ARTICLE IN PRESS

BRES-38892; No. of pages: 9; 4C: 3, 5

BR AIN RE S EA RCH XX ( 2 0 08 ) XXX –X XXa v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o mw w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Neutrophil elastase inhibitor attenuates hippocampal neuronaldamage after transient forebrain ischemia in rats
Hiroaki Matayoshi a , Takao Hirata a , Satoshi Yamashita a , Kazuyoshi Ishida a ,Yoichi Mizukami b , Toshikazu Gondo c , Mishiya Matsumoto a , Takefumi Sakabe a,⁎a

Department of Anesthesiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube,
Yamaguchi, 755-8505 Japanb
Center for Gene Research, Yamaguchi University, Japanc
Department of Surgical Pathology, Yamaguchi University Hospital, Japan

ARTICLE INFOABS T R AC T

Article history:

Inflammatory responses have been known to contribute to the development of neuronal

Accepted 29 December 2008

damage after brain ischemia in experimental animals. Also, neutrophil elastase activity inthe plasma has been elevated in the patients with acute cerebral infarction. In order to clarify
whether neutrophil elastase distributes into the brain parenchyma and exacerbates

Keywords:

neuronal damage following ischemia, we examined the effects of specific neutrophil

Neutrophil elastaseelastase inhibitor, ONO-5046, on hippocampal CA1 neuronal death in relation to neutrophil

Neutrophil elastase inhibitor

elastase activity in the plasma and its distribution in the brain and to caspase-3/7 activity.

Forebrain ischemia

ONO-5046 (5 and 10 mg/kg) or saline (control group) was administrated after 8 min of

Caspaseforebrain ischemia in rats. Ratio of surviving neurons (median, [range]) in hippocampal CA1

Hippocampal CA1seven days after ischemia was significantly higher in the ONO-5046 5 mg/kg (31% [12–57%]),

Neuronal deathand 10 mg/kg groups (69% [39–76%]) than in the control group (3.2% [0–10%]). Plasma
neutrophil elastase activity in the ONO-5046 10 mg/kg group was significantly lower than in
the control group (14 [11–25] vs. 41 [35–68] nmol/ml). Neutrophil elastase distributed in theextracellular space in the hippocampal CA1 neuronal layer in the control group, while, in the
ONO-5046 10 mg/kg group, trace of neutrophil elastase was detected only in the endothelium.
Caspase-3/7 activity was elevated after ischemia over 8 h in the control group, while, in the
ONO-5046 10 mg/kg group, no elevation was observed. The results suggest that neutrophil
elastase may contribute to neuronal death in hippocampal CA1 following forebrain ischemia
and that neutrophil elastase inhibitor attenuates neuronal death.
© 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Transient global cerebral ischemia causes delayed neuronal
death in vulnerable regions such as hippocampal CA1. A

complex series of events has been suggested as the mechanisms, including excessive release of glutamate, accumulationof intracellular calcium, formation of free radicals anddegradation of membrane lipids (Siesjö, 1988; Siesjö and⁎ Corresponding author. Fax: +81 836 22 2292.
E-mail address: takefumi@yamaguchi-u.ac.jp (T. Sakabe).
Abbreviations: MABP, mean arterial blood pressure; ANOVA, analysis of variance; IL, interleukin; TNF, tumor necrosis factor;
MMP, matrix metalloproteinase
0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2008.12.070

Please cite this article as: Matayoshi, H., et al., Neutrophil elastase inhibitor attenuates hippocampal neuronal damage after
transient forebrain ischemia in rats, Brain Res. (2008), doi:10.1016/j.brainres.2008.12.070

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Table 1 – Physiological variables for the histopathological outcome study

Pre-ischemia
Control
ONO-5046 5 mg/kg
ONO-5046 10 mg/kg
Post-ischemia
Control
ONO-5046 5 mg/kg
ONO-5046 10 mg/kg

pH

PaCO2 (mmHg)

PaO2 (mmHg)

Hb (g/dl)

Glucose (mg/dl)

Temperature (°C)

