Reactivity of microglia and astrocytes after an excitotoxic injury induced by kainic acid in the rat spinal cord

Tissue and Cell 56 (2019) 31–40

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Reactivity of microglia and astrocytes after an excitotoxic injury induced by kainic acid in the rat spinal cord

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Carolina Natalia Zanuzzia,c, ,1, Fabián Nishidaa,c,1, María Susana Sistia,c, Claudio Gustavo Barbeitob,c, Enrique Leo Portianskya,c a

Image Analysis Laboratory, School of Veterinary Sciences, National University of La Plata (UNLP), Buenos Aires, Argentina Laboratory of Descriptive, Experimental and Comparative, Histology and Embriology, Argentina c National Research Council of Science and Technology (CONICET), Argentina b

A R T I C LE I N FO

A B S T R A C T

Keywords: Gliosis Astrocytes Microglia Neurodegeneration Spinal cord Kainic acid

After injury of the nervous system glial cells react according to the stimuli by modifying their morphology and function. Glia activation was reported in different kainic acid (KA)-induced neurodegeneration models. Here, we describe glial morphometric changes occurring in an excitotoxic KA-induced cervical spinal cord injury model. Concomitant degenerative and apoptotic processes are also reported. Male rats injected at the spinal cord C5 segment either with KA or saline were euthanized at post-injection (PI) days 1, 2, 3 or 7. Anti-IBA-1 and antiGFAP antibodies were used to identify microglia and activated astrocytes, respectively, and to morphometrically characterized them. Fluoro-Jade B staining and TUNEL reaction were used to determine neuronal and glial degeneration and apoptosis. KA-injected group showed a significant increase in microglia number at the ipsilateral side by PI day 3. Different microglia reactive phenotypes were observed. Reactive microglia was still present by PI day 7. Astrocytes in KA-injected group showed a biphasic increase in number at PI days 1 and 3. Degenerative and apoptotic events were only observed in KA-injected animals, increasing mainly by PI day 1. Understanding the compromise of glia in different neurodegenerative processes may help to define possible common or specific therapeutic approaches directed towards neurorestorative strategies.

1. Introduction It is well-known that glial cells are involved in the pathogenesis of many Central Nervous System (CNS) disorders. Any damage to the CNS triggers a reactive gliosis, which includes the participation of astrocytes, microglia, oligodendrocytes and ependymal cells (Verkhratsky et al., 2014). After injury microglia become active by changing their morphology, secreting pro and anti-inflammatory mediators, and enhancing their motility and phagocytic activity (Christensen et al., 2006) in response to cytokines, growth factors, sexual hormones and other molecules (Arevalo et al., 2013; Saijo et al., 2013; Sousa et al., 2017; YanguasCasás et al., 2018). Reactive astrogliosis is a defensive mechanism used to isolate a damaged area, to recover the neuronal circuits and to regenerate tissues after injury (Anderson et al., 2016). Despite the considerable heterogeneity displayed by reactive astrocytes, a hallmark of their activation is the expression of intermediate filament proteins, such as glial

fibrillary acidic protein (GFAP) and vimentin (Hol and Pekny, 2015). Mature astrocytes may become reactive cells under the influence of damage associated signals, which would induce their proliferation or differentiation from ependymal cells (Barnabé-Heider et al., 2010; Silver et al., 2014). In addition, under neurotoxic insults astrocytes synthetize local steroids with neuroprotective and regenerative effects (Garcia-Segura et al., 1999). Although each glial cell type has specific functions, there is a crosstalk between the activation of astrocytes and microglial cells under pathological conditions (García-Ovejero et al., 2005; Pekny and Pekna, 2014; Silver et al., 2014). The injection of kainic acid (KA), an agonist of glutamate, can elicit selective excitotoxicity in in vivo and in vitro models of neurological diseases (Kuzhandaivel et al., 2010; Mazzone et al., 2010; Mitra et al., 2013). The intraparenchymal injection of KA was validated as a useful model to induce tissue damage in discrete regions of the CNS without affecting directly the axons passages (Pisharodi and Nauta, 1985). In previous studies we injected KA at the Lamina VII of the C5 cervical

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Corresponding author at: Image Analysis Laboratory, School of Veterinary Sciences, National University of La Plata, Calles 60 y 118, 1900, La Plata, Argentina. E-mail address: [email protected] (C.N. Zanuzzi). 1 Identical contribution. https://doi.org/10.1016/j.tice.2018.11.007 Received 13 September 2018; Received in revised form 16 November 2018; Accepted 30 November 2018 Available online 01 December 2018 0040-8166/ © 2018 Published by Elsevier Ltd.

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2.3. Spinal cord collection and processing

segment of the rat spinal cord (Nishida et al., 2014, 2015) and shown motor and sensitive alterations probably related, among other factors, to the observed neuronal loss and changes in the expression of neuronal intermediate filaments (Nishida et al., 2017). Glia activation was reported in different KA-induced neurodegeneration models (Jorgensen et al., 1993; Ding et al., 2000; Chen et al., 2005; Chiu et al., 2016; Yoon et al., 2016). The effects of glutamate (Liu, 1994) and of the intraparenchymal injection into the brain and spinal cord of different glutamate-agonistic neurotoxins such as KA, quisqualic acid and N-methyl aspartate were reported several decades ago (Coyle et al., 1978; Pisharodi and Nauta, 1985; Liu, 1994). Although in those studies gliosis was mentioned no counting of those cells was performed nor detailed description of their morphology was reported. Knowledge of the morphological changes of glial cells could serve as a predictor of the evolution of the lesions induced by excitotoxicity. Likewise, it also allows to establish the impact of different aggressors on the spinal cord. Moreover, understanding the participation of glial cells in different neurodegenerative experimental models could contribute to the proposal of different therapeutic strategies for their treatment. Here we described the morphological reaction of astrocytes and microglia, and the degenerative and apoptotic processes observed in vivo after the injection of KA in the rat spinal cord.

