Hematopoietic Growth Factors Regulate the Entry of Monocytes into the Adult Brain via Chemokine Receptor CCR5.

Monocytes are circulating macrophage precursors generated from bone marrow hematopoietic stem cells. In adults, monocytes continuously replenish cerebral border-associated macrophages under physiological conditions. Monocytes also rapidly infiltrate the brain in pathological settings. The mechanisms of recruiting monocyte-derived macrophages into the brain under pathological conditions have been extensively studied. However, it remains unclear how monocytes enter the brain to renew border-associated macrophages under physiological conditions. Using both in vitro and in vivo approaches, this study reveals that a combination of two hematopoietic growth factors, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF), complementarily and synergistically enhances the adhesion of monocytes to cerebral endothelial cells in a dose-dependent manner. Cysteine-cysteine chemokine receptor 5 (CCR5) in brain endothelial cells, but not the cell adhesion molecules mediating neuroinflammation-related infiltration of monocyte-derived macrophages, modulates SCF+G-CSF-enhanced monocyte-endothelial cell adhesion. Blocking CCR5 or genetically deleting CCR5 reduces monocyte-endothelial cell adhesion induced by SCF+G-CSF. The SCF+G-CSF-enhanced recruitment of bone marrow-derived monocytes/macrophages into the cerebral perivascular space is also reduced in adult CCR5 knockout mice. This study demonstrates the role of SCF and G-CSF in regulating the entry of monocytes into the adult brain to replenish perivascular macrophages.

Single-Cell RNA Sequencing Reveals Immunomodulatory Effects of Stem Cell Factor and Granulocyte Colony-Stimulating Factor Treatment in the Brains of Aged APP/PS1 Mice.

Alzheimer's disease (AD) leads to progressive neurodegeneration and dementia. AD primarily affects older adults with neuropathological changes including amyloid-beta (Aß) deposition, neuroinflammation, and neurodegeneration. We have previously demonstrated that systemic treatment with combined stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) reduces the Aß load, increases Aß uptake by activated microglia and macrophages, reduces neuroinflammation, and restores dendrites and synapses in the brains of aged APPswe/PS1dE9 (APP/PS1) mice. However, the mechanisms underlying SCF+G-CSF-enhanced brain repair in aged APP/PS1 mice remain unclear. This study used a transcriptomic approach to identify the potential mechanisms by which SCF+G-CSF treatment modulates microglia and peripheral myeloid cells to mitigate AD pathology in the aged brain. After injections of SCF+G-CSF for 5 consecutive days, single-cell RNA sequencing was performed on CD11b+ cells isolated from the brains of 28-month-old APP/PS1 mice. The vast majority of cell clusters aligned with transcriptional profiles of microglia in various activation states. However, SCF+G-CSF treatment dramatically increased a cell population showing upregulation of marker genes related to peripheral myeloid cells. Flow cytometry data also revealed an SCF+G-CSF-induced increase of cerebral CD45high/CD11b+ active phagocytes. SCF+G-CSF treatment robustly increased the transcription of genes implicated in immune cell activation, including gene sets that regulate inflammatory processes and cell migration. The expression of S100a8 and S100a9 was robustly enhanced following SCF+G-CSF treatment in all CD11b+ cell clusters. Moreover, the topmost genes differentially expressed with SCF+G-CSF treatment were largely upregulated in S100a8/9-positive cells, suggesting a well-conserved transcriptional profile related to SCF+G-CSF treatment in resident and peripherally derived CD11b+ immune cells. This S100a8/9-associated transcriptional profile contained notable genes related to pro-inflammatory and anti-inflammatory responses, neuroprotection, and Aß plaque inhibition or clearance. Altogether, this study reveals the immunomodulatory effects of SCF+G-CSF treatment in the aged brain with AD pathology, which will guide future studies to further uncover the therapeutic mechanisms.

Single cell RNA sequencing reveals immunomodulatory effects of stem cell factor and granulocyte colony-stimulating factor treatment in the brains of aged APP/PS1 mice.

