Journal of Neuroscience Methods 216 (2013) 22–27

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Journal of Neuroscience Methods

journal homepage: www.elsevier.com/locate/jneumeth

Basic Neuroscience

Complexity in differentiating the expression of truncated or matured forms of

MMP-2 and MMP-9 through zymography in rat brain tissues after acute
ischaemic stroke
Mustafa Alam, Ashfaq Shuaib ∗
Stroke Research Laboratory, Department of Medicine-Neurology, University of Alberta, Canada

h i g h l i g h t s

• Both MMP-2 and MMP-9 expression level increased in ischaemic hemisphere of rat brain tissue.
• MMP-9 is more efficient to degrade gelatin than MMP-2.
• Mature form of MMP-2 and MMP-9 are almost equal in size.
• Mature form of MMP-2 and MMP-9 determination should not be interpreted based on zymographic analysis.

a b s t r a c t
a r t i c l e i n f o
Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of ischaemic stroke. In
Article history:
particular, the mature forms of MMPs 2 and 9 have similar sizes and share gelatine as a common substrate.
Received 8 March 2013
Accepted 12 March 2013 Both MMPs are upregulated in ischaemic stroke and play detrimental roles during stroke pathogenesis.
Throughout this study, we demonstrated that pro-MMP-2 and pro-MMP-9 from ischaemic rat brain tissue
homogenate is detected either through immunoblotting or zymography because of the remarkable size
difference between these enzymes (72 versus 95 kDa, respectively). However, the mature MMP-2 and
MMP-9 cannot be discriminated through zymography because of the almost identical sizes of these forms
(66 and 67 kDa, respectively). The use of gelatine zymography on ischaemic rat brain tissue homogenate
revealed a 65-kDa MMP band, corresponding to the heterogeneous band of mature MMP-2 and/or MMP-9.
Furthermore, we also detected mature MMPs of 65 kDa generated from both recombinant human MMP-2
and MMP-9. Using a pull down assay in rat brain tissue homogenate with gelatine–agarose beads, we
showed increased activities for both the pro and mature forms of MMP-2 and MMP-9. However, we
could not determine the origin of the respective mature MMPs from the heterogeneous band. Thus, in
this study, we demonstrated that the identification and quantification of mature MMP-2 and MMP-9
could not be achieved using zymography alone. Therefore, the development of a reliable technique to
identify and measure the respective MMPs is needed to test new stroke therapies targeting MMP-2 and
MMP-9.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction proteases and cytokines, and the increased expression of matrix

In ischaemic stroke, when blood flow ceases in specific cere-
bral vessels, the downstream microvasculature develops vascular
dysfunction and ischaemic infarction (del Zoppo et al., 1998). As
a result, the integrity of the microvasculature becomes seriously
compromised because of the increased expression of different
∗ Corresponding author at: Stroke Research Laboratory, 533 HMRC Building, Uni-
versity of Alberta, Edmonton, Alberta, Canada T6G 2S2. Tel.: +1 780 407 3254;
fax: +1 780 407 1325.
E-mail address: ashfaq.shuaib@ualberta.ca (A. Shuaib).
metalloproteinases (MMPs) is the most deleterious. MMPs play
important roles in physiological and multiple pathological events,
such as wound healing, tissue remodelling, angiogenesis, tumouri-
genesis and metastasis (Birkedal-Hansen et al., 1993). During
ischaemic stroke events, the expression of both MMPs 2 and 9 is
up-regulated in the ischaemic region, and a cascade of molecular
events, including the degradation of the structural components of
the basement membrane (laminin, fibronectin and type IV colla-
gen), is initiated, leading to the disruption of the blood brain barrier
(BBB), haemorrhagic transformation and eventually neuronal cell
death (Clark et al., 1997; Planas et al., 2001; Rosenberg et al., 1998).
Conversely, MMPs also play a role in modulating the regrowth of

