Neuroprotective effects of N-acetylcysteine via inhibition of matrix metalloproteinase in a mouse model of transient global cerebral ischemia

Journal Pre-proof Neuroprotective effects of N-acetylcysteine via inhibition of matrix metalloproteinase in a mouse model of transient global cerebral ischemia Yoon-Hyung Lee, Seong-Ryong Lee

PII:

S0361-9230(19)30316-8

DOI:

https://doi.org/10.1016/j.brainresbull.2019.10.004

Reference:

BRB 9786

To appear in:

Brain Research Bulletin

Received Date:

23 April 2019

Revised Date:

6 October 2019

Accepted Date:

15 October 2019

Please cite this article as: Yoon-Hyung L, Seong-Ryong L, Neuroprotective effects of N-acetylcysteine via inhibition of matrix metalloproteinase in a mouse model of transient global cerebral ischemia, Brain Research Bulletin (2019), doi: https://doi.org/10.1016/j.brainresbull.2019.10.004

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Neuroprotective effects of N-acetylcysteine via inhibition of matrix metalloproteinase in a mouse model of transient global cerebral ischemia

Yoon-Hyung Lee a,b, Seong-Ryong Lee a, *

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Department of Pharmacology, and ODR center, and Brain Research Institute, School of

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Medicine, Keimyung University, Daegu, 42601, South Korea Department of Urology, Fatima Hospital, Daegu, 42601, South Korea

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* Corresponding author: Seong-Ryong Lee, M.D., Ph.D.

Department of Pharmacology and ODR center,

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School of medicine, Keimyung University 1095 Dalgubeoldaeru, Dalseo-gu,

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Daegu, 42601, South Korea

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e-mail: [email protected]

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Highlights  MMP-9 destructs extracellular matrix protein resulting in anoikis in hippocampu s  NAC inhibits MMP-9 activity after global brain ischemia.  NAC reduces breakdown of extracellular matrix protein in hippocampus.  NAC protects ischemic neuronal damage after global brain ischemia.

ABSTRACT

N-acetylcysteine (NAC) is known to serve many biological functions including acting as an antioxidant, and electing antiinflammatory effects. Previous reports have revealed that NAC may have neuroprotective effects against the deleterious effects of brain ischemia. Despite of

this, the mechanism by which NAC prevents neuronal damage after brain ischemia remains unclear. The current study aimed to investigate this mechanism in a mouse model of transient global brain ischemia. In the present study, mice were subjected to 20 min of transient global brain ischemia, proceeded by intraperitoneal administration of NAC (150 mg/kg) in one group. The mice were then euthanized 72 h after this ischemic insult for collection of experimental tissues. The effect of NAC on neuronal damage and matrix metalloproteinase

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(MMP)-9 activity were assessed and immunofluorescence, and hippocampal terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay experiments were conducted and results compared between NAC- and vehicle-treated groups.

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damage was primarily observed in the hippocampal CA1 and CA2 regions. In NAC-treated

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mice, neuronal damage was significantly reduced after ischemia when compared to vehicle-

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treated animals. NAC also inhibited increased MMP-9 activity after global brain ischemia. NAC increased laminin and NeuN expression and inhibited increases in TUNEL-positive

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cells, all in the hippocampus. These results suggest that NAC reduces hippocampal neuronal

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damage following transient global ischemia, potentially via reductions in MMP-9 activity.

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Keywords: N-acetylcysteine; Global brain ischemia; Hippocampus; Matrix metalloproteinase

1. Introduction

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Transient global brain ischemia is often induced by profound hypotension, cardiac arrest,

resuscitation, or carbon monoxide intoxication, among other things. Transient global cerebral ischemia leads to selective neuronal death in vulnerable brain regions including the hippocampus and striatum (Kreisman et al., 2000; Nitatori et al., 1995). Although this selective sensitivity to ischemia has been recognized in many reports, the mechanisms that underlie this process remain unclear. Various mechanisms by which fundamental neuronal

death pathways may be engaged have been proposed to explain this phenomenon, including excitatory neurotoxicity, oxidative stress, neuronal apoptosis, and neuroinflammation (Lipton, 1999; MacManus et al., 1993; Pulsinelli et al., 1982).

In addition to the engagement of cell death pathways, extracellular matrix proteolytic enzymes such as, matrix metalloproteinases (MMPs) can degrade extracellular matrix protein

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and induce anoikis-like neuronal cell death (Gu et al., 2002). MMPs comprise an important family of proteolytic enzyme that are associated with the cellular basement membrane and are involved in extracellular matrix remodeling in both physiological and pathological processes in central nervous system processes (Mun-Bryce et al., 2002; Rosenberg, 1995).

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MMPs can modulate cell-to-cell and cell-to-extracellular matrix interactions.

