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Brain 2012: 135; 2375–2389 A JOURNAL OF NEUROLOGY Fluoxetine inhibits matrix metalloproteaseactivation and prevents disruption of blood–spinalcord barrier after spinal cord injury Jee Y. Lee,1,2 Hwang S. Kim,1,3 Hye Y. Choi,1 Tae H. Oh1 and Tae Y. Yune1,2,3,4 1 Age-Related and Brain Diseases Research Centre, School of Medicine, Kyung Hee University, Seoul 130-701, Korea2 Neurodegeneration Control Research Centre, School of Medicine, Kyung Hee University, Seoul 130-701, Korea 3 Graduate Programme for Neuroscience, School of Medicine, Kyung Hee University, Seoul 130-701, Korea4 Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Seoul 130-701, Korea Correspondence to: Tae Y. Yune,Department of Biochemistry and Molecular Biology,and Age-Related and Brain Diseases Research Centre,School of Medicine, Kyung Hee University,Medical Building 10th Floor,Dongdaemun-gu, Hoegi-dong 1,Seoul 130-701, Korea, E-mail: [email protected] After spinal cord injury, the disruption of blood–spinal cord barrier by activation of matrix metalloprotease is a critical eventleading to infiltration of blood cells, inflammatory responses and neuronal cell death, contributing to permanent neurologicaldisability. Recent evidence indicates that fluoxetine, an anti-depressant drug, is shown to have neuroprotective effects inischaemic brain injury, but the precise mechanism underlying its protective effects is largely unknown. Here, we show thatfluoxetine prevented blood–spinal cord barrier disruption via inhibition of matrix metalloprotease activation after spinal cordinjury. After a moderate contusion injury at the T9 level of spinal cord with an infinite horizon impactor in the mouse, fluoxetine(10 mg/kg) was injected intraperitoneally and further administered once a day for indicated time points. Fluoxetine treatmentsignificantly inhibited messenger RNA expression of matrix metalloprotease 2, 9 and 12 after spinal cord injury. By zymographyand fluorimetric enzyme activity assay, fluoxetine also significantly reduced matrix metalloprotease 2 and matrix metallopro-tease 9 activities after injury. In addition, fluoxetine inhibited nuclear factor kappa B-dependent matrix metalloprotease 9expression in bEnd.3, a brain endothelial cell line, after oxygen–glucose deprivation/reoxygenation. Fluoxetine also attenuatedthe loss of tight junction molecules such as zona occludens 1 and occludin after injury in vivo as well as in bEnd.3 cultures. Byimmunofluorescence staining, fluoxetine prevented the breakdown of the tight junction integrity in endothelial cells of bloodvessel after injury. Furthermore, fluoxetine inhibited the messenger RNA expression of chemokines such as Groa, MIP1a and 1b,and prevented the infiltration of neutrophils and macrophages, and reduced the expression of inflammatory mediators afterinjury. Finally, fluoxetine attenuated apoptotic cell death and improved locomotor function after injury. Thus, our results indicatethat fluoxetine improved functional recovery in part by inhibiting matrix metalloprotease activation and preventing blood–spinalcord barrier disruption after spinal cord injury. Furthermore, our study suggests that fluoxetine may represent a potentialtherapeutic agent for preserving blood–brain barrier integrity following ischaemic brain injury and spinal cord injury in humans.
Keywords: blood–brain barrier; fluoxetine; matrix metalloprotease; spinal cord injury; tight junction Received February 13, 2012. Revised April 25, 2012. Accepted May 16, 2012. Advance Access publication July 13, 2012ß The Author (2012). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
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Abbreviations: MMP = matrix metalloprotease; TUNEL = terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate-biotin nick end labelling In addition, fluoxetine is known to alleviatepost-stroke depression helps The blood–brain barrier including blood–spinal cord barrier is a motor recovery in stroke patients highly specialized brain endothelial structure of the fully differen- and facilitates cognition after traumatic brain tiated neurovascular system. The blood–brain barrier is primarily injury Recent reports also show that flu- formed by brain endothelial cells, which form a tight seal due to oxetine provides neuroprotective effect via its anti-inflammatory the presence of well-developed tight junction limiting the entry of effect after middle cerebral artery occlusion plasma components and blood cells into the brain or spinal cord.
and by inhibiting microglial activation in ischaemic injury and When damaged by various causes including traumatic spinal cord MPTP-induced Parkinson disease in animal models injury, the blood–brain barrier or blood–spinal cord barrier disrup- In addition, fluoxetine prevents lipopolysacchar- tion generates neurotoxic products that can compromise synaptic ide-induced degeneration of nigral dopaminergic neurons by in- and neuronal functions hibiting microglial activation followed oxidative stress and induces the ‘programmed death' of Based on these observations, fluoxetine appears neurons and glia, leading to permanent neurological deficits to have neuroprotective effects after ischaemic brain injury, but the mechanism of its action is unknown. Thus, we hypothesized preventing the blood–spinal cord barrier disruption should be con- that fluoxetine might exert neuroprotective effects by preserving sidered as a potential approach for therapeutic interventions after blood–spinal cord barrier integrity after spinal cord injury. Here, spinal cord injury.
we examined whether fluoxetine would prevent blood–spinal Matrix metalloproteases (MMPs), a family of zinc endopeptid- cord barrier disruption, inhibit MMP activity, attenuate cell ases, are known to degrade extracellular matrix and other extra- death and improve functional recovery after injury.
cellular proteins and are essential for remodelling of the extracellularmatrix, Materials and methods However, excessive proteolytic activity of MMP can bedetrimental, leading to numerous pathological conditions includ- Spinal cord injury ing blood–brain barrier or blood–spinal cord barrier disruption Adult male C57BL/6 (18–22 g, Samtako) mice were anaesthetized with after ischaemic brain injury and spinal cord injury chloral hydrate (500 mg/kg) and a laminectomy was performed at the T9 level, exposing the cord beneath without disrupting the dura.