0165-0270/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jneumeth.2013.03.007
 M. Alam, A. Shuaib / Journal of Neuroscience Methods 216 (2013) 22–27
neurons and blood vessels during the post-stroke recovery stage
(Zhao et al., 2006).
Although the role of MMPs in stroke pathogenesis and recov-
ery is well known, the detection of the mature forms of MMP-2
and MMP-9 in stroke-induced brain tissue is difficult due to the
overlapping characteristics, particularly the almost equal sizes of
these proteins. Both MMP-2 and MMP-9 are gelatinases or type IV
collagenases with gelatine-binding sites in the fibronectin-related
regions of these multi-domain proteins (Banyai and Patthy, 1991;
Collier et al., 1992). Although proMMP-9 (95 kDa) is larger than
proMMP-2 (72 kDa), the mature forms of MMP-9 and MMP-2 are
nearly equal in size, with molecular weights of 67 and 66 kDa,
respectively (Parsons et al., 1997). In fact, the analysis of the mature
forms of both MMP-2 and MMP-9 using gel zymography reveals
gelatinase bands at the same position due to the similar sizes of
these enzymes. Therefore, researchers are unable to precisely dis-
tinguish the mature forms of MMP-2 and MMP-9.
Although MMP-2 and MMP-9 are classified as gelatinases based
on their substrate specificity towards gelatine (Murphy and Nagase,
2008), it has also been reported that these enzymes play specific
roles in stroke pathogenesis and recovery (Liu et al., 2012; Lucivero
et al., 2007). Notably, the mature forms of MMP-2 and MMP-9 are
enzymatically more active in stroke pathogenesis compared with
the full-length pro-forms of these proteins.
The aim of this study was to demonstrate the limitation of
gel zymographic analysis in measuring the activities of mature
MMP-2 and MMP-9. However, this method generates imprecise
information and should be treated with caution when used to
extrapolate the activity of mature MMPs in stroke-induced rat brain
tissue homogenate. The reduced expression, presence of different
isoforms and susceptibility to proteolysis in the brain present chal-
lenges for the study of MMP regulation. One of the major obstacles is
the unavailability of effective and specific antibodies. Therefore, the
development of strategies to identify and measure the expression
of mature MMP-2 and MMP-9 in stroke is highly desirable. In this
study, we showed that the analysis of mature MMP-2 and MMP-9
expression in stroke-induced brain tissues using a conventional gel
zymography-based technique generates misleading results.
2. Materials and methods
2.1. Reagents
Bovine thrombin, obtained from the John Pharma Incorpora-
tion (Bristol, VA), was used to make a blood clot for embolic
stroke induction in rats. The gelatine–agarose resin, phosphatase
inhibitors, 2,3,5-triphenyltetrazolium chloride (TTC) stain, Ponceau
S stain and gelatine A and B were obtained from Sigma–Aldrich,
and the molecular weight marker proteins for SDS-PAGE, Biosafe
Coomassie Brilliant Blue G-250 (CBB-250) for gel staining and
nitrocellulose membrane were obtained from Bio-Rad Laborato-
ries (Hercules, CA). All other reagents were of the highest analytical
grade available. Purified human recombinant MMP-2 and MMP-9
were purchased from Calbiochem (USA).
2.2. Animals
The experimental protocols used in the present study were
approved by the Institutional Animal Ethics Committee of the
University of Alberta according to the standards of the Canadian
Council on Animal Care (CCAC). Male Sprague–Dawley rats weigh-
ing 250–300 g (Bioscience Animal Facility, U of A) were housed at
21 ◦ C under a light–dark cycle. All animals were provided with free
access to food and water.
23
2.3. Embolic rat stroke model
Middle cerebral artery occlusion (MCAO) was performed as pre-
viously described (Alam et al., 2011; Noor et al., 2003; Wang et al.,
2001). Briefly, the animals were anaesthetised using inhaled isoflu-
rane (2.5%). The body temperature was maintained at 37 ◦ C during
surgery. A midline incision was made on the ventral side of the
neck to expose the common carotid artery (CCA). After the identifi-
cation of the bifurcation between the external and internal carotid
arteries, the external carotid artery (ECA) was ligated and dissected
at the distal portion. Approximately 50 ␮L of blood was collected
through the ECA using a PE-50 catheter prefilled with thrombin, and
the blood sample was incubated at room temperature for 15 min to
facilitate clot formation. We subsequently inserted the blood clot
towards the MCA from the incision made on the ECA proceeding
through the ICA. The total length of the 17-mm PE-10 catheter was
inserted inside the artery. The incision was closed using a surgi-
cal stapler, and the animals were allowed to regain consciousness
after surgery. The rats were sacrificed under anaesthetic conditions
24 h after surgery, and the rat brains were collected for staining and
biochemical analysis.
2.4. TTC staining
Twenty-four hours after surgery, the rats were deeply anaes-
thetised and decapitated using a small animal guillotine; the brains
were carefully dissected en bloc. After briefly (1–2 min) cooling on
ice, the brains were sectioned at 2-mm intervals from the frontal
portion of the brain. The first 3 slices were collected and stained
with 2,3,5-triphenyltetrazolium chloride (TTC) to verify infarction
in the brain. The remaining brain tissue was snap frozen using cold
(−80 ◦ C) isopentane and stored at −80 ◦ C until further analysis. For
staining, the individual slices were soaked for 15 minutes in a 2%
solution of 2,3,5-triphenyltetrazolium chloride (TTC) in 0.9% NaCl at
37 ◦ C. The slices were evenly covered with TTC solution and gently
stirred to ensure the complete exposure of the surfaces to the TTC
solution. The brain sections were scanned and documented after
30 min.
2.5. Tissue analysis
The frozen rat brain tissues were separated into non-ischaemic
and ischaemic hemispheres and homogenised in a buffer contain-
ing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid,
1.0% NP-40, 1 mM DTT and protease inhibitors (Sigma–Aldrich, Cat
#P8340) using a hand homogeniser. The homogenate was cen-
trifuged at 800 × g for 10 min to remove debris. The supernatants
were subsequently assayed to determine the protein concentration
and adjusted to contain an equal concentration of protein. Equal
amounts of the proteins from these samples were used for both
immunoblot and zymographic analyses.
2.6. Western blotting
Immunoblots were prepared using the appropriate brain tis-
sue homogenates. The samples were resolved through SDS-PAGE
and transferred to nitrocellulose membranes. The membrane trans-
fer was assessed through staining with Ponceau S solution. The
membranes were probed with anti-MMP-2 (Santa Cruz Biotech-
nology, cat #sc-13594, mouse polyclonal from human source,
diluted 1:1000, recognising pro-form), anti-MMP-9 (Calbiochem
Cat #IM09L, mouse monoclonal human origin, diluted 1:1000,
recognising both the pro and mature forms), and anti-actin
(Santa Cruz Biotechnology, cat #sc-1616, rabbit polyclonal from
C-terminal human origin, diluted 1:1000) antibodies. The antibod-
ies were diluted with 5% skim milk in TTBS (Tris buffered saline
24 M. Alam, A. Shuaib / Journal of Neuroscience Methods 216 (2013) 22–27

