Experimental and Toxicologic Pathology 63 (2011) 467–471

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Experimental and Toxicologic Pathology

0940-29

doi:10.1

n Corr

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journal homepage: www.elsevier.de/etp

Melatonin ameliorates neocortical neuronal dendritic impairment inducedby toluene inhalation in the rat

Rodrigo Pascual a,n, S. Pilar Zamora-Leon b, Natalia Perez a, Tatiana Rojas a, Anamarıa Rojo a,Marıa Jose Salinas a, Alvaro Reyes a, Carlos Bustamante a

a Laboratorio de Neurociencias, Escuela de Kinesiologıa, Facultad de Ciencias, Pontificia Universidad Catolica de Valparaıso, Avenida Brasil 2950, Valparaıso, Chileb Laboratorio de Biologıa Molecular, Instituto de Ciencias Basicas, Universidad Catolica del Maule, Talca, Chile

a r t i c l e i n f o

Article history:

Received 11 November 2009

Accepted 14 March 2010

Keywords:

Toluene

Melatonin

Pyramidal neurons

Dendritic development

Golgi impregnation

93/$ - see front matter & 2010 Elsevier Gmb

016/j.etp.2010.03.006

esponding author. Tel.: +56 71 203162; fax:

ail address: rodrigo.pascual@ucv.cl (R. Pascua

a b s t r a c t

The present study investigated the effects of toluene inhalation and the restorative effects of melatonin

on branching and basal dendritic outgrowth of superficial pyramidal neurons in rat’s frontal, parietal,

and occipital cortices. At postnatal day 21 (P21), Sprague-Dawley male rats were randomly assigned to

either an air-only group or a toluene group. From P22 to P32 the animals were exposed to either clean

air or toluene vapors (5000–6000 ppm) for 10 min/day. This strategy simulated common toluene abuse

in humans, which consists of 15–20 rapid inhalations of highly concentrated solvent. Once the

inhalation period was over (P32), toluene exposed animals were randomly reassigned to one of following

experimental groups: (i) air-control/saline; (ii) toluene/saline; (iii) toluene/melatonin 0.5 mg/kg;

(iv) toluene/melatonin 1.0 mg/kg; (v) toluene/melatonin 5.0 mg/kg; and (vi) toluene/melatonin 10 mg/

kg. Seven days after the last inhalation (P39), all the animals were sacrificed under deep anesthesia;

brains were dissected out and stained according to the Golgi-Cox-Sholl procedure. Layer II/III pyramidal

neurons were morphologically analyzed by measuring their basilar dendritic length and the number of

branches. The results obtained revealed that (i) toluene inhalation significantly reduced dendritic

outgrowth and branching in all cortical areas studied, and (ii) intraperitoneal administration of melatonin

(0.5–10 mg/kg) was able to restore the dendritic impairment induced by toluene exposure.

& 2010 Elsevier GmbH. All rights reserved.

1. Introduction

It has been previously described that chronic tolueneinhalation produces diverse brain and behavioral abnormalities,including alterations in regional brain blood flow, cerebral andcerebellar atrophies, white matter abnormalities, and variousneurological deficits, such as memory loss, tremor, gait imbalance,and symptoms of frontal lobe release (Aydin et al., 2002; Deleuand Hanssens, 2000; Fornazzari et al., 1983; Hormes et al., 1986;Kamran and Bakshi, 1998; Kucuk et al., 2000; Lazar et al., 1983;Ryu et al., 1998). Animal models have confirmed these neurotoxiceffects, showing that repeated toluene inhalation during pre- orearly postnatal periods, resulted in reductions of brain weight,neurogenesis, number of cortical neurons, and Purkinje celldendritic outgrowth (Bowen et al., 2005, 2007; Burry et al.,2003; Gospe and Zhou, 2000; Pascual et al., 1996a). Even thoughneocortical neurons are thought to be involved in severalneurological and neuropsychological symptoms described insolvent abusers, the effect of toluene on cerebrocortical neuron

H. All rights reserved.

56 71 242 890.

l).

morphology has not yet been characterized. Then, the first aim ofthe present study focused on defining the impact of tolueneinhalation on dendritic outgrowth and branching in rat neocor-tical pyramidal neurons of frontal, parietal, and occipital cortices.

