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Inflammatory responses in the rat superior colliculus after eye enucleation
Brain Research Bulletin 101 (2014) 1–6
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Inflammatory responses in the rat superior colliculus after eye enucleation夽 Marina S. Hernandes ∗ , Luiz R.G. Britto Department of Physiology and Biophysics, Institute of Biomedical Sciences, USP, SP, Brazil
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Article history: Received 19 September 2013 Received in revised form 26 November 2013 Accepted 2 December 2013 Available online 12 December 2013 Keywords: Eye enucleation Glial cells Visual system COX-2
a b s t r a c t Ocular enucleation induces profound morphological alterations in central visual areas. However, little is known about the response of glial cells and possible inflammatory processes in visual brain areas resulting from eye enucleation. In this study, immunoblotting and immunostaining assays revealed increased expression of astrocyte and microglia markers in the rat superior colliculus (SC) between 1 and 15 days after contralateral enucleation. A transient increase of neuronal COX-2 protein expression was also found in the SC. To evaluate the role of an anti-inflammatory drug in attenuating both COX-2 and glial cell activation, the synthetic glucocorticoid dexamethasone (DEX) was administered (1 mg/kg i.p., for 3 days) to enucleated rats. Immunoblotting data revealed that DEX treatment significantly inhibited COX-2 protein expression. Postlesion immunostaining for astrocyte and microglia markers was also significantly reduced by DEX treatment. These findings suggest that the removal of retinal ganglion cell input generates inflammatory responses in central retinorecipient structures. © 2013 Elsevier Inc. All rights reserved.
1. Introduction The visual system provides a suitable model to study the cellular and molecular mechanisms involved in the neuroplasticity of the adult central nervous system. In the rat visual system, the main primary retinal projection areas are the superior colliculus (SC) and the dorsal lateral geniculate nucleus (DLG). More than 90% of the retinal ganglion cells cross at the optic chiasm and only a small amount of the retinocollicular fibers (from 5 to 3%) had been shown to project to the ipsilateral hemisphere of the brain (Sefton et al., 2004). The experimental manipulation of the visual system has attracted particularly interest because the retinal inputs to retinorecipient areas are highly arranged topographically. The lesions are also easy to perform and completely reproducible. Unilateral loss of the retinal inputs can be induced by sectioning the optic nerve or by eye enucleation. The morphological, functional and biochemical alterations specifically induced by eye enucleation have significantly extended our knowledge of
夽 Grant info: Work supported by grants from FAPESP (Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). M.S.H. is the recipient of a fellowship from FAPESP. L.R.G.B. is a research fellow from CNPq. ∗ Corresponding author at: Laboratory of Cellular Neurobiology, Institute of Biomedical Sciences, University of São Paulo, Av. Professor Lineu Prestes, 1524, CEP 05508-900 São Paulo, Brazil. Tel.: +55 11 3091 7242; fax: +55 11 3091 7426. E-mail address: [email protected] (M.S. Hernandes). 0361-9230/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2013.12.001
mechanisms involved in the neuroplasticity events in several structures of the visual system (Smith and Bedi, 1997). The removal of the retinal input leads to the degeneration of the corresponding optic nerve terminals and induces a series of morphological changes predominantly in the contralateral retinorecipient structures (Cajal, 1911; Nauta and van Straaten, 1947). However, anatomical studies on the projection targets of the first-order (SC and DLG) and second-order (e.g. visual cortices) centers revealed that both contralateral and ipsilateral hemispheres are deeply affected by this intervention (Wree and Schleicher, 1988). Several studies have suggested that ocular enucleation results in profound alterations in the SC. In the rat, it has been demonstrated that eye enucleation causes selective decrease in the immunoreactivity for glutamate receptors into the contralateral SC (Kim and Jeon, 1999; Ortega et al., 1995). The volume of stratum griseum superficiale (SGS), one of the superficial layers of the SC, was reduced by about 40% on the side contralateral to the enucleated eye, in the same way as the numerical density of neurons (Smith and Bedi, 1997). Using a microarray enzymatic assay to detect amino acid content, it was demonstrated that the glutamate content exhibited a significant reduction into the SC after deafferentation, whereas neither GABA nor aspartate content was affected by that lesion (Sakurai and Okada, 1992). It has been also demonstrated, by means of ligand binding quantitative autoradiography, that unilateral enucleation induces a selective reduction in the serotonin receptor binding activity in the deprived SC and DLG. A significant reduction of the GABA receptor binding was only observed into DLG and visual cortex (Chalmers and McCulloch, 1991).
