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EFFECTS OF MINOCYCLINE ON ENDOGENOUS NEURAL STEM CELLS AFTER EXPERIMENTAL STROKE M. A. RUEGER, a,b* S. MUESKEN, a M. WALBERER, a,b S. U. JANTZEN, a,b K. SCHNAKENBURG, a H. BACKES, b R. GRAF, b B. NEUMAIER, b M. HOEHN, b G. R. FINK a,c AND M. SCHROETER a,b a
Department of Neurology, University Hospital of Cologne, Germany
b
Max Planck Institute for Neurological Research, Cologne, Germany
tion by therapeutic agents. We found minocycline, previously implied in attenuating microglial activation, to have positive effects on endogenous NSC survival. These findings hold promise for the development of novel treatments in stroke therapy. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
c
Institute of Neuroscience and Medicine (INM-3), Cognitive Neurology Section, Research Centre Juelich, Germany
Key words: neural stem cells, minocycline, Positron Emission Tomography, [18F]FLT, [11C]PK11195.
Abstract—Minocycline has been reported to reduce infarct size after focal cerebral ischemia, due to an attenuation of microglia activation and prevention of secondary damage from stroke-induced neuroinflammation. We here investigated the effects of minocycline on endogenous neural stem cells (NSCs) in vitro and in a rat stroke model. Primary cultures of fetal rat NSCs were exposed to minocycline to characterize its effects on cell survival and proliferation. To assess these effects in vivo, permanent cerebral ischemia was induced in adult rats, treated systemically with minocycline or placebo. Imaging 7 days after ischemia comprised (i) Magnetic Resonance Imaging (MRI), assessing the extent of infarcts, (ii) Positron Emission Tomography (PET) with [11C]PK11195, characterizing neuroinflammation, and (iii) PET with 3’-deoxy-3’-[18F]fluoro-L-thymidine ([18F]FLT), detecting proliferating endogenous NSCs. Immunohistochemistry was used to verify ischemic damage and characterize cellular inflammatory and repair processes in more detail. In vitro, specific concentrations of minocycline significantly increased NSC numbers without increasing their proliferation, indicating a positive effect of minocycline on NSC survival. In vivo, endogenous NSC activation in the subventricular zone (SVZ) measured by [18F]FLT PET correlated well with infarct volumes. Similar to in vitro findings, minocycline led to a specific increase in endogenous NSC activity in both the SVZ as well as the hippocampus. [11C]PK11195 PET detected neuroinflammation in the infarct core as well as in peri-infarct regions, with both its extent and location independent of the infarct size. The data did not reveal an effect of minocycline on stroke-induced neuroinflammation. We show that multimodal PET imaging can be used to characterize and quantify complex cellular processes occurring after stroke, as well as their modula-
INTRODUCTION Focal cerebral ischemia elicits various cellular responses that occur in the ‘chronic phase’ of stroke, i.e. days to weeks after the onset of ischemia (reviewed by Onteniente and Polentes, 2011). These include both (neuro-)inflammatory processes, e.g. activation of resident microglia and recruitment of monocytes/macrophages, as well as regenerative responses, involving the mobilization of endogenous neural stem cells (NSCs). Stroke-induced cellular neuroinflammation involves the rapid activation of glial cells (microglia, astrocytes) as well as recruitment of hematogenous cells (granulocytes, T-cells, monocytes/macrophages) from the blood stream. These multi-faceted responses are triggered by the upregulation of cell adhesion molecules, chemokines and cytokines and follow specific temporospatial patterns (Hallenbeck et al., 1986; Schroeter et al., 1994, 1999; Mabuchi et al., 2000; Schroeter et al., 2001; Wang et al., 2007). In vivo, neuroinflammation can be visualized by Positron Emission Tomography (PET) using the radiotracer [11C]PK11195 that selectively binds to the translocator protein-18 kDa expressed on inflammatory cells (Benavides et al., 1983), thereby allowing for the noninvasive quantification of neuroinflammation in a living animal in a longitudinal experimental setting (Rojas et al., 2007; Schroeter et al., 2009). Cerebral ischemia also leads to a robust expansion of endogenous NSCs, as has been shown for various ischemia models including transient global ischemia (Liu et al., 1998), transient focal ischemia (Jin et al., 2001; Arvidsson et al., 2002), or permanent focal ischemia (Takasawa et al., 2002). NSCs seem to contribute to regeneration after stroke by providing neuroprotection and trophic support, reducing neuroinflammation and inducing remodeling, rather than by replacing lost neurons (reviewed by Chopp et al., 2009). Endogenous NSC activation can be assessed in vivo using the PET-tracer 30 -deoxy-30 -[18F]fluoro-L-thymidine
*Correspondence to: M. A. Rueger, Department of Neurology, University Hospital of Cologne, Kerpener Strasse 62, 50924 Cologne, Germany. Tel: +49-221-478-89144; fax: +49-221-478-89143. E-mail address:
[email protected] (M. A. Rueger). Both authors contributed equally to this work. Abbreviations: BrdU, Bromodeoxyuridine; CCA, common carotid artery; DCX, doublecortin; EAE, encephalomyelitis; ECA, external carotid artery; ICA, internal carotid artery; mAb, monoclonal antibody; NSCs, neural stem cells; PET, Positron Emission Tomography; SVZ, subventricular zone.