On the other hand, it has been demonstrated that melatonin,the main substance produced by the pineal gland, exertsprotective effects in neuronal damage induced by eitherexperimental cerebral ischemia or nigrostriatal degeneration(Garcıa-Chavez et al., 2008; Lin et al., 2008). Considering thatoxidative stress is one of the most important pathophysiologicalmechanism involved in the neuronal damage induced by thesepathologies (Khusnutdinova et al., 2008), and since melatonin is apotent free radical scavenger, it has been suggested that theneuroprotective action of melatonin is accomplished by itspowerful antioxidant effects (Manda et al., 2008). In the samevein, chronic toluene exposure seems to exert its deleteriouseffects on neuronal tissue through oxidative stress. In fact, it hasbeen shown that exposure to toluene, both in vitro and in vivo,increases free radical production, and consequently furtherincreasing the concentration of reactive oxygen species (Edelforset al., 2002; Mattia et al., 1993). Interestingly, administration ofmelatonin to toluene-exposed animals diminished the levels oftissue lipid peroxidation in hippocampus, cerebellum and cerebral

R. Pascual et al. / Experimental and Toxicologic Pathology 63 (2011) 467–471468

cortex, supporting its antioxidant and cell-protective actions(Baydas et al., 2003). Considering the above findings, it is possiblethat the administration of melatonin could promote the recoveryof structural neuronal damage induced by early toluene exposure.Thus, the second aim of the present study was to evaluate thepotential restorative effect of melatonin on neuronal dendriticimpairment induced by toluene inhalation.

Fig. 1. Golgi-Cox-Sholl impregnated pyramidal neurons (400� ), showing a clear

staining of dendritic processes. Bar: 20 mm.

2. Materials and methods

2.1. Animals and experimental design

Adult female Sprague-Dawley rats were mated withcolony-breeder male rats. One week before birth, dams wereindividually housed in laboratory cages with sawdust as beddingin a room under controlled environmental laboratory conditions(12:12 h light/dark cycle; 2172 1C), and free access to pellets andwater. Approximately 12 h after delivery (postnatal day 0, P0),litters were culled to 8 pups/dam (5 males and 3 females) andhoused together with a mother in standard laboratory cages(50�30�20 cm3). Pups were left with their biological dam,weaned at postnatal day 21 (P21) and randomly assigned to eitherthe air-control group (AC; n¼10,) or the toluene group (T; n¼42).Between P22 and P32, each male rat was gently placed into asealed glass chamber with a vent in the floor, and exposed toeither toluene vapors or clean air for 10 min/day. Toluene vaporswere generated by placing a toluene embedded filter paper at thebottom of the exposure chamber and volatilized by a fun. In orderto avoid an excessive accumulation of CO2, the animals wereplaced in a large chamber (25 l) for short periods (10 min). Due tothe chamber volume and the physicochemical properties oftoluene, animals were exposed to 5000–6000 ppm of vaporizedtoluene (Shelton, 2007). Under these conditions, toluene bloodconcentration should be around 81 mg/ml (Shelton and Slavova-Hernandez, 2009), like the concentration found in toluene abusers(Thiesen et al., 2007). Furthermore, this strategy simulated theusual abuse performed by humans, which consists of 15–20 rapidinhalations of highly concentrated solvent (‘‘sniffing’’ or ‘‘bagging’’behavior; Wilkins-Haug, 1997). Control animals were similarlymanipulated, but exposed to clean air only.

Once the inhalation period was over (P32), toluene exposedanimals were reassigned to one of the following experimental groups:(i) air-control/saline (AC; n: 10); (ii) toluene/saline (T; n: 10);(iii) toluene/melatonin 0.5 mg/kg (T/M0.5; n: 8); (iv) toluene/melato-nin 1.0 mg/kg (T/M1.0; n: 8); (v) toluene/melatonin 5.0 mg/kg (T/M5.0;n: 8); and (vi) toluene/melatonin 10 mg/kg (T/M10; n: 8). Melatonin(Sigma-Aldrich) was diluted in 10% ethanol–saline (0.9% NaCl and0.1% ethanol; Merck) and injected intraperitoneally (0.1 ml; Carloniet al., 2008; Garcıa-Chavez et al., 2008; Letechipıa-Vallejo et al., 2007).Air-control animals were treated similarly but without melatonin.Melatonin and saline solutions were daily administered between P32and P38.

To evaluate the potential impact of solvent inhalation on bodyweight, all animals were weighed every 3 days throughout theexperimental period.

Animal treatments and their care conditions were inaccordance with international guidelines and were also approvedby the local ethics committee. In addition, this study wasconducted using a minimal number of animals.

