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Plasmodium berghei ANKA: Erythropoietin activates neural stem cells in an experimental cerebral malaria model
Experimental Parasitology 127 (2011) 500–505
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Plasmodium berghei ANKA: Erythropoietin activates neural stem cells in an experimental cerebral malaria model Andrew Core a,b,1, Casper Hempel a,c,d,⇑,1, Jørgen A.L. Kurtzhals c,d, Milena Penkowa a a
Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark Faculty of Medicine, National University of Ireland, Galway, Ireland c Centre for Medical Parasitology, Department of Clinical Microbiology, Copenhagen University Hospital (Rigshospitalet), Denmark d Centre for Medical Parasitology, Department of International Health, Immunology, and Microbiology, Faculty of Health Sciences, University of Copenhagen, Denmark b
a r t i c l e
i n f o
Article history: Received 1 June 2010 Received in revised form 31 August 2010 Accepted 22 September 2010 Available online 30 October 2010 Keywords: Cerebral malaria Adult neurogenesis Neural stem cells Plasmodium berghei ANKA Erythropoietin (EPO) Neuroregeneration
a b s t r a c t Cerebral malaria (CM) causes substantial mortality and neurological sequelae in survivors, and no neuroprotective regimens are currently available for this condition. Erythropoietin (EPO) reduces neuropathology and improves survival in murine CM. Using the Plasmodium berghei model of CM, we investigated if EPO’s neuroprotective effects include activation of endogenous neural stem cells (NSC). By using immunohistochemical markers of different NSC maturation stages, we show that EPO increased the number of nestin+ cells in the dentate gyrus and in the sub-ventricular zone of the lateral ventricles, relative to control-treatment. 75% of the EPO-treated CM mice displayed migration as nestin+ NSC. The NSC showed differentiation towards a neural cell lineage as shown by PSA-NCAM binding and NSC maturation and lineage commitment was significantly affected by exogenous EPO and by CM in the sub ventricular zone. These results indicate a rapid, EPO-dependent activation of NSC during CM pathology. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Malaria continues to cause substantial morbidity and mortality, mainly in Sub-Saharan Africa. In particular, cerebral malaria (CM), characterised by coma often associated with convulsions, has a high case fatality rate despite a relatively low parasitemia. Among surviving patients neurological sequelae of shorter or longer duration are relatively common (Molyneux et al., 1989). A marginally increased survival due to improved anti-parasitic treatment appears associated with higher risk of neurological sequelae, highlighting the need for adjunct neuroprotective treatment for CM (PrayGod et al., 2008; Taylor et al., 1998). The pathogenesis of CM remains unresolved but is suspected to arise from a combination of an increased level of pro-inflammatory cytokines, sequestering infected erythrocytes, leukocytes, and platelets in the brain microvasculature, as well as a dysregulated coagulation cascade (Medana and Turner, 2006; Van Der Heyde et al., 2006).
⇑ Corresponding author at: Panum Instituttet, Blegdamsvej 3, 2200 Copenhagen N, Denmark. E-mail addresses: [email protected] (A. Core), [email protected] (C. Hempel), [email protected] (J.A.L. Kurtzhals), [email protected] (M. Penkowa). 1 These authors contributed equally to this work. 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.09.010
CM results in breakdown of the blood–brain barrier, downstream oedemas and multifocal, cerebral, petechial haemorrhages (Brown et al., 1999; Newton et al., 1998). Additionally, perivascular activation of glia cells and axonal injury is seen in patients with cerebral involvement. However, the parasite itself is not seen within the brain in CM patients (Medana et al., 2002; Medana and Turner, 2006). Murine malaria models share several characteristics with human disease and are useful when studying CM pathogenesis (Hunt and Grau, 2003; Medana and Turner, 2006). Despite differences between human and murine CM pathology (White et al., 2010) in particular relating to the importance of sequestering, infected erythrocytes, the experimental studies are advantageous in allowing manipulations of the model and for performing pre-clinical treatment trials (Hunt et al., 2010). Several animal studies have explored various adjunct treatments of CM, one of these being erythropoietin (EPO) (CasalsPascual et al., 2009; Kaiser et al., 2006; Wiese et al., 2008). When correctly dosed and timed, this treatment can reduce the CMinduced mortality in the mouse model significantly (Kaiser et al., 2006; Wiese et al., 2008). In a descriptive, post-mortem study in Asian adults the expression of EPO, EPO receptor (EPOR) and CD131 was assessed in the medulla. There was a significant, positive correlation between markers of hypo-oxygenation and EPO/ EPOR expression in glia cells (Medana et al., 2009). EPO is a
A. Core et al. / Experimental Parasitology 127 (2011) 500–505
pleiotropic cytokine which possibly acts via the heterodimeric EPOR resulting in anti-apoptotic, anti-inflammatory and anti-oxidatory effects in several neuropathological conditions (Brines et al., 2004; Hasselblatt et al., 2006). All these properties are potentially beneficial in CM. In addition, EPO is involved in survival and signalling in adult, neural stem cells (NSC) (Ransome and Turnley, 2007; Shingo et al., 2001). NSC are undifferentiated, multipotent cells residing in the sub granular layer of the dentate gyrus and in the sub ventricular zone (SVZ) of the lateral ventricle (Ma et al., 2009). A recent paper also demonstrated the presence of NSC in the circumventricular organ (Bennett et al., 2009). When stimulated by neurotrophic factors or with EPO, NSC differentiate into mature neurons or glia cells (Jessberger et al., 2008; Ma et al., 2009). The plasticity of NSC makes them therapeutically relevant in several neurodegenerative diseases (Ma et al., 2009; Richardson et al., 2009). A comprehensive palette of antibodies makes the visualization of the different stages of maturation possible in situ (Doetsch et al., 1997; Doetsch, 2003; Ma et al., 2009; Shingo et al., 2001). In vitro studies typically use neurospheres; free-floating clusters cells derived from NSC (Bez et al., 2003). Neurospheres express EPOR in higher levels than in mature neurons and administration of EPO to a culture of neurospheres induced proliferation in a dose-dependent manner (Chen et al., 2007; Shingo et al., 2001). Neurospheres express EPO upon a hypoxic insult and applying anti-EPO antibodies to the culture medium blocked the hypoxiainduced neurogenesis suggesting an autocrine–paracrine function of EPO on neurospheres (Shingo et al., 2001). In vivo studies using brain-specific EPOR knock out (KO) mice suggest that EPOR is fundamental for neurogenesis. EPOR KO mice had decreased number of dividing NSC in the SVZ and failed to display the continuous migration of NSC to the infarct area after stroke that is normally seen in this experimental model (Tsai et al., 2006). In healthy mice, systemic EPO administration for 7 days gives rise to increased neurogenesis and an increased number of proliferating neuroblasts in the hippocampus (Ransome and Turnley, 2007). Intracerebroventricular EPO infusions over 6 days cause migration of NSC from the SVZ along the rostral migratory stream (RMS) and stimulate the maturation into neuronal progenitors (Shingo et al., 2001). The neurogenic effects of