Fluoxetine and vitamin C synergistically inhibits blood-spinal cord barrier disruption and improves functional recovery after spinal cord injury

Accepted Manuscript Fluoxetine and vitamin C synergistically inhibits blood-spinal cord barrier disruption and improves functional recovery after spinal cord injury Jee Y. Lee, Hae Y. Choi, Tae Y. Yune PII:

S0028-3908(16)30216-7

DOI:

10.1016/j.neuropharm.2016.05.018

Reference:

NP 6319

To appear in:

Neuropharmacology

Received Date: 14 January 2016 Revised Date:

23 May 2016

Accepted Date: 24 May 2016

Please cite this article as: Lee, J.Y., Choi, H.Y., Yune, T.Y., Fluoxetine and vitamin C synergistically inhibits blood-spinal cord barrier disruption and improves functional recovery after spinal cord injury, Neuropharmacology (2016), doi: 10.1016/j.neuropharm.2016.05.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

ACCEPTED MANUSCRIPT

Fluoxetine and vitamin C synergistically inhibits blood-spinal

spinal cord injury

RI PT

cord barrier disruption and improves functional recovery after

1

Age-Related

and

Brain

M AN U

SC

Jee Y. Lee1, Hae Y. Choi1, and Tae Y. Yune1, 2

Diseases

Research

Center,

2

Department

of

Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University,

TE D

Seoul, 02447, Korea

Correspondence should be addressed to:

EP

Tae Y. Yune, Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Medical Building 10th Floor, 26, Kyungheedae-

AC C

ro, Dongdaemun-gu, Seoul, 02447, Korea. Phone: +82-02-961-0968; Fax: +8202-969-6343. E-mail address: [email protected]

1

ACCEPTED MANUSCRIPT

Abstract

RI PT

Recently we reported that fluoxetine (10 mg/kg) improves functional recovery by attenuating blood spinal cord barrier (BSCB) disruption after spinal cord injury (SCI).

Here we investigated whether a low-dose of

SC

fluoxetine (1 mg/kg) and vitamin C (100 mg/kg), separately not possessing

recovery when combined.

M AN U

any protective effect, prevents BSCB disruption and improves functional After a moderate contusion injury at T9 in rat,

a low-dose of fluoxetine and vitamin C, or the combination of both was administered intraperitoneally immediately after SCI and further treated Co-treatment with fluoxetine and vitamin C

TE D

once a day for 14 d.

significantly attenuated BSCB permeability at 1 d after SCI.

When only

EP

fluoxetine or vitamin C was treated after injury, however, there was no effect on BSCB disruption. Co-treatment with fluoxetine and vitamin C

AC C

also significantly inhibited the expression and activation of MMP-9 at 8 h and 1 d after injury, respectively, and the infiltration of neutrophils (at 1 d) and macrophages (at 5 d) and the expression of inflammatory mediators (at 2 h, 6 h, 8 h or 24 h after injury) were significantly inhibited by cotreatment with fluoxetine and vitamin C.

2

Furthermore, the combination of

ACCEPTED MANUSCRIPT

fluoxetine and vitamin C attenuated apoptotic cell death at 1 d and 5 d

RI PT

after injury and improved locomotor function at 5 weeks after SCI. These results demonstrate the synergistic effect combination of low-dose

fluoxetine and vitamin C on BSCB disruption after SCI and furthermore

SC

support the effectiveness of the combination treatment regimen for the

M AN U

management of acute SCI.

Key words: blood spinal cord barrier, fluoxetine, vitamin C, matrix

Introduction

TE D

metalloprotease-9, inflammation

EP

Traumatic spinal cord injury (SCI) is a devastating condition that results in temporary or permanent loss of sensation, motor deficit or bowel/bladder Despite advances in understanding the pathophysiology of SCI,

AC C

dysfunction.

there is no cure at this time. The blood spinal cord barrier (BSCB) is the functional equivalent of the

blood-brain

barrier

(B-BB)

in

the

sense

of

providing

microenvironment for the cellular constituents of the spinal cord.

3

a

specialized When BSCB

ACCEPTED MANUSCRIPT

is damaged by an injury, blood cells are infiltrated into the injured parenchyma

2005;Abbott et al., 2006;Zlokovic, 2008).

RI PT

and contribute to secondary injuries such as inflammation (Hawkins and Davis, These secondary injuries induce

apoptotic cell death of neurons and glia, which results in permanent

SC

neurological deficits (Xu et al., 2001;Noble et al., 2002;Gerzanich et al., 2009).

M AN U

Emerging studies indicate that matrix metalloproteinase (MMPs) play critical roles in the BSCB disruption during acute SCI by degrading components of the extracellular matrix and tight junctions (TJ) in endothelial cells, leading to numerous pathological conditions including inflammation (Rosenberg et al.,

TE D

1994;Rosenberg and Navratil, 1997;Noble et al., 2002;Lee et al., 2012b). Therefore, MMPs can be projected as a potential therapeutic target for

EP

treatment in SCI.

Fluoxetine, a selective serotonin reuptake inhibitor, is a FDA-approved drug

AC C

widely used to treat depression, obsessive compulsive disorder, bulimia, and panic disorder (Mostert et al., 2008).

Accumulating evidences also suggest

that fluoxetine provides neuroprotective effects in various neurological disorders (Lim et al., 2009;Chung et al., 2011;Lee et al., 2012b;Scali et al., 2013;Lee et al., 2014).

Especially, our recent studies showed that fluoxetine treatment

4

ACCEPTED MANUSCRIPT

(immediately after injury and then daily injection for 14 d) significantly

RI PT

attenuates B-BB and BSCB disruption by inhibiting the expression and activation of MMP-9 and improves functional recovery in cerebral ischemia and SCI (Lee et al., 2012b;Lee et al., 2014).

SC

Vitamin C, a potential antioxidant, is known to have protective effects via

M AN U

reducing or preventing oxidative damage (Santos et al., 2009;Iwata et al., 2014). Recently, it has been shown that high dose vitamin C treatment (2,000 mg/kg, daily for 14 d or 200 mg/kg, daily for 28 d) significantly improves functional recovery in rats with acute SCI (Robert et al., 2012;Yan et al., 2014).

In

TE D

addition, recent evidences showed that vitamin C prevents the disruption of BBB and sustained the B-BB integrity in central nervous system (CNS) disease

EP

animal models such as sustained compression of primary somatosensory cortex, stroke, and Alzheimer’s disease (Lin et al., 2010;Kook et al.,

AC C

2014;Allahtavakoli et al., 2015). Here, we determined the effect of fluoxetine and vitamin C co-treatment (on

BSCB breakdown and functional recovery after SCI. For the combination of both agents, we examined the dose level for each compound which did not show any effect of each drug administered alone. Consequently we selected a

5

ACCEPTED MANUSCRIPT

low dose of fluoxetine (1 mg/kg) and vitamin C (100 mg/kg) and determined

RI PT

whether the combination treatment (daily for 14 d) exhibits synergistic effect on BSCB disruption, blood cell infiltration followed inflammation, apoptotic cell

SC

death, and neurological function after SCI.

