A novel histochemical method for the visualization of thrombin activity in the nervous system

Article abstract of DOI:10.1016/j.neuroscience.2016.01.065

Although thrombin has an important role in both central and peripheral nerve diseases, characterization of the anatomical distribution of its proteolytic activity has been limited by available methods. This study presents the development, challenges, validation and implementation of a novel histochemical method for visualization of thrombin activity in the nervous system. The method is based on the cleavage of the substrate, Boc-Asp(OBzl)-Pro-Arg-4MβNA by thrombin to liberate free 4-methoxy-2-naphthylamine (4MβNA). In the presence of 5-nitrosalicylaldehyde, free 4MβNA is captured, yielding an insoluble yellow fluorescent precipitate which marks the site of thrombin activity. The sensitivity of the method was determined in vitro using known concentrations of thrombin while the specificity was verified using a highly specific thrombin inhibitor. Using this method we determined the spatial distribution of thrombin activity in mouse brain following transient middle cerebral artery occlusion (tMCAo) and in mouse sciatic nerve following crush injury. Fluorescence microscopy revealed well-defined thrombin activity localized to the right ischemic hemisphere in cortical areas and in the striatum compared to negligible thrombin activity contralaterally. The histochemical localization of thrombin activity following tMCAo was in good correlation with the infarct areas per triphenyltetrazolium chloride staining and to thrombin activity measured biochemically in tissue punches (85 ± 35 and 20 ± 3 mU/ml, in the cortical and striatum areas respectively, compared to 7 ± 2 and 13 ± 2 mU/ml, in the corresponding contralateral areas; mean ± SEM; p < 0.05). In addition, 24 h following crush injury, focal areas of highly elevated thrombin activity were detected in teased sciatic fibers. This observation was supported by the biochemical assay and western blot technique. The histochemical method developed in this study can serve as an important tool for studying the role of thrombin in physiological and pathological conditions.

Full text of DOI:10.1016/j.neuroscience.2016.01.065

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Please cite this article in press as: Bushi D et al. A novel histochemical method for the visualization of thrombin activity in the nervous systemI
Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.01.065
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Neuroscience xxx (2016) xxx–xxx
A NOVEL HISTOCHEMICAL METHOD FOR THE VISUALIZATION
OF THROMBIN ACTIVITY IN THE NERVOUS SYSTEMI
2
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y
D. BUSHI, a,b
*
O. GERA, a,b,gy G. KOSTENICH, e
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5
6
cerebral artery occlusion (tMCAo) and in mouse sciatic nerve
following crush injury. Fluorescence microscopy revealed
well-defined thrombin activity localized to the right ischemic
hemisphere in cortical areas and in the striatum compared to
negligible thrombin activity contralaterally. The histochemi-
cal localization of thrombin activity following tMCAo was in
good correlation with the infarct areas per triphenyltetra-
zolium chloride staining and to thrombin activity measured
E. SHAVIT-STEIN, a R. WEISS, a,f J. CHAPMAN a,b,c,dà AND
D. TANNE a,cà
a Comprehensive Stroke Center, Department of Neurology and
The J. Sagol Neuroscience Center, Chaim Sheba Medical Center,
Tel HaShomer, Israel
b Department of Physiology and Pharmacology, Sackler Faculty
of Medicine, Tel Aviv University, Tel Aviv, Israel
c Department of Neurology, Sackler Faculty of Medicine, Tel
Aviv University, Tel Aviv, Israel
d Robert and Martha Harden Chair in Mental and
Neurological Diseases, Sackler Faculty of Medicine, Tel Aviv
University, Israel
e Advanced Technology Center, Chaim Sheba Medical Center,
Tel HaShomer, Israel
f Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel
g Department of Physical Therapy, Sackler Faculty of Medicine,
Tel Aviv University, Tel Aviv, Israel
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biochemically in tissue punches (85 35 and 20
3
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13
mU/ml, in the cortical and striatum areas respectively,
compared to 7 2 and 13 2 mU/ml, in the corresponding
contralateral areas; mean SE; p < 0.05). In addition, 24 h
following crush injury, focal areas of highly elevated
thrombin activity were detected in teased sciatic fibers. This
observation was supported by the biochemical assay and
western blot technique. The histochemical method devel-
oped in this study can serve as an important tool for studying
the role of thrombin in physiological and pathological condi-
tions. Ó 2016 Published by Elsevier Ltd. on behalf of IBRO.
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Abstract—Although thrombin has an important role in both
central and peripheral nerve diseases, characterization of
the anatomical distribution of its proteolytic activity has
been limited by available methods. This study presents the
development, challenges, validation and implementation of
a novel histochemical method for visualization of thrombin
activity in the nervous system. The method is based on the
cleavage of the substrate, Boc-Asp(OBzl)-Pro-Arg-4MbNA
by thrombin to liberate free 4-methoxy-2-naphthylamine
(4MbNA). In the presence of 5-nitrosalicylaldehyde, free
4MbNA is captured, yielding an insoluble yellow fluorescent
precipitate which marks the site of thrombin activity. The
sensitivity of the method was determined in vitro using
known concentrations of thrombin while the specificity
was verified using a highly specific thrombin inhibitor. Using
this method we determined the spatial distribution of
thrombin activity in mouse brain following transient middle
Key words: ischemic stroke, thrombin, transient middle
cerebral artery occlusion, crushed sciatic nerve, enzyme
histochemistry.
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INTRODUCTION
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Thrombin has, in addition to its role in thrombogenesis,
important hormone-like activities that affect various cells
in the brain through the activation of its receptor, the
protease-activated receptor 1 (PAR1) (Bar-Shavit et al.,
1986; Coughlin, 2000; Junge et al., 2003). Activation of
this receptor by low concentrations of thrombin may have
neuroprotective effects while at higher concentrations
thrombin has deleterious effects (Xue and Del Bigio,
2001; Noorbakhsh et al., 2003; Xi et al., 2003; Chen
et al., 2010; Kameda et al., 2012). Previously, using a
novel and specific biochemical method for direct quantita-
tive detection of thrombin activity in brain slices, we have
found that following both permanent (Bushi et al., 2013)
and transient (Bushi et al., 2015) middle cerebral artery
occlusion (MCAo) performed in mice, thrombin activity is
elevated throughout the ischemic hemisphere reaching
peak levels at the ischemic core. Spatial distribution anal-
ysis indicates that following transient MCAo transient mid-
dle cerebral artery occlusion (tMCAo) thrombin activity is
elevated in both the infarct area and peri-infarct areas
(Bushi et al., 2015). These results are in agreement with
the study performed by Chen et al. showing that during
acute ischemic stroke, disruption of the blood–brain
q
This work was performed in partial fulfillment of the requirements
for a Ph.D. degree by Doron Bushi and Orna Gera at the Sackler
Faculty of Medicine, Tel Aviv University, Israel.