Fig. 1. MMP-2 and MMP-9 are upregulated in the ischaemic hemisphere of embolic stroke-induced rat brain tissue. (A) TTC staining of representative brain slices to confirm
stroke induction. The infarcted region of the brain slices is shown in white. (B–D) Western blotting analysis of the tissues collected from the same animal using anti-MMP-2,
anti-MMP-9 and anti-actin antibodies. The non-ischaemic and ischaemic hemispheres from each animal were homogenised as described in the Materials and Methods. Sixty
micrograms of tissue homogenate from each brain tissue hemisphere was loaded per lane, followed by probing with anti-MMP-2, anti-MMP-9 and anti-actin antibodies.
(E) Zymographic analysis of equal amounts of protein from the same samples loaded onto a 0.2% gelatine A-containing 10% SDS-PAGE gel and processed as described in the
Materials and Methods. Two different animals were analysed in this study. All lanes are loaded in duplicate. R1 and R2 represent rat-1 and rat-2, respectively, subjected to
embolic stroke induction.

containing 0.1% Tween 20). The primary antibodies were detected
using the appropriate horseradish peroxidase-coupled secondary
antibodies (Santa-Cruz Biotechnology SC2054 or SC2055) for poly-
clonal and monoclonal primary antibodies. The immunoblots were
developed using an enhanced chemiluminescence reagent (Amer-
sham Biosciences).
2.7. Zymography
beads were washed 3–4 times with homogenisation buffer, and
subsequently, the bound proteins were eluted from the beads using
homogenisation buffer containing 10% DMSO.
The molecular mass and purity of the purified gelatine-binding
proteins were determined through SDS-PAGE with 3% (w/v)
stacking and 10% (w/v) separating gels, as previously described
(Laemmli, 1970). All analyses presented in this study were per-
formed using the purified gelatine-binding proteins.