systemically delivered EPO are possibly of an immediate character since hippocampal suppressor of cytokine signalling-3 (SOCS-3) expression is upregulated only 2 h after a single intra peritoneal (i.p.) EPO inoculation (Ransome and Turnley, 2007). We used the murine model of CM to investigate the possibility that the beneficial effects of EPO treatment may be associated with the maturation and migration of NSC. 2. Materials and methods 2.1. Animals Sixty female, 5-week old, pathogen-free C57BL/6j mice were purchased from Taconic (Ejby, Denmark). The mice were kept under standard conditions with free access to pellet food and water. All experiments complied with Danish and European guidelines for animal research and were approved by the national board for animal studies. All efforts were made to minimize animal suffering and to reduce the number of animals used. 2.2. Infection and treatment Prior to experimental infection Plasmodium berghei ANKA from a frozen stock were passed once in vivo in C57BL/6j mice. The experimental mice were split in four equal-sized groups (Table 1). At the commencement of the experiment, 30 mice were infected
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through i.p. injection of 104 parasitized erythrocytes diluted in 200 ll sterile isotonic (0.9%) NaCl (normal saline). Thirty mice served as uninfected controls receiving 200 ll normal saline i.p. Mice were supervised daily for body temperature, parasitemia and clinical signs of CM. The clinical signs included ataxia, paralysis (mono-, hemi-, para-, or tetraplegia), deviations of the head, convulsions, and coma. Body temperature was measured rectally (Digital thermometer DM852 with rectal probe, Ellab, Denmark). Parasitemia was determined by flow cytometry as described (Hein-Kristensen et al., 2009). Briefly, 2 ll blood from the tail vein was withdrawn using a sterile syringe and diluted in 98 ll heparinised PBS and stained with acridine orange. The parasitemia was estimated in percentage from 10,000 gated erythrocytes. Haematocrit was measured automatically from 20 ll blood diluted in a 500 ll cell pack mix (CPK-310A, Sysmex, Hamburg, Germany) using a Sysmex KX-21 N. Fifteen infected as well as 15 uninfected mice received 5000 IU/ kg recombinant human EPO (Eprex, Janssen-Cilag, Schaffhausen, Switzerland) diluted in normal saline by i.p. injection on day 4–7 post infection (p.i.). The 30 remaining mice received normal saline i.p. on the same days. All mice were euthanised on day 8 p.i. when the infected mice demonstrated clinical signs of CM, encompassing a body temperature below 32 °C. In deep Hypnorm/Dormicum anaesthesia (10 ll/g body weight, Wiese et al., 2006) mice were transcardially perfused briefly with heparinised (15,000 U/l, Leo Pharma, Denmark) normal saline followed by 5 min (45 ml/min) of perfusion-fixation with Zamboni’s fixative (4% paraformaldehyde, saturated picric acid solution, pH 7.4). Afterwards the brains were removed and immersed in Zamboni’s fixative for 2–4 h followed by dehydration according to standard procedures, embedded in paraffin and were cut in 5 lm thick sagittal sections for immunohistochemistry.
2.3. Immunohistochemistry Tissue was rehydrated according to standard procedures and heat induced epitope retrieval (HIER) was performed in a microwave oven. All sections were boiled in citrate buffer pH 6.0. Endogenous peroxidase activity was quenched by incubating for 20 min in 0.5% H2O2 (30% H2O2, Sigma–Aldrich, Brøndby, Denmark) dissolved in Tris-buffered saline (TBS) with 0.5% Tween-20 (Merck, Darmstadt, Germany). Non-specific binding was blocked by adding either 10% Goat serum (In vitro, Fredensborg, Denmark) or Proteinfree serum block (Dako, Glostrup, Denmark) when primary antibody was of mouse IgG type. Sections were incubated over night at 4 °C with one of the following primary antibodies: Mouse Anti-Poly Sialic Acid-neural cell adhesion molecule (PSA-NCAM) (300 diluted, cat. no. Ab C0019, AbCys SA-Paris, France), Mouse Anti-nestin (200 diluted, cat. no. MAB353, Chemicon, Millipore, MA, US), Mouse Anti-a-Internexin (100 diluted, cat. no. MAB5224, Chemicon, Millipore). The primary antibody was recognized with biotinylated secondary antibodies: goat anti-mouse IgG (200 diluted, cat. no. B8774, Sigma–Aldrich) and goat anti-mouse IgM (10 diluted, cat. no. 115-065-020, Jackson Immunoresearch, Suffolk, UK). Sections were incubated for 30 min with streptavidin–peroxidase (200 diluted, cat. no. S5512, Sigma–Aldrich). Staining intensity was amplified using biotinylated tyramide and streptavidin–peroxidase complex (cat. no. NEL700, Perkin Elmer, US) in goat serum following manufacturer’s recommendations in order to amplify the signal. Immunoreactions were visualized using 0.015% H2O2 (Sigma–Aldrich) in 3,3-diaminobenzidinetetrahydrochloride tablets (DAB, Kem-En-Tec Diagnostics, Taastrup, Denmark) completely dissolved in TBS for 10 min at room temperature. Sections were counterstained in Mayer’s hematoxyline (VWR, Herlev, Denmark) prior to mounting. Standard negative
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A. Core et al. / Experimental Parasitology 127 (2011) 500–505 Table 1 Overview of the four different groups in the study: uninfected, saline-treated (UninfSal), uninfected, EPO-treated (UninfEPO), infected, saline-treated (InfSal) and infected, EPO-treated (InfEPO). Body weight was measured immediately before the study was initiated (day 0 p.i.). Other parameters were measured at day 8 p.i. before mice were euthanised. Group
Mean body weight (SD) [g]
Mean body temperature at day 8 p.i. (SD) [°C]
Mean parasitemia day 8 p.i. (SD) [%]
Mean Haematocrit day 8 p.i (SD) [%]
UninfSal UninfEPO InfSal InfEPO
17.4 16.8 17.3 18.0
37.7 37.5 33.7 37.5
0 (0) 0 (0) 8.4 (3.2) 2.6 (0.77)
42.9 51.8 38.5 43.4
(1.2) (1.2) (1.5) (1.1)
(0.51) (0.84) (4.8) (0.61)
(5.9) (7.9) (4.7) (10.9)
control stainings, without any primary antibody, were performed simultaneously for each primary antibody. 2.4. Visualization and quantification Chromogenically stained samples were visualized on an Imager Z1 microscope fitted with an AxioCam MRc5 Camera (Carl Zeiss, Germany). All assessments were carried out blinded and randomly. Sections chromogenically stained with nestin and PSA-NCAM were examined within stem cell niches in the hippocampal and lateral ventricular region (Doetsch, 2003; von Bohlen Und Halbach, 2007). Nestin+ and PSA-NCAM+ cells were counted in the sub granular zone of the dentate gyrus and in the lining of the lateral ventricle at an equal distance from the midline. If present, cells leaving the SVZ of the lateral ventricles were noted for the construction of a contingency table. Thus, the inclusion of cells being NSC depended on both morphology and the site of detection. Neurite outgrowth and growth cones were analysed qualitatively on a-internexin-stained sections for all treatment groups in the hippocampus. 2.5. Statistical analysis Survival analysis was performed with log-rank statistics. Body temperature and parasitemia followed a normal distribution and inter-group variation was analysed by one-way analysis of variance (ANOVA). Post-tests were performed using pair-wise t-tests with the Holm correction. Cell counts were log transformed (x0 = log(x + 1)) and analysed using a two-way ANOVA for the factors: infection (yes or no) and treatment (saline or EPO). The contingency table based on migration from the SVZ was analysed using a log linear model. All statistical analyses were performed using R (version 2.6.1 for Windows) and results considered significant if p < 0.05.
