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Abstract Background The blood brain barrier (BBB) is the first line of defence of the central nervous system (CNS) against circulating pathogens, such as HIV. The cytotoxic HIV protein, gp120, damages endothelial cells of the BBB, thereby compromising its integrity, which may lead to migration of HIV-infected cells into the brain. Fibroblast growth factor 2 (FGF2), produced primarily by astrocytes, promotes endothelial cell fitness and angiogenesis. We hypothesized that treatment of human umbilical vein endothelial cells (HUVEC) with FGF2 would protect the cells from gp120-mediated toxicity via endothelial cell survival signalling. Results Exposure of HUVEC to gp120 resulted in dose- and time-dependent cell death; whereas, pre-treatment of endothelial cells with FGF2 protected cells from gp120 angiotoxicity. Treatment of HUVEC with FGF2 resulted in dose- and time-dependent activation of the extracellular regulated kinase (ERK), with moderate effects on phosphoinositol 3 kinase (PI3K) and protein kinase B (PKB), also known as AKT, but no effects on glycogen synthase kinase 3 (GSK3β) activity. Using pharmacological approaches, gene transfer and kinase activity assays, we show that FGF2-mediated angioprotection against gp120 toxicity is regulated by crosstalk among the ERK, PI3K-AKT and PKC signalling pathways. Conclusions Taken together, these results suggest that FGF2 may play a significant role in maintaining the integrity of the BBB during the progress of HIV associated cerebral endothelial cell damage.

Ischemic stroke is the leading cause of serious, long-term adult disability and is associated with sensorimotor and cognitive impairments due to neuronal degeneration. Currently, recombinant tissue plasminogen activator (rTPA) is the only FDA-approved medical therapy for treatment of patients with acute ischemic stroke. However, rTPA can only be given within 3 hours of symptom onset, and only 2% of patients are eligible. Therefore, there is an urgent need for novel neuroprotective treatment options for ischemic stroke. An emerging treatment for a diverse range of neurological disorders associated with neurodegeneration is rapamycin, a key modulator of the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway is the primary regulator of the cellular response to nutrient availability, changes in energy status and stress as seen following ischemia and reperfusion. However, rapamycin’s effects on mTORC1 and mTORC2 are poorly understood in neurons. In the current study we show that rapamycin can prevent the activation of both mTORC1 and mTORC2 in cortical neurons and improve cell survival following oxygen glucose deprivation (OGD), an in vitro model of ischemic stroke. This work further supports the investigation of rapamycin as a novel neuroprotectant for ischemic stroke. [ABSTRACT FROM AUTHOR]

Background Insulin-like growth factor binding protein-2 (IGFBP-2) regulates the bioavailability, transportation, and localization of insulin-like growth factor-I (IGF-I), an effective neuroprotectant in animal stroke models especially when administered intranasally. Therefore, determining IGFBP-2's endogenous distribution in the normal and ischemic brain is essential in maximizing the neuroprotective potential of the intranasal IGF-I treatment approach. However, current data on IGFBP-2 is limited to mRNA and in situ hybridization studies. The purpose of this study was to determine if there are any changes in IGFBP-2 protein levels and distribution in ischemic brain and also to determine if IGFBPs play a role in the transportation of intranasally administered IGF-I into the brain. Results Using an in vitro approach, we show that ischemia causes changes in the distribution of IGFBP-2 in primary cortical neurons and astrocytes. In addition, we show using the transient middle cerebral artery occlusion (MCAO) model in mice that there is a significant increase in IGFBP-2 levels in the stroke penumbra and core after 72 h. This correlated with an overall increase in IGF-I after stroke, with the highest levels of IGF-I in the stroke core after 72 h. Brain sections from stroke mice indicate that neurons and astrocytes located in the penumbra both have increased expression of IGFBP-2, however, IGFBP-2 was not detected in microglia. We used binding competition studies to show that intranasally administered exogenous IGF-I uptake into the brain is not receptor mediated and is likely facilitated by IGFBPs. Conclusions The change in protein levels indicates that IGFBP-2 plays an IGF-I-dependent and - independent role in the brain's acute (neuroprotection) and chronic (tissue remodeling) response to hypoxic-ischemic injury. Competition studies indicate that IGFBPs may have a role in rapid transportation of exogenous IGF-I from the nasal tissue to the site of injury. [ABSTRACT FROM AUTHOR]