7.36 ± 0.02
7.36 ± 0.03
7.35 ± 0.04

41 ± 241 ± 241 ± 2108 ± 4108 ± 5109 ± 712.4 ± 1
13.4 ± 1
12.9 ± 1

119 ± 17125 ± 14116 ± 1037.0 ± 0.2
37.0 ± 0.1
37.0 ± 0.1

7.35 ± 0.04
7.36 ± 0.04
7.37 ± 0.02

42 ± 241 ± 340 ± 3110 ± 6115 ± 9112 ± 611.9 ± 1
12.7 ± 1
11.5 ± 1

127 ± 14135 ± 15126 ± 1037.1 ± 0.2
37.1 ± 0.2
37.0 ± 0.1

Values presented mean ± SD. Post-ischemic values were obtained 30 min after forebrain ischemia. There were no significant differences inpre- and post-ischemic values in each group and no differences among three groups.

Bengtsson, 1989). In addition, the contribution of proinflammatory cytokines (e.g., IL-1β, IL-6, TNFα) to delayed neuronal
death has been suggested (Saito et al., 1996). Neutrophil
elastase derived from activated neutrophils following inflammation or various stresses degrades a broad spectrum of
extracellular matrix and cell surface proteins (Janoff, 1985;
Mainardi et al., 1980; McDonald and Kelley, 1980). Intracerebral
injection of neutrophil elastase has been shown to disrupt
blood–brain barrier and to produce brain damage withintraparenchymal hemorrhage (Armao et al., 1997). It is ofgreat interest that plasma neutrophil elastase levels were
found to be increased in the patients with acute cerebral
infarction (Iwatsuki et al., 1998). Furthermore, recent animalstudies reported that specific neutrophil elastase inhibitor,
ONO-5046, (Sodium N-{2-[4-(2, 2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl} aminoacetate tetrahydrate), has aprotective effect against central nervous system damage
induced by ischemia (Shimakura et al., 2000; Yamauchi et al.,2006) and trauma (Tonai et al., 2001), suggesting that neutrophil elastase plays an important role in the development ofcentral nervous system damage. Thus, elucidation of the roleof neutrophil elastase in developing ischemic neuronaldamage and its modification by elastase inhibitor may provide
an important therapeutic strategy in cerebral ischemia. We
hypothesized that neutrophil elastase activated in plasma
following brain ischemia exacerbates neuronal damage by
distributing in the brain parenchyma. In the present study, wesought to investigate the involvement of inflammatory
responses in aggravating selective neuronal death in the
hippocampal CA1 following forebrain ischemia. We focused,in particular, on whether activation of neutrophil elastase andits distribution in the brain parenchyma is produced andwhether pharmacological inhibition of activated neutrophil
elastase attenuates neuronal death. Because the nature of theselective neuronal death in hippocampal CA1 is characterized,
though not exclusively, by apoptotic cell death, we measured
the chronological changes of caspase-3 activity which has been
known to be involved in apoptotic signaling (Cao et al., 2002).

2.

Results

2.1.

Physiological variables for pre- and post-ischemia

In all series of experiments, pericranial temperature wasmaintained close to 37 °C throughout the experiments. Meanarterial blood pressure (MABP) as well as PaCO2 and PaO2
was maintained in the normal ranges (except for MABP
during ischemia). Tables 1 and 2 show physiologicalvariables (pH, PaCO2, PaO2, hemoglobin, glucose, or pericranial temperature) and MABP in each group for the histopathological outcome study. There were no significant
differences in physiological variables and MABP among thethree groups. The physiological variables in another series ofstudies were comparable to those of the histopathologicaloutcome study (data not shown).

2.2.
Histopathological outcome 7 days after forebrainischemia
Fig. 1 shows the representative microphotographs of the
hippocampus in each group (7 days after ischemia) andpercentage of surviving neurons in each group is illustrated
in Fig. 2. Hippocampal CA1 neurons were selectively andextensively damaged in the control group, exhibiting only
limited number of normal neurons (3.2% [0–10%] (median[range]) of those in the age matched normal rats). Pyramidal
neuronal shrinkage and chromatin condensation of nuclei aswell as reactive gliosis were observed. In the ONO-5046
5 mg/kg and 10 mg/kg groups, neuronal damage wassignificantly less compared with that in the control group,
exhibiting normal neurons 31% [12–57%], and 69% [39–76%](p < 0.001), respectively. Administration of 10 mg/kg ONO-5046
most strongly attenuated hippocampal CA1 neuronal damage
induced by transient forebrain ischemia.

