Effects of combination of melatonin and dexamethasone onsecondary injury in an experimental mice model of spinal cordtrauma

Introduction

After acute injury, the spinal cord undergoes biochemicaland cellular sequential pathologic changes, including hem-orrhage, edema, axonal and neuronal necrosis, and demy-elination, followed by cyst formation and infarction [1].

Although the precise mechanisms of the secondary injuryprocesses leading to progressive necrosis and motor dys-function are not fully known, several experimental findings

suggest that vascular damage plays a major role [2, 3].Neutrophils are the first leukocytes to arrive within theinjured spinal cord [4, 5]. In this setting, neutrophils release

neutral proteases and reactive oxygen species (ROS) [6]. Inthis regard, the production of ROS such as superoxideanions, hydrogen peroxide, and peroxynitrite, is also

associated with local and systemic inflammatory responseas well as the neurodegenerative disease [7]. Melatonin, aproduct of tryptophan metabolism in the pineal gland, is apotent free radical scavenger and antioxidant [8]. Several

studies indicate that melatonin produces neuroprotectiveeffects in vitro and in vivo ischemia/hypoxia models [9]. Ithas been suggested that melatonin may also have strong

anti-inflammatory actions [10]. In previous study, we havedemonstrated that melatonin exerts a beneficial effect in anSCI model [11] as well as in the inflammatory bowel disease

(IBD) model [12] suggesting the possible use of melatoninas disease-modifying therapeutic agent in inflammatorydiseases. In addition, it is known that melatonin has directscavenging actions, the indole in fact promotes the activities

of several antioxidative enzymes [13, 14] and metabolitesthat are formed when melatonin interacts with free radicalsand radical products that are also excellent scavengers

[15–18]. Glucocorticoids (GC) have potent immunosup-pressive effects, and are widely used in the management ofinflammatory diseases. Glucocorticoids exert beneficial

effects after acute CNS injury in humans and experimentalanimals [19]. Important data show that GC may have adisease-modifying effect in addition to their well-documen-ted anti-inflammatory actions [20]. In fact, the therapeutic

management of long-term pathologies with steroids is oftenlinked to a series of unwanted side effects, involving thehypothalamus-pituitary-adrenal axis, the cardiovascular

system, as well as the fat and bone metabolism [21]. GCcan downregulate the expression of several inflammatory

Abstract: This study investigates the effects of combination therapy with

melatonin and dexamethasone on the degree of spinal cord injury caused by

the application of vascular clip in mice. Spinal cord injury in mice resulted in

severe trauma, characterized by edema, neutrophil infiltration, and apoptosis

(measured by terminal deoxynucleotidyltransferase-mediated UTP end

labeling staining, and immunoreaction of Bax, Bcl-2, and Fas Ligand).

Infiltration of the spinal cord tissue with neutrophils (measured as increase in

myeloperoxidase activity) was associated with enhanced immuno-

histochemical and functional alterations revealed, respectively, by an

increased of tumor necrosis factor (TNF)-a immunoreactivity, NOS as well

as nitrotyrosine and loss of hind leg movement in spinal cord injury (SCI)-

operated mice. In contrast, the degree of neutrophil infiltration at different

time points, cytokine expression, histologic damage iNOS expression,

apoptosis, was markedly reduced in the tissues obtained from SCI-treated

mice with the combination therapy, and the motor recovery was also

ameliorated. No anti-inflammatory effect was observed in animals treated

with melatonin (10 mg/kg) or with dexamethasone (0.025 mg/kg) alone. This

study shows that the combination therapy with melatonin and

dexamethasone reduces the degree of secondary damage associated with

spinal cord injury in mice, and supports the possible use of melatonin in

combination with steroids to reduce the dose and the side effects related with

the use of steroids for the management of inflammatory disease.

Tiziana Genovese1,2, EmanuelaMazzon1,2, Concetta Crisafulli 1,Emanuela Esposito 2,3, RosannaDi Paola 1,2, Carmelo Muia1, PaoloDi Bella 2, Placido Bramanti 2 andSalvatore Cuzzocrea 1,2

1Department of Clinical and Experimental

Medicine and Pharmacology, School of

Medicine, University of Messina, Messina;2IRCCS Centro Neurolesi �Bonino-Pulejo�,Messina; 3Dipartimento di Farmacologia

Sperimentale, Universita di Napoli �Federico II�,Napoli, Italy

Key words: acute inflammation, combination

therapy, dexamethasone, melatonin,

oxidative stress

Address reprint requests to Salva-

tore Cuzzocrea, Department of

Clinical and Experimental Medicine

and Pharmacology, School of

Medicine, University of Messina,

Torre Biologica, Policlinico Univer-

sitario Via C. Valeria, Gazzi, 98100

Messina, Italy.

E-mail: salvator@unime.it

Received February 20, 2007;

accepted March 26, 2007.

J. Pineal Res. 2007; 43:140–153Doi:10.1111/j.1600-079X.2007.00454.x

� 2007 The AuthorsJournal compilation � 2007 Blackwell Munksgaard

Journal of Pineal Research

140

genes, including those encoding cytokines, such as IL-1,tumor necrosis factor (TNF)-a, and IL-6. Steroids interferewith the function and expression of several transcription

factors, affecting, in this manner, the induction of numer-ous inflammatory genes [22, 23]. As the GC side effects arealso dose-dependent and GC are potent anti-inflammatoryagents available, therapeutic use would benefit greatly from

a reduced burden of the side effects, particularly thoseaffecting the bone compartment. Glucocorticoids areamong a variety of endogenous compounds that have been

suggested to influence melatonin production in variousvertebrate species, including humans, and the existence ofthe mutual relationship between the pineal gland and the

hypothalamo-pituitary-adrenal axis has been postulated bysome authors [e.g. 24]. Based on the previous observations,in the present study, we have investigated the effects ofcombination therapy with melatonin and dexamethasone

associated with SCI. In particular, we have investigated theeffect of the combination therapy on polymorphonuclear(PMN) infiltration [myeloperoxidase (MPO) activity],

TNF-a expression, histologic damage, the nitration oftyrosine residues (an indicator of the formation of peroxy-nitrite by immunohistochemistry), iNOS expression apop-

tosis frequency, and motor recovery.

