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Overexpression of kynurenic acid in stroke: An endogenous neuroprotector?
Annals of Anatomy 211 (2017) 33–38
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RESEARCH ARTICLE
Overexpression of kynurenic acid in stroke: An endogenous neuroprotector? ˜ c A. Mangas a,b,c,∗ , J. Yajeya d , N. González a,b , I. Ruiz a , S. Duleu b , M. Geffard a,b , R. Covenas a
Gemacbio, Saint Jean d’Illac, France Institut pour le Développement de la Recherche en Pathologie Humaine et Thérapeutique (IDRPHT), Talence, France Institute of Neurosciences of Castilla y León (INCYL), Laboratory of Neuroanatomy of the Peptidergic Systems, University of Salamanca, Salamanca, Spain d School of Medicine, Department of Physiology, University of Salamanca, Salamanca, Spain b c
a r t i c l e
i n f o
Article history: Received 27 July 2016 Received in revised form 8 November 2016 Accepted 4 January 2017 Keywords: Astrocyte Ischemia Glia Monoclonal antibody Neuroprotection Immunohistochemistry
a b s t r a c t It is known that kynurenic acid (KYNA) exerts a neuroprotective effect against the neuronal loss induced by ischemia; acting as a scavenger, and exerting antioxidant action. In order to study the distribution of KYNA, a highly specific monoclonal antibody directed against KYNA was developed. This distribution was studied in control rats and in animals in which a middle cerebral artery occlusion (stroke model) was induced. By double immunohistochemistry, astrocytes containing KYNA and GFAP were exclusively found in the ipsilateral cerebral cortex and/or striatum, at 2, 5 and 21 days after the induction of stroke. In control animals and in the contralateral side of the stroke animals, no immunoreactivity for KYNA was found. Under pathological conditions, the presence of KYNA is reported for the first time in the mammalian brain from early phases of stroke. The distribution of KYNA matches perfectly with the infarcted regions suggesting that, in stroke, this overexpressed molecule could be involved in neuroprotective/scavenger/antioxidant mechanisms. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction Stroke is a major health problem. The Framingham Study reported that 1 of 5 women and 1 of 6 men, aged from 55 to 75 years, will experience stroke at some time during their lives. In stroke, several phases have been described (Tatro, 2006). The first is called excitotoxicity, in which a high release of glutamate occurs as well as the generation of reactive oxygen species (ROS), whereas the second phase corresponds to inflammatory activation and apoptosis. The latter phase is characterized by the activation of cytokines, caspases and both indoleamine 2,3-dioxygenase (IDO) and nitric oxide (NO) pathways. In the IDO pathway, tryptophan is catabolised, leading to the generation of nicotinamide adenine dinucleotide (NAD) and several intermediate metabolites (e.g., kynurenic acid (KYNA)) are also generated. Thus, KYNA is an intermediate molecule of the catabolism of tryptophan and it is mainly synthesized by astrocytes (Guillemin et al., 2001; López et al., 2016).
∗ Corresponding author at: University of Salamanca, Institute of Neurosciences of Castilla y León (INCYL), Laboratory of Neuroanatomy of the Peptidergic Systems (Lab. 14), c/Pintor Fernando Gallego, 1, 37007 Salamanca, Spain. Fax: +34 923294549. E-mail address: [email protected] (A. Mangas). http://dx.doi.org/10.1016/j.aanat.2017.01.002 0940-9602/© 2017 Elsevier GmbH. All rights reserved.
It is known that KYNA exerts a neuroprotective effect against the neuronal loss induced by ischemia (Gellért et al., 2011); that helps the scavenger activity of astrocytes, and that exerts an antioxidant activity (Lugo-Huitrón et al., 2011). Due to the beneficial mentioned roles, it is important to study the distribution and/or function of KYNA in several pathologies (e.g., stroke). In order to carry out this aim, a highly specific monoclonal antibody directed against KYNA was firstly developed. Later, and in order to study the neuroanatomical expression of KYNA in the rat central nervous system, an immunohistochemical study was carried out in normal and operated animals. In this study, we sought to investigate the alterations in KYNA expression in a rat model of stroke. 2. Material and methods 2.1. Single transient middle cerebral artery occlusion (tMCAO) The experimental procedure of this work was performed under the guidelines of the ethics and legal recommendations of Spanish, French and European laws. This study was approved by the research commission of the University of Salamanca (Spain). Eighteen adult male Wistar rats (control: 6 animals; tMCAO procedure: 12 animals) weighing 350 gr were used. Animals were deeply anesthetized (with isoflurane (4% induction; 2.5% maintenance)) by
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means of a facial mask. As previously described (Longa et al., 1989; Qiao et al., 2009; Uluc¸ et al., 2011), tMCAO surgical procedures were carried out. Thus, a middle cerebral artery occlusion (50–55 min) was conducted by using a rounded tip monofilament (Doccol, USA). As previously described (Uluc¸ et al., 2011) and before the recovery from anesthesia, the analgesic buprenorphine (0.05 mg/kg) was subcutaneously administered to animals. Once the tMCAO experimental model was performed, animals were divided into three groups: 2D, 5D and 21D. Animals belonging to the 2D group were perfused two days after the middle cerebral artery occlusion; those belonging to the 5D group, five days after the occlusion, and those of the third group (21D), twenty-one days after the surgical procedure. In each experimental group, 6 animals were used: 2 control rats and 4 tMCAO animals. 