2.3.
Time course of neutrophil elastase activity in theplasma during reperfusion
Time course of neutrophil elastase activity in the plasma
after ischemia in the control and the ONO-5046 10 mg/kg
groups is shown in Fig. 3. In the control group, neutrophil
elastase activity was time-dependently elevated over
60 min. After peaking at 60 min (41 [35–68] nmol/ml(median [range]) vs. baseline), the activity was declined tobaseline at 4 and 8 h (0 [0–1] and 0 [0–4] nmol/ml,
respectively). In the ONO-5046 10 mg/kg group, neutrophil
elastase activity showed only slight insignificant increase at
60 min (14 [11–25] nmol/ml) and decreased to the baseline
values at 4 and 8 h (0 [0–7] and 0 [0–15] nmol/ml,
respectively) after ischemia. Neutrophil elastase activity at
60 min after ischemia in the ONO-5046 10 mg/kg group was

Please cite this article as: Matayoshi, H., et al., Neutrophil elastase inhibitor attenuates hippocampal neuronal damage after
transient forebrain ischemia in rats, Brain Res. (2008), doi:10.1016/j.brainres.2008.12.070

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Table 2 – Mean arterial blood pressure (MABP, mmHg) forthe histopathological outcome study
Preischemia
Control
ONO-5046
5 mg/kg
ONO-5046
10 mg/kg

Ischemia

Postischemia

4 min

8 min

97 ± 999 ± 747 ± 246 ± 247 ± 247 ± 399 ± 898 ± 799 ± 746 ± 247 ± 299 ± 9There were no significant differences in MABP among three groups.

significantly lower than in the control group (14 [11–25] vs.
41 [35–68] nmol/ml, p = 0.009).

2.4.

Distribution of neutrophil elastase in the brain

Fig. 4 shows the distribution of immunohistochemical
staining for neutrophil elastase in the area of hippocampus.
In the control group (ischemia), neutrophil elastase immu-

3

nostaining (intense brown-staining compared to the shamgroup (Figs. 4A, a)) was detected in endothelial cells and inperi-vascular space (Fig. 4c), but not in the hippocampal
neuronal layer (Fig. 4C) at 4 h after ischemia. At 8 h, diffuse
positive staining, as compared with negative control (Figs.
4B, b), was observed in the extracellular space adjacent tohippocampal CA1 neurons (Figs. 4D, d). In the ONO-5046
10 mg/kg group (Ischemia + ONO), elastase staining was
detected only in endothelial cells, not in the peri-vascularspace at 4 h (Fig. 4e) and not in the extracellular space
adjacent to hippocampal CA1 neurons at 8 h (Fig. 4f).

2.5.
Time-course changes in caspase-3/7 activity inthe hippocampus
Fig. 5 shows the changes in caspase-3/7 activity in the
hippocampal CA1 region after ischemia. In the control group,
caspase-3/7 activity was significantly elevated at 8 h (145%[114–190%] (median [range]) of baseline, p = 0.003) after
ischemia. In the ONO-5046 10 mg/kg group, no elevation of
caspase-3/7 activity was observed and the activity at 8 h (75%Fig. 1 – Histopathological outcome in hippocampal CA1 (at −3.8 mm from bregma) after forebrain ischemia (8 min)–reperfusion(7 days). Light microphotographs of the hippocampus with hematoxylin-eosin stain in the control group (A and a),
ONO-5046 5 mg/kg group (B and b) and ONO-5046 10 mg/kg group (C and c). Panels (A and a), control group: selectively andextensively damaged neurons in CA1 are observed. Pyramidal neuronal shrinkage and chromatin condensation of nuclei aswell as reactive gliosis is observed (a). Panels (B and b), ONO-5046 5 mg/kg group: Neurons with shrinkage and chromatincondensation with normal surviving neurons in pyramidal cell layer are observed. Panels (C and c), ONO-5046 10 mg/kg group:
neurons in pyramidal cell layer of hippocampal CA1 are markedly preserved. Arrows indicate the area examined with ahigh power micrograph. (Scale bar: A–C, 500 μm; a–c, 100 μm).
Please cite this article as: Matayoshi, H., et al., Neutrophil elastase inhibitor attenuates hippocampal neuronal damage after
transient forebrain ischemia in rats, Brain Res. (2008), doi:10.1016/j.brainres.2008.12.070

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[66–116%], p = 0.006) was significantly lower than that of thecontrol group.

