Application of Zymographic Methods to Study Matrix Enzymes Following Traumatic Brain Injury - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 188

Fig. 3. Application of inhibition and activation controls for gelatin zymography. When PMSF,
NEM, and EDTA are added to the development buffer and lytic activity is compared to that
present in calcium/zinc-treated TBI + BEC samples, only EDTA-specifi c MMP inhibition
blocks MMP-2 and MMP-9 activity ( a ). In ( b ), samples are incubated with APMA from 0 to
8 h prior to zymography. Lytic activity in tissue samples comigrates with MMP-2 standard
(68 and 62 kDa) and active MMP-2 (62 kDa) is visible after 3 h of APMA exposure.

gelatin zymography as described above. MMP-2 standard enzyme
(0.25 ng) was treated in parallel with 1 mM APMA for up to 3 h
at 37°C and run alongside the APMA-treated supernatant extracts
on zymography gels for 72 h at 37°C. Additional correlative support
for enzyme activation may be obtained by employing commercial
activity assays which use various detection systems for targeted sub-
strate lysis within tissue homogenates (Note 13).

In vivo application of therapeutic compounds for TBI, such as the
NMDA antagonist MK-801 (1.0 mg/kg, i.p., twice daily for 2 days
post injury), can produce detectable shifts on MMP lytic activity
within injured gray matter when visualized by gelatin zymography
(Fig. 4 ; Note 14). These alterations in lytic signal intensity may be
quantifi ed as described above and statistically analyzed for experi-
mental effect. Similarly, we have shown that the in vivo application
of MMP-9-targeted inhibitors, such as minocycline (45 mg/kg,
i.p., 30 min and 6 h post injury), can be used with zymography to
validate and discriminate unique MMP responses in injured white
matter ( 21 ).

3.5.3. Drug Manipulation