Materials and methods

Animals

Mice were anaesthetized using chloral hydrate (400 mg/kg

body weight). A longitudinal incision was made on themidline of the back, exposing the paravertebral muscles.These muscles were dissected away exposing the T5–T8

vertebrae. The spinal cord was exposed via a four-levelT6–T7 laminectomy, and SCI was produced by the extra-dural compression of the spinal cord, using an aneurysm

clip with a closing force of 24 g. Following surgery, 1.0 cm3

of saline was administered subcutaneously in order toreplace the blood volume lost during the surgery. Duringrecovery from anesthesia, the mice were placed on a warm

heating pad and covered with a warm towel. The mice weresingly housed in a temperature-controlled room at 27�C fora survival period of 10 days. Food and water were provided

to the mice ad libitum. During this time period, the animals�bladders were manually voided twice a day until the micewere able to regain the normal bladder function. In all

injured groups, the spinal cord was compressed for 1 min.Sham-injured animals were only subjected to laminectomy.

Experimental groups

Mice were randomly allocated into the following groups: (i)SCI + vehicle group – mice were subjected to SCI plus

administration of saline and ethanol 10% in saline 1 and4 hr after SCI via transperitoneally (N ¼ 40); (ii) dexa-methasone + melatonin group – same as the SCI + vehicle

group but in which dexamethasone and melatonin, at thedose of 10 mg/kg and 0.025 mg/kg, respectively, wereadministered 1 and 4 hr after SCI via transperitoneally

(N ¼ 40); (iii) dexamethasone group – same as theSCI + vehicle group, but were administered dexametha-

sone (0.025 mg/kg) 1 and 4 hr after SCI via transperiton-eally (N ¼ 40); (iv) melatonin group – same as theSCI + vehicle group but were administered melatonin(10 mg/kg i.p. bolus) 1 and 4 hr after SCI via transperi-

toneally (N ¼ 40); (v) sham + vehicle group – same as theSCI + vehicle group but except that the aneurysm clip wasnot applied (N ¼ 40); (vi) sham + dexamethasone + mela-

tonin group – identical to sham + saline group except forthe administration of dexamethasone + melatonin (N ¼40). In the experiments regarding the motor score, the

animals were treated with dexamethasone + melatonin 1and 4 hr after SCI, and daily until day 9. At different timepoints (see Fig. 1), the animals (n ¼ 10 mice from each

group for each time point) were killed in order to evaluatethe various parameters as described below.

Immunohistochemical localization of nitrotyrosine,iNOS, TNF-a, Fas Ligand, Bax, and Bcl-2

At the 24 hr after SCI, the tissues were fixed in 10% (w/v)

PBS-buffered formaldehyde and 8 lm sections were pre-pared from paraffin-embedded tissues. After deparaffiniza-tion, endogenous peroxidase was quenched with 0.3% (v/v)

hydrogen peroxide in 60% (v/v) methanol for 30 min. Thesections were permeabilized with 0.1% (w/v) Triton X-100in PBS for 20 min. Nonspecific adsorption was minimizedby incubating the section in 2% (v/v) normal goat serum in

PBS for 20 min. Endogenous biotin- or avidin-binding siteswere blocked by sequential incubation for 15 min withbiotin and avidin (DBA, Milan, Italy), respectively. Sec-

tions were incubated overnight with antinitrotyrosinerabbit polyclonal antibody (1:500 in PBS, v/v), with anti-TNF-a polyclonal antibody (1:500 in PBS, v/v), or with

anti-iNOS polyclonal antibody (1:500 in PBS, v/v), or anti-Fas Ligand, or anti-Bax rabbit polyclonal antibody (1:500in PBS, v/v) or with anti-Bcl-2 polyclonal antibody rat

(1:500 in PBS, v/v). Sections were washed with PBS, andincubated with secondary antibody. Specific labeling wasdetected with a biotin-conjugated goat antirabbit IgG andavidin–biotin peroxidase complex (DBA). To verify the

binding specificity for nitrotyrosine, TNF-a, iNOS, FasLigand, Bax, and Bcl-2, some sections were also incubatedwith only the primary antibody (no secondary) or with only

the secondary antibody (no primary). In these situations,no positive staining was found in the sections, indicatingthat the immunoreaction was positive in all the experiments

SCI

1 hr* 4 hr* 10 hr*

24 hr*

Daily *

Motor score

MPO ActivityNitrotyrosine, iNOS, Bax and Bcl-2expression immunohistochemistryTUNEL stainingHistologyBax, Bcl-2 western blotting analysis

Fig. 1. Mice were killed at different time points in order to evaluatevarious parameters. n ¼ 10 mice from each group for each timepoint (see Materials and methods for further explanations).

Dexamethasone and melatonin in SCI

141

carried out. Immunocytochemistry photographs (N ¼ 5)were assessed by densitometry, as previously described [25],by using Imaging Densitometer (AxioVision, Zeiss, Milan,Italy) and a computer program.

Terminal deoxynucleotidyltransferase-mediated UTPend labeling assay

Terminal deoxynucleotidyltransferase-mediated UTP endlabeling (TUNEL) assay was conducted by using a TUNEL

detection kit according to the manufacturer’s instructions(Apotag, HRP kit DBA, Milan, Italy). Briefly, sectionswere incubated with 15 lg/mL proteinase K for 15 min at

room temperature, and then washed with PBS. Endogenousperoxidase was inactivated by 3% H2O2 for 5 min at roomtemperature and then washed with PBS. Sections wereimmersed in terminal deoxynucleotidyltransferase (TdT)

buffer, containing deoxynucleotidyl transferase and biotin-ylated dUTP in TdT buffer, incubated in a humid atmo-sphere at 37�C for 90 min, and then washed with PBS. The

sections were incubated at room temperature for 30 minwith antihorseradish peroxidase-conjugated antibody, andthe signals were visualized with diaminobenzidine. The

number of TUNEL-positive cells/high-power field wascounted in five to 10 fields for each coded slide.

Total protein extraction and Western blot analysis forBax and Bcl-2

Tissue samples from animals subjected to SCI were

homogenized with an Ultra-turrax T8 homogenizer(IKA�-WERKE, Staufen, Germany) in a buffer containing20 mm HEPES pH 7.9, 1.5 mm MgCl2, 400 mm NaCl,

1 mm ethylenediaminetetraacetic acid (EDTA), 1 mm eth-yleneglycoltetraacetic acid (EGTA), 1 mm dithiothreitol(DTT), 0.5 mm phenylmethylsulphonyl fluoride (PMSF),

1.5 lg/mL trypsin inhibitor, 3 lg/mL pepstatin, 2 lg/mLleupeptin, 40 lm benzidamin, 1% NP-40, and 20% gly-cerol. The homogenates were centrifuged at 30,000 g, for15 min and at 4�C, the supernatant was collected to

evaluate contents of Bax and Bcl-2.Protein concentration was determined with the Bio-Rad

protein assay kit (Bio-Rad Laboratories, Munchen,

Germany). Proteins were mixed with the gel loading buffer[50 mm Tris, 10% (w/v), sodium dodecyl sulphate (SDS),10% (w/v) glycerol, 10% (v/v) 2-mercaptoethanol, 2 mg/

mL bromophenol], boiled for 3 min and centrifuged at10,000 rpm for few seconds. Protein concentration wasdetermined and equivalent amounts (75 lg) of each sampleelectrophoreses in a 12% (w/v) discontinuous polyacryla-

mide minigel. Proteins were separated electrophoretically

and transferred onto nitrocellulose membranes. For immu-noblotting, membranes were blocked with 10% nonfat drymilk in Tris-buffered saline (TBS) for 1 hr and incubatedwith primary antibodies against Bax and Bcl-2 (1:1000)

overnight at 4�C. The membranes were washed three timesfor 10 min in TBS with 0.1% Tween 20 and incubated withAffiniPure Goat Anti-rabbit IgG coupled to peroxidase

(1:2000). The immune complexes were visualized, using theSuperSignal West Pico chemiluminescence Substrate(PIERCE, Milan, Italy).