Euthanasia was performed according to the Guidelines for the Use of Animals in Neuroscience Research (the Society of Neuroscience) and the Research Laboratory Design Policy and Guidelines of NIH. Immediately before euthanasia, rats were placed under general anesthesia by injection of ketamine hydrochloride (40 mg/kg; i.p.) plus xylazine (8 mg/kg; i.m.) and then intracardially perfused with 4% paraformaldehyde in phosphate buffer saline (PBS). The vertebral column was removed and postfixed for 24 h in 10% buffered formaldehyde. The spinal cord was then dissected out, immersed in cryopreservation buffer (sucrose 30%; polyvinylpyrrolidone 1%; ethylene glycol 30% phosphate buffer 1 M 1%; distilled water to 100 ml) and stored at −20 °C until use. A coronal section of the C5 segment was obtained under a magnifying glass and then placed at the center of a well in a 48-well plate. The well was then filled with 0.5 ml jellifying solution (sucrose 10% in phosphate buffer 1 M; low melting point agarose [Sigma Chemical Co., St. Louis, MO] 4%) and stored at 4 °C until a jelly block was formed. Then, the block was serially cut into 20 μm thick coronal sections using a vibratome (Leica VT 1000S, Germany). From each block, three to five sections, 120 μm apart, were mounted on gelatin-coated slides (unflavored gelatin 6 g; KCr(SO4)2.12 H2O 0.5 g; distilled water to 300 ml), and stained either with the cresyl violet technique for histopathological analysis or immunohistochemistry and lectin-histochemistry for cell counting. The remaining sections were used for neuronal degeneration and apoptosis determinations.

2. Materials and methods 2.1. Animals

2.4. Neuronal degeneration. Fluoro-Jade B staining

Young (3–4 months old, 300–400 g b.w.) male Sprague-Dawley rats (n = 37), raised in a temperature-controlled room (22 ± 2 °C) on a 12:12 h light/dark cycle were used. Food and water were available ad libitum. All experiments with animals were performed according to the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was also approved by the Committee on the School of Veterinary Sciences, National University of Plata Institutional Committee for Care and Use of Laboratory Animals (CICUAL), code 49-8-15 P.

To determine degeneration after the excitotoxic activity of KA, the Fluoro-Jade® B (FJB) dye (Milipore, Temecula, CA) was used. Sections were rehydrated and immediately transferred into a 0.06% KMnO4 solution for 10 min. Sections were then rinsed for 2 min in distilled water and placed into a 0.0004% FJB solution (4 ml of 0.01% of FJB stock solution into 96 ml of 0.1% acetic acid plus 0.2% of the fluorescent dye 4',6-diamino-2-fenilindol - DAPI, Invitrogen Life Technologies, Eugene, OR, USA). After 20 min in the staining solution, sections were rinsed three times for 1 min with distilled water. Sections were then dried at 37 °C for at least 30 min and then cleared by immersion for at least 1 min in xylol before coverslipping. Sections were observed under a laser scanning confocal microscope (FV100, Olympus Co., Tokyo, Japan) using 473 nm and 405 nm solid state lasers for exciting FJB and DAPI, respectively. Morphometric counting was performed using a digital image analyzer software (cellSens Dimension, V1.7, Olympus Corporation, Japan).

2.2. Experimental groups and KA administration Before surgery, KA (Sigma-Aldrich, Inc., St. Louis, MO, USA) was dissolved in 0.9% saline and kept at 4 °C until use. On experimental day 0, rats were anesthetized with an intraperitoneal (i.p.) injection of ketamine hydrochloride (40 mg/kg) plus an intramuscular (i.m.) injection of xylazine (8 mg/kg) and placed in prone position. A group of animals were injected either with 1 mM KA (KA group) or saline (vehicle-injected group). The injection of 1 mM KA induced a reversible loss of motor activity of the ipsilateral forelimb, as previously reported (Nishida et al., 2015). Injected rats were euthanized either at post injection (PI) day 1, 2, 3 or 7. For each time point, five KA-injected and three vehicle-injected rats were studied. One non-operated rat (intact control group) was euthanized per testing day, starting at day 0. Injections were performed following the protocol described by Nishida et al. (2014). Briefly, trepanation at the C4–C5 fibrous joint 1 mm lateral from the midline (dorsal spinal process) was performed to gain access to the C5 segment. KA or vehicle (saline) were injected using a 10 μl Hamilton® syringe with a 26 G needle. The needle was vertically introduced 1.5 mm down on the right side of the spinal cord to reach the Lamina-VI of that side (ipsilateral). Then it was left in place for 2 min before 5 μl of either the KA solution or saline were discharged at a rate of 1 μl/min. Before withdrawal, the needle was held in place for additional 2 min to avoid leaking of the solution. After surgery animals were kept on a heating pad and checked periodically until they woke up. Thereafter, they were returned to their cages. In no case animals required manual emptying of the bladder.

2.5. Apoptosis. TUNEL technique The terminal deoxynucleotidyltransferase dUTP nick end labelling (TUNEL) technique was used to study apoptosis (Tunel in situ Cell Death Detection Kit, TMR red; Roche, Indianapolis, IN, USA), as previously described (Scrochi et al., 2017). Briefly, sections were hydrated to eliminate the rest of the jellifying solution and incubated with proteinase K for antigen retrieval. After washing with PBS containing 0.5% Tween 20 (Merck, Schuchardt OHG, Hohenbrunn, Germany), slides were incubated with the reaction mixture containing modified nucleotides (TMR-dUTP) and the enzyme terminal deoxynucleotidyltransferase (TdT), that catalyzes the template-independent polymerization of deoxyribonucleotides to the 3’-end of single and doublestranded DNA. After washing for three times, nuclei were counterstained with 5 μg ml−1 DAPI, following the manufacturer’s protocol. Coverslips were mounted on slides using the aqueous medium FluoroSave® reagent (Calbiochem, La Jolla, CA, USA) and then examined under confocal microscopy. Non-apoptotic cells will appear blue due to DAPI labeling, while apoptotic cells will appear red due to the labelling 32

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fields of both ipsilateral and contralateral sides of the spinal cord segment was performed using the cellSens Dimension (V1.7, Olympus Corporation, Japan) image analyzer. Color segmentation was used to identify TMR red stained nuclei in the corresponding channel of the multidimensional image. Automatic counting of thresholded cells was then performed. Red stained or colocalized (red + blue) nuclei were considered after color segmentation. Fragments of the same nucleus were considered as a unity for the counting. For identifying positively labelled FJB positive cells, color segmentation was applied. Automatic counting of thresholded cells was then performed. Morphometry and cell counting were carried out by investigators who were blind to the experimental conditions.