*
Correspondence to: D. Bushi, Neuroscience Research Lab, Joseph
Sagol Neuroscience Center, Chaim Sheba Medical Center,
Tel HaShomer, Israel. Tel: +972-3-5304409; fax: +972-3-5304752.
E-mail address: doron.bushi@gmail.com (D. Bushi).
y
à
Equally contributing first authors.
Equally contributing last authors.
Abbreviations: 4MbNA, 4-methoxy-2-naphthylamine; BBB, blood–brain
barrier; CNS, central nervous system; MCAo, middle cerebral artery
occlusion; mTBI, minimal traumatic brain injury; NAPAP, Na-(2-Napht
hylsulfonylglycyl)-4-Amidino-(D,L)-Phenylalanine Piperidide Acetate;
NSA, 5-nitrosalicylaldehyde; OGD, oxygen glucose deprivation;
PAR1, proteases- activated receptor 1; PBS, phosphate- buffered
saline; tMCAo, transient middle cerebral artery occlusion; TTC,
triphenyltetrazolium chloride.
http://dx.doi.org/10.1016/j.neuroscience.2016.01.065
0306-4522/Ó 2016 Published by Elsevier Ltd. on behalf of IBRO.
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barrier (BBB) allows higher concentrations of blood-
derived thrombin to enter the brain (Chen et al., 2012).
Moreover, several in vitro studies have shown that throm-
bin is synthesized in the brain upon oxygen glucose depri-
vation (OGD) simulating ischemia (Thevenet et al., 2009;
Stein et al., 2015). In this context, thrombin has been
shown to cause synaptic dysfunction (Maggio et al.,
2008; Maggio et al., 2013a,b; Becker et al., 2014;
Prabhakaran et al., 2015) and later on neuronal damage
(Junge et al., 2003; Suo et al., 2004; Hamill et al.,
2009), through the activation of PAR1 (Chen et al.,
2012). Indeed, the toxic effects of thrombin in the central
nervous system (CNS) have been shown in various
ischemic models, inflammatory and neurodegenerative
brain diseases (Yin et al., 2010; Rami, 2012; Chapman,
2013; Davalos et al., 2014), where neuroprotection could
be achieved either by PAR1 deletion as shown by the
group of Traynelis (Junge et al., 2003; Hamill et al.,
2009) or with thrombin inhibitors (McColl et al., 2004;
Chen et al., 2012; Lyden et al., 2014).
EXPERIMENTAL PROCEDURES
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The histochemical method is based on the landmark work
of Dolbeare and Smith (1977). In the early stage of the
development we used the commercial thrombin substrate
Z-Gly-Pro-Arg-4MbNA (J-1120, Bachem, Switzerland).
However, as described in the results section, we found that
the specificity and sensitivity of this substrate does not fulfill
the requirements for an appropriate histochemical method
to localize thrombin activity. Thus, the substrate that we
finally used is Boc-Asp(OBzl)-Pro-Arg-4MbNA, a thrombin
substrate that was specially synthesized per our request
(GL Biochem, Shanghai, China), containing the sequence
Asp(OBzl)-Pro-Arg that was found to be one of the most
sensitive sequences for cleavage by thrombin (Kawabata
et al., 1988).
4-methoxy-b-naphthylamine (4MbNA) is a known
soluble fluorophore that produces a blue fluorescence
(emission = 425 nm [nm]) following excitation by
wavelength of 340 [nm]. However, when 4MbNA
interacts with 5-nitrosalicylaldehyde (NSA) it produces a
schiff-base complex with a shift in fluorescence from
yellow to orange which is water insoluble and potentially
trapped in the tissue (Dolbeare and Smith, 1977; Smith,
1983; Back and Gorenstein, 1989; Cataldo and Nixon,
1990; Rudolphus et al., 1992; Kamiya et al., 1998)
(Fig. 1). In our previous work we have found that thrombin
activity is raised dramatically in the ischemic hemisphere
24 h following both permanent (Bushi et al., 2013) and
transient (Bushi et al., 2015) MCAo. In addition, thrombin
is elevated in sciatic nerves following crush injury
(Smirnova et al., 1996; Friedmann et al., 1999). Thus,
we assumed that thrombin that is generated in the
ischemic tissue and in crushed nerve will potentially
In addition to the CNS, there are many lines of
evidence suggesting that thrombin and its receptors
have important roles in peripheral nerve diseases.
Significant increase in thrombin-like activity was
observed in sciatic nerves that were taken from rat and
mice
1 and 2 days respectively after crush injury
(Smirnova et al., 1996; Friedmann et al., 1999). More-
over, we have described the localization of PAR1 at the
nodes of ranvier and functionally activation of these
receptors results in conduction block (Shavit et al., 2008).
In contrast to immunohistochemical methods that
localize the enzyme protein whether it is active or not,
histochemical visualization of the activity of an enzyme
is a powerful approach to study whether an enzyme is
functionally involved in a pathophysiological process
because it links the enzyme activity to cell and tissue
structure. Although thrombin has an important role in
cleave
the
thrombin
substrate
Boc-Asp(OBzl)-
Pro-Arg-4MbNA releasing free 4MbNA that can react with
NSA to yield insoluble 4MbNA–NSA yellow fluorescent
complexes marking the site of thrombin activity (Fig. 1).
both
central
and
peripheral
nerve
diseases,
characterization of the anatomical distribution of its
proteolytic activity in the nervous system has been
limited by available methods. With the rapid growth in
the field of thrombin-based therapy it is becoming
increasingly clear that the histochemical study of
thrombin activity will be important in understanding the
mechanisms that regulate its function during both
central and peripheral nerve diseases. Recently, Chen
et al. elegantly detected the location of thrombin activity
in rat brains following ischemic stroke using a novel cell-
penetrating peptide-imaging probe that was infused into
the blood stream and then entered brain tissue through
vascular disruption (Chen et al., 2012). Our study pre-
sents a new alternative method to visualize the sites of
thrombin activity in all brain regions and it is not depen-
dent on reperfusion as in the methodology of Chen
et al. Moreover, this fluorescent histochemical method is
relatively simple, uses commercially available products,
and can be performed ex vivo with different tissue types.