The primary antibodies directed at Bax and Bcl-2 wereobtained from Santa Cruz Biotechnology, Inc. (Santa Cruz,CA, USA). The secondary antibody was obtained from

Jackson Immuno Research, Laboratories, Inc. (Jackson,CA, USA).

Myeloperoxidase activity

Myeloperoxidase activity, an indicator of PMN leukocyteaccumulation, was determined as previously described [26]

4 hr after SCI. The time of 4 hr after SCI was chosen inagreement with other studies [27]. At the specified timefollowing SCI, spinal cord tissues were obtained and

weighed; then each piece was homogenized in a solutioncontaining 0.5% (w/v) hexadecyltrimethyl-ammonium bro-mide dissolved in 10 mm potassium phosphate buffer (pH7) and centrifuged for 30 min at 20,000 g at 4�C. An aliquot

of the supernatant was then allowed to react with a solutionof 1.6 mm tetramethylbenzidine and 0.1 mm H2O2. Therate of change in absorbance was measured spectrophoto-

metrically at 650 nm. MPO activity was defined as thequantity of enzyme degrading 1 lmol of peroxide per minat 37�C and was expressed in milliunits/g of wet tissue.

Light microscopy

Spinal cord biopsies were taken at 24 hr following trauma.Tissue segments containing the lesion (1 cm on each side ofthe lesion) were paraffin-embedded and cut into 5-lm-thicksections. Tissue sections (thickness 5 lm) were deparaffi-

nized with xylene, stained with hematoxylin/eosin (H&E)and methyl green pyronin staining (used to simultaneouslyDNA and RNA localization) and studied using light

microscopy (Dialux 22 Leitz, Milan, Italy).The segments of each spinal cord were evaluated by an

experienced histopathologist. Damaged neurons were

counted and the histopathologic changes of the gray matterwere scored on a 6-point scale [28]: 0, no lesion observed; 1,gray matter contained 1–5 eosinophilic neurons; 2, graymatter contained 5–10 eosinophilic neurons; 3, gray matter

contained more than 10 eosinophilic neurons; 4, small

Fig. 2. Effect of combination therapy on histologic alteration of the spinal cord tissue 24 hr after injury. No histologic alteration wasobserved in spinal cord tissues obtained from sham-operated mice (A). 24 hr after the trauma, a significant damage to the spinal cord fromSCI + vehicle-treated mice at the perilesional, as assessed by the presence of edema as well as an alteration of the white matter (B). Notably,a significant protection of SCI was observed in the tissue collected from combination therapy-treated SCI mice (C). The simultaneouspresence of DNA and RNA was detected by methyl green pyronin staining in sham-operated animals (D), a significant loss of DNA andRNA presence in lateral and dorsal funiculi was observed in the spinal cord at 24 hr after the injury, in SCI + vehicle mice (E, see particleE1), a significant loss of myelin was observed. In contrast, in the tissue collected from the combination therapy-treated SCI mice (F, seeparticle F1) was attenuated in the central part of lateral and dorsal funiculi. This figure is representative of at least three experimentsperformed on different experimental days. wm: white matter; gm: gray matter.

Genovese et al.

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Dexamethasone and melatonin in SCI

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infarction (less than one-third of the gray matter area); 5,moderate infarction (one-third to one half of the graymatter area); and 6, large infarction (more than half of the

gray matter area). The scores from all the sections fromeach spinal cord were averaged to give a final score forindividual mice. All the histologic studies were performed ina blinded fashion.

Grading of motor disturbance

The motor function of the mice subjected to compressiontrauma was assessed once a day for 10 days after injury.Recovery from the motor disturbance was graded, using the

modified murine Basso, Beattie, and Bresnahan (BBB) [29]hind limb locomotor rating scale [30, 31].

Materials

Unless otherwise stated, all compounds were obtained fromSigma-Aldrich Company Ltd (Poole, Dorset, UK). All

other chemicals were of the highest commercial gradeavailable. All stock solutions were prepared in nonpyro-genic saline (0.9% w/v NaCl; Baxter Healthcare Ltd,

Thetford, Norfolk, UK).

Statistical evaluation

All values in the figures and text are expressed asmean ± S.E.M. of N observations. For the in vivo studiesN represents the number of animals studied. In the

experiments involving histology or immunohistochemistry,the figures shown are representative of at least threeexperiments performed on three different experimental

days. Data sets were examined by one- or two-way analysisof variance, and individual group mean values were thencompared with Student’s unpaired t-test. The BBB scale

data were analyzed by the Mann–Whitney U-test andconsidered significant when P-value was <0.05.

Results

The severity of the trauma at the level of the perilesionalarea, assessed as the presence of edema as well as alteration

of the white matter, was evaluated at 24 hr after injury. Asignificant damage to the spinal cord was observed in thespinal cord tissue of control mice subjected to SCI (Fig. 2B)

when compared with sham-operated mice (Fig. 2A). Mela-tonin or the dexamethasone treatment did not attenuate thedevelopment of tissue damage after SCI (data not shown).

