Live-cell imaging of human apurinic/apyrimidinic endonuclease 1 in the nucleus and nucleolus using a chaperone@DNA probe.

Human apurinic/apyrimidinic endonuclease 1 (APE1) plays crucial roles in repairing DNA damage and regulating RNA in the nucleus. However, direct visualization of nuclear APE1 in live cells remains challenging. Here, we report a chaperone@DNA probe for live-cell imaging of APE1 in the nucleus and nucleolus in real time. The probe is based on an assembly of phenylboronic acid modified avidin and biotin-labeled DNA containing an abasic site (named PB-ACP), which cleverly protects DNA from being nonspecifically destroyed while enabling targeted delivery of the probe to the nucleus. The PB-ACP construct specifically detects APE1 due to the high binding affinity of APE1 for both avidin and the abasic site in DNA. It is easy to prepare, biocompatible and allowing for long-term observation of APE1 activity. This molecular tool offers a powerful means to investigate the behavior of APE1 in the nuclei of various types of live cells, particularly for the development of improved cancer therapies targeting this protein.

Surface imprinted bio-nanocomposites for affinity separation of a cellular DNA repair protein.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional DNA repair protein localized in different subcellular compartments. The mechanisms responsible for the highly regulated subcellular localization and "interactomes" of this protein are not fully understood but have been closely correlated to the posttranslational modifications in different biological context. In this work, we attempted to develop a bio-nanocomposite with antibody-like properties that could capture APE1 from cellular matrices to enable the comprehensive study of this protein. By fixing the template APE1 on the avidin-modified surface of silica-coated magnetic nanoparticles, we first added 3-aminophenylboronic acid to react with the glycosyl residues of avidin, followed by addition of 2-acrylamido-2-methylpropane sulfonic acid as the second functional monomer to perform the first step imprinting reaction. To further enhance the affinity and selectivity of the binding sites, we carried out the second step imprinting reaction with dopamine as the functional monomer. After the polymerization, we modified the nonimprinted sites with methoxypoly (ethylene glycol) amine (mPEG-NH2 ). The resulting molecularly imprinted polymer-based bio-nanocomposite showed high affinity, specificity, and capacity for template APE1. It allowed for the extraction of APE1 from the cell lysates with high recovery and purity. Moreover, the bound protein could be effectively released from the bio-nanocomposite with high activity. The bio-nanocomposite offers a very useful tool for the separation of APE1 from various complex biological samples.

Programmable One-Pot Enzymatic Reaction for Direct Fluorescence Detection of Ultralow-Abundance Mutations in the DNA Duplex.

Sensitive detection of low-abundance driver mutations may provide valuable information for precise clinical treatment. Compared to next-generation sequencing and droplet digital PCR methods, fluorescent probes show great flexibility in rapid detection of specific mutations with high sensitivity and easily accessible instruments. However, existing approaches with fluorescent probes need an additional step to convert duplex DNA to single-stranded DNA (ssDNA) before the detection step, which increases the time, cost, and risk of loss of low-input target strands. In this work, we attempt to integrate the ssDNA-generation step with the subsequent detection into a programable one-pot reaction by employing lambda exonuclease (λ exo), a versatile nanopore nuclease which exercises different functions on different substrates. The capability of λ exo in discrimination of mismatched bases in 5'- FAM-ended 2 nt-unpaired DNA duplexes was first demonstrated. Specific fluorescent probes were developed for EGFR exon 19 E746-A750del and PIK3CA E545K mutations with discrimination factors as high as 8470 and 884, respectively. By mixing the probes and λ exo with the PCR products of cell-free circulating DNA extracted from plasma samples, the reaction was immediately initiated, which allowed sensitive detection of the two types of mutations at an abundance as low as 0.01% within less than 2 h. Compared to existing approaches, the new method has distinct advantages in simplicity, low cost, and rapidity. It provides a convenient tool for companion diagnostic tests and other routine analysis targeting genetic mutations in clinical samples.