M AN U

Materials & Methods Spinal cord injury

Adult rats [Sprague Dawley, male, 250-300 g; Sam:TacN (SD) BR; Samtako, Osan, Korea] were maintained under a constant temperature (23 ± 1 °C) and

TE D

humidity (60 ± 10%) under a 12 h light/ dark cycle (light on 07:30–19:30 h) with ad libitum access to drinking water and food.

Prior to surgery, rats were

EP

weighed and anesthetized with chloral hydrate (500 mg/kg, intraperitoneal injection). An adequate level of anesthesia was determined by monitoring the

AC C

corneal and hindlimb withdrawal reflexes.

The back and neck regions were

then shaved and laminectomy was performed at the T9-T10 level, exposing the cord beneath without disrupting the dura.

The spinous processes of T8 and

T11 were then clamped to stabilize the spine, and the exposed dorsal surface of the cord was subjected to moderate contusion injury (10 g x 25 mm) using a

6

ACCEPTED MANUSCRIPT

New York University (NYU) impactor as described previously (Lee et al., 2010).

RI PT

For the sham-operated controls, the animals underwent a T9-T10 laminectomy without weight-drop injury. After the injury the muscles and skin were closed in

layers, and the rats were placed in a temperature and humidity-controlled

SC

chamber overnight. After spinal cord injury, the injured rats were randomly

M AN U

assigned to the placebo control or drug treated groups by a study assistant as a certain number of animals in each group. In this study, the following inclusion and exclusion criteria were adopted.

The inclusion criteria were SD rats,

weight between 250 and 300 g or general condition and normal motility. The

TE D

exclusion criteria were: Death before breaking under anesthesia, out of injury range based on NYU impactor which calculates the parameters of trauma (eg.

EP

drop height, impact velocity, cord compression distance, cord compression rates) or normal movement in the first post-injury evaluation (21 points on the

AC C

BBB scale of functional evaluation). Postoperatively, the rats were housed one per cage after injury with water and food easily accessible. Body weights and the remaining chow and water weight were recorded each morning for all animals. The bladder was emptied manually three times per day until reflexive bladder emptying was established.

All surgical interventions and post-

7

ACCEPTED MANUSCRIPT

operative animal care were performed in accordance with the Guidelines and

RI PT

Polices for Rodent Survival Surgery provided by the Animal Care Committee of the Kyung Hee University.

SC

Drug treatment

M AN U

Fluoxetine (Sigma, St. Louis, MO: 1, 5, or 10 mg/kg) and ascorbic acid (vitamin C, Sigma: 100, 200, 500, or 1,000 mg/kg) were dissolved in sterile PBS. Combination of fluoxetine (1 mg/kg) and vitamin C (100 mg/kg) was dissolved in sterile PBS and administered intraperitoneally (i.p) immediately after SCI and

TE D

then further treated once a day for 14 d for analysis of behavioral analysis or for the indicated time points as previously described (Lee et al., 2012b). PBS was For the sham-operated controls, animals

EP

administered for vehicle control.

underwent a T9-T10 laminectomy without contusion injury, and received no

AC C

pharmacological treatment. All data obtained in this study were collected by the authors who were blinded to all treatment.

Evans blue assay At 24 h after SCI, the permeability of the BSCB was investigated with Evans

8

ACCEPTED MANUSCRIPT

Blue dye extravasation as previously described (Lee et al., 2012a).

In brief, 5

RI PT

ml of 2% Evans blue dye (Sigma) solution in saline was administered intraperitoneally. Three hours later, animals were anesthetized and sacrificed by intra-cardiac perfusion with saline.

The T9 spinal cord segment was

SC

removed and homogenized in a 50% trichloroacetic acid solution.

After

M AN U

homogenization, samples were centrifuged at 10,000 x g for 10 min, supernatants were collected and its fluorescence was quantified using a spectrophotometer at an excitation wavelength of 620 nm and an emission wavelength of 680 nm. Dye in samples was determined as micrograms per

TE D

gram of tissue from a standard curve plotted using known amounts of dye.

EP

Tissue preparation

At indicated time points after SCI, rats were anesthetized with chloral hydrate

AC C

(500 mg/kg) and perfused via cardiac puncture initially with 0.1 M PBS and subsequently with 4% paraformaldehyde in 0.1 M PBS. A 20-mm section of the spinal cord, centered at the lesion site, was dissected out, post-fixed by immersion in the same fixative (4% paraformaldehyde) for 5 h and placed in 30% sucrose in 0.1 M PBS.

The segment was embedded in OCT for frozen

9

ACCEPTED MANUSCRIPT

sections, and serial transverse sections were then cut at 10 or 20 µm on a For molecular work, rats were perfused

RI PT

cryostat (CM1850; Leica, Germany).

with 0.1 M PBS and segments of spinal cord (10 mm) including the lesion site

SC

were isolated and frozen at -80°C.

M AN U

RNA isolation and RT-PCR

At 2 h, 6 h, 8 h and 24 h after injury, total RNA was isolated using TRIZOL Reagent (Invitrogen, Carlsbad, CA) and 0.5 µg of total RNA was reversetranscribed into first strand cDNA using MMLV according to the manufacturer’s The resulting cDNAs were subjected to real-time

TE D

instructions (Invitrogen).

PCR with a Stratagene Mx3000P (Agilent Technologies, Waldbronn, Germany)

EP

using QuantiMix SYBR (PhileKorea Technology, Seoul, Korea) as described (Lee et al., 2012c).

The primers were synthesized by the Genotech (Daejeon,

AC C

Korea) and the primer sequences were summarized in Table 1.

Western blot

Segments of spinal cord (1 cm) including the lesion site were isolated at 4 h (for caspase-3), 1 d (for ZO-1, COX-2, iNOS), and 5 d (for caspase-3, ED-1) after

10

ACCEPTED MANUSCRIPT

SCI.

Total protein was prepared and Western blot analysis was performed with

RI PT

the lysates from spinal cord as previously described (Lee et al., 2012a). The primary antibodies used in Western blot analysis are as follows; cleaved caspase-3 (1:1,000, Cell Signaling Technology, Danvers, MA), ZO-1 (1:1,000,

SC

Invitrogen), occludin (1:1,000, Invitrogen), iNOS (1:10,000, Transduction

M AN U

Laboratory, Lexington, KY), COX-2 (1:1,000, Cayman Chemicals Ann Arbor, MI), and ED-1 (1:1,000, Serotec, Raleigh, NC). (1:10,000; Sigma) was used.

As loading controls, β-tubulin

The densitometric values of the bands on

Western blots obtained by AlphaImager software (Alpha Innotech Corporation, Background in films

TE D

San Leandro, CA) were subjected to statistical analysis.

EP

was subtracted from the optical density measurements.

Gelatin zymography

AC C

At 1 d after injury the activity of MMP-2 and MMP-9 was examined by gelatin zymography as previously described (Lee et al., 2012a). Relative intensity of zymography (relative to sham) was measured and analyzed by AlphaImager software (Alpha Innotech Corporation). Background was subtracted from the optical density measurements. Experiments were repeated three times and

11

ACCEPTED MANUSCRIPT

the values obtained for the relative intensity were subjected to statistical

RI PT

analysis.