Alzheimers disease leads to progressive neurodegeneration and dementia. Alzheimers disease primarily affects older adults with neuropathological changes including amyloid beta deposition, neuroinflammation, and neurodegeneration. We have previously demonstrated that systemic treatment with combined stem cell factor, SCF, and granulocyte colony stimulating factor, GCSF, reduces amyloid beta load, increases amyloid beta uptake by activated microglia and macrophages, reduces neuroinflammation, and restores dendrites and synapses in the brains of aged APP-PS1 mice. However, the mechanisms underlying SCF-GCSF-enhanced brain repair in aged APP-PS1 mice remain unclear. This study used a transcriptomic approach to identify potential mechanisms by which SCF-GCSF treatment modulates microglia and peripheral myeloid cells to mitigate Alzheimers disease pathology in the aged brain. After injections of SCF-GCSF for 5 consecutive days, single cell RNA sequencing was performed on CD11b positive cells isolated from the brains of 28-month-old APP-PS1 mice. The vast majority of cell clusters aligned with transcriptional profiles of microglia in various activation states. However, SCF-GCSF treatment dramatically increased a cell population showing upregulation of marker genes related to peripheral myeloid cells. Flow cytometry data also revealed an SCF-GCSF-induced increase of cerebral CD45high-CD11b positive active phagocytes. SCF-GCSF treatment robustly increased the transcription of genes implicated in immune cell activation, including gene sets that regulate inflammatory processes and cell migration. Expression of S100a8 and S100a9 were robustly enhanced following SCF-GCSF treatment in all CD11b positive cell clusters. Moreover, the topmost genes differentially expressed with SCF-GCSF treatment were largely upregulated in S100a8-S100a9 positive cells, suggesting a well-conserved transcriptional profile related to SCF-GCSF treatment in resident and peripherally derived CD11b positive immune cells. This S100a8-S100a9-associated transcriptional profile contained notable genes related to proinflammatory and antiinflammatory responses, neuroprotection, and amyloid beta plaque inhibition or clearance. Altogether, this study reveals immunomodulatory effects of SCF-GCSF treatment in the aged brain with Alzheimers disease pathology, which will guide future studies to further uncover the therapeutic mechanisms.

Stem Cell Factor and Granulocyte Colony-Stimulating Factor Promote Remyelination in the Chronic Phase of Severe Traumatic Brain Injury.

Severe traumatic brain injury (TBI) causes long-term disability and death in young adults. White matter is vulnerable to TBI damage. Demyelination is a major pathological change of white matter injury after TBI. Demyelination, which is characterized by myelin sheath disruption and oligodendrocyte cell death, leads to long-term neurological function deficits. Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) treatments have shown neuroprotective and neurorestorative effects in the subacute and chronic phases of experimental TBI. Our previous study has revealed that combined SCF and G-CSF treatment (SCF + G-CSF) enhances myelin repair in the chronic phase of TBI. However, the long-term effect and mechanism of SCF + G-CSF-enhanced myelin repair remain unclear. In this study, we uncovered persistent and progressive myelin loss in the chronic phase of severe TBI. SCF + G-CSF treatment in the chronic phase of severe TBI enhanced remyelination in the ipsilateral external capsule and striatum. The SCF + G-CSF-enhanced myelin repair is positively correlated with the proliferation of oligodendrocyte progenitor cells in the subventricular zone. These findings reveal the therapeutic potential of SCF + G-CSF in myelin repair in the chronic phase of severe TBI and shed light on the mechanism underlying SCF + G-CSF-enhanced remyelination in chronic TBI.

Stem cell factor and granulocyte colony-stimulating factor promote remyelination in the chronic phase of severe traumatic brain injury.

Severe traumatic brain injury (TBI) causes long-term disability and death in young adults. White matter is vulnerable to TBI damage. Demyelination is a major pathological change of white matter injury after TBI. Demyelination which is characterized by myelin sheath disruption and oligodendrocyte cell death leads to long-term neurological function deficits. Stem cell factor (SCF) and granulocyte colonyâ€"stimulating factor (G-CSF) treatments have shown neuroprotective and neurorestorative effects in the subacute and chronic phases of experimental TBI. Our previous study has revealed that combined SCF and G-CSF treatment (SCF+G-CSF) enhances myelin repair in the chronic phase of TBI. However, the long-term effect and mechanism of SCF+G-CSF-enhanced myelin repair remain unclear. In this study, we uncovered persistent and progressive myelin loss in the chronic phase of severe TBI. SCF+G-CSF treatment in the chronic phase of severe TBI enhanced remyelination in the ipsilateral external capsule and striatum. The SCF+G-CSF-enhanced myelin repair is positively correlated with the proliferation of oligodendrocyte progenitor cells in the subventricular zone. These findings reveal the therapeutic potential of SCF+G-CSF in myelin repair in the chronic phase of severe TBI and shed light on the mechanism underlying SCF+G-CSF-enhanced remyelination in chronic TBI.

