Journal of Neuroscience Research 92:1457–1465 (2014)
Interleukin-1 Receptor Antagonist Promotes Survival of Ventral Horn Neurons and Suppresses Microglial Activation in Mouse Spinal Cord Slice Cultures N. Schizas,1* B. Andersson,1 J. Hilborn,2 and N.P. Hailer1 1
The SpineLab, Institute of Surgical Sciences, Department of Orthopaedics, Uppsala University, SE-751 85 Uppsala, Sweden 2 Division of Polymer Chemistry, Department of Materials Chemistry, The A˚ngstr€ om Laboratory, Uppsala University, SE-751 21 Uppsala, Sweden
Secondary damage after spinal cord injury (SCI) induces neuronal demise through neurotoxicity and inflammation, and interleukin (IL)-1b is a key inflammatory mediator. We hypothesized that IL-1b is released in spinal cord slice cultures (SCSC) and aimed at preventing the potentially neurotoxic effects of IL-1b by using interleukin-1 receptor antagonist (IL1RA). We hypothesized that IL1RA treatment enhances neuronal survival and suppresses microglial activation. SCSC were cultured up to 8 days in vitro (DIV) in the presence of IL1RA or without, either combined with trophic support using neurotrophin (NT)-3 or not. Four groups were studied: negative control, IL1RA, NT-3, and IL1RA 1 NT-3. IL-1b concentrations in supernatants were measured by ELISA. SCSC were immunohistochemically stained for NeuN and a-neurofilament, and microglial cells were visualized with isolectin B4. After 8 DIV, ventral horn neurons were significantly more numerous in the IL1RA, NT-3, and IL1RA 1 NT-3 groups compared with negative controls. Activated microglial cells were significantly less numerous in the IL1RA, NT-3, and IL1RA 1 NT-3 groups compared with negative controls. Axons expanded into the collagen matrix after treatment with IL1RA, NT-3, or IL1RA 1 NT-3, but not in negative controls. IL-1b release from cultures peaked after 6 hr and was lowest in the IL1RA 1 NT-3 group. We conclude that IL-1b is released in traumatized spinal cord tissue and that IL1RA could exert its neuroprotective actions by blocking IL-1receptors. IL1RA thereby sustains neuronal survival irrespective of the presence of additional trophic support. Microglial activation is suppressed in the presence of IL1RA, suggesting decreased inflammatory activity. IL1RA treatment approaches may have substantial impact following SCI. VC 2014 Wiley Periodicals, Inc.
prominent mediators of neurotoxicity (Woodroofe et al., 1991; Hailer, 2008; Spulber et al., 2009), and it has been suggested that it plays a regulatory role during neuroinflammation by promoting the expression of other proinflammatory mediators such as tumor necrosis factor (TNF)-a and cycloxygenase-2 (Gibson et al., 2004; Kaushik et al., 2013). IL-1 receptor antagonist (IL1RA) is an endogenous factor that antagonizes IL-1b effects by blocking the IL-1 receptor on the cell membrane (Dinarello, 1994). IL1RA has been studied mainly for its potential for neuroprotection in models of cerebral ischemia or haemorrhage, and it has been shown to reduce lesion size following middle cerebral artery occlusion (Loddick et al., 1997). IL1RA has been associated with reduced memory deficits in rats after IL-1b-induced neuroinflammation (Song et al., 2013), and it abolishes increased AMPA- and NMDAmediated currents in IL-1b-injured cerebral neurons in rats (Liu et al., 2013). Moreover, IL1RA inhibits microglial activation after excitotoxic injury in rat organotypic hippocampal slice cultures (Vogt et al., 2008). Neurotrophin (NT)-3 is a neurotrophic factor that has been extensively studied for its neuroprotective effects (Ma et al., 2012; Yu and Chuang, 1997). Briefly, NT-3 enhances neuronal survival of mechanically injured neurons in vitro (Ma et al., 2012) and improves neurological recovery followed ischemic brain damage in vivo (Zhang et al., 2012).
Key words: neuroprotection
neuroinflammation;
*Correspondence to: Nikos Schizas, MD, The SpineLab, Institute of Surgical Sciences, Department of Orthopaedics, Uppsala University, SE-751 85 Uppsala, Sweden. E-mail: [email protected]
Secondary damage after spinal cord injury (SCI) is a pathophysiological cascade of events that involves liberation of neurotoxic agents that are secreted by activated microglial cells. Interleukin (IL)-1b is one of the most
Received 25 October 2013; Revised 23 April 2014; Accepted 9 May 2014
interleukin-1;
C 2014 Wiley Periodicals, Inc. V
Contract grant sponsor: Regional agreement on medical training and clinical research (ALF) between Uppsala County Council and Uppsala University.
Published online 26 June 2014 in Wiley Online (wileyonlinelibrary.com). DOI: 10.1002/jnr.23429
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We have previously shown that spinal cord slice cultures (SCSC) maintained on polyethylene terephthalate (PET) inserts or collagen gel degenerate substantially after 4 days in vitro (DIV), whereas maintenance on a hyaluronic acid hydrogel improves tissue preservation, enhances neuronal survival, and reduces microglial activation (Schizas et al., 2013). It is reasonable to believe that both neuronal damage and microglial activation are at least in part mediated by IL-1b. Therefore, we sought to promote neuronal survival and to suppress microglial activation in the above-described model using IL1RA. We hypothesized that IL-1b is released in freshly prepared SCSC and that treatment of such cultures with IL1RA, either in the presence or in the absence of trophic support with NT-3, results in increased neuronal survival and supressed microglial activation. Neuronal survival, microglial activation, and axonal outgrowth in and from SCSC were thus examined. ELISA was used to measure the concentration of IL-1b in supernatants from SCSC. MATERIALS AND METHODS Experimental Design All experiments were conducted under aseptic conditions using sterile instruments and after approval of the local ethics committee. SCSC were obtained from postnatal day (P) 9 mice (C57/B6; Taconic Denmark) and incubated in vitro in a previously described 3D collagen matrix (Allodi et al., 2011). Sixtyfive P9 mice were used in the study, and approximately 15 cultures were obtained from each animal. All experiments were reproduced at least three times as independent experiments. Cultures were divided into four different groups (negative control, IL1RA, NT-3, and IL1RA 1 NT-3) and assessed after 2, 4, 6, and 8 DIV. Twenty SCSC were included in each group, and each culture was treated as an independent observation. The control group was incubated in culture medium and served as a negative control, the IL1RA group was incubated in culture medium containing 100 ng/ml soluble IL1RA (Prospec, Ness Ziona, Israel), the NT-3 group was incubated in the presence of 50 ng/ml NT-3 (Prospec), and the IL1RA 1 NT-3 group was incubated in the presence of both substances. A group of cultures that were directly fixed after preparation (0 DIV), i.e., without any treatment, served as a positive control to determine the number of neurons at the onset of the experiments. Neuronal survival was studied by using immunohistochemistry against neuronal nuclei (NeuN), the microglial response was studied by using Griffonia simplicifolia isolectin B4 (IB4), and axons were studied by using immunofluorescence against neurofilament-L (NF-L). Preparation and Maintenance of Collagen Gel Collagen gel was prepared following previously described methods, with some minor modifications (Allodi et al., 2011). Nine hundred microliters of rat-tail collagen 3.68 mg/ml (BD Biosciences) was mixed with 110 ll MEM and 4.4 ll 7.5% sodium bicarbonate, resulting in a final collagen concentration of 3.4 mg/ml. Five hundred microliters was applied onto each PET membrane and incubated at 37 C for 2 hr prior to the application of cultures.
SCSC Preparation P9 mice were euthanatized by decapitation, and the skin above the lower back was surgically removed to expose the lumbar and sacral region. The spine was detached from the sacrum, and a 23-gauge cannula was caudally inserted 223 mm inside the spinal canal. Subsequently, ice-cold preparation medium (MEM containing 1% glutamine, pH 7.35) was injected through the cannula, and the spinal cord was flushed out through the cervical spine. With a tissue chopper (McIlwain Tissue Chopper; Mickle Laboratory Engineering, Surrey, United Kingdom), 500lm slices were obtained and immediately transferred into petri dishes containing preparation medium. The slices were placed in PET culture inserts coated with a layer of collagen gel, and immediately a drop (30 ll) of collagen gel was placed on top of the slice culture, creating a 3D matrix. After 45 min of incubation in 37 C, the cultures continued incubation at 35 C in a 5% CO2-enriched atmosphere. Culture media consisting of 50% minimal essential medium (MEM; Statens Veterinarmedicinska Anstalt [SVA], Uppsala, Sweden), 25% Hank’s balanced salt solution (HBSS; Gibco Life Technologies, Stockholm, Sweden), 25% normal horse serum (NHS; Gibco), 2% glutamine (SigmaAldrich, Stockholm, Sweden), 1 lg/ml insulin (Sigma-Aldrich), 2.4 mg/ml glucose (Sigma-Aldrich), 0.1 mg/ml streptomycin (SVA), 100 U/ml penicillin (SVA), and 0.8 lg/ml vitamin C (Sigma-Aldrich), pH 7.4, were changed every other day. Cultures were subsequently fixed after 0, 2, 4, 6, and 8 DIV using a mixture of paraformaldehyde 4% and picric acid as previously described (Schizas et al., 2013). Immunohistochemistry Unsectioned SCSC were washed in PBS for 30 min and incubated in 10% normal goat serum (Vector Laboratories, J€arf€alla, Sweden) for 60 min. Incubation with NeuN primary antisera (1:500, rabbit polyclonal, ABN78; Millipore, Temecula, CA) or NF-L primary antisera (1:500, rabbit polyclonal; Millipore) for 24 hr, or FITC-conjugated Griffonia simplicifolia isolectin B4 (IB4; 1:20 lectin, L2895; Sigma-Aldrich) for 16 hr followed. The cultures incubated with NeuN or NF-L primary antisera underwent a 2-hr PBS washing and goat ant-rabbit secondary antibody (Vector Laboratories) followed for 16 hr. After PBS washing, they were incubated with DyLight 488 Streptavidin (Vector Laboratories) for 2 hr and washed for further 2 hr. The cultures were then transferred to Super Frost/Plus glass slides and mounted with Vectashield (Vector Laboratories). Image Analysis For image analysis of NeuN-, NF-L-, and IB4-positive cells, a Zeiss LSM 510 epifluorescence/confocal microscope and software were used. Images were scanned with a 488-nm argon laser and assessed by a blinded observer. For NeuN, four images were saved per culture, two taken in the ventral horns (one in each horn) and two in the dorsal horns (one in each horn). NeuNpositive neurons were manually counted in ImageJ (National Institutes of Health, Bethesda, MD). Only neurons showing a clear cytoplasmic along with nuclear staining were counted. For analysis of IB4 staining, four images were saved per culture, two within the median fissures representing parts of the white matter and two within the gray matter. Resting and Journal of Neuroscience Research
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Fig. 1. Number of neurons in the ventral and dorsal horns during days in vitro (DIV). Values denote mean number of neurons 6 SEM. After 8 DIV, the number of neurons in the ventral horn was significantly higher in all treatment groups compared with the negative control group (*P 0.05). The number of neurons in the IL1RA 1 NT-3 group was not statistically significantly different from the number of neurons determined immediately after culture preparation (“Direct”).