Mesenchymal stromal cell-derived exosomes ameliorate peripheral neuropathy in a mouse model of diabetes.

AIMS/HYPOTHESIS: Diabetic peripheral neuropathy (DPN) is one of the major complications of diabetes, which contributes greatly to morbidity and mortality. There is currently no effective treatment for this disease. Exosomes are cell-derived nanovesicles and play an important role in intercellular communications. The present study investigated whether mesenchymal stromal cell (MSC)-derived exosomes improve neurological outcomes of DPN. METHODS: Exosomes were isolated from the medium of cultured mouse MSCs by ultracentrifugation. Diabetic mice (BKS.Cg-m+/+Leprdb/J, db/db) at the age of 20 weeks were used as DPN models. Heterozygous mice (db/m) of the same age were used as the control. MSC-exosomes were administered weekly via the tail vein for 8 weeks. Neurological function was evaluated by testing motor and sensory nerve conduction velocities, and thermal and mechanical sensitivity. Morphometric analysis was performed by myelin sheath staining and immunohistochemistry. Macrophage markers and circulating cytokines were measured by western blot and ELISA. MicroRNA (miRNA) array and bioinformatics analyses were performed to examine the exosomal miRNA profile and miRNA putative target genes involved in DPN. RESULTS: Treatment of DPN with MSC-exosomes markedly decreased the threshold for thermal and mechanical stimuli and increased nerve conduction velocity in diabetic mice. Histopathological analysis showed that MSC-exosomes markedly augmented the density of FITC-dextran perfused blood vessels and increased the number of intraepidermal nerve fibres (IENFs), myelin thickness and axonal diameters of sciatic nerves. Western blot analysis revealed that MSC-exosome treatment decreased and increased M1 and M2 macrophage phenotype markers, respectively. Moreover, MSC-exosomes substantially suppressed proinflammatory cytokines. Bioinformatics analysis revealed that MSC-exosomes contained abundant miRNAs that target the Toll-like receptor (TLR)4/NF-κB signalling pathway. CONCLUSIONS/INTERPRETATION: MSC-derived exosomes alleviate neurovascular dysfunction and improve functional recovery in mice with DPN by suppression of proinflammatory genes.

Ablation of the microRNA-17-92 cluster in neural stem cells diminishes adult hippocampal neurogenesis and cognitive function.

Impairment of adult neurogenesis in the hippocampus causes cognitive deficits; however, the underlying molecular mechanisms have not been fully elucidated. microRNAs (miRNAs) regulate neural stem cell (NSC) function. With the use of a transgenic mouse line with conditional ablation of the miR-17-92 cluster in nestin lineage NSCs, we tested the hypothesis that the miR-17-92 cluster regulates adult neurogenesis and cognitive function in vivo. Compared with wild-type mice, ablation of the miR-17-92 cluster significantly reduced the number of proliferating NSCs and neuroblasts and neuronal differentiation in the dentate gyrus (DG) of the hippocampus and significantly impaired hippocampal-dependent learning and memory, as assayed by social recognition memory, novel object recognition, and Morris water-maze tests. Statistical analysis showed a highly significant correlation between newly generated neuroblasts in the DG and cognition deficits in miR-17-92 knockout (KO) mice. Western blot analysis showed that conditional KO of the miR-17-92 cluster significantly increased and reduced a cytoskeleton-associated protein, Enigma homolog 1 (ENH1), and its downstream transcription factor, inhibitor of differentiation 1 (ID1), respectively, as well as increased phosphatase and tensin homolog gene. These proteins are related to neuronal differentiation. Our study demonstrates that the miR-17-92 cluster in NSCs is critical for cognitive and behavioral function and regulates neurogenesis and that the miR-17-92 cluster may target ENH1/ID1 signaling.-Pan, W. L., Chopp, M., Fan, B., Zhang, R., Wang, X., Hu, J., Zhang, X. M., Zhang, Z. G., Liu, X. S. Ablation of the microRNA-17-92 cluster in neural stem cells diminishes adult hippocampal neurogenesis and cognitive function.