Among the MMPs, MMP-2 and MMP-9, so-called gelatinases, have been implicated

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specifically in cerebral ischemia (Lo et al., 2002). In particular, MMP-9 has been reported to be involved in blood-brain barrier breakdown, brain hemorrhage, neuroinflammation, and

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neuronal death (Gu et al., 2002; Rosenberg 2002). In previous reports, MMP-9 deficient mice demonstrated decreased neural injury following brain trauma, focal brain ischemia, or global

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brain ischemia (Asahi et al., 2001; Lee et al., 2004; Wang et al., 2000). N-acetylcysteine (NAC), an acetylated amino acid, has high lipid solubility and can easily

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pass across biomembranes (Zhang et al., 2008). NAC increases levels of glutathione (GSH) in various cells and tissues (Zhang et al., 2008). NAC, a precursor of GSH, acts as an antioxidant and has anti-inflammatory, anti-apoptotic effects which subserve its neuroprotective role (Wang et al., 2007). NAC may also inhibit neuronal damage induced by ischemia (Khan et al., 2004; Krzyzanowaska et al., 2017). In addition, previous reports

indicate that NAC also has inhibitory effect on MMP activity in various tissues (Liu et al., 2017; Riegger et al., 2016).

The purpose of the present study was to investigate whether NAC would protect against

2. Materials and Methods 2.1. Animals and transient global brain ischemia model

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neuronal cell damage via inhibition of MMP after transient global brain ischemia.

Male, 10-week-old C57BL/6 mice (25-30 g, Koatec-Harlan, Pyungtaek, Korea) were

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maintained under controlled conditions (22±1°C, 12 h light/dark cycle). Food and water were

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available ad libitum. All procedures were performed with prior approval from Keimyung University School of Medicine's institutional animal care and use committee. To achieve

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transient global cerebral ischemia, mice were anesthetized with isoflurane anesthesia. The common carotid arteries were occluded bilaterally for 20 min using micro-clips. To avoid the

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influence of hypothermia during surgery, animal rectal temperature was monitored and maintained at 37 ± 0.5 C using a feedback thermoregulator (CMA 150, CMA Microdialysis,

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Sweden). 72 h following surgical introduction of global ischemia, mice were sacrificed for further experimentation.

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2.2. The measurement of the cerebral perfusion A laser Doppler flowmeter (5000 system, Perimed, Sweden) was used to monitor cerebral

cortical microperfusion. A glass-fiber sensor tip was attached via cement at its point (4 mm lateral and 2 mm posterior to the bregma) to the skull surface. Only mice that showed less than 15% of baseline control microperfusion during the first minute of occlusion were used in subsequent experiments.

2.3. Drug administration NAC (Sigma, Korea) was dissolved in a normal saline and injected intraperitoneally (150 mg/kg) 30 min after the surgical induction of ischemia. This was followed by once-daily injections until sacrifice. According to the previous report (Cao et al, 2012), we used 150 mg/kg as a drug dosage of NAC. In control animals, NAC injections were replaced, in equal

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volume and according to the same schedule, with sterile saline.

2.4. Histological examination

72 h following the induction of ischemia, experimental animals were deeply anesthetized

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using ether. After opening the thoracic cage, they were intracardially perfused with

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phosphate-buffered saline (PBS, pH 7.2) injected via a syringe through the left ventricle. Frozen brains were then sectioned at a thickness of 14-μm using a cryostat. Coronal brain

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sections were fixed in a 10% formalin solution for approximately 1 hr and then stained with hematoxylin and eosin (H & E) dyes.

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The CA 1 and 2 regions of the hippocampus were examined the degree of neuronal damage to these regions. In making these assessment, we measured damage in the more

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severely affected cerebral hemisphere. Using the previous method, we performed a semiquantitative approach to assessing the number of damaged neuronal cells according to a four-

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point scale. Points were assigned as follows: 0 points (no ischemic injury to pyramidal neurons in the CA1 and CA2), 1 point (ischemic injury to less than 20% of pyramidal neurons in the CA1 and CA2), 2 points (ischemic injury to 20-50% of pyramidal neurons in the CA1 and CA2), 3 points (ischemic injury to more than 50% of pyramidal neurons in the CA1 and CA2), and 4 points (broad ischemic injury to the CA1 and CA2 as well as throughout the hippocampus) (Tsuchiya et al., 2002).

2.5. Gelatin gel zymography (SDS-PAGE zymography) At 8 h, 1 d, or 3 d after the global ischemic insult, mice were deeply anesthetized for sampling. After transcardial perfusion with ice-cold PBS, the brain was removed from the skull and the hippocampus was dissected out on ice condition and then immediately frozen (80 °C). The hippocampus was then homogenized, using a solution including protease

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inhibitors cooled with ice. After centrifugation, the supernatant was obtained and total protein assessed by Bradford assay (Bio-Rad, USA). Following this, 40 μg of protein was combined with sample buffer and then decoupled using a 10% Tris-glycine gel (0.1% gelatin) via

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electrophoresis. The gel was incubated for 24 h at 37 °C. Coomassie Blue R-250 (0.5%) was

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used to stain the gel for 30 min and then was bleached appropriately.