2.2. Antibody development As previously described (Mangas et al., 2012), a primary antiserum was developed in BALB/c mice after immunization with KYNA-bovine serum albumin (BSA) immunogen linked via ethylchloroformiate (ECF). Briefly, 10 mg of KYNA were dissolved in methanol; later, 20 mg of bovine serum albumin (BSA) were dissolved in water, and 40 l of triethylamine were added to both solutions. Then, the activation of the KYNA solution was carried out by adding an extemporaneous solution containing 375 l of dimethylformamide mixed with 25 l of ECF. After 10 min of activation, the solution containing KYNA was added drop by drop into a tube containing the BSA solution. Using dialysis membranes with cut-off limits between 12 and 16 KDa, the obtained conjugate (KYNA-BSA) was purified by dialysis. The purification was performed in one litre bucket at 4 ◦ C during 36 h, changing the bath every 2–3 h (Mangas et al., 2012). After the synthesis of KYNA-BSA, mice were immediately immunized by one injection every 2–3 weeks with the immunogen (containing KYNA-BSA). Each immunization was carried out by administering 50 l of an immunogenic NaCl solution and 50 l of complete (only used in the first immunization) or incomplete Freund adjuvant. After the second immunization, serum samples were collected and the antisera were pre-purified by immunoabsorption and tested by ELISA as previously described (Mangas et al., 2007, 2008, 2012). Once a highly specific polyclonal antibody was obtained, the fusion of SP2/O/Ag myeloma cells and mice splenocytes was carried out. Then, the screening and the selection of specific clones were performed. Once the highly specific monoclonal antibody against KYNA was obtained, cells were expanded in plastic flasks. Supernatant was collected every week, centrifuged and pre-purified with a saturated (NH4 )2 SO4 solution, dialyzed in PBS and finally purified in an HiTrap protein G HP column (170404-01, GE Healthcare). An Isotyping kit was used to determinate the type of immunoglobulin and chain (26179, ThermoScientific): the anti-KYNA antibody was characterized as an isotype Ig G1 and chain. The affinity estimated of the monoclonal anti-KYNA antibody was 10−10 M and its specificity was considered excellent because close molecules were not recognized by the antibody (Table 1). 2.3. Immunohistochemical study Once the tMCAO experimental model was performed (2, 5 or 21 days), the immunocytochemical study was conducted. As previously reported (Mangas et al., 2007, 2012), animals were anaesthetized and perfused and the brains were dissected out, post-fixed and cryoprotected for histological studies. Using a freezing microtome, 40–50 -thick brain sections were obtained and processed for immunohistochemistry. In order to avoid possible interference by endogenous peroxidase, sections were treated with methanol and H2 O2 . Later, sections were washed in PBS and
pre-incubated in PBS containing Triton X-100 and normal horse serum (mix solution). Sections were incubated overnight at 4 ◦ C in the mix solution containing the monoclonal anti-KYNA antibody (diluted 1/1000, Gemacbio), the monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (1/400, Abcam), the polyclonal rabbit anti-GFAP antibody (1/100, Dako) or the polyclonal goat anti-ionized calcium-binding adapter molecule 1 (IBA-1) antibody (1/1500, Abcam). Later, sections were washed in PBS and incubated with biotinylated anti-mouse/rabbit/goat immunogammaglobulin, diluted 1/200 in the mix solution. After a rinse in PBS, sections were incubated with the avidin-biotin-peroxidase complex (ABC) (1/100). Sections were washed in PBS and in Tris–HCl buffer and then the tissue-bound peroxidase was developed with H2 O2 , using 3,3 diaminobenzidine (DAB) as chromogen. Histological controls were carried out to confirm the specificity of the immunoreactivity: (1) omission of the primary and/or secondary antibodies; and (2) pre-absorption of the anti-KYNA antibody with an excess (100 g/ml) of KYNA. No residual immunoreactivity was found in either case. Sections, in which KYNA was detected by the DAB developing procedure, were prepared for double-labelling immunohistochemistry (GFAP: 1/100, Dako) according to a previous published protocol (Marcos et al., 2013). The product of the second immunohistochemical reaction was revealed using 4-chloro-1-naphol as the chromogen. This substance provides a blue precipitate (GFAP) easily distinguishable from the brown product (KYNA) of DAB (Marcos et al., 2013). The stereotaxic atlas of Paxinos and Watson (1982) was used for mapping and nomenclature. Photomicrographs were obtained with an Olympus DP50 digital camera attached to a Kyowa Unilux12 microscope. Using a Leica DMRB photomicroscope/Neurolucida system (8.0; Microbrightfield-bioscience, USA) photographs at low-power magnification were also obtained. Adobe Photoshop CS software was used: to improve the visualization of results, only the brightness and contrast of the images were adjusted. 3. Results Using ELISA tests, the monoclonal anti-KYNA antibody obtained was fully characterized (Table 1). This antibody showed a rather high affinity (the estimated IC50 was 10−10 M) and an excellent specificity. In these assays, the parameters studied were antibody titration, avidity and specificity versus other close structural analogues (Table 1), according to previously described protocols (Mangas et al., 2007, 2012). ELISA tests were carried out at a dilution of 1/30,000, corresponding to an optical density of 1, at 492 nm (Mangas et al., 2007). Accordingly, competition experiments were performed with different competitors (Table 1). All competition experiments (dilution: 1/30,000) were carried out with the same
Table 1 Affinity and specificity of antibodies against conjugated KYNA. Compounds
Cross-reactivity at half-displacement (IC50 )
Kynurenic acid-BSA l-Kynurenine-BSA Quinolinic acid-BSA Picolinic acid-BSA Xanthurenic acid-BSA 3-Hydroxy-anthranilic acid-BSA l-Phenylalanine-BSA l-Tryptophan-BSA
1 1/>50,000 1/>50,000 1/>50,000 1/>50,000 1/>50,000 1/>50,000 1/>50,000
Using competition ELISA tests, cross-reactivity was calculated from the displacement curves at half-displacement: the best recognized conjugate was Kynurenic acid-BSA, whose concentration was divided by the concentration of each of the other conjugates.