Exploring the Complex Impact of Proteins on Dopamine Polymerization: Mechanisms and Strategies for Modulation.

Polydopamine (pDA) is a valuable material with wide-ranging potential applications. However, the complex and debated nature of dopamine polymerization complicates our understanding. Specifically, the impact of foreign substances, especially proteins, on pDA formation adds an additional layer of subtlety and complexity. This study delves into specific surface features of proteins that predominantly shape their impact on dopamine polymerization. Notably, the biotin-binding site emerges as a critical region responsible for the pronounced inhibitory effect of avidin and neutravidin on the dopamine polymerization process. The binding of biotin successfully mitigates these inhibitory effects. Moreover, several nucleases demonstrated a significant hindrance to pDA formation, with their impact substantially alleviated through the introduction of DNA. It is speculated that hydrogen bonding, electrostatic, cation-π, and/or hydrophobic interactions may underlie the binding between protein surfaces and diverse oligomeric intermediates formed by the oxidation products of dopamine. Additionally, we observed a noteworthy blocking effect on the dopamine polymerization reaction induced by erythropoietin (EPO), a glycoprotein hormone known for its role in stimulating red blood cell production and demonstrating neuroprotective effects. The inhibitory influence of EPO persisted even after deglycosylation. These findings not only advance our comprehension of the mechanisms underlying dopamine polymerization but also provide strategic insights for manipulating the reaction to tailor desired biomaterials.

Cerebrolysin enhances neurogenesis in the ischemic brain and improves functional outcome after stroke.

Cerebrolysin is a peptide preparation mimicking the action of neurotrophic factors and has beneficial effects on neurodegenerative diseases and stroke. The present study investigated the effect of Cerebrolysin on neurogenesis in a rat model of embolic middle cerebral artery occlusion (MCAo). Treatment with Cerebrolysin at doses of 2.5 and 5 ml/kg significantly increased the number of bromodeoxyuridine-positive (BrdU(+)) subventricular zone (SVZ) neural progenitor cells and doublecortin (DCX) immunoreactivity (migrating neuroblasts) in the ipsilateral SVZ and striatal ischemic boundary 28 days after stroke when the treatment was initiated 24 hr after stroke. The treatment also reduced TUNEL(+) cells by ∼50% in the ischemic boundary. However, treatment with Cerebrolysin at a dose of 2.5 ml/kg initiated at 24 and 48 hr did not significantly reduce infarct volume but substantially improved neurological outcomes measured by an array of behavioral tests 21 and 28 days after stroke. Incubation of SVZ neural progenitor cells from ischemic rats with Cerebrolysin dose dependently augmented BrdU(+) cells and increased the number of Tuj1(+) cells (a marker of immature neurons). Blockage of the PI3K/Akt pathway abolished Cerebrolysin-increased BrdU(+) cells. Moreover, Cerebrolysin treatment promoted neural progenitor cell migration. Collectively, these data indicate that Cerebrolysin treatment when initiated 24 and 48 hr after stroke enhances neurogenesis in the ischemic brain and improves functional outcome and that Cerebrolysin-augmented proliferation, differentiation, and migration of adult SVZ neural progenitor cells contribute to Cerebrolysin-induced neurogenesis, which may be related to improvement of neurological outcome. The PI3K/Akt pathway mediates Cerebrolysin-induced progenitor cell proliferation.

Influence of Sex on Cognition and Peripheral Neurovascular Function in Diabetic Mice.

