- Email: [email protected]
Microglia activation during neuroregeneration in the adult vertebrate brain
Neuroscience Letters 497 (2011) 11–16
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Microglia activation during neuroregeneration in the adult vertebrate brain Matthew Kirkham ∗ , Daniel A. Berg, András Simon Department of Cell and Molecular Biology, Developmental Biology for Regenerative Medicine (DBRM), Karolinska Institute, Stockholm, Sweden
a r t i c l e
i n f o
Article history: Received 27 January 2011 Received in revised form 28 March 2011 Accepted 6 April 2011 Keywords: Neurogenesis Regeneration Salamander Newt Dopamine Microglia
a b s t r a c t Brain injury and neuronal loss leads to an inflammatory response, which is initiated by the innate immune system. To what extent this immune response is beneficial or detrimental for neurogenesis and regeneration is unclear. We addressed this question during regeneration of dopamine neurons in the adult salamander brain. In contrast to mammals, ablation of dopamine neurons evokes robust neurogenesis leading to complete histological and functional regeneration within four weeks in salamanders. Here we show that similarly to mammals, ablation of dopamine neurons causes microglia activation and an increase in microglia numbers in the ablated areas. Furthermore, microglia numbers remain elevated compared to the uninjured brain at least six weeks after ablation. Suppression of the microglia response results in enhanced regeneration, concomitant with reduced death of dopamine neurons during the regeneration phase. Thus neuroregeneration is not dependent on the absence of an innate immune response, but the suppression of this response may be a means to promote neurogenesis in the adult vertebrate brain. © 2011 Elsevier Ireland Ltd. All rights reserved.
Injury in the brain elicits an inflammatory response and activation of the resident microglia population, which may orchestrate neuronal replacement [29]. However, how the innate immune system relates to brain injury and neurodegenerative diseases remains uncertain. Acute inflammation after brain injury can result in increased neuronal loss and reduced neurogenesis [6,9,13,14,17,21]. On the other hand, growing evidence suggest that the effect of the inflammatory response is context dependent, and inflammation may support neurogenesis and recovery by for example promoting expression of neurotrophic factors or facilitating the migration of progenitors to lesion sites [1,2,10,16,18,27,28]. Naturally occurring regeneration in non-mammalian model organisms, such as in teleost fish and newts provides an opportunity to understand the role of the immune system and restoration of brain tissue. A microglia response was observed in teleost fish during neuronal regeneration in the corpus cerebelli and spinal cord [3,30]. Microglia have been identified in the normal and regenerating salamander CNS [22,26]. However the effect of the microglia response on regeneration has remained unclear. To start addressing the relationship between immune cells and the regeneration of individual neuronal subtypes in the adult brain, we examined the dynamics of microglia activation during regeneration of dopamine neurons in the brain of the adult
Abbreviations: TH, tyrosine hydroxylase; IBA1, ionized calcium binding adaptor molecule 1; MAC2, Galectin-3/Mac-2; PCNA, proliferating cell nuclear antigen. ∗ Corresponding author. Tel.: +46 8 52487183; fax: +46 8 324927. E-mail address: [email protected] (M. Kirkham). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.04.007
aquatic salamander, the red spotted newt. The newt brain responds uniquely among adult vertebrates to 6-OHDA-mediated ablation, as it regenerates all the lost neurons within four weeks [4,5,23,25]. Regeneration in newts leads to complete restoration of tissue architecture and locomotor performance, and is fuelled by the activation of quiescent ependymoglia cells, which divide and subsequently produce neurons [4,23]. Here we reveal that ablation of dopamine neurons causes a specific activation of microglia in the regions of neuronal loss, and the number of microglia remain elevated compared to the non-ablated brain throughout the entire regenerative phase. Inhibition of the microglia response by administrating dexamethasone, a glucocorticoid and a potent anti-inflammatory agent [6–8,11,17], caused enhanced neuronal regeneration as defined by the increase in the number of tyrosine hydroxylase (TH) positive neurons, which was paralleled with reduced death of TH+ neurons. Adult red spotted newts Notophthalmus viridescens were maintained as described earlier [4]. Immunostainings and TH quantification were performed as described earlier [4], for detecting the ionized calcium binding adaptor molecule 1 (IBA1), and galectin 3/MAC2 (MAC2) a rabbit anti-IBA1 (1:1000, Wako) and rat anti-MAC2–biotin (1:200 Cedarlane) were used, respectively. TUNEL staining was performed as described earlier [23]. Microglia cells were quantified in the ventricular zone and in the parenchyma within a 200 m radius from the TH+ cell populations. All images were acquired on a LSM 510 Meta laser microscope with LSM 5 Image Browser software (both Carl Zeiss MicroImaging Inc.). Projections of z series were processed using LSM 5 Image Browser software.
12
M. Kirkham et al. / Neuroscience Letters 497 (2011) 11–16
Fig. 1. Morphology and localisation of microglia in the uninjured newt brain. Majority of microglia in uninjured tissue are IBA1+ (green) and MAC2− (red) (A), with an irregular morphology and no protrusions. Box indicates region shown in (C)–(E). Arrow indicates non-specific ventricular labelling. A fraction of microglia are IBA1+ (green)/MAC2+ (red) (B), box indicates region shown in (F)–(H). Less frequently highly ramified IBA1+ (green) microglia were observed (I). Panels (C)–(E) and (I) are projections of confocal z series Scale bar 50 microm.