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Although many studies have described mechanisms of neuronal plasticity and structural remodeling induced by ocular enucleation, very little is known about possible inflammatory responses induced by this procedure into retinorecipient structures. However, such mechanisms are relevant, considering that inflammatory responses are likely to influence the repair process that occurs after neural lesions. We have previously demonstrated that the removal of retinal ganglion cell input leads to a transient nuclear-factor B (NF-B) activation into SC, a transcription factor widely accepted to be involved in the pathophysiology of inflammatory diseases. NF-B activation was effectively blocked by the treatment with dexamethasone (DEX). Therefore, the experiments described here were designed to directly test the hypothesis that there is a significant inflammatory component in the SC after unilateral eye enucleation in adult rats. The spatio-temporal protein expression of cyclooxygenase-2 (COX-2) and of astrocyte and microglia markers was evaluated in the SC after retinal removal. In addition, the effect of the synthetic glucocorticoid dexamethasone (DEX) in attenuating both COX-2 expression and glial cell activation was also determined. 2. Materials and methods 2.1. Animals Nine week-old male Wistar rats, weighing between 180 and 200 g, were used throughout this study. Animals were singly housed and maintained on a 12:12 h light/dark cycle. All procedures were approved by the Institutional Animal Care Committee of the Institute of Biomedical Sciences, University of São Paulo (protocol number 017/2007). 2.2. Unilateral enucleation The animals were anesthetized with ketamine hydrochloride (5 mg/100 g of body weight, i.m.) and xylazine (1 mg/100 g of body weight, i.m.). Lidocaine was applied around the eye before and after enucleation. A piece of absorbable gelatin (Gelfoam, Upjohn, Kalamazoo, MO, USA) was placed inside the orbit for better healing. The eyelids were sutured and the animals were returned to the animal facility. Animals used for immunoblotting (n = 35) were sacrificed after different survival times, namely 1 h and 1, 7, 10, 15, and 30 days after unilateral enucleation. An additional group of rats (n = 25) were unilaterally enucleated, allowed to survive for 7 days, and treated with DEX; their brains were used to evaluate COX-2 protein expression by means of immunoblotting assays and to analyze astrocyte (glial fibrillary acidic protein – GFAP) and microglia (OX42) immunostaining. Five other rats were unilaterally enucleated and their brains were used for COX-2 immunofluorescence assays. Intact animals were used as control groups. 2.3. Dexamethasone treatment Eye-enucleated animals received daily injections of DEX (1 mg/kg i.p., for 3 days), a synthetic glucocorticoid with antiinflammatory properties (Barel et al., 2010). DEX was dissolved in saline and thus the control groups received saline injections. Seven days after the lesion the brains were processed for both immunohistochemistry for GFAP and OX42 and for immunoblotting assays. 2.4. Western blotting In the various time-points after ocular enucleation, animals were decapitated and the SC quickly collected, frozen in liquid
nitrogen and stored at −70 ◦ C until use. The tissue was then homogenized at 4 ◦ C in extraction buffer (Tris, pH 7.4, 100 mM; EDTA 10 mM; PMSF 2 mM; aprotinin 0.01 mg/ml). The homogenates were centrifuged at 12,000 rpm at 4 ◦ C for 20 min, and the protein concentration of the supernatant was determined using a protein assay kit (Bio-Rad, San Diego, CA, USA). The material was stored in sample buffer (Tris/HCl 125 mM, pH 6.8; 2.5% (p/v) SDS; 2.5% 2-mercaptoethanol, 4 mM EDTA and 0.05% bromophenol blue) at −70 ◦ C until immunoblotting assay. Fifty to 75 g of total protein in Laemmli buffer were boiled for 5 min and separated by 6.5% acrylamide SDS gels (Bio-Rad, San Diego, CA, USA) at 25 mA and electrophoretically transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) at 15 V for 45 min using a Trans-Blot cell system (Bio-Rad, San Diego, CA, USA). The membranes were then blocked for 2 h at room temperature with phosphate buffered saline (PBS) containing 0.05% Tween-20 (TTBS) and 5% non-fat milk, and incubated overnight at 4 ◦ C with mouse anti-rat OX42 (CD11b/c, Biosciences, CA, USA), mouse anti-rat GFAP (Immunon, Pittsburgh, PA, USA) and with goat anti-rat COX-2 (Abcam, Cambridge, UK) at a concentration of 1:1000. The membranes were then incubated for 2 h with anti-mouse-HRP IgG for OX42 and GFAP (Santa Cruz, CA, USA), and anti-goat-HRP for COX-2 (Santa Cruz, CA, USA), diluted 1:1000 in TTBS with 1% non-fat milk. The probed proteins were developed by using a chemiluminescent kit (ECL, Amersham Biosciences, EUA). To detect multiple signals using a single membrane, the membrane was incubated for 30 min at room temperature with stripping buffer. An anti--actin antibody (Sigma, St Louis, MO) was used to quantify -actin as a loading control. The bound antibodies were visualized using radiographic films. The quantification of band intensity was performed with Image J (National Institutes of Health/USA). 2.5. Immunoperoxidase Seven days after ocular enucleation, rats were deeply anesthetized with ketamine hydrochloride and xylazine and subjected to transcardiac perfusion, with a buffered saline solution, followed by a fixative solution composed of 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer (PB, pH 7.4). The brain was collected, post-fixed in PFA for 4 h, and transferred to a 30% sucrose solution in PB to ensure cryoprotection, which lasted for 48 h. The brain sections (30 m) were obtained on a sliding microtome adapted for cryosectioning. The sections were incubated free-floating for 12–16 h with the same primary antibodies used for immunoblotting anti-GFAP and anti-OX42, both diluted 1:1000 in 0.3% of Triton X-100, containing 0.05% normal donkey serum. Following 3 washes of 10 min each with PB, sections were incubated for 2 h with the biotinylated secondary antibody (donkey anti-mouse IgG, Jackson ImmunoResearch, West Grove, PA, USA, 1:200), then with the avidin–biotin complex (1:100; ABC Elite kit, Vector Labs, Burlingame, CA, USA). After washing, the sections were reacted with 0.05% 3,3-diaminobenzidine and 0.01% hydrogen peroxide in PB. Intensification was conducted with 0.05% osmium tetroxide in water. The sections were mounted on gelatinized slides, dehydrated, cleared and coverslipped. Controls for immunostaining included the omission of the primary antibody and its substitution for normal mouse serum, which completely eliminated staining. The material was analyzed on a light microscope and digital images were collected. Quantitative image analysis was performed using ImageJ (National Institutes of Health/USA). GFAP and OX42 immunostaining was evaluated in terms of optical density within 0.4 mm2 areas of the superficial, visual portion of the SC. The mean optical density of SC labeled areas was compared with the mean density of neighboring, non-labeled areas in the same sections, to obtain a labeling index reflecting the mean signal-to-noise ratio. The resulting indexes