Immunohistochemistry

SC

Frozen sections were processed for immunohistochemistry with antibodies

M AN U

against ZO-1 (1:100, Invitrogen), RECA-1 (Serotec), myeloperoxidase (MPO, 1:100, Carpinteria, CA) at 1 d and ED-1 (1:300, Serotec) at 5 d as previously described (Yune et al., 2007). For quantification of MPO- or ED-1-intensity, serial transverse sections (20 µm thickness) were collected every 100 µm Digital

TE D

rostral and caudal 3,000 µm to the lesion site (total 60 sections).

images of MPO- or ED-1-stained tissues were obtained and we quantified the

EP

fluorescent intensity above threshold by using MetaMorph software (Molecular devices, Sunnyvale, CA) and averaged.

The threshold value was at least

AC C

three times the background and the background was quantified and normalized to the primary antibody omitted control.

TUNEL staining One and five days after injury, serial spinal cord sections (10 µm thickness)

12

ACCEPTED MANUSCRIPT

were collected every 100 µm and processed for terminal deoxynucleotidyl deoxyuridine

triphosphate-biotin

nick

end

labeling

RI PT

transferase-mediated

(TUNEL) staining using an Apoptag in situ kit (Millipore, Billerica, MA).

Diaminobenzidine substrate kit (Vector Laboratories) was used for peroxidase

SC

staining, and the sections were then counterstained with methyl green. Control

M AN U

sections were incubated in the absence of TdT enzyme. Investigators who were blind as to the experimental conditions carried out all TUNEL analyses. For quantification, serial transverse sections (20 µm thickness) were collected every 100 µm section from 2 mm rostral to 2 mm caudal to the lesion epicenter

TE D

(total 40 sections for neurons at 1 d) or 5 mm rostral to 5 mm caudal to the lesion epicenter (total 100 sections for oligodendrocytes at 5 d) as described Only those cells showing morphological features of nuclear

EP

(Lee et al., 2010).

condensation and/or compartmentalization in the GM and WM were counted as Some sections were processed for TUNEL and then for

AC C

TUNEL-positive.

immunohistochemistry using specific cell type markers: NeuN (1:100, Millipore) for neurons; CC1 (1:100, Oncogene, Cambridge, MA) for oligodendrocytes. For double labeling, cyanine 3-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) was used and nuclei were labeled with

13

ACCEPTED MANUSCRIPT

RI PT

DAPI according to the protocol of the manufacturer (Invitrogen, Carlsbad, CA).

Behavioral tests

To test hindlimb locomotor function, open-field locomotion was evaluated at 1 d,

SC

7 d, 14 d, 28 d, and 35 d after injury by using the Basso–Beattie–Bresnahan

2007).

M AN U

(BBB) locomotion scale as previously described (Basso et al., 1995;Yune et al., BBB is a 22-point scale (scores 0–21) that systematically and logically

follows recovery of hindlimb function from a score of 0, indicative of no observed hindlimb movements, to a score of 21, representative of a normal BBB test was performed by trained investigators who were

TE D

ambulating rodent.

blind as to the experimental conditions. In this study, the rats that were used

EP

for behavioral studies were not used in the other assay.

AC C

Assessment of lesion volume Lesion volume, using rats employed for behavioral analyses, was assessed as described previously (Yune et al., 2008). Serial longitudinal sections (10 µm) through the dorsoventral axis of the spinal cord were used to determine lesion volume. Every 50 µm, sections were stained with Cresyl violet acetate and

14

ACCEPTED MANUSCRIPT

studied with light microscopy. With a low-power (1.25 X) objective, the lesion

RI PT

area was determined by MetaMorph software (Molecular devices). Areas at each longitudinal level were determined, and the total lesion volume derived by

SC

means of numerical integration of sequential areas.

M AN U

Statistical analysis

Data presented as the mean ± SEM. Comparisons between two groups were made by unpaired Student’s t-test.

Multiple comparisons between groups

were performed one-way ANOVA. Behavioral score was analyzed by repeated Tukey’s multiple comparison was used

TE D

measures ANOVA (time vs. treatment). as Post hoc analysis.

Statistical significance was accepted with p<0.05.

AC C

IL).

EP

Statistical analyses were performed using SPSS 15.0 (SPSS Science, Chicago,

Results

Co-treatment with vitamin C and fluoxetine synergistically inhibits BSCB disruption after SCI Previously, we reported that a high dose of fluoxetine significantly inhibits the

15

ACCEPTED MANUSCRIPT

increase of BSCB permeability in injured mouse spinal cord (Lee et al., 2012b).

RI PT

In addition, it has been shown that vitamin C prevents B-BB disruption in rat brain injury models (Lin et al., 2010;Allahtavakoli et al., 2015). To determine the effect of fluoxetine and vitamin C co-treatment on BSCB permeability after

SC

SCI, we assessed Evans blue assay at 1 d after injury. As shown in Fig. 1A,

M AN U

the amount of Evans blue dye extravasation increased after SCI when compared with the sham control, which indicates that SCI elicits BSCB disruption.

Furthermore, fluoxetine (5 or 10 mg/kg) treatment after injury

significantly reduced the amount of Evans blue dye extravasation when

TE D

compared with the vehicle control. In addition, vitamin C (200, 500 or 1,000 mg/kg) treatment also inhibits Evans blue dye extravasation in dose dependent

EP

manner (Fig. 1B). However, a low dose of fluoxetine (1 mg/kg) or vitamin C (100 mg/kg) treatment only did not inhibit Evans blue dye extravasation (Fig. 1A,

AC C

B).

Next, to determine the synergistic effect of fluoxetine and vitamin C co-

treatment, we divided SCI-injured rats into four groups as follows: control group (PBS injection), vitamin C group (100 mg/kg of vitamin C only injection), fluoxetine group (1 mg/kg of fluoxetine only injection), and vitamin C+ fluoxetine

16

ACCEPTED MANUSCRIPT

group [V+F, mixture of vitamin C (100 mg/kg) and fluoxetine (1 mg/kg) injection].

RI PT

At this concentration of fluoxetine (1 mg/kg) and vitamin C (100 mg/kg), there was no inhibition effect on BSCB disruption after SCI (Fig. 1A, B). As shown in

Fig. 1C and D, the Evans blue dye extravasation was not inhibited by only

SC

vitamin C or fluoxetine treatment, whereas co-treatment with vitamin C and

M AN U

fluoxetine (V+F) significantly inhibited BSCB permeability (Veh, 35.4 ± 1.9; Vit C only, 33.2 ± 2.6; Flu only, 31.6 ± 3.2; V+F, 18.6 ± 2.1, p < 0.05).

This result

suggests the synergistic effect of vitamin C and fluoxetine co-treatment on

TE D

BSCB disruption after SCI.

