Ccl11 promotes migration and proliferation of mouse neural progenitor cells – europe pmc article – europe pmc anoxia cerebral

Neonatal hypoxia-ischemia induces massive brain damage during the perinatal period, resulting in long-term consequences to central nervous system structural and functional maturation. Although neural progenitor cells (npcs) migrate through the parenchyma and home in to injury sites in the rodent brain, the molecular mechanisms are unknown. We examined the role of chemokines in mediating NPC migration after neonatal hypoxic-ischemic brain injury.Nine-day-old mice were exposed to a 120-minute hypoxia following unilateral carotid occlusion. Chemokine levels were quantified in mouse brain extract. Migration and proliferation assays were performed using embryonic and infant mouse npcs.The neonatal hypoxic-ischemic brain injury resulted in an ipsilateral lesion, which was extended to the cortical and striatal areas.Anoxia cerebral npcs migrated toward an injured area, where a marked increase of CC chemokines was detected. In vitro studies showed that incubation of npcs with recombinant mouse CCL11 promoted migration and proliferation. These effects were partly inhibited by a CCR3 antagonist, SB297006.Our data implicate an important effect of CCL11 for mouse npcs. The effective activation of npcs may offer a promising strategy for neuroregeneration in neonatal hypoxic-ischemic brain injury.


Perinatal hypoxia and ischemia can cause serious complications. In neonatal animal models, accumulating evidence suggests that hypoxic-ischemic brain damage influences the activation of endogenous neural progenitor cells (npcs) in the subventricular zone (SVZ) [ 1].Anoxia cerebral increased neurogenesis and migration of npcs toward the injury site has been observed in animal models of epilepsy, stroke, trauma, alzheimer’s disease, parkinson’s disease, and huntington’s disease [ 2]. Recent studies indicate that neurogenesis may play regenerative roles in response to central nervous system (CNS) injuries. Neurogenesis in the immature mammalian brain is highly modulated by excitotoxic insults [ 3, 4]. Moreover, npcs have been shown to be induced in the post-stroke human cerebral cortex for more than 1 month based on immunostaining of autopsied tissue from stroke patients [ 5, 6]. Although the origin of these npcs is uncertain, this raises the possibility of developing therapeutic strategies aimed at activating the neurogenic capacity in order to repair the damaged brain.Anoxia cerebral

Nevertheless, the molecular mechanisms for NPC activation and mobilization have not been clearly identified. It has been demonstrated in rodents that repulsive slit-robo signal orientated the migration of newly generated immature neurons through the rostral migratory stream (RMS) under normal conditions. RMS astrocytes express robo receptors and regulate the rapid migration of slit1-expressing new neurons [ 7]. Fibroblast growth factor 2 (FGF-2) and anosmin-1 increase SVZ NPC migration via epidermal growth factor receptor 1 (EGFR1) during pre- and postnatal rat development [ 8]. In contrast, in the injured brain, npcs from the SVZ migrate to injury sites through blood vessels for up to 1 year after injury [ 9, 10].Anoxia cerebral this directed NPC migration is considered the result of chemoattractive cues expressed from the injury site. Inflammatory mediators contribute to the pathological changes, which are sensitive to hypoxic-ischemic brain injury [ 11].

We hypothesized that inflammatory mediators were involved in the migration of npcs toward the injury site. The major role of chemokines is to act as a chemoattractant to guide the migration of immune cells. The CC chemokines induce recruitment of eosinophils, basophils, neutrophils, and monocytes, and play important roles in the regulation of many inflammatory conditions. In this study, we demonstrated that CCL11, one of CC chemokines highly expressed at brain injury sites, promoted the migration and proliferation of npcs.Anoxia cerebral these findings suggest that CCL11 may have important functions in the neonatal CNS.