Ablations were performed as described earlier [23]. Dexamethasone (Sigma, 2 mg/kg) was dissolved in 10% ETOH. 100 l dexamethasone or vehicle was injected intraperitoneally once daily between day 3 to day 14 or day 3 to day 7 after either 6-OHDA injection or sham injection. 50 l BrdU (Sigma 20 mg/kg) was administered intraperitoneally at 12 h intervals, between day 3 to day 14. First we identified microglia in the newt brain using antibodies against the IBA1 and MAC2. IBA1 marks resting as well as activated microglia, whereas MAC2 marks activated microglia [15,18]. We detected cells expressing IBA1 (Fig. 1A and C–E), which normally were devoid of MAC2-expression. Except for one animal, in which most IBA1+ cells with a range of morphologies expressed MAC2 (Fig. 1B and F–H), in all other cases (n = 5) IBA1+ cells were devoid of
MAC2 expression. A majority of the IBA1+ cells displayed irregular morphology, however a subset of IBA1+ showed highly ramified extensions, characteristic of resting microglia (Fig. 1I) [19]. Next we determined the dynamics of microglia number and activation after ablation of dopamine neurons in the dien-and mesencephalon. Fig. 2A–C shows the ablation and regeneration of TH+ neurons following 6-OHDA-injection. The lowest number of TH+ cells was observed 3 days after 6-OHDA-administration (degeneration phase; Fig. 2D), after which the number of TH+ cells gradually increased and reached normal levels within 4 weeks (regeneration phase; Fig. 2D) [23]. We found that ablation of dopamine neurons caused a persistent increase in the number of microglia cells compared to sham ablation (Fig. 2E). This increase was due to local proliferation of microglia as revealed by double immuno-
M. Kirkham et al. / Neuroscience Letters 497 (2011) 11–16
13
Fig. 2. Ablation of dopamine neurons causes activation of microglia. TH+ neurons (green) in the ventral tegmental region (A) are lost 3 days after 6-OHDA (B), which is followed by a regeneration phase. Partial regeneration is reached after 14 days (C). Schematic illustration of the ablation/regeneration paradigm (D). Increased number of IBA1+ cells 3 days and 14 days after 6-OHDA injection compared to sham injection (E). Increased number of IBA1+ (green)/PCNA+ (red) (F–H) and IBA1+ (green)/MAC2+ (red) cells (I–K). Sustained increase in the number of IBA1+ cells two weeks after restoration of the number of TH+ cells. Error bars represent SEM. Scale bars 50 M.
14
M. Kirkham et al. / Neuroscience Letters 497 (2011) 11–16
Fig. 3. Dexamethasone reduces the number of IBA+ cells and enhances neuronal regeneration. Dexamethasone administration reduces the number of IBA+ (A) and the number of IBA+/PCNA+ cells (B). Dexamethasone treatment enhances regeneration of TH+ neurons (red) (C). Dexamethasone decreases the number of apoptotic neurons (green) during the regeneration phase (D–F). Arrows indicate TUNEL+ TH+ neurons, the arrowhead highlights TUNEL+ TH- cell. Error bars represent SEM. Scale bars 50 M.
labelling for IBA1 and the proliferating cell nuclear antigen (PCNA) (Fig. 2F–H). Consistent with this we observed activated microglia indicated by increased number of IBA1+/MAC2+ cells (Fig. 2I–K and Suppl. Fig. 1). Furthermore, all ablated brains (Fig. 2K, n = 16) contained microglia with amoeboid morphology (Suppl. Fig. 1) compared to only 1 out of 5 control brains (data not shown), further indicating microglia activation [19]. Interestingly the number of IBA1+ cells remained significantly elevated at least two weeks after the restoration of the normal number of TH+ neuron (Fig. 2L). However we did not observe any significant increase in the numbers of IBA1+/PCNA+ and IBA1+MAC2+ cells compared to the uninjured brain at this time point (data not shown). To test whether microglia activation promotes or counteracts dopamine neuron regeneration we treated animals during the
first 10 days of the regeneration phase, i.e. between days 3 and 14, with dexamethasone, which is known to suppress inflammatory processes including microglia activation [6–8,11,17]. In accordance with these observations, dexamethasone reduced the total numbers of IBA1+ and IBA1+/PCNA+ cells (Fig. 3A and B), while proliferation of other cell types (IBA1−) was not reduced (Suppl. Fig. 2). Interestingly, dexamethasone treatment led to increased production of TH+ neurons by 147 ± 26% (Fig. 3C). The increased number of TH+ neurons correlated with decreased number of dying TH+ cells as revealed by the quantification of TH+/TUNEL+ cells after a 4 days treatment with dexamethosone starting from day 3 (Fig. 3D–F). These data indicate that suppression of microglia response promotes the survival of newly formed TH+ neurons.