The MMP activities were assessed using gelatine zymography
using previously described methods (Leber and Balkwill, 1997). The
proteins were separated on a non-reducing SDS/polyacrylamide
gel (10%) containing 2 mg/ml of gelatine. After electrophoresis in a
buffer containing 25 mM Tris–HCl, 250 mM glycine, and 1% SDS, the
gel was washed at room temperature with 2.5% Triton X-100, 5 mM
CaCl2 , 50 mM Tris–HCl (pH 7.5) and incubated 3 times in the same
buffer for 15 min each, followed by incubation overnight (16 h) at
37 ◦ C in 5 mM CaCl2 , 1 ␮M ZnCl2 , 50 mM Tris–HCl (pH 7.5). The
gel was stained with Coomassie Brilliant Blue G (0.5%, w/v) and
destained with methanol/acetic acid/water (45:10:45). The area of
gelatine degradation (gelatinase activity) on the gel zymograph was
depicted as clear bands against a blue background of undegraded
gelatine.
2.8. Gelatine–agarose binding analysis
The MMPs from rat brain tissue homogenate were isolated
through one-step batch purification using gelatine–agarose (Sigma,
cat #G5384) beads. The tissue homogenate containing gelatinase
activity was mixed with gelatine–agarose beads previously equili-
brated with homogenisation buffer and mixed for 1 h at 4 ◦ C. The
3. Results and discussion
In this study, we examined the expression of MMPs in embolic
stroke-induced rat brain tissues. We analysed tissue homogenates
using the immunoblotting and gel zymographic techniques rou-
tinely used in animal stroke research. The ischaemic hemispheres
were confirmed through TTC staining of the first three slices
of the frontal lobe (Fig. 1A). The remaining brain portions
were homogenised separately as non-ischaemic and ischaemic
hemispheres, followed by immunoblotting with anti-MMP-2, anti-
MMP-9 and anti-actin antibodies to compare the expression levels
of these enzymes in both hemispheres. Considering the deleteri-
ous role of MMP-2 and MMP-9 in the post-ischaemic disruption
of the blood brain barrier (BBB) and haemorrhagic transforma-
tion (Fukuda et al., 2004; Rosenberg et al., 1996), we measured
MMP-2 and MMP-9 levels in ischaemic rat brain tissue using
immunoblotting. Actin was used as an internal loading control.
Increased levels of pro-MMP-2 were detected in the ischaemic
hemisphere compared with the non-ischaemic hemisphere; how-
ever, pro-MMP-9 expression was detected only in the ischaemic
hemisphere (Fig. 1B and C). Notably, these observations were qual-
itative, and therefore, we could not determine the precise degree of
 M. Alam, A. Shuaib / Journal of Neuroscience Methods 216 (2013) 22–27
expression of either pro-MMP-2 or pro-MMP-9. These differential
levels of MMP expression might reflect the differential affinities
of the poly/monoclonal antibodies used to detect these antigens.
Another explanation for the difference in the level of MMPs might
be that MMP-2 is a constitutively expressed protease that plays
a role in ECM homeostasis, whereas MMP-9 is an inducible pro-
tease (Gaertner et al., 2005). The SDS-PAGE analysis using standard
molecular weight markers confirmed the sizes of these enzymes as
72 and 95 kDa for pro-MMP-2 and pro-MMP-9, respectively. Con-
sistent with the immunoblotting data, increases in the activities
of pro-MMP-9 and proMMP-2 in the ischaemic hemisphere were
detected at the same position in the lanes using gel zymographic
analysis (Fig. 1E). However, the zymographic data for pro-MMP-2
and pro-MMP-9 were not consistent with the immunoblotting data
(Fig. 1B and C) in terms of gelatine-degrading activities, suggesting
differential antigen-antibody affinities for MMP-2 and MMP-9, as
previously speculated. Interestingly, gel zymography (Fig. 1E) also
revealed a robust increase in a 65-kDa band, potentially represent-
ing the truncated (mature) form of either MMP-2 or MMP-9 or the
combination of both enzymes. This 65-kDa band was not detected
through immunoblotting with either anti-MMP-2 or anti-MMP-9
antibodies. Because we could not detect the 65-kDa MMP-2/MMP-9
band through immunoblotting, we could not exclude the possibility
that these gelatinase activity bands represented neither MMP-2 nor
MMP-9. However, this lack of detection might potentially reflect
the inability of the antibodies used to detect the mature forms of
MMP-2/MMP-9. Without immunoblotting, it is nearly impossible
to confirm whether these 65-kDa gelatinase bands represent the