activated microglial cells were counted separately within the gray and white matter in ImageJ. Moreover, the total numbers of activated and resting microglial cells were calculated per culture (in four images) as well as the total number of all microglial cells per culture. Microglial cells were assessed by computerized analysis; they were counted as separate objects through a defined threshold of color and intensity that remained unaltered during the analysis. To separate resting from activated microglial cells, we used the circularity index (FF1) that was calculated by the equation FF1 5 4p 3 (cell area)/(cell perimeter)2 (Heppner et al., 1998). To validate this method, 20 randomly chosen images were assessed by manual counts, followed by assessment via computerized analysis. Pearson’s correlation analysis was performed on the results obtained by these two different modes of analysis, and, because the results correlated highly significantly with each other (r2 5 0.96, P < 0.001), we felt confident in the continued use of the computerized assessment. Axonal outgrowth of NF-Limmunoreactive axonal structures was examined qualitatively in images taken within the gray and white matter. ELISA SCSC were obtained from P9 mice as described above and transferred into PET inserts (three cultures per insert) and hosted in culture wells containing 1 ml culture medium. Inserts did not contain a collagen matrix because of the risk that a considerable amount of IL-1b would bind to the collagen substrate, inhibiting its release into the medium. Apart from the absence of a collagen matrix, SCSC were incubated under the conditions described above. The experiments were independently reproduced three times. Supernatants were collected from three culture wells after 6, 24, and 72 hr in sterile low-proteinbinding tubes (VWR, Stockholm, Sweden) and immediately frozen at 280 C. ELISA was performed on the collected culture media using the Mouse IL-1b Instant ELISA Kit acquired from eBiosciences with a standard curve range between 3.9 and 250 pg/ml (AH Diagnostics, Stockholm, Sweden). Standard IL-1b concentrations in all samples were run in duplicate. Journal of Neuroscience Research
Native culture media that had not been in contact with SCSC were incubated under the same conditions as the cultures for 6, 24, and 72 hr, serving as a baseline investigation of IL-1b contents in culture media not exposed to spinal cord tissue. Absorbance was measured on a Thermo Multiscan Ascent plate reader, and IL-1b concentrations were calculated based on a standard curve that was generated in Ascent Software for Multiscan. Statistical Analysis For statistical analysis of NeuN-positive neurons and microglial cells, one-way ANOVA with planned contrasts and Dunnett’s test for multiple comparisons were applied. The assumption of equal variances between groups was tested by using Levene’s test. First, all groups at each time point were compared with cultures directly fixed after preparations. Planned contrasts were used to compare the IL1RA, NT-3, and IL1RA 1 NT-3 groups with negative controls at each time point with the directional hypothesis that treatment with IL1RA, either with or without trophic support by NT-3, would be superior to its appropriate control. Dunnett’s test was used for post hoc comparisons of treatment groups with negative controls. For comparisons of IL-1b concentrations in supernatants from the four experimental groups, we used the directional hypothesis that the concentration of IL-1b would be lowest in the NT3 1 IL1RA group, because neuronal survival was most pronounced in that group, and statistical analysis was therefore performed via one-way ANOVA combined with Dunnett’s test.
RESULTS Neurons in the Ventral Horn The number of NeuN-positive neurons in the ventral horn was reduced in SCSC in all groups and at all time points compared with cultures fixed directly after preparation (P 5 0.001, Fig. 1). Signs of degeneration with strong nuclear and faint cytoplasmic staining were observed already after 2 DIV in all groups, but these
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Fig. 2. Micrographs of SCSC through the ventral horn. SCSC were stained against NeuN, either directly after culture preparation (A) or after 8 days in vitro (DIV; B2E). The number of neurons in the negative control group (B) was significantly lower compared with the other groups (C: IL1RA group, D: NT-3 group, E: IL1RA 1 NT-3 group).
The number of neurons in the combination group (E) did not differ significantly from the number of neurons in cultures fixed directly after preparation (A). Boxplots show the mean number of neurons and error bars show SEM (*P < 0.05). Scale bar 5 50 lm.
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TABLE I. Number of Microglial Cells Within Gray and White Matter* Control Resting mgc DIV 4 Gray matter 5.5 6 0.8 White matter 0.9 6 0.2 Total counted 12 6 1.2 Active mgc DIV 4 Gray matter 2.7 6 0.8 White matter 13.6 6 1.6 Total counted 31.3 6 4.0 Resting mgc DIV 8 Gray matter 6.1 6 1.1 White matter 0.5 6 0.2 Total counted 12.5 6 2.4 Active mgc DIV 8 Gray matter 5.6 6 2.0 White matter 18.8 6 2.6 Total counted 47.6 6 8.8
IL1RA
NT-3
IL1RA and NT-3
5.8 6 0.7 0.7 6 0.2 13.2 6 1.6
8.3 6 2.3† 1.4 6 0.3 17.5 6 1.0
7.5 6 0.8 1.0 6 0.1 15.9 6 1.9
2.7 6 1.0 15.0 6 2.1 36.06 6 5.8
1.3 6 0.44 13.5 6 2.1 28.2 6 5.1
2.3 6 0.8 14.3 6 1.6 31.5 6 4.7
8.0 6 0.9 1.1 6 0.4 17.1 6 1.9
8.7 6 0.9 1.4 6 0.5 19.2 6 2.1
7.6 6 1.1 1.8 6 0.6 20.9 6 3.0
2.0 6 0.5† 11.0 6 1.0† 24.4 6 2.0†
1.6 6 0.9† 10.1 6 1.0† 20.9 6 2.7†
1.7 6 0.8† 9.6 6 1.2† 18.2 6 3.0†
*The number of microglial cells was determined by computerized analysis in Image J software. Mean value 6 SEM is presented. † P < 0.05 compared with the control.
degenerative changes were more pronounced in the control group. Statistically significant differences between the groups with respect to neuronal numbers were observed after 8 DIV. At that time point, the number of neurons in the IL1RA, NT-3, and IL1RA 1 NT-3 groups was higher compared with the negative control group (P 5 0.01, Fig. 2). The number of neurons in the IL1RA 1 NT-3 group did not differ significantly from the number of neurons in slices directly fixed after preparation (P 5 0.3), whereas the number of ventral horn neurons in all other groups was lower compared with the directly fixed preparations (P < 0.05 for all comparisons). The number of neurons in each group declined with increasing time in vitro in the negative control and in the IL1RA groups while it remained rather stable in the NT3 and IL1RA 1 NT-3 groups (Fig. 1). Neurons in the Dorsal Horn The degeneration in the dorsal horns was more pronounced than that within the ventral horns. Already after 2 DIV, the number of NeuN-positive neurons was significantly decreased in all treatment groups compared with cultures directly fixed after preparation (P < 0.001, Fig. 1). This difference was similarly observed at all later time points. Statistical analysis revealed no significant difference among the four groups (negative control, IL1RA, NT-3, IL1RA 1 NT-3) at any time point (Fig. 1). Microglial Cells Statistically significant differences between the four groups (negative control, IL1RA, NT-3, IL1RA 1 NT-3) were noted only after 8 DIV and concerned only activated, not resting, microglial cells. At this time point, the number of Journal of Neuroscience Research
Fig. 3. Micrographs of SCSC through the median fissure after 8 DIV. Microglial cells were labeled with IB4. The number of activated microglial cells in the control group (A) was significantly higher compared with all treatment groups (B: IL1RA group, C: NT-3 group, D: IL1RA 1 NT-3 group). Scale bar 5 50 lm.