M. Kirkham et al. / Neuroscience Letters 497 (2011) 11–16
In this study we showed the existence of resident microglia in the adult newt brain. We demonstrated that ablation of dopamine neurons leads to an increase in microglia number, concomitant with alternations of microglia morphology, which is a hallmark of microglia activation. Our data suggest that these processes have an overall inhibitory character for neuroregeneration since interference with microglia activation increases the number of regenerated TH+ neurons. Nevertheless the persistent elevation of microglia number beyond the time frame of full cellular and functional recovery suggests that they may exert a supportive role, possibly in the long-term survival of the newly formed neurons. Accumulating data suggest a potentially dual effect of microglia on neurogenesis [10]. While the acute microglia response, shortly after injury, has been assigned a negative impact on brain repair, the role of the subsequent chronic phase has been unsettled and strong evidence indicate that microglia have the potential of promoting neuroregeneration. The fact that persistent accumulation and activation of microglia accompanies a naturally occurring complete regeneration process in the adult newt brain could support this model. However, the observation that inhibition of microglia activation and reduction of microglia number further enhances the rate of neuroregeneration in newts supports the opposite view. Clearly a certain degree of inflammatory response mediated by microglia is not incompatible with efficient neuroregeneration. It is possible that while the microglia response is an evolutionary conserved trait in both regenerative and non-regenerative animals, a key difference may reside in the interplay of microglia and other brain cells. Recently a dynamic interplay between microglia and astroglia has been suggested, with microglia activating astrocytes by secreting cytokines acting in a paracrine fashion [24]. To what extent the newt brain harbours astrocytes is unclear but the restriction of GFAP expression to ventricular cells with radial glia features indicates the absence of parenchymal astrocytes. This may be one key difference and explain in part how an initial inflammatory process is blunted, and does not reach a magnitude that would effectively compromise neuroregeneration in newts. It has been hypothesized that the maturation of the immune system is one of the underlying reasons why extensive regeneration abilities are not present in most vertebrates [20]. However, elements of the immune system are not absent and could in fact regulate processes that are required for tissue regeneration in the newt [12], including the brain. Further examination of the inflammatory response in the regenerating newt brain could give further insights into these questions, and provide pointers for promoting regeneration in normally non-regenerative species. Here we have shown that ablation of TH+ neurons in the newt brain evokes microglia proliferation and activation. The overall effect of microglia is inhibitory on neuronal regeneration in the newt brain. However microglia may have a role in the long-term survival of the newly formed neurons. Acknowledgements This work was supported by grants from the Karolinska Institute, Swedish Research Council, Parkinsonfonden, Swedish Foundation for Strategic Research, Cancerfonden, AFA Försäkringar to Andras Simon. Matthew Kirkham was supported by a long-term postdoctoral fellowship from HFSP. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2011.04.007.
15
References [1] P.E. Batchelor, G.T. Liberatore, J.Y. Wong, M.J. Porritt, F. Frerichs, G.A. Donnan, D.W. Howells, Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, J. Neurosci. 19 (1999) 1708–1716. [2] K.D. Beck, H.X. Nguyen, M.D. Galvan, D.L. Salazar, T.M. Woodruff, A.J. Anderson, Quantitative analysis of cellular inflammation after traumatic spinal cord injury: evidence for a multiphasic inflammatory response in the acute to chronic environment, Brain 133 (2010) 433–447. [3] T. Becker, C.G. Becker, Regenerating descending axons preferentially reroute to the gray matter in the presence of a general macrophage/microglial reaction caudal to a spinal transection in adult zebrafish, J. Comp. Neurol. 433 (2001) 131–147. [4] D.A. Berg, M. Kirkham, A. Beljajeva, D. Knapp, B. Habermann, J. Ryge, E.M. Tanaka, A. Simon, Efficient regeneration by activation of neurogenesis in homeostatically quiescent regions of the adult vertebrate brain, Development (Cambridge, England) 137 (2010) 4127–4134. [5] D.A. Berg, M. Kirkham, H. Wang, J. Frisen, A. Simon, Dopamine controls neurogenesis in the adult salamander midbrain in homeostasis and during regeneration of dopamine neurons, Cell Stem Cell 8 (2011) 426–433. [6] A. Castano, A.J. Herrera, J. Cano, A. Machado, The degenerative effect of a single intranigral injection of LPS on the dopaminergic system is prevented by dexamethasone, and not mimicked by rh-TNF-alpha, IL-1beta and IFN-gamma, J. Neurochem. 