The spinous processes of T8 and T11 were then clamped to stabilize mation ). For example, the spine, and the exposed dorsal surface of the cord was subjected to MMP9 induces proteolytic degradation of blood–brain barrier moderate contusion injury (50 kdyn force per 500 to 600 mm displace- and white matter components leading to an increase in infarct ment) using an Infinite Horizons impactor (Infinite Horizons Inc.). The volume after transient cerebral ischaemia incision sites were then closed in layers and a topical antibiotic MMP9 also plays a key role in abnormal vascular permeability (Bacitracin) was applied to the incision site. For the sham-operatedcontrols, the animals underwent a T9 laminectomy without contusion and inflammation early after spinal cord injury, and blocking of injury. Surgical interventions and postoperative animal care were per- MMP9 activity inhibits vascular permeability, thereby improving formed in accordance with the Guidelines and Policies for Rodent Survival Surgery provided by the Animal Care Committee of the upregulation of MMP12 after spinal cord injury induces an in- Kyung Hee University.
crease of blood–spinal cord barrier permeability followed micro-glia/macrophage activation and blood cell infiltration, thereby hindering recovery of motor function Furthermore, MMPs have been implicated in neurodegenerative Fluoxetine (Sigma) dissolved in sterile PBS was immediately adminis- disorders such as multiple sclerosis and Alzheimer's disease tered into injured mice via intraperitoneal injection (10 mg/kg) hypothesized that blocking MMP activity soon after spinal after spinal cord injury and then further treated once aday for 2 weeks for behavioural test or for indicated time points for cord injury would prevent the blood–spinal cord barrier disrup- other experiments. PBS was administered for vehicle control. For the tion, attenuate inflammatory response, reduce cell death and sham-operated controls, the animals underwent a T9 laminectomy thereby improve functional recovery.
without contusion injury and received no pharmacological treatment.
Fluoxetine as a selective serotonin reuptake inhibitor is most Significant side effects resulting from fluoxetine treatment such as commonly prescribed as an anti-depressant. It also exerts anti- changes in body weight or an increase in mortality were not observed inflammatory and pain-relieving effects during our experiments.
Fluoxetine inhibits BBB disruption Brain 2012: 135; 2375–2389 incubated at 37C for 60 min. After incubation, 50 ml of stop solutionwas added and mixed, and then the fluorescence intensity was mea- A transformed mouse brain endothelial cell line, bEnd.3 cells were sured at an excitation wavelength of 490 nm and an emission wave- purchased from ATCC. Details on bEnd.3 culture are provided in the length of 520 nm.
Measurement of blood–spinal cord Tissue preparation barrier disruption Tissue preparation was performed as previously described ( The integrity of the blood–spinal cord barrier was investigated with Evan's Blue dye extravasation according to previous reports with a few modifications. At 1 day after spinal cord injury, 0.5 ml of 2% Evan's Blue dye (Sigma) solutionin saline was administered intraperitoneally. Three hours later, animals Total protein was prepared with a lysis buffer containing 50 mM were anaesthetized and killed by intracardiac perfusion with saline.
Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, The T9 spinal cord segment was removed and homogenized in a 0.1% SDS, 10 mM Na2P2O7, 10 mM NaF, 1 mg/ml aprotinin, 50% trichloroacetic acid solution. After homogenization, samples 10 mg/ml leupeptin, 1 mM sodium vanadate and 1 mM PMSF. Tissue were centrifuged at 10 000g for 10 min, supernatants were collected homogenates were incubated for 20 min at 4C, and centrifuged at and fluorescence was quantified at an excitation wavelength of 25 000g for 30 min at 4C. Protein extraction of both the nuclear and 620 nm and an emission wavelength of 680 nm. Dye in samples was cytosolic fractions was performed as previously described determined as mg/g of tissue from a standard curve plotted using The protein concentration was determined using the BCATM known amounts of dye For qualitative examin- assay kit (Pierce). Protein samples (10–40 mg) were separated on ation of Evan's Blue extravasation, the animals were perfused with PBS SDS–PAGE and transferred to nitrocellulose membrane (Millipore).
and subsequently with 4% formaldehyde, as described above. The The membranes were blocked in 5% non-fat skimmed milk or 5% spinal cords were sectioned 20-mm thick with a cryostat. The fluores- bovine serum albumin in Tris-buffered saline solution with Tween cence of Evan's Blue in spinal tissues was observed with a fluorescence (TBST) for 1 h at room temperature followed by incubation with anti- microscope and the relative fluorescence intensity was determined by bodies against cleaved caspase 3 (1:1000, Cell Signalling Technology), MetaMorph software (Molecular devices).
occludin (1:1000, Invitrogen), zona occludens-1 (ZO-1, 1:1000, Invitrogen), nuclear factor kappa B (NF-B, 1:500, Santa CruzBiotechnology), inducible nitric oxide synthase (iNOS, 1:10 000, Transduction Laboratory) and COX2 (1:1000, Cayman Chemicals).
Frozen sections were processed for immunofluorescence staining or double labelling with antibodies against ZO-1 (1:300, Invitrogen), CD31 (1:200, Invitrogen), MMP9 (1:100, Santa Cruz Biotechnology), ImmunoResearch). Immunoreactive bands were visualized by chemilu- neuron specific enolase (1:100, Dako Corporation), Gr-1 (1:100, Invitrogen), laminin -1 (1:100, Santa Cruz Biotechnology), or laminin (1:10 000; Sigma) or Histone 3 (1:1000, Cell Signalling Technology) -4 (1:100, Santa Cruz Biotechnology). FITC or cy3-conjugated sec- were used as an internal control. Experiments were repeated three ondary antibodies were used (Jackson ImmunoResearch). Nuclei were times and the densitometric values of the bands on western blots labelled with DAPI (406-diamidino-2-phenylindole) according to the obtained by AlphaImagerÕ software (Alpha Innotech Corporation) manufacturer's protocol (Molecular Probes). The immunohistochemis- were subjected to statistical analysis. Background in films was sub- try control study was performed by omission of the primary antibodies; tracted from the optical density measurements.
by replacement of the primary antibodies with non-immune, controlantibody, and by pre-absorption with an excess (10 mg/ml) of the re- Gelatin zymography spective antigens. Some serial sections were also stained for histolo-gical analysis with Cresyl violet acetate.
