The role of granulocyte-colony stimulating factor (G-CSF) in the healthy brain: a characterization of G-CSF-deficient mice.

Granulocyte-colony stimulating factor (G-CSF) is a hematopoietic growth factor that controls proliferation and differentiation of neural stem cells. Although recent studies have begun to explore G-CSF-related mechanisms of action in various disease models, little is known about its function in the healthy brain. In the present study, the effect of G-CSF deficiency on memory formation and motor skills was investigated. The impact of G-CSF deficiency on the structural integrity of the hippocampus was evaluated by analyzing the generation of doublecortin-expressing cells, the amount of bromodeoxyurine-labeled cells, the dendritic complexity in hippocampal neurons, the binding densities of NMDA and GABA(A) receptors and the induction of long-term potentiation (LTP). G-CSF deficiency caused a disruption in memory formation and in the development of motor skills. These impairments were associated with reduced ligand binding densities of NMDA receptors in hippocampal subfields CA3 and the dentate gyrus. The reduced excitation was potentiated by increased ligand binding densities of GABA(A) receptors resulting in a relative shift in favor of inhibition and impaired behavioral performance. These alterations were accompanied by impaired induction of LTP in the CA1 region. Moreover, G-CSF deficiency led to decreased dendritic complexity in hippocampal neurons in the dentate gyrus and the CA1 region. G-CSF deficiency also caused a reduction of neuronal precursor cells in the dentate gyrus. These findings confirm G-CSF as an essential neurotrophic factor, and point to a role in the proliferation, differentiation and functional integration of neural cells necessary for the structural and functional integrity of the hippocampal formation.

Prevention of an increase in cortical ligand binding to AMPA receptors may represent a novel mechanism of endogenous brain protection by G-CSF after ischemic stroke.

PURPOSE: Using G-CSF deficient mice we recently demonstrated neuroprotective properties of endogenous G-CSF after ischemic stroke. The present follow-up study was designed to check, whether specific alterations in ligand binding densities of excitatory glutamate or inhibitory GABAA receptors may participate in this effect. METHODS: Three groups of female mice were subjected to 45 minutes of MCAO: wildtype, G-CSF deficient and G-CSF deficient mice substituted with G-CSF. Infarct volumes were determined after 24 hours and quantitative in vitro receptor autoradiography was performed using [3H]MK-801, [3H]AMPA and [3H]muscimol for labeling of NMDA, AMPA and GABAA receptors, respectively. Ligand binding densities were analyzed in regions in the ischemic core, peri-infarct areas and corresponding contralateral regions. RESULTS: Infarct volumes did not significantly differ between the experimental groups. Ligand binding densities of NMDA and GABAA receptors were widely in the same range. However, AMPA receptor binding densities in G-CSF deficient mice were substantially enhanced compared to wildtype mice. G-CSF substitution in mice lacking G-CSF largely reversed this effect. CONCLUSIONS: Although infarct volumes did not differ 24 hours after ischemia the increase of AMPA receptor binding densities in G-CSF deficient mice may explain the bigger infarcts previously observed at later time-points with the same stroke model.

Sodium-dependent vitamin C transporter 2 (SVCT2) expression and activity in brain capillary endothelial cells after transient ischemia in mice.

Expression and transport activity of Sodium-dependent Vitamin C Transporter 2 (SVCT2) was shown in various tissues and organs. Vitamin C was shown to be cerebroprotective in several animal models of stroke. Data on expression, localization and transport activity of SVCT2 after cerebral ischemia, however, has been scarce so far. Thus, we studied the expression of SVCT2 after middle cerebral artery occlusion (MCAO) in mice by immunohistochemistry. We found an upregulation of SVCT2 after stroke. Co-stainings with Occludin, Von-Willebrand Factor and CD34 demonstrated localization of SVCT2 in brain capillary endothelial cells in the ischemic area after stroke. Time-course analyses of SVCT2 expression by immunohistochemistry and western blots showed upregulation in the subacute phase of 2-5 days. Radioactive uptake assays using (14)C-labelled ascorbic acid showed a significant increase of ascorbic acid uptake into the brain after stroke. Taken together, these results provide evidence for the expression and transport activity of SVCT2 in brain capillary endothelial cells after transient ischemia in mice. These results may lead to the development of novel neuroprotective strategies in stroke therapy.

Investigation of neuronal progenitor cell origin after transient focal cerebral ischemia in mice.