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Fig. 1. (A, C, E) Low-power magnification (21 days). Photographs were taken using a Leica microscope/neurolucida software. Note the overexpression of IBA-1, GFAP and KYNA in the infarcted region (arrows). B, D, F (ipsilateral side; cerebral cortex: 21 days). Infarcted region stained with IBA-1, a pan-microglia marker (B), GFAP (D) or KYNA (F) antibodies. In F, the border of the infarcted region is clearly observed. L: lateral; V: ventral.
amount of antibody, but using different concentrations of the conjugated antigens (Table 1). The monoclonal antibody used here recognized the specific target at very low concentrations and this means that its avidity was very high for the target (Mangas et al., 2004, 2007, 2008). In the performed assay, we tested the closest available analogues of KYNA and we observed a very low crossreactivity between the target molecule and the closest compounds (Table 1). In control animals and after the application of the immunohistochemical technique, no immunoreactivity was found for KYNA. However, as expected, in these animals IBA-1 was located in the microglia throughout the whole brain. Moreover, in control animals, the presence of astrocytes was observed in the whole brain when the immunohistochemical technique for GFAP was carried out. In the tMCAO model, a middle cerebral artery occlusion is carried out and then, after using immunocytochemical techniques, the ipsilateral and contralateral sides of the brain can be compared (Fig. 1A, C, E). In our study, the contralateral side of the brain of the animals belonging to the three experimental groups (2D, 5D, 21D) showed the same immunoreactive distribution pattern for KYNA, GFAP and IBA-1 as that observed in control animals. How-
ever, on the ipsilateral side (that is, in the infarcted region) there was an overexpression of KYNA, GFAP and IBA-1 in comparison to that observed in the contralateral side; these findings were identical in the three experimental groups (2D, 5D, 21D) (Fig. 1A-F). That is, in the whole brain KYNA was only observed in the ipsilateral infarcted region (Figs. 1E, F; 2E, F). It is important to note that the infarcted region was found in the ipsilateral striatum, in the ipsilateral cerebral cortex or in both ipsilateral striatum and cerebral cortex (Fig. 1A, C, E). In sum, KYNA was exclusively observed in the ipsilateral side of those animals in which a middle cerebral artery occlusion was carried out independently of the time after the stroke (2, 5 or 21 days); this immunoreactivity was exclusively found in the ipsilateral infarcted regions. Moreover, in these regions a perfect match (overexpression) of KYNA, GFAP and IBA-1 was observed in the three experimental groups (2D, 5D, 21D) (Figs. 1A–F; 2A–F). KYNA and GFAP were observed in astrocytes (cells containing KYNA or GFAP showed the same morphological characteristics) (Fig. 2C–F), whereas IBA-1 was found in microglia (Fig. 2A, B). In order to confirm the coexistence of KYNA and GFAP in astrocytes, a double-labelling immunohistochemistry technique was carried out. GFAP-immunoreactivity was observed in those astrocytes
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Fig. 2. Infarcted region (ipsilateral side; cerebral cortex: 21 days) stained with anti-IBA-1 (A, B), anti-GFAP (C, D) or anti-KYNA (E, F). IBA-1 was observed in microglia (A, B) and GFAP or KYNA in astrocytes (C–F). B. Inset at top right: a higher magnification of the region delimited by the rectangle. E. Inset at top right: a higher magnification of the region delimited by the rectangle. F. Inset at top left: a higher magnification of the region delimited by the rectangle. Arrows indicate blood vessels.
expressing KYNA (Fig. 3A–D). In sum, in the three experimental groups studied here, an overexpression of KYNA, GFAP and IBA-1 was observed in the ipsilateral side (infarcted region) compared with that found in the contralateral side. 4. Discussion This is the first study in which the presence of astrocytes containing KYNA has been reported under pathological conditions in the mammalian brain. The most important findings are that, in stroke, the expression of KYNA occurs from early stages and that this expression is exclusively found in the infarcted regions. Thus, there is a perfect anatomical relationship between the infarcted regions and the expression of KYNA. Regarding the development of the monoclonal anti-KYNA antibody, it is very important to take the chemical structure of the target into consideration. In this sense, the choice of the coupling agent (e.g., ECF, glutaraldehyde) is crucial in order to maintain the original chemical structure and conformation of the antigen, ensuring that the antibody specifically recognises the required target (e.g., KYNA) (Mangas et al., 2008, 2012). Thus, KYNA was linked to the protein carrier (BSA) by different chemical procedures (using
different coupling agents), in order to confirm that the chemical procedure followed did not affect the original structure of KYNA. In all cases, using a competition ELISA test, KYNA was specifically recognized by the antibody used in this study. Thus, it was concluded that different antigens (KYNA linked to BSA by different chemical procedures) were equally recognized by the antibody. This procedure is very important for the specific recognition of KYNA in the tissue, after the application of the immunohistochemical technique, and in order to exclude spurious signals (background). In sum, we have developed a monoclonal anti-KYNA antibody showing a high affinity (10−10 M) and specificity (the antibody does not recognize closest competitors). In addition, the histological controls carried out confirmed the specificity of the immunoreactivity observed. However, it seems that in the infarcted region, an overexpression of several molecules occurs and hence the specificity of the immunoreactivity for KYNA should be assessed by other experiments. The specificity of the immunoreactivity is never absolute and different methods must be tested in order to confirm it. In the tMCAO model, it is known that there is a considerable variability in the extension of the infarcted region (Lin et al., 2013). This is in agreement with our observations, since the infarcted regions were observed in the striatum and/or cerebral cortex. Astrocytes
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Fig. 3. (A–D) Infarcted region (ipsilateral side; cerebral cortex: 21 days). Double-labelling immunohistochemistry. In astrocytes, the coexistence of KYNA (brown labelling) and GFAP (blue labelling) (arrowheads) is observed.
containing KYNA were strictly observed in the infarcted regions. Thus, the regions showing KYNA-immunoreactive structures were variable in tMCAO animals. The different distribution pattern of the immunoreactivity (KYNA, GFAP, IBA-1) observed in the ipsilateral (overexpression) and contralateral sides of tMCAO animals demonstrated that the surgical procedures were correctly conducted. Thus, the infarcted area was recognized by the overexpression of IBA-1, GFAP and KYNA. For these markers, a perfect match occurs in the infarcted region. By double-labelling immunohistochemistry, we have demonstrated the coexistence of KYNA and GFAP in astrocytes. GFAP was revealed using 4-chloro-1-napthol and KYNA using DAB (Marcos et al., 2013). The blue precipitate due to the first chromogen is easily distinguishable from the brown product due to DAB, which enables a second immunohistochemical reaction on the same section without antibody removal. The DAB reaction product masks the antigen and catalytic sites of the first sequence of immunoreagents, preventing an interaction of the second sequence with the reagents (Marcos et al., 1997). It is important to note that two different anti-GFAP antibodies were used; in both cases, astrocytes showed an identical distribution. Both antibodies showed an overexpression of GFAP in the ipsilateral side of tMCAO animals. Moreover, it is important to note that KYNA was only found in those regions (striatum and/or cerebral cortex) in which an overexpression of GFAP and IBA-1 was observed (ipsilateral side). Finally, tMCAO animals were studied at different post-stroke phases: 2, 5 and 21 days. In the three experimental groups, KYNAimmunoreactive structures showed the same distribution pattern. This means that, in stroke, KYNA was expressed from early stages and that this expression continued 21 days post-stroke. In the tMCAO model, the temporal profile of the expression of IBA-1 has been previously reported (Ito et al., 2001). Fourteen days after the tMCAO, these authors found necrosis in the ischemic region and a cavity was observed; however, cells containing IBA-1 were visualized. Moreover, they found the higher immunoreactivity for IBA-1 on day 7 after the tMCAO (Ito et al., 2001). Our data are partially in agreement with these previous results (Ito et al., 2001).