The activity of MMP2 and 9 at 1 day after injury was examined bygelatin zymography based on a previously described method with minor modifications (Supplementary material).
Fluorimetric assay for matrix triphosphate-biotin nick end labelling metalloprotease 9 activity MMP9 activity was measured using the SensoLyteÕ 520 MMP-9 Assay One day after injury, serial spinal cord sections (10 mm thickness) were Kit according to the manufacturer's protocol (AnaSpec). Briefly, spinal collected every 100 mm and processed for terminal deoxynucleotidyl cords were homogenized with the assay buffer containing 0.1% TritonTMX-100, and centrifuged for 15 min at 10 000g at 4C. The labelling (TUNEL) staining using an ApopTagÕ in situ kit (Chemicon supernatant was collected and stored at 70C until use. To activate MMP prior to assay, the supernatants were incubated with Laboratories) was used for peroxidase staining, and the sections 4-aminophenylmercuric acetate for 1 h at 37C. Then, MMP-contain- were then counterstained with methyl green. Control sections were ing sample was added to a 96-well plate (50 ml/well) and MMP9 incubated in the absence of terminal deoxynucleotidyl transferase substrate (50 ml/well) was added to the sample and control wells.
enzyme. Investigators who were blind as to the experimental condi- The reagents were mixed by shaking the plate gently for 30 s and tions carried out all TUNEL analyses. TUNEL-positive cells in the grey Brain 2012: 135; 2375–2389 J. Y. Lee et al.
matter at 1 day after spinal cord injury (total 40 sections) were Behavioural tests counted and quantified using a 20  objective. Only those cells show-ing morphological features of nuclear condensation and/or compart- Locomotor outcome after spinal cord contusion injury was assessed mentalization in the grey matter were counted as TUNEL-positive.
using the Basso Mouse Scale Mice were scoredin an open-field environment by trained investigators who were blindto the experimental conditions. Consensus scores for each animal were RNA isolation and reverse transcriptase averaged at each time point for a maximum of nine points for the polymerase chain reaction Basso Mouse Scale score and 11 points for the subscore, whichassesses finer aspects of locomotion.
Complementary DNA synthesis and reverse transcriptase PCR were Statistical analysis performed as previously described (Supplementarymaterial).
Data, except behaviour tests, are presented as the mean  SD valuesand behavioural data presented as the mean  SEM. Comparisons be- tween vehicle and fluoxetine-treated groups were made by unpaired Enzyme-linked immunosorbent assay Student's t-test. Behavioural scores from the Basso Mouse Scale ana-lysis were analysed by repeated measures ANOVA (Time versus The levels of cytokines (TNF , IL1b and IL6) were assayed using cyto- Treatment). Tukey's multiple comparison was used as a post hoc ana- kine ELISA kits (BioSource Europe). Mice were deeply anaesthetized lysis. Statistical significance was accepted with P 5 0.05. Statistical with chloral hydrate (500 mg/kg, intraperitoneally) and cardinally per- analyses were performed using SPSS 15.0 (SPSS Science).
fused. The spinal cord was immediately removed after perfusion,weighed and frozen in liquid nitrogen for storage at 80C. To de-termine the plasma levels of cytokines, blood samples were collected from the femoral vein at 1 day and 5 days after injury, and serum wasisolated by centrifugation and stored at 80C. Tissues were alsohomogenized and the levels of TNF , IL1b and IL6 were assayed by Fluoxetine inhibits the expression and ELISA and determined according to the manufacturer's instructions. All activation of matrix metalloproteinases samples were analysed in triplicate.
2, 9 and 12 after spinal cord injury Mice were subjected to contusive injury (spinal cord injury) at T9 level and killed at indicated time points after injury. Total RNA andtissue extracts from spinal cord (8 mm in length) including lesion Flow cytometry was performed as previously described epicentre were prepared as described above. First, we examined Spinal cords (5 mm, one spinal cord per sample) in PBS MMP2, 9 and 12 messenger RNA expression after injury by re- were mechanically disrupted with a small glass Dounce homogenizer, verse transcriptase PCR (n = 3). As shown in messenger and single-cell suspensions were obtained by passing the solution RNA levels of MMP2, 9 and 12 increased after injury. In addition, through a wire mesh screen (70 mm in size; Sigma). Spinal cord sam- fluoxetine significantly inhibited MMP2 and MMP9 messenger ples were subjected to centrifugation at 4C at 200g for 10 min (low RNA expression at 4 and 8 h, and MMP12 at 3 and 5 days brake). Pellets were resuspended in foetal bovine serum staining buffer after injury as compared to vehicle controls Next, (BD Biosciences) and were subjected to centrifugation (1200g) for we analysed MMP2 and 9 activities by gelatin zymography. As 7 min, slow brake at 4C). Pellets were then resuspended in foetal shown in MMP9 activity was markedly increased at 8 h bovine serum staining buffer. Spinal cord samples were split into sev- and 1 day after injury, with bands corresponding to the MMP9 eral tubes, and cells alone and isotype-matched control samples weregenerated from 10 ml of each sample (mix of all spinal