activated microglial cells was significantly lower in all treatment groups compared with the negative control group, counted separately in the white and gray matter (P < 0.01 for both comparisons; Table I, Fig. 3). The total number of activated microglial cells counted per culture, i.e., the sum of activated microglial cells in both white and gray matter, was also significantly lower in the IL1RA, NT-3, and IL1RA 1 NT3 groups compared with the control group (Table I). The total number of all microglial cells, i.e., the sum of resting and activated microglial cells per culture, did not differ significantly among the groups (P > 0.9 for all comparisons). Axonal Outgrowth In the white matter of directly fixed cultures, NF-L immunoreactivity was seen predominantly in the form of dots, representing parts of axons and fiber tracts that had been transversally axotomized during culture preparation (Fig. 4). In spinal cord slice cultures, axonal sprouting was observed already after 2 DIV within the white matter. After 6 DIV, the majority of the white matter had been occupied by axons extending transversally, suggesting axonal sprouting. This observation of sprouting inside the white matter was made in all experimental groups. In contrast, sprouting axons that expanded outside the cultures and into the collagen matrix were seen only in the IL1RA, NT-3, and IL1RA 1 NT-3 groups (Fig. 4). IL-1b Concentration Comparisons of IL-1b concentrations in supernatants from SCSC from the four different experimental
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Fig. 4. Micrographs of SCSC through the white matter. SCSCs were stained against NF-L directly after culture preparation (A) and after 6 DIV (B: negative control, C: IL1RA, D: NT-3, E: LI1RA 1 NT-3). In directly fixed cultures, axons were observed as immunoreactive dots denoting axons that had been axotomized transversally during
culture preparation. After 6 DIV, axonal sprouting was observed in all groups within the white matter (B2E). However, only in the IL1RA, NT-3 and IL1RA 1 NT-3 groups did these axons expand outside the culture and into the surrounding collagen gel. Scale bar 5 50 lm.
centrations were significantly higher in the IL1RA group compared with the IL1RA 1 NT-3 group (Fig. 5). Mean concentrations in the negative control and NT-3 groups were also higher than in the IL1RA 1 NT-3 group, but this finding failed to reach the level of statistical significance. Control experiments indicated that IL-1b was present in native culture media incubated under standard culture conditions without exposure to spinal cord tissue; IL-1b conentrations in native media were at the low end of the detection range after 6, 24, and 72 hr and measured about 6 pg/ml.
Fig. 5. Concentration of IL-1b in culture medium in different groups at different time points. Concentration is expressed in pg/ml 6 SEM (*P 0.05).
groups (IL1RA, NT-3, IL1RA 1 NT-3, negative controls) were performed after 6, 24, and 72 hr. Apart from the IL1RA 1 NT-3 group, which displayed low concentrations at all time points, IL-1b concentrations peaked after 6 hr and declined thereafter in the remaining groups (Fig. 5). Statistically significant differences between groups were noted only after 6 hr. At this time point, IL-1b con-
DISCUSSION We found an early peak of IL-1b-release in SCSC, and treatment of SCSC with IL1RA induced enhanced preservation of neuronal populations and supported axonal sprouting, both with and without trophic support by NT-3. Microglial activation as a measure of inflammation was reduced after treatment with IL1RA. To our knowledge this is the first report on increased neuronal survival in spinal cord cultures after immunomodulatory treatment. Treatment of SCSC with either IL1RA or NT-3 alone seemed insufficient to preserve the neuronal population at the level present immediately after culture preparation; however, a combination of these two agents supported neuronal populations up to 8 DIV. Treatment with IL1RA alone or NT-3 alone also enhanced neuronal survival compared with negative controls, but both treatments were unable to reach the effects induced by combined treatment with IL1RA and NT-3. Journal of Neuroscience Research
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Whether the above-mentioned enhanced neuronal preservation is the result of a synergistic effect between IL1RA and NT-3 requires further investigation. IL1RA has been previously studied for its neuroprotective potential and was found to inhibit microglial activation and suppress excitotoxic neuronal injury in vitro (Vogt et al., 2008). IL1RA has an effect on cannabinoid receptors in vitro and enhances their neuroprotective anatgonization of glutamate receptors (MolinaHolgado et al., 2003). In vivo experiments on excitotoxic SCI show that IL1RA improves locomotor function (Liu et al., 2008), and in a different in vivo model of SCI treatment with IL1RA enhanced neuronal survival and improved locomotor function (Zong et al., 2012). On a molecular level, IL1RA has been shown to block the haem-induced IL-1b inflammatory pathway following subarachnoid hemorrhage and thus reduce blood2brain barrier (BBB) breakdown (Greenhalgh et al., 2012). IL-1b plays a key role in the inflammatory cascade after hemorrhage, and it is reasonable to assume that IL1RA could similarly play a neuroprotective role during secondary damage following CNS trauma. The IL1RA concentration of 100 ng/ml and the NT-3 concentration of 50 ng/ml used in our experiments were chosen on the basis of our previous in vitro experiments (Hailer et al., 2005; Vogt et al., 2008; Stavridis et al., 2009). Much higher concentrations of IL1RA were used in in vivo models of SCI: Continuous administration of IL1RA at a concentration of 750 ng/ml with a minipump was associated with a reduction of contusion-induced apoptosis (Nesic et al., 2001), and a considerably higher concentration of 2 mg/ml was used for a single local administration after experimental SCI (Zong et al., 2012). It is reasonable to assume that higher concentrations of IL1RA are needed to reach a therapeutic effect in in vivo experiments, especially if only a single dose is administered. NT-3 is a neurotrophic factor that has been extensively studied for its neuroprotective potential, and even though its mechanism of action is not fully understood it has been suggested that NT-3 exerts direct effects on neurons subjected to oxidative stress or exposed to neurotoxic substances (Yu and Chuang, 1997; Korsak et al., 2012; Ma et al., 2012). Inflammation, hypoxia, and excitotoxicity are conditions likely to be present in SCSC following culture preparation. Increased neuronal survival compared with controls was observed in the IL1RA and NT-3 groups, and the neuroprotective actions of these substances most probably involve different pathways. It seems that treatment of spinal cord slice cultures with NT-3 alone did not preserve neuronal numbers at the level observed immediately after culture preparation. However, when NT-3 was combined with IL1RA, the number of neurons after 8 DIV did not differ significantly from the number of neurons counted directly after culture preparation. Even more important, trophic support with NT-3 induces axonal sprouting (Bradbury et al., 1999; Kamei et al., 2007; Stavridis et al., 2009; Boato et al., 2011), but we have demonstrated here that even treatment with IL1RA alone was associated with axonal sprouting into the collagen matrix. Journal of Neuroscience Research
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The neuroprotection observed in our study was identified in the ventral parts of the slice culture, whereas the dorsal parts seem to be more prone to degeneration. We have previously shown that neurons within the dorsal horn of SCSC degenerate substantially and to a higher extent than neurons that reside in the ventral horn (Schizas et al., 2013). Concerning the dorsal parts of the culture, even the combined treatment with IL1RA and NT3 seemed not to have any statistically significant effect. This was unexpected because dorsal horn neurons should be more resistant to degeneration withj their expression of calbindin, a protein that regulates the concentration of intracellular Ca21 and that is considered an intrinsic antiexcitotoxic neuroprotectant in interneurons (Ren and Ruda, 1994; Sun et al., 2011; Yuan et al., 2013). The observed extensive degeneration in the dorsal horn suggests that the mechanisms underlying neurodegeneration in the dorsal parts of the culture are different from those in the ventral horn, being less accessible to antiinflammatory treatment with IL1RA. Since neurons in the ventral horn respond to IL1RA treatment, one could speculate that neuroinflammation is likely to be the main reason behind neuronal degeneration in the ventral horn. Microglial activation was suppressed in all treatment groups compared with negative controls after 8 DIV. The state of activation of microglial cells is regulated by both immunomodulatory and neurotrophic substances such as IL1RA and NT-3 (Tzeng and Huang, 2003; Tzeng et al., 2005; Vogt et al., 2008; Pradillo et al., 2012). It is unclear how the state of activation of microglial cells is affected by NT-3. Improved culture preservation and enhanced neuronal survival as a result of trophic support with NT-3 could indirectly reduce the number of activated microglial cells, but direct effects of NT-3 on microglial cells cannot be ruled out (Tzeng et al., 2005). SCSC undergo degenerative changes during in vitro incubation, most probably as a result of the substantial injury sustained during culture preparation and the loss of both afferent and efferent pathways. We have previously shown that SCSC derived from postnatal mice degenerate substantially already after 4 DIV (Schizas et al., 2013), which is in partial contrast to observations made on SCSC originating from postnatal rats (Stavridis et al., 2005). Although neurons degenerate under control conditions, microglial cells change their state of activation. However, as SCSC undergo degenerative changes, one would expect that the total number of microglial cells would increase. In fact, after chemical injury in vivo, microglial cells proliferate rapidly as a response to induced inflammation (Jorgensen et al., 1993; Hailer et al., 1999). One plausible explanation for our observation that the total number of microglial cells remained unchanged irrespective of treatment is that a large number of microglial cells undergo apoptotic changes, in line with the general degeneration that characterizes the cultures after a certain time in vitro. Axonal sprouting from slice cultures of neural tissue is seen after the use of NT-3 (Bradbury et al., 1999; Kamei et al., 2007; Stavridis et al., 2009; Boato et al., 2011). In our study, even cultures under control