2.6. In situ zymography

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While an in situ zymography cannot be used to differentiate between MMP-2 and MMP-9 activity in fresh tissue, this technique may be used to assess the activity and location of these

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enzymes (Oh et al., 1999). Following transcardial perfusion with PBS, the brain was removed promptly from the skull without fixation. The brain was then rapidly frozen in 2-

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methylbutane solution with liquid nitrogen. Frozen fresh brain samples were then sectioned using a cryostat at a thickness of 14 μm. Sections were then processed using an Enz-Check

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kit (Molecular Probe, USA) at 37 C for approximately 18 hours, after which we conducted fluorescent microscopy assessments. The activity of gelatinase in the tissue was measured via fluorescent microscopy and fluorescein isothiocyanate (FITC) signals, which are emitted when FITC-conjugated gelatin is degraded by the enzymes. This technique was used to determine the activity of gelatinase across hippocampal regions. The relative intensity of

FITC fluorescent signals in the medial portion of CA1 and the total CA2 were examined because both regions were found to be sensitive to transient global cerebral ischemia in our model. Signal analyses were performed according to previously reported method (Hong et al., 2012). In brief, in situ zymography micrographs were captured by a Leica DM 3000 microscope coupled to a CCD camera (Leica DFC 480). Fluorescence quantification was then performed using image analyzing software (Carl Zeiss LSM Image Examiner version

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4.2.0.121) by a blind examiner. Data are presented as a percentage of FITC fluorescent intensity.

2.7. Immunofluorescence

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Mice were sacrificed 72 h after the induction of global cerebral ischemia. Following deep

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anesthesia with isoflurane, animals were intracardially perfused with ice-cold PBS followed by 4% paraformaldehyde. Brains were then dissected out and fixed in 4% paraformaldehyde

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at 4 C overnight. This was followed by cryoprotective treatment with a 15% and then 30% sucrose solution, also at 4 C. Tissue sections were prepared a thickness of 14 μm using a

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cryostat and were mounted on the coated slides. Sections were then preserved at -80 C for further use. A PBS-based blocking solutions containing 0.2% Triton X-100 and 3% normal

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goat serum was applied to tissues. Slide-mounted tissues were then treated with primary antibodies including anti-laminin (1:60, Sigma, Korea) and anti-Neuronal Nuclei (NeuN)

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(1:100, Millipore, USA) at 4 C for 24 h. The slides were then washed with PBS, and treated with secondary antibodies including anti-rabbit FITC, 1:200 (Jackson ImmunoResearch, USA), and anti-mouse tetramethylrhodamine-5-(and-6)-isothiocyanate (TRITC), 1:100 (Jackson ImmunoResearch, USA) for 30 min. Tissues were then assessed on the microscope.

2.8. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)

staining To examine apoptotic processes resulting from ischemic neuronal cell damage, we performed TUNEL staining using an in situ cell death detection kit (Roche Diagnostic, USA) according to the manufacturer’s instructions. Following surgery to induce ischemia, animals were transcardially perfused using ice-chilled PBS with a syringe through the left ventricle of the heart. Following removal of the brain from the skull, tissues were promptly frozen using 2-methylbutane and liquid nitrogen. The brains were then sectioned via a cryostat at a

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thickness of 14 μm. Coronal brain section were then fixed using a 10% formalin solution and washed with PBS three times. Slide-mounted brain sections were then treated with the TUNEL reaction solution and then reacted with diaminobenzidine substrate solution. Samples

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were then imaged using a light microscope and TUNEL staining was assessed by a blinded

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examiner.

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2.9. Statistical analysis

All data in the present study are expressed as mean ± S.E. Statistical analyses were

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performed using an one-way analysis of variance followed by Bonferroni's post-hoc analysis or an unpaired Student’s t-test (comparison between single pairs of vehicle and NAC- treated

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samples). A P-value of < 0.05 was considered statistically significant.

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3. Results

3.1. Effect of NAC on hippocampal neuronal injury following transient global cerebral ischemia

Histological assessments of H&E staining were suggestive of greater ischemic injury among pyramidal neurons in the CA1 and CA2 regions of the hippocampus in cerebral ischemia animals than in sham-operated animals. Findings from these assessments included

neuronal atrophy and distortion, and neuronal cell loss. The degree of injury was also found to be markedly decreased in experimental animals to whom NAC had been administered (Fig. 1). Damage was evaluated using a semi-quantitative method. In the sham-operated group, there was a lack of cell damage. As compared to vehicle-treated animals (n=10), neuronal damage was significantly decreased in the CA1 and CA2 regions among NAC-treated

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animals (n=11) (P < 0.05) (Fig. 1G & 1H).

3.2. Effect of NAC on gelatinase (MMP-9 & MMP-2) activity following transient global cerebral ischemia

Very low levels of the active form of MMP-9 (97 kDa) and the latent form of MMP-2 (72

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kDa) were observed in the hippocampi of sham-operated animals. However, after transient

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global brain ischemia, MMP-9 was increased in the vehicle-treated animals over time and comparison sham-operated animals, increase in MMP-9 became statistically significant by 1

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d after global ischemic insult (Fig. 2A & 2B). For assessment of the effect of NAC treatment on MMP-9 enzyme, mice were killed 72 h after transient global brain ischemia and the

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increases of MMP-9 were significantly decreased in the NAC-treated animals (P < 0.05) (Fig. 2C & 2D). Although both MMP-2 and MMP-9 may contribute to gelatinase activity, MMP-2

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(72 kDa) was not remarkably changed after ischemia nor did NAC significantly influence MMP-2 activity in our model (Fig. 2C).