cord samples) to active form and the inactive zymogen (pro-MMP9). This activity control for non-specific binding and autofluorescence. The following decreased by 5 days after injury. Pro-MM2 appeared in all injured isotype control antibodies were used: phycoerythrin-labelled rat IgG2b, samples and active MMP2 was detected at 5 days after injury K, fluorescein isothiocyanate (FITC)-labelled rat IgG2b, K, and peridi- Furthermore, fluoxetine significantly decreased the level (Pharmingen). Cell counts were performed by adding 10 ml of trypan of pro-MMP9, active MMP9 and pro = MMP2 as compared to blue to 10 ml of each sample to optimize antibody dilutions. Fc block vehicle controls at 1 day after injury and C) (n = 4, (BD Biosciences) was added to each sample (10 min at 4C) to min- pro-MMP9, vehicle, 2.5  0.3 versus fluoxetine, 1.2  0.6; imize background staining. After incubation with combinations of anti- active MMP9, vehicle, 6.8  0.2 versus fluoxetine, 3.0  0.3; bodies for 30 min at 4C, the samples were washed twice in foetal pro-MMP2, vehicle, 3.2  0.44 versus fluoxetine, 1.9  0.3, bovine serum staining buffer and resuspended in 1% buffered forma- P 5 0.05). In addition, we confirmed the effect of fluoxetine on lin. The antibodies used were anti-Gr-1 FITC, anti-CD11b FITC and MMP2 and 9 activities by using the fluorimetric enzyme activity anti-CD45 PerCP (BD Bioscience). All samples were then immediatelyanalysed with a Becton Dickinson LSR Benchtop Flow Cytometer (BD assay kit. As shown in fluoxetine significantly inhibited Biosciences). Forward scatter was adjusted to minimize cellular debris, MMP9 activity at 1 day after injury as compared to vehicle con- and propidium iodide exclusion was used to determine cell viability. A trols. However, we were unable to show MMP2 activity at 1 day minimum of 250 000 cells from spinal cord samples and 50 000 cells after injury due to its low activity (data not shown). Double label- from blood samples were analysed.
ling shows that MMP9 was localized in neuron specific

Fluoxetine inhibits BBB disruption Brain 2012: 135; 2375–2389 Figure 1 Fluoxetine inhibits disruption of tight junction after spinal cord injury. Total spinal extracts were prepared at the time pointsindicated (8 h, 1 day, 3 days or 7 days after injury) as described in the Materials and methods. Mice were treated with fluoxetine (10 mg/kg) and total spinal extracts or tissue sections at 1 day after injury were prepared (n = 4/group). (A) Western blots of occludin and ZO-1 at8 h to 7 days after SCI. (B) Western blots of occludin and ZO-1 of vehicle or fluoxetine treated animals at 1 day after injury. (C)Densitometric analyses of western blots. Data represent mean  SD. *P 5 0.05 versus vehicle control. (D) Representative micrographsshowing double immunofluorescence with ZO-1 and CD31 antibodies (endothelial cell marker) at 500 mm caudal to the lesion epicentre.
Scale bar = 10 mm.
enolase-positive motor neurons of ventral horn, Gr-1-positive neu- increased by 1 h of reoxygenation after oxygen–glucose depriv- trophils and CD31-positive endothelial cells at 1 day after injury ation, whereas fluoxetine significantly inhibited NFB translocation However, MMP9 was not detected in uninjured, normal and B). To confirm the involvement of NFB, we exam- or sham spinal cord (data not shown).
ined the effect of MG-132 (a proteasome inhibitor that inhibitsdegradation of IB and suppresses NFB translocation to the nu-cleus) on MMP9 induction. In bEnd.3 cells, MMP9 messenger Fluoxetine inhibits nuclear factor B RNA expression was increased at 1 h reoxygenation after oxy- dependent matrix metalloproteinase 9 gen–glucose deprivation for 6 h as compared with controls, expression and loss of tight junction which was reversed by pretreatment with MG-132. In addition,fluoxetine treatment significantly inhibited MMP9 messenger RNA proteins in endothelial cells after expression These findings indicate that the induction of MMP9 by oxygen–glucose deprivation/reoxygenation occurs through NFB activation and fluoxetine inhibits MMP9 expressionin part by inhibiting the NFB pathway. Next, we examined NFB signalling pathway has been known as one of the MMP9 whether fluoxetine affects the alteration of tight junction proteins expression regulators To investigate how fluoxetine inhibits MMP9 expression in endothelial cells, we blood–brain barrier disruption is known to be associated with deg- applied oxygen–glucose deprivation and reoxygenation to bEnd.3 radation of endothelial tight junction proteins as well as decrease cells, a mouse brain endothelial cell line. As shown in in their expression after CNS injuries, including spinal cord injury western blots of nuclear extracts with anti-p65 antibody showed As shown in and E, that the level of translocated NFB into the nucleus was markedly the levels of occludin and ZO-1 decreased at 6 h after Brain 2012: 135; 2375–2389 J. Y. Lee et al.
Figure 2 Fluoxetine inhibits MMP2 and 9 activation after spinal cord injury. After spinal cord injury, mice were treated with fluoxetineand gelatin zymography was performed with protein lysates prepared at indicated time points (n = 4/group). (A) Gelatin zymographyshowing temporal profile of MMP2 and 9 activities after injury. (B) The effect of fluoxetine on MMP2 and 9 at 1 day after injury.