Background: The blood brain barrier (BBB) is impermeable to most drugs, impeding the establishment of novel neuroprotective therapies and strategies for many neurological diseases. Intranasal administration offers an alternative path for efficient drug delivery into the CNS. So far, the anatomical structures discussed to be involved in the transport of intranasally administered drugs into the CNS include the trigeminal nerve, olfactory nerve and the rostral migratory stream (RMS), but the relative contributions are debated. Methods and Findings: In the present study we demonstrate that surgical transection, and the resulting structural disruption of the RMS, in mice effectively obstructs the uptake of intranasally administered radioligands into the CNS. Furthermore, using a fluorescent cell tracer, we demonstrate that intranasal administration in mice allows agents to be distributed throughout the entire brain, including olfactory bulb, hippocampus, cortex and cerebellum. Conclusions: This study provides evidence of the vital role the RMS has in the CNS delivery of intranasally administered agents. The identification of the RMS as the major access path for intranasally administered drugs into the CNS may contribute to the development of treatments that are tailored for efficient transport within this structure. Research into the RMS needs to continue to elucidate its limitations, capabilities, mechanisms of transport and potential hazards before we are able to advance this technique into human research. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small, non-protein-coding RNA molecules that modulate gene translation. Their expression is altered in many central nervous system (CNS) injuries suggesting a role in the cellular response to stress. Current studies in brain tissue have not yet described the cell-specific temporal miRNA expression patterns following ischemic injury. In this study, we analyzed the expression alterations of a set of miRNAs in neurons and astrocytes subjected to 60 minutes of ischemia and collected at different time-points following this injury. To mimic ischemic conditions and reperfusion in vitro, cortical primary neuronal and astrocytic cultures prepared from fetal rats were first placed in oxygen and glucose deprived (OGD) medium for 60 minutes, followed by their transfer into normoxic pre-conditioned medium. Total RNA was extracted at different time-points after the termination of the ischemic insult and the expression levels of miRNAs were measured. In neurons exposed to OGD, expression of miR-29b was upregulated 2-fold within 6 h and up to 4-fold at 24 h post-OGD, whereas induction of miR-21 was upregulated 2-fold after 24 h when compared to expression in neurons under normoxic conditions. In contrast, in astrocytes, miR-29b and miR-21 were upregulated only after 12 h. MiR-30b, 107, and 137 showed expression alteration in astrocytes, but not in neurons. Furthermore, we show that expression of miR-29b was significantly decreased in neurons exposed to Insulin-Like Growth Factor I (IGF-I), a well documented neuroprotectant in ischemic models. Our study indicates that miRNAs expression is altered in neurons and astrocytes after ischemic injury. Furthermore, we found that following OGD, specific miRNAs have unique cell-specific temporal expression patterns in CNS. Therefore the specific role of each miRNA in different intracellular processes in ischemic brain and the relevance of their temporal and spatial expression patterns warrant further investigation that may lead to novel strategies for therapeutic interventions. [ABSTRACT FROM AUTHOR]

Objective Prolonged human immunodeficiency virus-1 (HIV-1) infection leads to neurological debilitation, including motor dysfunction and frank dementia. Although pharmacological control of HIV infection is now possible, HIV-associated neurocognitive disorders (HAND) remain intractable. Here, we report that chronic treatment with erythropoietin (EPO) and insulin-like growth factor-I (IGF-I) protects against HIV/gp120-mediated neuronal damage in culture and in vivo. Methods Initially, we tested the neuroprotective effects of various concentrations of EPO, IGF-I, or EPO+IGF-I from gp120-induced damage in vitro. To assess the chronic effects of EPO+IGF-I administration in vivo, we treated HIV/gp120-transgenic or wild-type mice transnasally once a week for 4 months and subsequently conducted immunohistochemical analyses. Results Low concentrations of EPO+IGF-I provided neuroprotection from gp120 in vitro in a synergistic fashion. In vivo, EPO+IGF-I treatment prevented gp120-mediated neuronal loss, but did not alter microgliosis or astrocytosis. Strikingly, in the brains of both humans with HAND and gp120-transgenic mice, we found evidence for hyperphosphorylated tau protein (paired helical filament-I tau), which has been associated with neuronal damage and loss. In the mouse brain following transnasal treatment with EPO+IGF-I, in addition to neuroprotection we observed increased phosphorylation/activation of Akt (protein kinase B) and increased phosphorylation/inhibition of glycogen synthase kinase (GSK)-3β, dramatically decreasing downstream hyperphosphorylation of tau. These results indicate that the peptides affected their cognate signaling pathways within the brain parenchyma. Interpretation Our findings suggest that chronic combination therapy with EPO+IGF-I provides neuroprotection in a mouse model of HAND, in part, through cooperative activation of phosphatidylinositol 3-kinase/Akt/GSK-3β signaling. This combination peptide therapy should therefore be tested in humans with HAND. ANN NEUROL 2010;68:342-352 [ABSTRACT FROM AUTHOR]