3.

Fig. 2 – Histopathological outcome in hippocampal CA1 after
forebrain ischemia (8 min)–reperfusion (7 days). Y axis
represents ratio of surviving neurons (percentage of normal
neurons in hippocampal CA1 in each group as referenced tothose of the age matched normal rats). Box plots show themedian (middle line of box), 25th–75th percentiles (box) andminimum/maximum values (lower/upper whiskers). In the
ONO-5046 5 mg/kg and 10 mg/kg groups, neuronal damage issignificantly less compared with the control group.
ONO-5046 10 mg/kg administration leads to a greaterattenuation of ischemic neuronal damage compared to thatwith 5 mg/kg administration. (*p < 0.01 vs. control group,#p < 0.01 vs. ONO-5046 5 mg/kg group, respectively).

Discussion

In the present study, we demonstrated that a specificneutrophil elastase inhibitor, ONO-5046, ameliorated
ischemic neuronal damage in hippocampal CA1 7 days after
8-min forebrain ischemia in rats. Ameliorating effect was
associated with an inhibition of activated neutrophil elastasein the plasma and distribution in the brain parenchyma as
well as an inhibition of caspase-3/7 activity. These results
suggest that elastase contributes to the development ofischemic neuronal damage in hippocampal CA1 induced bytransient forebrain ischemia.
There have been a few reports regarding the protective
effects of neutrophil elastase inhibitor, ONO-5046, in various
models of central nervous system damage (Shimakura et al.,
2000; Tonai et al., 2001; Yamauchi et al., 2006). Shimakura et al.
(2000) first demonstrated in focal cerebral ischemia, inducedby occlusion of middle cerebral artery (MCA), that ONO-5046,
given just after occlusion of MCA or from 3 h after ischemia,
reduced brain damage (as evaluated by the size of damaged
area and water content) 24 h following ischemia, while notaffecting the accumulation of neutrophils. Attenuating effectsof ONO-5046 have also been reported in spinal cord damage
induced either by ischemia (Yamauchi et al., 2006) orcompression (Tonai et al., 2001). Our finding that ONO-5046
attenuated neuronal damage is in accordance with thesereported results, despite the different experimental models.
Shimakura et al. (2000) suggested that the protective

Fig. 3 – Time-course changes of neutrophil elastase activity in plasma during reperfusion. Y axis represents neutrophil
elastase activity, quantified by spectrophotometrical analysis. Box plots show the median (middle line of box), 25th–75th
percentiles (box) and minimum/maximum values (lower/upper whiskers). In the control group (C), neutrophil elastaseactivity was significantly elevated at 60 min. In the ONO-5046 10 mg/kg group (ONO), elastase activity showed only slightinsignificant increase at 60 min and decreased to the baseline values at 4 and 8 h. At 60 min after ischemia, neutrophil elastaseactivity in the ONO-5046 10 mg/kg group (ONO) was significantly lower than in the control group. (*p < 0.01 vs. baseline,#p < 0.01 vs. control group, ○ = outliers (outside threefold of the whole box length).
Please cite this article as: Matayoshi, H., et al., Neutrophil elastase inhibitor attenuates hippocampal neuronal damage after
transient forebrain ischemia in rats, Brain Res. (2008), doi:10.1016/j.brainres.2008.12.070