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for the control and deafferented groups were then compared and subjected to statistical analysis using Graphpad Prism (3.02). 2.6. Immunofluorescence For co-localization studies, immunofluorescence tissue preparation was performed in the same conditions as in the immunoperoxidase assays, described above. Briefly, the brain sections were incubated free-floating for 12–16 h with the following primary antibodies: mouse anti-rat GFAP, mouse anti-rat OX42, mouse anti-rat NeuN (Chemicon, Billerica, MA, USA) and goat antirat COX-2 diluted 1:500 in 0.3% of Triton X-100. Anti-goat FITC conjugated (1:250; Abcam, Cambridge, UK) and anti-mouse TRITC conjugated (1:250; Abcam, Cambridge, UK) secondary antibodies were used. The sections were mounted on gelatinized slides and coverslipped. Negative controls were performed by the omission of primary antibody or secondary antibody. The material was analyzed on a confocal microscope and digital images were collected. 2.7. Statistical analysis Values are expressed as means ± S.E.M. Statistical comparisons were performed by one-way ANOVA, followed by Tukey’s multiple comparison test. Values of p ≤ 0.05 were considered significant. Data from the contralateral side of the brain to the removed eye was always compared to data obtained from intact animals. The number of rats (n) used in each group is referred to in the text. 3. Results 3.1. Immunoblotting Immunoblotting data revealed increased GFAP, OX42 and COX-2 protein expression after unilateral enucleation. As shown in Fig. 1A, densitometric analyses revealed an increase of GFAP protein levels in SC at days 7 (ca. 551%) and 15 postlesion (ca. 240%). Similar results were also observed for OX42, for which a significant increase was observed at day 7 (ca. 205%) postlesion (Fig. 2A). For COX-2, the increase of optical density started as early as 1 h (ca. 287%) and 1 day (ca. 258%), persisting until day 7 postlesion (ca. 218%). This effect appeared to decrease progressively thereafter (Fig. 3A). Immunoblotting data also revealed that DEX treatment significantly inhibited COX-2 protein expression in SC 7 days after unilateral enucleation (Fig. 3B). 3.2. Immunoperoxidase In agreement to our immunoblotting data, immunostaining for GFAP was markedly increased (ca. 165%) in the SC at 7 days postlesion (Fig. 1B and C). A consistent increase of OX42 immunostaining (ca. 130%) was also found in the superficial layers of the SC 7 days after eye enucleation, when compared to control animals (Fig. 2B and C). The treatment with DEX significantly decreased both GFAP (by about 162%) and OX42 immunostaining (by about 80%), as depicted in Figs. 1 and 2, respectively. 3.3. Immunofluorescence The observation that COX-2 increased protein expression was induced by ocular enucleation in the SC led us to test whether this protein is expressed in neurons, astrocytes or microglial cells. Consistent with the immunoblotting results, a significant increase of COX-2 expression was observed in the SC 7 days after ocular enucleation, in comparison to the control group (data not shown). COX-2 displayed coexpression with NeuN in the SC, indicating neuron-specific expression of COX-2 (Fig. 4). Neither astrocytes nor
Fig. 1. Effects of eye enucleation on GFAP protein levels in the SC. In (A), mean ratio of GFAP densitometric data in relation to beta-actin after different survival periods post-lesion. Significance compared to control at *p < 0.001 and **p < 0.05. In (B), digital images of GFAP immunoreactivity in the control, control + DEX, deafferented and deafferented + DEX conditions 7 days post-lesion. In (C), GFAP immunoreactivity optical density data. *p < 0.001 (control vs deafferented) and **p < 0.001 (deafferented vs deafferented + DEX). DEX: dexamethasone.
microglia expressed COX-2, as verified by co-immunostaining of COX-2 with GFAP (astrocytes) or OX42 (microglia) (Fig. 4). 4. Discussion In the present study we hypothesized that unilateral eye enucleation elicits inflammatory-mediated responses in a retinorecipient structure. Our principal findings demonstrate that (1) Deafferentation of the visual system resulted in increased transient glial cell activation and COX-2 protein expression in SC; (2) The systemic administration of dexamethasone, a synthetic glucocorticoid, attenuated both glial cell activation and COX-2 protein expression; (3) COX-2 was found to be expressed only in neurons. In rodents, visual information from the retina is predominantly sent to the SC through the optic nerve constituted by retinal ganglion cells (RGCs) axons (Sefton et al., 2004). The degeneration
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Fig. 3. Effects of ocular enucleation on COX-2 protein levels in the SC. In (A), mean ratio of COX-2 densitometric data in relation to beta-actin. Significance compared to control at *p < 0.001 and **p < 0.05. In (B), effect of DEX treatment on COX-2 protein expression in the SC from animals allowed to survive for 7 days postlesion. *p < 0.05 (control vs deafferented) and *p < 0.05 (deafferented vs deafferented + DEX). DEX: dexamethasone.
Fig. 2. Effects of ocular enucleation on OX42 protein levels in the SC. In (A), mean ratio of OX42 densitometric data in relation to beta-actin after different survival periods post-lesion. Significance compared to control at **p < 0.05. In (B), digital images of OX42 immunoreactivity in the control, control + DEX, deafferented and deafferented + DEX conditions 7 days post-lesion. In (C), OX42 immunoreactivity optical density data. *p < 0.001 (control vs deafferented) and **p < 0.05 (deafferented vs deafferented + DEX). DEX: dexamethasone.