mature (truncated) forms of either pro-MMP-2 or pro-MMP-9, as
these proteins have nearly equal sizes (Fig. 4). Highly specific anti-
bodies might facilitate the detection of hidden mature forms of
MMP-2/MMP-9. Gel zymography also revealed that the matured
MMPs are enzymatically more active than the full-length pro-forms
of these enzymes; the N-terminal pro-domain shields the catalytic
active site cleft in pro-forms of MMPs through a mechanism called
cysteine-switch (Morgunova et al., 1999; Van Wart and Birkedal-
Hansen, 1990). Therefore, it is likely that the mature MMPs, rather
than their pro-forms, are involved in stroke pathogenesis through
the disruption of the integrity of the BBB structure. These results
also showed that the differences in the immunoblot and zymo-
graphic analysis data for rat brain MMP-2 and MMP-9 are consistent
with the results observed with purified human recombinant MMP-
2 and MMP-9, as shown in Figs. 1E and 2. The gelatine-based
zymography technique is simple and straightforward to measure
MMP activity based on the size differences of these enzymes, but
this assay lacks specificity. Although MMP-2 and MMP-9 were cat-
egorised as gelatinase A and gelatinase B, respectively, almost all
MMPs degrade commercial gelatine to some extent and often show
overlapping substrate specificity. Accordingly, pure recombinant
human MMP-2 and MMP-9 were analysed using gelatine A and
B-based gel zymography. As shown in Fig. 2, no substrate prefer-
ence was observed with either gelatine A or B. Interestingly, the
mature forms of both rhMMP-2 and rhMMP-9 were nearly identical
in size, consistent with the 65-kDa heterogeneous band observed
in the ischaemic hemisphere of rat brain tissue (Fig. 1E). Although
the mature form of rhMMP-2 showed higher activity than that of
mature rhMMP-9, we could not rule out the possibility conclude
that mature MMP-2 possesses stronger activity than mature MMP-
9, as previously suggested. These results might also reflect the fact
that most of the loaded pro-rhMMP-2 (100 ng) was converted to the
mature form. When rhMMP-2 and rhMMP-9 run together in either
gelatine A- or gelatine B-based gel zymography, the mature forms
of these enzymes showed a heterogeneous band equivalent to the
65-kDa mature form of these MMPs, as observed in the ischaemic
rat brain (Fig. 1E). Thus, the data shown in Figs. 1E and 2 suggest that
the mature forms of both MMP-2 and MMP-9 are nearly the same
25
Fig. 2. Zymographic analysis of purified human recombinant MMP-2 and MMP-9 in
gelatine A- and B-containing gels. One hundred nanograms of each protein was sep-
arated on 10% SDS-PAGE gels containing 0.2% gelatine A or B. The gels were stained
with Coomassie Brilliant Blue G-250 and destained with water:methanol:acetic acid
(45:45:10). There were no distinctions between MMP-2 and MMP-9 in the degra-
dation of either gelatine A- or gelatine B-based gels. Subtle size differences between
the 67-kDa MMP-9 and 66-kDa MMP-2 mature forms were detected when both
MMP-2 and MMP-9 were analysed in parallel.
size and will not be distinguished through zymography. Notably,
the MMP-9 bands larger than 95 kDa (Fig. 2) likely represent mul-
timers, dimers or complexes with macroglobulin (Goldberg et al.,
1992; Triebel et al., 1992). The gelatine degradation bands smaller
than 66/67 kDa likely represent the degradation products of mature
MMPs. In addition, gelatine A and B are not the natural substrates
of MMPs, but rather these compounds are the derivatives of natu-
ral collagen from animal sources (usually skin and bone) extracted
after hydrolysis using either an acid (gelatine A) or alkali (gelatine
B) treatment, thereby changing the naturally occurring substrate
of MMPs. These data showed that MMP-2 and MMP-9 degrade
gelatine A and B to an equal extent. However, previous studies
have empirically identified MMP-2 and MMP-9 as gelatinase A and
gelatinase B, respectively, based on the specificity of these enzymes
for the respective gelatine derivatives. The data obtained in the
present study do not support the sub-classification of gelatinases
based on the commercial gelatine substrates. Thus, we conclude
that the mature forms of MMP-2 and MMP-9 cannot be discrimi-
nated through gel zymographic analysis. These two potent MMPs
are directly involved in the degradation of the ECM, and therefore,