SCF + G-CSF treatment in the chronic phase of severe TBI enhances axonal sprouting in the spinal cord and synaptic pruning in the hippocampus.

Traumatic brain injury (TBI) is a major cause of long-term disability in young adults. An evidence-based treatment for TBI recovery, especially in the chronic phase, is not yet available. Using a severe TBI mouse model, we demonstrate that the neurorestorative efficacy of repeated treatments with stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) (SCF + G-CSF) in the chronic phase is superior to SCF + G-CSF single treatment. SCF + G-CSF treatment initiated at 3 months post-TBI enhances contralesional corticospinal tract sprouting into the denervated side of the cervical spinal cord and re-balances the TBI-induced overgrown synapses in the hippocampus by enhancing microglial function of synaptic pruning. These neurorestorative changes are associated with SCF + G-CSF-improved somatosensory-motor function and spatial learning. In the chronic phase of TBI, severe TBI-caused microglial degeneration in the cortex and hippocampus is ameliorated by SCF + G-CSF treatment. These findings reveal the therapeutic potential and possible mechanism of SCF + G-CSF treatment in brain repair during the chronic phase of severe TBI.

Brain-derived CCR5 Contributes to Neuroprotection and Brain Repair after Experimental Stroke.

Chemokine (C-C motif) receptor 5 (CCR5) is expressed not only in the immune cells but also in cerebral cells such as neurons, glia, and vascular cells. Stroke triggers high expression of CCR5 in the brain. However, the role of CCR5 in stroke remains unclear. In this study, using bone marrow chimeras we have determined the involvement of brain-derived or bone marrow-derived CCR5 in neuroprotection and brain repair after experimental stroke. CCR5-/- mice that received either wild-type (WT) or CCR5-/- bone marrow transplantation showed larger infarction sizes than the WT mice that received either WT or CCR5-/- bone marrow transplantation in both the acute (48h) and subacute (2 months) phases after cerebral cortical ischemia, suggesting that the lack of CCR5 in the brain leads to severe brain damage after stroke. However, the lack of CCR5 in the bone marrow-derived cells did not affect infarction size. The impairments of somatosensory-motor function and motor coordination were exacerbated in the mice lacking CCR5 in the brain. At 2 months post-stroke, increased degenerative neurons, decreased dendrites and synapses, decreased Iba1+ microglia/ macrophages, reduced myelination and CNPase+ oligodendrocytes in the peri-infarct cortex were observed in the mice lacking CCR5 in the brain. These pathological changes are significantly correlated with the increased infarction size and exacerbated neurological deficits. These data suggest that brain-derived CCR5 plays a key role in neuroprotection and brain repair in the subacute phase of stroke. This study reveals a novel role of CCR5 in stroke, which sheds new light on post-stroke pathomechanism.

The contribution of stem cell factor and granulocyte colony-stimulating factor in reducing neurodegeneration and promoting neurostructure network reorganization after traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of death and disability in young adults worldwide. TBI-induced long-term cognitive deficits represent a growing clinical problem. Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) are involved in neuroprotection and neuronal plasticity. However, the knowledge concerning reparative efficacy of SCF + G-CSF treatment in post-acute TBI recovery remains incomplete. This study aims to determine the efficacy of SCF + G-CSF on post-acute TBI recovery in young adult mice. The controlled cortical impact model of TBI was used for inducing a severe damage in the motor cortex of the right hemisphere in 8-week-old male C57BL mice. SCF + G-CSF treatment was initiated 3 weeks after induction of TBI. Severe TBI led to persistent motor functional deficits (Rota-Rod test) and impaired spatial learning function (water maze test). SCF + G-CSF treatment significantly improved the severe TBI-impaired spatial learning function 6 weeks after treatment. TBI also caused significant increases of Fluoro-Jade C positive degenerating neurons in bilateral frontal cortex, striatum and hippocampus, and significant reductions in MAP2+ apical dendrites and overgrowth of SMI312+ axons in peri-TBI cavity frontal cortex and in the ipsilateral hippocampal CA1 at 24 weeks post-TBI. SCF + G-CSF treatment significantly reduced TBI-induced neurodegeneration in the contralateral frontal cortex and hippocampal CA1, increased MAP2+ apical dendrites in the peri-TBI cavity frontal cortex, and prevented TBI-induced axonal overgrowth in both the peri-TBI cavity frontal cortex and ipsilateral hippocampal CA1.These findings reveal a novel pathology of axonal overgrowth after severe TBI and demonstrate a therapeutic potential of SCF + G-CSF in ameliorating severe TBI-induced long-term neuronal pathology, neurostructural network malformation, and impairments in spatial learning.