0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.04.036 174
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([18F]FLT) that enables imaging and measuring of cell proliferation (Rueger et al., 2010). The tetracycline minocycline has recently emerged as a promising therapeutic agent for treating stroke in the ‘chronic phase’. In animal models of cerebral ischemia, it has been shown to be neuroprotective (Yrjanheikki et al., 1998) and to reduce infarct size (Xu et al., 2004), which has already resulted in first clinical trials on stroke patients (Lampl et al., 2007; Fagan et al., 2010). When investigating the anti-inflammatory properties of minocycline on various models of neurological disease, neurogenesis was detected to be increased in parallel to the reduction in inflammation that had been anticipated (Ekdahl et al., 2003; Rasmussen et al., 2011; Das et al., 2011). To date, however, the effects of minocycline on endogenous neural stem cells have not been evaluated systematically. We here show that minocycline has positive effects on the survival of endogenous NSC, adding to its therapeutic potential in stroke therapy.
EXPERIMENTAL PROCEDURES Cell culture NSCs were cultured from fetal rat cortex on embryonic day 14.5 according to the method of Johe et al. (1996) and as described previously (Rueger et al., 2010). Cells were expanded as monolayer cultures in serum-free DMEM/F12 medium (Gibco, Karlsruhe, Germany) with N2 supplement (Gibco, Karlsruhe, Germany) and FGF2 (10 ng/ml, Invitrogen, Karlsruhe, Germany) for 5 days and were re-plated fresh at 10,000 cells per cm2. FGF2 was included throughout the experiments. Minocycline was added to cultures 3 h after re-plating at concentrations of 0 nM, 10 nM, 100 nM, 1 lM, 10 lM, 50 lM, and 100 lM. 48 h later, MTT assay was performed using an MTT Cell Proliferation Assay Kit (Molecular Probes, #V-13154) according to manufacturer’s protocols. Optical density measured by MTT assay is proportional to the number of live cells, and was therefore used as a surrogate marker for NSC number. To determine the ratio of proliferating cells, 10 lM Bromodeoxyuridine (BrdU; Fluka, Munich, Germany) was added to cultures for 6 h, before cells were fixed with 4% PFA. Cells were stained with monoclonal antibody (mAb) against BrdU to identify proliferating cells (clone BU-33, dilution 1:100, Sigma, Munich, Germany). For antigen-retrieval prior to staining, sections were incubated in 2 N HCl for 30 min. For visualization, FITC-labeled anti-mouse IgG was used (Invitrogen, Karlsruhe, Germany). All cell culture experiments were performed in triplicate. To calculate the fraction of proliferating cells, total cell numbers as measured by MTT assay were multiplied by the ratio of proliferating cells assessed by BrdU staining. To assess the differentiation potential of minocycline-treated vs. control cells, mitogen was withdrawn during the expansion phase, followed by a differentiation phase of 10 days in the absence (control) or presence of 10 lM minocycline. Immunocytochemistry using markers for young neurons (TuJ1), astrocytes (GFAP) and oligodendrocytes (CNPase) was used to verify all three fates of NSCs.