MMP-9 expression and activation after SCI is inhibited by co-treatment

EP

with vitamin C and fluoxetine

It has been known that the excessive proteolytic activity of MMPs such as

AC C

MMP-2 and MMP-9 results in B-BB or BSCB disruption after CNS injury such as stroke and SCI (Rosenberg and Navratil, 1997;Rosenberg et al., 1998;Xu et al., 2001;Asahi et al., 2001;Noble et al., 2002).

Because co-treatment with vitamin

C and fluoxetine reduced BSCB disruption after SCI (Fig. 1), we examined whether co-treatment with vitamin C and fluoxetine would inhibit the expression

17

ACCEPTED MANUSCRIPT

and activity of MMP-2 and/or MMP-9 after injury. As shown in Fig. 2A, the

RI PT

levels of MMP-2 and MMP-9 mRNA increased at 8 h after injury compared with the sham control.

Furthermore, SCI-induced increase in Mmp-9 mRNA

expression was significantly inhibited by co-treatment with vitamin C and

SC

fluoxetine compared with the vehicle control, whereas Mmp-2 mRNA expression

M AN U

was not (Fig. 2A, B). Using gelatin zymography, co-treatment with vitamin C and fluoxetine also significantly inhibited an increase in the activity of MMP-9 (active MMP-9 band) after SCI (Fig. 2C) as compared with the vehicle control

TE D

(Fig. 2C, D) (active MMP-9; Veh, 8.7 ± 0.6 vs. V+F, 6.1 ± 0.7, p < 0.05).

Co-treatment with vitamin C and fluoxetine prevents the degradation of

EP

tight junction proteins after SCI

It is known that tight junctions (TJs) in the endothelial cells of the blood vessels

AC C

maintain B-BB integrity (Zlokovic, 2008).

The loss or degradation of TJ

proteins has been implicated to mediate the hyperpermeability of BSCB after SCI (Lee et al., 2012a;2012b).

Thus, we next examined the effect of co-

treatment with vitamin C and fluoxetine on the expression levels of the TJ proteins ZO-1 and occludin in the injured spinal lysates using Western blot

18

ACCEPTED MANUSCRIPT

analysis. As shown in Fig. 3A and B, the decrease of the levels of ZO-1 at 1 d

RI PT

and occludin at 5 d after SCI as in previous report (Lee et al., 2012a) was significantly attenuated by co-treatment with vitamin C and fluoxetine as

compared to vehicle control. In addition, double staining with ZO-1 and RECA-

However, co-treatment with vitamin C and fluoxetine

M AN U

increased after SCI.

SC

1 antibodies also showed that the fragmentation of capillary blood vessel was

significantly attenuated the capillary fragmentation (Fig. 3C), indicating that cotreatment with vitamin C and fluoxetine preserves TJ integrity by inhibiting loss

TE D

of TJ proteins after injury.

Co-treatment with vitamin C and fluoxetine inhibits the infiltration of

EP

neutrophils and macrophages after SCI Blood infiltration such as neutrophils and macrophages is followed by BSCB and

hemorrhage

AC C

disruption

1998a;1998b).

after

SCI

(Mun-Bryce

and

Rosenberg,

Since co-treatment with vitamin C and fluoxetine prevented

BSCB disruption after SCI, we examined the effect of co-treatment with vitamin C and fluoxetine on the infiltration of blood cells by immunohistochemistry and Western blot analysis with the antibodies of MPO and ED-1. As shown in Fig.

19

ACCEPTED MANUSCRIPT

4A, both MPO-positive neutrophils at 1 d and ED-1-positive macrophages at 5 d

RI PT

after injury were observed in the injured spinal cord. The relative fluorescence intensity analysis showed that co-treatment with vitamin C and fluoxetine

significantly inhibited the infiltration of neutrophils and macrophages compared

SC

with the vehicle control (MPO: Veh, 1.0 ± 0.06 vs. V+F, 0.63 ± 0.09%, ED-1: Veh,

M AN U

1.0 ± 0.08 vs. V+F, 0.52 ± 0.12, p < 0.05) (Fig. 4A, B). Western blot analysis also revealed that the increase of ED-1 level in the injured spinal cord lysates was significantly reduced by co-treatment with vitamin C and fluoxetine at 5 d after injury compared with the vehicle control (Fig. 4C, D).

These findings

TE D

suggest that the combination of a low dose of vitamin C and fluoxetine inhibits blood cell infiltration by preventing BSCB disruption after SCI.

EP

It has also been known that both neutrophils and macrophages infiltrated after SCI produce inflammatory mediators such as IL-1β, TNF-α, COX-2 and

AC C

iNOS and chemokines, such as Gro-α (CXCL-1), MCP-1 (CCL-2), MIP-1α

(CCL-3), MIP-1β (CCL-4), and MIP-2α (CXCL-2), which thereby facilitate inflammatory responses (McTigue et al., 1998;Mun-Bryce and Rosenberg,

1998a;Ghirnikar et al., 2001;Ousman and David, 2001;Pineau and Lacroix, 2007).

Thus, we next determined the effect of co-treatment with vitamin C and

20

ACCEPTED MANUSCRIPT

fluoxetine on the expression of inflammatory mediators and chemokines after Using real-time RT-PCR, the increase of TNF-α, IL-1β (at 2 h), iNOS, and

RI PT

SCI.

COX-2 (at 6 h) mRNA level after SCI were significantly inhibited by co-treatment with vitamin C and fluoxetine (Fig. 5A).

The protein levels of iNOS and COX-2

SC

at 1 d after injury were also significantly reduced by co-treatment with vitamin C

M AN U

and fluoxetine as compared with the vehicle control (Fig. 5B, C). In addition, co-treatment with vitamin C and fluoxetine significantly inhibited the increases in the mRNA levels of Gro-α, MIP- 1α and MIP- 1β at 8 h after injury (Fig. 5D).

TE D

Co-treatment with vitamin C and fluoxetine inhibits apoptotic cell death and improves functional recovery after SCI

EP

It is known that infiltrating blood cells produce and release free radicals, nitric oxide, and MMPs, which directly cause tissue damage and apoptosis of

AC C

neurons and oligodendrocytes after SCI (Hausmann, 2003).

By TUNEL

staining, we examined the effect of co-treatment with vitamin C and fluoxetine on apoptotic cell death in the GM at 1 d and in the WM at 5 d after SCI. Most TUNEL-positive cells in the GM at 1 d and in the WM at 5 d after SCI are neurons and oligodendrocytes respectively based on double-labeling (Fig. 6A’)

21

ACCEPTED MANUSCRIPT

as described (Lee et al., 2010).

As a result, any positive signal was not

RI PT

observed in uninjured sham control (data not shown) and the co-treatment with vitamin C and fluoxetine significantly reduced the number of TUNEL-positive neurons and oligodendrocytes as compared to vehicle control (GM at 1 d: Veh,

Consistently, the increase of the cleaved (activated) forms of

M AN U

(Fig. 6B).

SC

323 ± 27; V+F, 203 ± 22; WM at 5 d: Veh, 205 ± 22; V+F, 122 ± 26, p ≤ 0.05)

caspase-3 level at 4 h and 5 d after SCI was significantly alleviated by cotreatment with vitamin C and fluoxetine as compared to vehicle control (Fig. 6C, D).