81 (2002) 150–157. [7] J.Y. Chang, L.Z. Liu, Inhibition of microglial nitric oxide production by hydrocortisone and glucocorticoid precursors, Neurochem. Res. 25 (2000) 903–908. [8] C.C. Chao, S. Hu, K. Close, C.S. Choi, T.W. Molitor, W.J. Novick, P.K. Peterson, Cytokine release from microglia: differential inhibition by pentoxifylline and dexamethasone, J. Infect. Dis. 166 (1992) 847–853. [9] C.T. Ekdahl, J.H. Claasen, S. Bonde, Z. Kokaia, O. Lindvall, Inflammation is detrimental for neurogenesis in adult brain, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 13632–13637. [10] C.T. Ekdahl, Z. Kokaia, O. Lindvall, Brain inflammation and adult neurogenesis: the dual role of microglia, Neuroscience 158 (2009) 1021–1029. [11] S. Ganter, H. Northoff, D. Mannel, P.J. Gebicke-Harter, Growth control of cultured microglia, J. Neurosci. Res. 33 (1992) 218–230. [12] J.W. Godwin, J.P. Brockes, Regeneration, tissue injury and the immune response, J. Anat. 209 (2006) 423–432. [13] B.D. Hoehn, T.D. Palmer, G.K. Steinberg, Neurogenesis in rats after focal cerebral ischemia is enhanced by indomethacin, Stroke 36 (2005) 2718–2724. [14] R.E. Iosif, C.T. Ekdahl, H. Ahlenius, C.J. Pronk, S. Bonde, Z. Kokaia, S.E. Jacobsen, O. Lindvall, Tumor necrosis factor receptor 1 is a negative regulator of progenitor proliferation in adult hippocampal neurogenesis, J. Neurosci. 26 (2006) 9703–9712. [15] D. Ito, Y. Imai, K. Ohsawa, K. Nakajima, Y. Fukuuchi, S. Kohsaka, Microgliaspecific localisation of a novel calcium binding protein, Iba1, Brain Res. Mol. Brain Res. 57 (1998) 1–9. [16] B.J. Kim, M.J. Kim, J.M. Park, S.H. Lee, Y.J. Kim, S. Ryu, Y.H. Kim, B.W. Yoon, Reduced neurogenesis after suppressed inflammation by minocycline in transient cerebral ischemia in rat, J. Neurol. Sci. 279 (2009) 70–75. [17] I. Kurkowska-Jastrzebska, T. Litwin, I. Joniec, A. Ciesielska, A. Przybylkowski, A. Czlonkowski, A. Czlonkowska, Dexamethasone protects against dopaminergic neurons damage in a mouse model of Parkinson’s disease, Int. Immunopharmacol. 4 (2004) 1307–1318. [18] M. Lalancette-Hebert, G. Gowing, A. Simard, Y.C. Weng, J. Kriz, Selective ablation of proliferating microglial cells exacerbates ischemic injury in the brain, J. Neurosci. 27 (2007) 2596–2605. [19] E. Lehrmann, T. Christensen, J. Zimmer, N.H. Diemer, B. Finsen, Microglial and macrophage reactions mark progressive changes and define the penumbra in the rat neocortex and striatum after transient middle cerebral artery occlusion, J. Comp. Neurol. 386 (1997) 461–476. [20] A.L. Mescher, A.W. Neff, Limb regeneration in amphibians: immunological considerations, Sci. World J. 6 (Suppl. 1) (2006) 1–11. [21] M.L. Monje, H. Toda, T.D. Palmer, Inflammatory blockade restores adult hippocampal neurogenesis, Science (New York, NY) 302 (2003) 1760–1765. [22] C. Naujoks-Manteuffel, U. Niemann, Microglial cells in the brain of Pleurodeles waltl (Urodela, Salamandridae) after wallerian degeneration in the primary visual system using Bandeiraea simplicifolia isolectin B4-cytochemistry, Glia 10 (1994) 101–113. [23] C.L. Parish, A. Beljajeva, E. Arenas, A. Simon, Midbrain dopaminergic neurogenesis and behavioural recovery in a salamander lesion-induced regeneration model, Development (Cambridge, England) 134 (2007) 2881–2887. [24] K. Saijo, B. Winner, C.T. Carson, J.G. Collier, L. Boyer, M.G. Rosenfeld, F.H. Gage, C.K. Glass, A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death, Cell 137 (2009) 47–59. [25] A. Simon, D. Berg, M. Kirkham, Not lost in translation sensing the loss and filling the gap during regeneration, Semin. Cell Dev. Biol. 20 (2009) 691–696. [26] L.J. Stensaas, The ultrastructure of astrocytes, oligodendrocytes, and microglia in the optic nerve of urodele amphibians (A. punctatum, T. pyrrhogaster, T. viridescens), J. Neurocytol. 6 (1977) 269–286. [27] P. Thored, U. Heldmann, W. Gomes-Leal, R. Gisler, V. Darsalia, J. Taneera, J.M. Nygren, S.E. Jacobsen, C.T. Ekdahl, Z. Kokaia, O. Lindvall, Long-term
16
M. Kirkham et al. / Neuroscience Letters 497 (2011) 11–16
accumulation of microglia with proneurogenic phenotype concomitant with persistent neurogenesis in adult subventricular zone after stroke, Glia 57 (2009) 835–849. [28] Y. Ziv, H. Avidan, S. Pluchino, G. Martino, M. Schwartz, Synergy between immune cells and adult neural stem/progenitor cells promotes functional recovery from spinal cord injury, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 13174–13179.