Thus, we observed an overexpression of IBA-1 in the infarcted area, but the degree of the immunoreactivity was similar 2, 5 or 21 days after the tMCAO. This discrepancy could be due to the time of the transient ischemia: 90 min in the study of Ito et al. (2001) and 50–55 min in our study. It is clear that the tissue damage was higher in the former study, but it is important to note that 14 days after the tMCAO, cells containing IBA-1 were still observed (Ito et al., 2001). In our study, in the infarcted region, a higher number of IBA1-immunoreactive cells were observed because the tissue damage was lower following ischemic conditions. In the IDO pathway, the essential amino acid tryptophan is catabolised, several intermediate metabolites (e.g., KYNA, kynurenine, quinolinic acid, 3-OH-anthranilic) are generated, and at the end of route NAD is also generated. It is known that this pathway also regulates the immune response (Moffett and Namboodiri, 2003) and that it is altered in several pathologies (e.g., stroke, Alzheimer, amyotrophic lateral sclerosis, depression, Huntington, multiple sclerosis, Parkinson, schizophrenia) (Gold et al., 2011; Bohár et al., 2015; Lim et al., 2016; Lovelace et al., 2017). In stroke, using HPLC, it has been reported a relationship between the kynurenine/tryptophan balance (IDO pathway) and the post-stroke cognitive impairment (Gold et al., 2011). In astrocytes, KYNA is synthesized by kynurenine aminotransferases (KAT) I, II and III and hence it is important to know the activators/inhibitors of those enzymes (Baran et al., 2010). For example, it is known that the glia depressing factor controls the levels of both KAT and KYNA, as well as regulates the proliferation of glial cells (Baran et al., 2010). Metabolites of the IDO pathway exert opposite physiological effects. KYNA has a neuroprotective action, whereas quinolinic (mainly synthesized in microglia) and picolinic acids exert a neurotoxic effect (Guillemin et al., 2004, 2007; Lim et al., 2016). Thus, it seems that the overexpression of KYNA, exclusively observed here in the infarcted regions, could be the response of astrocytes against the ischemic damage induced in the tMCAO model. Thus, KYNA, acting as a neuropro-
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tective/scavenger/antioxidant factor, could counteract the tissue damage produced after stroke. In sum, in a tMCAO stroke model, (i) KYNA was visualized for the first time in the mammalian astrocytes; (ii) KYNA was exclusively observed in the infarcted regions; (iii) an overexpression of KYNA occurred from early stages; (iv) there was a perfect anatomical relationship between the expression of KYNA and the infarcted region, and (v) KYNA could play a neuroprotective/scavenger/antioxidant role. Acknowledgements This work has been supported by the European Union FP7 Collaborative Grant TargetBraIn (number 279017), Gemacbio S.A. Laboratories (Saint Jean d’Illac, France), and by the IDRPHT (Talence, France). We wish to thank Professors Miguel Merchán, Margarita Heredia and Adelaida Riolobos (INCYL, Salamanca, Spain) for technical facilities and Miss Marianny Pernìa for technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aanat.2017.01. 002. References Baran, H., Kepplinger, B., Draxler, M., 2010. Endogenous kynurenine aminotransferases inhibitor is proposed to act as glia depressing factor (GDF). Int. J. Tryptophan Res. 3, 13–22. Bohár, Z., Toldi, J., Fülöp, F., Vécsei, L., 2015. Changing the face of kynurenines and neurotoxicity: therapeutic considerations. Int. J. Mol. Sci. 16, 9772–9793. Gellért, L., Fuzik, J., Göblös, A., Sárközi, K., Marosi, M., Kis, Z., Farkas, T., Szatmári, I., Fülöp, F., Vécsei, L., Toldi, J., 2011. Neuroprotection with a new kynurenic acid analog in the four-vessel occlusion model of ischemia. Eur. J. Pharmacol. 667, 182–187. Gold, A.B., Herrmann, N., Swardfager, W., Black, S.E., Aviv, R.I., Tennen, G., Kiss, A., Lanctôt, K.L., 2011. The relationship between indoleamine 2,3-dioxygenase activity and post-stroke cognitive impairment. J. Neuroinflamm. 8, 17. Guillemin, G.J., Kerr, S.J., Smythe, G.A., Smith, D.G., Kapoor, V., Armati, P.J., Croitoru, J., Brew, B.J., 2001. Kynurenine pathway metabolism in human astrocytes: a paradox for neuronal protection. J. Neurochem. 78, 842–853. Guillemin, G.J., Smythe, G., Takikawa, O., Brew, B.J., 2004. Expression of indoleamine 2, 3-dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons. Glia 49, 15–23. Guillemin, G.J., Cullen, K.M., Lim, C.K., Smythe, G.A., Garner, B., Kapoor, V., Takikawa, O., Brew, B.J., 2007. Characterization of the kynurenine pathway in human neurons. J. Neurosci. 27, 12884–12892. Ito, D., Tanaka, K., Suzuki, S., Dembo, T., Fukuuchi, Y., 2001. Enhanced expression of Iba1, ionized calcium-binding adapter molecule 1, after transient focal cerebral ischemia in rat brain. Stroke 32, 1208–1215.