Neonatal ischemia-reperfusion brain injury

The NOD/SCID (NOD.CB17-prkdc scid/J) mice were purchased from charles river laboratories japan inc. (kanagawa, japan). Experimental mouse models of neonatal ischemia-reperfusion brain injury were used based on a modified rice-vannucci model as previously described [ 12]. Briefly, 9-day-old postnatal NOD/SCID mice of both sexes (n = 48) were anesthetized with 2% isoflurane. The right common carotid artery (CCA) was occluded with an aneurysm clip (mizuho, tokyo, japan). The pups were placed in a hypoxia chamber held at 8% O 2 for 120 minutes. Reperfusion was achieved by unclamping the artery and exposing the pups to normoxic conditions.Anoxia cerebral pups were returned to their dams after the procedure. Sham-operated mice (n = 8) were subjected to CCA isolation alone and no brain injury. NOD/SCID mice aged 1, 2, 4, 6, and 8 weeks were used as normal control (n = 30). All animal experiments were performed under an institutionally approved protocol for the use of animal research at kochi university (approval number: J-00002).

Isolation and culture neural stem/progenitor cells

Embryonic npcs were isolated from pregnant NOD/SCID mice (n = 3) on E13.5 day of gestation. Striata were dissected from each embryo in phosphate-buffered saline (PBS) containing 0.6% glucose (sigma-aldrich) and penicillin-streptomycin (50 U/ml; life technologies). After dissection, the tissue was triturated to a single cell suspension using a transfer pipette.Anoxia cerebral cells were cultured in neurocult™ proliferation medium containing 20 ng/ml epidermal growth factor (EGF) (stemcell technologies inc., vancouver, BC, canada). NPCs from infant NOD/SCID mice were isolated from the ipsilateral and contralateral SVZ at 1 week post-injury (n = 5). The SVZ tissue was triturated with neural tissue dissociation solution (sumitomo dainippon pharma co., ltd, tokyo, japan). Cells were cultured in neurocult™ proliferation medium containing 20 ng/ml EGF, 10 ng/ml fibroblast growth factor (FGF) and 2 μl/ml heparin (stemcell technologies inc.). Cells were proliferated to generate neurospheres.

NPCs migration in the mouse brain

The experimental design is showed in fig. 1a. The mouse model of neonatal ischemia-reperfusion injury was induced at post-natal day 9.Anoxia cerebral MRI was performed 1 week after the brain injury. T2 hyperintensity abnormalities reflecting tissue edema were observed in the cortex and striatum of the mouse brain (fig. 1b). We evaluated the migration of npcs (dcx-positive cells) in the neonatal mice brain (fig. 1c). In the injured SVZ side, the number of dcx-positive cells began to increase rapidly at 1 week, and was stable thereafter for up to 7 weeks following the brain injury (fig. 1c, f). At 5 weeks post-injury, dcx-positive cells had migrated a long distance and had reached the core injury area (fig. 1d). In contrast, in the intact SVZ side, the number of dcx-positive cells was diminished with advancing age (fig. 1e). The number of PSA-NCAM-positive cells was also significantly increased in the injured side (additional file 2: figure s2a, b).Anoxia cerebral in the sham-operated mice, there was no difference in the number of dcx-positive cells between the intact side and the injury side (additional file 2: figure s2c, d).

In vitro migration assay

Neurospheres were generated from the embryo and infant mice brains, and these showed high expression of sex-determining region Y-box 2 (SOX2), ki-67, and no expression of the mature neuronal marker, neun (fig. 3a). NPCs cultured on poly-D-lysine expressed the neuroblast maker, PSA-NCAM (PSA-NCAM + cells: 68.7 ± 5.2%, PSA-NCAM – cells: 30.2 ± 2.9%) and dcx (fig. 3b, c). We then evaluated the effect of chemokines on NPC migration. Cell tracking with CCL11 over 24 hours of monitoring is shown in fig. 3d. The minus (leftward) direction of cell movement was defined as chemokine-induced migration (fig. 3e).Anoxia cerebral each color represents the trajectory of an individual cell. NPCs actively proliferated and migrated toward the CCL11 injected side (fig. 3e, additional file 1: figure S1). When migration distance and end points of embryonic npcs between each chemokine were compared, CCL11 significantly promoted embryonic npcs migration compared with PBS or other chemokines (fig. 3g, PBS: -0.77 ± 4.3 μm; CCL11: -65.9 ± 11.6 μm). Although CCL11 also promoted migration of npcs derived from the brain-injured infant mice, the end points of migration were decreased in the infant npcs compared to embryonic npcs. There was no significant difference between the injury side and intact side of npcs from infant brain (fig. 3f, h).Anoxia cerebral