Fig. 1. Progression of parasitemia and body temperature during the course of infection. Parasitemia rose gradually, though at a lower speed in the InfEPO group (solid triangles) compared to the InfSal (solid circles). Body temperature dropped suddenly in terminal ill mice. This was observed in the InfSal group (open grey circles) but not in the InfEPO group (open grey triangles). Clinical signs were also correspondingly less frequently observed in the InfEPO group compared to the InfSal group. A list of typical CM-related signs are listed as the number of positive mice in each group at a given day p.i. (ntotal = 15).
survival significantly (p < 0.001), as consistently seen in this model. Similarly, the decrease in body temperature in terminally ill InfSal mice was significantly lower than in the other groups including the InfEPO mice (Table 1, ANOVA, p < 0.001; Welsh test (InfSal vs. InfEPO): p = 0.008). Parasitemia (mean ± SD) was significantly decreased in InfEPO mice (2.6% ± 0.8) compared to InfSal (8.4% ± 3.2) (Table 1, p < 0.001). Since EPO induces erythrocytosis and thus dilutes the quantity of parasitized erythrocytes this could be a confounder in the analysis. However, taking haematocrit into account did not change the statistical significance (p < 0.001).
3. Results 3.2. Neural stem cells (nestin+ cells) 3.1. Clinical, descriptive results The four groups of mice were age-matched and had similar body weight (Table 1, p = 0.08). The clinical course of the infection in the infected groups (infected saline-treated mice (InfSal) and infected EPO-treated mice (InfEPO)) is summarized in Fig. 1. Parasitemia rose gradually in both groups from day 4. On day 6 and 7 p.i., infected, the InfSal group started showing clinical signs of CM. On day 8 p.i. all mice were still alive but the majority of the InfSal group was terminally ill with low body temperature and neurological disturbances. Two mice were paralysed but none of the mice were comatose. In order to get comparable treatment groups the study was then terminated and all 60 mice were euthanised. As an estimate of differences in survival, we used a drop in body temperature below 32 °C as proxy of death, since for ethical reasons mice could not be followed beyond this point of disease progression (Curfs et al., 1989). EPO treatment increased
Nestin is an intermediate filament expressed in pluripotent NSC, which can differentiate into neural or glial type progeny. However, recent data point towards a neuronal fate (Lagace et al., 2007). Nestin is also a marker of ependymal cells and astrocytes (Doetsch et al., 1997), but NSC are morphologically distinct. Nestin+ cells displayed comparable morphology in all four groups and were localized in the NSC niches. In the dentate gyrus, nestin+ cells were detectable in the sub-granular layer in all four groups. In the SVZ, the nestin+ round cells lined the ventricular wall (Fig. 2). A significant increase was seen in the SVZ in EPO-treated mice (Fig. 2(a)–(e), 2-way ANOVA, p = 0.03). InfEPO mice had a significantly higher stem cell numbers than InfSal mice (p = 0.006), saline-treated control (UninfSal) mice (p = 0.03) and EPO-treated control (UninfEPO) mice (p = 0.05). Stimulation of NSC promotes differentiation as well as migration (Iwai et al., 2002; Tsai et al., 2006). The NSC derived from
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Fig. 2. Nestin+ neural stem cells in the SVZ of the lateral ventricle and in the dentate gyrus. The number of cells in the granular layer of the dentate gyrus was counted. In the SVZ, only cells lining the ventricle wall were counted. It was noted if cells had left the SVZ and started migration. Micrographs (a)–(d) are representatives of nestin+ cells in the SVZ. LV – lateral ventricle. Arrowhead – migrating cells. Quantifications are shown in the bar chart in (e) (whiskers show standard error of mean). Micrographs (e)–(i) are representatives of nestin+ cells in the dentate gyrus. PoDG – polymorph dentate gyrus. Quantifications are shown in the bar charts in (j) (whiskers show standard error of mean). Scale bar: 50 lm.
SVZ showed migrational activity in response to EPO, as they were observed inside the cerebral parenchyme in EPO-treated mice but rarely in placebo-treated mice. Thus, migrating nestin+ cells were found in 75% of InfEPO mice, but not in the InfSal mice and only in 12% of UninfSal mice and in 14% of UninfEPO (v2, p < 0.001). The log linear model showed a significant interaction between migration, EPO treatment and infection (p = 0.04) underscoring the combined effect of EPO and infection. Also in the dentate gyrus, EPO treatment significantly increased the number of nestin+ cells in both uninfected and in infected mice (Fig. 1(f)–(j), 2-way ANOVA, p = 0.007).
were observed in the same niches as nestin+ cells in the dentate gyrus and SVZ. In the SVZ, PSA-NCAM+ cells lined the ventricles in all mice. Both EPO treatment and CM increased the number of PSA-NCAM+ cells in this area (Fig. 3(a)–(e), 2-way ANOVA, p = 0.04 and p = 0.002, respectively). In contrast, the number of PSA-NCAM+ cells in the dentate gyrus was comparable in all four groups disregarding infection or treatment (Fig. 3(f)–(j), ANOVA, p = 0.2).
3.3. Neuronal progenitor cells (PSA-NCAM+ cells)
During later stages of NSC maturation the cells show neuritogenesis and axonal outgrowth as they evolve along the neuronal cell lineage (Shea and Beermann, 1999). To assess the level of differentiation in the dentate gyrus, a-internexin expression, a
PSA-NCAM is a late stage marker of neural-type stem cells (Iwai et al., 2003) and of glia cells (Fox et al., 2001). PSA-NCAM+ cells
3.4. Neurite outgrowth (a-internexin+ cells)
Fig. 3. PSA-NCAM+ neuronal progenitor cells in the SVZ of the lateral ventricle and in the dentate gyrus. The number of cells in the granular layer of the dentate gyrus was counted. In the SVZ, only cells lining the ventricle wall were counted. It was noted if cells had left the SVZ and started migration. Micrographs (a)–(d) are representatives of PSA-NCAM+ cells in the SVZ. LV – lateral ventricle. Arrowhead – migrating cells. Quantifications are shown in the bar chart in e (whiskers show standard error of mean). Micrographs (f)–(i) are representatives of PSA-NCAM+ cells in the dentate gyrus. PoDG – polymorph dentate gyrus. Quantifications are shown in the bar chart in e (whiskers show standard error of mean). Scale bar: 50 lm.
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marker of neurite outgrowth and regeneration, was analysed (McGraw et al., 2002). When present in the dentate gyrus, ainternexin was localized in neurons in mice from all four groups. Only few infected mice disregarding grouping had a-internexin+ cells in the sub-granular layer in the dentate gyrus. In some of these mice, the a-internexin+ cells had a pyramidal structure indicating outgrowth and ramification in the hippocampal area. However, no significant differences were found between the groups (data not shown, 2-way ANOVA, p > 0.2).