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RESEARCH ARTICLE
Overexpression of kynurenic acid in stroke: An endogenous neuroprotector? ˜ c A. Mangas a,b,c,∗ , J. Yajeya d , N. González a,b , I. Ruiz a , S. Duleu b , M. Geffard a,b , R. Covenas a
Gemacbio, Saint Jean d’Illac, France Institut pour le Développement de la Recherche en Pathologie Humaine et Thérapeutique (IDRPHT), Talence, France Institute of Neurosciences of Castilla y León (INCYL), Laboratory of Neuroanatomy of the Peptidergic Systems, University of Salamanca, Salamanca, Spain d School of Medicine, Department of Physiology, University of Salamanca, Salamanca, Spain b c
a r t i c l e
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Article history: Received 27 July 2016 Received in revised form 8 November 2016 Accepted 4 January 2017 Keywords: Astrocyte Ischemia Glia Monoclonal antibody Neuroprotection Immunohistochemistry
a b s t r a c t It is known that kynurenic acid (KYNA) exerts a neuroprotective effect against the neuronal loss induced by ischemia; acting as a scavenger, and exerting antioxidant action. In order to study the distribution of KYNA, a highly specific monoclonal antibody directed against KYNA was developed. This distribution was studied in control rats and in animals in which a middle cerebral artery occlusion (stroke model) was induced. By double immunohistochemistry, astrocytes containing KYNA and GFAP were exclusively found in the ipsilateral cerebral cortex and/or striatum, at 2, 5 and 21 days after the induction of stroke. In control animals and in the contralateral side of the stroke animals, no immunoreactivity for KYNA was found. Under pathological conditions, the presence of KYNA is reported for the first time in the mammalian brain from early phases of stroke. The distribution of KYNA matches perfectly with the infarcted regions suggesting that, in stroke, this overexpressed molecule could be involved in neuroprotective/scavenger/antioxidant mechanisms. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction Stroke is a major health problem. The Framingham Study reported that 1 of 5 women and 1 of 6 men, aged from 55 to 75 years, will experience stroke at some time during their lives. In stroke, several phases have been described (Tatro, 2006). The first is called excitotoxicity, in which a high release of glutamate occurs as well as the generation of reactive oxygen species (ROS), whereas the second phase corresponds to inflammatory activation and apoptosis. The latter phase is characterized by the activation of cytokines, caspases and both indoleamine 2,3-dioxygenase (IDO) and nitric oxide (NO) pathways. In the IDO pathway, tryptophan is catabolised, leading to the generation of nicotinamide adenine dinucleotide (NAD) and several intermediate metabolites (e.g., kynurenic acid (KYNA)) are also generated. Thus, KYNA is an intermediate molecule of the catabolism of tryptophan and it is mainly synthesized by astrocytes (Guillemin et al., 2001; López et al., 2016).
∗ Corresponding author at: University of Salamanca, Institute of Neurosciences of Castilla y León (INCYL), Laboratory of Neuroanatomy of the Peptidergic Systems (Lab. 14), c/Pintor Fernando Gallego, 1, 37007 Salamanca, Spain. Fax: +34 923294549. E-mail address: [email protected] (A. Mangas). http://dx.doi.org/10.1016/j.aanat.2017.01.002 0940-9602/© 2017 Elsevier GmbH. All rights reserved.
It is known that KYNA exerts a neuroprotective effect against the neuronal loss induced by ischemia (Gellért et al., 2011); that helps the scavenger activity of astrocytes, and that exerts an antioxidant activity (Lugo-Huitrón et al., 2011). Due to the beneficial mentioned roles, it is important to study the distribution and/or function of KYNA in several pathologies (e.g., stroke). In order to carry out this aim, a highly specific monoclonal antibody directed against KYNA was firstly developed. Later, and in order to study the neuroanatomical expression of KYNA in the rat central nervous system, an immunohistochemical study was carried out in normal and operated animals. In this study, we sought to investigate the alterations in KYNA expression in a rat model of stroke. 2. Material and methods 2.1. Single transient middle cerebral artery occlusion (tMCAO) The experimental procedure of this work was performed under the guidelines of the ethics and legal recommendations of Spanish, French and European laws. This study was approved by the research commission of the University of Salamanca (Spain). Eighteen adult male Wistar rats (control: 6 animals; tMCAO procedure: 12 animals) weighing 350 gr were used. Animals were deeply anesthetized (with isoflurane (4% induction; 2.5% maintenance)) by
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means of a facial mask. As previously described (Longa et al., 1989; Qiao et al., 2009; Uluc¸ et al., 2011), tMCAO surgical procedures were carried out. Thus, a middle cerebral artery occlusion (50–55 min) was conducted by using a rounded tip monofilament (Doccol, USA). As previously described (Uluc¸ et al., 2011) and before the recovery from anesthesia, the analgesic buprenorphine (0.05 mg/kg) was subcutaneously administered to animals. Once the tMCAO experimental model was performed, animals were divided into three groups: 2D, 5D and 21D. Animals belonging to the 2D group were perfused two days after the middle cerebral artery occlusion; those belonging to the 5D group, five days after the occlusion, and those of the third group (21D), twenty-one days after the surgical procedure. In each experimental group, 6 animals were used: 2 control rats and 4 tMCAO animals. 