Long-term beneficial effects of hematopoietic growth factors on brain repair in the chronic phase of severe traumatic brain injury.

Severe traumatic brain injury (TBI) is the major cause of long-term, even life-long disability and cognitive impairments in young adults. The lack of therapeutic approaches to improve recovery in the chronic phase of severe TBI is a big challenge to the medical research field. Using a single severe TBI model in young adult mice, this study examined the restorative efficacy of two hematopoietic growth factors, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF), on brain repair in the chronic phase of TBI. SCF and G-CSF alone or combination (SCF + G-CSF) treatment was administered at 3 months post-TBI. Functional recovery was evaluated by neurobehavioral tests during the period of 21 weeks after treatment. Neuropathology was examined 22 weeks after treatment. We observed that severe TBI caused persistent impairments in spatial learning/memory and somatosensory-motor function, long-term and widespread neuropathology, including dendritic reduction, decrease and overgrowth of axons, over-generated excitatory synapses, and demyelination in the cortex, hippocampus and striatum. SCF, G-CSF, and SCF + G-CSF treatments ameliorated severe TBI-induced widespread neuropathology. SCF + G-CSF treatment showed superior efficacy in improving long-term functional outcome, enhancing neural plasticity, rebalancing neural structure networks disturbed by severe TBI, and promoting remyelination. These novel findings demonstrate the therapeutic potential of SCF and G-CSF in enhancing recovery in the chronic phase of severe TBI .

S100 Calcium-Binding Protein A9 Knockout Contributes to Neuroprotection and Functional Improvement after Traumatic Brain Injury.

S100 calcium-binding protein A9 (S100a9), a proinflammatory protein, has been shown to be involved in the development of neuroinflammatory disorders and neurodegenerative diseases. Upregulation of S100a9 in the brain during acute brain injury has been proposed to be associated with acute neuroinflammation. However, it remains unclear whether eliminating S100a9 expression will show beneficial outcomes after traumatic brain injury (TBI). Using S100a9 knockout mice, this study has demonstrated that S100a9 deletion ameliorates post-TBI anxiety, improves TBI-impaired motor and cognitive function, reduces lesion size, prevents perilesional neuron loss and neurodegeneration, diminishes neuroinflammation and TBI-induced neurogenesis, and enhances perilesional expression of neuroplasticity protein. These findings suggest that S100a9 plays a detrimental role in TBI. Genetic deletion of S100a9 enhances neuroprotection and improves functional outcome after TBI. This study sheds light on the pathological involvement of S100a9 in TBI, which would provide a new therapeutic target to minimize TBI-induced brain damage.

Stem cell factor and granulocyte colony-stimulating factor promote brain repair and improve cognitive function through VEGF-A in a mouse model of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy (CADASIL) is a cerebral small vascular disease caused by NOTCH3 gene mutation in vascular smooth muscle cells (VSMCs), leading to ischemic stroke and vascular dementia. To date, the pathogenesis of CADASIL remains poorly understood, and there is no treatment that can slow the progression of CADASIL. Using a transgenic mouse model of CADASIL (TgNotch3R90C), this study reveals novel findings for understanding CADASIL pathogenesis that decreased cerebral vascular endothelial growth factor (VEGF/VEGF-A) is linked to reduced cerebral blood vessel density. Reduced endothelial cell (EC) proliferation and angiogenesis are seen in TgNotch3R90C mouse brain-isolated ECs. Decreased dendrites, axons, and synapses in the somatosensory and motor cortex layer 2/3 and in the hippocampal CA1, and reduced neurogenesis in both the subventricular zone and subgranular zone occur in 15-month-old TgNotch3R90C mice. These reductions in neuron structures, synapses, and neurogenesis are significantly correlated to decreased cerebral vasculature in the corresponding areas. Impaired spatial learning and memory in TgNotch3R90C mice are significantly correlated with the reduced cerebral vasculature, neuron structures, and synapses. Repeated treatment of stem cell factor and granulocyte colony-stimulating factor (SCF+G-CSF) at 9 and 10 months of age improves cognitive function, increases cerebral VEGF/VEGF-A, restores cerebral vasculature, and enhances regeneration of neuronal structures, synaptogenesis and neurogenesis in TgNotch3R90C mice. Pretreatment with Avastin, an angiogenesis inhibitor by neutralizing VEGF-A, completely eliminates the SCF+G-CSF-enhanced cognitive function, vascular and neuronal structure regeneration, synaptogenesis and neurogenesis in TgNotch3R90C mice. SCF+G-CSF-enhanced EC proliferation and angiogenesis in TgNotch3R90C mouse brain-isolated ECs are also blocked by Avastin pretreatment. These data suggest that SCF+G-CSF treatment may repair Notch3R90C mutation-damaged brain through the VEGF-A-mediated angiogenesis. This study provides novel insight into the involvement of VEGF/VEGF-A in the pathogenesis of CADASIL and sheds light on the mechanism underlying the SCF+G-CSF-enhanced brain repair in CADASIL.