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3.3. Effect of NAC on in situ gelatinase activity following transient global cerebral ischemia Three days following surgery to induce cerebral ischemia, we assessed the activity of

MMP in the hippocampus. MMP activity was increased, primarily in the hippocampus, following induction of ischemia. In addition, gelatinase activity seemed also to be localized to the hippocampal region, and especially to CA1 and CA2, upon histological observation. NAC-treated animals' CA1 and CA2 regions exhibited significantly decreased gelatinase

enzyme activity compared to controls (P < 0.05) upon quantification of in situ gelatinase via fluorescent intensity (Fig. 3I & 3J). Furthermore, to confirm whether NAC directly inhibits gelatinase activity, NAC (1 mM) was added to the incubation reaction solution for in situ zymography of fresh, post-ischemic hippocampal tissues (Fig. 4). Reduced gelatinase activity following NAC application was observed (Fig. 4C & 4D).

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3.4. Effect of NAC on NeuN and laminin expression following transient global cerebral ischemia

In the control, proteins such as laminin and NeuN were normally expressed in the hippocampus. Following transient global cerebral ischemia, however, laminin expression was

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reduced and NeuN expression was also reduced in the hippocampal CA1 and CA2. That is,

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ischemia produced the breakdown of laminin and loss of neuronal cells. These reductions

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were both inhibited by NAC administration to the CA1 (Fig. 5A) and CA2 (Fig. 5B).

3.5. Effect of NAC on TUNEL staining following transient global ischemia

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As demonstrated histologically, TUNEL-positive neuronal cells were present even in control animals' hippocampal CA1 and CA2 (Fig. 6). This indicated that normative apoptosis

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occurs in both regions. Furthermore TUNEL-positive neuronal cells area corresponded to that revealed by H & E staining. In the NAC-treated group, the number of TUNEL-positive

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neuronal cells was markedly reduced in the CA1 and CA2 (P > 0.05, Fig. 6G & 6H, respectively).

4. Discussion Our results demonstrated that NAC, a potent anti-oxidant and an anti-inflammatory agent,

reduced neuronal injury due to transient global cerebral ischemia in a mouse model. The protective effect of NAC appears to be due to its ability to attenuate the activation of gelatinase, such as MMP-9, following ischemic insult. We further demonstrate that this protective effect is based on decreased MMP activity that is otherwise increased following ischemic insult.

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Hippocampal pyramidal cells are highly sensitive to transient global ischemic injury (Ito et al., 1975; Kreisman et al., 2000; Pulsinelli et al., 1982). Various mechanisms have been proposed which might underlie the relationship between neuronal injury and global cerebral ischemia including excitotoxicity, free radical injury, and apoptosis (Chen et al., 1996; Choi,

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1996; Endoh et al., 1994). Additionally, transient global ischemia results in delayed

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hippocampal neuronal cell death where apoptosis, rather than necrosis, which is usually seen in focal ischemia, is most evident (MacManus et al., 1993; Nitatori et al., 1995). Several

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reports have concluded that the breakdown of extracellular matrix component is a further mechanism underlying apoptotic neuronal death in brain ischemia (Tsirka et al., 1997;

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Zalewska et al., 2002).

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MMP has been reported to play an essential role in the pathophysiology of cerebral ischemia (Gidday et al., 2005; Lo et al., 2002; Rosenberg, 1995). It is further known that

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gelatinase, MMP-2 and MMP-9 play critical roles in the regulation of brain ischemia's effects on the brain. In a murine model, various types of MMPs, including MMP-9, have also been identified as playing an essential role in the mediation of these processes (Lo et al., 2002). It has also been reported, however, that ischemic neuronal death is not inhibited in MMP-2 knock-out (KO) mice (Asahi et al., 2001). In our study, there were significant increase in MMP-9 activity of gelatin gel zymography after transient global brain ischemia. However,

there were no significant changes in MMP-2 activity after transient global brain ischemia. Given these conflicting findings, the precise role of MMP-2 in brain ischemia remains controversial.

Another possible mechanism by which cellular injury might occur following global cerebral ischemia involves edema or bleeding due to the destruction of blood-brain-barrier

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(BBB). If this is the case, the role of MMP in BBB maintenance and response to injury should be assessed as it may mediate the effects of ischemia on hippocampal neuron death. One possibility is that MMP is involved in triggering anoikis-like cell death by interfering with the interaction between the cells and the extracellular matrix. Anoikis is defined as

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apoptosis due to the degradation of extracellular matrix materials. In addition, it has also been

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reported that the MMP-9-mediated degradation of the extracellular matrix has been reported to plays a key role in neuronal apoptosis (Gu et al., 2002; Lee et al., 2008). Presumably, these

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mechanisms might also be involved in the current experimental study. Further studies are

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warranted, however, to examine the process of anoikis in this model.

