An extended window of opportunity for G-CSF treatment in cerebral ischemia

BACKGROUND: Granulocyte-colony stimulating factor (G-CSF) is known as a powerful regulator of white blood cell proliferation and differentiation in mammals. We, and others, have shown that G-CSF is effective in treating cerebral ischemia in rodents, both relating to infarct size as well as functional recovery. G-CSF and its receptor are expressed by neurons, and G-CSF regulates apoptosis and neurogenesis, providing a rational basis for its beneficial short- and long-term actions in ischemia. In addition, G-CSF may contribute to re-endothelialisation and arteriogenesis in the vasculature of the ischemic penumbra. In addition to these trophic effects, G-CSF is a potent neuroprotective factor reliably reducing infarct size in different stroke models. RESULTS: Here, we have further delayed treatment and studied effects of G-CSF on infarct volume in the middle cerebral artery occlusion (MCAO) model and functional outcome in the cortical photothrombotic model. In the MCAO model, we applied a single dose of 60 μg/kg bodyweight G-CSF in rats 4 h after onset of ischemia. Infarct volume was determined 24 h after onset of ischemia. In the rat photothrombotic model, we applied 10 μg/kg bodyweight G-CSF daily for a period of 10 days starting either 24 or 72 h after induction of ischemia. G-CSF both decreased acute infarct volume in the MCAO model, and improved recovery in the photothrombotic model at delayed timepoints. CONCLUSION: These data further strengthen G-CSF's profile as a unique candidate stroke drug, and provide an experimental basis for application of G-CSF in the post-stroke recovery phase

Enhancement of endogenous neurogenesis in ephrin-B3 deficient mice after transient focal cerebral ischemia

Cerebral ischemia stimulates endogenous neurogenesis. However, the functional relevance of this phenomenon remains unclear because of poor survival and low neuronal differentiation rates of newborn cells. Therefore, further studies on mechanisms regulating neurogenesis under ischemic conditions are required, among which ephrin-ligands and ephrin-receptors (Eph) are an interesting target. Although Eph/ephrin proteins like ephrin-B3 are known to negatively regulate neurogenesis under physiological conditions, their role in cerebral ischemia is largely unknown. We therefore studied neurogenesis, brain injury and functional outcome in ephrin-B3−/− (knockout) and ephrin-B3+/+ (wild-type) mice submitted to cerebral ischemia. Induction of stroke resulted in enhanced cell proliferation and neuronal differentiation around the lesion site of ephrin-B3−/− compared to ephrin-B3+/+ mice. However, prominent post-ischemic neurogenesis in ephrin-B3−/− mice was accompanied by significantly increased ischemic injury and motor coordination deficits that persisted up to 4 weeks. Ischemic injury in ephrin-B3−/− mice was associated with a caspase-3-dependent activation of the signal transducer and activator of transcription 1 (STAT1). Whereas inhibition of caspase-3 had no effect on brain injury in ephrin-B3+/+ animals, infarct size in ephrin-B3−/− mice was strongly reduced, suggesting that aggravated brain injury in these animals might involve a caspase-3-dependent activation of STAT1. In conclusion, post-ischemic neurogenesis in ephrin-B3−/− mice is strongly enhanced, but fails to contribute to functional recovery because of caspase-3-mediated aggravation of ischemic injury in these animals. Our results suggest that ephrin-B3 might be an interesting target for overcoming some of the limitations of further cell-based therapies in stroke

Growth hormone responsive neural precursor cells reside within the adult mammalian brain

The detection of growth hormone (GH) and its receptor in germinal regions of the mammalian brain prompted our investigation of GH and its role in the regulation of endogenous neural precursor cell activity. Here we report that the addition of exogenous GH significantly increased the expansion rate in long-term neurosphere cultures derived from wild-type mice, while neurospheres derived from GH null mice exhibited a reduced expansion rate. We also detected a doubling in the frequency of large (i.e. stem cell-derived) colonies for up to 120 days following a 7-day intracerebroventricular infusion of GH suggesting the activation of endogenous stem cells. Moreover, gamma irradiation induced the ablation of normally quiescent stem cells in GH-infused mice, resulting in a decline in olfactory bulb neurogenesis. These results suggest that GH activates populations of resident stem and progenitor cells, and therefore may represent a novel therapeutic target for age-related neurodegeneration and associated cognitive decline