The method is shown to demonstrate the distribution of
thrombin activity in mice brain following tMCAo and in
mice sciatic nerve following crush injury.
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Optimized substrate concentration
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Since in this study we used for the first time the custom-
made thrombin substrate Boc-Asp(OBzl)-Pro-Arg-4MbNA,
our first step was to determine its most effective
concentration. We determined this by comparing the
cleavage rates of different substrate concentrations by
50 mU/ml of bovine thrombin (Sigma–Aldrich, Israel).
Different concentrations of Boc-Asp(OBzl)-Pro-Arg-
4MbNA substrate (0.01, 0.05, 0.1, 0.4, 0.8 mM) were
added to a 96-well black microplate (Nunc, Denmark),
each well in the microplate contained thrombin buffer
(50 mM TRIS/HCl, pH 8.0, 0.15M NaCL, 1 mM CaCl2),
0.1% BSA and 50 mU/ml of bovine thrombin. Thrombin
cleaved the substrate and released free 4MbNA that was
measured by a fluorescence reader (Molecular Devices,
USA) with excitation and emission filters of 340 and
425 nm respectively. Cleavage rate was measured by the
linear slope of the fluorescence intensity vs. time. As
shown in the results section, the optimal substrate
concentration was found to be 0.1 mM.
.
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(BP 300-385, BA420, DM400, Olympus, Japan). The
spectra of the 4MbNA–NSA complexes were determined
using a spectrometer (Glacier, BWTEK Inc, USA) at
excitation k = 420 nm (multi led light source (Prizmatix,
Israel)). As shown in the results section, the 4MbNA–NSA
complexes were generated in the tested thrombin range
(25–2000 mU/ml).
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Assessing thrombin activity levels following tMCAo
and sciatic crush injury
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In order to implement the histochemical method for
thrombin activity staining in the nervous system we first
verified using a quantitative method that the thrombin
activity levels that are generated in mice brain following
tMCAo and in mice sciatic nerves following crush injury
are in the sensitivity range of the suggested method.
Animal handling as well as all described experiments
were performed in accordance and approved by the
Institutional Animal Care and Use Committee of The
Chaim Sheba Medical Center (Tel HaShomer, Israel),
which adheres to the Israeli law on the use of laboratory
animals and NIH rules.
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Thrombin activity level in mouse brain following
tMCAo
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Studies were carried out on eight-week-old male C57BL6
mice (n = 6, purchased from Harlan Laboratories).
Anesthesia was performed with 2.5% isoflurane mixed
in oxygen and delivered through a facemask. TMCAo
was performed based on our previously reported
technique (Bushi et al., 2013). Briefly, a silicone-coated
filament (Doccol Corp, CA, USA) was inserted through a
small hole in the right external carotid artery. The filament
was carefully advanced upto 11 mm from the carotid
artery bifurcation or until resistance was felt. The filament
was then left in place for about 90 min and then removed
to achieve reperfusion. Throughout the procedure, the
body temperature was kept constant at ꢀ37 °C using a
heating pad. Following surgery until recovery from anes-
thesia animals were kept in a closed chamber heated with
a lamp. Mice were sacrificed 24 h after the procedure
using pental (100 ll) by I.P. administration.
As described in our previous studies (Bushi et al.,
2013), thrombin enzymatic activity was measured using a
fluorometric assay based on the cleavage rate of the
synthetic substrate Boc-Asp(OBzl)-Pro-Arg-AMC (I-1560,
Bachem, Switzerland) and defined by the linear slope of
the fluorescence intensity vs. time. Following sacrifice,
the brain of each animal was immediately removed and
placed in a steel brain matrix (1 mm, Coronal, Stoelting,
USA). The coronal slice that is located 1 mm posterior to
the bregma was removed and spatial distribution of throm-
bin activity in this slice was measured using the thrombin
activity assay and 1-mm diameter tissue punches that were
taken from 4 different locations (named A–D; Fig. 6). Each
tissue sample was placed in a separate well in the 96 black
microplate and thrombin activity was measured using the
thrombin activity assay. Measurements were carried out
using a microplate reader (Infinite 2000, Tecan, Switzer-
land) with excitation and emission filters of 360 35 and
Fig. 1. Schematic diagram of the fluorescent enzyme histochemical
method for the localization of thrombin activity. (I) The substrate
Boc-Asp(OBzl)-Pro-Arg-4MbNA, (II) Thrombin cleaves the substrate,
releases the fluorophore 4MbNA. Free 4MbNA (III) reacts with
5-nitrosalicylaldehyde (IV) to form a Schiff-base 4MbNA-NSA
insoluble yellow fluorescent complexes (V) marking the sites of
thrombin activity.
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Assessment of the method sensitivity
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In order to estimate the sensitivity of the method, we
examined whether 4MbNA–NSA complexes are
generated when different thrombin concentrations
cleaved the Boc-Asp(OBzl)-Pro-Arg-4MbNA substrate in
the presence of NSA. Eighty five microliters of thrombin
buffer (50 mM TRIS/HCl, pH 7.0, 0.15M NaCL, 1 mM
CaCl2) were mixed with 1 microliter (0.6 mM) of NSA
(Sigma–Aldrich, Israel) and 10 microliters of a range of
different thrombin concentrations (2000, 250,100, 50,
25 mU/ml). As control, we used a solution containing
2000 mU/ml thrombin and NAPAP (60 lM, Na-(2-Naphthyl
sulfonylglycyl)-4-Amidino-(D,L)-Phenylalanine Piperidide
Acetate, Pefabloc TH, Sigma–Aldrich, Israel) one of the
most potent and selective competitive inhibitors of
thrombin. Next, 5 microliters (0.1 mM) of the Boc-Asp
(OBzl)-Pro-Arg-4MbNA substrate were added to the
95 microliter solution that was placed on microscopic
glass slides (Superfrost Plus, Thermo Scientific, USA).