Antioxidants, such as NAC, may help to prevent apoptotic processes following cerebral

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ischemia. NAC has known neuroprotective effects, in addition to various pharmacological effects (Naziroglu et al., 2013; Yang et al., 2012). According to previous reports, NAC likely

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has a protective effect against the neuronal damage induced by brain ischemia. These neuroprotective effects have primarily been studied in the context of animal models of focal cerebral ischemia, however (Khan et al., 2004; Krzyzanowska et al., 2017). To date, only a negligible number of studies have examined the biological effects of NAC on the neuronal injury induced by transient global cerebral ischemia. Moreover, there are no reports on the effects of NAC on MMPs, specially, which inhibiting neuronal damage induced by transient

global cerebral ischemia. In the present study we do not identify the exact mechanism by which modulates MMP activation after transient global brain ischemia. We hypothesize, however, that NAC's neuroprotective effects in global brain ischemia may be due to its antioxidative or anti-inflammatory properties which reduce MMP-9 activity following ischemia. This may occur because oxidative stress, and the associated generation of reactive oxygen and nitrogen species, potentially activates the MMP enzyme (Arun et al., 2016; Cavdar et al.,

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2017). For example, NAC has been shown to significantly reduce the levels of reactive oxygen species and inhibit apoptotic cell death (Lin et al., 1997). Furthermore, NAC inhibits MMP enzyme activity in various other pathological conditions (Liu et al., 2017; Riegger et al.,

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2016).

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As mentioned above, our previous report finds that MMP-9 KO mice exhibit significantly reduced hippocampal neuronal damage that MMP-9 WT control mice following transient

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global brain ischemia (Lee et al., 2004). In the present study, we expand on these findings and report that the MMP-mediated destruction of the extracellular matrix played a key role in

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neuronal injury due to global cerebral ischemia. While we didn't describe the exact mechanism by which NAC inhibits MMP enzyme activity here, it can inferred that NAC's

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anti-oxidative properties may help to preserve neuroprotective interactions between neurons and the extracellular matrix. In support of this conclusion, we report that NAC reduced

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laminin degradation and preserve pyramidal neurons in a murine model of global brain ischemia. Based on these findings, NAC likely has various neuroprotective effects via mechanisms including the inhibition of anoikis.

These results indicate that administration of NAC, a potent antioxidant, reduced the apoptosis of hippocampal neurons in a mouse model of transient global cerebral ischemia.

These findings may be due to the inhibitory effects of NAC on the activity of the MMP enzyme, as well as NAC's previously reported neuroprotective roles. Our data also support the conclusion that MMP inhibition in a transient global cerebral ischemia model may underlie additional, beneficial effects of NAC in the management of ischemic stroke.

Conflict of Interest: The authors declare no conflict of interest.

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Funding & closure This work was supported by the research promoting grant from the Keimyung

University Dongsan Medical Center in 2012, the National Research Foundation of Korea

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Grant funded by the Korean Government (MSIP) (No. 2014R1A5A2010008), and KBRI

Science and ICT (18-BR-03-01).

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Acknowledgements

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basic research program through Korea Brain Research Institute funded by Ministry of

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We would thank Dr. J.H. Park for his excellent contributions as a blinded examiner.

References Arun, M.Z., Reel, B., Sala-Newby, G.B., Bond, M., Tsaousi, A., Maskell, P. 2016. Newby AC2.Zoledronate upregulates MMP-9 and -13 in rat vascular smooth muscle cells by inducing

oxidative

stress.

Drug

Des.

Devel

Ther.

10,

1453-1460.

https://doi:10.2147/DDDT.S103124. Asahi, M., Wang, X., Mori, T., Sumii, T., Jung, J.C., Moskowitz, M.A., Fini, M.E., Lo, E.H. 2001. Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood brain

ro of

barrier and white matter components after cerebral ischemia. J. Neurosci. 21, 7724-7732. https://doi.org/10.1523/JNEUROSCI.21-19-07724.2001.

Cavdar, Z., Ural, C., Celik, A., Arslan, S., Terzioglu, G., Ozbal, S., Yildiz, S., Ergur, U.B., Guneli, E., Camsari, T., Akdogan, G. 2017. Protective effects of taurine against renal

mitogen-activated

protein

kinase

signaling.

Biotech.

Histochem.

12,

1-

re

12.http://doi:10.1080/10520295.2017.1367033.

-p

ischemia/reperfusion injury in rats by inhibition of gelatinases, MMP-2 and MMP- 9, and p38

lP

Cao, L., Li, L., Zuo, Z. 2012. N-acetylcysteine reverses existing cognitive impairment and increased oxidative stress in glutamate transporter type 3 deficient mice. Neuroscience 220, 85-89. https://doi:10.1016/j.neuroscience.2012.06.044.

na

Chan, P. H., 2001. Reactive oxygen radicals in signaling and damage in the ischemic Brain. J. Cereb. Blood Flow Metab. 21, 2-14. https://doi.org/10.1097/00004647-200101000-00002.

ur

Chen, J., Zhu, R.L., Nakayama, M., Kawaguchi, K., Jin, K., Stetler, R.A., Simon, R.P., Graham, S.H. 1996. Expression of the apoptosis-effector gene, Bax, is up-regulated in

Jo

vulnerable hippocampal CA1 neurons following global ischemia. J. Neurochem. 67, 64-71. https://doi.org/10.1046/j.1471-4159.1996.67010064.x. Choi, D.W. 1996. Ischemia-induced neuronal apoptosis. Curr. Opin. Neurobiol. 6, 667-672. https://doi.org/10.1016/S0959-4388(96)80101-2. Endoh, M., Maiese, K., Wagner, J.A. 1994. Expression of the neural form of nitric oxide synthase by CA1 hippocampal neurons and other central nervous system neurons. Neuroscience 63, 679-689. https://doi.org/10.1016/0306-4522(94)90514-2.