4. Discussion In this study using a murine model of CM we demonstrated a significant, EPO-dependent increase in the number of NSC in the dentate gyrus and in the SVZ as well as a pathology-dependant migration from the SVZ. This coincided with a significant increase in survival and decreased clinical signs of CM relative to untreated mice. It has been suggested that EPO could be a useful, adjunct therapy for CM (Casals-Pascual et al., 2009; Picot et al., 2009), though this view has been challenged recently (Medana et al., 2009). Publications from several groups have shown its neuroprotective and life-saving properties in the murine CM model (Kaiser et al., 2006; Wiese et al., 2008). Here, we show that EPO also had regenerative potential in this model. A therapy that increases neuronal repair has potential to improve the neurological sequelae often experienced by survivors of CM (Carter et al., 2005; Idro et al., 2004; Kihara et al., 2006; Molyneux et al., 1989; Mung’Ala-Odera et al., 2004). In our model, EPO treatment resulted in an increased number of nestin+ NSC in the specific niches in both infected and healthy control mice. This finding contrasts with findings from a stroke model using unilateral middle cerebral artery occlusion in mice. In the stroke model EPO-treatment only resulted in an increased number of nestin+ NCS on the ipsilateral side but not in the contralateral unaffected hemisphere. Several factors in the experimental design could explain the discrepancy between our studies. In particular Wang et al. (2004) assessed the late effects, whereas our experimental setup focused on immediate effects of EPO treatment. The response to EPO treatment appears highly time dependent. Thus, Mash1, a transcription factor involved in neurogenesis, is upregulated immediately after EPO treatment and returns to normal levels within 24 h (Shingo et al., 2001), suggesting that some processes are only seen immediately after treatment. The upregulation of PSA-NCAM+ cells (Fig. 3) implies that stem cells were stimulated to mature into neurons in EPO treated CM mice (Bonfanti, 2006). This was detectable after only 4 days underscoring the regenerative potential induced by EPO treatment in acute, neurological syndromes (Lacerda-Queiroz et al., 2010; Lackner et al., 2006). In the stroke model the number of doublecortin+ neuronal precursor cells in the SVZ was increased 21 days after the last inoculation of EPO (Wang et al., 2004), and in a different model, PSA-NCAM+ cells in the SVZ had increased in numbers on day 10 post ischemia and at a later time point in the RMS and in the olfactory lobe (Iwai et al., 2003). Similarly, a-internexin levels peaked 7 days post injury (McGraw et al., 2002). Thus, we may only have seen the initial regenerative steps in our experimental setup, which may explain the lack of discernable changes in PSA-NCAM and a-internexin expression in the dentate gyrus. EPO administration increases the migration of neural stem cells after hypoxia (Shingo et al., 2001). This is in line with our findings, showing EPO stimulated migration only in mice with clinical signs of cerebral pathology. Knocking out the EPO receptor in the brain causes impaired NSC proliferation in the SVZ and results in reduced continuous migration of neuroblasts to the infarct area (Tsai et al., 2006). Another study has shown that EPO increases the number of
neurons generated from neurospheres in vitro whereas EPO had no effect on the number of astrocytes (Wang et al., 2004). EPOs antiapoptotic effects on neurons is hypothesised to be the driving force for reducing other types of neurodegeneration directly or indirectly (Ghezzi and Brines, 2004). Together with our data this suggests that EPO is a pro-survival factor decreasing the constant turn-over rate of NSC that normally cause the majority of the generated stem cells to undergo apoptosis before maturation (von Bohlen Und Halbach, 2007). Carbamylated EPO that does not bind to the EPOR (Leist et al., 2004) has shown equal neurogenic characteristics as EPO in vitro. Furthermore, without a functional EPOR, Mash1 was involved in neurogenesis (Wang et al., 2007), and one study suggests that only haematopoietic cell lines express EPOR (Sinclair et al., 2010). Thus, EPO-induced neurogenesis may at least partly be governed via its pleiotropic actions rather than interaction with the EPOR. In addition, the ultimate fate of EPO-induced stem cell maturation and migration in vivo remains to be clarified. Our model appears to be suitable for further studies of the signalling events and the down stream effects responsible for the neuroprotective properties of EPO. Overall the present data suggest that EPO causes proliferation of NSC and neuronal precursor cells in the SVZ in both healthy and malaria-infected mice. However, CM-associated pathology (Kaiser et al., 2006; Wiese et al., 2008), was needed to initiate stem cell migration. Administering EPO as adjunct treatment for human CM could potentially improve recovery from neurological sequelae in survivors. Acknowledgments The statistical advice from Biostatistical Department at University of Copenhagen is acknowledged. Also critical review by Professor David Arnot (University of Edinburgh) is appreciated. Funding from the Danish Agency for Science, Technology and Innovation, the Danish Medical Association Foundation, The Medical Society of Copenhagen, Carl og Ellen Hertzs Legat til Dansk Læge- og Naturvidenskab, Harboefonden, Hestehandler Ole Jacobsens Mindelegat, Hørslev-Fonden, Aase og Ejnar Danielsens Fond, Kathrine og Vigo Skovgaards Fond, Karen A. Tolstrups Fond, Fonden af 17-12-1981, Direktør Ib Henriksens Fond, and Th. Maigaard´s Eftf. Fru Lily Benthine Lunds Fond is highly appreciated. References Bennett, L., Yang, M., Enikolopov, G., Iacovitti, L., 2009. Circumventricular organs: a novel site of neural stem cells in the adult brain. Mol. Cell Neurosci. 41 (3), 337– 347. Bez, A., Corsini, E., Curti, D., Biggiogera, M., Colombo, A., Nicosia, R.F., Pagano, S.F., Parati, E.A., 2003. Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization. Brain Res. 993 (1,2), 18–29. Bonfanti, L., 2006. PSA-NCAM in mammalian structural plasticity and neurogenesis. Prog. Neurobiol. 80 (3), 129–164. Brines, M., Grasso, G., Fiordaliso, F., Sfacteria, A., Ghezzi, P., Fratelli, M., Latini, R., Xie, Q.W., Smart, J., Su-Rick, C.J., Pobre, E., Diaz, D., Gomez, D., Hand, C., Coleman, T., Cerami, A., 2004. Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor. Proc. Natl. Acad. Sci. USA 101 (41), 14907–14912. Brown, H., Hien, T.T., Day, N., Mai, N.T., Chuong, L.V., Chau, T.T., Loc, P.P., Phu, N.H., Bethell, D., Farrar, J., Gatter, K., White, N., Turner, G., 1999. Evidence of blood– brain barrier dysfunction in human cerebral malaria. Neuropathol. Appl. Neurobiol. 25 (4), 331–340. Carter, J.A., Mung’Ala-Odera, V., Neville, B.G., Murira, G., Mturi, N., Musumba, C., Newton, C.R., 2005. Persistent neurocognitive impairments associated with severe falciparum malaria in Kenyan children. J. Neurol. Neurosurg. Psychiatry 76 (4), 476–481. Casals-Pascual, C., Idro, R., Picot, S., Roberts, D.J., Newton, C.R., 2009. Can erythropoietin be used to prevent brain damage in cerebral malaria? Trends Parasitol. 25 (1), 30–36. Chen, Z.Y., Asavaritikrai, P., Prchal, J.T., Noguchi, C.T., 2007. Endogenous erythropoietin signaling is required for normal neural progenitor cell proliferation. J. Biol. Chem. 282 (35), 25875–25883.