with tetramethylrhodamine (TMR red) and DAPI (blue). The TUNEL reaction mixture replaced by the label solution of the kit was used as a negative control. 2.6. Microglia and astrocyte identification Immunohistochemistry/fluorescence (IHC/IF) and lectin-histochemistry (LHC) techniques were used to evaluate the morphology of microglia and astrocytes as well as to determine their number. For IHC and LHC, spinal cord sections were washed with PBS containing 0.05% Tween 20 (PBST), to eliminate the rest of the jellifying solution, and then incubated for 30 min at room temperature with 0.03% H2O2 in PBS, to block endogenous peroxidase. Sections were then rinsed twice in PBST. At this point, sections used for IHC were exposed to microwave antigen retrieval using a citrate buffer solution, pH 6.0 and then washed twice with PBST. Later, all sections were incubated with 1% bovine serum albumin (BSA) for 30 min, followed by overnight incubation either with rabbit anti-IBA-1 polyclonal antibody (Wako Chemicals USA, Inc.; diluted 1:1000), rabbit anti-GFAP polyclonal antibody (DakoCytomation, Carpinteria, CA, USA; ready to use) for IHC analysis or the biotinylated lectin GS-IB4 from Griffonia simplicifolia (Vector Laboratories Inc., CA, USA) for LHC study. Sections were then rinsed with PBST and incubated with the EnVision® detection system + HRP system labeled anti-rabbit polymer (DakoCytomation, Carpinteria, CA, USA) for 45 min (IHC) or streptavidin for 30 min (LHC). Finally, sections were rinsed in PBS and the reaction was revealed with the liquid chromogen 3,3-diaminobenzidine tetrahydrochloride (Vector Laboratories Inc., CA, USA). Hill’s hematoxylin was used for counterstaining. Control negative sections were prepared by omitting the primary antibody or the lectin for IHC and LHC, respectively. Some sections were used for double immunofluorescence to study microglia or astrocyte proliferation after injury. For this purpose, sections were incubated for 30 min with 1% BSA in PBS, followed by overnight incubation with anti-PCNA monoclonal antibody (clone PC 10, ascites fluid, Sigma Chemical Co., St. Louis, MO, USA; 1:3000 dilution), in combination either with anti-GFAP or anti-IBA-1 antibodies. Sections were then rinsed threefold in PBS and incubated for 45 min either with 1:1000 Alexa Fluor 488-conjugated anti-mouse or Alexa Fluor 555-conjugated anti-rabbit secondary antibodies (Invitrogen, Thermo Fisher Scientific Inc.). Then, sections were rinsed threefold in PBS and counterstained for 15 min with DAPI. Control negative sections were prepared by omitting the primary antibody.

2.8. Cell counting For all applied techniques, the ipsilateral and contralateral sides of each spinal cord segment were considered as separated. The average counting of positively stained cells in each side was compared among groups. To estimate the number of glial cells per segment the following formula was used (Portiansky et al., 2004):

N=

d ns

n

∑x i=1

where, N = total estimated number of cellular bodies; d = length (μm) of the rostro-caudal axis of the cervical segment being assessed (2 mm); n = number of non-contiguous (120 μm apart) slices counted per cervical segment (n = 5); s = thickness of the section (20 μm); x = number of nuclei counted per non-contiguous slice assessed. Therefore, N represents approximately the total number of glial cells present at the C5 cervical segment. As it is normally done in studies of cell counting, Abercrombie’s formula (Abercrombie, 1946) was introduced to correct any overestimation of counts due to section thickness, as was suggested by Guillery (2002). In this case, the mean diameter of glial cells present in both sides of the spinal cord was used to calculate the correction formula. Apoptosis and cellular degeneration were evaluated at both sides of the spinal cord segment and expressed as the percentage of positive cells over all DAPI stained nuclei, in each group. 2.9. Morphological/morphometric characterization of glial cells

2.7. Image analysis

Morphological study of microglia was performed on IBA-1 IHC stained sections, according to Diz-Chaves et al. (2012). For each animal 100 cells per spinal cord segment side (ipsilateral and contralateral) were evaluated. Briefly, five morphological types were considered: type I, cells with few cellular processes (two or less); type II, cells showing three to five short branches; type III, cells with numerous (> 5) and longer cell processes and a small cell body; type IV, cells with large somas and retracted and thicker processes and type V, cells with an amoeboid cell body, numerous short processes and intense IBA-1 immunostaining. The morphometric study of astrocytes was based on their branching complexity. For this purpose, GFAP immunoreactive cells were submitted to Sholl analysis (Sholl, 1953) by superimposing a digital mask with concentric rings distributed at equal distances (5 μm). Care was taken no to consider those curved processes that may intersect many times with the same circle (del Cerro et al., 1995). The mask was constructed using FIJI software (NIH) and was centered on cell somas. The number of process intersections per ring i (an index of branching complexity) was computed and the total length of the processes was estimated by the sum of the i values for each ring multiplied by 5. For each animal 100 astrocytes per spinal cord segment side were randomly chosen, and their branching complexity measured. The average for every rat and group was then calculated and used for the statistical

Images of spinal cord sections studied by IHC or LHC were captured using a digital RGB video camera (Olympus DP73, Japan) attached to a microscope (Olympus BX53, Japan). To create a map of the entire segment, images captured with a 40x objective were stitched using the Multiple Image Alignment (MIA) function of a digital image analyzer (cellSens Dimension, V1.7, Olympus Corporation, Japan) and stored in TIFF format. No further processing was necessary after obtaining the original images. Images were then analyzed using an image analyzer (ImagePro Plus - v6.3, Media Cybernetics, MA, USA). To determine the total number of glial cell bodies per section, color segmentation was performed (Portiansky, 2018). Briefly, a color range of positively stained cells was considered for selecting cells to count. After automatic counting of thresholded cells only those with a visible nucleus were considered. Immunofluorescent images were captured using a laser scanning confocal microscope (Olympus FV1000, Japan). Apoptotic cells stained with TMR red and glial cells stained with Alexa 555 were excited using a 559 nm solid state laser. Alexa 488 and Fluoro Jade B stained cells were excited with a 473 nm solid state laser while all nuclei stained with DAPI were excited using a 405 nm solid state laser. Apoptotic cell counting in ten, 20x magnified randomly selected 33

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Fig. 1. Histological and immunohistochemical aspect of the spinal cord after the injection of vehicle or KA. a. Cresyl violet staining of a spinal cord section one day after the injection of vehicle (saline). Although there is no neuronal loss, some neurons are swollen while others are retracted (arrows). An increase in glial reaction is observed. Bar = 500 μm. b. Cresyl violet staining of a spinal cord sections one day after the injection of KA. Almost no neurons are seen at the ipsilateral side. An increase of glial cells is also observed. Bar = 500 μm. c. IBA-1 immunoreactivity in the spinal cord 3 days after the injection of KA. Distribution of immunoreactive cells is observed along the entire ipsilateral side. Bar = 500 μm. d. Higher magnification of C, showing details of the IBA-1 immunoreactive cells. Bar = 100 μm. d. GFAP immunoreactive astrocytes 3 days after the injection of KA. Distribution of positive cells is observed along the entire ipsilateral side. Bar = 200 μm. f. Higher magnification of E showing a network of astrocytes. Bar = 50 μm.

analysis.

of P < 0.05. Data are expressed as mean ± S.E.M.