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conditions displayed axonal sprouting within the culture tissue. Sprouting axons, however, expanded outside the tissue and into the collagen matrix only when cultures were treated either with IL1RA, NT-3, or a combination of both substances. To our knowledge, this is the first observation that treatment of slice cultures with IL1RA promotes axonal sprouting. Apart from NT-3, numerous other molecules such L1CAM, GAP-43 and IL-6 have been implicated in axonal sprouting, although their mechanism of action is not fully understood (Grasselli et al., 2011; Schafer and Frotscher, 2012; Yang et al., 2012; Allegra Mascaro et al., 2013). It is unclear by which mechanisms IL1RA affects axonal sprouting, and further detailed experiments are needed in order to draw mechanistic conclusions. Nonetheless, our observation is exciting and represents further evidence of the cross-talk between the immune and the nervous systems in the context of plasticity and regeneration. The presence of IL-1b in supernatants from SCSC suggests a release of IL-1b from SCSC, rendering IL-1b and its receptor potential targets for intervention with IL1RA. The concentration of IL-1b in culture media peaked after 6 hr in all groups except for IL1RA 1 NT-3, followed by a decline up to 72 hr. The concentration of IL1b in the IL1RA 1 NT-3 group was just above detection limits at all time points. The significantly higher IL-1b concentration in the IL1RA group after 6 hr may be a combined effect of IL-1 receptors being blocked by IL1RA and a slightly greater liberation of IL-1b in the absence of NT3, leading to a subsequent increase in free and detectable IL-1b (Dinarello, 1994). However, it has to be taken into account that our investigations on the release of IL-1b are confounded by the fact that small amounts of IL-1b were present in native culture media, probably derived from the relatively large proportion of serum. In addition, the variation in measured IL-1b- concentrations close to the lower end of the detection range was rather large. Nonetheless, concentrations in culture supernatants exceeded those in native culture media, and the concentrations of IL-1b in culture supernatants were lowest in the combined presence of IL1RA and NT-3, i.e., the group with the highest degree of neuronal preservation. Taken together, this set of experiments primarily indicates that IL-1b was released from SCSC and that the kinetics of IL-1b-release in vitro were similar to those described after experimental SCI, in which an early peak of IL-1b concentrations is also observed 6 hr after injury (Bartholdi and Schwab, 1997). In conclusion, we demonstrate that treatment of spinal cord slice cultures with IL1RA resulted in an increased neuronal survival, irrespective of the presence of additional trophic support. The combination of immunomodulatory treatment with IL1RA and the neurotrophic factor NT-3 improved neuronal survival such that the number of ventral horn neurons after 8 DIV in treated cultures was similar to the number of ventral horn neurons in slices fixed directly after preparation, an effect that NT-3 alone was unable to produce. In addition to these neuroprotective effects of IL1RA, microglial activation was suppressed, but whether microglial activation is indirectly attenuated as a
consequence of enhanced neuronal survival and whether microglial activation is directly inhibited remain unclear. IL1RA treatment, combined with trophic support, is an interesting SCI treatment approach. The novel observation of enhanced axonal sprouting after treatment with IL1RA merits further investigation. ACKNOWLEDGMENTS Amanda Barrow is acknowledged for her contribution in data collection and analysis. Michael Petrides is acknowledged for his contribution in statistical analyses concerning the ELISA experiments. Imaging was performed with equipment maintained by the Science for Life Lab BioVis Platform Uppsala, Sweden. The authors acknowledge the assistance and support of Dirk Pacholsky and Matyas Molnar, staff of the Science for Life Lab BioVis Platform. REFERENCES Allegra Mascaro AL, Cesare P, Sacconi L, Grasselli G, Mandolesi G, Maco B, Knott GW, Huang L, De Paola V, Strata P, Pavone FS. 2013. In vivo single branch axotomy induces GAP-43-dependent sprouting and synaptic remodeling in cerebellar cortex. Proc Natl Acad Sci U S A 110:10824210829. Allodi I, Guzman-Lenis MS, Hernandez J, Navarro X, Udina E. 2011. In vitro comparison of motor and sensory neuron outgrowth in a 3D collagen matrix. J Neurosci Methods 198:53261. Bartholdi D, Schwab ME. 1997. Expression of pro-inflammatory cytokine and chemokine mRNA upon experimental spinal cord injury in mouse: an in situ hybridization study. Eur J Neurosci 9: 142221438. Boato F, Hechler D, Rosenberger K, Ludecke D, Peters EM, Nitsch R, Hendrix S. 2011. Interleukin-1 beta and neurotrophin-3 synergistically promote neurite growth in vitro. J Neuroinflamm 8:183. Bradbury EJ, Khemani S, Von R, King, Priestley JV, McMahon SB. 1999. NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord. Eur J Neurosci 11:387323883. Dinarello CA. 1994. The interleukin-1 family: 10 years of discovery. FASEB J 8:131421325. Gibson RM, Rothwell NJ, Le Feuvre RA. 2004. CNS injury: the role of the cytokine IL-1. Vet J 168:2302237. Grasselli G, Mandolesi G, Strata P, Cesare P. 2011. Impaired sprouting and axonal atrophy in cerebellar climbing fibres following in vivo silencing of the growth-associated protein GAP-43. PLoS One 6: e20791. Greenhalgh AD, Brough D, Robinson EM, Girard S, Rothwell NJ, Allan SM. 2012. Interleukin-1 receptor antagonist is beneficial after subarachnoid haemorrhage in rat by blocking haem-driven inflammatory pathology. Dis Model Mech 5:8232833. Hailer NP. 2008. Immunosuppression after traumatic or ischemic CNS damage: it is neuroprotective and illuminates the role of microglial cells. Prog Neurobiol 84:2112233. Hailer NP, Grampp A, Nitsch R. 1999. Proliferation of microglia and astrocytes in the dentate gyrus following entorhinal cortex lesion: a quantitative bromodeoxyuridine-labelling study. Eur J Neurosci 11: 335923364. Hailer NP, Vogt C, Korf HW, Dehghani F. 2005. Interleukin-1beta exacerbates and interleukin-1 receptor antagonist attenuates neuronal injury and microglial activation after excitotoxic damage in organotypic hippocampal slice cultures. Eur J Neurosci 21:234722360. Heppner FL, Roth K, Nitsch R, Hailer NP. 1998. Vitamin E induces ramification and downregulation of adhesion molecules in cultured microglial cells. Glia 22:1802188. Journal of Neuroscience Research
Neuroprotection With IL1RA Jorgensen MB, Finsen BR, Jensen MB, Castellano B, Diemer NH, Zimmer J. 1993. Microglial and astroglial reactions to ischemic and kainic acid-induced lesions of the adult rat hippocampus. Exp Neurol 120:70288. Kamei N, Tanaka N, Oishi Y, Hamasaki T, Nakanishi K, Sakai N, Ochi M. 2007. BDNF, NT-3, and NGF released from transplanted neural progenitor cells promote corticospinal axon growth in organotypic cocultures. Spine 32:127221278. Kaushik DK, Thounaojam MC, Kumawat KL, Gupta M, Basu A. 2013. Interleukin-1beta orchestrates underlying inflammatory responses in microglia via Kruppel-like factor 4. J Neurochem (in press). Korsak K, Dolatshad NF, Silva AT, Saffrey MJ. 2012. Ageing of enteric neurons: oxidative stress, neurotrophic factors and antioxidant enzymes. Chem Central J 6:80. Liu S, Xu GY, Johnson KM, Echetebu C, Ye ZS, Hulsebosch CE, McAdoo DJ. 2008. Regulation of interleukin-1beta by the interleukin1 receptor antagonist in the glutamate-injured spinal cord: endogenous neuroprotection. Brain Res 1231:63274. Liu T, Jiang CY, Fujita T, Luo SW, Kumamoto E. 2013. Enhancement by interleukin-1beta of AMPA and NMDA receptor-mediated currents in adult rat spinal superficial dorsal horn neurons. Mol Pain 9:16. Loddick SA, Wong ML, Bongiorno PB, Gold PW, Licinio J, Rothwell NJ. 1997. Endogenous interleukin-1 receptor antagonist is neuroprotective. Biochem Biophys Res Commun 234:2112215. Ma YH, Zeng X, Zhang K, Zeng YS. 2012. A new in vitro injury model of mouse neurons induced by mechanical scratching. Neurosci Lett 510:14219. Molina-Holgado F, Pinteaux E, Moore JD, Molina-Holgado E, Guaza C, Gibson RM, Rothwell NJ. 2003. Endogenous interleukin-1 receptor antagonist mediates anti-inflammatory and neuroprotective actions of cannabinoids in neurons and glia. J Neurosci 23:647026474. Nesic O, Xu GY, McAdoo D, High KW, Hulsebosch C, Perez-Pol R. 2001. IL-1 receptor antagonist prevents apoptosis and caspase-3 activation after spinal cord injury. J Neurotrauma 18:9472956. Pradillo JM, Denes A, Greenhalgh AD, Boutin H, Drake C, McColl BW, Barton E, Proctor SD, Russell JC, Rothwell NJ, Allan SM. 2012. Delayed administration of interleukin-1 receptor antagonist reduces ischemic brain damage and inflammation in comorbid rats. J Cereb Blood Flow Metab 32:181021819. Ren K, Ruda MA. 1994. A comparative study of the calcium-binding proteins calbindin-D28K, calretinin, calmodulin and parvalbumin in the rat spinal cord. Brain Res Brain Res Rev 19:1632179. Schafer MK, Frotscher M. 2012. Role of L1CAM for axon sprouting and branching. Cell Tissue Res 349:39248. Schizas N, Rojas R, Kootala S, Andersson B, Pettersson J, Hilborn J, Hailer NP. 2013. Hyaluronic acid-based hydrogel enhances neuronal survival in spinal cord slice cultures from postnatal mice. J Biomater Appl (in press). Song C, Zhang Y, Dong Y. 2013. Acute and subacute IL-1beta administrations differentially modulate neuroimmune and neurotrophic systems:
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possible implications for neuroprotection and neurodegeneration. J Neuroinflamm 10:59. Spulber S, Bartfai T, Schultzberg M. 2009. IL-1/IL-1ra balance in the brain revisited—evidence from transgenic mouse models. Brain Behav Immun 23:5732579. Stavridis SI, Dehghani F, Korf HW, Hailer NP. 2005. Characterisation of transverse slice culture preparations of postnatal rat spinal cord: preservation of defined neuronal populations. Histochem Cell Biol 123: 3772392. Stavridis SI, Dehghani F, Korf HW, Hailer NP. 2009. Cocultures of rat sensorimotor cortex and spinal cord slices to investigate corticospinal tract sprouting. Spine 34:249422499. Sun S, Li F, Gao X, Zhu Y, Chen J, Zhu X, Yuan H, Gao D. 2011. Calbindin-D28K inhibits apoptosis in dopaminergic neurons by activation of the PI3-kinase-Akt signaling pathway. Neuroscience 199: 3592367. Tzeng SF, Huang HY. 2003. Downregulation of inducible nitric oxide synthetase by neurotrophin-3 in microglia. J Cell Biochem 90: 2272233. Tzeng SF, Huang HY, Lee TI, Jwo JK. 2005. Inhibition of lipopolysaccharide-induced microglial activation by preexposure to neurotrophin-3. J Neurosci Res 81:6662676. Vogt C, Hailer NP, Ghadban C, Korf HW, Dehghani F. 2008. Successful inhibition of excitotoxic neuronal damage and microglial activation after delayed application of interleukin-1 receptor antagonist. J Neurosci Res 86:331423321. Woodroofe MN, Sarna GS, Wadhwa M, Hayes GM, Loughlin AJ, Tinker A, Cuzner ML. 1991. Detection of interleukin-1 and interleukin-6 in adult rat brain, following mechanical injury, by in vivo microdialysis: evidence of a role for microglia in cytokine production. J Neuroimmunol 33:2272236. Yang P, Wen H, Ou S, Cui J, Fan D. 2012. IL-6 promotes regeneration and functional recovery after cortical spinal tract injury by reactivating intrinsic growth program of neurons and enhancing synapse formation. Exp Neurol 236:19227. Yu O, Chuang DM. 1997. Neurotrophin protection against toxicity induced by low potassium and nitroprusside in cultured cerebellar granule neurons. J Neurochem 68:68277. Yuan HH, Chen RJ, Zhu YH, Peng CL, Zhu XR. 2013. The neuroprotective effect of overexpression of calbindin-D(28k) in an animal model of Parkinson’s disease. Mol Neurobiol 47: 1172122. Zhang J, Shi Q, Yang P, Xu X, Chen X, Qi C, Zhang J, Lu H, Zhao B, Zheng P, Zhang P, Liu Y. 2012. Neuroprotection of neurotrophin-3 against focal cerebral ischemia/reperfusion injury is regulated by hypoxia-responsive element in rats. Neuroscience 222:129. Zong S, Zeng G, Wei B, Xiong C, Zhao Y. 2012. Beneficial effect of interleukin-1 receptor antagonist protein on spinal cord injury recovery in the rat. Inflammation 35:5202526.
Interleukin-1 Receptor Antagonist Promotes Survival of Ventral Horn Neurons and Suppresses Microglial Activation in Mouse Spinal Cord Slice Cultures N. Schizas,1* B. Andersson,1 J. Hilborn,2 and N.P. Hailer1 1
The SpineLab, Institute of Surgical Sciences, Department of Orthopaedics, Uppsala University, SE-751 85 Uppsala, Sweden 2 Division of Polymer Chemistry, Department of Materials Chemistry, The A˚ngstr€ om Laboratory, Uppsala University, SE-751 21 Uppsala, Sweden
Secondary damage after spinal cord injury (SCI) induces neuronal demise through neurotoxicity and inflammation, and interleukin (IL)-1b is a key inflammatory mediator. We hypothesized that IL-1b is released in spinal cord slice cultures (SCSC) and aimed at preventing the potentially neurotoxic effects of IL-1b by using interleukin-1 receptor antagonist (IL1RA). We hypothesized that IL1RA treatment enhances neuronal survival and suppresses microglial activation. SCSC were cultured up to 8 days in vitro (DIV) in the presence of IL1RA or without, either combined with trophic support using neurotrophin (NT)-3 or not. Four groups were studied: negative control, IL1RA, NT-3, and IL1RA 1 NT-3. IL-1b concentrations in supernatants were measured by ELISA. SCSC were immunohistochemically stained for NeuN and a-neurofilament, and microglial cells were visualized with isolectin B4. After 8 DIV, ventral horn neurons were significantly more numerous in the IL1RA, NT-3, and IL1RA 1 NT-3 groups compared with negative controls. Activated microglial cells were significantly less numerous in the IL1RA, NT-3, and IL1RA 1 NT-3 groups compared with negative controls. Axons expanded into the collagen matrix after treatment with IL1RA, NT-3, or IL1RA 1 NT-3, but not in negative controls. IL-1b release from cultures peaked after 6 hr and was lowest in the IL1RA 1 NT-3 group. We conclude that IL-1b is released in traumatized spinal cord tissue and that IL1RA could exert its neuroprotective actions by blocking IL-1receptors. IL1RA thereby sustains neuronal survival irrespective of the presence of additional trophic support. Microglial activation is suppressed in the presence of IL1RA, suggesting decreased inflammatory activity. IL1RA treatment approaches may have substantial impact following SCI. VC 2014 Wiley Periodicals, Inc.
prominent mediators of neurotoxicity (Woodroofe et al., 1991; Hailer, 2008; Spulber et al., 2009), and it has been suggested that it plays a regulatory role during neuroinflammation by promoting the expression of other proinflammatory mediators such as tumor necrosis factor (TNF)-a and cycloxygenase-2 (Gibson et al., 2004; Kaushik et al., 2013). IL-1 receptor antagonist (IL1RA) is an endogenous factor that antagonizes IL-1b effects by blocking the IL-1 receptor on the cell membrane (Dinarello, 1994). IL1RA has been studied mainly for its potential for neuroprotection in models of cerebral ischemia or haemorrhage, and it has been shown to reduce lesion size following middle cerebral artery occlusion (Loddick et al., 1997). IL1RA has been associated with reduced memory deficits in rats after IL-1b-induced neuroinflammation (Song et al., 2013), and it abolishes increased AMPA- and NMDAmediated currents in IL-1b-injured cerebral neurons in rats (Liu et al., 2013). Moreover, IL1RA inhibits microglial activation after excitotoxic injury in rat organotypic hippocampal slice cultures (Vogt et al., 2008). Neurotrophin (NT)-3 is a neurotrophic factor that has been extensively studied for its neuroprotective effects (Ma et al., 2012; Yu and Chuang, 1997). Briefly, NT-3 enhances neuronal survival of mechanically injured neurons in vitro (Ma et al., 2012) and improves neurological recovery followed ischemic brain damage in vivo (Zhang et al., 2012).
Key words: neuroprotection
neuroinflammation;
*Correspondence to: Nikos Schizas, MD, The SpineLab, Institute of Surgical Sciences, Department of Orthopaedics, Uppsala University, SE-751 85 Uppsala, Sweden. E-mail: [email protected]
Secondary damage after spinal cord injury (SCI) is a pathophysiological cascade of events that involves liberation of neurotoxic agents that are secreted by activated microglial cells. Interleukin (IL)-1b is one of the most
Received 25 October 2013; Revised 23 April 2014; Accepted 9 May 2014
interleukin-1;
C 2014 Wiley Periodicals, Inc. V
Contract grant sponsor: Regional agreement on medical training and clinical research (ALF) between Uppsala County Council and Uppsala University.
Published online 26 June 2014 in Wiley Online (wileyonlinelibrary.com). DOI: 10.1002/jnr.23429
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We have previously shown that spinal cord slice cultures (SCSC) maintained on polyethylene terephthalate (PET) inserts or collagen gel degenerate substantially after 4 days in vitro (DIV), whereas maintenance on a hyaluronic acid hydrogel improves tissue preservation, enhances neuronal survival, and reduces microglial activation (Schizas et al., 2013). It is reasonable to believe that both neuronal damage and microglial activation are at least in part mediated by IL-1b. Therefore, we sought to promote neuronal survival and to suppress microglial activation in the above-described model using IL1RA. We hypothesized that IL-1b is released in freshly prepared SCSC and that treatment of such cultures with IL1RA, either in the presence or in the absence of trophic support with NT-3, results in increased neuronal survival and supressed microglial activation. Neuronal survival, microglial activation, and axonal outgrowth in and from SCSC were thus examined. ELISA was used to measure the concentration of IL-1b in supernatants from SCSC. MATERIALS AND METHODS Experimental Design All experiments were conducted under aseptic conditions using sterile instruments and after approval of the local ethics committee. SCSC were obtained from postnatal day (P) 9 mice (C57/B6; Taconic Denmark) and incubated in vitro in a previously described 3D collagen matrix (Allodi et al., 2011). Sixtyfive P9 mice were used in the study, and approximately 15 cultures were obtained from each animal. All experiments were reproduced at least three times as independent experiments. Cultures were divided into four different groups (negative control, IL1RA, NT-3, and IL1RA 1 NT-3) and assessed after 2, 4, 6, and 8 DIV. Twenty SCSC were included in each group, and each culture was treated as an independent observation. The control group was incubated in culture medium and served as a negative control, the IL1RA group was incubated in culture medium containing 100 ng/ml soluble IL1RA (Prospec, Ness Ziona, Israel), the NT-3 group was incubated in the presence of 50 ng/ml NT-3 (Prospec), and the IL1RA 1 NT-3 group was incubated in the presence of both substances. A group of cultures that were directly fixed after preparation (0 DIV), i.e., without any treatment, served as a positive control to determine the number of neurons at the onset of the experiments. Neuronal survival was studied by using immunohistochemistry against neuronal nuclei (NeuN), the microglial response was studied by using Griffonia simplicifolia isolectin B4 (IB4), and axons were studied by using immunofluorescence against neurofilament-L (NF-L). Preparation and Maintenance of Collagen Gel Collagen gel was prepared following previously described methods, with some minor modifications (Allodi et al., 2011). Nine hundred microliters of rat-tail collagen 3.68 mg/ml (BD Biosciences) was mixed with 110 ll MEM and 4.4 ll 7.5% sodium bicarbonate, resulting in a final collagen concentration of 3.4 mg/ml. Five hundred microliters was applied onto each PET membrane and incubated at 37 C for 2 hr prior to the application of cultures.