Gidday, J.M., Gasche, Y.G., Copin, J.C., Shah, A.R., Perez, R.S., Shapiro, S.D., Chan, P.H., Park, T.S. 2005. Leukocyte-derived matrix metalloproteinase-9 mediates blood-brain barrier breakdown and is proinflammatory after transient focal cerebral ischemia. Am. J. Physiol. Heart Circ. Physiol. 289, H558-568. https://doi.org/10.1152/ajpheart.01275.2004. Gu, Z., Kaul, M., Yan, B., Kridel, S.J., Cui, J., Strongin, A., Smith, J.W., Liddington, R.C., Lipton, S.A., 2002. S-nitrosylation of matrix metalloproteinases: signaling pathway to

ro of

neuronal cell death. Science 297, 1186-1190. https://doi:10.1126/science.1073634. Hong, J.S., Chu, Y.K., Lee, H., Ahn, B.H., Park, J.H., Kim, M.J., Lee, S., Ryoo, H.S., Jang, J.H., Lee, S.R., Park, J.W. 2012. Effects of berberine on hippocampal neuronal damage and

Res. 90, 489-497. https://doi.org/10.1002/jnr.22756.

-p

matrix metalloproteinase-9 activity following transient global cerebral ischemia. J. Neurosci.

Ito, U., Spatz, M., Walker, J.T. Jr, Klatzo, I. 1975. Experimental cerebral ischemia in

lP

https://doi.org/10.1007/BF00696570.

re

mongolian gerbils. I. light microscopic observations. Acta Neuropathol. 32, 209- 223.

Khan, M., Sekhon, B., Jatana, M., Giri, S., Gilg, A.G., Sekhon, C., Singh, I., Singh, A.K. 2004. Administration of N-acetylcysteine after focal cerebral ischemia protects brain and

na

reduces inflammation in a rat model of experimental stroke. J. Neurosci. Res. 76, 519-527. https://doi.org/10.1002/jnr.20087.

and

ur

Kreisman, N.R., Soliman, S., Gozal, D., 2000. Regional differences in hypoxic depolarization swelling

in

hippocampal

slices.

J.

Neurophysiol.

83,

1031-1038.

Jo

https://doi.org/10.1152/jn.2000.83.2.1031.

Krzyżanowska, W., Pomierny, B., Bystrowska, B., Pomierny-Chamioło, L., Filip, M., Budziszewska, B., Pera, J. 2017. Ceftriaxone- and N-acetylcysteine-induced brain tolerance to ischemia: Influence on glutamate levels in focal cerebral ischemia. PLoS One 18, e0186243. https://doi:10.1371/journal.pone.0186243.

Lee, S.R., Tsuji, K., Lee, S.R., Lo, E.H. 2004. Role of matrix metalloproteinases in delayed neuronal damage after transient global cerebral ischemia. J. Neurosci. 24, 671-678. https://doi.org/10.1523/JNEUROSCI.4243-03.2004. Lee, K.J., Jang, Y.H., Lee, H., Yoo, H.S., Lee, S.R. 2008. PPARgamma agonist pioglitazone reduces [corrected] neuronal cell damage after transient global cerebral ischemia through matrix

metalloproteinase

inhibition.

Eur.

J.

Neurosci.

27,

334-242.

https://doi.org/10.1111/j.1460-9568.2007.06007.x.

ro of

Lin, K.T., Xue, J.Y., Sun, F.F., Wong, P.Y. 1997. Reactive oxygen species participate in peroxynitrite-induced apoptosis in HL-60 cells. Biochem. Biophys. Res. Commun. 230, 115119. https://doi.org/10.1006/bbrc.1996.5897.

Lipton, P., 1999. Ischemic cell death in brain neurons. Physiol. Rev. 79, 1431-1568.

-p

https://doi.org/10.1152/physrev.1999.79.4.1431.

Liu, Y., Ni, Y., Zhang, W., Sun, Y.E., Ma, Z., Gu, X. 2017. N-acetyl-cysteine attenuates

re

remifentanil-induced postoperative hyperalgesia via inhibiting matrix metalloproteinase-9 in

lP

dorsal root ganglia. Oncotarget 8, 16988-17001. https://doi:10.18632/oncotarget.15217. Lo, E.H., Wang, X., Cuzner, M.L. 2002. Extracellular proteolysis in brain injury and inflammation: role for plasminogen activators and matrix metalloproteinases. J. Neurosci.

na

Res. 69, 1-9. https://doi.org/10.1002/jnr.10270.