Background: The blood brain barrier (BBB) is the first line of defence of the central nervous system (CNS) against circulating pathogens, such as HIV. The cytotoxic HIV protein, gp120, damages endothelial cells of the BBB, thereby compromising its integrity, which may lead to migration of HIV-infected cells into the brain. Fibroblast growth factor 2 (FGF2), produced primarily by astrocytes, promotes endothelial cell fitness and angiogenesis. We hypothesized that treatment of human umbilical vein endothelial cells (HUVEC) with FGF2 would protect the cells from gp120-mediated toxicity via endothelial cell survival signalling. Results: Exposure of HUVEC to gp120 resulted in dose- and time-dependent cell death; whereas, pre-treatment of endothelial cells with FGF2 protected cells from gp120 angiotoxicity. Treatment of HUVEC with FGF2 resulted in dose- and time-dependent activation of the extracellular regulated kinase (ERK), with moderate effects on phosphoinositol 3 kinase (PI3K) and protein kinase B (PKB), also known as AKT, but no effects on glycogen synthase kinase 3 (GSK3β) activity. Using pharmacological approaches, gene transfer and kinase activity assays, we show that FGF2- mediated angioprotection against gp120 toxicity is regulated by crosstalk among the ERK, PI3KAKT and PKC signalling pathways. Conclusions: Taken together, these results suggest that FGF2 may play a significant role in maintaining the integrity of the BBB during the progress of HIV associated cerebral endothelial cell damage. [ABSTRACT FROM AUTHOR]

Presents information on a study which evaluated the role of integrin-linked kinase (ILK) in nerve growth factor-stimulated neurite outgrowth. Cell culture and drug exposure; Assessment of neurite growth; Kinase assay and immunoprecipitations; Immunocytochemical localization of ILK in neuronal cells.

Presents information on a study that showed that preconditioning with erythropoietin, a kidney cytokine regulating haematopoiesis, protects neurons in models of ischaemic and degenerative damage due to excitotoxins. Research methods; Results and discussion on the study.

After traumatic brain injury (TBI), multiple ongoing processes contribute to worsening and spreading of the primary injury to create a secondary injury. One major process involves disrupted fluid regulation to create vascular and cytotoxic edema in the affected area. Although understanding of factors that influence edema is incomplete, the astrocyte water channel Aquaporin 4 (AQP4) has been identified as an important mediator and therefore attractive drug target for edema prevention. The FDA-approved drug acetazolamide has been administered safely to patients for years in the United States. To test whether acetazolamide altered AQP4 function after TBI, we utilized in vitro and in vivo models of TBI. Our results suggest that AQP4 localization is altered after TBI, similar to previously published reports. Treatment with acetazolamide prevented AQP4 reorganization, both in human astrocyte in vitro and in mice in vivo. Moreover, acetazolamide eliminated cytotoxic edema in our in vivo mouse TBI model. Our results suggest a possible clinical role for acetazolamide in the treatment of TBI.

The mechanistic target of rapamycin (mTOR) is an intracellular protein kinase that functions as an energy and nutrient sensor in the cellular microenvironment of neurons. Modulation of mTOR is vital when nutrient and energy sources become limited. Hypoxia, traumatic brain injury, cellular energy states, and growth factors all regulate the phosphorylation and total levels of mTOR in cells. Alterations in the microenvironment induce transduction of signals to downstream proteins by mTOR allowing for cells to make the necessary adjustments to counteract stressors and survive. Progesterone, a hydrophobic steroid hormone, has been shown in studies of non-neural tissue to be a suppressor of mTOR and modulator of mTOR phosphorylation. Our study tested the effects of progesterone on mTOR expression following traumatic brain injury. C57BL/6 mice were treated with progesterone (8 mg/kg) at 1 (intraperitoneal), 6 (subcutaneous), 24 (subcutaneous), and 48 (subcutaneous) hours post closed skull traumatic brain injury. The hippocampus was then harvested 72 hours post injury and prepared for western blot analysis. We found that progesterone significantly decreased total mTOR levels in all groups compared to sham treated with vehicle. This was further confirmed by immunostaining showing decreased cytoplasmic mTOR levels compared to sham. Our study shows progesterone is a significant modulator of mTOR levels in the hippocampus of mice following traumatic brain injury., Competing Interests: There are no conflicts of interest to declare