SCSC Preparation P9 mice were euthanatized by decapitation, and the skin above the lower back was surgically removed to expose the lumbar and sacral region. The spine was detached from the sacrum, and a 23-gauge cannula was caudally inserted 223 mm inside the spinal canal. Subsequently, ice-cold preparation medium (MEM containing 1% glutamine, pH 7.35) was injected through the cannula, and the spinal cord was flushed out through the cervical spine. With a tissue chopper (McIlwain Tissue Chopper; Mickle Laboratory Engineering, Surrey, United Kingdom), 500lm slices were obtained and immediately transferred into petri dishes containing preparation medium. The slices were placed in PET culture inserts coated with a layer of collagen gel, and immediately a drop (30 ll) of collagen gel was placed on top of the slice culture, creating a 3D matrix. After 45 min of incubation in 37 C, the cultures continued incubation at 35 C in a 5% CO2-enriched atmosphere. Culture media consisting of 50% minimal essential medium (MEM; Statens Veterinarmedicinska Anstalt [SVA], Uppsala, Sweden), 25% Hank’s balanced salt solution (HBSS; Gibco Life Technologies, Stockholm, Sweden), 25% normal horse serum (NHS; Gibco), 2% glutamine (SigmaAldrich, Stockholm, Sweden), 1 lg/ml insulin (Sigma-Aldrich), 2.4 mg/ml glucose (Sigma-Aldrich), 0.1 mg/ml streptomycin (SVA), 100 U/ml penicillin (SVA), and 0.8 lg/ml vitamin C (Sigma-Aldrich), pH 7.4, were changed every other day. Cultures were subsequently fixed after 0, 2, 4, 6, and 8 DIV using a mixture of paraformaldehyde 4% and picric acid as previously described (Schizas et al., 2013). Immunohistochemistry Unsectioned SCSC were washed in PBS for 30 min and incubated in 10% normal goat serum (Vector Laboratories, J€arf€alla, Sweden) for 60 min. Incubation with NeuN primary antisera (1:500, rabbit polyclonal, ABN78; Millipore, Temecula, CA) or NF-L primary antisera (1:500, rabbit polyclonal; Millipore) for 24 hr, or FITC-conjugated Griffonia simplicifolia isolectin B4 (IB4; 1:20 lectin, L2895; Sigma-Aldrich) for 16 hr followed. The cultures incubated with NeuN or NF-L primary antisera underwent a 2-hr PBS washing and goat ant-rabbit secondary antibody (Vector Laboratories) followed for 16 hr. After PBS washing, they were incubated with DyLight 488 Streptavidin (Vector Laboratories) for 2 hr and washed for further 2 hr. The cultures were then transferred to Super Frost/Plus glass slides and mounted with Vectashield (Vector Laboratories). Image Analysis For image analysis of NeuN-, NF-L-, and IB4-positive cells, a Zeiss LSM 510 epifluorescence/confocal microscope and software were used. Images were scanned with a 488-nm argon laser and assessed by a blinded observer. For NeuN, four images were saved per culture, two taken in the ventral horns (one in each horn) and two in the dorsal horns (one in each horn). NeuNpositive neurons were manually counted in ImageJ (National Institutes of Health, Bethesda, MD). Only neurons showing a clear cytoplasmic along with nuclear staining were counted. For analysis of IB4 staining, four images were saved per culture, two within the median fissures representing parts of the white matter and two within the gray matter. Resting and Journal of Neuroscience Research
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Fig. 1. Number of neurons in the ventral and dorsal horns during days in vitro (DIV). Values denote mean number of neurons 6 SEM. After 8 DIV, the number of neurons in the ventral horn was significantly higher in all treatment groups compared with the negative control group (*P 0.05). The number of neurons in the IL1RA 1 NT-3 group was not statistically significantly different from the number of neurons determined immediately after culture preparation (“Direct”).
activated microglial cells were counted separately within the gray and white matter in ImageJ. Moreover, the total numbers of activated and resting microglial cells were calculated per culture (in four images) as well as the total number of all microglial cells per culture. Microglial cells were assessed by computerized analysis; they were counted as separate objects through a defined threshold of color and intensity that remained unaltered during the analysis. To separate resting from activated microglial cells, we used the circularity index (FF1) that was calculated by the equation FF1 5 4p 3 (cell area)/(cell perimeter)2 (Heppner et al., 1998). To validate this method, 20 randomly chosen images were assessed by manual counts, followed by assessment via computerized analysis. Pearson’s correlation analysis was performed on the results obtained by these two different modes of analysis, and, because the results correlated highly significantly with each other (r2 5 0.96, P < 0.001), we felt confident in the continued use of the computerized assessment. Axonal outgrowth of NF-Limmunoreactive axonal structures was examined qualitatively in images taken within the gray and white matter. ELISA SCSC were obtained from P9 mice as described above and transferred into PET inserts (three cultures per insert) and hosted in culture wells containing 1 ml culture medium. Inserts did not contain a collagen matrix because of the risk that a considerable amount of IL-1b would bind to the collagen substrate, inhibiting its release into the medium. Apart from the absence of a collagen matrix, SCSC were incubated under the conditions described above. The experiments were independently reproduced three times. Supernatants were collected from three culture wells after 6, 24, and 72 hr in sterile low-proteinbinding tubes (VWR, Stockholm, Sweden) and immediately frozen at 280 C. ELISA was performed on the collected culture media using the Mouse IL-1b Instant ELISA Kit acquired from eBiosciences with a standard curve range between 3.9 and 250 pg/ml (AH Diagnostics, Stockholm, Sweden). Standard IL-1b concentrations in all samples were run in duplicate. Journal of Neuroscience Research
Native culture media that had not been in contact with SCSC were incubated under the same conditions as the cultures for 6, 24, and 72 hr, serving as a baseline investigation of IL-1b contents in culture media not exposed to spinal cord tissue. Absorbance was measured on a Thermo Multiscan Ascent plate reader, and IL-1b concentrations were calculated based on a standard curve that was generated in Ascent Software for Multiscan. Statistical Analysis For statistical analysis of NeuN-positive neurons and microglial cells, one-way ANOVA with planned contrasts and Dunnett’s test for multiple comparisons were applied. The assumption of equal variances between groups was tested by using Levene’s test. First, all groups at each time point were compared with cultures directly fixed after preparations. Planned contrasts were used to compare the IL1RA, NT-3, and IL1RA 1 NT-3 groups with negative controls at each time point with the directional hypothesis that treatment with IL1RA, either with or without trophic support by NT-3, would be superior to its appropriate control. Dunnett’s test was used for post hoc comparisons of treatment groups with negative controls. For comparisons of IL-1b concentrations in supernatants from the four experimental groups, we used the directional hypothesis that the concentration of IL-1b would be lowest in the NT3 1 IL1RA group, because neuronal survival was most pronounced in that group, and statistical analysis was therefore performed via one-way ANOVA combined with Dunnett’s test.
RESULTS Neurons in the Ventral Horn The number of NeuN-positive neurons in the ventral horn was reduced in SCSC in all groups and at all time points compared with cultures fixed directly after preparation (P 5 0.001, Fig. 1). Signs of degeneration with strong nuclear and faint cytoplasmic staining were observed already after 2 DIV in all groups, but these
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Fig. 2. Micrographs of SCSC through the ventral horn. SCSC were stained against NeuN, either directly after culture preparation (A) or after 8 days in vitro (DIV; B2E). The number of neurons in the negative control group (B) was significantly lower compared with the other groups (C: IL1RA group, D: NT-3 group, E: IL1RA 1 NT-3 group).
The number of neurons in the combination group (E) did not differ significantly from the number of neurons in cultures fixed directly after preparation (A). Boxplots show the mean number of neurons and error bars show SEM (*P < 0.05). Scale bar 5 50 lm.