2.2. Antibody development As previously described (Mangas et al., 2012), a primary antiserum was developed in BALB/c mice after immunization with KYNA-bovine serum albumin (BSA) immunogen linked via ethylchloroformiate (ECF). Briefly, 10 mg of KYNA were dissolved in methanol; later, 20 mg of bovine serum albumin (BSA) were dissolved in water, and 40 l of triethylamine were added to both solutions. Then, the activation of the KYNA solution was carried out by adding an extemporaneous solution containing 375 l of dimethylformamide mixed with 25 l of ECF. After 10 min of activation, the solution containing KYNA was added drop by drop into a tube containing the BSA solution. Using dialysis membranes with cut-off limits between 12 and 16 KDa, the obtained conjugate (KYNA-BSA) was purified by dialysis. The purification was performed in one litre bucket at 4 ◦ C during 36 h, changing the bath every 2–3 h (Mangas et al., 2012). After the synthesis of KYNA-BSA, mice were immediately immunized by one injection every 2–3 weeks with the immunogen (containing KYNA-BSA). Each immunization was carried out by administering 50 l of an immunogenic NaCl solution and 50 l of complete (only used in the first immunization) or incomplete Freund adjuvant. After the second immunization, serum samples were collected and the antisera were pre-purified by immunoabsorption and tested by ELISA as previously described (Mangas et al., 2007, 2008, 2012). Once a highly specific polyclonal antibody was obtained, the fusion of SP2/O/Ag myeloma cells and mice splenocytes was carried out. Then, the screening and the selection of specific clones were performed. Once the highly specific monoclonal antibody against KYNA was obtained, cells were expanded in plastic flasks. Supernatant was collected every week, centrifuged and pre-purified with a saturated (NH4 )2 SO4 solution, dialyzed in PBS and finally purified in an HiTrap protein G HP column (170404-01, GE Healthcare). An Isotyping kit was used to determinate the type of immunoglobulin and chain (26179, ThermoScientific): the anti-KYNA antibody was characterized as an isotype Ig G1 and chain. The affinity estimated of the monoclonal anti-KYNA antibody was 10−10 M and its specificity was considered excellent because close molecules were not recognized by the antibody (Table 1). 2.3. Immunohistochemical study Once the tMCAO experimental model was performed (2, 5 or 21 days), the immunocytochemical study was conducted. As previously reported (Mangas et al., 2007, 2012), animals were anaesthetized and perfused and the brains were dissected out, post-fixed and cryoprotected for histological studies. Using a freezing microtome, 40–50 -thick brain sections were obtained and processed for immunohistochemistry. In order to avoid possible interference by endogenous peroxidase, sections were treated with methanol and H2 O2 . Later, sections were washed in PBS and
pre-incubated in PBS containing Triton X-100 and normal horse serum (mix solution). Sections were incubated overnight at 4 ◦ C in the mix solution containing the monoclonal anti-KYNA antibody (diluted 1/1000, Gemacbio), the monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (1/400, Abcam), the polyclonal rabbit anti-GFAP antibody (1/100, Dako) or the polyclonal goat anti-ionized calcium-binding adapter molecule 1 (IBA-1) antibody (1/1500, Abcam). Later, sections were washed in PBS and incubated with biotinylated anti-mouse/rabbit/goat immunogammaglobulin, diluted 1/200 in the mix solution. After a rinse in PBS, sections were incubated with the avidin-biotin-peroxidase complex (ABC) (1/100). Sections were washed in PBS and in Tris–HCl buffer and then the tissue-bound peroxidase was developed with H2 O2 , using 3,3 diaminobenzidine (DAB) as chromogen. Histological controls were carried out to confirm the specificity of the immunoreactivity: (1) omission of the primary and/or secondary antibodies; and (2) pre-absorption of the anti-KYNA antibody with an excess (100 g/ml) of KYNA. No residual immunoreactivity was found in either case. Sections, in which KYNA was detected by the DAB developing procedure, were prepared for double-labelling immunohistochemistry (GFAP: 1/100, Dako) according to a previous published protocol (Marcos et al., 2013). The product of the second immunohistochemical reaction was revealed using 4-chloro-1-naphol as the chromogen. This substance provides a blue precipitate (GFAP) easily distinguishable from the brown product (KYNA) of DAB (Marcos et al., 2013). The stereotaxic atlas of Paxinos and Watson (1982) was used for mapping and nomenclature. Photomicrographs were obtained with an Olympus DP50 digital camera attached to a Kyowa Unilux12 microscope. Using a Leica DMRB photomicroscope/Neurolucida system (8.0; Microbrightfield-bioscience, USA) photographs at low-power magnification were also obtained. Adobe Photoshop CS software was used: to improve the visualization of results, only the brightness and contrast of the images were adjusted. 3. Results Using ELISA tests, the monoclonal anti-KYNA antibody obtained was fully characterized (Table 1). This antibody showed a rather high affinity (the estimated IC50 was 10−10 M) and an excellent specificity. In these assays, the parameters studied were antibody titration, avidity and specificity versus other close structural analogues (Table 1), according to previously described protocols (Mangas et al., 2007, 2012). ELISA tests were carried out at a dilution of 1/30,000, corresponding to an optical density of 1, at 492 nm (Mangas et al., 2007). Accordingly, competition experiments were performed with different competitors (Table 1). All competition experiments (dilution: 1/30,000) were carried out with the same
Table 1 Affinity and specificity of antibodies against conjugated KYNA. Compounds
Cross-reactivity at half-displacement (IC50 )
Kynurenic acid-BSA l-Kynurenine-BSA Quinolinic acid-BSA Picolinic acid-BSA Xanthurenic acid-BSA 3-Hydroxy-anthranilic acid-BSA l-Phenylalanine-BSA l-Tryptophan-BSA
1 1/>50,000 1/>50,000 1/>50,000 1/>50,000 1/>50,000 1/>50,000 1/>50,000
Using competition ELISA tests, cross-reactivity was calculated from the displacement curves at half-displacement: the best recognized conjugate was Kynurenic acid-BSA, whose concentration was divided by the concentration of each of the other conjugates.