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Plasmodium berghei ANKA: Erythropoietin activates neural stem cells in an experimental cerebral malaria model Andrew Core a,b,1, Casper Hempel a,c,d,⇑,1, Jørgen A.L. Kurtzhals c,d, Milena Penkowa a a
Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark Faculty of Medicine, National University of Ireland, Galway, Ireland c Centre for Medical Parasitology, Department of Clinical Microbiology, Copenhagen University Hospital (Rigshospitalet), Denmark d Centre for Medical Parasitology, Department of International Health, Immunology, and Microbiology, Faculty of Health Sciences, University of Copenhagen, Denmark b
a r t i c l e
i n f o
Article history: Received 1 June 2010 Received in revised form 31 August 2010 Accepted 22 September 2010 Available online 30 October 2010 Keywords: Cerebral malaria Adult neurogenesis Neural stem cells Plasmodium berghei ANKA Erythropoietin (EPO) Neuroregeneration
a b s t r a c t Cerebral malaria (CM) causes substantial mortality and neurological sequelae in survivors, and no neuroprotective regimens are currently available for this condition. Erythropoietin (EPO) reduces neuropathology and improves survival in murine CM. Using the Plasmodium berghei model of CM, we investigated if EPO’s neuroprotective effects include activation of endogenous neural stem cells (NSC). By using immunohistochemical markers of different NSC maturation stages, we show that EPO increased the number of nestin+ cells in the dentate gyrus and in the sub-ventricular zone of the lateral ventricles, relative to control-treatment. 75% of the EPO-treated CM mice displayed migration as nestin+ NSC. The NSC showed differentiation towards a neural cell lineage as shown by PSA-NCAM binding and NSC maturation and lineage commitment was significantly affected by exogenous EPO and by CM in the sub ventricular zone. These results indicate a rapid, EPO-dependent activation of NSC during CM pathology. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Malaria continues to cause substantial morbidity and mortality, mainly in Sub-Saharan Africa. In particular, cerebral malaria (CM), characterised by coma often associated with convulsions, has a high case fatality rate despite a relatively low parasitemia. Among surviving patients neurological sequelae of shorter or longer duration are relatively common (Molyneux et al., 1989). A marginally increased survival due to improved anti-parasitic treatment appears associated with higher risk of neurological sequelae, highlighting the need for adjunct neuroprotective treatment for CM (PrayGod et al., 2008; Taylor et al., 1998). The pathogenesis of CM remains unresolved but is suspected to arise from a combination of an increased level of pro-inflammatory cytokines, sequestering infected erythrocytes, leukocytes, and platelets in the brain microvasculature, as well as a dysregulated coagulation cascade (Medana and Turner, 2006; Van Der Heyde et al., 2006).
⇑ Corresponding author at: Panum Instituttet, Blegdamsvej 3, 2200 Copenhagen N, Denmark. E-mail addresses: [email protected] (A. Core), [email protected] (C. Hempel), [email protected] (J.A.L. Kurtzhals), [email protected] (M. Penkowa). 1 These authors contributed equally to this work. 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.09.010
CM results in breakdown of the blood–brain barrier, downstream oedemas and multifocal, cerebral, petechial haemorrhages (Brown et al., 1999; Newton et al., 1998). Additionally, perivascular activation of glia cells and axonal injury is seen in patients with cerebral involvement. However, the parasite itself is not seen within the brain in CM patients (Medana et al., 2002; Medana and Turner, 2006). Murine malaria models share several characteristics with human disease and are useful when studying CM pathogenesis (Hunt and Grau, 2003; Medana and Turner, 2006). Despite differences between human and murine CM pathology (White et al., 2010) in particular relating to the importance of sequestering, infected erythrocytes, the experimental studies are advantageous in allowing manipulations of the model and for performing pre-clinical treatment trials (Hunt et al., 2010). Several animal studies have explored various adjunct treatments of CM, one of these being erythropoietin (EPO) (CasalsPascual et al., 2009; Kaiser et al., 2006; Wiese et al., 2008). When correctly dosed and timed, this treatment can reduce the CMinduced mortality in the mouse model significantly (Kaiser et al., 2006; Wiese et al., 2008). In a descriptive, post-mortem study in Asian adults the expression of EPO, EPO receptor (EPOR) and CD131 was assessed in the medulla. There was a significant, positive correlation between markers of hypo-oxygenation and EPO/ EPOR expression in glia cells (Medana et al., 2009). EPO is a
A. Core et al. / Experimental Parasitology 127 (2011) 500–505
pleiotropic cytokine which possibly acts via the heterodimeric EPOR resulting in anti-apoptotic, anti-inflammatory and anti-oxidatory effects in several neuropathological conditions (Brines et al., 2004; Hasselblatt et al., 2006). All these properties are potentially beneficial in CM. In addition, EPO is involved in survival and signalling in adult, neural stem cells (NSC) (Ransome and Turnley, 2007; Shingo et al., 2001). NSC are undifferentiated, multipotent cells residing in the sub granular layer of the dentate gyrus and in the sub ventricular zone (SVZ) of the lateral ventricle (Ma et al., 2009). A recent paper also demonstrated the presence of NSC in the circumventricular organ (Bennett et al., 2009). When stimulated by neurotrophic factors or with EPO, NSC differentiate into mature neurons or glia cells (Jessberger et al., 2008; Ma et al., 2009). The plasticity of NSC makes them therapeutically relevant in several neurodegenerative diseases (Ma et al., 2009; Richardson et al., 2009). A comprehensive palette of antibodies makes the visualization of the different stages of maturation possible in situ (Doetsch et al., 1997; Doetsch, 2003; Ma et al., 2009; Shingo et al., 2001). In vitro studies typically use neurospheres; free-floating clusters cells derived from NSC (Bez et al., 2003). Neurospheres express EPOR in higher levels than in mature neurons and administration of EPO to a culture of neurospheres induced proliferation in a dose-dependent manner (Chen et al., 2007; Shingo et al., 2001). Neurospheres express EPO upon a hypoxic insult and applying anti-EPO antibodies to the culture medium blocked the hypoxiainduced neurogenesis suggesting an autocrine–paracrine function of EPO on neurospheres (Shingo et al., 2001). In vivo studies using brain-specific EPOR knock out (KO) mice suggest that EPOR is fundamental for neurogenesis. EPOR KO mice had decreased number of dividing NSC in the SVZ and failed to display the continuous migration of NSC to the infarct area after stroke that is normally seen in this experimental model (Tsai et al., 2006). In healthy mice, systemic EPO administration for 7 days gives rise to increased neurogenesis and an increased number of proliferating neuroblasts in the hippocampus (Ransome and Turnley, 2007). Intracerebroventricular EPO infusions over 6 days cause migration of NSC from the SVZ along the rostral migratory stream (RMS) and stimulate the maturation into neuronal progenitors (Shingo et al., 2001). The neurogenic effects of systemically delivered EPO are possibly of an immediate character since hippocampal suppressor of cytokine signalling-3 (SOCS-3) expression is upregulated only 2 h after a single intra peritoneal (i.p.) EPO inoculation (Ransome and Turnley, 2007). We used the murine model of CM to investigate the possibility that the beneficial effects of EPO treatment may be associated with the maturation and migration of NSC. 2. Materials and methods 2.1. Animals Sixty female, 5-week old, pathogen-free C57BL/6j mice were purchased from Taconic (Ejby, Denmark). The mice were kept under standard conditions with free access to pellet food and water. All experiments complied with Danish and European guidelines for animal research and were approved by the national board for animal studies. All efforts were made to minimize animal suffering and to reduce the number of animals used. 2.2. Infection and treatment Prior to experimental infection Plasmodium berghei ANKA from a frozen stock were passed once in vivo in C57BL/6j mice. The experimental mice were split in four equal-sized groups (Table 1). At the commencement of the experiment, 30 mice were infected