Notably, a significant protection against the SCI wasobserved in dexamethasone + melatonin-treated mice(Fig. 2C). The simultaneous presence of DNA and RNAwas detected by methyl green pyronin staining (Fig. 2-

D,E,E1,F,F1). In sham animals (Fig. 2D), the simultaneouspresence of DNA and RNA was clearly stained by methylgreen pyronin staining in both lateral and dorsal funiculi of

the spinal cord. At 24 hr after the injury, a significant lossof the DNA and RNA presence in lateral and dorsalfuniculi was observed in the control mice subjected to SCI

(Fig. 2E,E1). Melatonin or the dexamethasone treatmentdid not attenuate the development of tissue damage after

SCI (data not shown). In contrast, in the dexametha-sone + melatonin-treated mice, the DNA and RNA deg-radation was attenuated in the central part of lateral and

dorsal funiculi (Fig. 2F,F1). In order to evaluate whetherhistologic damage to the spinal cord was associated with aloss of motor function, the modified BBB hind limblocomotor rating scale score was evaluated. While the

motor function was only slightly impaired in sham mice,those subjected to SCI had significant deficits in the hindlimb movement (Fig. 3). Melatonin or the dexamethasone

treatment did not attenuate the degree of score after SCI(data not shown). On the contrary, a significant amelior-ation of the hind limb motor disturbances was observed in

the dexamethasone + melatonin-treated mice (Fig. 3). Theabove-mentioned histologic pattern of SCI appeared to becorrelated with the influx of leukocytes into the spinal cord.Therefore, we investigated the role of dexametha-

sone + melatonin on the neutrophil infiltration, by meas-uring tissue MPO activity. MPO activity was significantly

3

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SCI + Vehicle

SCI + DEX + MEL

* ** * *

BB

B m

oto

r sc

ore

7 8 9 10

Fig. 3. Effect of combination therapy on hind limb motor distur-bance after SCI. The degree of motor disturbance was assessedevery day until 10 days after SCI by Basso, Beattie, and Bresnahancriteria. The administration of combination therapy reduces themotor disturbance after SCI. Values shown are mean ± S.E.M. of10 mice for each group.*P < 0.01 versus SCI vehicle mice.

700Vehicle

DEX + MEL *

°

SHAM SCI

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O L

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Fig. 4. Effects of combination therapy on myeloperoxidase (MPO)activity. Following the injury, MPO activity in spinal cord ofSCI + vehicle-treated mice significantly increased at 24 hr afterthe damage, in comparison with sham mice. Combination therapyreduces the SCI-induced increase in MPO activity. Data aremean ± S.E.M. of 10 mice for each group. *P < 0.05 versussham; �P < 0.01 versus SCI.

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elevated in the spinal cord at 24 hr after injury in thecontrol mice, subjected to SCI when compared with sham-operated mice (Fig. 4). Melatonin or the dexamethasone

treatment did not attenuate the development of neutrophilinfiltration after SCI (data not shown). In the dexameth-asone + melatonin-treated mice, on the contrary, theMPO activity in the spinal cord at 24 hr after injury was

significantly attenuated in comparison with that observedin SCI controls (Fig. 4). To determine the localization of

�peroxynitrite formation� and/or other nitrogen derivativesproduced during SCI, nitrotyrosine, a specific marker ofnitrosative stress, was measured by the immunohistochem-

ical analysis in the spinal cord sections at 24 hr after SCI.Sections of the spinal cord were taken at the same hourafter SCI in order to determine also, the immunohistologicstaining for iNOS. Spinal cord sections obtained from

SCI + vehicle-operated mice exhibited positive staining foriNOS (Figs 5B and 6) and nitrotyrosine (Figs 5E and 6),

(A)wm

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Fig. 5. Immunohistochemical localization of iNOS. No positive staining for iNOS (A) was observed in spinal cord tissues obtained from thesham group of mice. Administration of combination therapy to SCI-operated mice produced a marked reduction in the immunostaining foriNOS (C) in spinal cord tissue, when compared with positive staining for iNOS (B) observed in the spinal cord tissue of mice 24 hr after theinjury. In addition, no positive staining for nitrotyrosine (D) was observed in spinal cord tissues obtained from the sham group of mice. SCIcaused, at 24 hr, an increase in the nitrotyrosine formation (E). Treatment with combination therapy significantly inhibited the SCI-inducednitrotyrosine formation (F). Figure is representative of at least three experiments performed on different experimental days. wm: whitematter; gm: gray matter.

Dexamethasone and melatonin in SCI

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localized in inflammatory cells as well as in nuclei ofSchwann cells in the white and gray matter of the spinalcord tissues. Melatonin or the dexamethasone treatment

did not attenuate the immunostaining for nitrotyrosine andiNOS after SCI (data not shown). In contrast, in thedexamethasone + melatonin-treated SCI-operated mice,

the degree of positive staining for iNOS (Figs 5C and 6)and nitrotyrosine (Figs 5F and 6) was significantly reduced.Note that there was no staining for iNOS or nitrotyrosinein the spinal cord section from the sham-operated mice

(Figs 5A,D and 6, respectively). To test whether dexameth-asone + melatonin may modulate the inflammatory pro-cess through the regulation of the secretion of other

cytokines, we analyzed the spinal cord-positive stainingfor proinflammatory cytokines TNF-a in control micesubjected to SCI, and the dexamethasone + melatonin-

treated mice. A substantial increase of TNF-a staining wasfound in sections collected from control mice subjected toSCI at 24 hr after injury (Figs 6 and 7B,B1). Positivestaining for TNF-a was significantly higher in control mice

subjected to SCI in comparison with those of the dexa-methasone + melatonin-treated mice (Figs 6 and 7C).Melatonin or the dexamethasone treatment did not

attenuate the immunostaining for TNF-a after SCI (datanot shown). There was no staining for either TNF-a insections obtained from the sham group of mice (Figs 6 and

7A). Immunohistologic staining for Fas Ligand in thespinal cord was also determined 24 hr after the injury.Sections of spinal cord from sham-operated mice did not

8

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Nitrotyrosine FAS-I TNF-a BAX

Sham + vehicle

SCI + vehicleSham + DEX + MEL

SCI + DEX + MEL

Bcl-2

Fig. 6. Typical densitometry evaluation. Densitometry analysis ofimmunocytochemistry photographs (n ¼ 5 photos from eachsample collected from all mice in each experimental group) foriNOS, nitrotyrosine, tumor necrosis factor -a, Fas Ligand, Bax,and Bcl-2, from spinal cord tissues was assessed. The assay wascarried out by using optilab graftek software on a Macintoshpersonal computer (CPU G3-266). Data are expressed as percent-age of total tissue area. *P < 0.01 versus sham; �P < 0.01 versusSCI. ND: not detectable.

(A) wm wm

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Fig. 7. Immunohistochemical localization of tumor necrosis factor (TNF)-a. No positive staining for TNF-a (A) was obtained in spinalcord tissues from the sham-operated mice. A substantial increase of TNF-a (B, see particle B1) formation was found in spinal cord samplescollected from SCI + vehicle-operated mice at 24 hr after SCI. The positive staining for TNF-a was significantly attenuated in combinationtherapy-treated mice (C). Figure is representative of at least three experiments performed on different experimental days. wm: white matter;gm: gray matter.