MacManus, J.P., Buchan, A.M., Hill, I.E., Rasquinha, I., Preston, E., 1993. Global

ischemia

ur

can cause DNA fragmentation indicative of apoptosis in rat brain. Neurosci. Lett. 164, 89-92. https://doi.org/10.1016/0304-3940(93)90864-H.

Jo

Mun-Bryce, S., Lukes, A., Wallace, J., Lukes-Marx, M., Rosenberg GA. 2002. Stromelysin-1 and gelatinase A are upregulated before TNF-alpha in LPS-stimulated Neuroinflammation. Brain Res. 933, 42-49. https://doi.org/10.1016/S0006-8993(02)02303-X. Nazıroğlu, M., Ciğ, B., Ozgül, C. 2013. Neuroprotection induced by N-acetylcysteine against cytosolic glutathione depletion-induced Ca2+ influx in dorsal root ganglion neurons of

mice:

role

of

TRPV1

channels.

https://doi:10.1016/j.neuroscience.2013.03.032.

Neuroscience

242,

151-160.

Nitatori, T., Sato, N., Waguri, S., Karasawa, Y., Araki, H., Shibanai, K., Kominami, E., Uchiyama, Y., 1995. Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis. J. Neurosci. 15, 10011011. https://doi.org/10.1523/JNEUROSCI.15-02-01001.1995. Oh, L.Y., Larsen, P.H., Krekoski, C.A., Edwards, D.R., Donovan, F., Werb, Z., Yong, V.W. 1999. Matrix metalloproteinase-9/gelatinase B is required for process outgrowth by oligodendrocytes. J. Neurosci. 19, 8464-8475. https://doi.org/10.1523/JNEUROSCI.19-

ro of

19-08464.1999. Pulsinelli, W.A., Brierley, J.B., Plum, F., 1982. Temporal profile of neuronal damage in a model

of

transient

forebrain

ischemia.

Ann.

https://doi.org/10.1002/ana.410110509.

Neurol.

11,

491-498.

-p

Riegger, J., Joos, H., Palm, H.G., Friemert, B., Reichel, H., Ignatius, A., Brenner, R.E. 2016. Antioxidative therapy in an ex vivo human cartilage trauma-model: attenuation of trauma-

re

induced cell loss and ECM-destructive enzymes by N-acetylcysteine. Osteoarthritis Cartilage

lP

24, 2171-2180. https://doi:10.1016/j.joca.2016.07.019.

Rosenberg, G. A., 1995. Matrix metalloproteinases in brain injury. J. Neurotrauma 12, 833842. https://doi.org/10.1089/neu.1995.12.833.

na

Rosenberg, G.A., 2002. Matrix metalloproteinases in neuroinflammation. Glia 39, 279-91. https://doi.org/10.1002/glia.10108.

ur

Tsirka, S.E., Rogove, A.D., Bugge, T.H., Degen, J.L., Strickland, S. 1997. An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus. J.

Jo

Neurosci. 17, 543-552. https://doi.org/10.1523/JNEUROSCI.17-02-00543.1997.

Tsuchiya, D., Hong, S., Suh, S.W., Kayama, T., Panter, S.S., Weinstein, P.R. 2002. Mild hypothermia reduces zinc translocation, neuronal cell death, and mortality after transient global

ischemia

in

mice.

J.

Cereb.

Blood

Flow

Metab.

22,

1231-1238.

https://doi.org/10.1097/01.wcb.0000037995.34930.F5. Wang, X., Jung, J., Asahi, M., Chwang, W., Russo, L., Moskowitz, M.A., Dixon, C.E., Fini, M.E., Lo E.H. 2000. Effects of matrix metalloproteinase-9 gene knock-out on morphological

and motor outcomes after traumatic brain injury. J. Neurosci. 20, 7037-7042. https://doi.org/10.1523/JNEUROSCI.20-18-07037.2000. Wang, X., Svedin, P., Nie, C., Lapatto, R., Zhu, C., Gustavsson, M., Sandberg, M., Karlsson, J.O., Romero, R., Hagberg, H., Mallard, C. 2007. N-acetylcysteine reduces lipopolysaccharide-sensitized hypoxic-ischemic brain injury. Ann. Neurol. 61, 263-271. https://doi.org/10.1002/ana.21066. Yang, L., Tan, P., Zhou, W., Zhu, X., Cui, Y., Zhu, L., Feng, X., Qi, H., Zheng, J., Gu, P.,

ro of

Fan, X., Chen, H. 2012. N-acetylcysteine protects against hypoxia mimetic-induced autophagy by targeting the HIF-1α pathway in retinal ganglion cells. Cell Mol. Neurobiol. 32, 1275-1285. https://doi:10.1007/s10571-012-9852-0.

-p

Zalewska, T., Ziemka-Nałecz, M., Sarnowska, A., Domańska-Janik, K. 2002. Involvement of MMPs in delayed neuronal death after global ischemia. Acta Neurobiol.

re

Exp. (Wars) 62, 53-61.

lP

Zhang, H., Bosch-Marce, M., Shimoda, L.A., Tan, Y.S., Baek, J.H., Wesley, J.B., Gonzalez, F.J., Semenza, G.L. 2008. Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic

response

to

hypoxia.