Brain repair by hematopoietic growth factors in the subacute phase of traumatic brain injury.

OBJECTIVETraumatic brain injury (TBI) is a major cause of long-term disability and death in young adults. The lack of pharmaceutical therapy for post-acute TBI recovery remains a crucial medical challenge. Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF), which are 2 key hematopoietic growth factors, have shown neuroprotective and neurorestorative effects in experimental stroke. The objective of this study was to determine the therapeutic efficacy of combined treatment (SCF + G-CSF) in subacute TBI.METHODSYoung-adult male C57BL mice were subject to TBI in the cortex of the right hemisphere. After TBI induction, mice were randomly divided into 2 groups: a vehicle control group and an SCF + G-CSF treatment group. Mice without TBI served as sham operative controls. Treatment was initiated 2 weeks after TBI induction. SCF (200 µg/kg) and G-CSF (50 µg/kg) or an equal volume of vehicle solution was subcutaneously injected daily for 7 days. A battery of neurobehavioral tests for evaluation of memory and cognitive function (water maze and novel object recognition tests), anxiety (elevated plus maze test), and motor function (Rota-Rod test) was performed during the period of 2-9 weeks after treatment. Neurodegeneration and dendritic density in both hemispheres were determined through histochemistry and immunohistochemistry at 11 weeks posttreatment.RESULTSWater maze testing showed that TBI-impaired spatial learning and memory was restored by SCF + G-CSF treatment. The findings from the elevated plus maze tests revealed that SCF + G-CSF treatment recovered TBI-caused anxiety and risk-taking behavior. There were no significant differences between the treated and nontreated TBI mice in both the Rota-Rod test and novel object recognition test. In the brain sections, the authors observed that widespread degenerating neurons were significantly increased in both hemispheres in the TBI-vehicle control mice. TBI-induced increases in neurodegeneration were significantly reduced by SCF + G-CSF treatment in the contralateral hemisphere, making it no different from that of the sham controls. Dendritic density in the frontal cortex of the contralateral hemisphere was significantly reduced in the TBI-vehicle control mice, whereas SCF + G-CSF-treated TBI mice showed significant increases of the dendritic density in the same brain region. SCF + G-CSF-treated TBI mice also showed a trend toward increasing dendritic density in the contralateral hippocampus.CONCLUSIONSSCF + G-CSF treatment in the subacute phase of TBI restored TBI-impaired spatial learning and memory, prevented posttraumatic anxiety and risk-taking behavior, inhibited TBI-induced neurodegeneration, and enhanced neural network remodeling. These findings suggest the therapeutic potential of hematopoietic growth factors for brain repair in the subacute phase of TBI.

Repairing the Brain by SCF+G-CSF Treatment at 6 Months Postexperimental Stroke: Mechanistic Determination of the Causal Link Between Neurovascular Regeneration and Motor Functional Recovery.