ARTICLE IN PRESSBR AIN RE S EA RCH XX ( 2 0 08 ) XXX –X XX

5Fig. 4 – Distribution of neutrophil elastase in the brain. Panels (A and a), sham group. Panels (B and b), negative control(reperfusion 8 h after ischemia), shows lack of neutrophil elastase staining in peri-vascular area (B) and in the hippocampal
neuronal layer (b). Panels (C and c), 4 h after ischemia in the control group: immunohistochemical staining is observed in theendothelial cell and peri-vascular area (c, arrow), not in the hippocampal neuronal layer (C). Panels (D and d), 8 h after ischemia
in the control group: diffuse staining is observed in the extracellular space adjacent to hippocampal CA1 neurons (d, arrow).
Panels (E and e), 4 h after ischemia in ONO-5046 10 mg/kg group: elastase staining is detected in endothelial cells, not in theperi-vascular space (e, arrow). Panels (F and f), 8 h after ischemia in ONO-5046 10 mg/kg group: elastase staining localized
within endothelial cells (F, arrow), not in the extracellular space adjacent to hippocampal CA1 neurons (f). Panels (a–f)
respectively show high power magnification of the boxed area in corresponding low power picture. All photographs were taken
under same setting (shutter speed; 1/40 under low-magnification and 1/30 s under high-magnification view). (Scale bar: A–F,
100 μm; a–f, 25 μm).
Please cite this article as: Matayoshi, H., et al., Neutrophil elastase inhibitor attenuates hippocampal neuronal damage after
transient forebrain ischemia in rats, Brain Res. (2008), doi:10.1016/j.brainres.2008.12.070

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Fig. 5 – Caspase-3/7 activity (% sham) in the hippocampal
CA1 region after ischemia. The values are expressed aspercent of those values obtained in the normal (sham) rats(% sham). Box plots show the median (middle line of box),
25th–75th percentiles (box) and minimum/maximum
values (lower/upper whiskers). In the control group (C),
caspase activity was significantly elevated at 8-hour
reperfusion. In the ONO-5046 10 mg/kg group (ONO),
caspase activity was significantly inhibited at 8-hour
reperfusion. (*p < 0.01 vs. sham group, #p < 0.01 vs. control
group, respectively).

mechanism of ONO-5046 against ischemic damage may berelated to an inhibition of various profiles induced by
infiltrating neutrophils, such as an adherence of neutrophils
to the endothelium, production of toxic metabolites, proinflammatory cytokines and elastase, or activation of phospholipase. In the spinal cord compression model (Tonai et al.,2001), protective effect was suggested to be the result ofblocking cytokine-induced neutrophil chemo-attractant-1
mRNA and protein, preventing neutrophil activation–infiltration and vascular endothelial injury. It has been known that,in addition to proinflammatory cytokines (Owen et al., 1997),
reactive oxygen species (Demopoulos et al., 1980) andproteolytic enzymes (Taoka et al., 1997) were produced with
infiltrated leukocytes, which all can exacerbate ischemic
neuronal damage. However, in the transient global forebrainischemia model used in the present study, characteristic
neuronal damage is selective and delayed neuronal death inhippocampal CA1 without recruitment of hematogenous
inflammatory cells (Stoll et al., 1998). In the present study,
significant increase in activated neutrophil elastase in theplasma and its distribution in the brain parenchyma wasobserved in the control group. A specific neutrophil elastase
inhibitor, ONO-5046 (Kawabata et al., 1991) inhibited plasma
elastase activity and elastase presence in the extracellularmatrix after ischemia. It is therefore suggested that neutrophil
elastase contributes to the development of ischemic neuronaldamage by elastase distribution in the matrix component.
Armao et al. (1997) demonstrated that intracerebral injectionof neutrophil elastase disrupted blood–brain barrier andprovoked brain tissue damage with intraparenchymal hemorrhage. Our immunohistochemical examination showed neutrophil elastase in the endothelium of hippocampal vessels,peri-vascular regions and even in parenchyma in the hippocampal CA1 region. Although the nature of brain damage
differs between the studies by Armao et al. (1997) and ours, it ispossible that neutrophil elastase, when transferred into
parenchyma, can exacerbate ischemia-induced brain damage.
The present study is the first to demonstrate in forebrainischemia that neutrophil elastase inhibitor, ONO-5046, significantly ameliorates neuronal damage in hippocampal CA1,
when given immediately after reperfusion.
The mechanism for delayed neuronal death has beenargued from the intracellular signaling pathways such as
glutamate acting via N-methyl-D-aspartate receptors, disintegration of cellular ion metabolism with subsequent intracellular calcium accumulation or caspase-mediated apoptosis(MacManus et al., 1993; Siesjö, 1988; Siesjö and Bengtsson,1989). Interestingly, in a recent study in spinal cord ischemia,
Yamauchi et al. (2006) reported that attenuation of delayed
motor neuron death with ONO-5046 was accompanied by an
inhibition of caspase-3 expression. Our result that attenuationof neuronal death in hippocampal CA1 with ONO-5046 wasaccompanied by an inhibition of caspase-3/7 activity is inaccordance with the result reported by Yamauchi et al. (2006)
in spinal cord ischemia. The present study demonstrated thatthe elevation of caspase-3/7 activity in hippocampal CA1 in
untreated (control) rats was accompanied by expression ofneutrophil elastase not only in the peri-vascular space but also
into the extracellular space in the neuronal layer of hippocampal CA1. In contrast, in the rats treated with ONO-5046, no
staining was detected for neutrophil elastase in the extracellular space and caspase-3/7 activity was significantly
inhibited at 8 h after ischemia. The attenuation of neuronal
death in hippocampal CA1 with a specific neutrophil elastase
inhibitor ONO-5046 was accompanied by an inhibition of
caspase-3/7 activity and by inhibition of neutrophil elastaseactivity in the plasma and of its distribution in the brain
parenchyma. Although it is undetermined whether thedecreased activities of caspase-3/7 is the cause of or a resultof attenuated damage, the results of the present study
indicate that activation of neutrophil elastase is involved in
the development of ischemic neuronal damage in hippocampal CA1 induced by transient forebrain ischemia. Neutrophil
elastase is likely to operate both directly and indirectly inmodulating fibrotic extracellular matrix at different levels(Chua and Laurent, 2006). Matrix metalloproteinase-9 (MMP-9)
was suggested to be representative measurements of extracellular proteolysis. Our preliminary investigation showedthat MMP-9 activity at 1 day-reperfusion (n = 4, in each group)in the ONO-5046 group tended to be decreased compared tothe control group (see Supplementary Fig. 1).
There are some limitations of the present study. First,
because the neutrophil elastase inhibitor was given immediately after reperfusion, clinical relevance is limited. Second,
the assessment of neuropathological outcome 7 days after
ischemia may not be long enough and the possibility that the
drug may only delay neuronal injury cannot completely be
excluded. Third, current experimental design is not sufficient
to claim the source of the elastase present in the ischemic
brain because the possibility that microglia can secrete
elastase-like protease (Nakajima et al., 1992). Although theselimitations need to be addressed in the future work, thePlease cite this article as: Matayoshi, H., et al., Neutrophil elastase inhibitor attenuates hippocampal neuronal damage after
transient forebrain ischemia in rats, Brain Res. (2008), doi:10.1016/j.brainres.2008.12.070