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TABLE I. Number of Microglial Cells Within Gray and White Matter* Control Resting mgc DIV 4 Gray matter 5.5 6 0.8 White matter 0.9 6 0.2 Total counted 12 6 1.2 Active mgc DIV 4 Gray matter 2.7 6 0.8 White matter 13.6 6 1.6 Total counted 31.3 6 4.0 Resting mgc DIV 8 Gray matter 6.1 6 1.1 White matter 0.5 6 0.2 Total counted 12.5 6 2.4 Active mgc DIV 8 Gray matter 5.6 6 2.0 White matter 18.8 6 2.6 Total counted 47.6 6 8.8
IL1RA
NT-3
IL1RA and NT-3
5.8 6 0.7 0.7 6 0.2 13.2 6 1.6
8.3 6 2.3† 1.4 6 0.3 17.5 6 1.0
7.5 6 0.8 1.0 6 0.1 15.9 6 1.9
2.7 6 1.0 15.0 6 2.1 36.06 6 5.8
1.3 6 0.44 13.5 6 2.1 28.2 6 5.1
2.3 6 0.8 14.3 6 1.6 31.5 6 4.7
8.0 6 0.9 1.1 6 0.4 17.1 6 1.9
8.7 6 0.9 1.4 6 0.5 19.2 6 2.1
7.6 6 1.1 1.8 6 0.6 20.9 6 3.0
2.0 6 0.5† 11.0 6 1.0† 24.4 6 2.0†
1.6 6 0.9† 10.1 6 1.0† 20.9 6 2.7†
1.7 6 0.8† 9.6 6 1.2† 18.2 6 3.0†
*The number of microglial cells was determined by computerized analysis in Image J software. Mean value 6 SEM is presented. † P < 0.05 compared with the control.
degenerative changes were more pronounced in the control group. Statistically significant differences between the groups with respect to neuronal numbers were observed after 8 DIV. At that time point, the number of neurons in the IL1RA, NT-3, and IL1RA 1 NT-3 groups was higher compared with the negative control group (P 5 0.01, Fig. 2). The number of neurons in the IL1RA 1 NT-3 group did not differ significantly from the number of neurons in slices directly fixed after preparation (P 5 0.3), whereas the number of ventral horn neurons in all other groups was lower compared with the directly fixed preparations (P < 0.05 for all comparisons). The number of neurons in each group declined with increasing time in vitro in the negative control and in the IL1RA groups while it remained rather stable in the NT3 and IL1RA 1 NT-3 groups (Fig. 1). Neurons in the Dorsal Horn The degeneration in the dorsal horns was more pronounced than that within the ventral horns. Already after 2 DIV, the number of NeuN-positive neurons was significantly decreased in all treatment groups compared with cultures directly fixed after preparation (P < 0.001, Fig. 1). This difference was similarly observed at all later time points. Statistical analysis revealed no significant difference among the four groups (negative control, IL1RA, NT-3, IL1RA 1 NT-3) at any time point (Fig. 1). Microglial Cells Statistically significant differences between the four groups (negative control, IL1RA, NT-3, IL1RA 1 NT-3) were noted only after 8 DIV and concerned only activated, not resting, microglial cells. At this time point, the number of Journal of Neuroscience Research
Fig. 3. Micrographs of SCSC through the median fissure after 8 DIV. Microglial cells were labeled with IB4. The number of activated microglial cells in the control group (A) was significantly higher compared with all treatment groups (B: IL1RA group, C: NT-3 group, D: IL1RA 1 NT-3 group). Scale bar 5 50 lm.
activated microglial cells was significantly lower in all treatment groups compared with the negative control group, counted separately in the white and gray matter (P < 0.01 for both comparisons; Table I, Fig. 3). The total number of activated microglial cells counted per culture, i.e., the sum of activated microglial cells in both white and gray matter, was also significantly lower in the IL1RA, NT-3, and IL1RA 1 NT3 groups compared with the control group (Table I). The total number of all microglial cells, i.e., the sum of resting and activated microglial cells per culture, did not differ significantly among the groups (P > 0.9 for all comparisons). Axonal Outgrowth In the white matter of directly fixed cultures, NF-L immunoreactivity was seen predominantly in the form of dots, representing parts of axons and fiber tracts that had been transversally axotomized during culture preparation (Fig. 4). In spinal cord slice cultures, axonal sprouting was observed already after 2 DIV within the white matter. After 6 DIV, the majority of the white matter had been occupied by axons extending transversally, suggesting axonal sprouting. This observation of sprouting inside the white matter was made in all experimental groups. In contrast, sprouting axons that expanded outside the cultures and into the collagen matrix were seen only in the IL1RA, NT-3, and IL1RA 1 NT-3 groups (Fig. 4). IL-1b Concentration Comparisons of IL-1b concentrations in supernatants from SCSC from the four different experimental
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Fig. 4. Micrographs of SCSC through the white matter. SCSCs were stained against NF-L directly after culture preparation (A) and after 6 DIV (B: negative control, C: IL1RA, D: NT-3, E: LI1RA 1 NT-3). In directly fixed cultures, axons were observed as immunoreactive dots denoting axons that had been axotomized transversally during
culture preparation. After 6 DIV, axonal sprouting was observed in all groups within the white matter (B2E). However, only in the IL1RA, NT-3 and IL1RA 1 NT-3 groups did these axons expand outside the culture and into the surrounding collagen gel. Scale bar 5 50 lm.
centrations were significantly higher in the IL1RA group compared with the IL1RA 1 NT-3 group (Fig. 5). Mean concentrations in the negative control and NT-3 groups were also higher than in the IL1RA 1 NT-3 group, but this finding failed to reach the level of statistical significance. Control experiments indicated that IL-1b was present in native culture media incubated under standard culture conditions without exposure to spinal cord tissue; IL-1b conentrations in native media were at the low end of the detection range after 6, 24, and 72 hr and measured about 6 pg/ml.
Fig. 5. Concentration of IL-1b in culture medium in different groups at different time points. Concentration is expressed in pg/ml 6 SEM (*P 0.05).
groups (IL1RA, NT-3, IL1RA 1 NT-3, negative controls) were performed after 6, 24, and 72 hr. Apart from the IL1RA 1 NT-3 group, which displayed low concentrations at all time points, IL-1b concentrations peaked after 6 hr and declined thereafter in the remaining groups (Fig. 5). Statistically significant differences between groups were noted only after 6 hr. At this time point, IL-1b con-
DISCUSSION We found an early peak of IL-1b-release in SCSC, and treatment of SCSC with IL1RA induced enhanced preservation of neuronal populations and supported axonal sprouting, both with and without trophic support by NT-3. Microglial activation as a measure of inflammation was reduced after treatment with IL1RA. To our knowledge this is the first report on increased neuronal survival in spinal cord cultures after immunomodulatory treatment. Treatment of SCSC with either IL1RA or NT-3 alone seemed insufficient to preserve the neuronal population at the level present immediately after culture preparation; however, a combination of these two agents supported neuronal populations up to 8 DIV. Treatment with IL1RA alone or NT-3 alone also enhanced neuronal survival compared with negative controls, but both treatments were unable to reach the effects induced by combined treatment with IL1RA and NT-3. Journal of Neuroscience Research
Neuroprotection With IL1RA
Whether the above-mentioned enhanced neuronal preservation is the result of a synergistic effect between IL1RA and NT-3 requires further investigation. IL1RA has been previously studied for its neuroprotective potential and was found to inhibit microglial activation and suppress excitotoxic neuronal injury in vitro (Vogt et al., 2008). IL1RA has an effect on cannabinoid receptors in vitro and enhances their neuroprotective anatgonization of glutamate receptors (MolinaHolgado et al., 2003). In vivo experiments on excitotoxic SCI show that IL1RA improves locomotor function (Liu et al., 2008), and in a different in vivo model of SCI treatment with IL1RA enhanced neuronal survival and improved locomotor function (Zong et al., 2012). On a molecular level, IL1RA has been shown to block the haem-induced IL-1b inflammatory pathway following subarachnoid hemorrhage and thus reduce blood2brain barrier (BBB) breakdown (Greenhalgh et al., 2012). IL-1b plays a key role in the inflammatory cascade after hemorrhage, and it is reasonable to assume that IL1RA could similarly play a neuroprotective role during secondary damage following CNS trauma. The IL1RA concentration of 100 ng/ml and the NT-3 concentration of 50 ng/ml used in our experiments were chosen on the basis of our previous in vitro experiments (Hailer et al., 2005; Vogt et al., 2008; Stavridis et al., 2009). Much higher concentrations of IL1RA were used in in vivo models of SCI: Continuous administration of IL1RA at a concentration of 750 ng/ml with a minipump was associated with a reduction of contusion-induced apoptosis (Nesic et al., 2001), and a considerably higher concentration of 2 mg/ml was used for a single local administration after experimental SCI (Zong et al., 2012). It is reasonable to assume that higher concentrations of IL1RA are needed to reach a therapeutic effect in in vivo experiments, especially if only a single dose is administered. NT-3 is a neurotrophic factor that has been extensively studied for its neuroprotective potential, and even though its mechanism of action is not fully understood it has been suggested that NT-3 exerts direct effects on neurons subjected to oxidative stress or exposed to neurotoxic substances (Yu and Chuang, 1997; Korsak et al., 2012; Ma et al., 2012). Inflammation, hypoxia, and excitotoxicity are conditions likely to be present in SCSC following culture preparation. Increased neuronal survival compared with controls was observed in the IL1RA and NT-3 groups, and the neuroprotective actions of these substances most probably involve different pathways. It seems that treatment of spinal cord slice cultures with NT-3 alone did not preserve neuronal numbers at the level observed immediately after culture preparation. However, when NT-3 was combined with IL1RA, the number of neurons after 8 DIV did not differ significantly from the number of neurons counted directly after culture preparation. Even more important, trophic support with NT-3 induces axonal sprouting (Bradbury et al., 1999; Kamei et al., 2007; Stavridis et al., 2009; Boato et al., 2011), but we have demonstrated here that even treatment with IL1RA alone was associated with axonal sprouting into the collagen matrix. Journal of Neuroscience Research