The slides with the 100 microliter solution were placed in
a closed tray (Humid Chamber, H-6644, Sigma–Aldrich,
Israel) filled with a thin layer of water and placed at room
temperature for 24 h. Later, the yellow fluorescence
reactions of the generated 4MbNA–NSA complexes were
photographed on an inverted fluorescence microscope
(Ix81, Olympus, Japan) with a filter cube U-MWU2
.
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460 35 nm, respectively. For calibration, known concen-
trations of bovine thrombin (Sigma–Aldrich, Israel) were
used in the same assay. Following tissue samples, the
punctured slices were stained using triphenyltetrazolium
chloride (TTC) for infarct assessment.
enhanced chemiluminescence (ECL) assay kit (Thermo
Scientific, USA).
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Histochemical visualization of thrombin activity in
mice brain following tMCAo
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TMCAo was performed in eight-week-old male C57BL6
mice as described above. Mice were sacrificed 24 h after
the procedure using pental (100 ll) by I.P. administration.
Immediately after sacrifice the mice brains were removed
and inserted into 30% sucrose solution for 24 h at 4 °C.
Thereafter, the brains were cut into 20-lm coronal slices
using a cryostat (Leica CM1850, Leica, Germany), and
the cut sections were picked up on microscopic glass
slides (Superfrost Plus, Thermo Scientific, USA). The
glass slides with the fresh frozen sections were placed in
a closed tray (Humid Chamber, H-6644, Sigma–Aldrich,
Israel) filled with a thin water layer. For thrombin activity
staining, slices that were taken from the ischemic core
(ꢀ1 mm posterior to the bregma) were used. The slices
were incubated in a solution containing 93 microliters of
thrombin buffer (50 mM TRIS/HCl, pH 7.0, 0.15M NaCL,
1 mM CaCl2), 1 microliter (0.6 mM) of NSA, 1 microliter of
an aminopeptidase inhibitor bestatin (0.1 mg/ml, Cayman
Chemical Company, USA) and 5 microliters (0.1 mM) of
the thrombin substrate Boc-Asp(OBzl)-Pro-Arg-4MbNA.
The tray with the stained slices was placed at room
temperature for 24 h. In order to ensure that the
localization of the histochemical reaction product
specifically results from thrombin activity cleavage of the
substrate the following controls were used: (1) sections
were incubated in the histochemical solution containing
60 lM of the thrombin inhibitor NAPAP; (2) sections were
incubated in the histochemical solution from which the
NSA was omitted. The reaction was terminated by rinsing
the sections in cold 50 mM TRIS/HCl, pH 7.0. Next, the
sections were fixed using 4% paraformaldehyde in 0.1 M
PBS, pH 7.4. Twenty minutes later, the sections were
washed with PBS and 0.1% Triton X-100 (PBST) and
incubated for 10 min with Hoechst (1:1000, hoe-33342,
Sigma–Aldrich, Israel) for nuclear staining. After
additional washing with PBST the sections were air dried
and closed with mounting media and cover glass.
Thrombin activity localization and hoechst staining were
visualized using an inverted fluorescence microscope
(Ix81, Olympus, Japan) with a filter cube U-MWU2
(BP 300-385, BA420, DM400, Olympus, Japan).
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Thrombin activity level in mice sciatic nerves
following crush injury
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Previous studies have reported that thrombin-like activity
that was measured using an absorption detection method,
was elevated in rat and mice sciatic nerves 1 and 2 days
respectively after crush injury (Smirnova et al., 1996;
Friedmann et al., 1999). In this study we used a mouse
model, so we verified using a highly sensitive fluorometric
method, that 24 h following sciatic crush injury thrombin
activity levels in mice sciatic nerves are elevated above
the sensitivity limit of the histochemical method.
Mice (n = 8) were anesthetized with a mixture of
100 mg/kg ketamine and 10 mg/kg xylazine that was
injected I.P. The left thigh was cleaned by 70% ethanol,
and then a small incision was made in the thigh through
the connective tissue between the gluteus and the
biceps femoris muscles. The sciatic nerve was exposed
and tightly compressed with flattened forceps for 30 s.
Immediately thereafter, the skin was sutured and the
animals were placed in a closed heated chamber until
recovery from anesthesia. All the surgical procedures
were performed under a binocular operating microscope,
while throughout the procedure, the mouse’s body
temperature was kept constant at ꢀ37 °C using a
heating pad. Mice were sacrificed 24 h after the
procedure using pental (100 ll) by I.P. administration.
Twenty four hours following the crush procedure, both
injured and contralateral uninjured sciatic nerves were
isolated from the treated mice and washed with ice-cold
phosphate-buffered saline (PBS). The epineurium was
gently rolled-up and the fibers were placed in 96 black
microplate wells. Thrombin activity was measured, as
mentioned above, using thrombin activity assay based on
the Boc-Asp(OBzl)-Pro-Arg-AMC substrate. Sciatic
nerves that were taken from healthy control mice served
as
a
second control. For calibration, known
concentrations of bovine thrombin (Sigma–Aldrich, Israel)
were used in the same assay.
Differences in thrombin activity levels between injured
and contralateral uninjured nerves were confirmed using
western blot technique. The epineurium of isolated
sciatic nerves was gently rolled-up and the fibers were
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Histochemical visualization of thrombin activity in
mice sciatic nerve following crush injury
homogenized using
a pestle motor mixer (Argos
technologies, USA). Proteins from the sciatic
homogenates (20 lg total proteins) were separated by
polyacrylamide gel electrophoresis and transferred onto
nitrocellulose membranes for western blot analysis.
Membranes were incubated with primary goat anti
thrombin antibody (1:300, sc-23335, Santa Cruz, USA)
overnight with gentle agitation at 4 °C. Each injured
sciatic nerve had its uninjured contralateral one serving
as control. Membranes were incubated at room
temperature with horseradish peroxidase-conjugated
donkey anti-goat antibody (Jackson Immunoresearch
Laboratories, USA) and bound antibody detected using
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Crush injury was performed in eight-week-old male
C57BL6 mice as described above. Mice were sacrificed
24 h after the procedure using pental by I.P.
administration. Sciatic nerves were isolated from mice
and washed with ice-cold PBS. The epineurium was
gently rolled-up and the fibers were teased into single
fibers on microscopic glass slides (Superfrost Plus,
Thermo Scientific, USA). The glass slides with the
sciatic fibers were placed in a closed tray (Humid
Chamber, H-6644, Sigma–Aldrich, Israel) filled with thin
water layer and stained for thrombin activity as
.
