A method to accurately inject tumor cells into the caudate/putamen nuclei of the mouse brain

To improve currently used techniques to implant tumor cells into the parenchyma of the mouse brain.

The stereotactic injection of 0.5 to 5 microl of indigo carmine over 5 to 40 minutes into the caudate/putamen nuclei of the mouse was done followed by sacrifice and examination of the brain injection site. 1 microl containing 10(5) U87MG glioma cells were stereotactically implanted into the caudate/ putamen nuclei over 20 minutes. The animals were sacrificed from one hour to 63 days after implantation and the brain examined and tumor size measured.

An injection of 1 microl of indigo carmine over 20 minutes produced a spherical deposit of dye within the caudate/putamen nuclei. Larger volumes of indigo carmine or shorter injection times resulted in dye spreading along the injection tract or into the ventricles or subarachnoid space. Using the results of the dye studies, the same parameters were used to successfully inject and confine the glioma cells to the caudate/putamen nuclei in 30 of 32 mice. No tumor was found in 2 animals and appears to be explained by obstruction of the injection cannula. The tumor cells appeared viable an hour after injection. However by day three, considerable necrosis of tumor cells were noted, the effects of which resolved by day five. On day six, the injection site was comparable to that at one hour. In the early phase, until the fifth week, tumor volume doubling time was ten days while afterward it was only five days.

The technique described allows the highly accurate and reproducible introduction of a given number of cells into a specific area of the mouse brain. This should reduce the intragroup variability, be it control or therapeutic, allowing better assessment of outcome with fewer number of mice.