Granulocyte-colony stimulating factor (G-CSF) has a multimodal neuroprotective profile and the cumulative preclinical data from numerous translational studies statistically confirmed the efficacy of G-CSF as a treatment option in ischemic stroke. G-CSF activates anti-apoptotic, antioxidative, and anti-inflammatory signaling pathways and stimulates angiogenesis and neurogenesis. In this review, we summarize the role of G-CSF and the corresponding signal transduction pathways regulated by G-CSF in neuroprotection and discuss its potential as a new drug for stroke treatment.

Ischemic stroke is the leading cause of serious, long-term adult disability and is associated with sensorimotor and cognitive impairments due to neuronal degeneration. Currently, recombinant tissue plasminogen activator (rTPA) is the only FDA-approved medical therapy for treatment of patients with acute ischemic stroke. However, rTPA can only be given within 3 hours of symptom onset, and only 2% of patients are eligible. Therefore, there is an urgent need for novel neuroprotective treatment options for ischemic stroke. An emerging treatment for a diverse range of neurological disorders associated with neurodegeneration is rapamycin, a key modulator of the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway is the primary regulator of the cellular response to nutrient availability, changes in energy status and stress as seen following ischemia and reperfusion. However, rapamycin's effects on mTORC1 and mTORC2 are poorly understood in neurons. In the current study we show that rapamycin can prevent the activation of both mTORC1 and mTORC2 in cortical neurons and improve cell survival following oxygen glucose deprivation (OGD), an in vitro model of ischemic stroke. This work further supports the investigation of rapamycin as a novel neuroprotectant for ischemic stroke.

The search for potential drugs to treat neurodegenerative diseases has been intense in the last two decades. Among many candidates, erythropoietin (EPO) was identified as a potent protectant of neurons suffering from various adverse conditions. A wide array of literature indicates that endogenous or exogenous recombinant human erythropoietin and its variants activate cell signaling that initiates survival-promoting events in neurons and neuronal cells. This chapter gives an overview of the pro-survival signaling induced by endogenous and exogenous erythropoietin in vitro and in vivo and provides methods to further investigate the intracellular signaling. It is important to know that EPO is neuroprotective, but it will greatly enhance our chances to establish EPO as a new drug candidate if we know how EPO protects neurons.The descriptions below summarize our current knowledge in non-neuronal and neuronal signaling pathways induced by EPO. The signaling pathways involved in EPO are multiple; some are well known whereas others are still under intense investigation and few are observed in very specific cell types. It is important to note that neuronal signaling events triggered by EPO are still incomplete and require further research. Therefore, excellent review articles that explore specific EPO-signaling events are referenced.

Traumatic brain injury (TBI) is the leading cause of death and disability in children and young adults. Neuroprotective agents that may promote repair or counteract damage after injury do not currently exist. We recently reported that stimulation of the purinergic receptor subtype P2Y(1)R using 2-methylthioladenosine 5' diphosphate (2MeSADP) significantly reduced cytotoxic edema induced by photothrombosis. Here, we tested whether P2Y(1)R stimulation was neuroprotective after TBI. A controlled closed head injury model was established for mice using a pneumatic impact device. Brains were harvested at 1, 3, or 7 days post-injury and assayed for morphological changes by immunocytochemistry, Western blot analysis, and wet/dry weight. Cerebral edema and expression of both aquaporin type 4 and glial fibrillary acidic protein were increased at all time points examined. Immunocytochemical measurements in both cortical and hippocampal slices also revealed significant neuronal swelling and reactive gliosis. Treatment of mice with 2MeSADP (100 μM) or MRS2365 (100 μM) 30 min after trauma significantly reduced all post-injury symptoms of TBI including edema, neuronal swelling, reactive gliosis, and AQ4 expression. The neuroprotective effect was lost in IP(3)R2-/- mice treated with 2MeSADP. Immunocytochemical labeling of brain slices confirmed that P2Y(1)R expression was defined to cortical and hippocampal astrocytes, but not neurons. Taken together, the data show that stimulation of astrocytic P2Y(1)Rs significantly reduces brain injury after acute trauma and is mediated by the IP(3)-signaling pathway. We suggest that enhancing astrocyte mitochondrial metabolism offers a promising neuroprotective strategy for a broad range of brain injuries.