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stain for the Fas Ligand (Figs 6 and 8A), whereas spinalcord sections obtained from SCI control mice exhibitedpositive staining for Fas Ligand (Figs 6 and 8B) mainlylocalized in inflammatory cells as well as in the nuclei of

Schwann cells in the white and gray matter of the spinalcord tissues. Melatonin or the dexamethasone treatmentdid not attenuate the immunostaining for Fas Ligand after

SCI (data not shown). The treatment of mice subjected toSCI with dexamethasone + melatonin reduced the degreeof positive staining for Fas Ligand (Figs 6 and 8C) in the

spinal cord. To test whether spinal cord damage wasassociated with cell death by apoptosis, we measuredTUNEL-like staining in the perilesional spinal cord tissue.

Almost no apoptotic cells were detected in the spinal cordfrom the sham-operated mice (Fig. 9A). At 24 hr after thetrauma tissues obtained from SCI control mice demonstra-ted a marked appearance of dark brown apoptotic cells and

intercellular apoptotic fragments (TUNEL-positive cellswere 2.1 ± 0.12 per field, Fig. 9B,B1). In contrast, tissuesobtained from the mice treated with dexametha-

sone + melatonin (TUNEL-positive cells were 0.2 ± 0.05per field, Fig. 9C) demonstrated a small number of apop-totic cells or fragments. Melatonin or the dexamethasone

treatment did not attenuate the number of apoptotic cellsor fragments after SCI (data not shown). The appearanceof Bax in the homogenates of the spinal cord wasinvestigated by Western blot at 24 hr after SCI. A basal

level of Bax was detected in the spinal cord from sham-operated animals (Fig. 10A,A1). The Bax levels weresubstantially increased in the spinal cord from control micesubjected to SCI (Fig. 10A,A1). On the contrary, the

dexamethasone + melatonin treatment prevented the SCI-induced Bax expression (Fig. 10A,A1). Melatonin or dex-amethasone treatment did not prevent the SCI-induced Bax

expression (data not shown).To detect Bcl-2 expression, whole extracts from the

spinal cord of each mouse were also analyzed by the

Western blot analysis. A low basal level of the Bcl-2expression was detected in the spinal cord from sham-operated mice (Fig. 10B,B1). And, 24 hr after SCI, the Bcl-

2 expression was significantly reduced in whole extractsobtained from spinal cord of SCI control mice(Fig. 10B,B1). The treatment of mice with dexametha-sone + melatonin significantly reduced the SCI-induced

inhibition of Bcl-2 expression (Fig. 10B,B1). Melatonin orthe dexamethasone treatment did not reduce the SCI-induced inhibition of the Bcl-2 expression (data not shown).

Moreover, samples of the spinal cord tissue were taken at24 hr after SCI in order to determine the immunohistologicstaining for Bax and Bcl-2. Sections of the spinal cord from

sham-operated mice did not stain for Bax (Figs 6 and 11A),whereas spinal cord sections obtained from SCI controlmice exhibited a positive staining for Bax (Figs 6 and11B,B1), mainly localized in inflammatory cells as well as in

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Fig. 8. Immunohistochemical localization of Fas-L. No positive staining for Fas Ligand was observed in spinal cord tissues obtained fromthe sham group of mice (A). SCI caused, at 24 hr, an increase in the Fas Ligand expression (B). Treatment with combination therapy (C)significantly inhibited the SCI-induced Fas Ligand expression. Figure is representative of at least three experiments performed on differentexperimental days. wm: white matter; gm: gray matter.

Dexamethasone and melatonin in SCI

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the nuclei of Schwann cells in the white and gray matter ofthe spinal cord tissues. The dexamethasone + melatonintreatment reduced the degree of positive staining for Bax inthe spinal cord of those mice subjected to SCI (Figs 6 and

11C). Melatonin or the dexamethasone treatment did notreduce the degree of positive staining for Bax in the spinalcord of mice subjected to SCI (data not shown). In

addition, sections of the spinal cord from the sham-operated mice demonstrated positive staining for Bcl-2(Figs 6 and 11D,D1) while in SCI control mice, the staining

for Bcl-2 was significantly reduced (Figs 6 and 11E). The

dexamethasone + melatonin treatment attenuated the lossof positive staining for Bcl-2 in the spinal cord in micesubjected to SCI (Figs 6 and 11F,F1). Melatonin ordexamethasone treatment did not attenuate the loss of

positive staining for Bcl-2 in the spinal cord in micesubjected to SCI (data not shown).

Discussion

Much of the damage that occurs in the spinal cord

following traumatic injury is due to the secondary effects

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Fig. 9. Almost no apoptotic cells were detectable in the spinal cord tissue of the sham-operated mice (A). The number of apoptotic cells (seearrows) increased at 24 hr after spinal cord injury (B see particle B1). In contrast, only a few apoptotic cells were seen in spinal cord tissuefrom combination therapy-treated mice (C). Section D demonstrates the positive staining in the Kit-positive control tissue (normal rodentmammary gland). Figure is representative of at least three experiments performed on different experimental days. wm: white matter; gm:gray matter.

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of oxidative stress, which take part in a spiraling interactivecascade, ending in neuronal dysfunction and death [3, 32,

33]. The role of reactive oxygen and nitrogen species,including nitric oxide, superoxide, and the product of theirreaction, peroxynitrite, which are involved in free radical-

induced oxidative damage to spinal cord tissue was alsostrongly supported in our previous works [11, 34–37].Moreover, various pieces of evidence challenge the prevail-ing view that NO is independently toxic and propose that

much of the NO-related cytotoxicity and oxidant reactionsare, in fact, due to ONOO– formation [38–40]. In fact,potent novel, cell-permeable superoxide dismutase (SOD)

mimetics prevent the peroxynitrite formation and protectagainst the cellular injury in various models of inflamma-

tion [41–43]. In this regard, we previously demonstratedthat melatonin (at 50 mg/kg) exerts a protective effect in arat model of SCI [11]. This study provides the first evidence

that the combination therapy with melatonin (10 mg/kg)and dexamethasone (0.025 mg/kg), used at a dose which arenot effective when administered as single treatment, attenu-ates: (i) the tissue damage, (ii) the infiltration of the spinal

cord with PMN, (iii) TNF-a expression, (iv) the nitration oftyrosine residues, (v) iNOS expression, (vi) apoptosis level,and (vii) motor recovery.