[29] Y. Ziv, M. Schwartz, Orchestrating brain-cell renewal: the role of immune cells in adult neurogenesis in health and disease, Trends Mol. Med. 14 (2008) 471–478. [30] G.K. Zupanc, S.C. Clint, N. Takimoto, A.T. Hughes, U.M. Wellbrock, D. Meissner, Spatio-temporal distribution of microglia/macrophages during regeneration in the cerebellum of adult teleost fish, Apteronotus leptorhynchus: a quantitative analysis, Brain Behav. Evol. 62 (2003) 31–42.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Microglia activation during neuroregeneration in the adult vertebrate brain Matthew Kirkham ∗ , Daniel A. Berg, András Simon Department of Cell and Molecular Biology, Developmental Biology for Regenerative Medicine (DBRM), Karolinska Institute, Stockholm, Sweden
a r t i c l e
i n f o
Article history: Received 27 January 2011 Received in revised form 28 March 2011 Accepted 6 April 2011 Keywords: Neurogenesis Regeneration Salamander Newt Dopamine Microglia
a b s t r a c t Brain injury and neuronal loss leads to an inflammatory response, which is initiated by the innate immune system. To what extent this immune response is beneficial or detrimental for neurogenesis and regeneration is unclear. We addressed this question during regeneration of dopamine neurons in the adult salamander brain. In contrast to mammals, ablation of dopamine neurons evokes robust neurogenesis leading to complete histological and functional regeneration within four weeks in salamanders. Here we show that similarly to mammals, ablation of dopamine neurons causes microglia activation and an increase in microglia numbers in the ablated areas. Furthermore, microglia numbers remain elevated compared to the uninjured brain at least six weeks after ablation. Suppression of the microglia response results in enhanced regeneration, concomitant with reduced death of dopamine neurons during the regeneration phase. Thus neuroregeneration is not dependent on the absence of an innate immune response, but the suppression of this response may be a means to promote neurogenesis in the adult vertebrate brain. © 2011 Elsevier Ireland Ltd. All rights reserved.
Injury in the brain elicits an inflammatory response and activation of the resident microglia population, which may orchestrate neuronal replacement [29]. However, how the innate immune system relates to brain injury and neurodegenerative diseases remains uncertain. Acute inflammation after brain injury can result in increased neuronal loss and reduced neurogenesis [6,9,13,14,17,21]. On the other hand, growing evidence suggest that the effect of the inflammatory response is context dependent, and inflammation may support neurogenesis and recovery by for example promoting expression of neurotrophic factors or facilitating the migration of progenitors to lesion sites [1,2,10,16,18,27,28]. Naturally occurring regeneration in non-mammalian model organisms, such as in teleost fish and newts provides an opportunity to understand the role of the immune system and restoration of brain tissue. A microglia response was observed in teleost fish during neuronal regeneration in the corpus cerebelli and spinal cord [3,30]. Microglia have been identified in the normal and regenerating salamander CNS [22,26]. However the effect of the microglia response on regeneration has remained unclear. To start addressing the relationship between immune cells and the regeneration of individual neuronal subtypes in the adult brain, we examined the dynamics of microglia activation during regeneration of dopamine neurons in the brain of the adult
Abbreviations: TH, tyrosine hydroxylase; IBA1, ionized calcium binding adaptor molecule 1; MAC2, Galectin-3/Mac-2; PCNA, proliferating cell nuclear antigen. ∗ Corresponding author. Tel.: +46 8 52487183; fax: +46 8 324927. E-mail address: [email protected] (M. Kirkham). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.04.007
aquatic salamander, the red spotted newt. The newt brain responds uniquely among adult vertebrates to 6-OHDA-mediated ablation, as it regenerates all the lost neurons within four weeks [4,5,23,25]. Regeneration in newts leads to complete restoration of tissue architecture and locomotor performance, and is fuelled by the activation of quiescent ependymoglia cells, which divide and subsequently produce neurons [4,23]. Here we reveal that ablation of dopamine neurons causes a specific activation of microglia in the regions of neuronal loss, and the number of microglia remain elevated compared to the non-ablated brain throughout the entire regenerative phase. Inhibition of the microglia response by administrating dexamethasone, a glucocorticoid and a potent anti-inflammatory agent [6–8,11,17], caused enhanced neuronal regeneration as defined by the increase in the number of tyrosine hydroxylase (TH) positive neurons, which was paralleled with reduced death of TH+ neurons. Adult red spotted newts Notophthalmus viridescens were maintained as described earlier [4]. Immunostainings and TH quantification were performed as described earlier [4], for detecting the ionized calcium binding adaptor molecule 1 (IBA1), and galectin 3/MAC2 (MAC2) a rabbit anti-IBA1 (1:1000, Wako) and rat anti-MAC2–biotin (1:200 Cedarlane) were used, respectively. TUNEL staining was performed as described earlier [23]. Microglia cells were quantified in the ventricular zone and in the parenchyma within a 200 m radius from the TH+ cell populations. All images were acquired on a LSM 510 Meta laser microscope with LSM 5 Image Browser software (both Carl Zeiss MicroImaging Inc.). Projections of z series were processed using LSM 5 Image Browser software.
12
M. Kirkham et al. / Neuroscience Letters 497 (2011) 11–16
Fig. 1. Morphology and localisation of microglia in the uninjured newt brain. Majority of microglia in uninjured tissue are IBA1+ (green) and MAC2− (red) (A), with an irregular morphology and no protrusions. Box indicates region shown in (C)–(E). Arrow indicates non-specific ventricular labelling. A fraction of microglia are IBA1+ (green)/MAC2+ (red) (B), box indicates region shown in (F)–(H). Less frequently highly ramified IBA1+ (green) microglia were observed (I). Panels (C)–(E) and (I) are projections of confocal z series Scale bar 50 microm.