2.10. Statistical analysis

3. Results

Statistical analyses were performed using the NCSS software (v.07.1.20, NCSS, LLT.). All data were compared by Student t-test and one-way analysis of variance (ANOVA) and for multiple comparisons, Fisher's LSD and Tukey Kramer multiple-comparison test was used as a post-hoc test. For non-parametric values, the Mann–Whitney test and the Kruskall-Wallis test were used. Significance was assumed at values

3.1. Histological analysis Using Cresyl violet staining it was observed that the intraparenchymal injection of KA caused extensive neuronal loss at the ipsilateral side, as previously reported (Nishida et al., 2015). This change was accompanied by increased cellularity, observed mainly 34

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Fig. 2. Morphometric analysis of positive IBA-1 immunoreactive cells. a). Number of IBA-1 immunoreactive cells. Numbers were calculated after introducing the Abercrombie’s formula for correcting overestimation of counts due to section thickness. For the ipsilateral side, significant differences were found at all post-injection time points between vehicle and KA-injected groups in comparison to the control group (#). The KA group showed higher cell counting at PI day 3 in comparison to vehicle group at both sides of the spinal cord (*). For the contralateral side, the number of IBA-1 immunoreactive cells at PI day 3 was significantly higher in vehicle and KA group in comparison to control group (#). Data are expressed as mean ± S.E.M. #,* (P < 0.05). b). Percentage of morphological subtypes of IBA-1 immunoreactive cells. Significant differences between vehicle and KA-injected rats were found at the ipsilateral and contralateral sides at different PI days * (P < 0.05). c). Different microglial cell phenotypes. C1: type I; C2: type II; C3: type III; C4: type IV; C5: type V. Bar = 10 μm for all cell types.

during the first three PI days (Fig. 1a-b). Samples from animals injected with vehicle showed no neuronal loss but revealed an increased cellularity at the ipsilateral side as compared to the samples from non-

injected animals.

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higher global branching length in the ipsilateral side in comparison to those of the KA group at PI days 1 and 2, whereas by PI day 3 KAinjected group showed the highest length of branches of all the tested groups.

3.2. Number and morphometric characteristics of microglia after KA injection GS-IB4 lectin and anti-IBA-1 antibody were used to analyze microglial cell number and morphology (Fig. 1c-d). Microglia morphology and number were different depending on the experimental group and days after injection. Thus, counting of positive IBA-1 immunoreactive cells in the C5 cervical segment (including both white matter and grey matter) of the control (non-injected) group showed an average of 424 ± 81 and 409 ± 94 cells for ipsilateral and contralateral side, respectively (Fig. 2a). In the vehicle-injected group, the number of IBA1 immunoreactive cells at the ipsilateral side was significantly increased from PI day 1 towards PI day 7 in comparison to that of the non-injected control group (P < 0.05), whereas at the contralateral side a significant increase was detected at PI day 3 (P < 0.05) (Fig. 2a). In the KA-injected group, the number of IBA-1 immunoreactive cells was significantly increased in the ipsilateral side from PI day 1 towards PI day 7 in comparison to non-injected control group (P < 0.05), whereas a significant increase at PI day 3 (P < 0.05) was observed at the contralateral side (Fig. 2a). At PI day 3, animals injected with KA showed a significant increase in the number of IBA-1 immunoreactive cells in comparison to vehicle-injected animals at both sides of the spinal cord (P < 0.05) (Fig. 2a). In control non-injected animals, most of IBA-1 immunoreactive cells were of type I (76.69%) (Fig. 2b). Types IV and V were not identified in these animals. In vehicle-injected animals euthanized at PI day 1, 20–33% of the cells at the ipsilateral side were of types II, III, IV and V (Fig. 2c), whereas at the contralateral side most of them were of types II (46.78%) and III (38.13%). By PI day 2, most of the glial cells were of type II (47.22%) and III (35.87%) at the ipsilateral side, while at the contralateral side the percentages changed to 65.21% and 19.84%, respectively. At PI day 3, types II and III were still the most frequent glial cells at both sides. At PI day 7 the predominant cell types were I (40.61%) and II (48.82%) in the ipsilateral side, and 30.32% and 49.30%, respectively, at the contralateral side. As for the KA group, at PI day 1, types II (43.37%) and III (39.71%) were the most representative phenotypes at the ipsilateral side. At PI day 2, 13.5–32.3% of the cells were of types II, III, IV and V at the ipsilateral side, whereas by PI day 3 they represented the 13.60–30.35% of IBA-1 immunoreactive cells. At PI day 7, there was a predominance of types II (50.61%) and III (35.47%) at the ipsilateral side. Considering the contralateral side, types I, II and III were the predominant cell phenotypes at all the studied times.

3.4. Double immunofluorescence for proliferating cells Double immunofluorescence technique revealed the existence of IBA-1 positive cells and GFAP positive cells colocalizing PCNA in the ipsilateral and contralateral sides of the spinal cord of vehicle and KAinjected animals (Fig. 4). 3.5. Neuronal degeneration and apoptosis Fluoro-Jade B (FJB) staining and TUNEL technique were used to identify degenerative neurons and apoptotic cells, respectively. No evidence of FJB or TUNEL labeling was observed in samples from control or vehicle-injected animals at any studied PI time, whereas positive cells for both techniques were found in KA group samples (Fig. 5a). Counting of FJB positive cells revealed the highest percentage (35%) on PI day 1 at the ipsilateral side. In the same PI day, the percentage of positive TUNEL cells was 1% (Fig. 5b). The remaining days, the average percentage for both markers was always below that registered on PI day 1. 4. Discussion Glial reactivity in response to excitotoxicity induced by KA has been described in different regions of the CNS (Dusart et al., 1991; Ding et al., 2000; Chen et al., 2005; Milenkovic et al., 2005; Christensen et al., 2006; Zhang and Zhu, 2011). In the present work we described the changes occurring in the number and morphology of microglia and astrocytes in an excitotoxic model of spinal cord injury in rats (Nishida et al., 2015) where degenerative and apoptotic processes were observed. As it was also reported by Pisharodi and Nauta, (1985) we found extensive gray matter damage in the ipsilateral side of KA-injected rats, with no compromise of axon bundles. Although in that study it was reported that KA, quisqualic acid or aspartate caused loss of neurons and gliosis, neither identification, number nor the phenotype of involved glial cells was defined. Here, we found that after exposure to KA, microglia and astrocytes increased in number. According to the double immunofluorescence staining with PCNA-IBA-1 and PCNA-GFAP we may suggest that the recorded increase may be due to glial cell proliferation. This is also supported by observations of Marty et al. (1991) who describe that resident microglia in the thalamus of rats injected with KA increase their number due to proliferation of that population. In KA-injected animals, the number of microglial cells increased at the ipsilateral side of the C5 cervical segment. Vehicle-injected animals also showed an increase of IBA-1 immunoreactive cells with the phenotype of activated microglia. However, differences in the number of microglial cells between the latter and those injected with KA was only significant on PI day 3, which may indicate that in KA-injected rats there is a dual effect: a) reaction against the mechanical action induced by the needle, a stressful factor, as observed in the vehicle-injected groups, and b) increasing vulnerability to KA-excitotoxicity (Liu et al., 1999). In an in vitro model simulating a traumatic spinal cord injury (SCI) environment Yoon et al. (2016) showed that KA combined with a mechanical lesion (scratch) induced a significantly higher damage, in comparison to the individual effects of either KA or scratch alone. However, it should be noted that in the mentioned study interactions with other cells and components of the extracellular matrix, as occurs in vivo, were not considered. Although we recorded a variable response in some rats within the KA-injected group individual differences in the sensitivity to the excitotoxic effect of KA, as was stated by Miltiadous et al. (2010), should be considered.

