The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Summary

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Abstract

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved ¹. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

Protocol

1. Preparation

Before starting, assemble the required surgical instruments, dissection and culture media, and 7 day old rat or mouse pups. The protocol is the same for mouse and rat preparations.

(i) Materials and equipment

• Operating scissors (straight length 6", cat# RS6818, Roboz, Gaithersburg, MD)
• Micro dissecting scissors, (length 4'", cat# RS5910. Roboz, Gaithersburg, MD)
• Carbon Steel Dumont Tweezers (cat# RS-5045.Roboz, Gaithersburg, MD)
• Modified glass "transfer" pipettes (bottom of Pasteur pipette removed and edge smoothed)
• Aclar plastic film cut to....

Discussion

There are two important points to follow to ensure reproducible and consistent results when replicating experiments. First, it is important to let the hippocampal slices age for 12-14 days before experimentation. With slice isolation, there is a significant microglial response, and microglia remain activated for some time, with a return to a resting ramified state occurring by day 10 in vitro ^3,6. Secondly, mean PI fluorescence intensity is proportional to the number of injured cells ⁴. .......