of RGC results in irreversible blindness found in several major vision-threatening diseases such as glaucoma and diabetic retinopathy (Yokota et al., 2011). Moreover, several clinical and experimental studies have demonstrated that RGC degeneration in glaucoma shares some common features with axonopathies of several pathologic processes such as Alzheimer and Parkinson’s diseases, although the mechanisms driving these phenomena are far from being understood (McKinnon, 2003; Guo et al., 2007). Previous studies in animal models of glaucoma have demonstrated that the distal transport depletion in the SC is an early manifestation of the disease onset. Since then, the SC has attracted increased attention in experimental glaucoma research as it may represent a promising target to reduce glaucomatous neurodegeneration
(Crish et al., 2010). Thus, the investigation of specific inflammatory pathways elicited by RGC degeneration in a retinorecipient structure such as SC may represent a new direction in the pursuit of effective therapies for glaucoma and other neurodegenerative conditions. The initial experiments performed in this study demonstrated that deafferentation resulted in increased glial cell activation in SC. These results are in accordance with earlier studies in which it was demonstrated that unilateral optic nerve lesions change the glial cell population in the SC (Rao and Lund, 1989; Schmidt-Kastner et al., 1993). A response of the glial cell population was also reported in the adult rat hippocampus deafferented by unilateral lesion of the entorhinal cortex (Gall et al., 1979). It has been suggested that increased glial activation may be a consequence of the central inflammatory tissue reaction induced by the deafferentation of the brainstem vestibular nuclei (Campos-Torres et al., 1999). In line with this, we therefore suggest that the reactive gliosis observed in the SC after eye enucleation may also represent an inflammatory response induced by the lesion. Several in vitro and in vivo experimental models of brain inflammation have demonstrated that not only microglia, but also astrocytes, blood inflammatory cells, and even neurons participate in the modulation of the inflammatory response. Astrocyte activation in the nervous system is frequently associated with brain inflammation (Campos-Torres et al., 1999). Astrocytes produce several anti-inflammatory factors (Kim et al., 2010) and chemokines that recruit monocytes (Kim et al., 2011). In addition, astrocytes induce the expression of antioxidant enzymes such as heme oxygenase-1, which are able to inhibit microglial activation (Min et al., 2006). Morphologically, reactive astrocytes display hypertrophy of their cellular processes, elongation of cytoplasmic
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Fig. 4. Effects of ocular enucleation on COX-2 expression in the SC. Representative confocal microscopy images illustrating COX-2 immunostaining in the SC of control (left column) and deafferented (right column) mice 7 days after the lesion. Immunostaining was carried out using antibodies for COX-2 (shown in green) together with NeuN (top), GFAP (center) and OX42 (bottom). Microglia, astrocyte, and neuronal markers are shown in red. COX-2 positive cells are neurons.
processes, hyperplasia and an increased GFAP immunostaining. GFAP is an intermediate filament protein specifically produced by astrocytes, and its increased expression is considered to be a marker of reactive astrocytes (Malhotra et al., 1990). Microglial cells, in turn, represent the resident immune cells of the brain, and their activation seems to coordinate the inflammatory response in the nervous system (Kato et al., 1996; Lynch, 2009). Activation of microglia is linked to the secretion of a number of inflammatory mediators including cytokines, chemokines, eicosanoids, reactive oxygen species (ROS) and nitric oxide (Gehrmann et al., 1995). Similarly to astrocytes, microglial cells also change their morphology and function in response to neural lesions. Activated microglia show an increased cell body size, a thickening of proximal processes and a decrease in the ramification of distal branches (Raivich et al., 1999). Corroborating our results, three days after the transection of the facial nerve, an increased immunoreactivity for a microglial marker was observed into the facial motor nucleus (Raivich et al., 1999). It has been also demonstrated that the pro and anti-inflammatory cytokines produced by activated microglial cells could promote the survival of deafferented vestibular neurons, contributing to the vestibular compensation process (Liberge et al., 2010). Besides astrocyte and microglial cell involvement in the inflammatory response, two other aspects involving its activation deserve consideration. First, it has been demonstrated that reactive glial cells act by supporting several aspects of regeneration such
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as collateral sprouting and synaptic recovery, and maintaining homeostasis of extracellular fluid by taking up glutamate (Pekny and Nilsson, 2005). Since high levels of glutamate are present in the retinocollicular synaptic terminals (Li et al., 1996) and the deafferentation may cause a massive release of this neurotransmitter shortly after the lesion (Choi, 1988; Dye and Karten, 1996), the astrocyte and microglial activation may represent a protective mechanism against glutamate excitotoxicity and, consequently, against the deafferentation trauma. Second, both astrocytes and microglial cells have been described to be phagocytically active both in vitro and in vivo conditions (Ito et al., 2007; Kalmar et al., 2001). Therefore, it has been speculated that increased expression of astrocytes and microglia after deafferentation may reflect their function as phagocytotic cells to remove the debris generated by the degeneration of the retinocollicular fibers and axon terminals. Having in mind that eye enucleation may induce an inflammatory response into the SC, we further investigated whether glial cell activation could be inhibited by the treatment with an antiinflammatory. DEX is a synthetic glucocorticoid able to inhibit glial activation induced by different inflammatory stimuli (Felts et al., 2005; Nadeau and Rivest, 2003; Pan et al., 2001). The results showed that DEX was able to significantly decrease both microglia and astrocytic activation into the SC. Similar results have also been demonstrated in different experimental models, such as in cultured microglia (Ganter et al., 1992), in the suprachiasmatic nucleus of adrenalectomized rats (Maurel et al., 2000), and after the insertion of neural prosthetic devices in the rat neocortex (Spataro et al., 2005). COX-2 is one of the most important components of the inflammatory response (Nogawa et al., 1997). Neuroinflammatory processes involving increased expression of COX-2 have been associated with several neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease and amyotrophic lateral sclerosis (Minghetti, 2004). In the present study, upregulation of COX-2 was observed as early as 1 h after the lesion, and it was found to be intense for one to seven days and then declined until day 30. We also demonstrated that COX-2 was found only in neurons. Similarly, COX-2 was found to be co-localized predominantly in the spinal dorsal horn neurons after spinal nerve ligation (Lau et al., 2013). The beneficial effect of the anti-inflammatory DEX on decreasing COX2 protein expression suggests that COX-2 plays a proinflammatory role in deafferentation-induced neuroinflammation. Similar results have also been recently described following partial hepatectomy. The treatment with a COX-2 inhibitor decreased the hippocampal inflammation induced by that procedure (Peng et al., 2013). In summary, the findings of the present study reveal that eye enucleation induces glial cell activation in the rat SC. Additionally, the lesion also leads to COX-2 upregulation in neurons, and both glial cell activation and COX-2 upregulation can be inhibited by treatment with an anti-inflammatory. Our results suggest that the removal of retinal ganglion cell input generates inflammatory responses in central retinorecipient structures, which may play an important role in neuronal degeneration and deafferentationrelated neuroplasticity of the rat visual system.
Conflict of interest None declared.
Acknowledgments Thanks are due to Adilson S. Alves for technical assistance.