Ablations were performed as described earlier [23]. Dexamethasone (Sigma, 2 mg/kg) was dissolved in 10% ETOH. 100 l dexamethasone or vehicle was injected intraperitoneally once daily between day 3 to day 14 or day 3 to day 7 after either 6-OHDA injection or sham injection. 50 l BrdU (Sigma 20 mg/kg) was administered intraperitoneally at 12 h intervals, between day 3 to day 14. First we identified microglia in the newt brain using antibodies against the IBA1 and MAC2. IBA1 marks resting as well as activated microglia, whereas MAC2 marks activated microglia [15,18]. We detected cells expressing IBA1 (Fig. 1A and C–E), which normally were devoid of MAC2-expression. Except for one animal, in which most IBA1+ cells with a range of morphologies expressed MAC2 (Fig. 1B and F–H), in all other cases (n = 5) IBA1+ cells were devoid of
MAC2 expression. A majority of the IBA1+ cells displayed irregular morphology, however a subset of IBA1+ showed highly ramified extensions, characteristic of resting microglia (Fig. 1I) [19]. Next we determined the dynamics of microglia number and activation after ablation of dopamine neurons in the dien-and mesencephalon. Fig. 2A–C shows the ablation and regeneration of TH+ neurons following 6-OHDA-injection. The lowest number of TH+ cells was observed 3 days after 6-OHDA-administration (degeneration phase; Fig. 2D), after which the number of TH+ cells gradually increased and reached normal levels within 4 weeks (regeneration phase; Fig. 2D) [23]. We found that ablation of dopamine neurons caused a persistent increase in the number of microglia cells compared to sham ablation (Fig. 2E). This increase was due to local proliferation of microglia as revealed by double immuno-
M. Kirkham et al. / Neuroscience Letters 497 (2011) 11–16
13
Fig. 2. Ablation of dopamine neurons causes activation of microglia. TH+ neurons (green) in the ventral tegmental region (A) are lost 3 days after 6-OHDA (B), which is followed by a regeneration phase. Partial regeneration is reached after 14 days (C). Schematic illustration of the ablation/regeneration paradigm (D). Increased number of IBA1+ cells 3 days and 14 days after 6-OHDA injection compared to sham injection (E). Increased number of IBA1+ (green)/PCNA+ (red) (F–H) and IBA1+ (green)/MAC2+ (red) cells (I–K). Sustained increase in the number of IBA1+ cells two weeks after restoration of the number of TH+ cells. Error bars represent SEM. Scale bars 50 M.
14
M. Kirkham et al. / Neuroscience Letters 497 (2011) 11–16
Fig. 3. Dexamethasone reduces the number of IBA+ cells and enhances neuronal regeneration. Dexamethasone administration reduces the number of IBA+ (A) and the number of IBA+/PCNA+ cells (B). Dexamethasone treatment enhances regeneration of TH+ neurons (red) (C). Dexamethasone decreases the number of apoptotic neurons (green) during the regeneration phase (D–F). Arrows indicate TUNEL+ TH+ neurons, the arrowhead highlights TUNEL+ TH- cell. Error bars represent SEM. Scale bars 50 M.
labelling for IBA1 and the proliferating cell nuclear antigen (PCNA) (Fig. 2F–H). Consistent with this we observed activated microglia indicated by increased number of IBA1+/MAC2+ cells (Fig. 2I–K and Suppl. Fig. 1). Furthermore, all ablated brains (Fig. 2K, n = 16) contained microglia with amoeboid morphology (Suppl. Fig. 1) compared to only 1 out of 5 control brains (data not shown), further indicating microglia activation [19]. Interestingly the number of IBA1+ cells remained significantly elevated at least two weeks after the restoration of the normal number of TH+ neuron (Fig. 2L). However we did not observe any significant increase in the numbers of IBA1+/PCNA+ and IBA1+MAC2+ cells compared to the uninjured brain at this time point (data not shown). To test whether microglia activation promotes or counteracts dopamine neuron regeneration we treated animals during the
first 10 days of the regeneration phase, i.e. between days 3 and 14, with dexamethasone, which is known to suppress inflammatory processes including microglia activation [6–8,11,17]. In accordance with these observations, dexamethasone reduced the total numbers of IBA1+ and IBA1+/PCNA+ cells (Fig. 3A and B), while proliferation of other cell types (IBA1−) was not reduced (Suppl. Fig. 2). Interestingly, dexamethasone treatment led to increased production of TH+ neurons by 147 ± 26% (Fig. 3C). The increased number of TH+ neurons correlated with decreased number of dying TH+ cells as revealed by the quantification of TH+/TUNEL+ cells after a 4 days treatment with dexamethosone starting from day 3 (Fig. 3D–F). These data indicate that suppression of microglia response promotes the survival of newly formed TH+ neurons.