ARTICLE IN PRESSBR AIN RE S EA RCH XX ( 2 0 08 ) XXX –X XX

current study suggests that activation of neutrophil elastase
following ischemia is involved in exacerbating selective
neuronal damage in hippocampal CA1.
In summary the results suggest that neutrophil elastasepossibly, but may not exclusively, derived from activatedleukocytes contributes to the development of selective
neuronal death in hippocampal CA1 after 8 min-forebrainischemia in rats and that a specific neutrophil elastase
inhibitor, ONO-5046, ameliorates neuronal damage via inhibition of neutrophil elastase distribution in the extracellularmatrix after ischemia. Further study may be needed todetermine the therapeutic time window for ONO-5046 andthe effects of higher or repeated doses as well as the detailed
mechanisms for interaction between neutrophil elastase andactivation of caspase.

7

4.

Experimental procedures

hemorrhagic hypotension. Before starting ischemia the following criteria were assured: PaO2 90–140 mmHg; PaCO2 35–
45 mmHg; pH 7.35–7.45; and blood glucose level 90–140 mg/dl.
Forebrain ischemia of 8 min duration was induced by themethod of Smith et al. (1984) with a slight modification (Hirata
et al., 2007). First, hypotension was induced by 0.01 mg ofphentolamine and then bilateral carotid arteries were
occluded with vascular clips and MABP was maintained at
45–50 mmHg by withdrawing or infusing blood using heparinized syringe (see Results, Table 2). After 8 min ischemia,
vascular clips were removed, and withdrawn blood wasinfused. The vascular catheters were removed and wounds
were sutured. Thereafter anesthesia was discontinued andanimals were extubated after recovery of spontaneous
respiration and placed in a temperature controlled room.
The transient forebrain ischemia model used in the present
study is the one that produces selective and delayed neuronal
death in hippocampal CA1 with no mortality.

4.1.

Drugs

4.4.