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through i.p. injection of 104 parasitized erythrocytes diluted in 200 ll sterile isotonic (0.9%) NaCl (normal saline). Thirty mice served as uninfected controls receiving 200 ll normal saline i.p. Mice were supervised daily for body temperature, parasitemia and clinical signs of CM. The clinical signs included ataxia, paralysis (mono-, hemi-, para-, or tetraplegia), deviations of the head, convulsions, and coma. Body temperature was measured rectally (Digital thermometer DM852 with rectal probe, Ellab, Denmark). Parasitemia was determined by flow cytometry as described (Hein-Kristensen et al., 2009). Briefly, 2 ll blood from the tail vein was withdrawn using a sterile syringe and diluted in 98 ll heparinised PBS and stained with acridine orange. The parasitemia was estimated in percentage from 10,000 gated erythrocytes. Haematocrit was measured automatically from 20 ll blood diluted in a 500 ll cell pack mix (CPK-310A, Sysmex, Hamburg, Germany) using a Sysmex KX-21 N. Fifteen infected as well as 15 uninfected mice received 5000 IU/ kg recombinant human EPO (Eprex, Janssen-Cilag, Schaffhausen, Switzerland) diluted in normal saline by i.p. injection on day 4–7 post infection (p.i.). The 30 remaining mice received normal saline i.p. on the same days. All mice were euthanised on day 8 p.i. when the infected mice demonstrated clinical signs of CM, encompassing a body temperature below 32 °C. In deep Hypnorm/Dormicum anaesthesia (10 ll/g body weight, Wiese et al., 2006) mice were transcardially perfused briefly with heparinised (15,000 U/l, Leo Pharma, Denmark) normal saline followed by 5 min (45 ml/min) of perfusion-fixation with Zamboni’s fixative (4% paraformaldehyde, saturated picric acid solution, pH 7.4). Afterwards the brains were removed and immersed in Zamboni’s fixative for 2–4 h followed by dehydration according to standard procedures, embedded in paraffin and were cut in 5 lm thick sagittal sections for immunohistochemistry.
2.3. Immunohistochemistry Tissue was rehydrated according to standard procedures and heat induced epitope retrieval (HIER) was performed in a microwave oven. All sections were boiled in citrate buffer pH 6.0. Endogenous peroxidase activity was quenched by incubating for 20 min in 0.5% H2O2 (30% H2O2, Sigma–Aldrich, Brøndby, Denmark) dissolved in Tris-buffered saline (TBS) with 0.5% Tween-20 (Merck, Darmstadt, Germany). Non-specific binding was blocked by adding either 10% Goat serum (In vitro, Fredensborg, Denmark) or Proteinfree serum block (Dako, Glostrup, Denmark) when primary antibody was of mouse IgG type. Sections were incubated over night at 4 °C with one of the following primary antibodies: Mouse Anti-Poly Sialic Acid-neural cell adhesion molecule (PSA-NCAM) (300 diluted, cat. no. Ab C0019, AbCys SA-Paris, France), Mouse Anti-nestin (200 diluted, cat. no. MAB353, Chemicon, Millipore, MA, US), Mouse Anti-a-Internexin (100 diluted, cat. no. MAB5224, Chemicon, Millipore). The primary antibody was recognized with biotinylated secondary antibodies: goat anti-mouse IgG (200 diluted, cat. no. B8774, Sigma–Aldrich) and goat anti-mouse IgM (10 diluted, cat. no. 115-065-020, Jackson Immunoresearch, Suffolk, UK). Sections were incubated for 30 min with streptavidin–peroxidase (200 diluted, cat. no. S5512, Sigma–Aldrich). Staining intensity was amplified using biotinylated tyramide and streptavidin–peroxidase complex (cat. no. NEL700, Perkin Elmer, US) in goat serum following manufacturer’s recommendations in order to amplify the signal. Immunoreactions were visualized using 0.015% H2O2 (Sigma–Aldrich) in 3,3-diaminobenzidinetetrahydrochloride tablets (DAB, Kem-En-Tec Diagnostics, Taastrup, Denmark) completely dissolved in TBS for 10 min at room temperature. Sections were counterstained in Mayer’s hematoxyline (VWR, Herlev, Denmark) prior to mounting. Standard negative
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A. Core et al. / Experimental Parasitology 127 (2011) 500–505 Table 1 Overview of the four different groups in the study: uninfected, saline-treated (UninfSal), uninfected, EPO-treated (UninfEPO), infected, saline-treated (InfSal) and infected, EPO-treated (InfEPO). Body weight was measured immediately before the study was initiated (day 0 p.i.). Other parameters were measured at day 8 p.i. before mice were euthanised. Group
Mean body weight (SD) [g]
Mean body temperature at day 8 p.i. (SD) [°C]
Mean parasitemia day 8 p.i. (SD) [%]
Mean Haematocrit day 8 p.i (SD) [%]
UninfSal UninfEPO InfSal InfEPO
17.4 16.8 17.3 18.0
37.7 37.5 33.7 37.5
0 (0) 0 (0) 8.4 (3.2) 2.6 (0.77)
42.9 51.8 38.5 43.4
(1.2) (1.2) (1.5) (1.1)
(0.51) (0.84) (4.8) (0.61)
(5.9) (7.9) (4.7) (10.9)
control stainings, without any primary antibody, were performed simultaneously for each primary antibody. 2.4. Visualization and quantification Chromogenically stained samples were visualized on an Imager Z1 microscope fitted with an AxioCam MRc5 Camera (Carl Zeiss, Germany). All assessments were carried out blinded and randomly. Sections chromogenically stained with nestin and PSA-NCAM were examined within stem cell niches in the hippocampal and lateral ventricular region (Doetsch, 2003; von Bohlen Und Halbach, 2007). Nestin+ and PSA-NCAM+ cells were counted in the sub granular zone of the dentate gyrus and in the lining of the lateral ventricle at an equal distance from the midline. If present, cells leaving the SVZ of the lateral ventricles were noted for the construction of a contingency table. Thus, the inclusion of cells being NSC depended on both morphology and the site of detection. Neurite outgrowth and growth cones were analysed qualitatively on a-internexin-stained sections for all treatment groups in the hippocampus. 2.5. Statistical analysis Survival analysis was performed with log-rank statistics. Body temperature and parasitemia followed a normal distribution and inter-group variation was analysed by one-way analysis of variance (ANOVA). Post-tests were performed using pair-wise t-tests with the Holm correction. Cell counts were log transformed (x0 = log(x + 1)) and analysed using a two-way ANOVA for the factors: infection (yes or no) and treatment (saline or EPO). The contingency table based on migration from the SVZ was analysed using a log linear model. All statistical analyses were performed using R (version 2.6.1 for Windows) and results considered significant if p < 0.05.