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described above using the histochemical solution. As
controls, some fibers from the crushed sciatic nerves
were stained with the histochemical solution and
NAPAP (60 lM). In addition, fibers from the
contralateral uninjured nerve were also stained for
thrombin activity and served as controls.
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Statistics analysis
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Statistical analyses were conducted using SPSS v. 22 for
Windows (IBM, NY, USA). Student t test was used to
analyze differences in thrombin activity levels. All
numerical data are expressed as mean SEM, unless
otherwise indicated. P values of <0.05 were considered
significant.
Fig. 2. Unspecific fluorescence signals with the Z-Gly-Pro-Arg-
4MbNA substrate. Enzyme activity that was measured in brain slices
taken from infarct core of mouse brain 24 h following MCAo. The
raised signals are accumulation of free 4MbNA that were librated
from the Z-Gly-Pro-Arg-4MbNA substrate. NAPAP, a specific throm-
bin inhibitor, was added to each well after 45 min of detection. The
initial values of fluorescence intensity at time zero, related to auto
fluorescence of the tested tissue. Slice thickness = 1 mm; FI = Flu-
orescence Intensity.
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RESULTS
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Limitation of the thrombin substrate
Z-Gly-Pro-Arg-4MbNA for histochemical method
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The specificity of the commercially available substrate
Z-Gly-Pro-Arg-4MbNA for thrombin was studied using the
fluorometric assay for measuring thrombin activity in the
brain and brain slices taken from the infarct core 24 h
following MCAo. Briefly, mouse brain was immediately
removed following sacrifice and cut into 1-mm coronal
slices. Slices from the infarct core (slice # 5, #6 (1 mm
posterior to the bregma) and #7) with potentially the
highest amount of thrombin following MCAo were used
(Bushi et al., 2013, 2015). The slices were placed into a
96-well black microplate (Nunc, Denmark) containing the
substrate buffer as previously described (Bushi et al.,
2013, 2015) and the Z-Gly-Pro-Arg-4MbNA substrate
(0.8 mM). The thrombin in the ischemic tissues cleaved
the Z-Gly-Pro-Arg-4MbNA substrate and released free
4MbNA that was measured by the fluorescence reader
(Molecular Devices, USA) with excitation and emission
filters of 340 and 425 nm respectively. As shown in Fig. 2,
during the first 45 min a significant increase in fluorescence
signal was observed. After 45 min the thrombin inhibitor -
NAPAP (1 lM) was added to each well, and the fluores-
cence signalswere notcompletely inhibited, demonstrating
that the measured signals do not represent only thrombin
activity. This experiment demonstrates the lower specificity
of the substrate Z-Gly-Pro-Arg-4MbNA for thrombin. More-
over, no yellow 4MbNA–NSA complexes were generated
when the Z-Gly-Pro-Arg-4MbNA substrate was incubated
with NSA and high concentrations of commercial thrombin
(data not shown). Based on these results, we decided to
change the target substrate to a custom-made substrate
containing the sequence Asp(OBzl)-Pro-Arg which has
higher sensitivity (Kawabata et al., 1988) and better
specificity (Bushi et al., 2013) to thrombin cleavage.
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inhibit the reaction. For comparison, a lower cleavage
rate was measured when 50 mU/ml of thrombin cleaved
the thrombin substrate Z-Gly-Pro-Arg-4MbNA (using its
optimal concentration-0.8 mM), indicating the higher
sensitivity of Boc-Asp(OBzl)-Pro-Arg-4MbNA for thrombin
(Fig. 3).
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Assessment of the method sensitivity
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The yellow fluorescence reaction products of the
4MbNA–NSA complexes are presented in Fig. 4.
Different concentrations of thrombin (25–2000 mU/ml)
cleaved the Boc-Asp(OBzl)-Pro-Arg-4MbNA substrate
and liberated the 4MbNA fluorophore. Then, the
4MbNA coupled with free NSA to generate together
4MbNA–NSA insoluble complexes. As previously
described, we found that the 4MbNA–NSA complexes
appeared as small, discrete, yellow-orange fluorescent,
needle- shaped crystals (Dolbeare and Smith, 1977;
Back and Gorenstein, 1989; Cataldo and Nixon, 1990;
Rudolphus et al., 1992; Kamiya et al., 1998) (Fig. 4b).
The densities of the generated 4MbNA–NSA complexes
are positively correlated with the thrombin concentration
(Fig. 4a, d–g). Based on these findings we defined
25 mU/ml as the detection limit of the method. NAPAP, a
potent and selective competitive inhibitor of thrombin,
completely inhibited the reaction (Fig. 4c).
The measured fluorescence spectra of the
4MbNA–NSA complex are shown in Fig. 5. The resulting
fluorescence spectrum (peaks at 530 and 595 nm) is in
good agreement with the known data that the
4MbNA–NSA complex produces a shift in fluorescence of
the 4MbNA to 530 and 595 nm (Dolbeare and Smith,
1977). The 530 nm peak is a blue-green nonspecific
fluorescencethatprobably resultedfromthenonenzymatic
reaction of NSA with amino groups to form Schiff-base
adducts (Dolbeare and Smith, 1977).
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Optimized substrate concentration
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The cleavage rates of the thrombin substrate Boc-Asp
(OBzl)-Pro-Arg-4MbNA by 50 mU/ml of thrombin are
presented as
a
function of different substrate
concentrations (Fig. 3). The maximum cleavage rate was
achieved with a substrate concentration of 0.1 mM.
Interestingly, higher concentrations of the substrate
.
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the histochemical method developed in this study. In con-
trast, lower thrombin activity levels were measured in the
right striatum (location B) that included peri-infarct areas
per TTC staining. These lower values are around the sen-
sitivity limit of the histochemical method. In the contralat-
eral hemisphere even lower levels of thrombin activity
were measured in all regions and they were below the
detection limit of the histochemical method.
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Histochemical visualization of thrombin activity in
mice brain following tMCAo
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Fig. 7 presents the topographic distribution of thrombin
activity following tMCAo. This a representative slide out
of 15 experiments performed in a similar fashion. High
density of small, discrete, yellow-orange fluorescent,
needle-shaped crystals was observed in the right
ischemic hemisphere in all cortical and preoptic areas
(Fig. 7e, g, i) and in the striatum (Fig. 7c) compared to
negligible thrombin activity contralaterally (Fig. 7b, d, f).
