Application of Zymographic Methods to Study Matrix Enzymes Following Traumatic Brain Injury - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 182

done by either targeted in vitro enzyme activity assays or separation
and activation of enzyme in a fi xed gel system using zymographic
methods. While we ( 11 ) and other laboratories ( 19 ) have utilized
in vitro MMP activity assays with some success, many of these
methods retain relatively low specifi city among MMP family mem-
bers and require additional control experiments to verify separa-
tion of MMP signal from nonspecifi c substrate lysis, which naturally
occurs in brain tissue homogenates. Further comments regarding
the benefi ts of using zymography vs. in vitro assays for MMP activity
assessment in brain tissue are given in the Notes section below.
Given these issues, most laboratories choose zymographic methods
in order to ascertain relative changes in MMP enzyme activity and
the involvement of endogenous tissue inhibitors of metalloprotei-
nase (TIMPs) in CNS injury-induced processes (see again Table 1 ).
Zymography utilizes substrate- or enzyme-impregnated SDS gels
as a platform in which MMPs are separated by molecular weight
and fi xed at a specifi c site within the gel system. Under renaturing
and activating buffer conditions, the MMPs can be induced to lyse
the substrate present in the gel. If gelatin-based zymography is
used, bound MMPs will digest the gelatin protein at their site of
migration, leaving a clear band when the slab is stained with a pro-
tein binding dye, such as Coomassie blue. Imaging then allows a
relative densitometric measurement of digestion which is corre-
lated with enzyme activity. A distinct advantage of this method is
its ability to discriminate pro or latent forms from cleaved or active
forms of enzyme, principally due to their different molecular
weights in gel systems. Since denaturation/renaturation permits
activation of both pro and active forms under method conditions,
a more detailed MMP assessment is possible. Two variants of this
method are also applied. Reverse zymography targets the discrimi-
nation of sites, where endogenous TIMPs are bound to MMPs in
a tissue sample. This can be done in two ways. Gels separating tissue
proteins by kDa are incubated with a buffer containing active MMPs
or, alternatively, conditioned media containing active MMPs can
be infused into the gel along with the impregnated gelatin. The
latter is the preferred method, which generates a clear gel image
at all sites, except where TIMPs are bound. In this way, the extent
of TIMP binding, as well as the kDa of binding partners, can be
assessed. A third approach utilizing enzyme/substrate interaction,
in situ zymography, utilizes a DQ TM gelatin and fl uorescein conjugate,
which releases FITC signal when the gelatin is degraded by MMP
gelatinases (see again Table 1 ). This application layers the DQ TM
over tissue slices, generating a fl uorescent signal, where endogenous
MMPs cleave the tagged gelatin.
While several excellent overviews of these zymographic methods
already exist for general reference ( 3, 20 ), we have found that
direct application of these methods to tissue samples generated by
traumatic brain injury (TBI) models may be problematic. In working