3.3. Number and morphometric characteristics of astrocytes after KA injection Astrocytes were identified by GFAP immunostaining. Differences in astrocyte morphology (Fig. 1e-f) and cell counting were detected among groups. Thus, in the control (non-injected) group, cell counts showed an average of 3980 ± 542 and 4555 ± 603 cells at the ipsilateral and contralateral sides, respectively (Fig. 3a). In the vehicleinjected group, positive GFAP cells showed a significant increase in number by PI day 3 in comparison to the control group at both sides of the spinal cord (P < 0.05). Animals injected with KA also showed a significant increase in the number of positive GFAP cells at PI day 3 in comparison to the control non-injected animals (P < 0.05). In addition, KA- injected animals showed a significant increase in the number of GFAP immunoreactive cells in the ipsilateral side at PI day 1 compared to vehicle-injected animals (P < 0.05). GFAP immunoreactive cells exhibited small cell body and highly thin-branched, star-like processes in control non-injected rats. In the vehicle and KA-injected groups, astrocytes showed a significant increase of the global branching length, both at the ipsilateral and contralateral sides, in comparison to the control group, from PI day 1 to 3 (Fig. 3b). In addition, astrocytes in the vehicle-injected rats showed a 36

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Fig. 3. Morphometric analysis of GFAP immunoreactive cells. (a). Number of GFAP immunoreactive cells. Numbers were calculated after introducing the Abercrombie’s formula for correcting overestimation of counts due to section thickness. Significant differences (#) were found at pi day 3 between KA and vehicleinjected groups in comparison to control group at both ipsilateral and contralateral sides. At pi day 1 significant differences (*) were observed between vehicleinjected and KA-injected groups. Data are expressed as mean ± S.E.M. #,* (P < 0.05). (b). Branching complexity study. The global branch lengths of GFAP positive cells were measured following the sholl analysis. Significant differences were found in astrocytes of KA group in comparison to vehicle-injected rats. Astrocytes showed a more complex branching at PI day 3 at the ipsilateral side.

neurotoxic role induced by the secretion of proinflammatory cytokines that would turn into a neuroprotective role due to the secretion of other molecules that contribute to tissue restauration or protection. In a previous study, we observed that 7 days after an intraparenchymal injection of 1 mM KA animals recovered motor and sensorial performances together with tissue restoration of the spinal cord (Nishida et al., 2015). Interestingly, in the present study we recorded a general reduction in the number of microglia by PI day 7 as compared to PI day 3, and particularly, a reduction in the percentage of activated microglia-phenotypes (types IV and V), which may be related to a neurorestorative or neuroprotective function. The increased expression of GFAP is one of the hallmarks of the activation of astrocytes (Ridet et al., 1997). Once activated, beneficial and detrimental molecules are secreted by these cells, and their effects depend on the nature, exposure time of the injury and the CNS region compromised. Jeong et al. (2010; 2014), proposed that inflammatory responses in the injured brain and spinal cord are neuroprotective and necessary to repair the tissue. In a spinal cord injury model induced by contusion, the number of astrocytes were reduced on day 1 post impact, but they proliferated and reached a peak by day 3 (Nicola et al., 2017), confirming a typical response of the neural tissue to the injury associated with a glial scar formation. After a spinal cord stroke, astrocytes

Marty et al. (1991) reported an initial activation of microglia in the thalamus of rats injected with KA, followed by a second reaction observed for 15 days. In an epilepsy model induced by the intracerebral administration of KA, a biphasic increase in microglia number was also found, the first being one day after the injection of KA and the second at PI day 5 (Tzeng et al., 2013). Besides, these authors described a significant decrease of microglia by PI day 3, between these two peaked phases (Tzeng et al., 2013). In our model, on the contrary, the number of microglia significantly increased by PI day 3. Such differences in microglia response might be related to the anatomical region and the time points being studied after KA injection. Differences in the extent of the response was also shown depending on the CNS region considered; e.g. in the thalamus, microglial reactivity did not decrease until a month or even a year after the injection of KA (Marty et al., 1991). It is unclear, however, how microglia activation participates in the lesions induced by KA, or by other excitotoxic amino acids (EAA) glutamate-agonists. EAA are involved in the secondary damage that followed traumatic or ischemic spinal cord injury (Liu et al., 1994; 1999). It is known that EAA are an additional regulator of microglia activation and resultant phenotypes, owing to the presence of AMPA receptors localized on their cell membrane. (Christensen et al., 2006). Wu et al. (2005) showed that reactive microglia may have a possible initial 37

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Fig. 4. Double immunostaining for detecting proliferating glial cells. Confocal immunofluorescence detected at the central canal (a) and at the ventral horn (b) of spinal cord sections of KA-injected animals after 3 PI days. Arrows point to colocalization sites for PCNA (green) and IBA-1 (red) antibodies. Arrowheads point to non-colocalizing microglia. Lamina X area including the central canal (c) showing positive reaction for GFAP (red) and PCNA (green) antibodies. (d) Higher magnification of a GFAP positive cell colocalizing PCNA. Bar = 30 μm for A, B and C. Bar = 120 μm for D (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 5. Degeneration and apoptosis. (a). Percentage of positive Fluoro-Jade B cells over whole positive DAPI nuclei at both sides (ipsilateral and contralateral) of the spinal cord C5 segment, in KA-injected rats at different PI days. On its side, a representative image of degenerated cells is observed. The DAPI channel was ommited (b). Percentage of TUNEL positive cells over whole positive DAPI nuclei at the ipsilateral side of the spinal cord segment of the KA group, at different PI days. On its side, a representative image of apoptotic cells is observed.