(C) Densitometric analyses of zymography. (D) MMP9 activity was measured fluorometrically using a 5-carboxyfluorescein/QXL520fluorescence resonance energy transfer peptide. The change in fluorescence for 60 min was measured using a luminescence spectrometer (n = 5/group) and indicated as relative fluorescence units (RFU). (E) Representative fluorescence microscopic photographs showing thatneuron specific enolase-positive neurons, Gr-1-positive neutrophils and CD31-positive endothelial cells are positive for MMP9 at 1 dayafter injury. Scale bar = 30 mm. All data represent mean  SD. *P 5 0.05 versus vehicle control. NSE = neuron specific enolase.
reoxygenation. However, the decrease in occludin or ZO-1 level after also showed that the fluorescence intensity of ZO-1 and CD31 oxygen–glucose deprivation/reoxygenation was significantly re- immunoreactivity was decreased after injury as compared to versed by fluoxetine treatment. Immunocytochemistry also revealed sham controls, and fluoxetine treatment attenuated the decrease that the intensity of ZO-1 expression decreased by oxygen–glucose in its intensity These data indicate that fluoxetine pre- deprivation/reoxygenation as compared to oxygen–glucose depriv- serves tight junction integrity by inhibiting degradation of tight ation/reoxygenation-treated control cells, and fluoxetine treatment junction molecules, and thereby prevents blood–spinal cord barrier prevented the loss of ZO-1 expression disruption after spinal cord injury.
Fluoxetine inhibits tight junction Fluoxetine inhibits the increase of disruption after spinal cord injury blood–spinal cord barrier permeabilityafter spinal cord injury Next, we examined the alterations of spinal cord injury-inducedtight junction proteins and the effect of fluoxetine on these alter- It is well known that the tight junction in the endothelial cells of ations by western blot. We previously found that the antibodies blood vessels is involved in the integrity of blood–brain barrier or against occludin and ZO-1 showed specific immunoreactivity at blood–spinal cord barrier After spinal cord injury, the disruption of blood–spinal cord barrier by MMP activation is 220 kDa for ZO-1) (data not shown). In addition, the level of also well documented Since fluoxet- occludin or ZO-1 was decreased and the decrease was especially ine inhibited MMP expression and activities and and prominent at 1 and 3 days after injury Furthermore, preserved ZO-1 and occludin in endothelial cells we ex- fluoxetine significantly attenuated the decrease in occludin and pected that fluoxetine would inhibit blood–spinal cord barrier per- ZO-1 levels at 1 day after injury as compared with vehicle controls meability after spinal cord injury. Thus, we examined the effect of and C) (n = 4, occludin, vehicle, 0.27  0.12 versus flu- fluoxetine on blood–spinal cord barrier permeability at 1 day after oxetine, 0.83  0.07; ZO-1, vehicle, 0.15  0.08 versus fluoxet- injury by Evan's Blue assay (n = 5). As shown in spinal ine, 0.65  0.05; P 5 0.05). Double labelling immunofluorescence cord injury caused a marked increase in the amount of Evan's Blue Fluoxetine inhibits BBB disruption Brain 2012: 135; 2375–2389 Figure 3 Fluoxetine inhibits NFB dependent MMP9 expression and loss of tight junction proteins in endothelial cells after oxygen–glucose deprivation/reoxygenation. bEnd.3 cells were pretreated with vehicle, fluoxetine (50 mm) or MG-132 (10 mm) for 30 min beforeoxygen–glucose deprivation. (A) Western blot analysis of NFB in nuclear and cytoplasmic extracts using NFB p65 antibody at 1 hreoxygenation after oxygen–glucose deprivation for 6 h. Beta-tubulin and histone 3 were used as internal controls (CTR) of protein loadingfor cytosol and nuclear fraction, respectively. (B) Densitometric analyses of western blots for translocated (activated) NFB. (C) Reverse transcriptase PCR for MMP9 at 1 h reoxygenation after oxygen–glucose deprivation (OGD) for 6 h. (D) Western blots for ZO-1 andoccludin in bEnd.3 cell lysate treated with vehicle or fluoxetine (Flu) at 6 h reoxygenation after oxygen–glucose deprivation for 6 h.
(E) Densitometric analyses of western blots. (F) Immunocytochemistry from fixed bEnd.3 cells at 6 h reoxygenation after oxygen–glucosedeprivation for 6 h. Scale bar = 30 mm. All data represent mean  SD from five separate experiments. *P 5 0.05 versus vehicle control.
dye extravasation compared with the uninjured, sham controls, examined the effect of fluoxetine treatment on blood cell infiltra- which implies blood–spinal cord barrier leakage. Furthermore, flu- tion by fluorescence-activated cell sorting and expression of in- oxetine (10 mg/kg) treatment significantly reduced the amount of flammatory mediators by reverse transcriptase PCR, western Evan's Blue dye extravasation at 1 day after injury when compared blot, and ELISA assay at indicated time points after injury. In gen- with vehicle controls (n = 5, vehicle, 27.5  0.8 versus fluoxetine, eral, neutrophils at 1 day and macrophages at 5 days infiltrated 10.5  1.8, P 5 0.05) Qualitative analysis also shows after injury are known to mediate inflammatory responses that the fluorescence intensity of Evan's Blue in the injured Thus, we measured the number of blood cells infil- spinal cord (at 1 day) was higher than sham controls, and fluox- trated into injured spinal cord at 1 and 5 days after injury by etine significantly reduced the fluorescence intensity (n = 4, ve- fluorescence-activated cell sorting analysis. Only double positive hicle, 118  8.1 versus fluoxetine, 42  3.0, P 5 0.05) cells (Gr-1/CD45 for neutrophil at 1 day and CD45/CD11b for and D). These data indicate that fluoxetine inhibits the blood– macrophage at 5 days) were counted and analysed as described spinal cord barrier permeability after injury.
As shown in the number ofneutrophils and macrophages infiltrated was dramatically increased Fluoxetine inhibits blood cell infiltration after injury as compared with sham controls, and fluoxetine sig-nificantly attenuated the increase in the blood cell infiltration as and the expression of cytokines and compared with vehicle controls at indicated time points after injury inflammatory mediators after spinal (n = 4, CD45/Gr-1-positive cells, vehicle, 100  4.7 versus fluox- 99.5  10.7 versus fluoxetine, 32.2  2.9%; P 5 0.05). In add- After spinal cord injury, the blood cell infiltration following blood– ition, the expression levels of TNF , IL1b (at 2 h), IL6, inducible spinal cord barrier disruption initiates inflammatory responses, nitric oxide synthase and COX2 (at 6 h) messenger RNA were leading to the secondary injury cascade by producing inflamma- upregulated after injury, but fluoxetine treatment significantly tory mediators such as IL1b, IL6, TNF , COX2 and inducible nitric reduced their levels as compared with vehicle controls (n = 3) oxide synthase Therefore, we and B). ELISA assays also showed that fluoxetine Brain 2012: 135; 2375–2389 J. Y. Lee et al.