M. Kirkham et al. / Neuroscience Letters 497 (2011) 11–16
In this study we showed the existence of resident microglia in the adult newt brain. We demonstrated that ablation of dopamine neurons leads to an increase in microglia number, concomitant with alternations of microglia morphology, which is a hallmark of microglia activation. Our data suggest that these processes have an overall inhibitory character for neuroregeneration since interference with microglia activation increases the number of regenerated TH+ neurons. Nevertheless the persistent elevation of microglia number beyond the time frame of full cellular and functional recovery suggests that they may exert a supportive role, possibly in the long-term survival of the newly formed neurons. Accumulating data suggest a potentially dual effect of microglia on neurogenesis [10]. While the acute microglia response, shortly after injury, has been assigned a negative impact on brain repair, the role of the subsequent chronic phase has been unsettled and strong evidence indicate that microglia have the potential of promoting neuroregeneration. The fact that persistent accumulation and activation of microglia accompanies a naturally occurring complete regeneration process in the adult newt brain could support this model. However, the observation that inhibition of microglia activation and reduction of microglia number further enhances the rate of neuroregeneration in newts supports the opposite view. Clearly a certain degree of inflammatory response mediated by microglia is not incompatible with efficient neuroregeneration. It is possible that while the microglia response is an evolutionary conserved trait in both regenerative and non-regenerative animals, a key difference may reside in the interplay of microglia and other brain cells. Recently a dynamic interplay between microglia and astroglia has been suggested, with microglia activating astrocytes by secreting cytokines acting in a paracrine fashion [24]. To what extent the newt brain harbours astrocytes is unclear but the restriction of GFAP expression to ventricular cells with radial glia features indicates the absence of parenchymal astrocytes. This may be one key difference and explain in part how an initial inflammatory process is blunted, and does not reach a magnitude that would effectively compromise neuroregeneration in newts. It has been hypothesized that the maturation of the immune system is one of the underlying reasons why extensive regeneration abilities are not present in most vertebrates [20]. However, elements of the immune system are not absent and could in fact regulate processes that are required for tissue regeneration in the newt [12], including the brain. Further examination of the inflammatory response in the regenerating newt brain could give further insights into these questions, and provide pointers for promoting regeneration in normally non-regenerative species. Here we have shown that ablation of TH+ neurons in the newt brain evokes microglia proliferation and activation. The overall effect of microglia is inhibitory on neuronal regeneration in the newt brain. However microglia may have a role in the long-term survival of the newly formed neurons. Acknowledgements This work was supported by grants from the Karolinska Institute, Swedish Research Council, Parkinsonfonden, Swedish Foundation for Strategic Research, Cancerfonden, AFA Försäkringar to Andras Simon. Matthew Kirkham was supported by a long-term postdoctoral fellowship from HFSP. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2011.04.007.
15
References [1] P.E. Batchelor, G.T. Liberatore, J.Y. Wong, M.J. Porritt, F. Frerichs, G.A. Donnan, D.W. Howells, Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, J. Neurosci. 19 (1999) 1708–1716. [2] K.D. Beck, H.X. Nguyen, M.D. Galvan, D.L. Salazar, T.M. Woodruff, A.J. Anderson, Quantitative analysis of cellular inflammation after traumatic spinal cord injury: evidence for a multiphasic inflammatory response in the acute to chronic environment, Brain 133 (2010) 433–447. [3] T. Becker, C.G. Becker, Regenerating descending axons preferentially reroute to the gray matter in the presence of a general macrophage/microglial reaction caudal to a spinal transection in adult zebrafish, J. Comp. Neurol. 433 (2001) 131–147. [4] D.A. Berg, M. Kirkham, A. Beljajeva, D. Knapp, B. Habermann, J. Ryge, E.M. Tanaka, A. Simon, Efficient regeneration by activation of neurogenesis in homeostatically quiescent regions of the adult vertebrate brain, Development (Cambridge, England) 137 (2010) 4127–4134. [5] D.A. Berg, M. Kirkham, H. Wang, J. Frisen, A. Simon, Dopamine controls neurogenesis in the adult salamander midbrain in homeostasis and during regeneration of dopamine neurons, Cell Stem Cell 8 (2011) 426–433. [6] A. Castano, A.J. Herrera, J. Cano, A. Machado, The degenerative effect of a single intranigral injection of LPS on the dopaminergic system is prevented by dexamethasone, and not mimicked by rh-TNF-alpha, IL-1beta and IFN-gamma, J. Neurochem. 