Glucocorticoid, e.g. dexamethasone, are potent immu-nosuppressive and anti-inflammatory agents that are usedtherapeutically in several inflammatory pathologies. Long-

term therapy with GC is often necessary to control thesymptoms of different inflammatory conditions [e.g. rheu-matoid arthritis (RA) and IBD]. As the GC’s side effects arealso dose-dependent and GC are potent anti-inflammatory

agents available, it could be interesting to study the possiblebeneficial effects to doses for which they must not havesome therapeutic effect. In this regard, we recently demon-

strated that a combination with a SOD mimetic anddexamethasone exerts a beneficial effect in an arthritismodel [42]. The anti-inflammatory action observed with the

combination therapy is also related to the reduction of thedegree of iNOS protein. In addition, we demonstrated herethat the degree of staining for nitrotyrosine was signifi-

cantly reduced by the treatment with the combinationtherapy in SCI-treated mice. The findings obtained in thisstudy would provide strong evidence for a possible role forthe interaction of steroid by ROS. They suggest that the use

of a high dose of dexamethasone is needed in the clinic maybe explained by the fact that during the inflammatoryprocess (e.g. asthma, RA, or IBD), the patients basically

receive an anti-inflammatory agent that is partially deacti-vated through the in vivo generation of ROS.While the precise mechanisms responsible to damage in

SCI remain under study, there is evidence to show thatearly inflammatory events promote tissue damage in theacutely injured spinal cord [44]. An inflammatory response

develops within hours after the injury and is characterizedby the infiltration of neutrophils and the activation ofmicroglia [45]. This is followed by a second wave ofresponse to localize the inflammatory response within the

spinal cord tissue and to downmodulate this response. Inthis study, it was shown that the pharmacologic combina-tion of melatonin + dexamethasone reduced the inflam-

matory cell infiltration as assessed by the specificgranulocyte enzyme MPO. Consistent with this notion/hypothesis, in addition, Taoka et al. proved that activated

neutrophils were involved in compression trauma-inducedSCI in rats [46]. Neutrophils, recruited into the tissue, cancontribute to tissue destruction by the production ofreactive oxygen metabolites [4, 47], granule enzymes, and

cytokines that further amplify the inflammatory response[48]. Several authors have demonstrated in fact that theinjured environment during the acute phase of SCI is

dominated by the presence of the proinflammatory cytok-ines like TNF-a [49, 50]. Already, within 1 hr after SCI, anincreased synthesis and/or secretion of TNF-a, is detectableat the injury site [51]. We confirm in the present study thatthe model of SCI used here leads to a substantial increase in

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Fig. 10. Representative Western blot of Bax levels and Bcl-2.Western blot analysis was realized in spinal cord tissue collected at24 hr after injury. (A and A1) Sham: basal level of Bax was presentin the tissue from sham-operated mice; SCI: Bax band is moreevident in the tissue from spinal cord-injured mice; SCI + com-bination therapy: Bax band disappeared in the tissue from spinalcord-injured mice which received combination therapy. (B and B1)Sham: basal level of Bcl-2 was present in the tissue from sham-operated mice; SCI: Bcl-2 band disappeared in the tissue fromspinal cord-injured mice; SCI + combination therapy: Bcl-2 bandis more evident in the tissue from spinal cord-injured mice whichreceived combination therapy. (A1 and B1) The intensity ofretarded bands (measured by phosphoimager; Bio-Rad, Hercules,CA) in all the experimental groups. Immunoblotting in panels Aand B is representative of one spinal cord tissue out of five to sixanalyzed. The results in panels A1 and B1 are expressed asmean ± S.E.M. from 5 to 6 spinal cord tissues. *P < 0.01 versussham; �P < 0.01 versus SCI.

Dexamethasone and melatonin in SCI

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Fig. 11. Immunohistochemical expression of Bax and Bcl-2. No positive staining for Bax was observed in the tissue section from sham-operated mice (A). SCI in mice caused, at 24 hr, an increase in the release of Bax expression (B and B1). On the contrary, the degree ofpositive staining for Bax was significantly reduced in spinal cord tissues collected from combination therapy-treated mice (C). In addition,sections of spinal cord from sham-operated mice demonstrated positive staining for Bcl-2 (D). Spinal cord sections obtained from SCI-operated mice exhibited significantly less staining for Bcl-2 (E). The loss of positive staining for Bcl-2 was significantly reduced in spinal cordtissues collected from mice treated with combination therapy (F and F1). Figure is representative of at least three experiments performed ondifferent experimental days. wm: white matter; gm: gray matter.

Genovese et al.

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the levels of TNF-a in the spinal cord. Interestingly, thelevels of this proinflammatory cytokine are significantlylower in the tissues obtained from mice, treated with thecombination therapy. It was shown previously that, after

SCI, TNF-a might serve as an external signal, initiatingapoptosis in neurons and oligodendrocytes [51]. To furthercharacterize the apoptotic cascade initiated by TNF-aafter SCI, we examined the expression of the degree ofapoptosis, measured by the TUNEL detection kit, in thespinal cord after the damage. Apoptosis is an important

mediator of the secondary damage after SCI [52, 53]. Thegeneration of free radicals and nitric oxide by activatedmacrophages has been implicated in causing oligodendro-

cyte apoptosis [54]. In this study, we have demonstratedthat the combination therapy caused a lower expression ofapoptotic particles when compared with SCI nontreatedmice. Moreover, it is well known that Bax, a pro-

apoptotic gene, plays an important role in developmentalcell death [55] and in CNS injury [56]. Similarly, it wasshown that the administration of Bcl-xL fusion protein

(Bcl-xL FP; Bcl-2 is the most expressed antiapoptoticmolecule in adult central nervous system) into injuredspinal cords significantly increased neuronal survival,

suggesting that SCI-induced changes in Bcl-xL contributedconsiderably to neuronal death [57]. Based on thisevidence, we have identified proapoptotic transcriptionalchanges, including upregulation of the proapoptotic Bax

and the downregulation of antiapoptotic Bcl-2, by immu-nohistochemical staining. We report in the present studythat the co-treatment of melatonin with dexamethasone in

SCI experimental model record features of apoptotic celldeath after SCI, suggesting that protection from apoptosismay be a prerequisite for regenerative approaches to SCI.

In particular, we demonstrated that the co-administrationof melatonin with dexamethasone reduced Bax expression,on the contrary, Bcl-2 expressed much more in SCI-

treated mice. This result suggests that melatonin withdexamethasone prevents the loss of the antiapoptotic wayand reduces the pro-apoptotic pathway activation with amechanism still to be discovered.

Oxidative stress and apoptosis are two major character-istics of the progression of secondary damage after SCI. Fas(CD95)-mediated apoptosis is an essential mechanism for

the maintenance of homeostasis, and the disruption of thisdeath pathway contributes to many human diseases [58].