Fig. 1. Progression of parasitemia and body temperature during the course of infection. Parasitemia rose gradually, though at a lower speed in the InfEPO group (solid triangles) compared to the InfSal (solid circles). Body temperature dropped suddenly in terminal ill mice. This was observed in the InfSal group (open grey circles) but not in the InfEPO group (open grey triangles). Clinical signs were also correspondingly less frequently observed in the InfEPO group compared to the InfSal group. A list of typical CM-related signs are listed as the number of positive mice in each group at a given day p.i. (ntotal = 15).
survival significantly (p < 0.001), as consistently seen in this model. Similarly, the decrease in body temperature in terminally ill InfSal mice was significantly lower than in the other groups including the InfEPO mice (Table 1, ANOVA, p < 0.001; Welsh test (InfSal vs. InfEPO): p = 0.008). Parasitemia (mean ± SD) was significantly decreased in InfEPO mice (2.6% ± 0.8) compared to InfSal (8.4% ± 3.2) (Table 1, p < 0.001). Since EPO induces erythrocytosis and thus dilutes the quantity of parasitized erythrocytes this could be a confounder in the analysis. However, taking haematocrit into account did not change the statistical significance (p < 0.001).
3. Results 3.2. Neural stem cells (nestin+ cells) 3.1. Clinical, descriptive results The four groups of mice were age-matched and had similar body weight (Table 1, p = 0.08). The clinical course of the infection in the infected groups (infected saline-treated mice (InfSal) and infected EPO-treated mice (InfEPO)) is summarized in Fig. 1. Parasitemia rose gradually in both groups from day 4. On day 6 and 7 p.i., infected, the InfSal group started showing clinical signs of CM. On day 8 p.i. all mice were still alive but the majority of the InfSal group was terminally ill with low body temperature and neurological disturbances. Two mice were paralysed but none of the mice were comatose. In order to get comparable treatment groups the study was then terminated and all 60 mice were euthanised. As an estimate of differences in survival, we used a drop in body temperature below 32 °C as proxy of death, since for ethical reasons mice could not be followed beyond this point of disease progression (Curfs et al., 1989). EPO treatment increased
Nestin is an intermediate filament expressed in pluripotent NSC, which can differentiate into neural or glial type progeny. However, recent data point towards a neuronal fate (Lagace et al., 2007). Nestin is also a marker of ependymal cells and astrocytes (Doetsch et al., 1997), but NSC are morphologically distinct. Nestin+ cells displayed comparable morphology in all four groups and were localized in the NSC niches. In the dentate gyrus, nestin+ cells were detectable in the sub-granular layer in all four groups. In the SVZ, the nestin+ round cells lined the ventricular wall (Fig. 2). A significant increase was seen in the SVZ in EPO-treated mice (Fig. 2(a)–(e), 2-way ANOVA, p = 0.03). InfEPO mice had a significantly higher stem cell numbers than InfSal mice (p = 0.006), saline-treated control (UninfSal) mice (p = 0.03) and EPO-treated control (UninfEPO) mice (p = 0.05). Stimulation of NSC promotes differentiation as well as migration (Iwai et al., 2002; Tsai et al., 2006). The NSC derived from
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Fig. 2. Nestin+ neural stem cells in the SVZ of the lateral ventricle and in the dentate gyrus. The number of cells in the granular layer of the dentate gyrus was counted. In the SVZ, only cells lining the ventricle wall were counted. It was noted if cells had left the SVZ and started migration. Micrographs (a)–(d) are representatives of nestin+ cells in the SVZ. LV – lateral ventricle. Arrowhead – migrating cells. Quantifications are shown in the bar chart in (e) (whiskers show standard error of mean). Micrographs (e)–(i) are representatives of nestin+ cells in the dentate gyrus. PoDG – polymorph dentate gyrus. Quantifications are shown in the bar charts in (j) (whiskers show standard error of mean). Scale bar: 50 lm.
SVZ showed migrational activity in response to EPO, as they were observed inside the cerebral parenchyme in EPO-treated mice but rarely in placebo-treated mice. Thus, migrating nestin+ cells were found in 75% of InfEPO mice, but not in the InfSal mice and only in 12% of UninfSal mice and in 14% of UninfEPO (v2, p < 0.001). The log linear model showed a significant interaction between migration, EPO treatment and infection (p = 0.04) underscoring the combined effect of EPO and infection. Also in the dentate gyrus, EPO treatment significantly increased the number of nestin+ cells in both uninfected and in infected mice (Fig. 1(f)–(j), 2-way ANOVA, p = 0.007).
were observed in the same niches as nestin+ cells in the dentate gyrus and SVZ. In the SVZ, PSA-NCAM+ cells lined the ventricles in all mice. Both EPO treatment and CM increased the number of PSA-NCAM+ cells in this area (Fig. 3(a)–(e), 2-way ANOVA, p = 0.04 and p = 0.002, respectively). In contrast, the number of PSA-NCAM+ cells in the dentate gyrus was comparable in all four groups disregarding infection or treatment (Fig. 3(f)–(j), ANOVA, p = 0.2).
3.3. Neuronal progenitor cells (PSA-NCAM+ cells)
During later stages of NSC maturation the cells show neuritogenesis and axonal outgrowth as they evolve along the neuronal cell lineage (Shea and Beermann, 1999). To assess the level of differentiation in the dentate gyrus, a-internexin expression, a
PSA-NCAM is a late stage marker of neural-type stem cells (Iwai et al., 2003) and of glia cells (Fox et al., 2001). PSA-NCAM+ cells
3.4. Neurite outgrowth (a-internexin+ cells)
Fig. 3. PSA-NCAM+ neuronal progenitor cells in the SVZ of the lateral ventricle and in the dentate gyrus. The number of cells in the granular layer of the dentate gyrus was counted. In the SVZ, only cells lining the ventricle wall were counted. It was noted if cells had left the SVZ and started migration. Micrographs (a)–(d) are representatives of PSA-NCAM+ cells in the SVZ. LV – lateral ventricle. Arrowhead – migrating cells. Quantifications are shown in the bar chart in e (whiskers show standard error of mean). Micrographs (f)–(i) are representatives of PSA-NCAM+ cells in the dentate gyrus. PoDG – polymorph dentate gyrus. Quantifications are shown in the bar chart in e (whiskers show standard error of mean). Scale bar: 50 lm.
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marker of neurite outgrowth and regeneration, was analysed (McGraw et al., 2002). When present in the dentate gyrus, ainternexin was localized in neurons in mice from all four groups. Only few infected mice disregarding grouping had a-internexin+ cells in the sub-granular layer in the dentate gyrus. In some of these mice, the a-internexin+ cells had a pyramidal structure indicating outgrowth and ramification in the hippocampal area. However, no significant differences were found between the groups (data not shown, 2-way ANOVA, p > 0.2).