The histochemical localization of thrombin activity
corresponds to the infarct areas per TTC staining and to
areas with high thrombin activity levels as determined
using the thrombin activity assay and tissue punches
technique (Fig. 6). In addition, the relatively lower
thrombin activity levels that were measured using the
punch technique in location B (20 3 mU/ml, Fig. 6),
are probably due to measurement of thrombin activity in
borderzone areas that include both high and low
thrombin levels (Fig. 7a, dashed circle). No staining was
observed in the sections that were incubated with the
thrombin inhibitor NAPAP (Fig. 7h) and in the sections
that were incubated in the histochemical solution from
which the NSA was omitted (data not shown).
Fig. 3. The relation between the cleavage rates of the thrombin
substrate and substrate concentrations. Cleavage rates of thrombin
substrates (Boc-Asp(OBzl)-Pro-Arg-4MbNA, circles; Z-Gly-Pro-Arg-
4MbNA, triangle) by 50 mU/ml of bovine thrombin as a function of
different substrate concentrations. FI = Fluorescence Intensity.
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Thrombin activity levels in mice brain following
tMCAo
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Twenty four hours following 90-min tMCAo, the
distribution of thrombin activity levels was measured in
1-mm-thick fresh coronal slices that are located 1 mm
posterior to the bregma. Based on our previous studies,
in this brain area thrombin reached its maximal levels
following MCAo (Bushi et al., 2013, 2015). Tissue punches
were sampled from several locations in the slice including
the cortex and the striatum. High thrombin values were
found in locations A–D of the right ischemic hemisphere
and they were significantly higher compared to the corre-
sponding areas in the contralateral slices (61 25, 20
3, 73 35, 85 35 vs. 17 3, 13 2, 8 3, 7
2 mU/ml; mean SEM; n = 6; p = 0.025, 0.026,
0.026 and 0.025 respectively by paired Student t test;
Fig. 6). As expected, the highest thrombin activity values
were found in ischemic cortical areas (A, C and D) based
on TTC staining and they were above the sensitivity limit of
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Thrombin activity levels in mice sciatic nerves
following crush injury
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A significant increase in thrombin activity levels was
observed in mice sciatic nerves 24 h following crush
Fig. 4. Fluorescence reactions of the 4MbNA–NSA complexes as function of different thrombin concentrations. Different concentrations of bovine
thrombin (25–2000 mU/ml) cleaved the Boc-Asp(OBzl)-Pro-Arg-4MbNA substrate and liberated 4MbNA that coupled with free NSA to generate r
4MbNA–NSA insoluble yellow fluorescence complexes. The densities of the generated 4MbNA–NSA complexes are positively correlated with the
thrombin concentration: (a) 2000, (b) 2000 (magnification ꢁ400), (c) 2000+NAPAP (60 lM), (d) 250, (e) 100, (f) 50, (g) 25. All magnifications
(except b) are ꢁ100. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
.
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few reaction products were seen 1 h after the reaction
started (Fig. 9a) while optimal staining was observed
24 h later (Fig. 9b–e). In some cases large amounts of
thrombin that were generated in the fibers diffused out
to the surrounding areas (Fig. 9b, d). As expected, the
reaction products appear as small, discrete, yellow-
orange fluorescent, needle-shaped crystals (Fig. 9e). No
staining was seen in sciatic nerves that underwent crush
injury and were stained with the thrombin inhibitor
NAPAP (Fig. 9f, g) and in control uninjured nerves
(Fig. 9h).
DISCUSSION
541
Fig. 5. Fluorescence spectra of the 4MbNA–NSA complexes. Fluo-
rescence spectra of 4MbNA–NSA complexes that were generated
during hydrolysis of Boc-Asp(OBzl)-Pro-Arg-4MbNA by 2000 mU/ml
of bovine thrombin in the presence of NSA.
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The study of Dolbeare and Smith (1977) provided the
impetus for studies visualizing enzymes by histochemical
techniques that is based on the 4MbNA–NSA complexes.
This report presents a new fluorescent enzyme histo-
chemical method for fluorescent visualization of thrombin
activity. The method was used to demonstrate the local-
ization of thrombin activity in mice brain following tMCAo
and in mice sciatic nerve following crush injury.
The specificity of this histochemical method for
thrombin activity was demonstrated using NAPAP, one
of the most potent and selective competitive inhibitors of
thrombin (Bushi et al., 2013; Itzekson et al., 2014;
Itsekson-Hayosh et al., 2015; Stein et al., 2015). NAPAP
blocked the thrombin activity either when commercial
thrombin was used to determine the method’s sensitivity
(Fig. 4c), thrombin that was generated in ischemic brain
following tMCAo (Fig. 7h) or thrombin that was generated
in sciatic nerve following crush injury (Fig. 9f, g). Although
there are some data that trypsin-type serine proteases
can also cleave the substrate Boc-Asp(OBzl)-Pro-Arg-
4MbNA (Kawabata et al., 1988; Fukusen and Aoki,
1996), the sensitivity of this substrate for thrombin is
much higher compared to trypsin with higher Kcat/Km
ratio for thrombin (Kawabata et al., 1988). Moreover, the
fact that NAPAP that its inhibition constant for thrombin
is 2-fold lower than for trypsin (Sturzebecher et al.,
1984), completely inhibited the substrate’s cleavage, indi-
cates that reaction products results from thrombin activity
and not from trypsin. In addition, the similarity between
the sequence of the Boc-Asp(OBzl)-Pro-Arg-4MbNA sub-
strate and the amino acid order in the cleavage site of
thrombin in PAR1 (. . ..TLDPR/SFLLRNPN. . .) also pro-
vides support to the sensitivity of this substrate for throm-
bin. In contrast, trypsin preferentially cleaved PAR2 that
has a different amino acid sequence in its cleavage site
(. . ..SSKGR/SLIGKVDGTS. . .) (Kawabata and Kuroda,
2000).
Fig. 6. Spatial distribution of thrombin activity levels and infarct area
in mice brain following tMCA. Mean thrombin activity level (mU/ml of
tissue SEM) measured at different locations in the ischemic and
contralateral hemispheres following tMCAo. Typical TTC staining of
the relevant slice used in these analyses is presented (representative
of six slices developed by this method). Infarct regions are colored by
white and intact brain regions by red. (For interpretation of the
references to colour in this figure legend, the reader is referred to the
web version of this article.)