Many studies have linked apoptosis to thoracic SCI. Tosuch purpose, furthermore, some authors have also shownthat Fas and p75 receptors are expressed on oligodendro-cytes, astrocytes, and microglia in the spinal cord, following

SCI. Fas and p75 co-localize on many TUNEL-positivecells, suggesting that the Fas- and p75-initiated cell deathcascades may participate in the demise of some glia

following SCI [59].Our results, moreover, suggest that co-treatment in the

acute phase of SCImay decrease the extent of intramedullary

spinal cord hemorrhage and damage likely is demonstratedby the histologic tissue analysis. Thus, we have found in thepost-traumatic inflammatory combination therapy that

melatonin plus dexamethasone exerts a significant andimportant beneficial anti-inflammatory effect by blockingthe possible progression of secondary damage.In conclusion, the findings obtained in this study provide

strong evidence that the use of a high dose of dexameth-asone is needed in the clinic. This may be explained by thefact that SCI patients basically receive an anti-inflamma-

tory agent that is partially deactivated through the in vivogeneration of ROS. Thus, we have found that the combi-nation therapy with melatonin and dexamethasone exerts a

significant and important beneficial anti-inflammatoryeffect by blocking the possible progression of secondaryevents after SCI. Furthermore, our results suggest that invivo, the combination therapy allowed reducting 10-fold

the effective dexamethasone dose. These findings indicate anovel function of a possible combination therapy, whichprovides a key procedure for the control of the secondary

damage development after SCI.

Acknowledgments

This study was supported by a grant from a UniversityMinister grant. The authors would like to thank Giovanni

Pergolizzi and Carmelo La Spada for their excellenttechnical assistance during this study, Mrs Caterina Cut-rona for secretarial assistance and Miss Valentina Malvagnifor editorial assistance with the manuscript.

References

1. Kakulas BA. Pathology of spinal injuries. Cent Nerv Syst

Trauma 1984; 1:117–129.

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gm

wm

100 mm

(F1)

50 mm

Fig. 11. Continued

Dexamethasone and melatonin in SCI

151

2. Tator CH, Fehlings MG. Review of the secondary injury

theory of acute spinal cord trauma with emphasis on vascular

mechanisms. J Neurosurg 1991; 75:15–26.

3. Carlson SL, Parrish ME, Springer JE et al. Acute inflam-

matory response in spinal cord following impact injury. Exp

Neurol 1998; 151:77–88.

4. Mctigue DM, Tani M, Krivacic K et al. Selective chemo-

kine mRNA accumulation in the rat spinal cord after contu-

sion injury. J Neurosci Res 1998; 53:368–376.

5. Harlan JM. Consequences of leukocytes-vessel wall interac-

tions in inflammatory and immune reactions. Semin Thromb

Hemost 1987; 13:425–433.

6. Cuzzocrea S, Riley DP, Caputi AP et al. Antioxidant

therapy: a new pharmacological approach in shock, inflam-

mation, and ischemia/reperfusion injury. Pharmacol Rev 2001;

53:135–159.

7. Reiter RJ, Tan DX, QI W et al. Pharmacology and physiol-

ogy of melatonin in the reduction of oxidative stress in vivo.

Biol Signals Recept 2000; 9:160–171.

8. Maldonado MD, Murillo-Cabezas F, Terron MP et al.

The potential of melatonin in reducing morbidity-mortality

after craniocerebral trauma. J Pineal Res 2007; 42:1–11.

9. Carrillo-Vico A, Guerrero JM, Lardone PJ et al. A

review of the multiple actions of melatonin on the immune

system. Endocrine 2005; 27:189–200 (Review).

10. Genovese T, Mazzon E, Muia C et al. Attenuation in the

evolution of experimental spinal cord trauma by treatment

with melatonin. J Pineal Res 2005; 38:198–208.

11. Cuzzocrea S, Mazzon E, Serraino I et al. Melatonin

reduces dinitrobenzene sulfonic acid-induced colitis. J Pineal

Res 2001; 30:1–12.

12. Rodriguez C, Mayo JC, Sainz RM et al. Regulation of

antioxidant enzymes: a significant role for melatonin. J Pineal

Res 2004; 36:1–9.

13. Tomas-Zapico C, Coto-Montes A. A proposed mechanism

to explain the stimulatory effect of melatonin on antioxidative

enzymes. J Pineal Res 2005; 39:99–104.

14. Tan DX, Manchester LC, Terron MP et al. One molecule,

many derivatives: a never-ending interaction of melatonin with

reactive oxygen and nitrogen species? J Pineal Res 2007;

42:28–42.

15. Hardeland R. Antioxidative protection by melatonin: mul-

tiplicity of mechanisms from radical detoxification to radical

avoidance. Endocrine 2005; 27:119–130.

16. Guenther AL, Schmidt SI, Laatsch H et al. Reactions of

the melatonin metabolite AMK (N1-acetyl-5-methoxykynur-

amine) with reactive nitrogen species: formation of novel

compounds, 3-acetamidomethyl-6-methoxycinnolinone and

3-nitro-AMK. J Pineal Res 2005; 39:251–260.

17. Onuki J, Almeida EA, Medeiros MH et al. Inhibition of

5-aminolevulinic acid-induced DNA damage by melatonin,

N1-acetyl-N2-formyl-5-methoxykynuramine, quercetin or

resveratrol. J Pineal Res 2005; 38:107–115.

18. Gonzalez SL, Saravia F, Gonzalez Deniselle MC et al.

Glucocorticoid regulation of motoneuronal parameters in

rats with spinal cord injury. Cell Mol Neurobiol 1999;

19:597–611.

19. Kirwan JR. The effect of glucocorticoids on joint destruction

in rheumatoid arthritis. The Arthritis and Rheumatism

Council Low-dose Glucocorticoid Study Group. N Engl J Med

1995; 333:142–146.

20. Bond WS. Toxic reactions and side effects of glucocorticoids in

man. Am J Hosp Pharm 1977; 34:479–485.

21. Barnes PJ, Karin M. Nuclear factor-kappaB: a pivotal

transcription factor in chronic inflammatory diseases. N Engl J

Med 1997; 336:1066–1071.

22. Buttgereit F, Wehling M, Burmester GR. A new hypo-

thesis of modular glucocorticoid actions: steroid treatment of

rheumatic diseases revisited. Arthritis Rheum 1998; 41:761–767.

23. Benyassi A, Schwartz C, Ducouret B et al. Glucocorticoid

receptors and serotonin N-acetyltransferase activity in the fish

pineal organ. Neuroreport 2001; 12:889–892.

24. Shea TB. Technical report. An inexpensive densitometric

analysis system using a Macintosh computer and a desktop

scanner. Biotechniques 1994; 16:1126–1128.

25. Mullane KM, Kraemer R, Smith B. Myeloperoxidase

activity as a quantitative assessment of neutrophil infiltration

into ischemic myocardium. J Pharmacol Methods 1985;

14:157–167.