2.2. Histology and neuronal dendritic quantification

To study the impact of toluene inhalation and the potential‘‘therapeutic’’ effect of melatonin on pyramidal dendritic

development, animals were sacrificed under deep ether anesthe-sia. Brains were immediately dissected out, fixed and stained withthe Golgi-Cox-Sholl procedure (Sholl, 1953). After 30 days of slowmetallic mercury impregnation, the brains were dehydrated inethanol–acetone and ethanol–ether solutions (50–50% v/v;Merck), embedded in celoidin and hardened in chloroform vapors.Coronal sections (thickness: 120 mm) were cut using a sledgemicrotome, treated with potassium disulphide/oxalic acid (5%dilution; Merck) and coverslipped. To qualify for the basilardendritic morphometrical evaluations, pyramidal cells shouldfulfill the following criteria: (a) have a well-defined pyramidalshape; (b) show an adequate staining of the soma and dendrites;(c) have uninterrupted basilar dendritic processes; (d) have noextensive dendrites overlapping neighboring neurons; and (e) belocated in a cortical strip between 200 and 600 mm under the pialsurface of the frontal, parietal, and occipital cortices (carefullydelimited by the coordinates described in the Rat StereotaxicAtlas; Paxinos and Watson, 1998). Neurons were analyzed underan Olympus CX-3 light microscope (400� ). Selected neuronsmeeting the above criteria (Fig. 1) were captured with a digitalcamera attached to the microscope (Olympus CCD 5.0) andanalyzed using Micrometrics SE Premium V-2.8 software. Twodendritic variables were quantified: (i) basilar dendritic length/neuron (mm) and (ii) basilar dendritic branches/neuron. A total of1248 pyramidal cells were sampled from frontal, parietal andoccipital cortices (AC: 80; T: 80; T/M0.5: 64; T/M1.0: 64; T/M5.0: 64;T/M10.0: 64, for each cortical region). All slides were coded toavoid experimental bias and maximize reliability.

2.3. Statistical analysis

Experimental data were analyzed by the one-way ANOVA testand complemented post-hoc with the Scheffe test (STATA 9.1software) when significant differences (po0.05) were detected.

3. Results

In all cortical regions analyzed, animals exposed to toluenevapors revealed a significant reduction in dendritic length (Fig. 2;

Fig. 2. Basilar dendritic length per neuron in frontal (A), parietal (B), and occipital

(C) cortices of control, toluene, and toluene/melatonin (T/M) treated rats. All

comparisons were made in reference to the control group. Data are means7SEM;npo0.05, nnpo0.01 (one-way ANOVA test).

Fig. 3. Basilar dendritic branches per neuron in frontal (A), parietal (B), and

occipital (C) cortices of control, toluene, and toluene/melatonin (T/M) treated rats.

All comparisons were made in reference to the control group. Data are

means7SEM; npo0.05; nnpo0.01 (one-way ANOVA test).

R. Pascual et al. / Experimental and Toxicologic Pathology 63 (2011) 467–471 469

po0.01) and branching (Fig. 3; po0.01) compared to the agematched air-control group. Furthermore, as shown in Figs. 2 and3, all melatonin doses (0.5–10 mg/kg) were able to offset dendriticimpairment induced by toluene inhalation. Interestingly,pyramidal frontal neurons from animals treated with 1.0 or5.0 mg/kg melatonin exhibited between 33% and 40% increase indendritic length (Fig. 2A; po0.01) and branching (Fig. 3A;po0.01) compared to control animals. This trophic-like effectwas also observed in the length of dendrites of parietal neurons(Fig. 2B; po0.05) and in the dendritic branching of occipitalneurons (Fig. 3C; po0.05). As illustrated in Fig. 4, the rats fromdistinct experimental groups did not exhibit significant bodyweight differences throughout the experimental period.

Fig. 4. Mean body weight measurement (g) of control, toluene, and toluene/

melatonin (T/M) treated rats throughout the experimental period. AC: control;

TOL: toluene; T/M: toluene melatonin. Data are means7SEM (one-way ANOVA

test).

4. Discussion

In this study, we have demonstrated that daily subchronictoluene inhalation (5000–6000 ppm; 10 min/day) during theearly postweaning period (P22–P32) severely altered basilardendritic outgrowth in superficial pyramidal neurons in frontal,

parietal, and occipital cortices. Furthermore, we observed thatwith all doses of melatonin, the dendritic damage inducedby toluene exposure was significantly recovered. In addition,

R. Pascual et al. / Experimental and Toxicologic Pathology 63 (2011) 467–471470

melatonin showed to have dendritic trophic-like effects, mainly infrontal neurons.