Figure 4 Fluoxetine inhibits disruption of tight junction after spinal cord injury. After injury, mice were treated with fluoxetine (10 mg/kg)and total spinal extracts or tissue sections at 1 day after injury were prepared as described (n = 4/group). (B) Western blots of occludin andZO-1 at 1 d after injury. (C) Densitometric analyses of western blots. Data represent mean  SD. *P 5 0.05 versus vehicle control.
(D) Representative micrographs showing double immunofluorescence with ZO-1 and CD31 (endothelial cell marker) at 500 mm caudal tothe lesion epicentre. Scale bar = 10 mm.
n = 5, P 5 0.05) By western blot, the protein levels ofinducible nitric oxide synthase and COX2 at 1 day after injurywere significantly decreased by fluoxetine treatment as comparedwith vehicle controls (n = 5, P 5 0.05) and E). In addition,the plasma levels of cytokines were increased after spinal cord injury, whereas fluoxetine did not affect the plasma levels ofthese cytokines (Supplementary Fig. 1) (1 day: TNF , IL1b andIL6 of sham group, 1.7  0.3 pg/ml, 0.4  0.4 pg/ml, 3.3 0.99 pg/ml; vehicle group, 2.3  0.8 pg/ml, 1.4  0.56 pg/ml,17.5  3.3 pg/ml; 0.6 pg/ml, 16.8  2.7 pg/ml, respectively; n = 10, P 5 0.05). Thissuggests that fluoxetine may not affect systemic inflammationresponses after injury.
Fluoxetine inhibits the expression ofchemokines induced after spinalcord injury The expression of chomokines such as Gro- (CXCL1), MCP1 Figure 5 Fluoxetine inhibits the increase in blood–spinal cord (CCL2) and MIP1 (CCL3) is known to increase early after barrier permeability after spinal cord injury. After spinal cord spinal cord injury and induces the infiltration of blood cells such injury, mice were treated with fluoxetine and blood–spinal cord as neutrophils and macrophages, thereby facilitating inflammatory barrier permeabitliy was measured at 1 day after injury by usingEvan's Blue dye (n = 5/group). (A) Representative whole spinal cords showing Evan's Blue dye permeabilized into spinal cord at Since our data showed that fluoxetine reduced 1 day. (B) Quantification of the amount of Evan's Blue.
the number of infiltrated blood cells after spinal cord injury, (C) Representative confocal images of an Evan's Blue extrava- we anticipated that fluoxetine would decrease chemokine expres- sation at 1 mm caudal to the lesion epicentre at 1 day after spinal sion after spinal cord injury. Thus, we examined the effect of cord injury. (D) Quantification of the fluorescence intensity of fluoxetine on the expression of chemokines such as Gro- , Evan's Blue. All data represent mean  SD. *P 5 0.05 versus MIP2 (CXCL2), MCP1, MIP1 and MIP1b (CCL4) by reverse vehicle controls.
transcriptase PCR (n = 3). As shown in the expressionof Gro- , MIP2 and MCP1 messenger RNA increased at 4 h, significantly inhibited the production of TNF , IL1b and IL6 1 day 8 h and 1 day after injury, respectively, and then decreased after injury (TNF , IL1b, and IL6 of vehicle group: 39  4.4 pg/ml, while the expression of MIP1 and MIP1b messenger RNA increased 4 h after injury and then maintained up to 5 days after injury, as reported Fluoxetine inhibits BBB disruption Brain 2012: 135; 2375–2389 Figure 6 Fluoxetine inhibits infiltration of neutrophils and macrophages after spinal cord injury. After spinal cord injury, mice were treated with fluoxetine (10 mg/kg) and blood cells infiltrated into injured spinal cord at 1 and 5 days were prepared and analysed by flowcytometry after staining with Gr-1/CD45 (neutrophil markers) and CD11b/CD45 (macrophage markers) antibodies (n = 4/group).
Density plots of the gated region without fluorescent-conjugated antibodies (A, G) or with isotype-matched control antibodies (B, C, H, I)as control experiments, and density plot of representative sham (D, J), vehicle (E, K) and fluoxetine (F, L) treated spinal cord samples.
(M–N) Quantification of Gr-1high/CD45high for neutrophils (M) at 1 day and CD11bhigh/CD45high for macrophages (N) at 5 days afterinjury as a percentage of vehicle controls. Data represent mean  SD. *P 5 0.05 versus vehicle controls.
Furthermore, messenger RNA expression of Gro- , TUNEL-positive cells were observed mostly near and within the MIP1 and MIP1b at 8 h after injury was significantly inhibited by lesion area in the grey matter at 1 day Fluoxetine treat- fluoxetine treatment and C, n = 3). However, MIP2 and ment significantly decreased the number of TUNEL-positive cells MCP1 messenger RNA expression was not affected by fluoxetine when compared with the vehicle-treated controls (vehicle, (data not shown). These data suggest that fluoxetine might reduce 133.5  12.3 versus fluoxetine, 51.5  8.5 cells, n = 5, P 5 0.05) the number of infiltrating blood cells by attenuating the expression In this regard, double-labelling confirms that most of chemokines such as Gro- , MIP1 and MIP1b after spinal cord TUNEL-positive cells in the grey matter were neurons at 1 day after injury (data not shown) as previously reported In addition, the level of cleaved (acti- Fluoxetine inhibits caspase 3 activation vated) forms of caspase 3 increased at 4 h after injury and apoptotic cell death after spinal and fluoxetine significantly decreased the levelof activated caspase 3 at 4 h after injury when compared with vehicle controls and D) (vehicle, 4.3  0.4 versus fluox-etine, 2.6  0.25, n = 3, P 5 0.05). Thus, our results indicate that Recent reports show that fluoxetine provides a neuroprotective fluoxetine inhibits apoptotic cell death after injury.
