Application of Zymographic Methods to Study Matrix Enzymes Following Traumatic Brain Injury - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 179

Chapter 12
Application of Zymographic Methods to Study Matrix
Enzymes Following Traumatic Brain Injury
Lesley K. Harris , Raiford T. Black , Thomas M. Reeves,
and Linda L. Phillips
Abstract
Assays for proteolytic activity are often used to confi rm a functional role for enzymes involved with CNS
pathobiology. In studies focused on matrix metalloproteinases (MMPs), specifi cally those investigating the
role of gelatinases (MMP-2, MMP-9), gelatin zymography is used to assess relative changes in enzyme
activity. The advantages of this method lie in its gel separation of pro and active enzyme and the option for
generation of semiquantitative data. Gelatin zymography is applied with success in studies of traumatic
brain injury (TBI), but may require modifi cations for best results. In this chapter, we fi rst present an overview
of different zymographic approaches taken to assess the role of gelatinases in various forms of TBI and
related CNS pathologies. Next, we focus on the specifi c method developed in our laboratory to optimize
zymographic signal from injured CNS tissue. Finally, we include points of technique modifi cation which
have worked well for us, emphasizing tissue dissection, extraction method, and gel incubation period. This
information is presented to offer support for those seeking to apply gelatin zymography with samples from
injured brain tissue, where pathology can vary considerably. Individual models and experimental design may
require further zymographic modifi cations for optimization of MMP detection.
Key words: Gelatin zymography, Matrix metalloproteinase, Traumatic brain injury, Deafferentation
injury, Hippocampus, Corpus callosum
1. Introduction
The role of extracellular matrix (ECM) molecules in brain injury
pathophysiology and recovery has gained signifi cant interest in
recent years (see Table 1 ). This investigation has naturally included
the documentation of major ECM sculpting enzymes, the matrix
metalloproteinases (MMPs). In this approach, it is important to
establish that alterations in MMP expression level are accompanied
by functionally related changes in enzyme activity. Typically, this is