Following cerebral ischemia both neuronal precursors and hematogenous cells migrate along chemokine gradients towards the injured tissue. Bone marrow derived cells are involved in the stroke related inflammatory and restaurative processes and newly born neurons are known to proliferate and migrate from the subventricular zone to the ischemic lesion. In the present study, we investigated whether hematogenous cells contribute to subpopulations of neuronal precursors using green fluorescent protein-transgenic bone marrow chimeric mice. In our experiments we found no blood-borne neuronal precursors within the ischemic site indicating that detected neuronal progenitor cells are only of brain parenchymal origin.

Effects of G-CSF treatment on neutrophil mobilization and neurological outcome after transient focal ischemia.

Several recent studies demonstrated beneficial effects of G-CSF treatment (granulocyte colony-stimulating factor) in various CNS disease. Possible mechanisms underlying this activity are neuroprotection, anti-apoptosis, angiogenesis and anti-inflammation. Hence, we investigated the efficacy of G-CSF administration in experimental stroke by determining infarct volume and neurological score in wildtype, G-CSF-deficient and G-CSF-treated G-CSF-deficient mice. Besides, cerebral ischemia is followed by an upregulation of endothelial adhesion molecules which promote leukocyte recruitment to the injured area. In combination with G-CSF-induced leukocytosis, increased peripheral neutrophils could aggregate within microvasculature and additionally impair blood perfusion of the ischemic tissue. Therefore, we analyzed the neutrophil counts in both vessel and tissue compartment 2 and 5 days post-stroke by immunohistochemistry. Here we show that G-CSF deficiency leads to increased infarct volumes, whereas G-CSF substitution revokes detrimental effects by reducing lesion size and enhancing neurological outcome compared to untreated animals. Administration of G-CSF is accompanied by significant increase of circulating neutrophils 2 days post-ischemia but leukocytosis is restricted to the vessel compartment and has no deleterious effect on lesion formation and functional recovery. These observations are likely to be important for therapeutic targeting of G-CSF-mediated neuroprotection in stroke.

Granulocyte-colony stimulating factor for stroke treatment: mechanisms of action and efficacy in preclinical studies.

G-CSF is widely employed for the treatment of chemotherapy-induced neutropenia. Recently, neuroprotective effects of G-CSF in animal stroke models were discovered including infarct size reduction and enhancement of functional recovery. The underlying mechanisms of action of G-CSF in ischemia appear to be a direct anti-apoptotic activity in neurons and a neurogenesis inducing capacity. Additional effects may be based on the stimulation of new blood-vessel formation, the stimulation of immunocompetence and -modulation as well as on bone marrow mobilization. In addition to a discussion of these mechanisms, we will review the available preclinical studies and analyze their impact on the overall efficacy of G-CSF in experimental stroke.

Endogenous brain protection by granulocyte-colony stimulating factor after ischemic stroke.

Several lines of evidence have demonstrated beneficial effects of the hematopoietic factor G-CSF in experimental stroke. A conclusive demonstration of this effect in G-CSF deficient mice is, however, lacking. We therefore investigated the effect of G-CSF deficiency on infarct volumes, functional recovery, mRNA and protein expression of the matrix metalloproteinase 9 (MMP-9) after stroke. Furthermore we tested the efficacy of G-CSF substitution in G-CSF deficient animals to prevent the potential consequences of G-CSF deficiency. In the present study experimental stroke was induced in female non-treated wildtype (wt), G-CSF deficient mice and G-CSF substituted G-CSF deficient mice followed by assessment of infarct volumes, neurological outcome and sensorimotor function. In addition, immunohistochemistry and real-time PCR of the peri-ischemic area were performed. G-CSF deficient mice showed increased infarct volumes, whereas G-CSF substituted mice had a remarkable reduction in lesion size compared to wt mice. These findings are accompanied by an improvement in neurological and sensorimotor function. G-CSF deficiency resulted in an upregulation of MMP-9 in the direct peri-ischemic tissue. Treatment with G-CSF suppressed the upregulation of MMP-9. Taken together, G-CSF deficiency clearly resulted in enlarged infarct volumes, and worsened neurological outcome. G-CSF substitution abolished these negative effects, led to significant reduced lesion volumes, and improved neurological outcome. G-CSF mediated suppression of MMP-9 further demonstrates that endogenous G-CSF plays a significant role in brain protective mechanisms. We have shown for the first time that endogenous G-CSF is required for brain recovery mechanisms after stroke.