the accurate identification of these enzymes is highly desirable.
Because the level of MMPs expressed in brain is low, the detec-
tion of the expression of these MMPs in crude tissue homogenate
is not always possible. Fractionation or partial purification, fol-
lowed by immunoblotting or zymography might represent better
methods for the discrimination of MMP-2 and MMP-9. Therefore,
we partially purified MMP-2 and MMP-9 from rat brain tissues
through gelatine–agarose affinity binding, followed by elution with
a buffer containing 10% DMSO. SDS-PAGE, followed by Ponceau S
staining of the nitrocellulose membrane containing the partially
purified transferred proteins in the gelatin bound fractions, showed
two major protein bands with apparent molecular weights of 72
and 55 kDa (Fig. 3B). The 72-kDa band corresponded to pro-MMP-
2, as determined through immunoblotting using a monoclonal
anti-MMP-2 antibody (Fig. 3C). This observation suggests that the
MMP-2 expressed during ischaemic stroke can be partially puri-
fied using a one-step gelatine–agarose column. We also observed
increased pro-MMP-9 (95 kDa) in the ischaemic hemisphere, as
shown in Figs. 1C and E and 3D. However, pro-MMP-9 protein
was undetected in the fraction eluted from gelatine–agarose beads
26 M. Alam, A. Shuaib / Journal of Neuroscience Methods 216 (2013) 22–27
Fig. 3. Purification of gelatine-binding proteins/gelatinases from the tissue
homogenate of embolic stroke-induced rat brain tissues. Approximately 1 mg of tis-
sue homogenate from both non-ischaemic and ischaemic hemispheres was mixed
with an equal amount of gelatine–agarose beads pre-equilibrated with homogeni-
sation buffer. After mixing for one hour, followed by extensive washing with
homogenisation buffer, the bound proteins were eluted from the spin column using
homogenisation buffer containing 10% DMSO and were analysed as follows. (A)
SDS-PAGE analysis of crude rat brain tissue homogenate, followed by staining with
Coomassie Brilliant Blue G-250. (B) Ponceau S-stained nitrocellulose membrane of
partially purified proteins eluted from gelatine–agarose beads. (C) Immunoblot-
ting analysis of gelatine–agarose eluted proteins using an anti-MMP-2 antibody.
(D) Gelatine zymography analysis of proteins eluted from gelatine–agarose beads.
The arrows indicate the estimated gelatine-binding proteins/gelatinases based on
estimated sizes.
using immunoblotting, potentially reflecting the low affinity of
pro-MMP-9 towards the gelatine–agarose beads, suggesting that
the amount of pro-MMP-9 in the eluted fraction was insufficient
for antibody detection but adequate to show gelatinase activity
because of the sensitivity of zymographic technique or the robust
Fig. 4. Schematic diagram of the primary structure of pro-MMP-2 and pro-MMP-9. The pro-forms of both MMP-2 and MMP-9 are activated through proteolysis at the
interdomain linker regions. The interdomain linker regions are susceptible to proteolysis, as indicated with arrows.
activity of this enzyme in gelatine degradation. Consistently, the
mature forms of MMP-2 and MMP-9 could not be detected through
immunoblotting, reflecting the inadequacy of the antibody used to
recognise the mature forms of the respective MMPs.
As previously suggested, the 65-kDa band detected using
zymography (Figs. 1E and 3D) might represent either mature puri-
fied recombinant human MMP-2 or MMP-9, which are 66 and
67 kDa, respectively (Fig. 2). The 65-kDa proteins possess high
gelatinolytic activity, although they were not detectable in Pon-
ceau S staining (Figs. 1E and 3B). Intact pro-MMP-2 and pro-MMP-9
are not as active as their mature forms due to the steric hin-
drance of their structural folding, where N-terminal propeptide
blocks the active site through Cys-Zn binding in the pro-MMP
forms of these enzymes. During in zymography, this blockage is
partially removed during SDS-PAGE step but could also be com-
pletely removed through in vivo proteolysis under pathogenic
conditions (Springman et al., 1990; Van Wart and Birkedal-Hansen,
1990). Interestingly, although the 55-kDa bands eluted from the
gelatine beads were visible on the Ponceau S-stained membrane
(Fig. 3B), no gelatinase activity was detected. It is likely that these
55-kDa bands were not generated from upregulated pro-MMP-
2 or pro-MMP-9, as these bands were endogenously present in
both non-ischaemic and ischaemic hemispheres. Thus, these bands