NPCs migration and chemokines in an injured brain

A number of studies have shown activation and recruitment of npcs in animal models of ischemic brain injury [ 14– 16]. The number of npcs correlated with the volume of brain injury in the mouse model of stroke [ 17]. While low oxygen increased the proliferation of neural stem cells in vitro [ 18], oxygen glucose deprivation (OGD) decreased the growth of npcs and also decreased the phosphorylation of extracellular signal regulated kinase (ERK) [ 19]. The hypoxic-ischemic condition depletes the ATP content of the cells, compromising their metabolic activity. Various inflammatory mediators (cytokines, chemokines, and adhesion molecules) are produced from immune cells, endothelial cells, fibroblasts, and brain cells after ischemic injury.Anoxia cerebral directed NPC migration is considered to be the result of chemoattractive cues expressed from the injury site. Our chemokine quantification data demonstrated that CC chemokines were markedly elevated at 24 hours after injury, and were gradually reduced over 3 weeks post-injury in the infant mouse brain. Peculiarly, CCL2 and CCL11 were significantly upregulated in the injury side compared with the intact side up to 3 weeks post-injury. Chemokines are 8–14 kda small molecules mainly regulating immune cells trafficking during inflammatory responses. Chemokine-mediated signaling leads to cytoskeletal rearrangements that allow cell polarization toward the chemokine gradient. In recent years, chemokines have been reported to have non-immunological effects in the CNS, including regulation of cell proliferation, migration, survival, and synaptic activity [ 20].Anoxia cerebral adult npcs have been shown to express chemokine receptors, including CCR1-8, 10, and CXCR1-6 [ 21]. Among chemokine receptors, CXCL12/CXCR4 signaling is important for neural progenitor cell guidance and orientation in the developing mammalian brain [ 22, 23]. Following neural damage, CXCL12/CXCR4 signaling mediates specific migration of npcs to the ischemic damaged site [ 24]. Several signaling pathways may mediate CXCL12-induced NPC migration, including inositol 1,4,5-triphosphate, extracellular signal-regulated kinases 1/2, akt, c-jun N-terminal kinase, and intracellular calcium [ 25]. CCL2, CCL3, and CXCL1 also promote NPC migration to the striatum following quinolinic acid-induced lesion [ 26].Anoxia cerebral IFN-γ and IFN-β both inhibit cultured adult npcs proliferation, but only IFN-γ promotes neuronal differentiation [ 27]. In the present study, CCL2 and CCL3 were upregulated in the injury side in the hypoxic-ischemic brain of neonatal mice. The migration assay showed that CCL2 and CCL3 slightly promoted NPC migration. Since npcs are placed into the gap of μ − slide chemotaxis 3D and exposed to linear concentration gradients, the concentration of chemokines in the NPC area might be quite diluted. In consideration of a local inflammatory response, the induction effect may vary with the concentration of each chemokine in the injured mouse brain. The results of in vivo neutralizing assay suggests that the activation and migration of npcs seems to be controlled by an orchestration of cytokines include interleukins, interferons, growth factors and chemokines in the brain-injured mice.Anoxia cerebral

In another study, induction of striatal neurogenesis by the intraventricular administration of brain-derived neurotrophic factor (BDNF) and EGF promoted functional recovery in a mouse model of neonatal hypoxic-ischemic brain injury. In contrast, this BDNF/EGF-associated functional recovery was abolished in mice receiving a co-infusion of 2% cytosine-b-D-arabinofuranoside (ara-C), a mitotic inhibitor. These results indicate that the effect of functional recovery may be the result of newly generated neurons [ 28]. Identification of more factors responsible for recruitment of npcs to the injury site may lead to the development of therapies designed to promote functional recovery after neonatal ischemia-reperfusion brain injury.Anoxia cerebral