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Inflammatory responses in the rat superior colliculus after eye enucleation夽 Marina S. Hernandes ∗ , Luiz R.G. Britto Department of Physiology and Biophysics, Institute of Biomedical Sciences, USP, SP, Brazil
a r t i c l e
i n f o
Article history: Received 19 September 2013 Received in revised form 26 November 2013 Accepted 2 December 2013 Available online 12 December 2013 Keywords: Eye enucleation Glial cells Visual system COX-2
a b s t r a c t Ocular enucleation induces profound morphological alterations in central visual areas. However, little is known about the response of glial cells and possible inflammatory processes in visual brain areas resulting from eye enucleation. In this study, immunoblotting and immunostaining assays revealed increased expression of astrocyte and microglia markers in the rat superior colliculus (SC) between 1 and 15 days after contralateral enucleation. A transient increase of neuronal COX-2 protein expression was also found in the SC. To evaluate the role of an anti-inflammatory drug in attenuating both COX-2 and glial cell activation, the synthetic glucocorticoid dexamethasone (DEX) was administered (1 mg/kg i.p., for 3 days) to enucleated rats. Immunoblotting data revealed that DEX treatment significantly inhibited COX-2 protein expression. Postlesion immunostaining for astrocyte and microglia markers was also significantly reduced by DEX treatment. These findings suggest that the removal of retinal ganglion cell input generates inflammatory responses in central retinorecipient structures. © 2013 Elsevier Inc. All rights reserved.
1. Introduction The visual system provides a suitable model to study the cellular and molecular mechanisms involved in the neuroplasticity of the adult central nervous system. In the rat visual system, the main primary retinal projection areas are the superior colliculus (SC) and the dorsal lateral geniculate nucleus (DLG). More than 90% of the retinal ganglion cells cross at the optic chiasm and only a small amount of the retinocollicular fibers (from 5 to 3%) had been shown to project to the ipsilateral hemisphere of the brain (Sefton et al., 2004). The experimental manipulation of the visual system has attracted particularly interest because the retinal inputs to retinorecipient areas are highly arranged topographically. The lesions are also easy to perform and completely reproducible. Unilateral loss of the retinal inputs can be induced by sectioning the optic nerve or by eye enucleation. The morphological, functional and biochemical alterations specifically induced by eye enucleation have significantly extended our knowledge of
夽 Grant info: Work supported by grants from FAPESP (Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). M.S.H. is the recipient of a fellowship from FAPESP. L.R.G.B. is a research fellow from CNPq. ∗ Corresponding author at: Laboratory of Cellular Neurobiology, Institute of Biomedical Sciences, University of São Paulo, Av. Professor Lineu Prestes, 1524, CEP 05508-900 São Paulo, Brazil. Tel.: +55 11 3091 7242; fax: +55 11 3091 7426. E-mail address: [email protected] (M.S. Hernandes). 0361-9230/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2013.12.001
mechanisms involved in the neuroplasticity events in several structures of the visual system (Smith and Bedi, 1997). The removal of the retinal input leads to the degeneration of the corresponding optic nerve terminals and induces a series of morphological changes predominantly in the contralateral retinorecipient structures (Cajal, 1911; Nauta and van Straaten, 1947). However, anatomical studies on the projection targets of the first-order (SC and DLG) and second-order (e.g. visual cortices) centers revealed that both contralateral and ipsilateral hemispheres are deeply affected by this intervention (Wree and Schleicher, 1988). Several studies have suggested that ocular enucleation results in profound alterations in the SC. In the rat, it has been demonstrated that eye enucleation causes selective decrease in the immunoreactivity for glutamate receptors into the contralateral SC (Kim and Jeon, 1999; Ortega et al., 1995). The volume of stratum griseum superficiale (SGS), one of the superficial layers of the SC, was reduced by about 40% on the side contralateral to the enucleated eye, in the same way as the numerical density of neurons (Smith and Bedi, 1997). Using a microarray enzymatic assay to detect amino acid content, it was demonstrated that the glutamate content exhibited a significant reduction into the SC after deafferentation, whereas neither GABA nor aspartate content was affected by that lesion (Sakurai and Okada, 1992). It has been also demonstrated, by means of ligand binding quantitative autoradiography, that unilateral enucleation induces a selective reduction in the serotonin receptor binding activity in the deprived SC and DLG. A significant reduction of the GABA receptor binding was only observed into DLG and visual cortex (Chalmers and McCulloch, 1991).
