3-D reconstruction display the microvasculature of optic nerve proximal to globe their alignment are curved and gradually become straight along the optic nerve axis towards optic chiasm.In the same time,considering the anatomy structure of OA and CRA,we think the visual prosthesis should be implanted in upper surfaces of midpiece segment,which is postmedian of intraobital optic nerve,and the array should be implanted longitudinally along optic nerve axis.