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injury. Thrombin activity in the injured nerve was elevated
to levels of 245 41 mU/ml and it was significantly
higher compared to thrombin activity levels that were
measured in both the uninjured contralateral sciatic
nerves (27 7 mU/ml, n = 8, p < 0.0001, by paired
Student t test) and in control sciatic nerves that were
taken from healthy control mice (28 7 mU/ml, n = 8,
p < 0.0001, by unpaired Student t test) (Fig. 8a).
Western blotting of injured sciatic nerve samples
confirmed the significantly elevated level of thrombin
activity (n = 4, p < 0.05, by paired Student t test)
(Fig. 8b).
Even though the rate of coupling reaction of 4MbNA
and NSA is pH dependent, we found that there is
apparently sufficient formation of a stable adduct of
4MbNA and NSA at pH 7.0 to permit visualization of the
thrombin activity reaction product. Although the 4MbNA–
NSA complexes methodology is usually used for
visualization of enzymes with optimal activity in acidic
pH (Kamiya et al., 1998), in some studies (Back and
Gorenstein, 1989; Rudolphus et al., 1992) this methodol-
ogy was used in more neutral pH environments similar to
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Histochemical visualization of thrombin activity in
mice sciatic nerves following crush injury
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Fig. 9a–e depicts different examples of visualization of
thrombin activity staining by histochemical staining of
sciatic nerves of mice 24 h following crush injury. Only a
.
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Fig. 7. Histochemical visualization of thrombin activity following tMCAo. (a) Fluorescence photomicrographs of coronal section of mouse brain
following tMCAo that was incubated with Boc-Asp(OBzl)-Pro-Arg-4MbNA. Mosaic was formed by merging 20 pictures, each picture magnification
ꢁ40. Higher power photomicrograph of the typical appearance and distribution of the thrombin activity reaction product at selected areas in the
ischemic hemispheres (c, e, g, i, magnification ꢁ100). The small, discrete, yellow-orange fluorescent, needle-shaped crystals can clearly be seen in
the inset picture in Fig. 7c (taken from the right ischemic striatum, magnification ꢁ400). Negligible staining was observed in the contralateral side
(b, d, f, magnification ꢁ100). Cell nucleus was stained by hoechst and appeared as blue spots. (h) Absence of thrombin activity reaction product in
tissue incubated in histochemical staining solution containing NAPAP (Mosaic was formed by merging 20 pictures, each picture magnification ꢁ40).
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
.
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7-amino-4-methylcoumarin. We used this substrate in a
quantitative and specific method that we developed for
measurement of thrombin activity levels in brain slices
(Bushi et al., 2013). We have shown that NAPAP com-
pletely inhibited the fluorescence signals that were gener-
ated when thrombin activity was measured in ischemic
slices using Boc-Asp(OBzl)-Pro-Arg-AMC substrate
(Bushi et al., 2013). Moreover, the Asp(OBzl)-Pro-Arg
sequence is highly analogous to the known sequence of
PAR1 cleaved by thrombin and may better represent the
PAR1 cleaving potential of thrombin activity.
Optimal staining was observed with samples that were
incubated for 24 h in room temperature. These conditions
are in agreement with the work of Beck and Gorenstein
that developed the histochemical method, based on
4MbNA–NSA complexes, for visualization of enkephali-
nase activity in rat brain (Back and Gorenstein, 1989). In
contrast, different incubation conditions were used in other
studies in the same field that used cells or lung tissues sam-
ples (Dolbeare and Smith, 1977; Rudolphus et al., 1992;
Kamiya et al., 1998).
In the present study, we confirm our previous finding
(Bushi et al., 2015) that upon a tMCAo, thrombin activity
rises significantly in the ischemic hemisphere. Further-
more, spatial distribution analysis using tissue punches
indicated that the highest thrombin activity levels were
found in infarct areas based on TTC staining (Fig. 6). This
finding also confirms a positive correlation between
thrombin activity level and infarct size (Bushi et al.,
2013). It seems meaningful that we have found the distri-
bution of thrombin activity in the tested slices was similar
in both the punch method and the new higher resolution
histochemical method. In both methods high thrombin
activity levels were observed in the right ischemic hemi-
sphere in all cortical areas and in the striatum compared
to negligible thrombin activity levels contralaterally.
In addition to the CNS, we have demonstrated the
capability of this new histochemical staining to localize
excess thrombin activity in the sciatic nerve following
crush injury that simulates peripheral nerve disease.
First, we verified using the quantitative thrombin activity
assay that 24 h following crush injury, thrombin activity
levels in mice sciatic nerve are elevated above the
detection limit of the histochemical method (Fig. 8a).
This increase in thrombin was also confirmed by western
blot technique (Fig. 8b). Following staining for thrombin
activity localization we have found distinct yellow–orange
fluorescent products covering the nerve surfaces, while
in some cases some thrombin spilled out into the
surrounding areas (Fig. 9). The latter observation is in
agreement with our previous finding that thrombin
diffuses out from its source tissue and is detected in the
surrounding buffer solution (unpublished data).
Fig. 8. Thrombin activity increased in mice sciatic nerves following
crush injury. (a) Thrombin activity levels that were measured in mice
sciatic nerves using thrombin activity assay (*p < 0.0001). (b)
Western blots showed a significant increase of thrombin level in
crushed sciatic nerves compared with uninjured contralateral sciatic
nerves.
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that presented here. In the same context, the relative high
detection limit of the suggested method (ꢀ25 mU/ml,
Fig. 4), is probably due to the conflict between the optimal
conditions of thrombin activity (ꢀpH 8.0) to the optimal
conditions for 4MbNA–NSA coupling (acid pH). We did
consider modifying the method to carry out the enzymatic
reaction in two stages, the thrombin substrate reaction at
pH 8.0 followed by a second reaction in which NSA was
added to the reaction mixture acidified to pH ꢀ6.0 by
the addition of concentrated HCl. The results of these
assays (not shown) revealed that in order to obtain a
specific signal, long incubation times were necessary for
the thrombin reaction which would greatly reduce the spa-
tial resolution of the method and this approach was there-
fore abandoned.