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Fig. 1. (A, C, E) Low-power magnification (21 days). Photographs were taken using a Leica microscope/neurolucida software. Note the overexpression of IBA-1, GFAP and KYNA in the infarcted region (arrows). B, D, F (ipsilateral side; cerebral cortex: 21 days). Infarcted region stained with IBA-1, a pan-microglia marker (B), GFAP (D) or KYNA (F) antibodies. In F, the border of the infarcted region is clearly observed. L: lateral; V: ventral.
amount of antibody, but using different concentrations of the conjugated antigens (Table 1). The monoclonal antibody used here recognized the specific target at very low concentrations and this means that its avidity was very high for the target (Mangas et al., 2004, 2007, 2008). In the performed assay, we tested the closest available analogues of KYNA and we observed a very low crossreactivity between the target molecule and the closest compounds (Table 1). In control animals and after the application of the immunohistochemical technique, no immunoreactivity was found for KYNA. However, as expected, in these animals IBA-1 was located in the microglia throughout the whole brain. Moreover, in control animals, the presence of astrocytes was observed in the whole brain when the immunohistochemical technique for GFAP was carried out. In the tMCAO model, a middle cerebral artery occlusion is carried out and then, after using immunocytochemical techniques, the ipsilateral and contralateral sides of the brain can be compared (Fig. 1A, C, E). In our study, the contralateral side of the brain of the animals belonging to the three experimental groups (2D, 5D, 21D) showed the same immunoreactive distribution pattern for KYNA, GFAP and IBA-1 as that observed in control animals. How-
ever, on the ipsilateral side (that is, in the infarcted region) there was an overexpression of KYNA, GFAP and IBA-1 in comparison to that observed in the contralateral side; these findings were identical in the three experimental groups (2D, 5D, 21D) (Fig. 1A-F). That is, in the whole brain KYNA was only observed in the ipsilateral infarcted region (Figs. 1E, F; 2E, F). It is important to note that the infarcted region was found in the ipsilateral striatum, in the ipsilateral cerebral cortex or in both ipsilateral striatum and cerebral cortex (Fig. 1A, C, E). In sum, KYNA was exclusively observed in the ipsilateral side of those animals in which a middle cerebral artery occlusion was carried out independently of the time after the stroke (2, 5 or 21 days); this immunoreactivity was exclusively found in the ipsilateral infarcted regions. Moreover, in these regions a perfect match (overexpression) of KYNA, GFAP and IBA-1 was observed in the three experimental groups (2D, 5D, 21D) (Figs. 1A–F; 2A–F). KYNA and GFAP were observed in astrocytes (cells containing KYNA or GFAP showed the same morphological characteristics) (Fig. 2C–F), whereas IBA-1 was found in microglia (Fig. 2A, B). In order to confirm the coexistence of KYNA and GFAP in astrocytes, a double-labelling immunohistochemistry technique was carried out. GFAP-immunoreactivity was observed in those astrocytes
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Fig. 2. Infarcted region (ipsilateral side; cerebral cortex: 21 days) stained with anti-IBA-1 (A, B), anti-GFAP (C, D) or anti-KYNA (E, F). IBA-1 was observed in microglia (A, B) and GFAP or KYNA in astrocytes (C–F). B. Inset at top right: a higher magnification of the region delimited by the rectangle. E. Inset at top right: a higher magnification of the region delimited by the rectangle. F. Inset at top left: a higher magnification of the region delimited by the rectangle. Arrows indicate blood vessels.
expressing KYNA (Fig. 3A–D). In sum, in the three experimental groups studied here, an overexpression of KYNA, GFAP and IBA-1 was observed in the ipsilateral side (infarcted region) compared with that found in the contralateral side. 4. Discussion This is the first study in which the presence of astrocytes containing KYNA has been reported under pathological conditions in the mammalian brain. The most important findings are that, in stroke, the expression of KYNA occurs from early stages and that this expression is exclusively found in the infarcted regions. Thus, there is a perfect anatomical relationship between the infarcted regions and the expression of KYNA. Regarding the development of the monoclonal anti-KYNA antibody, it is very important to take the chemical structure of the target into consideration. In this sense, the choice of the coupling agent (e.g., ECF, glutaraldehyde) is crucial in order to maintain the original chemical structure and conformation of the antigen, ensuring that the antibody specifically recognises the required target (e.g., KYNA) (Mangas et al., 2008, 2012). Thus, KYNA was linked to the protein carrier (BSA) by different chemical procedures (using
different coupling agents), in order to confirm that the chemical procedure followed did not affect the original structure of KYNA. In all cases, using a competition ELISA test, KYNA was specifically recognized by the antibody used in this study. Thus, it was concluded that different antigens (KYNA linked to BSA by different chemical procedures) were equally recognized by the antibody. This procedure is very important for the specific recognition of KYNA in the tissue, after the application of the immunohistochemical technique, and in order to exclude spurious signals (background). In sum, we have developed a monoclonal anti-KYNA antibody showing a high affinity (10−10 M) and specificity (the antibody does not recognize closest competitors). In addition, the histological controls carried out confirmed the specificity of the immunoreactivity observed. However, it seems that in the infarcted region, an overexpression of several molecules occurs and hence the specificity of the immunoreactivity for KYNA should be assessed by other experiments. The specificity of the immunoreactivity is never absolute and different methods must be tested in order to confirm it. In the tMCAO model, it is known that there is a considerable variability in the extension of the infarcted region (Lin et al., 2013). This is in agreement with our observations, since the infarcted regions were observed in the striatum and/or cerebral cortex. Astrocytes
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Fig. 3. (A–D) Infarcted region (ipsilateral side; cerebral cortex: 21 days). Double-labelling immunohistochemistry. In astrocytes, the coexistence of KYNA (brown labelling) and GFAP (blue labelling) (arrowheads) is observed.
containing KYNA were strictly observed in the infarcted regions. Thus, the regions showing KYNA-immunoreactive structures were variable in tMCAO animals. The different distribution pattern of the immunoreactivity (KYNA, GFAP, IBA-1) observed in the ipsilateral (overexpression) and contralateral sides of tMCAO animals demonstrated that the surgical procedures were correctly conducted. Thus, the infarcted area was recognized by the overexpression of IBA-1, GFAP and KYNA. For these markers, a perfect match occurs in the infarcted region. By double-labelling immunohistochemistry, we have demonstrated the coexistence of KYNA and GFAP in astrocytes. GFAP was revealed using 4-chloro-1-napthol and KYNA using DAB (Marcos et al., 2013). The blue precipitate due to the first chromogen is easily distinguishable from the brown product due to DAB, which enables a second immunohistochemical reaction on the same section without antibody removal. The DAB reaction product masks the antigen and catalytic sites of the first sequence of immunoreagents, preventing an interaction of the second sequence with the reagents (Marcos et al., 1997). It is important to note that two different anti-GFAP antibodies were used; in both cases, astrocytes showed an identical distribution. Both antibodies showed an overexpression of GFAP in the ipsilateral side of tMCAO animals. Moreover, it is important to note that KYNA was only found in those regions (striatum and/or cerebral cortex) in which an overexpression of GFAP and IBA-1 was observed (ipsilateral side). Finally, tMCAO animals were studied at different post-stroke phases: 2, 5 and 21 days. In the three experimental groups, KYNAimmunoreactive structures showed the same distribution pattern. This means that, in stroke, KYNA was expressed from early stages and that this expression continued 21 days post-stroke. In the tMCAO model, the temporal profile of the expression of IBA-1 has been previously reported (Ito et al., 2001). Fourteen days after the tMCAO, these authors found necrosis in the ischemic region and a cavity was observed; however, cells containing IBA-1 were visualized. Moreover, they found the higher immunoreactivity for IBA-1 on day 7 after the tMCAO (Ito et al., 2001). Our data are partially in agreement with these previous results (Ito et al., 2001).