may first contribute to the formation of an inhibitory glial scar, but then they also participate in neural repair (Liu et al., 2014). Astrogliosis induced by excitotoxicity has been described after the injection of EEA in brain and spinal cord (Yezierski et al., 1993; Zhang and Zhu, 2011). In addition, Mitra et al. (2013) showed an increase in the astrocytic response in the spinal cord of rats after intra-cisternal administration of KA. Here, we focused on the effect of the intraparenchymal injection of KA into the spinal cord (Nishida et al., 2014, 2015) to further characterize and explain some changes reported

in other chemical spinal cord injury models (Liu et al., 1999; Urca and Urca, 1990). We found an increase in positive GFAP cells in the KA group at PI day 1 in comparison to control and vehicle-injected rats, followed by a reduction by PI day 2, and a new peak at PI day 3, but only in comparison to control group. In addition, a gradual increase in the global branching length of astrocytes was also found in the KA group, from PI days 1 to 3. Yezierski et al. (1993) reported positive GFAP staining in all regions where neuronal degeneration was observed and surrounding the central canal in the spinal cord of rats injected 38

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rule out such possibility that may also explain the results here described. In conclusion, our study described glial participation in an excitotoxic KA-induced spinal cord injury model. Here, we provided novel information about the phenotypical changes occurring on these cells after the excitotoxic effect of KA when it is applied by intraparenchymal route into the spinal cord. Understanding the involvement of microglia and astrocytes in different KA-induced models may help to define possible common or specific therapeutic targets for neurorestorative strategies.

with quisqualic acid. Regional heterogeneity of reactive gliosis may be associated with different neuronal sensitivity, as it was also observed for microglia, in addition to differences in the KA administration routes used. In this regard, Ding et al. (2000) injected KA in the hippocampus and showed that GFAP levels did not change by PI day 1, whereas in the amygdala/pyriform cortex there was a decrease. On PI day 2 they found a similar increase in both regions, which was approximately 140% higher than that initially recorded. This increase continued up to PI day 9. However, in the frontal cerebral cortex and the striatum, GFAP expression significantly raised on PI day 1 reaching up to 200%, in relation to the initial value, by PI day 3. From there on, no further increase of GFAP was detected. On the other hand, Milenkovic et al. (2005) did not find a demarcating glial scar surrounding the lesion induced by KA at the cerebellum, in contrast to the usual astrogliotic reaction observed in control groups. Dusart et al. (1991) found the existence of a complex glial response in the thalamus after KA injection. It was firstly characterized by an increase in the number of microglia, followed by an increase in the number and hypertrophy of astrocytes. On the other hand, the reduction in GFAP immunoreactivity observed by PI day 2 in KA-injected rats may not correspond to a reduction in cell number, but it may be the result of a transitory cytoskeletal rearrangement of reactive astrocytes, caused by an increase of the levels of K+ or glutamate, or by the proteolysis of intermediate filaments induced by the activation of astrocytic KA receptors (Ding et al., 2000; Milenkovic et al., 2005). Proteolysis of inner filaments structure might justify the shortness of the branching length in the KA-injected rats as compared to the vehicle-injected animals, observed in the first two days of the experiment. Studies of neuronal death and degeneration induced by KA were performed in mice and rats using systemic and intraparenchymal injections of the drug in the brain, showing great variability in the response (Schwob et al., 1980; Hu et al., 1998; Baik et al., 1999; Zemlan et al., 2003). In mice subcutaneously injected with 30 mg/kg KA, several positive FJB neurons were found after 24 h in the dentate gyrus, whereas few cells were identified in the CA3 and CA1 regions (Bluthé et al., 2005). On the other hand, the intraperitoneal injection of 50 mg/ kg KA in mice induced an increase in TUNEL-positive cells in the CA3 region of the hippocampus up to PI days 3 (Baik et al., 1999). Our results established that spinal cord neuronal degeneration and apoptotic death after intraparenchymal injection of KA had its highest peak on PI day 1. In other regions of the CNS changes in cell death and degeneration were also reported between PI days 1 and 3 (Hu et al., 1998; Tzeng et al., 2013). In a mice model of neuronal stress induced by the intraperitoneal administration of KA, DNA fragmentation was transitory and disappeared one week after treatment, as it was observed in our study (Hu et al., 1998). Low percentage values of apoptotic events in KA-injected rats were recorded at the ipsilateral side along the experiment. Low percentage values were also detected for degenerative processes except for those registered on PI day 1. According to the results of Jeong et al. (2014) in the brain, we infer that degeneration and apoptosis of cells could have been greater in the first few hours after the injection with KA. Since our analysis began at PI day 1 this change could not be assessed. The low percentage values of positive FJB and TUNEL cells registered may be attributable to their removal by microglia. At the contralateral sides of vehicle and KA-injected groups variations in the glial response and cell degeneration and apoptosis were also observed although it was not statistically significant in comparison to those registered at the ipsilateral side. The existence of a tissue response at the contralateral side was also reported by other authors, who considered that it may correspond to a secondary generalized response to limit or protect the still undamaged tissue (Jorgensen et al., 1993). It could also obey to anatomical connections as it was suggested by Miltiadous et al. (2010) or to KA local diffusion (Schwob et al., 1980). Although we found that diffusion of KA would be directed to the central canal via of the Virchow-Robbin spaces (Nishida et al., 2015) we cannot