Animals and surgery All animal procedures were in accordance with the German Laws for Animal Protection and were approved by the local animal care committee and local governmental authorities. Spontaneously
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breathing male Wistar rats weighing 290–330 g were anesthetized with 5% isoflurane and maintained with 2.5% isoflurane in 65%/35% nitrous oxide/oxygen. Focal cerebral ischemia. Permanent focal ischemia was produced by intra-arterial injection of TiO2 spheres into the middle cerebral artery (MCA) of n = 21 rats as described elsewhere in detail (Gerriets et al., 2004). In brief, after exposure of the left common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA), the ECA and the pterygopalatine branch of the ICA were ligated. Polyethylene tubing filled with saline and four TiO2 macrospheres 0.315–0.355 mm in diameter was advanced through the CCA into the ICA until the tip of the tube was placed distal to the origin of the pterygopalatine artery. The macrospheres were advanced into the ICA by slow injection of approximately 0.2 ml saline. After surgery, all animals were allowed to recover from anesthesia and were put back into their home cages, where they were given access to food and water ad libitum. Intraperitoneal injections. Prior to stroke induction, animals were randomized into treatment groups. Starting 3 days before induction of ischemia, n = 11 rats received daily intraperitoneal (i.p.) injections of 50 mg/kg/d minocycline (as described by Rasmussen et al., 2011) for 12 days, while n = 10 rats were sham-treated with i.p. injections of equal volumes of saline. In all animals, and starting on day 4 after induction of ischemia, the tracer BrdU was injected i.p. once daily for 5 days at a dose of 50 mg/kg/d to label dividing cells as described previously (Rueger et al., 2010).
Magnetic Resonance Imaging In all 21 animals, MRI was performed 6 days after induction of ischemia to characterize the extent of the ischemic lesions and the disruption of the blood–brain-barrier (BBB). Rats were anesthetized with isoflurane, and experiments were conducted on a 4.7 T BioSpec system (Bruker BioSpin, Ettlingen, Germany) with a 30-cm-bore horizontal magnet, equipped with a self-shielded gradient system (max gradient 100 mT/m; rise time 100 mm3 on MRI), involving cortical and subcortical structures. Four out of seven minocycline-treated rats
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Fig. 2. Proliferative activity was visualized by [18F]FLT PET in vivo and by BrdU-staining ex vivo in a large infarct (A and B), as well as in a subcortical infarct (C and D). Co-registration of [18F]FLT PET data on T2-weighted MRI one week after stroke (A and C) displayed tracer binding within the lesion (outlined in white) as well as in neurogenic niches (arrows on SVZ and hippocampus). BrdU immunoreactivity confirmed proliferative activity in the infarct core and neurogenic niches (B and D: 1; E: 40). Proliferating cells in part stained positive for DCX, indicating they underwent neurogenesis (F).
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with an ischemic lesion, and three out of eight placeboinjected rats with an ischemic lesion, developed large hemispheric infarcts. Overall, infarct volumes of minocycline-treated animals did not differ from those of placebo-treated ones as assessed by MRI (129 ± 44 vs. 133 ± 59 mm3, p = 0.95). Neural stem cell (NSC) activation after stroke In a subset of nine stroke rats (three minocycline-treated, six placebo-treated), endogenous NSC activation was visualized using the surrogate marker [18F]FLT, enabling the visualization of cell proliferation in vivo. Co-registration of PET data on T2-weighted MRI revealed [18F]FLT accumulation both in the infarct core as well as in neurogenic niches (SVZ, hippocampus; Fig. 2A and C). Immunohistochemical analyses staining for proliferating cells after BrdU incorporation revealed similar patterns of BrdU accumulation in the infarct core, SVZ, and dentate gyrus of the hippocampus (Fig. 2B, D and E). Part of the proliferating cells seemed to undergo neurogenesis, staining positive for doublecortin (DCX; Fig. 2F). This distribution of proliferating cells was observed for both large and small ischemic lesions. Proliferation in the neurogenic niches seemed more pronounced ipsilaterally (Fig. 2), but this difference was not statistically significant (data not shown). NSC activation in the SVZ as measured by [18F]FLT accumulation in vivo correlated well to infarct volumes determined by T2-weighted MRI one week after stroke (R2 = 0.74, p = 0.007; Fig. 3A). Systemic treatment with minocycline in vivo increased the mean width of the SVZ (Fig. 3B); however, this effect was not statistically significant. This increase in endogenous NSCs could also be detected in vivo by [18F]FLT PET in both the SVZ as well as in the hippocampus (Fig. 3C), but again, statistical significance was not reached. Proliferative activity within the ischemic core itself was unaffected by infarct size, as measured by [18F]FLT PET in vivo, with Standard Uptake Values for [18F]FLT of 0.2 (±0.04) in small infarcts compared to 0.24 (±0.02) in large infarcts >100 mm3 (n.s.).