Thus, we observed an overexpression of IBA-1 in the infarcted area, but the degree of the immunoreactivity was similar 2, 5 or 21 days after the tMCAO. This discrepancy could be due to the time of the transient ischemia: 90 min in the study of Ito et al. (2001) and 50–55 min in our study. It is clear that the tissue damage was higher in the former study, but it is important to note that 14 days after the tMCAO, cells containing IBA-1 were still observed (Ito et al., 2001). In our study, in the infarcted region, a higher number of IBA1-immunoreactive cells were observed because the tissue damage was lower following ischemic conditions. In the IDO pathway, the essential amino acid tryptophan is catabolised, several intermediate metabolites (e.g., KYNA, kynurenine, quinolinic acid, 3-OH-anthranilic) are generated, and at the end of route NAD is also generated. It is known that this pathway also regulates the immune response (Moffett and Namboodiri, 2003) and that it is altered in several pathologies (e.g., stroke, Alzheimer, amyotrophic lateral sclerosis, depression, Huntington, multiple sclerosis, Parkinson, schizophrenia) (Gold et al., 2011; Bohár et al., 2015; Lim et al., 2016; Lovelace et al., 2017). In stroke, using HPLC, it has been reported a relationship between the kynurenine/tryptophan balance (IDO pathway) and the post-stroke cognitive impairment (Gold et al., 2011). In astrocytes, KYNA is synthesized by kynurenine aminotransferases (KAT) I, II and III and hence it is important to know the activators/inhibitors of those enzymes (Baran et al., 2010). For example, it is known that the glia depressing factor controls the levels of both KAT and KYNA, as well as regulates the proliferation of glial cells (Baran et al., 2010). Metabolites of the IDO pathway exert opposite physiological effects. KYNA has a neuroprotective action, whereas quinolinic (mainly synthesized in microglia) and picolinic acids exert a neurotoxic effect (Guillemin et al., 2004, 2007; Lim et al., 2016). Thus, it seems that the overexpression of KYNA, exclusively observed here in the infarcted regions, could be the response of astrocytes against the ischemic damage induced in the tMCAO model. Thus, KYNA, acting as a neuropro-
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tective/scavenger/antioxidant factor, could counteract the tissue damage produced after stroke. In sum, in a tMCAO stroke model, (i) KYNA was visualized for the first time in the mammalian astrocytes; (ii) KYNA was exclusively observed in the infarcted regions; (iii) an overexpression of KYNA occurred from early stages; (iv) there was a perfect anatomical relationship between the expression of KYNA and the infarcted region, and (v) KYNA could play a neuroprotective/scavenger/antioxidant role. Acknowledgements This work has been supported by the European Union FP7 Collaborative Grant TargetBraIn (number 279017), Gemacbio S.A. Laboratories (Saint Jean d’Illac, France), and by the IDRPHT (Talence, France). We wish to thank Professors Miguel Merchán, Margarita Heredia and Adelaida Riolobos (INCYL, Salamanca, Spain) for technical facilities and Miss Marianny Pernìa for technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aanat.2017.01. 002. References Baran, H., Kepplinger, B., Draxler, M., 2010. Endogenous kynurenine aminotransferases inhibitor is proposed to act as glia depressing factor (GDF). Int. J. Tryptophan Res. 3, 13–22. Bohár, Z., Toldi, J., Fülöp, F., Vécsei, L., 2015. Changing the face of kynurenines and neurotoxicity: therapeutic considerations. Int. J. Mol. Sci. 16, 9772–9793. Gellért, L., Fuzik, J., Göblös, A., Sárközi, K., Marosi, M., Kis, Z., Farkas, T., Szatmári, I., Fülöp, F., Vécsei, L., Toldi, J., 2011. Neuroprotection with a new kynurenic acid analog in the four-vessel occlusion model of ischemia. Eur. J. Pharmacol. 667, 182–187. Gold, A.B., Herrmann, N., Swardfager, W., Black, S.E., Aviv, R.I., Tennen, G., Kiss, A., Lanctôt, K.L., 2011. The relationship between indoleamine 2,3-dioxygenase activity and post-stroke cognitive impairment. J. Neuroinflamm. 8, 17. Guillemin, G.J., Kerr, S.J., Smythe, G.A., Smith, D.G., Kapoor, V., Armati, P.J., Croitoru, J., Brew, B.J., 2001. Kynurenine pathway metabolism in human astrocytes: a paradox for neuronal protection. J. Neurochem. 78, 842–853. Guillemin, G.J., Smythe, G., Takikawa, O., Brew, B.J., 2004. Expression of indoleamine 2, 3-dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons. Glia 49, 15–23. Guillemin, G.J., Cullen, K.M., Lim, C.K., Smythe, G.A., Garner, B., Kapoor, V., Takikawa, O., Brew, B.J., 2007. Characterization of the kynurenine pathway in human neurons. J. Neurosci. 27, 12884–12892. Ito, D., Tanaka, K., Suzuki, S., Dembo, T., Fukuuchi, Y., 2001. Enhanced expression of Iba1, ionized calcium-binding adapter molecule 1, after transient focal cerebral ischemia in rat brain. Stroke 32, 1208–1215.
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