Application of Zymographic Methods to Study Matrix Enzymes Following Traumatic Brain Injury - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 183

with these methods, we have modifi ed the standard gelatin zymography
protocol in order to optimize assessment of MMP activity following
brain injury. This chapter focuses on these modifi cations, which we
have used to more confi dently track gelatinase activity following
TBI. We have not yet applied reverse zymography or in situ zymog-
raphy to our models; however, others have done so successfully to
reveal details of ECM response after TBI, subarachnoid hemorrhage,
and spinal cord injury ( 8, 12, 13 ). The reader is referred to these
publications for details as to how this additional methodology may
be adapted for brain injury analysis. In the present chapter, the major
procedural steps that we have applied to produce reliable results
with gelatin zymography are described fi rst. These include (1) sample
preparation, (2) gel electrophoresis and incubation, (3) visualization
of lysis, (4) measurement of signal, and (5) considerations of exper-
imental design. Additionally, we discuss several aspects of zymo-
graphic analysis in brain injury models which deserve consideration
in advance of experimentation. Foremost, depending upon model
and level of injury, there may be challenges in pulling out detectable
signal from standard tissue extracts. Moreover, different brain
regions may also vary considerably in the extent or pattern of MMP
signal. As described below, we have varied extraction procedures
and extended the lysis development phase of the protocol to aid in
signal detection. In our studies, at least two additional benefi ts for
zymographic analysis have been observed relative to TBI. First, we
have used the method to assay ECM-related mechanisms which
might be part of specifi c therapeutic manipulations. Our data sup-
ports clear differences in MMP activity as a function of specifi c
postinjury drug treatment. Second, the separation of pro and active
enzyme species may reveal interesting spatio-temporal changes
during injury-recovery cycles. These data can be combined with
protein and RNA profi les to signifi cantly enhance the extent of
interpretation.

2. Materials

2.1. Sample
Preparation

All extraction and zymography buffers are prepared just prior to
use. All reagents are of ACS grade.

1. Standard dissection tools, including razor blades for blocking,
forceps, microtip probes, and fi ne tip iridectomy scissors
2. Platform for dissection chilled over ice
3. Sterile Ringer's solution

2.1.1. Tissue Dissection

1. Pellet pestles with microtubes and pellet pestle cordless motor
(Kimble Chase Life Science and Research Products, Vineland,
NJ, #749520-0090)

2.1.2. Hippocampal
Tissue Extraction