Fig. 3. (A) NSC proliferation as measured by [18F]FLT accumulation in the SVZ in vivo correlated well with infarct volumes determined by T2-weighted MRI one week after stroke (R2 = 0.74, p = 0.007). (B) Systemic treatment with minocycline increased the width of the SVZ assessed immunohistochemically by staining for BrdU. Note: this effect did not reach statistical significance. (C) Systemic treatment with minocycline led to an increase in NSC activity in the neurogenic niches as measured by [18F]FLT PET in vivo. Note: this effect did not reach statistical significance (all values displayed as means ± SEM).
Neuroinflammation Cellular neuroinflammatory processes were visualized in vivo using the radiotracer [11C]PK11195 PET in a subset of six rats with ischemic lesions (three minocyclinetreated, three placebo-treated). Co-registration of [11C]PK11195 PET data on T2-weighted MRI revealed an accumulation of TSPO-expressing, inflammatory cells not only in the infarct core but also in peri-infarct regions (Fig. 4A and C). CD11b immunoreactivity was used to assess microglial activity in a more detailed histological analysis of neuroinflammatory processes. Areas of [11C]PK11195 accumulation in vivo unequivocally depicted microglial activation as verified by immunohistochemistry (Fig. 4B and D). Cellular neuroinflammatory processes were quantified based on in vivo imaging data collected in the subgroup of six animals that underwent PET. [11C]PK11195 accumulation occurred in both the infarct core as well as the surrounding region of all infarcts, and neither location nor
extent of neuroinflammatory processes was affected by infarct size (Fig. 5A). In those rats undergoing PETimaging, systemic treatment with minocycline did not result in a significant change in stroke-induced neuroinflammation as measured by [11C]PK11195 PET (Fig. 5B). Likewise, CD11b immunoreactivity as surrogate parameter of microglia activation did not reveal any difference between minocycline-treated and control brains (Fig. 5C). In addition, the number of Iba1-positive microglia was similar in the infarct border of minocycline-treated and untreated rats (Fig. 5D; note that necrotic tissue in the infarct core prohibited counting cells there).
DISCUSSION We investigated the effects of minocycline, a promising agent to treat stroke in the ‘chronic phase’, on endogenous NSCs in vitro and in vivo using PET. Our data
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Fig. 4. Neuroinflammation was visualized by [11C]PK11195 PET in vivo and by CD11b-staining ex vivo in a large infarct (A and B), as well as in a subcortical infarct (C and D). Co-registration of [11C]PK11195 PET data on T2-weighted MRI one week after stroke revealed inflammatory processes in the infarct core (outlined in white) as well as in peri-infarct regions (A and C). CD11b immunoreactivity co-localized well with tracer accumulation in vivo (B and D; 1). High magnification of CD11b in infarct subareas show activated microglia with stellate (arrow) or ameboid morphology (arrowhead), while resting microglia in the contralateral hemishpere displayed a ramified shape and faint CD11b-immunoreactivity (E).
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suggest a positive effect of minocycline on NSC survival in culture, a finding that could be reproduced in vivo by trend. The tetracycline minocycline has recently been used as a neuroprotective agent in a variety of experimental neurological disorders, including hypoxic-ischemic injury (Arvin et al., 2002), focal ischemia (Yrjanheikki et al., 1998; Xu et al., 2004), Huntington’s disease (Chen et al., 2000), and amyotrophic lateral sclerosis (Zhu et al., 2002). Several mechanisms of action have been described to account for this effect, including antiinflammatory (Kloppenburg et al., 1996; Yrjanheikki et al., 1999; Wixey et al., 2011) and antiapoptotic properties (Wang et al., 2004), as well as inhibition of both polyadenosine diphosphate ribose polymerase-1 and matrix metalloproteinases (Brundula et al., 2002; Alano et al., 2006). In experimental cerebral ischemia, minocycline has been demonstrated to (i) reduce infarct size (Xu et al., 2004), and (ii) reduce the harmful bleeding effects of tissue plasminogen activator used in thrombolysis (Murata et al., 2008; Machado et al., 2009). Those effects in particular have helped to initiate clinical trials to translate these findings from bench to bedside. A first open-label, evaluator-blinded study showed a better clinical outcome of minocycline-treated stroke patients (Lampl et al., 2007), and a recent phase I trial systematically demonstrated its safety in concentrations up to 10 mg/kg (Fagan et al., 2010). Our finding that minocycline has positive effects on NSC numbers confirms and extends previous findings suggesting clinically useful properties of minocycline in stroke therapy, since the mobilization of NSCs is associated with better functional recovery from motor deficits (Nakatomi et al., 2002; Androutsellis-Theotokis et al., 2006), with the degree of behavioral recovery correlating with the number of stem cells surviving to reach the target tissue (Guzman et al., 2008). Recently, research groups applying minocycline in the treatment of other models of neurological disorders have found an interesting ‘side effect’: when microglia activation was