Cognition impairment and peripheral neuropathy (DPN) are two major complications of diabetes. The aim of the present study is to investigate the effect of sex differences on cognition and DPN in diabetic mice. Male and female BKS.Cg-m+/+Leprdb/J (db/db) and db/m mice were used. At ages of 20 and 30 weeks, all animals were subjected to learning, memory and neurological function tests. Regional blood flow in footpad and sciatic nerves were measured using laser Doppler flowmetry. Our data showed that male db/db mice aged 20 weeks and 30 weeks spent significantly more time to locate the hidden platform in the correct quadrant and spent significantly less time exploring the cage with a new stranger mouse compared to aged-matched female db/db mice. Electrophysiological recordings showed that male db mice aged 30 weeks had significantly reduced motor and sensory nerve conduction velocity compared with females. Hot plate and tactile allodynia tests revealed that males exhibited significantly higher thermal and mechanical latency than females. Male db mice aged 30 weeks displayed significantly reduced blood perfusion in sciatic nerve and footpad tissues compared with females. In addition, compared with male and female non-diabetic db/m mice, db/db mice exhibited increased time spent on locating the hidden platform, decreased time spent on exploring the novel odor bead and an unfamiliar mouse, as well as showed significantly lower levels of blood flow, lower velocity of MCV and SCV, higher thermal and mechanical latencies. Blood glucose levels and body weight were not significantly different between male and female diabetic animals (age 30 weeks), but male db mice showed a higher serum total cholesterol content. Together, our data suggest that males develop a greater extent of diabetes-induced cognition deficits and peripheral neurovascular dysfunction than females.

MicroRNA-146a Mimics Reduce the Peripheral Neuropathy in Type 2 Diabetic Mice.

MicroRNA-146a (miR-146a) regulates multiple immune diseases. However, the role of miR-146a in diabetic peripheral neuropathy (DPN) has not been investigated. We found that mice (db/db) with type 2 diabetes exhibited substantial downregulation of miR-146a in sciatic nerve tissue. Systemic administration of miR-146a mimics to diabetic mice elevated miR-146a levels in plasma and sciatic nerve tissue and substantially increased motor and sensory nerve conduction velocities by 29 and 11%, respectively, and regional blood flow by 50% in sciatic nerve tissue. Treatment with miR-146a mimics also considerably decreased the response in db/db mice to thermal stimuli thresholds. Histopathological analysis showed that miR-146a mimics markedly augmented the density of fluorescein isothiocyanate-dextran-perfused blood vessels and increased the number of intraepidermal nerve fibers, myelin thickness, and axonal diameters of sciatic nerves. In addition, miR-146a treatment reduced and increased classically and alternatively activated macrophage phenotype markers, respectively. Analysis of miRNA target array revealed that miR-146a mimics greatly suppressed expression of many proinflammatory genes and downstream related cytokines. Collectively, our data indicate that treatment of diabetic mice with miR-146a mimics robustly reduces DPN and that suppression of hyperglycemia-induced proinflammatory genes by miR-146a mimics may underlie its therapeutic effect.

Ischemic cerebral tissue response to subventricular zone cell transplantation measured by iterative self-organizing data analysis technique algorithm.

To investigate the changes of the ischemic lesion in rat brain after subventricular zone (SVZ) cell transplantation and the influence of the grafted cells on the appearance of angiogenesis, SVZ cells, superparamagnetically labeled, were intracisternally transplanted into the rat brain 48 h after onset of embolic stroke. A complete set of magnetic resonance (MR) images was acquired for all animals with (n=8) and without (n=3) cell grafting at approximately 24 h, 72 h, and weekly for 6 weeks after stroke. Transplanted cells were tracked by high-resolution three-dimensional gradient-echo images and the interaction between the cells and ischemic lesion was detected by ISODATA (Iterative Self-Organizing Data Analysis Technique Algorithm) calculated from T(1), T(2) and T(1sat) maps. Tissue status from ISODATA was characterized by a specific signature, which represents the deviation from normal tissue in the feature space. Transplanted SVZ cells selectively migrated towards the ischemic side of the rat brain and approached the lesion boundary within 1-week after grafting. Cell treated rats exhibited a significant reduction of average lesion size compared with control rats (P<0.05). A significant reduction of tissue signature (P<0.001) induced by cell transplantation was localized to the position of grafted cells, and these sites exhibited stably restored cerebral blood flow (CBF) (approximately 85% of normal CBF). Angiogenesis was present in sites either immediately adjacent to or surrounded by the grafted cells. Our data indicate that map-ISODATA accurately and dynamically characterizes the ischemic lesion and its response to cell therapy.