Transcranial Low-Level Laser Therapy Improves Neurological Performance in Traumatic Brain Injury in Mice: Effect of Treatment Repetition Regimen

Low-level laser (light) therapy (LLLT) has been clinically applied around the world for a spectrum of disorders requiring healing, regeneration and prevention of tissue death. One area that is attracting growing interest in this scope is the use of transcranial LLLT to treat stroke and traumatic brain injury (TBI). We developed a mouse model of severe TBI induced by controlled cortical impact and explored the effect of different treatment schedules. Adult male BALB/c mice were divided into 3 broad groups (a) sham-TBI sham-treatment, (b) real-TBI sham-treatment, and (c) real-TBI active-treatment. Mice received active-treatment (transcranial LLLT by continuous wave 810 nm laser, 25 mW/cm[superscript 2], 18 J/cm[superscript 2], spot diameter 1 cm) while sham-treatment was immobilization only, delivered either as a single treatment at 4 hours post TBI, as 3 daily treatments commencing at 4 hours post TBI or as 14 daily treatments. Mice were sacrificed at 0, 4, 7, 14 and 28 days post-TBI for histology or histomorphometry, and injected with bromodeoxyuridine (BrdU) at days 21–27 to allow identification of proliferating cells. Mice with severe TBI treated with 1-laser Tx (and to a greater extent 3-laser Tx) had significant improvements in neurological severity score (NSS), and wire-grip and motion test (WGMT). However 14-laser Tx provided no benefit over TBI-sham control. Mice receiving 1- and 3-laser Tx had smaller lesion size at 28-days (although the size increased over 4 weeks in all TBI-groups) and less Fluoro-Jade staining for degenerating neurons (at 14 days) than in TBI control and 14-laser Tx groups. There were more BrdU-positive cells in the lesion in 1- and 3-laser groups suggesting LLLT may increase neurogenesis. Transcranial NIR laser may provide benefit in cases of acute TBI provided the optimum treatment regimen is employed.National Institutes of Health (U.S.) (Grant R01AI050875)Center for Integration of Medicine and Innovative Technology (DAMD17-02-2-0006)United States. Dept. of Defense. Congressionally Directed Medical Research Programs (W81XWH-09-1-0514)United States. Air Force Office of Scientific Research. Military Photomedicine Program (FA9550-11-1-0331

Absence of Colony Stimulation Factor-1 Receptor Results in Loss of Microglia, Disrupted Brain Development and Olfactory Deficits

The brain contains numerous mononuclear phagocytes called microglia. These cells express the transmembrane tyrosine kinase receptor for the macrophage growth factor colony stimulating factor-1 (CSF-1R). Using a CSF-1R-GFP reporter mouse strain combined with lineage defining antibody staining we show in the postnatal mouse brain that CSF-1R is expressed only in microglia and not neurons, astrocytes or glial cells. To study CSF-1R function we used mice homozygous for a null mutation in the Csflr gene. In these mice microglia are >99% depleted at embryonic day 16 and day 1 post-partum brain. At three weeks of age this microglial depletion continues in most regions of the brain although some contain clusters of rounded microglia. Despite the loss of microglia, embryonic brain development appears normal but during the post-natal period the brain architecture becomes perturbed with enlarged ventricles and regionally compressed parenchyma, phenotypes most prominent in the olfactory bulb and cortex. In the cortex there is increased neuronal density, elevated numbers of astrocytes but reduced numbers of oligodendrocytes. Csf1r nulls rarely survive to adulthood and therefore to study the role of CSF-1R in olfaction we used the viable null mutants in the Csf1 (Csf1op) gene that encodes one of the two known CSF-1R ligands. Food-finding experiments indicate that olfactory capacity is significantly impaired in the absence of CSF-1. CSF-1R is therefore required for the development of microglia, for a fully functional olfactory system and the maintenance of normal brain structure

Human Mesenchymal Stem Cells Prolong Survival and Ameliorate Motor Deficit through Trophic Support in Huntington's Disease Mouse Models