Therefore, our results indicate that the combination of vitamin C and

TE D

fluoxetine may inhibit the apoptotic cell death of neurons and oligodendrocytes after SCI via the attenuation of BSCB disruption and blood cell infiltration.

EP

To evaluate the effect of co-treatment with vitamin C and fluoxetine on functional recovery after SCI, rats were immediately treated with a mixture of

AC C

vitamin C (100 mg/kg) and fluoxetine (1 mg/kg) and further treated once a day for 2 weeks after SCI. Functional recovery was then evaluated using the 21point BBB scores (Basso et al., 1995) for locomotion. As a result, co-treatment with vitamin C and fluoxetine significantly increased the hindlimb locomotor

function from 28 d to 35 d after injury as compared to that observed in vehicle-

22

ACCEPTED MANUSCRIPT

treated control (At 35 d, Veh, 9.2 ± 0.7 vs. V+F 11.5 ± 0.8, p < 0.05) (Fig. 6E).

RI PT

When tissue loss with serial longitudinal sections after Cresyl violet staining was evaluated, the total lesion volume was significantly decreased upon cotreatment with vitamin C and fluoxetine as compared with that of behicle

M AN U

SC

treatment (Veh, 10.2 ± 1.3; V+F, 5.4 ± 0.9 mm3, p ≤ 0.05) (Fig. 6F, G).

Discussion

In the present study, we for the first time demonstrated that a low-dose combination of fluoxetine (1 mg/kg) and vitamin C (100 mg/kg) synergistically

TE D

exerts neuroprotective effects by inhibiting MMP-9 activation and thereby preventing BSCB disruption after SCI.

Co-treatment with fluoxetine and

EP

vitamin C also reduced the number of infiltrating blood cells such as neutrophils and macrophage and inhibited pro-inflammatory cytokine and chemokines,

AC C

thereby resulting in reduced inflammatory responses.

In addition, apoptotic

cell death of neurons and oligodendrocytes was inhibited and functional recovery was improved after SCI by post-treatment of the combination of fluoxetine and vitamin C.

Thus, our findings provide a possibility that the

combination therapy with fluoxetine and vitamin C can be effective in

23

ACCEPTED MANUSCRIPT

neurological diseases accompanying B-BB or BSCB disruption such as

RI PT

traumatic or ischemic brain injury including SCI.

It has been known that a high dose of fluoxetine (10 mg/kg) or vitamin C

SC

(500 mg/kg) inhibits B-BB or BSCB disruption after brain ischemia and SCI (Lee et al., 2012b;Lee et al., 2014). In addition, vitamin C (500 mg/kg) treatment B-BB

disruption

in

rat

brain

injury

models

(Lin

et

al.,

M AN U

prevented

2010;Allahtavakoli et al., 2015).

Furthermore, a high-dose of vitamin C

supplementation (33 g/l) was reported to reduce amyloid plaque burden and

TE D

ameliorate pathological change in the brain of 5XFAD mice (Kook et al., 2014). As shown in fig. 1A and B, BSCB disruption after SCI was not inhibited by a low dose of fluoxetine (1 mg/kg) or vitamin C (100 mg/kg) alone treatment.

EP

However, we found that co-treatment with fluoxetine and vitamin C significantly

AC C

reduced the extravasation of Evan blue dye after SCI, indicating the synergistic effect of co-treatment with vitamin C and fluoxetine on BSCB disruption.

It has

also been known that B-BB or BSCB disruption under various pathological conditions such as stroke and SCI results in increased cerebrovascular permeability with subsequent development of tissue edema (Utepbergenov et al., 1998).

Especially, the upregulation of MMP-9 has been implicated in 24

ACCEPTED MANUSCRIPT

BSCB disruption followed SCI-induced secondary damage by degrading the

RI PT

basal components of BSCB and facilitating immune cell infiltration (Noble et al., 2002), although many factors are known to contribute to B-BB or BSCB disruption. Our data also showed that co-treatment with fluoxetine and vitamin

SC

C significantly inhibited MMP-9 expression and activity after SCI (see Fig. 2).

M AN U

Additionally, blood infiltration and loss of TJ proteins were significantly attenuated by co-treatment with vitamin C and fluoxetine.

These results

suggest that the attenuation of BSCB disruption by co-treatment with vitamin C and fluoxetine is in part mediated by inhibiting MMP-9 expression and activation

TE D

after SCI.

Reactive oxygen species (ROS) as well as reactive nitrogen species such as

B-BB

EP

nitric oxide and peroxynitrite have been known to play an important role in the disruption

and

endothelial

AC C

2006;Schreibelt et al., 2007).

cell

permeability

(Parathath

et

al.,

Additionally, superoxide anion, one of the major

ROS has been known to regulate the endothelial TJ proteins and increase the B-BB permeability through RhoA, phosphatidylinositol 3 kinase, and the protein kinase B (PKB/AKT) signaling pathway (Sheth et al., 2003;Schreibelt et al., 2007).

The report by Lin et al. (2010) also showed that B-BB disruption

25

ACCEPTED MANUSCRIPT

induced by transient compression of somatosensory cortex was ameliorated by

RI PT

vitamin C treatment (500 mg/kg) by inhibiting ROS-mediated oxidative stress. Therefore, we can’t exclude a possibility that the inhibitory effect by the

combination of a low dose of fluoxetine and vitamin C on BSCB disruption and

M AN U

C and further study about this view is needed.

SC

functional recovery after SCI may be mediated by anti-oxidant effect of vitamin

Blood vessels including capillaries are ruptured immediately by mechanical injury itself and further fragmented by various factors such as metalloproteases

TE D

and sulfonylurea receptor 1 induced after SCI (Simard et al., 2007;Simard et al., 2010;Zhang et al., 2011), thereby, blood infiltration is increased further. In this study, we found that the fragmentation of blood vessels was increased after SCI

EP

as previously reported (Simard et al., 2010). Furthermore, co-treatment with

AC C

vitamin C and fluoxetine significantly inhibited the fragmentation of capillaries as compared with vehicle control (see Fig. 3B).

However, SCI-induced

sulfonylurea receptor 1 expression was not inhibited by co-treatment with vitamin C and fluoxetine (data not shown), indicating the inhibition of the fragmentation of capillary blood vessels by co-treatment with vitamin C and

26

ACCEPTED MANUSCRIPT

fluoxetine after SCI was mediated in part by attenuating MMP-9 expression and

RI PT

activation.