Nitric oxide enhances angiogenesis via the synthesis of vascular endothelial growth factor and cGMP after stroke in the rat.

We investigated the effects of NO on angiogenesis and the synthesis of vascular endothelial growth factor (VEGF) in a model of focal embolic cerebral ischemia in the rat. Compared with control rats, systemic administration of an NO donor, DETANONOate, to rats 24 hours after stroke significantly enlarged vascular perimeters and increased the number of proliferated cerebral endothelial cells and the numbers of newly generated vessels in the ischemic boundary regions, as evaluated by 3-dimensional laser scanning confocal microscopy. Treatment with DETANONOate significantly increased VEGF levels in the ischemic boundary regions as measured by ELISA. A capillary-like tube formation assay was used to investigate whether DETANONOate increases angiogenesis in ischemic brain via activation of soluble guanylate cyclase. DETANONOate-induced capillary-like tube formation was completely inhibited by a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ). Blocking VEGF activity by a neutralized antibody against VEGF receptor 2 significantly attenuated DETANONOate-induced capillary-like tube formation. Moreover, systemic administration of a phosphodiesterase type 5 inhibitor (Sildenafil) to rats 24 hours after stroke significantly increased angiogenesis in the ischemic boundary regions. Sildenafil and an analog of cyclic guanosine monophosphate (cGMP) also induced capillary-like tube formation. These findings suggest that exogenous NO enhances angiogenesis in ischemic brain, which is mediated by the NO/cGMP pathway. Furthermore, our data suggest that NO, in part via VEGF, may enhance angiogenesis in ischemic brain.

Biphasic effects of exogenous VEGF on VEGF expression of adult neural progenitors.

Vascular endothelial growth factor (VEGF) regulates neurogenesis. The present study investigated the direct effect of VEGF on the enhancement of proliferation and differentiation of the adult mouse subventricular zone (SVZ) neural progenitors in vitro. A high dose (500 ng/ml) of VEGF significantly downregulated endogenous VEGF receptors 1 and 2, which was associated with significantly reduced neural progenitor cell proliferation and enhancement of neuronal differentiation. A low dose (50 ng/ml) of VEGF significantly upregulated endogenous VEGF receptors 1 and 2 but did not increase proliferation and differentiation. These data suggest that exogenous VEGF has a biphasic effect on the expression of endogenous VEGF receptors, and the high dose of VEGF enhances adult neural progenitor cell differentiation into neurons.

Function of neural stem cells in ischemic brain repair processes.

Hypoxic/ischemic injury is the single most important cause of disabilities in infants, while stroke remains a leading cause of morbidity in children and adults around the world. The injured brain has limited repair capacity, and thereby only modest improvement of neurological function is evident post injury. In rodents, embryonic neural stem cells in the ventricular zone generate cortical neurons, and adult neural stem cells in the ventricular-subventricular zone of the lateral ventricle produce new neurons through animal life. In addition to generation of new neurons, neural stem cells contribute to oligodendrogenesis. Neurogenesis and oligodendrogenesis are essential for repair of injured brain. Much progress has been made in preclinical studies on elucidating the cellular and molecular mechanisms that control and coordinate neurogenesis and oligodendrogenesis in perinatal hypoxic/ischemic injury and the adult ischemic brain. This article will review these findings with a focus on the ventricular-subventricular zone neurogenic niche and discuss potential applications to facilitate endogenous neurogenesis and thereby to improve neurological function post perinatal hypoxic/ischemic injury and stroke.

Stroke transiently increases subventricular zone cell division from asymmetric to symmetric and increases neuronal differentiation in the adult rat.