Conflict of interest The authors declare that there is no conflict of interests. Acknowlegements We thank Dr. Luis Miguel García Segura for revising the manuscript and for his helpful critical suggestions. To Mr. Héctor Enrique for his help with the animals. This work was supported by grant PICT 20152087 from the National Agency for Promotion of Science and Technology (ANPCyT) to FN; and grant V235 from National University of La Plata to ELP. References Abercrombie, M., 1946. Estimation of nuclear population from microtome sections. Anat. Rec. 94, 239–247. Anderson, M.A., Burda, J.E., Ren, Y., Ao, Y., O’Shea, T.M., Kawaguchi, R., Coppola, G., Khakh, B.S., Deming, T.J., Sofroniew, M.V., 2016. Astrocyte scar formation aids central nervous system axon regeneration. Nature. 532, 195–200. Arevalo, M.A., Santos-Galindo, M., Acaz-Fonseca, E., Azcoitia, I., Garcia-Segura, L.M., 2013. Gonadal hormones and the control of reactive gliosis. Horm. Behav. 63, 216–221. Baik, E.J., Kim, E.J., Lee, S.H., Moon, C., 1999. Cyclooxygenase-2 selective inhibitors aggravate kainic acid induced seizure and neuronal cell death in the hippocampus. Brain Res. 843, 118–129. Barnabé-Heider, F., Göritz, C., Sabelström, H., Takebayashi, H., Pfrieger, F.W., Meletis, K., Frisén, J., 2010. Origin of new glial cells in intact and injured adult spinal cord. Cell Stem Cell 7, 470–482. Bluthé, R.M., Frenois, F., Kelley, K.W., Dantzer, R., 2005. Pentoxifylline and insulin-like growth factor-I (IGF-I) abrogate kainic acid-induced cognitive impairment in mice. J. Neuroimmunol. 169, 50–58. Chen, Z., Duan, R.S., Quezada, H.C., Mix, E., Nennesmo, I., Adem, A., Winblad, B., Zhu, J., 2005. Increased microglial activation and astrogliosis after intranasal administration of kainic acid in C57BL/6 mice. J. Neurobiol. 62, 207–218. Chiu, K.M., Lu, C.W., Lee, M.Y., Wang, M.J., Lin, T.Y., Wang, S.J., 2016. Neuroprotective and anti-inflammatory effects of lidocaine in kainic acid-injected rats. Neuroreport. 27, 501–507. Christensen, R.N., Ha, B.K., Sun, F., Bresnahan, J.C., Beattie, M.S., 2006. Kainate induces rapid redistribution of the actin cytoskeleton in ameboid microglia. J. Neurosci. Res. 84, 170–181. Coyle, J.T., Molliver, M.E., Kuhar, M.J., 1978. In situ injection of kainic acid: a new method for selectively lesioning neural cell bodies while sparing axons of passage. J. Comp. Neurol. 180, 301–323. Del Cerro, S., Garcia, E.J., Garcia Segura, L.M., 1995. Neuroactive steroids regulate astroglia morphology in hippocampal cultures from adult rat. Glia 14, 65–71. Ding, M., Haglid, K.G., Hamberger, A., 2000. Quantitative immunochemistry on neuronal loss, reactive gliosis and BBB damage in cortex/striatum and hippocampus/amygdale after systemic kainic acid administration. Neurochem. Int. 36, 313–318. Diz-Chaves, Y., Pernía, O., Carrero, P., García-Segura, L.M., 2012. Prenatal stress causes alterations in the morphology of microglia and the inflammatory response of the hippocampus of adult female mice. J. Neuroinflammation 9, 71. https://doi.org/10. 1186/1742-2094-9-71. Dusart, I., Marty, S., Peschanski, M., 1991. Glial changes following an excitotoxic lesion in the CNS-II. Astrocytes. Neuroscience. 45, 541–549. García-Ovejero, D., Azcoitia, I., Doncarlos, L.L., Melcangi, R.C., Garcia-Segura, L.M., 2005. Glia-neuron crosstalk in the neuroprotective mechanisms of sex steroid hormones. Brain Res. Brain. 48, 273–286. Garcia-Segura, L.M., Naftolin, F., Hutchison, J.B., Azcoitia, I., Chowen, J.A., 1999. Role of astroglia in estrogen regulation of synaptic plasticity and brain repair. J. Neurobiol. 40, 574–584. Guillery, R.W., 2002. On counting and counting errors. J. Comp. Neurol. 447, 1–7. Hol, E.M., Pekny, M., 2015. Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system. Curr. Opin. Cell Biol. 32, 121–130. Hu, R.Q., Koh, S., Torgerson, T., Cole, A.J., 1998. Neuronal stress and injury in C57/BL mice after systemic kainic acid administration. Brain Res. 810, 229–240.

39

Tissue and Cell 56 (2019) 31–40

C.N. Zanuzzi et al.

benefits. Physiol. Rev. 94, 1077–1098. Portiansky, E.L., Barbeito, C.G., Goya, R.G., Gimeno, E.J., Zuccolilli, G.O., 2004. Morphometry of cervical segments grey matter in the male rat spinal cord. J. Neurosci. Methods 139, 217–229. Portiansky, E.L., 2018. Análisis Multidimensional De Imágenes Digitales. Universidad Nacional de La Plata, La Plata, Argentina. http:/sedici.unlp.edu.ar/handle/10915/ 70938. Ridet, J.L., Malhotra, S.K., Privat, A., Gage, F.H., 1997. Reactive astrocytes: cellular and molecular cues to biological function. Trends Neurosci. 20, 570–577. Saijo, K., Crotti, A., Glass, C.K., 2013. Regulation of microglia activation and deactivation by nuclear receptors. Glia 61, 104–111. Scrochi, M.R., Zanuzzi, C.N., Fuentealba, N., Nishida, F., Bravi, M.E., Pacheco, M.E., Sguazza, G.H., Gimeno, E.J., Portiansky, E.L., Muglia, C.I., Galosi, C.M., Barbeito, C.G., 2017. Investigation of apoptosis in cultured cells infected with equine herpesvirus 1. Biotech. Histochem. 92, 560–568. Schwob, J.E., Fuller, T., Price, J.L., Olney, J.W., 1980. Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: a histological study. Neuroscience 5, 991–1014. Sholl, D.A., 1953. Dendritic organization in the neurons of the visual and motor cortices of the cat. J. Anat. 87, 387–406. Silver, J., Schwab, M.E., Popovich, P.G., 2014. Central nervous system regenerative failure: role of oligodendrocytes, astrocytes, and microglia. Cold Spring Harb. Perspect. Biol. 7, 020602. Sousa, C., Biber, K., Michelucci, A., 2017. Cellular and molecular characterization of microglia: a unique immune cell population. Front. Immunol. 8, 198. https://doi.org/ 10.3389/fimmu.2017.00198. eCollection 2017. Tzeng, T.T., Tsay, H.J., Chang, L., Hsu, C.L., Lai, T.H., Huang, F.L., Shiao, Y.J., 2013. Caspase 3 involves in neuroplasticity, microglial activation and neurogenesis in the mice hippocampus after intracerebral injection of kainic acid. J. Biomed. Sci. 20, 90. https://doi.org/10.1186/1423-0127-20-90. Urca, G., Urca, R., 1990. Neurotoxic effects of excitatory amino acids in the mouse spinal cord: quisqualate and kainate but not N-methyl-D-aspartate induce permanent neural damage. Brain Res. 529, 7–15. Verkhratsky, A., Parpura, V., Pekna, M., Pekny, M., Sofroniew, M., 2014. Glia in the pathogenesis of neurodegenerative diseases. Biochem. Soc. Trans. 42, 1291–1301. Wu, D., Miyamoto, O., Shibuya, S., Okada, M., Igawa, H., Janjua, N.A., Norimatsu, H., Itano, T., 2005. Different expression of macrophages and microglia in rat spinal cord contusion injury model at morphological and regional levels. Acta Med. Okayama 59, 121–127. Yanguas-Casás, N., Crespo-Castrillo, A., de Ceballos, M.L., Chowen, J.A., Azcoitia, I., Arevalo, M.A., Garcia-Segura, L.M., 2018. Sex differences in the phagocytic and migratory activity of microglia and their impairment by palmitic acid. Glia 522–537. https://doi.org/10.1002/glia.23263. Yezierski, R.P., Santana, M., Park, S.H., Madsen, P.W., 1993. Neuronal degeneration and spinal cavitation following intraspinal injections of quisqualic acid in the rat. J. Neurotrauma Winter. 10, 445–456. Yoon, J.Y., Hwang, C.H., Hong, H.N., 2016. A Model of glial scarring snalogous to the environment of a traumatically injured spinal cord using kainate. Ann. Rehabil. Med. 40, 757–768. Zemlan, F.P., Mulchahey, J.J., Gudelsky, G.A., 2003. Quantification and localization of kainic acid-induced neurotoxicity employing a new biomarker of cell death: cleaved microtubule-associated protein-tau (C-tau). Neuroscience 121, 399–409. Zhang, X.M., Zhu, J., 2011. Kainic Acid-induced neurotoxicity: targeting glial responses and glia-derived cytokines. Curr. Neuropharmacol. 9, 388–398.