It has been known that B-BB or BSCB disruption followed infiltration of blood

SC

cells including macrophages also results in tissue damage after SCI and the inhibition of blood cells infiltration ameliorates apoptotic cell death and improves

M AN U

functional recovery (Hamada et al., 1996;Taoka et al., 1997;Saiwai et al., 2010). Co-treatment with vitamin C and fluoxetine also significantly attenuated apoptotic cell death (see Fig. 6A-D), and improved functional recovery after SCI

TE D

(see Fig. 6E). Thus, our data suggest that the neuroprotective effect of cotreatment with vitamin C and fluoxetine might be mediated in part by preventing BSCB disruption followed blood cells infiltration after SCI. Oxidative stress by

EP

ROS produced after SCI also is known to play an important role in apoptotic cell

AC C

death. The report by Chen et al. (Chen et al., 2014) showed that the combined administration of vitamin C (240 mg/kg) and E (200 mg/kg) prevents programmed cell death after SCI through an anti-oxidant effect. In addition, it was known that a high dose of vitamin C (200 or 2,000 mg/kg) improves functional recovery after SCI in rats (Liao et al., 2001;Robert et al., 2012;Yan et al., 2014). Thus, there is a possibility that vitamin C as an anti-oxidant can 27

ACCEPTED MANUSCRIPT

protect apoptotic cell death after SCI, although the concentration of vitamin C

RI PT

(100 mg/kg) used in this study was low. However, it should be pointed out that the precise action mechanism of how low dose of vitamin C potentiate the effect

SC

of low dose of fluoxetine is still unknown.

Collectively, the present study indicated that co-treatment with a low-dose

M AN U

vitamin C and fluoxetine exhibits the synergistic neuroprotective effect after SCI, which is mediated by the inhibition of MMP-9 expression and activation followed BSCB disruption, thereby attenuating inflammatory response. Considering that

TE D

fluoxetine is currently used clinically as an antidepressant and vitamin C has no cytotoxic effect in human, our results suggest that the combination therapy with vitamin and fluoxetine may provide a potential therapeutic intervention for

AC C

EP

preventing BSCB or B-BB disruption after SCI and ischemic injury.

Acknowledgment This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number:

28

ACCEPTED MANUSCRIPT

HI13C1460). This research was also supported by Basic Science Research

the

Ministry

of

Science,

2015R1A2A2A01003795).

ICT

and

future

RI PT

Program through the National Research Foundation of Korea (NRF) funded by Planning

(grant

number:

All authors have read the journal’s policy on

SC

conflicts of interest and all authors declare no conflict of interest.

M AN U

References

Abbott NJ, Ronnback L, Hansson E (2006) Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci 7:41-53.

TE D

Allahtavakoli M, Amin F, Esmaeeli-Nadimi A, Shamsizadeh A, KazemiArababadi M, Kennedy D (2015) Ascorbic Acid Reduces the Adverse Effects of Delayed Administration of Tissue Plasminogen Activator in a Rat Stroke Model.

EP

Basic Clin Pharmacol Toxicol.

AC C

Asahi M, Wang X, Mori T, Sumii T, Jung JC, Moskowitz MA, Fini ME, Lo EH (2001) Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrier and white matter components after cerebral ischemia. J Neurosci 21:7724-7732.

Basso DM, Beattie MS, Bresnahan JC (1995) A sensitive and reliable locomotor

29

ACCEPTED MANUSCRIPT

rating scale for open field testing in rats. J Neurotrauma 12:1-21.

RI PT

Chen HC, Hsu PW, Tzaan WC, Lee AW (2014) Effects of the combined administration of vitamins C and E on the oxidative stress status and

SC

programmed cell death pathways after experimental spinal cord injury. Spinal Cord 52:24-28.

M AN U

Chung YC, Kim SR, Park JY, Chung ES, Park KW, Won SY, Bok E, Jin M, Park ES, Yoon SH, Ko HW, Kim YS, Jin BK (2011) Fluoxetine prevents MPTPinduced loss of dopaminergic neurons by inhibiting microglial activation.

TE D

Neuropharmacology 60:963-974.

Gerzanich V, Woo SK, Vennekens R, Tsymbalyuk O, Ivanova S, Ivanov A, Geng

EP

Z, Chen Z, Nilius B, Flockerzi V, Freichel M, Simard JM (2009) De novo expression of Trpm4 initiates secondary hemorrhage in spinal cord injury. Nat

AC C

Med 15:185-191.

Ghirnikar RS, Lee YL, Eng LF (2001) Chemokine antagonist infusion promotes axonal sparing after spinal cord contusion injury in rat. J Neurosci Res 64:582589.

30

ACCEPTED MANUSCRIPT

Hamada Y, Ikata T, Katoh S, Nakauchi K, Niwa M, Kawai Y, Fukuzawa K (1996)

RI PT

Involvement of an intercellular adhesion molecule 1-dependent pathway in the pathogenesis of secondary changes after spinal cord injury in rats. J

SC

Neurochem 66:1525-1531.

Hausmann ON (2003) Post-traumatic inflammation following spinal cord injury.

M AN U

Spinal Cord 41:369-378.

Hawkins BT, Davis TP (2005) The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev 57:173-185.

TE D

Iwata N, Okazaki M, Xuan M, Kamiuchi S, Matsuzaki H, Hibino Y (2014) Orally administrated ascorbic acid suppresses neuronal damage and modifies

EP

expression of SVCT2 and GLUT1 in the brain of diabetic rats with cerebral

AC C

ischemia-reperfusion. Nutrients 6:1554-1577.

Kook SY, Lee KM, Kim Y, Cha MY, Kang S, Baik SH, Lee H, Park R, Mook-Jung I (2014) High-dose of vitamin C supplementation reduces amyloid plaque burden and ameliorates pathological changes in the brain of 5XFAD mice. Cell Death Dis 5:e1083.

31

ACCEPTED MANUSCRIPT

Lee JY, Chung H, Yoo YS, Oh YJ, Oh TH, Park S, Yune TY (2010) Inhibition of

RI PT

apoptotic cell death by ghrelin improves functional recovery after spinal cord injury. Endocrinology 151:3815-3826.

attenuates

blood-spinal

cord

barrier

SC

Lee JY, Kim HS, Choi HY, Oh TH, Ju BG, Yune TY (2012a) Valproic acid disruption

by

inhibiting

matrix

M AN U

metalloprotease-9 activity and improves functional recovery after spinal cord injury. J Neurochem 121:818-829.

Lee JY, Kim HS, Choi HY, Oh TH, Yune TY (2012b) Fluoxetine inhibits matrix

TE D

metalloprotease activation and prevents disruption of blood-spinal cord barrier after spinal cord injury. Brain 135:2375-2389.

EP

Lee JY, Lee HE, Kang SR, Choi HY, Ryu JH, Yune TY (2014) Fluoxetine inhibits transient global ischemia-induced hippocampal neuronal death and memory

AC C

impairment by preventing blood-brain barrier disruption. Neuropharmacology 79:161-171.

Lee K, Na W, Lee JY, Na J, Cho H, Wu H, Yune TY, Kim WS, Ju BG (2012c) Molecular mechanism of Jmjd3-mediated interleukin-6 gene regulation in endothelial cells underlying spinal cord injury. J Neurochem 122:272-282. 32

ACCEPTED MANUSCRIPT

Liao SL, Chen WY, Raung SL, Kuo JS, Chen CJ (2001) Association of immune

RI PT

responses and ischemic brain infarction in rat. Neuroreport 12:1943-1947.

Lim CM, Kim SW, Park JY, Kim C, Yoon SH, Lee JK (2009) Fluoxetine affords

SC

robust neuroprotection in the postischemic brain via its anti-inflammatory effect. J Neurosci Res 87:1037-1045.