Similar to what was previously reported (Dolbeare and
Smith, 1977), we have found that NSA has some inhibi-
tory effect on the activity of thrombin (data not shown).
In order to minimize this inhibitory effect, we used NSA
concentration of 0.6 [mM] which is slightly above the min-
imal NSA concentration that is required by the NSA to trap
all liberated 4MbNA (Back and Gorenstein, 1989).
Due to its relative lower specificity (Fig. 2) and
sensitivity (Fig. 3) to thrombin, we found that the
substrate Z-Gly-Pro-Arg-4MbNA is not suitable for this
histochemical method. Instead, we successfully used the
Boc-Asp(OBzl)-Pro-Arg-4MbNA with amino acids
sequence that was found to be most sensitive for highly
purified human a thrombin compared to other blood-
clotting proteases (Kawabata et al., 1988). Previously, we
successfully used as thrombin substrate the same Boc-
Asp(OBzl)-Pro-Arg sequence coupled to the fluorophore
The main advantage of the histochemical method
presented in this study is the ability to detect the location
of thrombin activity at the cellular level. The broad
emission spectrum of the 4MbNA and NSA complex
(Fig. 5), makes it possible to visualize both thrombin
activity and the nuclear staining by exciting both
fluorophores simultaneously in the same tissue section
(e.g. Fig. 7c). Localization of thrombin activity at the
.
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Fig. 9. Histochemical visualization of thrombin activity following crush injury. (a) Fluorescence photomicrographs of sciatic nerves of mice 24 h
following crush injury and 1 h following incubation with the substrate Boc-Asp(OBzl)-Pro-Arg-4MbNA. The few yellow reaction products that were
generated are indicated by arrows (magnification ꢁ40). (b–e) Massive staining was observed 24 h following incubation (magnification: ꢁ40 (b–d),
ꢁ100 (e)). Higher power photomicrographs of pictures c–e are present in the upper right corner of each picture (magnification ꢁ100 (c, d), ꢁ400
(e)). (f and g) Sciatic nerves that underwent crush injury and were incubated in histochemical staining solution containing NAPAP (magnification
ꢁ40 (f), ꢁ100 (g)). (h) Uninjured contralateral sciatic nerves that were incubated for 24 h with the substrate Boc-Asp(OBzl)-Pro-Arg-4MbNA
(magnification ꢁ100).
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cellular level can serve as a strong tool for examining the
links between thrombin activity and brain cells. Further
experiments using the histochemical method presented
here combined with immunohistochemistry techniques
are needed to better understand these associations.
In this study we used unfixed cryostat sections for the
visualization of thrombin activity in brain slices following
tMCAo. We found that in order to retain appropriate
levels of thrombin activity in these thin frozen slices, it is
required to store the brain in sucrose solution prior to its
cutting. This observation is in agreement with relevant
literature finding loss of enzyme activity due to freezing,
dehydration (Carpenter et al., 1993; Prestrelski et al.,
1993; Anchordoquy et al., 2001) and storage at low tem-
peratures for long durations (Paveena et al., 2010, 2011).
Sucrose was found to be one of the effective additives for
improving the stability of freeze-dried enzymes (Paveena
et al., 2010, 2011). In contrast to the brain tissue, the rel-
ative rigid structure of the sciatic nerve enables the use of
fresh sciatic samples, thus, keeping the initial levels of
thrombin activity in the tissue in addition to its original
shape.
Chen et al. recently elegantly detected the location of
thrombin activity in rat brains following ischemic stroke
using a patented cell-penetrating peptide-imaging probe.
The probe was infused into the common carotid artery
where anterograde flow carries the infused probe into
the internal carotid and ultimately to the brain tissue
through breakthrough in the BBB (Chen et al., 2012). In
contrast to this technique, the histochemical method pre-
sented in this study is not dependent on blood flow and
reperfusion, thus allowing detection of thrombin activity
that is located also in brain tissue supplied by occluded
vessels. It is a simple histological method that uses com-
mercially available products and can be performed ex vivo
with different tissue types.
In the present study we focused on thrombin due to its
highly important role in the nervous system. Thrombin
.
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activity was detected early in experimental autoimmune
encephalomyelitis model indicating its potential as a
marker for neuroinflammation (Davalos et al., 2014). Fur-
thermore, thrombin activity was significantly elevated in
mice brains that underwent minimal traumatic brain injury
(mTBI), while injection of PAR1 antagonist completely
blocked the amnestic effects of mTBI (Itzekson et al.,
2014). Thrombin message and protein were highly
expressed in microvessels that were isolated from Alzhei-
mer’s disease patient brains but were not detectable in
control vessels (Yin et al., 2010). In a rat glioblastoma
model thrombin activity was significantly elevated in the
tumor and positively correlated with tumor-induced brain
edema (Itsekson-Hayosh et al., 2015). In the peripheral
system, low concentrations of thrombin were found to
be responsible for regeneration of mouse peripheral
nerve after its crushing (Balezina et al., 2005) while high
concentrations had deleterious effects (Lee et al., 1998).
Moreover, mice lacking the thrombin inhibitor protease
nexin-1 showed delayed structural and functional recov-
ery after sciatic nerve crush (Lino et al., 2007).
As a key player in the nervous system, thrombin is an
attractive target for drug therapy. Argatroban, a specific
thrombin inhibitor, has been shown to reduce cell injury
and ischemic lesion size after focal cerebral ischemia
(McColl et al., 2004; Chen et al., 2012; Lyden et al.,
2014). Moreover, administration of nafamostat mesilate
during ischemia and reperfusion in a rat model of MCAo
reduced thrombin activity and neurological deficit (Chen
et al., 2014). In addition, as a coagulation factor, thrombin
Efrat Shavit-Stein and Ronen Weiss helped with the
prepared sections for staining and with the thrombin
activity assay.
Joab Chapman and David Tanne supervised the
project and revised the manuscript critically for
important intellectual content.
All authors gave their approval to the manuscript.
784
785
Acknowledgment—The research was supported by institution
funding.
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Please cite this article in press as: Bushi D et al. A novel histochemical method for the visualization of thrombin activity in the nervous systemI
Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.01.065

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(Accepted 29 January 2016)
(Available online xxxx)
.
Please cite this article in press as: Bushi D et al. A novel histochemical method for the visualization of thrombin activity in the nervous systemI
Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.01.065