effect by its anti-inflammatory effect after middle cerebral arteryocclusion and by inhibiting microglial activationin ischaemic and MPTP-induced Parkinson disease model Fluoxetine improves functional recovery Thus, we examined the effect of fluoxetine on apop- after spinal cord injury totic cell death in the grey matter at 1 day after spinal cord injuryby TUNEL staining. Serial transverse sections (10 mm thickness) After spinal cord injury, mice were immediately treated with flu- were collected every 100 mm from 2 mm rostral to 2 mm caudal oxetine (10 mg/kg, intraperitoneally) and further treated once a day for 2 weeks. Functional recovery was then evaluated using the

Brain 2012: 135; 2375–2389 J. Y. Lee et al.
Figure 8 Fluoxetine inhibits the expression of chemokinesinduced after spinal cord injury. Total RNA from vehicle or Figure 7 Fluoxetine inhibits the expression of cytokines and fluoxetine-treated samples at indicated time points after injury inflammatory mediators after spinal cord injury. Total RNA and were prepared (n = 3/group). (A) Reverse transcriptase PCR of protein extracts from vehicle or fluoxetine-treated spinal cords at Gro- , MIP2 , MCP1, MIP1 and MIP1b messenger RNA ex- indicated time points after injury were prepared. (A) Reverse pression after injury. (B) The effect of fluoxetine on Gro- , transcriptase PCR of TNF , IL1b (at 2 h), IL6, COX2 and indu- MIP1 and MIP1b expression at 1 day after injury.
cible nitric oxide synthase (iNOS) (at 6 h) in sham, (C) Quantitative analysis of reverse transcriptase PCR. Data vehicle-treated and fluoxetine-treated spinal cords after injury represent mean  SD. *P 5 0.05 versus vehicle controls.
(n = 3/group). (B) Quantitative analysis of reverse transcriptasePCR. (C) ELISA of TNF , IL1b, IL6 at 1 day in sham,vehicle-treated and fluoxetine-treated spinal cords after injury(n = 5/group). (D) Western blots of inducible nitric oxide syn- neutrophils and macrophages, after injury by inhibiting expression thase and COX2 at 1 day in sham, vehicle-treated and of chemokines such as Gro- , MIP1 and MIP1b, resulting in fluoxetine-treated spinal cords after injury (n = 5/group).
(E) Quantitative analyses of western blots. Data represent reduced inflammatory responses. Furthermore, post-injury treat- mean  SD. *P 5 0.05 versus vehicle controls.
ment with fluoxetine inhibited apoptotic cell death and improvedfunctional recovery after spinal cord injury. Here, we present clearevidence for the mechanism of action of fluoxetine on the expres-sion and/or activity of MMP, which affects the blood–brain 9-point Basso Mouse Scale score and 11-point Basso Mouse Scale barrier/blood–spinal cord barrier integrity after injury. Our findings subscore for locomotion. As a result, fluoxet- have important implications in traumatic and ischaemic brain inju- ine treatment significantly increased the hindlimb locomotor func- ries and spinal cord injury in which the disruption of the blood– tion 10 to 28 days after injury, compared with that observed in brain barrier integrity triggers a secondary degenerative cascade vehicle-treated controls (n = 15/group, 28 days, Basso Mouse including inflammation in pathological processes Scale score, fluoxetine, 7.0  0.26 versus vehicle 4.3  0.31; Basso Mouse Scale subscore, fluoxetine, 4.5  0.3 versus vehicle 0.9  0.22, P 5 0.05) and B).
Under physiological conditions, the blood–brain barrier/blood– spinal cord barrier represents a tight barrier between the circulat-ing blood and CNS and is formed by dense tight junction proteins, which seal the space between adjacent brain endothelial cells.
Disruption of the blood–brain barrier occurs under various patho- In the present study, we demonstrate that fluoxetine, known as a logical conditions such as stroke and spinal cord injury, leading to representative anti-depressant drug, exerts neuroprotective effects an increased cerebrovascular permeability with subsequent devel- by preventing blood–spinal cord barrier disruption and inhibiting opment of tissue oedema Although MMP activation after spinal cord injury. In addition, we show that many factors are known to contribute to blood–brain barrier dis- fluoxetine reduces the number of infiltrating blood cells, such as ruption, MMPs play a critical role in the blood–brain barrier/

Fluoxetine inhibits BBB disruption Brain 2012: 135; 2375–2389 Figure 9 Fluoxetine inhibits caspase 3 activation and apoptotic cell death after spinal cord injury. After spinal cord injury, mice were treated with fluoxetine and spinal tissues and extracts were prepared for TUNEL staining and western blot. (A) Representative images ofTUNEL staining at 1 day after spinal cord injury. Scale bar = 20 mm. (B) Quantitative analysis of TUNEL-positive cells (n = 5/group).
(C) Western blots of cleaved caspase 3 at 4 h after spinal cord injury. (D) Quantitative analyses of western blots (n = 3/group). Datarepresent means  SD. *P 5 0.05 versus vehicle controls.