Jo

ur

na

https://doi:10.1074/jbc.M800102200.

J.

Biol.

Chem.

283,

10892-10903.

Figure Legends Fig. 1. Effect of N-acetylcysteine (NAC) on the hippocampal neuronal cell damage following global cerebral ischemia. Sham-operated animals show normal neuronal cells in hippocampal CA1 and CA2 (A & B). The medial portion of CA1 and all of CA2 were sensitive to transient global cerebral ischemia. Neuronal damage was most remarkable in these areas (C & D). H & E-stained sections revealed reduced neuronal damage after NAC administration (E & F).

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Insets show high resolution images (A-F). Semi-quantitative scores of neuronal damage reveal significantly reduced damage in NAC-treated animals (n = 11) compared to vehicletreated animals (n = 10) (G & H). Data are expressed as mean ± S.E. * P < 0.05. Sham: shamoperated animals; vehicle: vehicle-treated animals; NAC: NAC (150 mg/kg)-treated animals.

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Scale bars: A, 100 μm; inset, 25 μm.

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Fig. 2. Gelatin gel zymography of the hippocampus following global cerebral ischemia. Representative gel zymogram gel showing changes of MMP-9 (A & C). The active form of MMP-9 (97 kDa) was increased after transient global cerebral ischemia over time (B). Nacetylcysteine (NAC, 150 mg/kg) administration inhibited the active form of MMP-9. MMP2 was unchanged after transient global cerebral ischemia (C). A relative comparison of

MMP-9 activity after global brain ischemia (D). Sham: sham-operated control animal; vehicle: vehicle-treated animals; NAC: NAC-treated animals. Data are expressed as mean ±

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S.E. # P <0.05 vs. sham; * P <0.05 vs. vehicle.

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Fig. 3. Representative in situ gelatin zymograms in the hippocampus following global cerebral ischemia. Sham-operated animals show very weak gelatinase activity in the

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hippocampus (A & B). Hippocampal CA1 and CA2 showed increased gelatinase activity following transient global cerebral ischemia (C & D). N-acetylcysteine (NAC) administration

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inhibited gelatinase activity compared to vehicle-treated animals (E & F). The fluorescent signal intensity was measured in the medial part of CA1 and all of CA2 (G & H). White

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squares indicate areas of image analysis (each square has an area of 1175 μm2, 35 X 35 μm). Quantification was done across five squares within 500 μm of the medial portion of the CA1 (G) and across four square areas within 150 μm of the CA2 (H). NAC significantly inhibited the transient global cerebral ischemia-induced increase of in situ gelatinase activity in the CA1 and CA2 (I & J). Sham: sham-operated control animals; vehicle: vehicle-treated animals (n=10); NAC: NAC-treated animals (n=11). Scale bar = 100 μm.

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Fig. 4. Effect of direct inhibition of N-acetylcysteine (NAC) on the activation of the gelatinase enzyme following global cerebral ischemia assessed via in situ gelatin zymography.

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NAC (1 mM) was added to an incubation reaction solution on a post-ischemic hippocampus (A – D). Quantification of relative fluorescence intensity was done as same manner of figure

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3G & 3H. NAC (vs vehicle controls) directly inhibits gelatinase activity in CA1 and CA2, respectively (E & F). Vehicle: vehicle-treated animals (n=8); NAC: NAC-treated animals

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(n=8). Scale bar = 200 μm.

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Fig. 5. Effect of N-acetylcysteine (NAC) on the expression of laminin and NeuN in the hippocampal CA1 and CA2 following transient global cerebral ischemia. Laminin (green) and NeuN (red) expression are normal in sham-operated animals' hippocampal CA1 and CA2 (A & B). Vehicle-treated animals show degradation of laminin and loss of NeuN-positive

neuronal cells. White triangles indicate regions of degraded laminin and NeuN-positive neuronal cell loss (A & B). NAC administration inhibited ischemia-induced degradation of laminin and loss of NeuN-positive cells in the CA1 and CA2 (A & B). Sham: sham-operated

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Fig. 6. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay in the hippocampus after transient global cerebral ischemia. Sham-operated animals showed no TUNEL positive staining in neuronal cells in the hippocampal CA1 and CA2 (A

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& B). TUNEL positive neuronal cells are shown in the medial part of CA1 and across CA2 after transient global cerebral ischemia (C & D). N-acetylcysteine (NAC) administration

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reduced TUNEL positive neuronal cells (E & F). Quantification was done over a 500-μm

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long, medial aspect of the CA1 and across the entire CA2, both of which are known to be ischemia-sensitive regions. NAC administration significantly reduced TUNEL- positive neuronal cells in the hippocampal CA1 and CA2 compared with vehicle-treated animals (G & H). Data are expressed as the mean ± S.E. Sham: sham-operated animals; vehicle: vehicletreated animals; NAC: NAC-treated animals. * P < 0.05 vs. vehicle-treated animals. Scale bar = 100 μm.

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