Sodium N-{2-[4-(2, 2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl} aminoacetate tetrahydrate (ONO-5046) was
kindly provided by Ono Pharmaceutical Co., Ltd. (Osaka,
Japan). The elimination half life [t1/2 (2–60 min), and t1/2 (240–480 min)] of ONO-5046 (evaluated as radioactivity) has beenreported to be 15.6 ± 1.8 and 164 ± 51 min, respectively after asingle intravenous administration of 10 mg/kg 14C-ONO-5046
in male rats.

4.2.

Animals and experimental design

The study protocol was reviewed and approved by theCommittee of the Ethics on Animal Experiment and carriedout under the control of Guidelines for Animal Experiment inYamaguchi University Graduate School of Medicine. All
experiments were carried out in male Wistar rats aged 8 to
9 weeks (280–330 g, Kyudo, Kumamoto, Japan) subjected to
8 min period of forebrain ischemia produced by bilateralcarotid artery occlusion and hemorrhagic hypotension. We
sought to evaluate neuroprotective effect of the post-ischemic
treatment with neutrophil elastase inhibitor, ONO-5046, inhippocampal CA1 and the mechanism for neuroprotection, if
any, in relation to neutrophil elastase activity in the plasma
and its distribution in the brain as well as to the temporalprofile of caspase activity.

4.3.

Forebrain ischemia

Fasted rats for 12 h were anesthetized with 3% isoflurane andtheir trachea intubated, and mechanically ventilated with agas mixture of 2% isoflurane in O2 and nitrous oxide (30:70). A
polyethylene catheter was placed into the tail artery forcontinuously monitoring blood pressure and for obtaining
arterial blood samples. Pericranial temperature (temporalis
muscle temperature) was monitored and maintained at close
to 37 °C throughout the experiments. Bilateral carotid arteries
were carefully isolated for later temporal occlusion. A vascularcatheter was placed via right external jugular vein with its tip
at the right atrium for drug administration and for inducing

Histopathological outcome study

Thirty six rats were randomly assigned to one of the following
three groups: control group, ONO-5046 5 mg/kg group, andONO-5046 10 mg/kg, respectively (n = 12 in each). Rats in thetreated groups received a single intravenous injection of
ONO-5046 at doses of 5 or 10 mg/kg, dissolved in 1 ml ofsaline, immediately after reperfusion. In the control group,
1 ml of saline was administered in the same manner. We
have attempted to evaluate the effect of three doses, 5 mg/kg,
10 mg/kg and 30 mg/kg. However, 30 mg/kg of ONO-5046 was
not adequately dissolved in 1 ml of saline. Seven days after
ischemia, the rats were reanesthetized with 4% isoflurane
and their brains were fixed by trans-cardiac perfusion withsaline containing heparin (4 U/ml) followed by 4% paraformaldehyde. Coronal sections (6 μm thickness) of the brain
embedded in paraffin including the dorsal hippocampus (atthe level of − 3.8 mm from bregma) were stained withhematoxylin and eosin (HE). The number of intact neurons
was counted in a blind fashion along the pyramidal layer ofthe hippocampal CA1 region for 1 mm using light microscopy.
The neuronal densities (cell/mm) obtained in both right andleft hemispheres were averaged and presented as thepercentage of the normal neurons referenced to those of theage-matched eight normal rats. Normal neurons were definedas the neurons with no shrinkage and no chromatincondensation of nuclei.

4.5.
Time-course changes in neutrophil elastase activity inthe plasma
In fifty rats, we investigated the time-course changes inneutrophil elastase activity after ischemia (15, 30, 60 min, 4,8-h of reperfusion, n = 5 at each time-point) in the control andONO-5046 10 mg/kg groups, respectively. Preparation andischemia protocol are the same as the first series of experiment. ONO-5046 10 mg/kg was selected because this dose
showed greater neuroprotective effect than with 5 mg/kg (seeResults, Fig. 2).
Blood samples (1 ml/rat) were obtained through the venous
catheter placed in the right atrium. Because the blood volume

Please cite this article as: Matayoshi, H., et al., Neutrophil elastase inhibitor attenuates hippocampal neuronal damage after
transient forebrain ischemia in rats, Brain Res. (2008), doi:10.1016/j.brainres.2008.12.070