81 (2002) 150–157. [7] J.Y. Chang, L.Z. Liu, Inhibition of microglial nitric oxide production by hydrocortisone and glucocorticoid precursors, Neurochem. Res. 25 (2000) 903–908. [8] C.C. Chao, S. Hu, K. Close, C.S. Choi, T.W. Molitor, W.J. Novick, P.K. Peterson, Cytokine release from microglia: differential inhibition by pentoxifylline and dexamethasone, J. Infect. Dis. 166 (1992) 847–853. [9] C.T. Ekdahl, J.H. Claasen, S. Bonde, Z. Kokaia, O. Lindvall, Inflammation is detrimental for neurogenesis in adult brain, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 13632–13637. [10] C.T. Ekdahl, Z. Kokaia, O. Lindvall, Brain inflammation and adult neurogenesis: the dual role of microglia, Neuroscience 158 (2009) 1021–1029. [11] S. Ganter, H. Northoff, D. Mannel, P.J. Gebicke-Harter, Growth control of cultured microglia, J. Neurosci. Res. 33 (1992) 218–230. [12] J.W. Godwin, J.P. Brockes, Regeneration, tissue injury and the immune response, J. Anat. 209 (2006) 423–432. [13] B.D. Hoehn, T.D. Palmer, G.K. Steinberg, Neurogenesis in rats after focal cerebral ischemia is enhanced by indomethacin, Stroke 36 (2005) 2718–2724. [14] R.E. Iosif, C.T. Ekdahl, H. Ahlenius, C.J. Pronk, S. Bonde, Z. Kokaia, S.E. Jacobsen, O. Lindvall, Tumor necrosis factor receptor 1 is a negative regulator of progenitor proliferation in adult hippocampal neurogenesis, J. Neurosci. 26 (2006) 9703–9712. [15] D. Ito, Y. Imai, K. Ohsawa, K. Nakajima, Y. Fukuuchi, S. Kohsaka, Microgliaspecific localisation of a novel calcium binding protein, Iba1, Brain Res. Mol. Brain Res. 57 (1998) 1–9. [16] B.J. Kim, M.J. Kim, J.M. Park, S.H. Lee, Y.J. Kim, S. Ryu, Y.H. Kim, B.W. Yoon, Reduced neurogenesis after suppressed inflammation by minocycline in transient cerebral ischemia in rat, J. Neurol. Sci. 279 (2009) 70–75. [17] I. Kurkowska-Jastrzebska, T. Litwin, I. Joniec, A. Ciesielska, A. Przybylkowski, A. Czlonkowski, A. Czlonkowska, Dexamethasone protects against dopaminergic neurons damage in a mouse model of Parkinson’s disease, Int. Immunopharmacol. 4 (2004) 1307–1318. [18] M. Lalancette-Hebert, G. Gowing, A. Simard, Y.C. Weng, J. Kriz, Selective ablation of proliferating microglial cells exacerbates ischemic injury in the brain, J. Neurosci. 27 (2007) 2596–2605. [19] E. Lehrmann, T. Christensen, J. Zimmer, N.H. Diemer, B. Finsen, Microglial and macrophage reactions mark progressive changes and define the penumbra in the rat neocortex and striatum after transient middle cerebral artery occlusion, J. Comp. Neurol. 386 (1997) 461–476. [20] A.L. Mescher, A.W. Neff, Limb regeneration in amphibians: immunological considerations, Sci. World J. 6 (Suppl. 1) (2006) 1–11. [21] M.L. Monje, H. Toda, T.D. Palmer, Inflammatory blockade restores adult hippocampal neurogenesis, Science (New York, NY) 302 (2003) 1760–1765. [22] C. Naujoks-Manteuffel, U. Niemann, Microglial cells in the brain of Pleurodeles waltl (Urodela, Salamandridae) after wallerian degeneration in the primary visual system using Bandeiraea simplicifolia isolectin B4-cytochemistry, Glia 10 (1994) 101–113. [23] C.L. Parish, A. Beljajeva, E. Arenas, A. Simon, Midbrain dopaminergic neurogenesis and behavioural recovery in a salamander lesion-induced regeneration model, Development (Cambridge, England) 134 (2007) 2881–2887. [24] K. Saijo, B. Winner, C.T. Carson, J.G. Collier, L. Boyer, M.G. Rosenfeld, F.H. Gage, C.K. Glass, A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death, Cell 137 (2009) 47–59. [25] A. Simon, D. Berg, M. Kirkham, Not lost in translation sensing the loss and filling the gap during regeneration, Semin. Cell Dev. Biol. 20 (2009) 691–696. [26] L.J. Stensaas, The ultrastructure of astrocytes, oligodendrocytes, and microglia in the optic nerve of urodele amphibians (A. punctatum, T. pyrrhogaster, T. viridescens), J. Neurocytol. 6 (1977) 269–286. [27] P. Thored, U. Heldmann, W. Gomes-Leal, R. Gisler, V. Darsalia, J. Taneera, J.M. Nygren, S.E. Jacobsen, C.T. Ekdahl, Z. Kokaia, O. Lindvall, Long-term
16
M. Kirkham et al. / Neuroscience Letters 497 (2011) 11–16
accumulation of microglia with proneurogenic phenotype concomitant with persistent neurogenesis in adult subventricular zone after stroke, Glia 57 (2009) 835–849. [28] Y. Ziv, H. Avidan, S. Pluchino, G. Martino, M. Schwartz, Synergy between immune cells and adult neural stem/progenitor cells promotes functional recovery from spinal cord injury, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 13174–13179.
[29] Y. Ziv, M. Schwartz, Orchestrating brain-cell renewal: the role of immune cells in adult neurogenesis in health and disease, Trends Mol. Med. 14 (2008) 471–478. [30] G.K. Zupanc, S.C. Clint, N. Takimoto, A.T. Hughes, U.M. Wellbrock, D. Meissner, Spatio-temporal distribution of microglia/macrophages during regeneration in the cerebellum of adult teleost fish, Apteronotus leptorhynchus: a quantitative analysis, Brain Behav. Evol. 62 (2003) 31–42.