26. Hamada Y, Ikata T, Katoh S et al. Involvement of an intra-

cellular adhesion molecule 1-dependent pathway in the path-

ogenesis of secondary changes after spinal cord injury in rats.

J Neurochem 1996; 66:1525–1531.

27. Sirin BH, Ortac R, Cerrahoglu M et al. Ischaemic pre-

conditioning reduces spinal cord injury in transient ischaemia.

Acta Cardiol 2002; 57:279–285.

28. Basso DM, Beattie MS, Bresnahan JC. A sensitive and

reliable locomotor rating scale for open field testing in rats.

J Neurotrauma 1995; 12:1–21.

29. Joshi M, Fehlings MG. Development and characterization of

a novel, graded model of clip compressive spinal cord injury in

the mouse: Part 1. Clip design, behavioral outcomes, and his-

topathology. J Neurotrauma 2002; 19:175–190.

30. Joshi M, Fehlings MG. Development and characterization of

a novel, graded model of clip compressive spinal cord injury in

the mouse: Part 2. Quantitative neuroanatomical assessment

and analysis of the relationships between axonal tracts, resid-

ual tissue, and locomotor recovery. J Neurotrauma 2002;

19:191–203.

31. Anderson DK, Hall ED. Pathophysiology of spinal cord

trauma. Ann Emerg Med 1993; 22:987–992.

32. Lynch DR, Dawson TM. Secondary mechanisms in neuronal

trauma. Curr Opin Neurol 1994; 7:510–516.

33. La Rosa G, Cardali S, Genovese T et al. Inhibition of the

nuclear factor-kappaB activation with pyr-

rolidinedithiocarbamate attenuating inflammation and oxida-

tive stress after experimental spinal cord trauma in rats.

J Neurosurg Spine 2004; 1:311–321.

34. Scott GS, Cuzzocrea S, Genovese T et al. Uric acid protects

against secondary damage after spinal cord injury. Proc Natl

Acad Sci U S A 2005; 102:3483–3488.

35. Genovese T, Mazzon E, Mariotto S et al. Modulation of

nitric oxide homeostasis in a mouse model of spinal cord in-

jury. J Neurosurg Spine 2006; 4:145–153.

36. Salvemini D, Wang ZQ, Bourdon DM et al. Evidence of

peroxynitrite involvement in the carrageenan-induced rat paw

edema. Eur J Pharmacol 1996; 303:217–220.

37. Crow JP, Beckman JS. Reactions between nitric oxide,

superoxide, and peroxynitrite: footprints of peroxynitrite in

vivo. Adv Pharmacol 1995; 34:17–43.

38. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and

peroxynitrite: the good, the bad, and ugly. Am J Physiol 1996;

271:1424–1437.

39. Szabo C. The role of peroxynitrite in the pathophysiology of

shock, inflammation and ischemia-reperfusion injury. Shock

1996; 6:79–88.

Genovese et al.

152

40. Cuzzocrea S, Zingarelli B, Gilad E et al. Protective effect

of melatonin in carrageenan-induced models of locar inflam-

mation: relationship to its inhibitory effect on nitric oxide

production and its peroxinitrite scavenging activity. J Pineal

Res 1997; 23:106–116.

41. Cuzzocrea S, Mazzon E, Paola RD et al. Effect of combi-

nation M40403 and dexamethasone therapy on joint disease in

a rat model of collagen-induced arthritis. Arthritis Rheum

2004; 52:1929–1940.

42. Cuzzocrea S, Reiter RJ. Pharmacological actions of mela-

tonin in acute and chronic inflammation. Curr Top Med Chem

2002; 2:153–165

43. Popovich PG, Jones TB. Manipulating neuroinflammatory

reactions in the injured spinal cord: back to basics. Trends

Pharmacol Sci 2003; 24:13–17.

44. Mctigue DM, Popovich PG, Jakeman LB et al. Strategies

for spinal cord injury repair. Prog Brain Res 2000; 128:3–8.

45. Taoka Y, Okajima K, Uchiba M et al. Role of neutrophils in

spinal cord injury in the rat. Neuroscience 1997; 79:1177–1182.

46. Tator CH. Update on the pathophysiology and pathology of

acute spinal cord injury. Brain Pathol 1995; 5:407–413.

47. Chatham WW, Swaim R, Frohsin H et al. Degradation of

human articular cartilage by neutrophils in synovial fluid.

Arthritis Rheum 1993; 36:51–58.

48. Tyor WR, Avgeropoulos N, Ohlandt G et al. Treatment of

spinal cord impact injury in the rat with transforming growth

factorbeta. J Neurol Sci 2002; 200:33–41.

49. Klusman I, Schwab ME. Effects of pro-inflammatory

cytokines in experimental spinal cord injury. Brain Res 1997;

762:173–184.

50. Yune TY, Chang MJ, Kim SJ et al. Increased production of

tumor necrosis factor-alpha induces apoptosis after traumatic

spinal cord injury in rats. J Neurotrauma 2003; 20:207–219.

51. Beattie MS, Farooqui AA, Bresnahan JC. Review of cur-

rent evidence for apoptosis after spinal cord injury. J Neuro-

trauma 2000; 17:915–925.

52. Beattie MS, Hermann GE, Rogers RC et al. Cell death in

models of spinal cord injury. Prog Brain Res 2002; 137:37–47.

53. Merrill JE, Ignarro LJ, Sherman MP et al. Microglial cell

cytotoxicity of oligodendrocytes is mediated through nitric

oxide. J Immunol 1993; 151:2132–2141.

54. Chittenden T, Harrington EA, O’connor R et al. Induc-

tion of apoptosis by the Bcl-2 homologue Bak. Nature 1995;

374:733–736.

55. Bar-Peled O, Knudson M, Korsmeyer SJ et al. Motor

neuron degeneration is attenuated in Bax-deficient neurons in

vitro. J Neurosci Res 1999; 55:542–556.

56. Nesic-Taylor O, Cittelly D, Ye Z et al. Exogenous Bcl-xL

fusion protein spares neurons after spinal cord injury. J Neu-

rosci Res 2005; 79:628–637.

57. Lu B, Wang L, Stehlik C et al. Phosphatidylinositol 3-kin-

ase/Akt positively regulates Fas (CD95)-mediated apoptosis in

epidermal Cl41 cells. J Immunol 2006; 176:6785–6793.

58. Casha S, Yu WR, Fehlings MG. Oligodendroglial apoptosis

occurs along degenerating axons and is associated with FAS

and p75 expression following spinal cord injury in the rat.

Neuroscience 2001; 103:203–218.

59. Wallace MC, Tator CH, Lewis AJ. Chronic regenerative

changes in the spinal cord after cord compression injury in

rats. Surg Neurol 1984; 27:209–219.

Dexamethasone and melatonin in SCI

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