The orientation of mitotic cleavage regulates neurogenesis during neural development. We examined the orientation of mitotic cleavage of dividing progenitor cells in the subventricular zone (SVZ) of adult rats subjected to stroke. In nonstroke rats, 55% of dividing cells were oriented horizontally, whereas 40% were oriented vertically. Horizontal and vertical cleavage orientations produce asymmetric and symmetric divisions, respectively. Four days after stroke, the number of dividing cells increased twofold, whereas the proportion of symmetric dividing cells significantly (p < 0.01) increased from 40% before stroke to 60%. Fourteen days after stroke, the percentage of symmetric dividing cells was 47%. Stroke-increased numbers of dividing cells in M-phase were confirmed by immuostaining. In nonstroke rats, 37 and 33% of symmetric and asymmetric dividing cells, respectively, exhibited a neuronal marker (TuJ1). Four days after stroke, rats exhibited a significant (p < 0.05) augmentation of the frequency (47%) of neuronal distribution showing TuJ1 immunoreactivity in cells with symmetric division but not cells with asymmetric division (33%). Numb immunoreactivity was detected in SVZ cells of nonstroke rats. Stroke did not change Numb distribution. Our data suggest that neurons are produced by both asymmetric and symmetric cell divisions in the adult SVZ, and the transient increases in symmetric division and neuronal differentiation may result in stroke-induced neurogenesis.

Adjuvant treatment with a glycoprotein IIb/IIIa receptor inhibitor increases the therapeutic window for low-dose tissue plasminogen activator administration in a rat model of embolic stroke.

BACKGROUND: Platelet aggregation and fibrin deposition are key events leading to microvascular thrombosis and progressive impairment of downstream microvascular perfusion after stroke. We tested the hypothesis that inhibition of platelet function with a GP IIb/IIIa receptor antagonist would increase the efficacy and safety and increase the time window for thrombolytic therapy for stroke with full- and half-dose tissue plasminogen activator (tPA). METHODS AND RESULTS: Rats were subjected to embolic middle cerebral artery occlusion. Four hours after ischemia, rats were treated with 7E3 F(ab')2 (6 mg/kg) in combination with tPA at doses of 10 and 5 mg/kg, tPA alone at a dose of 10 or 5 mg/kg, 7E3 F(ab')2 (6 mg/kg) alone, or saline. Combination treatment with 7E3 F(ab')2 and tPA (full- or half-dose) significantly (P<0.05) reduced infarct volume and neurological deficits compared with saline-treated rats. However, treatment with 7E3 F(ab')2 or tPA (full- or half-dose) alone did not reduce infarct volume. Quantitative measurements of cerebral microvessels perfused by FITC-dextran revealed that combination treatment with 7E3 F(ab')2 and full-dose tPA significantly (P<0.05) increased the percentage of FITC-dextran-perfused vessels compared with saline and full-dose tPA-treated rats. In addition, treatment with 7E3 F(ab')2 in combination with full-dose tPA significantly (P<0.05) decreased microvascular platelet accumulation and matrix metalloproteinase 9 immunoreactivity and protected against loss of collagen IV immunoreactivity. CONCLUSIONS: Combination treatment with 7E3 F(ab')2 with full- and half-dose tPA at 4 hours after ischemia significantly reduces infarct volume and improves neurological outcome. Enhancement of patency and integrity of cerebral microvessels most likely contributes to the benefits observed with this combination therapy.

Therapeutic Benefit of Extended Thymosin ß4 Treatment Is Independent of Blood Glucose Level in Mice with Diabetic Peripheral Neuropathy.

Peripheral neuropathy is a chronic complication of diabetes mellitus. To investigated the efficacy and safety of the extended treatment of diabetic peripheral neuropathy with thymosin ß4 (Tß4), male diabetic mice (db/db) at the age of 24 weeks were treated with Tß4 or saline for 16 consecutive weeks. Treatment of diabetic mice with Tß4 significantly improved motor (MCV) and sensory (SCV) conduction velocity in the sciatic nerve and the thermal and mechanical latency. However, Tß4 treatment did not significantly alter blood glucose levels. Treatment with Tß4 significantly increased intraepidermal nerve fiber density. Furthermore, Tß4 counteracted the diabetes-induced axon diameter and myelin thickness reductions and the g-ratio increase in sciatic nerve. In vitro, compared with dorsal root ganglia (DRG) neurons derived from nondiabetic mice, DRG neurons derived from diabetic mice exhibited significantly decreased neurite outgrowth, whereas Tß4 promoted neurite growth in these diabetic DRG neurons. Blockage of the Ang1/Tie2 signaling pathway with a neutralized antibody against Tie2 abolished Tß4-increased neurite outgrowth. Our data demonstrate that extended Tß4 treatment ameliorates diabetic-induced axonal degeneration and demyelination, which likely contribute to therapeutic effect of Tß4 on diabetic neuropathy. The Ang1/Tie2 pathway may mediate Tß4-induced axonal remodeling.

