Комбинация лучевой терапии в Ортотопическая модель опухоли головного мозга мыши

Summary

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Abstract

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors¹. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment². As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure³. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 μl every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse’s body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

Protocol

I. Cell Preparation The cells are grown to confluence in DMEM with 10% FBS about one 80% confluent 225 cm3 flask for every 5 mice to be implanted. Aspirate media off of the cells; wash with 10 ml of PBS without Calcium or Magnesium. Aspirate PBS off cells. Trypsinize cells with 3 ml of trypsin, wait 10-15 min, and neutralize the trypsin with 15 ml of media. Pipet all cells and media into a 50 ml conical. Two large flasks fit into one 50 ml conical. Spin cells…

Discussion

Malignant gliomas, such as glioblastoma multiforme, represent the most common primary brain tumors and have a dismal prognosis⁴. Survival of patients affected by GBM has remained virtually unchanged during the last decade (i.e., 9-12 months post-diagnosis) despite advances in surgery, radiation, and chemotherapy⁵. The features of an appropriate model for the study of glioma treatment should include a reproducible tumor location and growth of the cells as a relatively discrete tumor; the cells should…

Materials

Lab Standard Stereotaxic Instrutment	Stoelting Mouse and Rat Adaptor
Betadine	100% EtOH
70% EtOH	50% Acetone
6″ Q-tips	Alcohol swabs
Hamilton syringe 80008	Ethicon 5-0 PD
1701 SN 31-33G/1″/PT3 needle	1 ml syringes
25G needles	Heating pad
Artificial Tears Ointment	Very fine tipped permanent marker