M AN U

Lin JL, Huang YH, Shen YC, Huang HC, Liu PH (2010) Ascorbic acid prevents blood-brain barrier disruption and sensory deficit caused by sustained compression of primary somatosensory cortex. J Cereb Blood Flow Metab

TE D

30:1121-1136.

McTigue DM, Tani M, Krivacic K, Chernosky A, Kelner GS, Maciejewski D, Maki

EP

R, Ransohoff RM, Stokes BT (1998) Selective chemokine mRNA accumulation

AC C

in the rat spinal cord after contusion injury. J Neurosci Res 53:368-376.

Mostert JP, Koch MW, Heerings M, Heersema DJ, De KJ (2008) Therapeutic potential of fluoxetine in neurological disorders. CNS Neurosci Ther 14:153-164.

Mun-Bryce

S,

Rosenberg

GA

(1998b)

Matrix

metalloproteinases

cerebrovascular disease. J Cereb Blood Flow Metab 18:1163-1172.

33

in

ACCEPTED MANUSCRIPT

Mun-Bryce S, Rosenberg GA (1998a) Gelatinase B modulates selective

RI PT

opening of the blood-brain barrier during inflammation. Am J Physiol 274:R1203-R1211.

SC

Noble LJ, Donovan F, Igarashi T, Goussev S, Werb Z (2002) Matrix

metalloproteinases limit functional recovery after spinal cord injury by

M AN U

modulation of early vascular events. J Neurosci 22:7526-7535.

Ousman SS, David S (2001) MIP-1alpha, MCP-1, GM-CSF, and TNF-alpha control the immune cell response that mediates rapid phagocytosis of myelin

TE D

from the adult mouse spinal cord. J Neurosci 21:4649-4656.

Parathath SR, Parathath S, Tsirka SE (2006) Nitric oxide mediates

EP

neurodegeneration and breakdown of the blood-brain barrier in tPA-dependent

AC C

excitotoxic injury in mice. J Cell Sci 119:339-349.

Pineau I, Lacroix S (2007) Proinflammatory cytokine synthesis in the injured mouse spinal cord: multiphasic expression pattern and identification of the cell types involved. J Comp Neurol 500:267-285.

Robert AA, Zamzami M, Sam AE, Al JM, Al MS (2012) The efficacy of

34

ACCEPTED MANUSCRIPT

antioxidants in functional recovery of spinal cord injured rats: an experimental

RI PT

study. Neurol Sci 33:785-791.

Rosenberg GA, Dencoff JE, McGuire PG, Liotta LA, Stetler-Stevenson WG

SC

(1994) Injury-induced 92-kilodalton gelatinase and urokinase expression in rat brain. Lab Invest 71:417-422.

M AN U

Rosenberg GA, Estrada EY, Dencoff JE (1998) Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening after reperfusion in rat brain. Stroke 29:2189-2195.

TE D

Rosenberg GA, Navratil M (1997) Metalloproteinase inhibition blocks edema in intracerebral hemorrhage in the rat. Neurology 48:921-926.

EP

Saiwai H, Ohkawa Y, Yamada H, Kumamaru H, Harada A, Okano H, Yokomizo T,

AC C

Iwamoto Y, Okada S (2010) The LTB4-BLT1 axis mediates neutrophil infiltration and secondary injury in experimental spinal cord injury. Am J Pathol 176:23522366.

Santos IM, Tome AR, Saldanha GB, Ferreira PM, Militao GC, Freitas RM (2009) Oxidative stress in the hippocampus during experimental seizures can be

35

ACCEPTED MANUSCRIPT

ameliorated with the antioxidant ascorbic acid. Oxid Med Cell Longev 2:214-221.

RI PT

Scali M, Begenisic T, Mainardi M, Milanese M, Bonifacino T, Bonanno G, Sale A, Maffei L (2013) Fluoxetine treatment promotes functional recovery in a rat

SC

model of cervical spinal cord injury. Sci Rep 3:2217.

Schreibelt G, Kooij G, Reijerkerk A, van DR, Gringhuis SI, van der PS, Weksler

M AN U

BB, Romero IA, Couraud PO, Piontek J, Blasig IE, Dijkstra CD, Ronken E, de Vries HE (2007) Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling. FASEB J 21:3666-3676.

TE D

Sheth P, Basuroy S, Li C, Naren AP, Rao RK (2003) Role of phosphatidylinositol 3-kinase in oxidative stress-induced disruption of tight junctions. J Biol Chem

EP

278:49239-49245.

AC C

Simard JM, Tsymbalyuk O, Ivanov A, Ivanova S, Bhatta S, Geng Z, Woo SK, Gerzanich V (2007) Endothelial sulfonylurea receptor 1-regulated NC Ca-ATP channels mediate progressive hemorrhagic necrosis following spinal cord injury. J Clin Invest 117:2105-2113.

Simard JM, Woo SK, Norenberg MD, Tosun C, Chen Z, Ivanova S, Tsymbalyuk

36

ACCEPTED MANUSCRIPT

O, Bryan J, Landsman D, Gerzanich V (2010) Brief suppression of Abcc8

RI PT

prevents autodestruction of spinal cord after trauma. Sci Transl Med 2:28ra29.

Taoka Y, Okajima K, Uchiba M, Murakami K, Kushimoto S, Johno M, Naruo M,

SC

Okabe H, Takatsuki K (1997) Role of neutrophils in spinal cord injury in the rat. Neuroscience 79:1177-1182.

(1998)

Nitric

oxide

M AN U

Utepbergenov DI, Mertsch K, Sporbert A, Tenz K, Paul M, Haseloff RF, Blasig IE protects

blood-brain

barrier

in

vitro

from

hypoxia/reoxygenation-mediated injury. FEBS Lett 424:197-201.

TE D

Xu J, Kim GM, Ahmed SH, Xu J, Yan P, Xu XM, Hsu CY (2001) Glucocorticoid receptor-mediated suppression of activator protein-1 activation and matrix

EP

metalloproteinase expression after spinal cord injury. J Neurosci 21:92-97.

AC C

Yan M, Yang M, Shao W, Mao XG, Yuan B, Chen YF, Ye ZX, Liang W, Luo ZJ (2014) High-dose ascorbic acid administration improves functional recovery in rats with spinal cord contusion injury. Spinal Cord 52:803-808.

Yune TY, Lee JY, Jung GY, Kim SJ, Jiang MH, Kim YC, Oh YJ, Markelonis GJ, Oh TH (2007) Minocycline alleviates death of oligodendrocytes by inhibiting pro-

37

ACCEPTED MANUSCRIPT

nerve growth factor production in microglia after spinal cord injury. J Neurosci

RI PT

27:7751-7761.

Zhang H, Trivedi A, Lee JU, Lohela M, Lee SM, Fandel TM, Werb Z, Noble-

SC

Haeusslein LJ (2011) Matrix metalloproteinase-9 and stromal cell-derived

factor-1 act synergistically to support migration of blood-borne monocytes into

Zlokovic

BV

(2008)

The

M AN U

the injured spinal cord. J Neurosci 31:15894-15903.

blood-brain

barrier

in

health

and

chronic

Figure legends

TE D

neurodegenerative disorders. Neuron 57:178-201.