To our knowledge, this is the first report that quantitativelyanalyzes the effect of early toluene inhalation on dendriticmorphology among different neocortical regions (frontal, parietaland occipital cortices). These results are in agreement with ourprior findings on dendritic maturation of cerebellar Purkinje cells.In that study, rats exposed to a mixture of toluene and n-hexaneduring the preweaning period, exhibited a significant decrease indendritic processes (Pascual et al., 1996a). The fact that tolueneinhalation significantly reduced pyramidal cell dendritic field inall the analyzed neocortical regions confirms its widespreadneurotoxic effect (Lo and Chen, 2005). Results mean that thereduced basilar dendritic outgrowth may lead to (or be associatedwith) the cortical alterations including the hippocampal altera-tions, extensive brain atrophy and loss of white/gray mattermaturation (Aydin et al., 2002, 2009; Rosenberg et al., 1988;Slomianka et al., 1990).

Toluene exposure altered both branching and dendritic length.This is consistent with several studies demonstrating that dendriticoutgrowth is vulnerable to different manipulations, such as under-nutrition, postweaning social isolation stress, and maternal depriva-tion (Pascual et al., 1996b; Pascual and Zamora-Leon, 2007).Considering that toluene exposure increases the expression of glialfibrillary acidic protein (GFAP), and since GFAP is a key indicator ofastrocytic activation as a consequence of neurodegenerative altera-tions (Gotohda et al., 2000), it is possible that the reduced dendriticoutgrowth reported in the present study could be induced byneuronal damage rather than inhibition of normal maturationalevents. Further immunohistochemical studies should be performedin order to elucidate whether toluene actually exerted dendriticdamage or simply altered the dendritic outgrowth program ofneocortical pyramidal neurons.

Since the exact mechanisms involved in the neurotoxic effectof toluene on dendritic process outgrowth are still unknown, wepostulate few possible pathways. First, given that N-methyl-D-aspartate (NMDA) receptors play important roles in dendriticmaturation (Rajan and Cline, 1998; Espinosa et al., 2009), andsince toluene exposure alters the functionality of this glutama-tergic receptor (Bale et al., 2005; Cruz et al., 1998), it is possiblethat the stunted dendritic branching reported in the current studycould be generated, at least in part, via NMDA receptoralterations. A second probable mechanism involved in tolueneneurotoxicity is oxidative stress, since it has been previouslydemonstrated that synaptosomal fractions from toluene exposedrats presented higher levels of H2O2 (Edelfors et al., 2002), whichcould damage neuronal membranes and structural proteins.Interestingly, the administration of substances with radical-scavenging properties, such as melatonin, protects neural tissuefrom toluene-based neurotoxicity (Baydas et al., 2003), demon-strating the neurotoxic effect induced by oxidative stress. Third,given that toluene alters membrane fluidity throughout a changein ganglioside concentrations (Engelke et al., 1992; Von Euleret al., 1987), and that glycosphingolipids are involved inpromoting neuronal dendritic outgrowth (Walkley et al., 2000),the observed dendritic impairments induced by toluene exposurecould also be a consequence of dendritic membrane alterations.

On the other hand, melatonin administration during the weekfollowing the last toluene inhalation significantly offset the toxiceffect of toluene on neuronal differentiation. The current restorativeeffect observed in melatonin-treated animals is consistent withseveral studies performed in animal models of neural damage. Forexample, melatonin at 10 mg/kg/day protected prefrontal andhippocampal pyramidal neurons (dendritic spines and branching)from experimental hypoxia-ischemia damage (Gonzalez-Burgoset al., 2007; Garcıa-Chavez et al., 2008; Letechipıa-Vallejo et al.,

2007), and prevented nigrostriatal degeneration and a-synucleinaggregation in rotenone animal models of Parkinson’s disease (Linet al., 2008). Similar neuroprotective effects were observed inhypoxic-ischemic brain injury in preweaning rats treated with5.0–15 mg/kg melatonin, 30 min before experimental brain damage(Carloni et al., 2008). Then, the above-mentioned data, together withour results, support the suggested action of melatonin as aneuroprotective and neurorescue agent. Interestingly, a dramaticincrease in dendritic branching and length was observed in animalstreated with 1.0 or 5.0 mg/kg melatonin, mainly in frontal pyramidalneurons, compared to air-control animals. This could be explainedby the fact that melatonin stimulates growth hormone secretion(Meeking et al., 1999), which could subsequently promote dendriticoutgrowth.

In conclusion, the results obtained in the present studyindicated that early subchronic toluene inhalation severelyaltered branching and dendritic outgrowth in layer II/III of frontal,parietal, and occipital pyramidal neurons. Additionally, melatoninadministration, at doses frequently used in animal models ofexperimental brain damage, was able to recover the observeddendritic impairments.

Acknowledgments

This research was supported by Grant PUCV-VRIEA 127.706/2008.

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