Role of CCL11 in CNS

Whereas chemokine signaling in immune cells has been studied in detail, the molecular mechanisms of chemokine-induced NPC activation are not completely understood. This study has shown that a chemokine, CCL11, could activate npcs in vitro. The CCL11 activity for npcs was exerted under EGF- and FGF-free conditions. CCL11 is traditionally associated with eosinophil recruitment and pro-inflammatory responses [ 29, 30]. The mechanism of how CCL11 affects the brain has not been determined. Recently, increased blood levels of CCL11 in aging have been shown to negatively regulate adult hippocampal neurogenesis. Systemic CCL11 administration decreased the numbers of dcx-positive cells in the dentate gyrus of young mice.Anoxia cerebral young mice that received CCL11 exhibited impaired learning and memory deficits [ 31]. In aged mice, plasma CCL11 protein levels were significantly increased in 18-month-old mice than in 21-month-old mice [ 32]. In the mice model of experimental cerebral malaria, CCL11 was elevated in the hippocampus and frontal cortex. Dcx-positive cells in the dentate gyrus were decreased in parallel with increased CCL11 [ 33]. Furthermore, CCL11 promoted the migration of microglia, and induced microglial production of reactive oxygen species. CCL11 triggers oxidative stress via microglial activation and potentiates glutamate-mediated neurotoxicity, which may be involved in the pathogenesis of various neurological disorders [ 34].Anoxia cerebral in one physiological study, the striatum had an early rapid-uptake phase for CCL11, which was the fastest among different brain regions [ 35]. CCL11 in the circulation can cross many regions of the blood-brain barrier (BBB). This suggests that blood-borne CCL11 may have important physiological functions in the CNS and implicates the BBB as an important regulator of the physiological versus pathological effects of this chemokine. Our results may reflect opposite effects of CCL11 on npcs, as recently reported in previous studies. In our study, the proliferation of npcs was promoted at a much higher concentration (1–5 μg/ml) of CCL11 compared to villeda’s study (10 ng/ml) [ 31]. Moreover, CCL11 might have differential effects in SVZ npcs compared to hippocampal npcs.Anoxia cerebral

Meanwhile, positive effects of CCL11 have also been reported in several studies. Adzemovic et al . Demonstrated that the CCL11/CCR3 pathway was associated with immune response modulation in a rat model of multiple sclerosis (MS). The milder EAE phenotype in the eae18b congenic strain of rat was accompanied by significantly lower myelin loss and reduced cumulative microglia activation. The congenic rats significantly upregulated CCL11 mrna in lymph nodes and spinal cord tissue [ 36]. Moreover, CCL11/CCR3 promotes the proliferation of oligodendrocyte precursor cells (opcs) and migration of smooth muscle cells (smcs) [ 37, 38]. Both of these cells are involved in the regeneration of damaged tissue.Anoxia cerebral in demyelinating diseases such as MS, remyelination is preceded by the division of endogenous NG2-positive opcs. The opcs migrate to sites of injury and differentiate into mature oligodendrocytes [ 39, 40]. In atherosclerosis, smcs proliferate and migrate to form part of the intimal plaque [ 41]. Furukawa et al. Showed that IL-10, an anti-inflammatory cytokine elevation was correlated with milder symptoms and that CCL11 elevation was correlated with slower disease progression in patients with amyotrophic lateral sclerosis (ALS), suggesting that those cytokines may confer neuroprotection against ALS [ 42]. Therefore, CCL11 may have both beneficial and harmful effects in the CNS that depend on factors such as concentration, anatomical location, and pathological condition.Anoxia cerebral

Additional files

Additional file 1: figure S1. (141M, zip)

The time-lapse movie of NPC migration. Images were captured every 15 minutes for 24 hours. (A) npcs did not migrate toward the PBS-injected side. (B) after CCL11 injection, npcs actively proliferated and migrated toward the CCL11-injected side. (ZIP 144651 kb) additional file 2: figure S2. (655K, pdf)

The immunostaining in the model mice of neonatal hypoxic-ischemic brain injury and the sham-operated mice. (A) PSA-NCAM immunostaining image of SVZ in the model mouse. Red: PSA-NCAM; blue: DAPI. Scale bar = 50 μm. (B) number of PSA-NCAM-positive cells per slice. N = 8. The data are presented as the mean numbers of PSA-NCAM-positive cells in the injury side and the intact side ± SD. * P 0.05. (C) dcx immunostaining image of SVZ in the sham-operated mouse.Anoxia cerebral N = 6. Green: dcx; blue: DAPI. Scale bar = 50 μm. (D) number of dcx-positive cells per slice. N = 8. The data are presented as the mean numbers of dcx-positive cells in the sham-operated side and the intact side ± SD. N.S. Not significant. (PDF 655 kb)