4. Discussion In this study using a murine model of CM we demonstrated a significant, EPO-dependent increase in the number of NSC in the dentate gyrus and in the SVZ as well as a pathology-dependant migration from the SVZ. This coincided with a significant increase in survival and decreased clinical signs of CM relative to untreated mice. It has been suggested that EPO could be a useful, adjunct therapy for CM (Casals-Pascual et al., 2009; Picot et al., 2009), though this view has been challenged recently (Medana et al., 2009). Publications from several groups have shown its neuroprotective and life-saving properties in the murine CM model (Kaiser et al., 2006; Wiese et al., 2008). Here, we show that EPO also had regenerative potential in this model. A therapy that increases neuronal repair has potential to improve the neurological sequelae often experienced by survivors of CM (Carter et al., 2005; Idro et al., 2004; Kihara et al., 2006; Molyneux et al., 1989; Mung’Ala-Odera et al., 2004). In our model, EPO treatment resulted in an increased number of nestin+ NSC in the specific niches in both infected and healthy control mice. This finding contrasts with findings from a stroke model using unilateral middle cerebral artery occlusion in mice. In the stroke model EPO-treatment only resulted in an increased number of nestin+ NCS on the ipsilateral side but not in the contralateral unaffected hemisphere. Several factors in the experimental design could explain the discrepancy between our studies. In particular Wang et al. (2004) assessed the late effects, whereas our experimental setup focused on immediate effects of EPO treatment. The response to EPO treatment appears highly time dependent. Thus, Mash1, a transcription factor involved in neurogenesis, is upregulated immediately after EPO treatment and returns to normal levels within 24 h (Shingo et al., 2001), suggesting that some processes are only seen immediately after treatment. The upregulation of PSA-NCAM+ cells (Fig. 3) implies that stem cells were stimulated to mature into neurons in EPO treated CM mice (Bonfanti, 2006). This was detectable after only 4 days underscoring the regenerative potential induced by EPO treatment in acute, neurological syndromes (Lacerda-Queiroz et al., 2010; Lackner et al., 2006). In the stroke model the number of doublecortin+ neuronal precursor cells in the SVZ was increased 21 days after the last inoculation of EPO (Wang et al., 2004), and in a different model, PSA-NCAM+ cells in the SVZ had increased in numbers on day 10 post ischemia and at a later time point in the RMS and in the olfactory lobe (Iwai et al., 2003). Similarly, a-internexin levels peaked 7 days post injury (McGraw et al., 2002). Thus, we may only have seen the initial regenerative steps in our experimental setup, which may explain the lack of discernable changes in PSA-NCAM and a-internexin expression in the dentate gyrus. EPO administration increases the migration of neural stem cells after hypoxia (Shingo et al., 2001). This is in line with our findings, showing EPO stimulated migration only in mice with clinical signs of cerebral pathology. Knocking out the EPO receptor in the brain causes impaired NSC proliferation in the SVZ and results in reduced continuous migration of neuroblasts to the infarct area (Tsai et al., 2006). Another study has shown that EPO increases the number of
neurons generated from neurospheres in vitro whereas EPO had no effect on the number of astrocytes (Wang et al., 2004). EPOs antiapoptotic effects on neurons is hypothesised to be the driving force for reducing other types of neurodegeneration directly or indirectly (Ghezzi and Brines, 2004). Together with our data this suggests that EPO is a pro-survival factor decreasing the constant turn-over rate of NSC that normally cause the majority of the generated stem cells to undergo apoptosis before maturation (von Bohlen Und Halbach, 2007). Carbamylated EPO that does not bind to the EPOR (Leist et al., 2004) has shown equal neurogenic characteristics as EPO in vitro. Furthermore, without a functional EPOR, Mash1 was involved in neurogenesis (Wang et al., 2007), and one study suggests that only haematopoietic cell lines express EPOR (Sinclair et al., 2010). Thus, EPO-induced neurogenesis may at least partly be governed via its pleiotropic actions rather than interaction with the EPOR. In addition, the ultimate fate of EPO-induced stem cell maturation and migration in vivo remains to be clarified. Our model appears to be suitable for further studies of the signalling events and the down stream effects responsible for the neuroprotective properties of EPO. Overall the present data suggest that EPO causes proliferation of NSC and neuronal precursor cells in the SVZ in both healthy and malaria-infected mice. However, CM-associated pathology (Kaiser et al., 2006; Wiese et al., 2008), was needed to initiate stem cell migration. Administering EPO as adjunct treatment for human CM could potentially improve recovery from neurological sequelae in survivors. Acknowledgments The statistical advice from Biostatistical Department at University of Copenhagen is acknowledged. Also critical review by Professor David Arnot (University of Edinburgh) is appreciated. Funding from the Danish Agency for Science, Technology and Innovation, the Danish Medical Association Foundation, The Medical Society of Copenhagen, Carl og Ellen Hertzs Legat til Dansk Læge- og Naturvidenskab, Harboefonden, Hestehandler Ole Jacobsens Mindelegat, Hørslev-Fonden, Aase og Ejnar Danielsens Fond, Kathrine og Vigo Skovgaards Fond, Karen A. Tolstrups Fond, Fonden af 17-12-1981, Direktør Ib Henriksens Fond, and Th. Maigaard´s Eftf. Fru Lily Benthine Lunds Fond is highly appreciated. References Bennett, L., Yang, M., Enikolopov, G., Iacovitti, L., 2009. Circumventricular organs: a novel site of neural stem cells in the adult brain. Mol. Cell Neurosci. 41 (3), 337– 347. Bez, A., Corsini, E., Curti, D., Biggiogera, M., Colombo, A., Nicosia, R.F., Pagano, S.F., Parati, E.A., 2003. Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization. Brain Res. 993 (1,2), 18–29. Bonfanti, L., 2006. PSA-NCAM in mammalian structural plasticity and neurogenesis. Prog. Neurobiol. 80 (3), 129–164. Brines, M., Grasso, G., Fiordaliso, F., Sfacteria, A., Ghezzi, P., Fratelli, M., Latini, R., Xie, Q.W., Smart, J., Su-Rick, C.J., Pobre, E., Diaz, D., Gomez, D., Hand, C., Coleman, T., Cerami, A., 2004. Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor. Proc. Natl. Acad. Sci. USA 101 (41), 14907–14912. Brown, H., Hien, T.T., Day, N., Mai, N.T., Chuong, L.V., Chau, T.T., Loc, P.P., Phu, N.H., Bethell, D., Farrar, J., Gatter, K., White, N., Turner, G., 1999. Evidence of blood– brain barrier dysfunction in human cerebral malaria. Neuropathol. Appl. Neurobiol. 25 (4), 331–340. Carter, J.A., Mung’Ala-Odera, V., Neville, B.G., Murira, G., Mturi, N., Musumba, C., Newton, C.R., 2005. Persistent neurocognitive impairments associated with severe falciparum malaria in Kenyan children. J. Neurol. Neurosurg. Psychiatry 76 (4), 476–481. Casals-Pascual, C., Idro, R., Picot, S., Roberts, D.J., Newton, C.R., 2009. Can erythropoietin be used to prevent brain damage in cerebral malaria? Trends Parasitol. 25 (1), 30–36. Chen, Z.Y., Asavaritikrai, P., Prchal, J.T., Noguchi, C.T., 2007. Endogenous erythropoietin signaling is required for normal neural progenitor cell proliferation. J. Biol. Chem. 282 (35), 25875–25883.
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