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Although many studies have described mechanisms of neuronal plasticity and structural remodeling induced by ocular enucleation, very little is known about possible inflammatory responses induced by this procedure into retinorecipient structures. However, such mechanisms are relevant, considering that inflammatory responses are likely to influence the repair process that occurs after neural lesions. We have previously demonstrated that the removal of retinal ganglion cell input leads to a transient nuclear-factor B (NF-B) activation into SC, a transcription factor widely accepted to be involved in the pathophysiology of inflammatory diseases. NF-B activation was effectively blocked by the treatment with dexamethasone (DEX). Therefore, the experiments described here were designed to directly test the hypothesis that there is a significant inflammatory component in the SC after unilateral eye enucleation in adult rats. The spatio-temporal protein expression of cyclooxygenase-2 (COX-2) and of astrocyte and microglia markers was evaluated in the SC after retinal removal. In addition, the effect of the synthetic glucocorticoid dexamethasone (DEX) in attenuating both COX-2 expression and glial cell activation was also determined. 2. Materials and methods 2.1. Animals Nine week-old male Wistar rats, weighing between 180 and 200 g, were used throughout this study. Animals were singly housed and maintained on a 12:12 h light/dark cycle. All procedures were approved by the Institutional Animal Care Committee of the Institute of Biomedical Sciences, University of São Paulo (protocol number 017/2007). 2.2. Unilateral enucleation The animals were anesthetized with ketamine hydrochloride (5 mg/100 g of body weight, i.m.) and xylazine (1 mg/100 g of body weight, i.m.). Lidocaine was applied around the eye before and after enucleation. A piece of absorbable gelatin (Gelfoam, Upjohn, Kalamazoo, MO, USA) was placed inside the orbit for better healing. The eyelids were sutured and the animals were returned to the animal facility. Animals used for immunoblotting (n = 35) were sacrificed after different survival times, namely 1 h and 1, 7, 10, 15, and 30 days after unilateral enucleation. An additional group of rats (n = 25) were unilaterally enucleated, allowed to survive for 7 days, and treated with DEX; their brains were used to evaluate COX-2 protein expression by means of immunoblotting assays and to analyze astrocyte (glial fibrillary acidic protein – GFAP) and microglia (OX42) immunostaining. Five other rats were unilaterally enucleated and their brains were used for COX-2 immunofluorescence assays. Intact animals were used as control groups. 2.3. Dexamethasone treatment Eye-enucleated animals received daily injections of DEX (1 mg/kg i.p., for 3 days), a synthetic glucocorticoid with antiinflammatory properties (Barel et al., 2010). DEX was dissolved in saline and thus the control groups received saline injections. Seven days after the lesion the brains were processed for both immunohistochemistry for GFAP and OX42 and for immunoblotting assays. 2.4. Western blotting In the various time-points after ocular enucleation, animals were decapitated and the SC quickly collected, frozen in liquid
nitrogen and stored at −70 ◦ C until use. The tissue was then homogenized at 4 ◦ C in extraction buffer (Tris, pH 7.4, 100 mM; EDTA 10 mM; PMSF 2 mM; aprotinin 0.01 mg/ml). The homogenates were centrifuged at 12,000 rpm at 4 ◦ C for 20 min, and the protein concentration of the supernatant was determined using a protein assay kit (Bio-Rad, San Diego, CA, USA). The material was stored in sample buffer (Tris/HCl 125 mM, pH 6.8; 2.5% (p/v) SDS; 2.5% 2-mercaptoethanol, 4 mM EDTA and 0.05% bromophenol blue) at −70 ◦ C until immunoblotting assay. Fifty to 75 g of total protein in Laemmli buffer were boiled for 5 min and separated by 6.5% acrylamide SDS gels (Bio-Rad, San Diego, CA, USA) at 25 mA and electrophoretically transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) at 15 V for 45 min using a Trans-Blot cell system (Bio-Rad, San Diego, CA, USA). The membranes were then blocked for 2 h at room temperature with phosphate buffered saline (PBS) containing 0.05% Tween-20 (TTBS) and 5% non-fat milk, and incubated overnight at 4 ◦ C with mouse anti-rat OX42 (CD11b/c, Biosciences, CA, USA), mouse anti-rat GFAP (Immunon, Pittsburgh, PA, USA) and with goat anti-rat COX-2 (Abcam, Cambridge, UK) at a concentration of 1:1000. The membranes were then incubated for 2 h with anti-mouse-HRP IgG for OX42 and GFAP (Santa Cruz, CA, USA), and anti-goat-HRP for COX-2 (Santa Cruz, CA, USA), diluted 1:1000 in TTBS with 1% non-fat milk. The probed proteins were developed by using a chemiluminescent kit (ECL, Amersham Biosciences, EUA). To detect multiple signals using a single membrane, the membrane was incubated for 30 min at room temperature with stripping buffer. An anti--actin antibody (Sigma, St Louis, MO) was used to quantify -actin as a loading control. The bound antibodies were visualized using radiographic films. The quantification of band intensity was performed with Image J (National Institutes of Health/USA). 2.5. Immunoperoxidase Seven days after ocular enucleation, rats were deeply anesthetized with ketamine hydrochloride and xylazine and subjected to transcardiac perfusion, with a buffered saline solution, followed by a fixative solution composed of 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer (PB, pH 7.4). The brain was collected, post-fixed in PFA for 4 h, and transferred to a 30% sucrose solution in PB to ensure cryoprotection, which lasted for 48 h. The brain sections (30 m) were obtained on a sliding microtome adapted for cryosectioning. The sections were incubated free-floating for 12–16 h with the same primary antibodies used for immunoblotting anti-GFAP and anti-OX42, both diluted 1:1000 in 0.3% of Triton X-100, containing 0.05% normal donkey serum. Following 3 washes of 10 min each with PB, sections were incubated for 2 h with the biotinylated secondary antibody (donkey anti-mouse IgG, Jackson ImmunoResearch, West Grove, PA, USA, 1:200), then with the avidin–biotin complex (1:100; ABC Elite kit, Vector Labs, Burlingame, CA, USA). After washing, the sections were reacted with 0.05% 3,3-diaminobenzidine and 0.01% hydrogen peroxide in PB. Intensification was conducted with 0.05% osmium tetroxide in water. The sections were mounted on gelatinized slides, dehydrated, cleared and coverslipped. Controls for immunostaining included the omission of the primary antibody and its substitution for normal mouse serum, which completely eliminated staining. The material was analyzed on a light microscope and digital images were collected. Quantitative image analysis was performed using ImageJ (National Institutes of Health/USA). GFAP and OX42 immunostaining was evaluated in terms of optical density within 0.4 mm2 areas of the superficial, visual portion of the SC. The mean optical density of SC labeled areas was compared with the mean density of neighboring, non-labeled areas in the same sections, to obtain a labeling index reflecting the mean signal-to-noise ratio. The resulting indexes