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The neuroprotection observed in our study was identified in the ventral parts of the slice culture, whereas the dorsal parts seem to be more prone to degeneration. We have previously shown that neurons within the dorsal horn of SCSC degenerate substantially and to a higher extent than neurons that reside in the ventral horn (Schizas et al., 2013). Concerning the dorsal parts of the culture, even the combined treatment with IL1RA and NT3 seemed not to have any statistically significant effect. This was unexpected because dorsal horn neurons should be more resistant to degeneration withj their expression of calbindin, a protein that regulates the concentration of intracellular Ca21 and that is considered an intrinsic antiexcitotoxic neuroprotectant in interneurons (Ren and Ruda, 1994; Sun et al., 2011; Yuan et al., 2013). The observed extensive degeneration in the dorsal horn suggests that the mechanisms underlying neurodegeneration in the dorsal parts of the culture are different from those in the ventral horn, being less accessible to antiinflammatory treatment with IL1RA. Since neurons in the ventral horn respond to IL1RA treatment, one could speculate that neuroinflammation is likely to be the main reason behind neuronal degeneration in the ventral horn. Microglial activation was suppressed in all treatment groups compared with negative controls after 8 DIV. The state of activation of microglial cells is regulated by both immunomodulatory and neurotrophic substances such as IL1RA and NT-3 (Tzeng and Huang, 2003; Tzeng et al., 2005; Vogt et al., 2008; Pradillo et al., 2012). It is unclear how the state of activation of microglial cells is affected by NT-3. Improved culture preservation and enhanced neuronal survival as a result of trophic support with NT-3 could indirectly reduce the number of activated microglial cells, but direct effects of NT-3 on microglial cells cannot be ruled out (Tzeng et al., 2005). SCSC undergo degenerative changes during in vitro incubation, most probably as a result of the substantial injury sustained during culture preparation and the loss of both afferent and efferent pathways. We have previously shown that SCSC derived from postnatal mice degenerate substantially already after 4 DIV (Schizas et al., 2013), which is in partial contrast to observations made on SCSC originating from postnatal rats (Stavridis et al., 2005). Although neurons degenerate under control conditions, microglial cells change their state of activation. However, as SCSC undergo degenerative changes, one would expect that the total number of microglial cells would increase. In fact, after chemical injury in vivo, microglial cells proliferate rapidly as a response to induced inflammation (Jorgensen et al., 1993; Hailer et al., 1999). One plausible explanation for our observation that the total number of microglial cells remained unchanged irrespective of treatment is that a large number of microglial cells undergo apoptotic changes, in line with the general degeneration that characterizes the cultures after a certain time in vitro. Axonal sprouting from slice cultures of neural tissue is seen after the use of NT-3 (Bradbury et al., 1999; Kamei et al., 2007; Stavridis et al., 2009; Boato et al., 2011). In our study, even cultures under control
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conditions displayed axonal sprouting within the culture tissue. Sprouting axons, however, expanded outside the tissue and into the collagen matrix only when cultures were treated either with IL1RA, NT-3, or a combination of both substances. To our knowledge, this is the first observation that treatment of slice cultures with IL1RA promotes axonal sprouting. Apart from NT-3, numerous other molecules such L1CAM, GAP-43 and IL-6 have been implicated in axonal sprouting, although their mechanism of action is not fully understood (Grasselli et al., 2011; Schafer and Frotscher, 2012; Yang et al., 2012; Allegra Mascaro et al., 2013). It is unclear by which mechanisms IL1RA affects axonal sprouting, and further detailed experiments are needed in order to draw mechanistic conclusions. Nonetheless, our observation is exciting and represents further evidence of the cross-talk between the immune and the nervous systems in the context of plasticity and regeneration. The presence of IL-1b in supernatants from SCSC suggests a release of IL-1b from SCSC, rendering IL-1b and its receptor potential targets for intervention with IL1RA. The concentration of IL-1b in culture media peaked after 6 hr in all groups except for IL1RA 1 NT-3, followed by a decline up to 72 hr. The concentration of IL1b in the IL1RA 1 NT-3 group was just above detection limits at all time points. The significantly higher IL-1b concentration in the IL1RA group after 6 hr may be a combined effect of IL-1 receptors being blocked by IL1RA and a slightly greater liberation of IL-1b in the absence of NT3, leading to a subsequent increase in free and detectable IL-1b (Dinarello, 1994). However, it has to be taken into account that our investigations on the release of IL-1b are confounded by the fact that small amounts of IL-1b were present in native culture media, probably derived from the relatively large proportion of serum. In addition, the variation in measured IL-1b- concentrations close to the lower end of the detection range was rather large. Nonetheless, concentrations in culture supernatants exceeded those in native culture media, and the concentrations of IL-1b in culture supernatants were lowest in the combined presence of IL1RA and NT-3, i.e., the group with the highest degree of neuronal preservation. Taken together, this set of experiments primarily indicates that IL-1b was released from SCSC and that the kinetics of IL-1b-release in vitro were similar to those described after experimental SCI, in which an early peak of IL-1b concentrations is also observed 6 hr after injury (Bartholdi and Schwab, 1997). In conclusion, we demonstrate that treatment of spinal cord slice cultures with IL1RA resulted in an increased neuronal survival, irrespective of the presence of additional trophic support. The combination of immunomodulatory treatment with IL1RA and the neurotrophic factor NT-3 improved neuronal survival such that the number of ventral horn neurons after 8 DIV in treated cultures was similar to the number of ventral horn neurons in slices fixed directly after preparation, an effect that NT-3 alone was unable to produce. In addition to these neuroprotective effects of IL1RA, microglial activation was suppressed, but whether microglial activation is indirectly attenuated as a
consequence of enhanced neuronal survival and whether microglial activation is directly inhibited remain unclear. IL1RA treatment, combined with trophic support, is an interesting SCI treatment approach. The novel observation of enhanced axonal sprouting after treatment with IL1RA merits further investigation. ACKNOWLEDGMENTS Amanda Barrow is acknowledged for her contribution in data collection and analysis. Michael Petrides is acknowledged for his contribution in statistical analyses concerning the ELISA experiments. Imaging was performed with equipment maintained by the Science for Life Lab BioVis Platform Uppsala, Sweden. The authors acknowledge the assistance and support of Dirk Pacholsky and Matyas Molnar, staff of the Science for Life Lab BioVis Platform. REFERENCES Allegra Mascaro AL, Cesare P, Sacconi L, Grasselli G, Mandolesi G, Maco B, Knott GW, Huang L, De Paola V, Strata P, Pavone FS. 2013. In vivo single branch axotomy induces GAP-43-dependent sprouting and synaptic remodeling in cerebellar cortex. Proc Natl Acad Sci U S A 110:10824210829. Allodi I, Guzman-Lenis MS, Hernandez J, Navarro X, Udina E. 2011. In vitro comparison of motor and sensory neuron outgrowth in a 3D collagen matrix. J Neurosci Methods 198:53261. Bartholdi D, Schwab ME. 1997. Expression of pro-inflammatory cytokine and chemokine mRNA upon experimental spinal cord injury in mouse: an in situ hybridization study. Eur J Neurosci 9: 142221438. Boato F, Hechler D, Rosenberger K, Ludecke D, Peters EM, Nitsch R, Hendrix S. 2011. Interleukin-1 beta and neurotrophin-3 synergistically promote neurite growth in vitro. J Neuroinflamm 8:183. Bradbury EJ, Khemani S, Von R, King, Priestley JV, McMahon SB. 1999. NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord. Eur J Neurosci 11:387323883. Dinarello CA. 1994. The interleukin-1 family: 10 years of discovery. FASEB J 8:131421325. Gibson RM, Rothwell NJ, Le Feuvre RA. 2004. CNS injury: the role of the cytokine IL-1. Vet J 168:2302237. Grasselli G, Mandolesi G, Strata P, Cesare P. 2011. Impaired sprouting and axonal atrophy in cerebellar climbing fibres following in vivo silencing of the growth-associated protein GAP-43. PLoS One 6: e20791. Greenhalgh AD, Brough D, Robinson EM, Girard S, Rothwell NJ, Allan SM. 2012. Interleukin-1 receptor antagonist is beneficial after subarachnoid haemorrhage in rat by blocking haem-driven inflammatory pathology. Dis Model Mech 5:8232833. Hailer NP. 2008. Immunosuppression after traumatic or ischemic CNS damage: it is neuroprotective and illuminates the role of microglial cells. Prog Neurobiol 84:2112233. Hailer NP, Grampp A, Nitsch R. 1999. Proliferation of microglia and astrocytes in the dentate gyrus following entorhinal cortex lesion: a quantitative bromodeoxyuridine-labelling study. Eur J Neurosci 11: 335923364. Hailer NP, Vogt C, Korf HW, Dehghani F. 2005. Interleukin-1beta exacerbates and interleukin-1 receptor antagonist attenuates neuronal injury and microglial activation after excitotoxic damage in organotypic hippocampal slice cultures. Eur J Neurosci 21:234722360. Heppner FL, Roth K, Nitsch R, Hailer NP. 1998. Vitamin E induces ramification and downregulation of adhesion molecules in cultured microglial cells. Glia 22:1802188. Journal of Neuroscience Research
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