Jeong, H.K., Jou, I., Joe, E.H., 2010. Systemic LPS administration induces brain inflammation but not dopaminergic neuronal death in the substantia nigra. Exp. Mol. Med. 42, 823–832. Jeong, H.K., Ji, K.M., Min, K.J., Choi, I., Choi, D.J., Jou, I., Joe, E.H., 2014. Astrogliosis is a possible player in preventing delayed neuronal death. Mol. Cells 37, 345–355. Jorgensen, M.B., Finsen, B.R., Jensen, M.B., Castellano, B., Diemer, N.H., Zimmer, J., 1993. Microglial and astroglial reactions to ischemic and kainic acid-induced lesions of the adult rat hippocampus. Exp. Neurol. 120, 70–88. Kuzhandaivel, A., Nistri, A., Mladinic, M., 2010. Kainate-mediated excitotoxicity induces neuronal death in the rat spinal cord in vitro via a PARP-1 dependent cell death pathway (Parthanatos). Cell. Mol. Neurobiol. 30, 1001–1012. Liu, D., 1994. An experimental model combining microdialysis with electrophysiology, histology, and neurochemistry for studying excitotoxicity in spinal cord injury. Effect of NMDA and kainate. Mol. Chem. Neuropathol. 23, 77–92. Liu, D., Xu, G.Y., Pan, E., McAdoo, D.J., 1999. Neurotoxicity of glutamate at the concentration released upon spinal cord injury. Neuroscience 93, 1383–1389. Liu, Z., Li, Y., Cui, Y., Roberts, C., Lu, M., Wilhelmsson, U., Pekny, M., Chopp, M., 2014. Beneficial effects of gfap/vimentin reactive astrocytes for axonal remodeling and motor behavioral recovery in mice after stroke. Glia 62, 2022–2033. Marty, S., Dusart, I., Schanski, M., 1991. Glial changes following an excitotoxic lesion in the CNS-I. Microglia/macrophages. Neurosci. 45, 529–539. Mazzone, G.L., Margaryan, G., Kuzhandaivel, A., Nasrabady, S.E., Mladinic, M., Nistri, A., 2010. Kainate-induced delayed onset of excitotoxicity with functional loss unrelated to the extent of neuronal damage in the in vitro spinal cord. Neuroscience 168, 451–462. Milenkovic, I., Nedeljkovic, N., Filipovic, R., Pekovic, S., Culic, M., Rakic, L., Stojiljkovic, M., 2005. Pattern of glial fibrillary acidic protein expression following kainate-induced cerebellar lesion in rats. Neurochem. Res. 30, 207–213. Miltiadous, P., Stamatakis, A., Stylianopolou, F., 2010. Neuroprotective effects of IGF-I following kainic acid induced hippocampal degeneration in the rat. Cell. Mol. Neurobiol. 30, 347–360. Mitra, N.K., Goh, T.E., Bala Krishnan, T., Nadarajah, V.D., Vasavaraj, A.K., Soga, T., 2013. Effect of intra-cisternal application of kainic acid on the spinal cord and locomotor activity in rats. Int. J. Clin. Exp. Pathol. 6, 1505–1515. Nicola, F.D.C., Marques, M.R., Odorcyk, F., Arcego, D.M., Petenuzzo, L., Aristimunha, D., Vizuete, A., Sanches, E.F., Pereira, D.P., Maurmann, N., Dalmaz, C., Pranke, P., Netto, C.A., 2017. Neuroprotector effect of stem cells from human exfoliated deciduous teeth transplanted after traumatic spinal cord injury involves inhibition of early neuronal apoptosis. Brain Res. https://doi.org/10.1016/j.brainres.2017.03.015. pii: S0006-8993(17)30123-3. Nishida, F., Zanuzzi, C.N., Marquez, M., Barbeito, C.G., Portiansky, E.L., 2014. La trepanación vertebral como un método alternativo para la inoculación intraparenquimatosa de diversas suspensiones dentro de la médula espinal. Analecta Vet. 34, 11–17. Nishida, F., Zanuzzi, C.N., Martínez, A., Barbeito, C.G., Portiansky, E.L., 2015. Functional and histopathological changes induced by intraparenchymal injection of kainic acid in the rat cervical spinal cord. Neurotoxicol. 49, 68–78. Nishida, F., Sisti, M.S., Zanuzzi, C.N., Barbeito, C.G., Portiansky, E.L., 2017. Neurons of the rat cervical spinal cord express vimentin and neurofilament after intraparenchymal injection of kainic acid. Neurosci. Lett. 643, 103–110. Pisharodi, M., Nauta, H.J., 1985. An animal model for neuron-specific spinal cord lesions by the microinjection of N-methylaspartate, kainic acid, and quisqualic acid. Appl. Neurophysiol. 48, 226–233. Pekny, M., Pekna, M., 2014. Astrocyte reactivity and reactive astrogliosis: costs and

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