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required for assay is rather large, only one time point
measurement for each rat was done. Plasma samples wereisolated from blood anticoagulated with EDTA by centrifugation (3000 ×g for 3 min) and were stored frozen. Assay ofneutrophil elastase activity was performed at the central
laboratory (Mitsubishi Chemical Medience Co. Ltd., Tokyo,
Japan). Briefly, neutrophil elastase activity was determinedwith the synthetic substrate N-methoxysuccinyl-Ara-Ara-ProVal p-nitroanilide, which is highly specific for neutrophil
elastase (Yoshimura et al., 1994). Samples were incubated in
0.1 M Tris–HCl buffer (pH 8.0) containing 0.5 M NaCl and 1 mM
substrate for 24 h at 37 °C. After incubation, p-nitroaniline wasmeasured spectrophotometrically at 405 nm, and absorbance,corrected for baseline activity, was used as an index ofneutrophil elastase activity.

5 mM MgCl2, 1 mM EGTA, and 1 μg/ml each pepstatin,
leupeptin, and aprotinin, and 1 mM phenylmethanesulfonyl
fluoride) with a Mixer Mill MM300 (Rescht, Haan, Germany) at
30 Hz for 30 s. The lysates were centrifuged (12 000 ×g, 15 min,4 °C), and the supernatants were used for protein determination. The protein concentrations of supernatant were adjusted
to 1 mg/ml with extraction buffer. The extracted protein(10 μg/ml) was added to equal volume of Caspase-Glo reagentsin a white-walled 96-well plate and incubated at room
temperature for 1 hour. The luminescence of each samples
was measured in a plate-reading luminometer (MicroLumat
LB 96P, EG and G Berthold, Bad Wildbad, Germany). The values
are expressed as percent of those values obtained in thenormal rats (sham group, n = 6).

4.8.
4.6.
brain

In two groups of rats (control and ONO-5046 10 mg/kg),
distribution of activated neutrophil elastase in the brain wasassessed at two time points, 4 and 8 h after ischemia (n = 6 ateach time-point). Sham group of rats (n = 6) were preparedwithout subjecting to ischemia. The brains were perfusedtranscardially with ice-cold Tris-buffered saline (TBS), pH 7.4,
followed with ice-cold 4% paraformaldehyde in TBS. The
brains were removed, immersed with the same fixative
overnight. The brains were cryoprotected in 30% sucrose in
PBS, and frozen with hexane cooled in liquid nitrogen.
Frozen coronal sections (10 μm thick) were prepared using acryostat. After blocking endogenous peroxidase with 0.3%hydrogen peroxide in methanol and blocking nonspecificbackground with TBS containing 3% normal rabbit serum,
sections were incubated overnight at 4 °C with neutrophil
elastase antibody (diluted in 1:100; Santa Cruz Biotechnology, Santa Cruz, CA). Detection of the primary antibody was
performed by the supersensitive Multilink-HRP/DAB detection kit (BioGenex, San Ramon, CA) using the biotin–
streptavidin-peroxidase technique, followed by counterstaining with hematoxylin. The staining procedures were carriedout using an automated immunostainer, Optimax plus
(BioGenex).

4.7.

Statistical analysis

Immunohistochemistry of neutrophil elastase in the
Physiological parameters analyzed for one-way ANOVA followed by post hoc Bonferroni–Dunn test were used to detectsignificant difference and were expressed as mean ± SD.
Statistical analysis for ratio of surviving neurons, elastaseactivity, and caspase-3/7 activity was performed by nonparametric test (Mann–Whitney U-test for two-independent samples and Kruskal–Wallis for multiple comparison) to
determine significance. Results were expressed as medians[range] and box plots were used to graphically display data
distributions. Differences were considered significant with
p value less than 0.01.

Acknowledgments
This work was supported, in part, by the Japan Society for the
Promotion of Science, Grant-in-Aid for Scientific ResearchNo. 18791080.

Appendix A. Supplementary data
Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.brainres.2008.12.070.

Time-course changes in caspase activity in the brain
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Please cite this article as: Matayoshi, H., et al., Neutrophil elastase inhibitor attenuates hippocampal neuronal damage after
transient forebrain ischemia in rats, Brain Res. (2008), doi:10.1016/j.brainres.2008.12.070