EP

Table 1. Primer sequences used for real time RT-PCR

AC C

Figure 1. Co-treament with vitamin C and fluoxetine synergistically inhibits BSCB disruption after SCI. Vehicle (PBS), fluoxetine (1, 5, 10 mg/kg), vitamin C (100, 200, 500, or 1,000 mg/kg), and mixture of vitamin C (100 mg/kg) and fluoxetine (1 mg/kg) were administered by i.p. injection immediately after injury, and rats were sacrificed at 24 h (1 d) and processed for Evans blue assay as described in the Methods sections (n = 5). 38

(A) Quantification of the Evans blue

ACCEPTED MANUSCRIPT

extravasation by using fluorometer (excitation at 620 nm and emission at 680

RI PT

nm). Note that Evans blue extravasation was significantly reduced in 5 and 10 mg/kg of fluoxetine-treated group as compared with vehicle control.

(B)

Quantification of the Evans blue extravasation. Note that Evans blue

SC

extravasation was significantly reduced in 200, 500, and 1,000 mg/kg of vitamin

M AN U

C-treated group as compared with vehicle control. (C) Representative whole spinal cords demonstrating that Evans blue dye was permeabilized into spinal cord at 1 d after injury.

(D) Quantification of the Evans blue extravasation.

Note that Evans blue extravasation was significantly reduced in a mixture of

TE D

vitamin C (100 mg/kg) and fluoxetine (1 mg/kg)-treated rats (V+F) compared with vehicle-treated rats (Veh), whereas 100 mg/kg of vitamin C (Vit) and 1

EP

mg/kg of fluoxetine (Flu)-treated rats was not reduced Evans blue extravasation. The value is presented as amount of dye (µg)/tissue weight (g).

AC C

presented as the mean ± SEM.

Data are

*p < 0.05 vs. Veh.

Figure 2. Co-treament with vitamin C and fluoxetine inhibits the expression and activation of MMP-9 after SCI. (A) RT-PCR for Mmp-2 and Mmp-9 at 8 h after SCI.

(B) Densitometric analyses for Mmp-2 and Mmp-9 (n = 3).

39

(C) Gelatin

ACCEPTED MANUSCRIPT

zymography at 1 d after SCI. (D) Densitometric analysis for zymography (n = Note that V+F treatment significantly inhibited MMP-9 expression and

RI PT

4).

activation after SCI. Data are presented as the mean ± SEM. *p < 0.05 vs.

SC

Veh.

M AN U

Figure 3. Co-treament with vitamin C and fluoxetine prevents the degradation of tight junction proteins after SCI. To examine the degradation of tight junction proteins after SCI, vehicle or V+F were injected into the injured rats and total protein was extracted from the spinal cord at 1 d and 5 d after injury (n =

after SCI).

(A) Western blots for ZO-1 (at 1 d after SCI) and occludin (at 5 d

TE D

3/group).

(B) Quantitative analysis of the Western blots.

The data are

Veh.

EP

presented as the mean ± SEM. The levels of significance was *p ≤ 0.05 vs (C) Representative micrographs showing double immunofluorescence

AC C

with ZO-1 and RECA-1 (endothelial cell marker) at 500 µm caudal to the lesion epicenter.

Scale bar, 50 µm.

Figure-4. Co-treament with vitamin C and fluoxetine attenuates the infiltration of neutrophils and macrophages after SCI.

40

After SCI, V+F were treated and

ACCEPTED MANUSCRIPT

blood cell infiltration was assessed as described in the Methods section. (A)

RI PT

Representative photographs of MPO-labeled neutrophils or ED-1-labeled macrophages in the dorsal column of injured spinal tissues treated with or without V+F are shown.

The representative sections were collected 2 mm

(B) Quantitative analysis of

SC

rostral to the lesion epicenter. Scale bar, 100 µm.

M AN U

immunoreactivity of MPO- or ED-1-positive cells (n = 5). (C, D) Western blot and densitometric analysis for ED-1 at 5 d after injury (n = 3). Note that V+F treatment significantly inhibited the infiltration of blood cells after SCI when compared with vehicle control. Data are presented as the mean ± SEM. *p <

TE D

0.05 vs. Veh.

EP

Figure-5. Co-treament with vitamin C and fluoxetine inhibits the expression of inflammatory factors and chemokines after SCI.

Total RNA and protein

AC C

extracts from vehicle or V+F-treated spinal cords were prepared at the indicated time points after injury. (A) real-time RT- PCR of IL-1β, Tnf-α (at 2 h), Cox-2, and iNos (at 6 h) after injury (n = 3). (B, C) Western blots (B) and quantitative analysis (C) of iNos and Cos-2 at 1 d after injury (n = 3). (D) real-time RT-PCR of Gro-α, Mip-2α, Mcp-1, Mip-1α, and Mip-1β (at 1 d) mRNA expression after

41

ACCEPTED MANUSCRIPT

RI PT

injury (n = 3). Data are presented as the mean ± SEM. *p < 0.05 vs. Veh.

Figure-6. Co-treament with vitamin C and fluoxetine inhibits apoptotic cell death and improves functional recovery after SCI. After SCI, rats were treated with

SC

vehicle or V+F and spinal tissues and extract were prepared for TUNEL staining

and 5 d (WM) after injury.

M AN U

and Western blot. (A) Representative image of TUNEL staining at 1 d (GM) (A’, upper panels) TUNEL-positive neurons (arrows)

in the GM at 1 d after injury.

(A’, bottom panels) TUNEL-positive

oligodendrocytes (arrows) in the WM at 5 d after SCI.

Representative images

TE D

were from the sections selected 1 mm (1 d, GM) or 5 mm (5 d, WM) rostral to the lesion epicenter.

after SCI.

(C) Western blots of cleaved caspase-3 at 4h and 5 d

EP

positive cells (n = 5).

Scale Bars, 30 µm. (B) Quantitative analysis of TUNEL-

(D) Quantitative analyses of Western blots (n = 3).

(E) Functional

AC C

recovery in injured rats was assessed with BBB locomotor score (n = 15). (F) Representative spinal cord tissues (1.2 mm from the dorsal surface) showing cavitation in the lesion site at 38 d after injury.

(G) Quantitative analysis of the

lesion area (n = 5). Note that co-treatment of vitamin C and fluoxetine significantly decreased the lesion volume at 38 d after injury. The data are

42

ACCEPTED MANUSCRIPT

presented as the mean ± SEM. The levels of significance was *p ≤ 0.05 vs.

AC C

EP

TE D

M AN U

SC

RI PT

Veh.

43

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Highlights
Combination of low-dose fluoxetine and vitamin C prevents BSCB disruption

RI PT

after SCI.
Combination of low-dose fluoxetine and vitamin C inhibits apoptosis after SCI.
Combination of low-dose fluoxetine and vitamin improves functional recovery

AC C

EP

TE D

M AN U

SC

after SCI.