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for the control and deafferented groups were then compared and subjected to statistical analysis using Graphpad Prism (3.02). 2.6. Immunofluorescence For co-localization studies, immunofluorescence tissue preparation was performed in the same conditions as in the immunoperoxidase assays, described above. Briefly, the brain sections were incubated free-floating for 12–16 h with the following primary antibodies: mouse anti-rat GFAP, mouse anti-rat OX42, mouse anti-rat NeuN (Chemicon, Billerica, MA, USA) and goat antirat COX-2 diluted 1:500 in 0.3% of Triton X-100. Anti-goat FITC conjugated (1:250; Abcam, Cambridge, UK) and anti-mouse TRITC conjugated (1:250; Abcam, Cambridge, UK) secondary antibodies were used. The sections were mounted on gelatinized slides and coverslipped. Negative controls were performed by the omission of primary antibody or secondary antibody. The material was analyzed on a confocal microscope and digital images were collected. 2.7. Statistical analysis Values are expressed as means ± S.E.M. Statistical comparisons were performed by one-way ANOVA, followed by Tukey’s multiple comparison test. Values of p ≤ 0.05 were considered significant. Data from the contralateral side of the brain to the removed eye was always compared to data obtained from intact animals. The number of rats (n) used in each group is referred to in the text. 3. Results 3.1. Immunoblotting Immunoblotting data revealed increased GFAP, OX42 and COX-2 protein expression after unilateral enucleation. As shown in Fig. 1A, densitometric analyses revealed an increase of GFAP protein levels in SC at days 7 (ca. 551%) and 15 postlesion (ca. 240%). Similar results were also observed for OX42, for which a significant increase was observed at day 7 (ca. 205%) postlesion (Fig. 2A). For COX-2, the increase of optical density started as early as 1 h (ca. 287%) and 1 day (ca. 258%), persisting until day 7 postlesion (ca. 218%). This effect appeared to decrease progressively thereafter (Fig. 3A). Immunoblotting data also revealed that DEX treatment significantly inhibited COX-2 protein expression in SC 7 days after unilateral enucleation (Fig. 3B). 3.2. Immunoperoxidase In agreement to our immunoblotting data, immunostaining for GFAP was markedly increased (ca. 165%) in the SC at 7 days postlesion (Fig. 1B and C). A consistent increase of OX42 immunostaining (ca. 130%) was also found in the superficial layers of the SC 7 days after eye enucleation, when compared to control animals (Fig. 2B and C). The treatment with DEX significantly decreased both GFAP (by about 162%) and OX42 immunostaining (by about 80%), as depicted in Figs. 1 and 2, respectively. 3.3. Immunofluorescence The observation that COX-2 increased protein expression was induced by ocular enucleation in the SC led us to test whether this protein is expressed in neurons, astrocytes or microglial cells. Consistent with the immunoblotting results, a significant increase of COX-2 expression was observed in the SC 7 days after ocular enucleation, in comparison to the control group (data not shown). COX-2 displayed coexpression with NeuN in the SC, indicating neuron-specific expression of COX-2 (Fig. 4). Neither astrocytes nor
Fig. 1. Effects of eye enucleation on GFAP protein levels in the SC. In (A), mean ratio of GFAP densitometric data in relation to beta-actin after different survival periods post-lesion. Significance compared to control at *p < 0.001 and **p < 0.05. In (B), digital images of GFAP immunoreactivity in the control, control + DEX, deafferented and deafferented + DEX conditions 7 days post-lesion. In (C), GFAP immunoreactivity optical density data. *p < 0.001 (control vs deafferented) and **p < 0.001 (deafferented vs deafferented + DEX). DEX: dexamethasone.
microglia expressed COX-2, as verified by co-immunostaining of COX-2 with GFAP (astrocytes) or OX42 (microglia) (Fig. 4). 4. Discussion In the present study we hypothesized that unilateral eye enucleation elicits inflammatory-mediated responses in a retinorecipient structure. Our principal findings demonstrate that (1) Deafferentation of the visual system resulted in increased transient glial cell activation and COX-2 protein expression in SC; (2) The systemic administration of dexamethasone, a synthetic glucocorticoid, attenuated both glial cell activation and COX-2 protein expression; (3) COX-2 was found to be expressed only in neurons. In rodents, visual information from the retina is predominantly sent to the SC through the optic nerve constituted by retinal ganglion cells (RGCs) axons (Sefton et al., 2004). The degeneration
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Fig. 3. Effects of ocular enucleation on COX-2 protein levels in the SC. In (A), mean ratio of COX-2 densitometric data in relation to beta-actin. Significance compared to control at *p < 0.001 and **p < 0.05. In (B), effect of DEX treatment on COX-2 protein expression in the SC from animals allowed to survive for 7 days postlesion. *p < 0.05 (control vs deafferented) and *p < 0.05 (deafferented vs deafferented + DEX). DEX: dexamethasone.
Fig. 2. Effects of ocular enucleation on OX42 protein levels in the SC. In (A), mean ratio of OX42 densitometric data in relation to beta-actin after different survival periods post-lesion. Significance compared to control at **p < 0.05. In (B), digital images of OX42 immunoreactivity in the control, control + DEX, deafferented and deafferented + DEX conditions 7 days post-lesion. In (C), OX42 immunoreactivity optical density data. *p < 0.001 (control vs deafferented) and **p < 0.05 (deafferented vs deafferented + DEX). DEX: dexamethasone.