Importance of the Field: Erythropoietin (EPO) is a growth hormone and cytokine that plays an important role in erythropoiesis and neuroprotection. However, EPO treatment for neurological diseases requires repeated injections or high-dose systemic administration, which may cause systemic side effects. The lack of any effective treatment of acute and chronic neurodegenerative diseases and the promising outcome by EPO in animal models in vivo demand a critical evaluation of intranasal EPO delivery to the brain as an alternative administration method., Areas Covered in This Review: The current use and intranasal administration of EPO and its derivatives in preclinical studies and recent clinical trials with EPO in neurological diseases., What the Reader Will Gain: This paper gives an overview of the therapeutic considerations of intranasal EPO and EPO derivatives for neuroprotection., Take Home Message: Intranasal delivery (ID) of neuroprotective drugs is an area of great interest. Among the administration strategies used at present, ID of EPO is the most promising. Further preclinical and clinical studies are needed to evaluate the potential significance of this alternative route for increasing EPO bioavailability and decreasing side effects.

Importance of the Field: Recombinant erythropoietin (rEPO) failed in a recent clinical study to protect from damages induced by ischemic stroke. The lack of acute treatments in ischemic stroke and the promising outcome in numerous preclinical studies in vivo demands a more critical evaluation of the future use of EPO as an acute treatment., Areas Covered in This Review: The current use and administration of rhEPO and its analogs in animal models and the future use of this cytokine in the treatment of ischemic stroke., What the Reader Will Gain: In this review the potential reasons for the failure of EPO in the clinical trial are analysed and whether the preclinical trials sufficiently evaluated the true potential of recombinant EPO and its analogs is assessed. Alternative methods for administration of EPO to enhance its potential as a neuroprotective drug in ischemic stroke are discussed., Take Home Message: Failure in clinical trial does not necessarily indicate the lack of therapeutic potential of EPO. This review encourages further investigation of the true potential of EPO as a candidate drug for the treatment of ischemic stroke by improved preclinical experimental design and utilization of alternative administration methods.

Object: Individually, the cytokines erythropoietin (EPO) and insulin-like growth factor-I (IGF-I) have both been shown to reduce neuronal damage significantly in rodent models of cerebral ischemia. The authors have previously shown that EPO and IGF-I, when administered together, provide acute and prolonged neuroprotection in cerebrocortical cultures against N-methyl-D-aspartate-induced apoptosis. The aim of this study was to determine whether intranasally applied EPO plus IGF-I can provide acute neuroprotection in an animal stroke model and to show that intranasal administration is more efficient at delivering EPO plus IGF-I to the brain when compared with intravenous, subcutaneous, or intraperitoneal administration., Methods: The EPO and IGF-I were administered intranasally to mice that underwent transient middle cerebral artery occlusion (MCAO). Stroke volumes were measured after 1 hour of MCAO and 24 hours of reperfusion. To evaluate the long-term effects of this treatment, behavioral outcomes were assessed at 3, 30, 60, and 90 days following MCAO. Radiography and liquid scintillation were used to visualize and quantify the uptake of radiolabeled 125I-EPO and 125I-IGF-I into the mouse brain after intranasal, intravenous, subcutaneous, or intraperitoneal administration., Results: Intranasal administration of EPO plus IGF-I reduced stroke volumes within 24 hours and improved neurological function in mice up to 90 days after MCAO. The 125I-EPO and 125I-IGF-I were found in the brain within 20 minutes after intranasal administration and accumulated within the injured areas of the brain. In addition, intranasal administration delivered significantly higher levels of the applied 125I-EPO and 125I-IGF-I to the brain compared with intravenous, subcutaneous, or intraperitoneal administration., Conclusions: The data demonstrate that intranasal EPO plus IGF-I penetrates into the brain more efficiently than other drug delivery methods and could potentially provide a fast and efficient treatment to prevent chronic effects of stroke.

Infection with human immunodeficiency virus (HIV)-1 can lead to neurological complications that range from mild cognitive and motor impairment to HIV-associated dementia (HAD). The mechanism of brain injury and dementia remains poorly understood. Interestingly, post mortem brain specimen from HAD patients and transgenic mice expressing the viral envelope protein gp120 present with similar neuropathological signs. The cytokine erythropoietin (EPO) is clinically used to treat anemia but has also been found to prevent neuronal death due to inflammation or excitotoxicity. Here we show that EPO protects cerebrocortical neurons against apoptosis induced by HIV-1/gp120.