We investigated the therapeutic potential of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in Huntington's disease (HD) mouse models. Ten weeks after intrastriatal injection of quinolinic acid (QA), mice that received hBM-MSC transplantation showed a significant reduction in motor function impairment and increased survival rate. Transplanted hBM-MSCs were capable of survival, and inducing neural proliferation and differentiation in the QA-lesioned striatum. In addition, the transplanted hBM-MSCs induced microglia, neuroblasts and bone marrow-derived cells to migrate into the QA-lesioned region. Similar results were obtained in R6/2-J2, a genetically-modified animal model of HD, except for the improvement of motor function. After hBM-MSC transplantation, the transplanted hBM-MSCs may integrate with the host cells and increase the levels of laminin, Von Willebrand Factor (VWF), stromal cell-derived factor-1 (SDF-1), and the SDF-1 receptor Cxcr4. The p-Erk1/2 expression was increased while Bax and caspase-3 levels were decreased after hBM-MSC transplantation suggesting that the reduced level of apoptosis after hBM-MSC transplantation was of benefit to the QA-lesioned mice. Our data suggest that hBM-MSCs have neural differentiation improvement potential, neurotrophic support capability and an anti-apoptotic effect, and may be a feasible candidate for HD therapy

CXCL12-Mediated Guidance of Migrating Embryonic Stem Cell-Derived Neural Progenitors Transplanted into the Hippocampus

Stem cell therapies for neurodegenerative disorders require accurate delivery of the transplanted cells to the sites of damage. Numerous studies have established that fluid injections to the hippocampus can induce lesions in the dentate gyrus (DG) that lead to cell death within the upper blade. Using a mouse model of temporal lobe epilepsy, we previously observed that embryonic stem cell-derived neural progenitors (ESNPs) survive and differentiate within the granule cell layer after stereotaxic delivery to the DG, replacing the endogenous cells of the upper blade. To investigate the mechanisms for ESNP migration and repair in the DG, we examined the role of the chemokine CXCL12 in mice subjected to kainic acid-induced seizures. We now show that ESNPs transplanted into the DG show extensive migration through the upper blade, along the septotemporal axis of the hippocampus. Seizures upregulate CXCL12 and infusion of the CXCR4 antagonist AMD3100 by osmotic minipump attenuated ESNP migration. We also demonstrate that seizures promote the differentiation of transplanted ESNPs toward neuronal rather than astrocyte fates. These findings suggest that ESNPs transplanted into the adult rodent hippocampus migrate in response to cytokine-mediated signals

Caveolin-1 Plays a Crucial Role in Inhibiting Neuronal Differentiation of Neural Stem/Progenitor Cells via VEGF Signaling-Dependent Pathway

In the present study, we aim to elucidate the roles of caveolin-1(Cav-1), a 22 kDa protein in plasma membrane invaginations, in modulating neuronal differentiation of neural progenitor cells (NPCs). In the hippocampal dentate gyrus, we found that Cav-1 knockout mice revealed remarkably higher levels of vascular endothelial growth factor (VEGF) and the more abundant formation of newborn neurons than wild type mice. We then studied the potential mechanisms of Cav-1 in modulating VEGF signaling and neuronal differentiation in isolated cultured NPCs under normoxic and hypoxic conditions. Hypoxic embryonic rat NPCs were exposed to 1% O2 for 24 h and then switched to 21% O2 for 1, 3, 7 and 14 days whereas normoxic NPCs were continuously cultured with 21% O2. Compared with normoxic NPCs, hypoxic NPCs had down-regulated expression of Cav-1 and up-regulated VEGF expression and p44/42MAPK phosphorylation, and enhanced neuronal differentiation. We further studied the roles of Cav-1 in inhibiting neuronal differentiation by using Cav-1 scaffolding domain peptide and Cav-1-specific small interfering RNA. In both normoxic and hypoxic NPCs, Cav-1 peptide markedly down-regulated the expressions of VEGF and flk1, decreased the phosphorylations of p44/42MAPK, Akt and Stat3, and inhibited neuronal differentiation, whereas the knockdown of Cav-1 promoted the expression of VEGF, phosphorylations of p44/42MAPK, Akt and Stat3, and stimulated neuronal differentiation. Moreover, the enhanced phosphorylations of p44/42MAPK, Akt and Stat3, and neuronal differentiation were abolished by co-treatment of VEGF inhibitor V1. These results provide strong evidence to prove that Cav-1 can inhibit neuronal differentiation via down-regulations of VEGF, p44/42MAPK, Akt and Stat3 signaling pathways, and that VEGF signaling is a crucial target of Cav-1. The hypoxia-induced down-regulation of Cav-1 contributes to enhanced neuronal differentiation in NPCs