Stroke, a leading cause of adult disability in the world, is a severe medical condition with limited treatment. Physical therapy, the only treatment available for stroke rehabilitation, appears to be effective within 6 months post-stroke. Here, we have mechanistically determined the efficacy of combined two hematopoietic growth factors, stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF; SCF + G-CSF), in brain repair 6 months after cortical infarct induction in the transgenic mice carrying yellow fluorescent protein in Layer V pyramidal neurons (Thy1-YFP-H). Using a combination of live brain imaging, whole brain imaging, molecular manipulation, synaptic and vascular assessments, and motor function examination, we found that SCF + G-CSF promoted mushroom spine formation, enlarged postsynaptic membrane size, and increased postsynaptic density-95 accumulation and blood vessel density in the peri-infarct cavity cortex; and that SCF + G-CSF treatment improved motor functional recovery. The SCF + G-CSF-enhanced motor functional recovery was dependent on the synaptic and vascular regeneration in the peri-infarct cavity cortex. These data suggest that a stroke-damaged brain is repairable by SCF + G-CSF even 6 months after the lesion occurs. This study provides novel insights into the development of new restorative strategies for stroke recovery.

Microglia in Glia-Neuron Co-cultures Exhibit Robust Phagocytic Activity Without Concomitant Inflammation or Cytotoxicity.

A simple method to co-culture granule neurons and glia from a single brain region is described, and microglia activation profiles are assessed in response to naturally occurring neuronal apoptosis, excitotoxin-induced neuronal death, and lipopolysaccharide (LPS) addition. Using neonatal rat cerebellar cortex as a tissue source, glial proliferation is regulated by omission or addition of the mitotic inhibitor cytosine arabinoside (AraC). After 7-8 days in vitro, microglia in AraC(-) cultures are abundant and activated based on their amoeboid morphology, expressions of ED1 and Iba1, and ability to phagocytose polystyrene beads and the majority of neurons undergoing spontaneous apoptosis. Microglia and phagocytic activities are sparse in AraC(+) cultures. Following exposure to excitotoxic kainate concentrations, microglia in AraC(-) cultures phagocytose most dead neurons within 24 h without exacerbating neuronal loss or mounting a strong or sustained inflammatory response. LPS addition induces a robust inflammatory response, based on microglial expressions of TNF-α, COX-2 and iNOS proteins, and mRNAs, whereas these markers are essentially undetectable in control cultures. Thus, the functional effector state of microglia is primed for phagocytosis but not inflammation or cytotoxicity even after kainate exposure that triggers death in the majority of neurons. This model should prove useful in studying the progressive activation states of microglia and factors that promote their conversion to inflammatory and cytotoxic phenotypes.

Mild exercise reduces cerebral vasospasm after aneurysm subarachnoid hemorrhage: a retrospective clinical study and correlation with laboratory investigation.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (SAH) is a leading cause of death and disability and is often complicated by cerebral vasospasm (CV). Conventional management to prevent CV includes bedrest; however, inactivity places the patient at risk for nonneurological complications. We investigated the effect of mild exercise after SAH in clinical and laboratory settings. METHODS: Clinical: Data from 80 patients with SAH were analyzed retrospectively. After aneurysms were secured, physical therapy was initiated as tolerated. CV and complications were compared by the timing of active physical therapy. Laboratory: 18 Rodents were divided into three groups: (1) control, (2) SAH without exercise, and (3) SAH plus mild exercise. On day 5, brainstems were removed and analyzed for the injury marker inducible nitric oxide synthase (iNOS). RESULTS: Clinical: Mild exercise before day 4 significantly lowered the incidence of symptomatic CV compared with the nonexercised group. There was no difference in the incidence of additional complications based upon exercise. Laboratory: Staining for iNOS was significantly higher in the SAH group than the control group, but there was no difference between exercised and nonexercised SAH groups, confirming that exercise did not promote neuronal injury. CONCLUSION: Early mobilization significantly reduced clinical CV. The relationship should be studied further in a prospective trial with defined exercise regimens.

A low mortality rat model to assess delayed cerebral vasospasm after experimental subarachnoid hemorrhage.

