Region-specific cell grafting into cervical and lumbar spinal cord in rat: a qualitative and quantitative stereological study.
In the present study, we have characterized an atraumatic grafting technique which permits multiple, segmental, and lamina-specific injections into cervical or lumbar spinal cord. Cell injections were performed in spinally mounted rats of different ages and spinal cord size, using a micromanipulator and glass microcapillary connected to a digital microinjector. For grafting, we used human neuroteratoma (hNT) cells, BrdU-labeled rat spinal precursors or primary embryonic spinal cord neurons isolated from E14 spinal cord of the eGFP+ rat. Systematic quantification of grafted cells was performed using stereological principles of systematic random sampling and semi-automated optical Disector software. Volume reconstruction was performed using serial sections from grafted areas and custom-developed software (Ellipse) which permits "two reference points" semi-automated alignment of images, as well as volume reconstruction and calculation. By coupling these techniques, it is possible to achieve a relatively precise and atraumatic cell delivery into multiple spinal cord segments and specific spinal laminae. Consistency of the multiple grafts position in the targeted laminar areas was verified by a systematic volume reconstruction. Good survival of implanted cells for the three different cell lines used indicate that this grafting technique coupled with a systematic analysis of the individual grafting sites can represent a valuable implantation-analytical system.
Kakinohana
Osamu
O
Anesthesiology Research Laboratory, University of California-San Diego-0818, La Jolla, CA 92093, USA.
Cizkova
Dasa
D
Tomori
Zoltan
Z
Hedlund
Eva
E
Marsala
Silvia
S
Isacson
Ole
O
Marsala
Martin
M
D000818:Animals; D030801:Animals, Genetically Modified; D002452:Cell Count; D002469:Cell Separation; D017690:Cell Transplantation / Q000379:methods*; D006085:Graft Survival; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D019044:Image Cytometry / Q000379:methods; D008161:Lumbosacral Region; D008845:Microinjections; D009043:Motor Activity / Q000502:physiology; D009333:Neck; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000637:transplantation; D009474:Neurons / Q000187:drug effects / Q000637:transplantation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000166:cytology / Q000196:embryology / Q000601:surgery; D033581:Stem Cell Transplantation / Q000379:methods; D013234:Stem Cells / Q000166:cytology; D013482:Superoxide Dismutase / Q000235:genetics
D049452:Green Fluorescent Proteins; C109415:SOD1 G93A protein; D013482:Superoxide Dismutase