might represent other endogenous non-MMP gelatine-binding pro-
teins or the degradation of other MMPs devoid of gelatinase activity.
Further investigation of these 55-kDa bands is required to deter-
mine whether these enzymes contribute to MMP-ECM interactions
and disease progression in ischaemic stroke and other neurodegen-
erative diseases. Moreover, the present study also showed that the
MMP-2 expression observed during immunoblotting is not con-
sistent with the gelatinase activity detected through zymography.
Similarly, the pro-MMP-9 expression and gelatinase activities of
mature MMP-9 are also not consistent. These observations suggest
that MMP-2 exhibits less gelatinase activity than MMP-9. However,
we cannot exclude the possibility that the inconsistency between
the expression level and gelatinase activity of pro-MMP-2 might
reflect the strong affinity of the antibody used to detect pro-MMP-2,
as previously suggested.
More than 30 MMPs have been identified; however, simple and
straightforward strategies, other than immunoblotting, zymogra-
phy and ELISA, for the identification of MMPs from tissue samples
have not been developed. Each of these techniques has advantages
and disadvantages. Western blotting is highly specific and therefore
presents a relatively easy method for distinguishing MMP-2 and
MMP-9. However, this technique is time consuming, and specific
commercial antibodies for each individual MMP are not available.
 M. Alam, A. Shuaib / Journal of Neuroscience Methods 216 (2013) 22–27
ELISA represents a rapid, high-throughput screening technique but
has a high background noise level, and the kits for different sources
of MMPs are not available. Zymography is a straightforward tech-
nique but lacks substrate specificity, as both MMP-2 and MMP-9
are capable of degrading both gelatine A and B. Real-time dual
zymography uses fluorescein-isothiocyanate (FITC)- and Texas Red
(TR)-labelled gelatine in the same gel to distinguished MMP-3 from
MMP-2 and MMP-9 (Watanabe and Hattori, 2002). No similar strat-
egy has been developed for the individual detection of MMP-2 and
MMP-9. A recent study suggested methodologies for the sensitive
quantitative detection of the inactive (pro) and active (mature)
forms of both MMP-2 and MMP-9 (Frankowski et al., 2012) but did
not discuss the use of these methodologies to discriminate mature
MMP-2 and MMP-9, as we have discussed in this manuscript.
In conclusion, for the accurate and discriminatory detection
of mature MMP-2 and MMP-9 in animal stroke research, it is
important to analyse brain tissues using immunoblotting rather
than relying only on traditional non-specific gel zymographic tech-
niques. Thus, specific antibodies that specifically recognise the
mature forms of MMP-2 or MMP-9 have not been developed.
Monoclonal antibodies against specific sites in mature MMP-2
and MMP-9 would be the best option to resolve this issue, and
zymography in combination with immunoblotting might provide
an additional advantage. Although immunohistochemistry is rou-
tinely used in brain tissue analysis, immunoblotting, followed by
zymography, will provide an advantage over any single technique,
such as immunohistochemistry or zymography alone. For exam-
ple, in the immunohistochemical analysis, the differences between
zymozen/full-length pro-MMP and the mature/truncated form of
MMPs could not be differentiated. The ratio of pro-MMP to mature
MMP greatly influences stroke outcome, which cannot be ignored.
Therefore, more specific, efficient and effective antibodies against
the deleterious MMPs should be developed for the precise iden-
tification of mature MMP-2 and MMP-9. To our knowledge, this
study is the first to address this important methodological issue and
report compelling data in this regard. The accurate identification
and expression analysis of the mature forms of MMP-2 and MMP-9
will be crucial to establish the mechanistic pathways of brain dam-
age mediated through the expression of mature MMP-2/MMP-9
and develop efficient and effective drugs for the treatment of stroke.
Conflict of interest
None.
Acknowledgements
This study was supported through grants to Dr. A. Shuaib from
The Canadian Institutes of Health Research (CIHR). We are grateful
to Kath McKenzie for proofreading this manuscript.
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