Figure 10 Fluoxetine improves functional recovery after spinal cord injury. After spinal cord injury, mice were treated with fluoxetine andfunctional recovery was assessed with the Basso Mouse Scale (BMS) score (A) and Basso Mouse Scale subscore (B). Each value representsthe mean  SEM (n = 15/group). *P 5 0.05, **P 5 0.01 versus vehicle controls.
blood–spinal cord barrier disruption in pathological conditions expression and activity of MMPs after injury and After spinal cord injury, upregulation of MMP9 has been impli- our results show that messenger RNA expression and enzyme cated in spinal cord injury-induced secondary damage and blood– activity of MMPs were upregulated after spinal cord injury.
spinal cord barrier disruption by degrading the basal components Furthermore, fluoxetine treatment significantly inhibited the of blood–brain barrier and facilitating immune cell infiltration Brain 2012: 135; 2375–2389 J. Y. Lee et al.
MMP9 is also shown to mediate Furthermore, fluoxetine treatment significantly in- hypoxia-induced vascular leakage in the brain via tight junction hibited fragmentation of capillaries as compared with vehicle con- rearrangement Several reports have also trol (Supplementary Fig. 2).
demonstrated that the upregulation of MMP2 contributes to the Chemokines are known to mediate chemotaxis and leukocyte initial opening of the blood–brain barrier by degrading the basal activation and induce blood cells lamina leading to neuronal injury such as neutrophils and macrophages to migrate along concentra- In addition, minocycline treatment protects the blood– tion gradients to the lesion site Extensive evidence has shown that inflammatory cells such as neu- haemorrhage in the rat, by inhibiting MMP12 messenger RNA trophils and macrophages are infiltrated via blood–spinal cord bar- expression MMP12 is also rier disruption, increase tissue damage, induce apoptotic cell death known to be upregulated after spinal cord injury and mechanis- and impair functional recovery after injury tically, there is decreased permeability of the blood–spinal cord barrier and reduced neutrophil and macrophage density in Our data show that chemokines such as Gro- , MIP1 , MIP1b, MMP12 null mice when compared with wild-type controls MIP2 and MCP1 were upregulated after injury and Thus, these results suggest that fluoxetine fluoxetine significantly inhibited upregulation of Gro- , MIP1 effectively prevents blood–spinal cord barrier disruption, in part and MIP1b and C) and thereby reduced the number of by inhibiting the expression and activity of MMPs after spinal infiltrating blood cells In parallel with this result, the ex- cord injury.
pression of inflammatory mediators such as TNF , IL1b, IL6, in- It is known that tight junction proteins such as occludin, ducible nitric oxide synthase, and COX2, was upregulated after claudin 5 and ZO-1 are essential components of the blood–brain injury, which was significantly reduced by fluoxetine Thus, barrier or blood–spinal cord barrier and known to be substrates of these results suggest that fluoxetine may inhibit inflammatory re- MMPs Our data show that occludin and ZO-1 sponses by attenuating expression of chemokines and reducing were rapidly degraded at soon (8 h to 1 day) after spinal cord blood cell infiltration following production of inflammatory medi- injury and fluoxetine significantly inhibited degradation ators after spinal cord injury.
of these molecules and C). It has been shown that blood– show that the number of infiltrating neu- brain barrier disruption induced by transient focal cerebral ischae- trophils significantly correlates with the amount of tissue damage mia is attenuated in MMP9 knockout mice by reducing degrad- after spinal cord injury. Several studies also demonstrate that sup- ation of ZO-1 protein compared with wild-type mice pression of neutrophil and macrophage infiltration after injury MMP2 and 9 secreted by leukaemic cells also increase the ameliorates apoptotic cell death and improves functional recovery permeability of the blood–brain barrier by disrupting tight junction proteins Thus, these results indicate that flu- this regard, our results also show that apoptotic cell death was oxetine might prevent blood–spinal cord barrier disruption by increased after injury and fluoxetine significantly attenu- reducing degradation of tight junction proteins via inhibition of ated apoptotic cell death and improved functional recovery MMP2 and 9 activities after spinal cord injury. On the other after spinal cord injury Thus, our data suggest that the hand, it is not known whether MMP12 could cleave tight junction neuroprotective effect of fluoxetine might be mediated in part by proteins, although it is shown to cleave elastin, one of the matrix preventing blood–spinal cord barrier disruption following infiltra- proteins However, we can't rule out the tion of neutrophils and macrophages after spinal cord injury.
possibility that upregulation of MMP12 may be involved in the The neuroprotective effect of fluoxetine can be mediated in part degradation or cleavage of tight junction proteins, thereby disrup- by either its primary effect on immune cells and their activation tingthe blood–brain barrier integrity after spinal cord injury.
outside of the brain, or its direct effect on brain cells expressing It is known that the infiltration of blood leukocytes is mediated MMPs, tight junction proteins and/or proinflammatory cytokines, through two passage processes: the passage of the vascular wall into perivascular spaces and the passage of the glia limitans. Since the respective basement membranes are composed of different shown in Supplementary Fig. 1, fluoxetine does not affect sys- laminin isoforms and only the parenchymal basement membrane temic inflammation after injury as indicated by the plasma levels is linked to dystroglycan the broad MMP of cytokines. Thus, we postulate that the direct effects of fluox- inhibitor BB-94 blocks the second step only etine on CNS cells expressing MMPs, tight junction proteins and Thus, it is very important to define these processes in vari- inflammatory cytokines are likely to be involved in its neuropro- ous vascular diseases, such as autoimmune diseases including mul- tective effect after spinal cord injury, although we can't exclude the possibility of a primary effect of fluoxetine on immune cells However, blood vessels including capillaries are ruptured outside of the CNS.
immediately by mechanical injury itself and further fragmented In conclusion, this study examined the protective effect of flu- by various factors such as metalloproteases and SUR1 induced oxetine on MMPs and blood–spinal cord barrier integrity after by spinal cord injury spinal cord injury. Our study shows that fluoxetine attenuated thereby, further increasing blood infiltration. In this study, blood–spinal cord barrier disruption and inhibited MMP2, 9 and we also found that the fragmentation of blood vessels was 12 activities and improved functional recovery after spinal cord increased after spinal cord injury as previously reported injury. Fluoxetine is currently used as an anti-depressant.
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