OBJECTIVE: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) in rats, in order to identify the initiating events, pathophysiological changes and potential targets for treatment. METHODS: Twenty-eight male Sprague-Dawley rats (250 - 300 g) were arbitrarily assigned to one of two groups - SAH or saline control. Rat subarachnoid hemorrhage in the SAH group (n=15) was induced by double injection of autologous blood, 48 hr apart, into the cisterna magna. Similarly, normal saline (n=13) was injected into the cisterna magna of the saline control group. Rats were sacrificed on day five after the second blood injection and the brains were preserved for histological analysis. The degree of vasospasm was measured using sections of the basilar artery, by measuring the internal luminal cross sectional area using NIH Image-J software. The significance was tested using Tukey/Kramer's statistical analysis. RESULTS: After analysis of histological sections, basilar artery luminal cross sectional area were smaller in the SAH than in the saline group, consistent with cerebral vasospasm in the former group. In the SAH group, basilar artery internal area (.056 µm ± 3) were significantly smaller from vasospasm five days after the second blood injection (seven days after the initial blood injection), compared to the saline control group with internal area (.069 ± 3; p=0.004). There were no mortalities from cerebral vasospasm. CONCLUSION: The rat double SAH model induces a mild, survivable, basilar artery vasospasm that can be used to study the pathophysiological mechanisms of cerebral vasospasm in a small animal model. A low and acceptable mortality rate is a significant criterion to be satisfied for an ideal SAH animal model so that the mechanisms of vasospasm can be elucidated. Further modifications of the model can be made to adjust for increased severity of vasospasm and neurological exams.

Targeted cancer gene therapy using a hypoxia inducible factor dependent oncolytic adenovirus armed with interleukin-4.

There is a need for novel therapies targeting hypoxic cells in tumors. These cells are associated with tumor resistance to therapy and express hypoxia inducible factor-1 (HIF-1), a transcription factor that mediates metabolic adaptation to hypoxia and activates tumor angiogenesis. We previously developed an oncolytic adenovirus (HYPR-Ad) for the specific killing of hypoxic/HIF-active tumor cells, which we now armed with an interleukin-4 gene (HYPR-Ad-IL4). We designed HYPR-Ad-IL4 by cloning the Ad E1A viral replication and IL-4 genes under the regulation of a bidirectional hypoxia/HIF-responsive promoter. The IL-4 cytokine was chosen for its ability to induce a strong host antitumor immune response and its potential antiangiogenic activity. HYPR-Ad-IL4 induced hypoxia-dependent IL-4 expression, viral replication, and conditional cytolysis of hypoxic, but not normoxic cells. The treatment of established human tumor xenografts with HYPR-Ad-IL4 resulted in rapid and maintained tumor regression with the same potency as that of wild-type dl309-Ad. HYPR-Ad-IL4-treated tumors displayed extensive necrosis, fibrosis, and widespread viral replication. Additionally, these tumors contained a distinctive leukocyte infiltrate and prominent hypoxia. The use of an oncolytic Ad that locally delivers IL-4 to tumors is novel, and we expect that HYPR-Ad-IL4 will have broad therapeutic use for all solid tumors that have hypoxia or active HIF, regardless of tissue origin or genetic alterations.

Anti-epidermal growth factor receptor monoclonal antibody cetuximab augments radiation effects in glioblastoma multiforme in vitro and in vivo.

OBJECTIVE: Previously, we demonstrated that the anti-epidermal growth factor receptor (EGFR) antibody cetuximab alone was effective against EGFR-amplified glioblastoma multiforme (GBM) cells in vivo and in vitro. The purpose of the present work was to study further the effectiveness of cetuximab as a monotherapy as well as combining it with radiation therapy or chemotherapy. METHODS: EGFR-amplified GBM cells were implanted either in the flanks of nude mice to determine the effectiveness of cetuximab on larger tumor burden or intracranially to assess the ability of cetuximab to cross the blood-brain barrier. Cells were also exposed to cetuximab in combination with radiation in vivo or chemotherapeutic agents in vitro. RESULTS: Increasing tumor burden in the flanks of mice decreased the amount of tumor growth inhibition. For the first two intracranial models using cetuximab for 5 weeks, the treated mice had a significant increase in median survival compared with controls. When cetuximab was given indefinitely, the results were encouraging, with an increase in median survival for the treated group not yet reached but at least 900%. Mice with flank GBM exposed to cetuximab and radiation had a larger increase in median survival than those with either treatment alone. Preliminary in vitro experiments using cetuximab and chemotherapeutic agents showed increased cytotoxicity. CONCLUSION: These results were encouraging, demonstrating the effectiveness of cetuximab against EGFR-amplified GBM. Surprisingly, cetuximab was effective when administered systemically in an intracranial model. Radiation augmented the effect of cetuximab on GBM in vitro and in vivo. In vitro analysis demonstrated additive effects for chemotherapeutic agents as well. These results confirm EGFR blockade with cetuximab as a potential treatment against human GBM.