suppressed, animals additionally displayed an increase of NSCs in the neurogenic niche (Ekdahl et al., 2003; Das et al., 2011; Rasmussen et al., 2011). Rasmussen et al. (2011) treated mice suffering from experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, with minocycline, and found an increase in the number of BrdU+ NSCs in the SVZ. Das et al. (2011) found similar results in a mouse model for encephalitis, and reproduced these results in vitro using microglia-conditioned media, while Ekdahl et al. (2003) observed similar effects for epilepsy. Our findings are in good accordance with those results, and extend them by the first demonstration that this effect is directly attributable to minocycline itself, and not (only) indirectly mediated by microglia. Although the effect of minocycline on NSC numbers in culture was evident, in vivo this finding could only be reproduced in trend using [18F]FLT-PET. Several issues may account for this discrepancy: Although the quantification of NSC mobilization with [18F]FLT-PET is possible, this method suffers from a reduced signal-to-noise ratio
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Fig. 5. (A) One week after stroke, [11C]PK11195 accumulation occurred both in the infarct core and the peri-infarct region, and was not affected by infarct size. (B) Systemic treatment with minocycline did not lead to a significant change in stroke-induced neuroinflammation as measured by [11C]PK11195 PET. (C) Accumulation of microglia as measured as optical density of CD11b immunoreactivity did not reveal any difference between minocyclinetreated and control brains. (D) The number of Iba1-positive microglia per field of view was the same in minocycline-treated and control brains (all values displayed as means ± SEM).
due to its poorer spatial resolution compared to histology, and partial volume effects (Rueger et al., 2010). Moreover, for the in vivo experiments we administered minocycline systemically, without (invasively) assessing the resulting brain tissue dose. It is therefore possible that the dose of the drug reaching target tissue was not high enough to evoke significant effects. Indeed, the systemic (i.p.) dose of minocycline that is required to reach a brain concentration of 36 lM was reported to be 90 mg/kg for mice (Milane et al., 2007), a dose that is associated with unpleasant side effects such as inflammatory reactions of the peritoneum (Fagan et al., 2004). Another aspect is that our macrosphere model of permanent focal ischemia produces infarcts of heterogenous size and severity. This is one of the few disadvantages of that particular stroke model (Walberer et al., 2010) and may have contributed to the heterogenous outcome following minocycline treatment. Admittedly, a more
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homogeneous stroke model such as the suture model of transient focal ischemia might be even more useful for future treatment studies. Interestingly, we found only the number of NSCs to be increased upon minocycline treatment, but not their proliferative activity. The data suggest that the increase in NSC number without an increase in proliferation might be due to an increase in cell survival; however, our study did not directly analyze the effect of minocycline on cell death. We found NSC proliferation as measured by BrdUincorporation using immunohistochemical methods to be rather decreased at higher concentrations of minocycline. These findings are in line with the data of Kim et al. (2009) who suggest that the number of BrdU+ cells in the SVZ may actually be decreased after stroke and minocycline treatment. This research group – unlike several others – did not observe the promoting effects of minocycline on NSC numbers, but rather the opposite effects, when using BrdU-staining. In light of our results that (i) only NSC survival is affected by minocycline while (ii) NSC proliferation might even be impaired at higher concentrations, these conflicting data on minocycline effects on NSC numbers could be resolved. It has to be noted, however, that very high doses of minocycline (100 lM) seem to be toxic on NSCs, reducing their numbers as well as their proliferative activity. We did not observe the often reported effect of minocycline on infarct size or stroke-induced neuroinflammation in our [11C]PK11195-PET study. We assume that the heterogeneous infarct sizes and -morphologies induced by our stroke model was insufficient to demonstrate this effect in vivo in this sample size. Future studies – potentially with a more homogeneous stroke model – will be needed to further evaluate this issue.
CONCLUSION We used multimodal PET imaging along with immunohistochemical validation to characterize and quantify complex cellular processes occurring after stroke, and used those methods as well as cell culture to test specific effects of the drug minocycline. We found that minocycline, previously implied in attenuating microglial activation and being a promising candidate in clinical stroke therapy, has a direct positive effect on the survival of endogenous NSCs in culture as well as in vivo. Acknowledgment—This work was supported by the Koeln Fortune Program/Faculty of Medicine, University of Cologne, Germany (143/2011).
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(Accepted 13 April 2012) (Available online 24 April 2012)