of RGC results in irreversible blindness found in several major vision-threatening diseases such as glaucoma and diabetic retinopathy (Yokota et al., 2011). Moreover, several clinical and experimental studies have demonstrated that RGC degeneration in glaucoma shares some common features with axonopathies of several pathologic processes such as Alzheimer and Parkinson’s diseases, although the mechanisms driving these phenomena are far from being understood (McKinnon, 2003; Guo et al., 2007). Previous studies in animal models of glaucoma have demonstrated that the distal transport depletion in the SC is an early manifestation of the disease onset. Since then, the SC has attracted increased attention in experimental glaucoma research as it may represent a promising target to reduce glaucomatous neurodegeneration
(Crish et al., 2010). Thus, the investigation of specific inflammatory pathways elicited by RGC degeneration in a retinorecipient structure such as SC may represent a new direction in the pursuit of effective therapies for glaucoma and other neurodegenerative conditions. The initial experiments performed in this study demonstrated that deafferentation resulted in increased glial cell activation in SC. These results are in accordance with earlier studies in which it was demonstrated that unilateral optic nerve lesions change the glial cell population in the SC (Rao and Lund, 1989; Schmidt-Kastner et al., 1993). A response of the glial cell population was also reported in the adult rat hippocampus deafferented by unilateral lesion of the entorhinal cortex (Gall et al., 1979). It has been suggested that increased glial activation may be a consequence of the central inflammatory tissue reaction induced by the deafferentation of the brainstem vestibular nuclei (Campos-Torres et al., 1999). In line with this, we therefore suggest that the reactive gliosis observed in the SC after eye enucleation may also represent an inflammatory response induced by the lesion. Several in vitro and in vivo experimental models of brain inflammation have demonstrated that not only microglia, but also astrocytes, blood inflammatory cells, and even neurons participate in the modulation of the inflammatory response. Astrocyte activation in the nervous system is frequently associated with brain inflammation (Campos-Torres et al., 1999). Astrocytes produce several anti-inflammatory factors (Kim et al., 2010) and chemokines that recruit monocytes (Kim et al., 2011). In addition, astrocytes induce the expression of antioxidant enzymes such as heme oxygenase-1, which are able to inhibit microglial activation (Min et al., 2006). Morphologically, reactive astrocytes display hypertrophy of their cellular processes, elongation of cytoplasmic
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Fig. 4. Effects of ocular enucleation on COX-2 expression in the SC. Representative confocal microscopy images illustrating COX-2 immunostaining in the SC of control (left column) and deafferented (right column) mice 7 days after the lesion. Immunostaining was carried out using antibodies for COX-2 (shown in green) together with NeuN (top), GFAP (center) and OX42 (bottom). Microglia, astrocyte, and neuronal markers are shown in red. COX-2 positive cells are neurons.
processes, hyperplasia and an increased GFAP immunostaining. GFAP is an intermediate filament protein specifically produced by astrocytes, and its increased expression is considered to be a marker of reactive astrocytes (Malhotra et al., 1990). Microglial cells, in turn, represent the resident immune cells of the brain, and their activation seems to coordinate the inflammatory response in the nervous system (Kato et al., 1996; Lynch, 2009). Activation of microglia is linked to the secretion of a number of inflammatory mediators including cytokines, chemokines, eicosanoids, reactive oxygen species (ROS) and nitric oxide (Gehrmann et al., 1995). Similarly to astrocytes, microglial cells also change their morphology and function in response to neural lesions. Activated microglia show an increased cell body size, a thickening of proximal processes and a decrease in the ramification of distal branches (Raivich et al., 1999). Corroborating our results, three days after the transection of the facial nerve, an increased immunoreactivity for a microglial marker was observed into the facial motor nucleus (Raivich et al., 1999). It has been also demonstrated that the pro and anti-inflammatory cytokines produced by activated microglial cells could promote the survival of deafferented vestibular neurons, contributing to the vestibular compensation process (Liberge et al., 2010). Besides astrocyte and microglial cell involvement in the inflammatory response, two other aspects involving its activation deserve consideration. First, it has been demonstrated that reactive glial cells act by supporting several aspects of regeneration such
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as collateral sprouting and synaptic recovery, and maintaining homeostasis of extracellular fluid by taking up glutamate (Pekny and Nilsson, 2005). Since high levels of glutamate are present in the retinocollicular synaptic terminals (Li et al., 1996) and the deafferentation may cause a massive release of this neurotransmitter shortly after the lesion (Choi, 1988; Dye and Karten, 1996), the astrocyte and microglial activation may represent a protective mechanism against glutamate excitotoxicity and, consequently, against the deafferentation trauma. Second, both astrocytes and microglial cells have been described to be phagocytically active both in vitro and in vivo conditions (Ito et al., 2007; Kalmar et al., 2001). Therefore, it has been speculated that increased expression of astrocytes and microglia after deafferentation may reflect their function as phagocytotic cells to remove the debris generated by the degeneration of the retinocollicular fibers and axon terminals. Having in mind that eye enucleation may induce an inflammatory response into the SC, we further investigated whether glial cell activation could be inhibited by the treatment with an antiinflammatory. DEX is a synthetic glucocorticoid able to inhibit glial activation induced by different inflammatory stimuli (Felts et al., 2005; Nadeau and Rivest, 2003; Pan et al., 2001). The results showed that DEX was able to significantly decrease both microglia and astrocytic activation into the SC. Similar results have also been demonstrated in different experimental models, such as in cultured microglia (Ganter et al., 1992), in the suprachiasmatic nucleus of adrenalectomized rats (Maurel et al., 2000), and after the insertion of neural prosthetic devices in the rat neocortex (Spataro et al., 2005). COX-2 is one of the most important components of the inflammatory response (Nogawa et al., 1997). Neuroinflammatory processes involving increased expression of COX-2 have been associated with several neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease and amyotrophic lateral sclerosis (Minghetti, 2004). In the present study, upregulation of COX-2 was observed as early as 1 h after the lesion, and it was found to be intense for one to seven days and then declined until day 30. We also demonstrated that COX-2 was found only in neurons. Similarly, COX-2 was found to be co-localized predominantly in the spinal dorsal horn neurons after spinal nerve ligation (Lau et al., 2013). The beneficial effect of the anti-inflammatory DEX on decreasing COX2 protein expression suggests that COX-2 plays a proinflammatory role in deafferentation-induced neuroinflammation. Similar results have also been recently described following partial hepatectomy. The treatment with a COX-2 inhibitor decreased the hippocampal inflammation induced by that procedure (Peng et al., 2013). In summary, the findings of the present study reveal that eye enucleation induces glial cell activation in the rat SC. Additionally, the lesion also leads to COX-2 upregulation in neurons, and both glial cell activation and COX-2 upregulation can be inhibited by treatment with an anti-inflammatory. Our results suggest that the removal of retinal ganglion cell input generates inflammatory responses in central retinorecipient structures, which may play an important role in neuronal degeneration and deafferentationrelated neuroplasticity of the rat visual system.
Conflict of interest None declared.
Acknowledgments Thanks are due to Adilson S. Alves for technical assistance.
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M.S. Hernandes, L.R.G. Britto / Brain Research Bulletin 101 (2014) 1–6
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