Sildenafil ameliorates long term peripheral neuropathy in type II diabetic mice.

Diabetic peripheral neuropathy is a common complication of long-standing diabetes mellitus. To mimic clinical trials in which patients with diabetes enrolled have advanced peripheral neuropathy, we investigated the effect of sildenafil, a specific inhibitor of phosphodiesterase type 5 enzyme, on long term peripheral neuropathy in middle aged male mice with type II diabetes. Treatment of diabetic mice (BKS.Cg-m+/+Leprdb/J, db/db) at age 36 weeks with sildenafil significantly increased functional blood vessels and regional blood flow in the sciatic nerve, concurrently with augmentation of intra-epidermal nerve fiber density in the skin and myelinated axons in the sciatic nerve. Functional analysis showed that the sildenafil treatment considerably improved motor and sensory conduction velocities in the sciatic nerve and peripheral thermal stimulus sensitivity compared with the saline treatment. In vitro studies showed that mouse dermal endothelial cells (MDE) cultured under high glucose levels exhibited significant down regulation of angiopoietin 1 (Ang1) expression and reduction of capillary-like tube formation, which were completely reversed by sildenafil. In addition, incubation of dorsal root ganglia (DRG) neurons with conditioned medium harvested from MDE under high glucose levels suppressed neurite outgrowth, where as conditional medium harvested from MDE treated with sildenafil under high glucose levels did not inhibit neurite outgrowth of DRG neurons. Moreover, blockage of the Ang1 receptor, Tie2, with a neutralized antibody against Tie2 abolished the beneficial effect of sildenafil on tube formation and neurite outgrowth. Collectively, our data indicate that sildenafil has a therapeutic effect on long term peripheral neuropathy of middle aged diabetic mice and that improvement of neurovascular dysfunction by sildenafil likely contributes to the amelioration of nerve function. The Ang1/Tie2 signaling pathway may play an important role in these restorative processes.

Treatment of stroke with erythropoietin enhances neurogenesis and angiogenesis and improves neurological function in rats.

BACKGROUND AND PURPOSE: Erythropoietin (EPO) promotes proliferation and differentiation of erythroid progenitors and the survival of maturing erythroid cells. Here, we investigated the role of EPO in brain repair after stroke. METHODS: Rats were treated with recombinant human EPO (rhEPO) at 24 hours after the onset of embolic stroke. An array of behavior tests was performed. Rats were euthanized 28 days after stroke for measurements of infarct volume, angiogenesis, and neurogenesis. In vitro, neurospheres derived from the subventricular zone (SVZ) of the rat and cerebral endothelial cells derived from the mouse were treated with rhEPO. Capillary-like tube formation and neuronal differentiation were measured. RESULTS: Treatment with rhEPO significantly improved functional recovery, along with increases in density of cerebral microvessels at the stroke boundary and numbers of BrdU, doublecortin, and nestin immunoreactive cells in the SVZ. rhEPO treatment significantly increased brain levels of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). In vitro, rhEPO enhanced capillary tube formation of cerebral endothelial cells, which was inhibited by a specific VEGF receptor 2 antagonist (SU1498). Incubation of neurospheres derived from stroke SVZ with anti-EPO neutralizing antibody inhibited neurogenesis, whereas incubation of stroke-derived neurospheres with rhEPO enhanced neurogenesis. CONCLUSIONS: Our data suggest that EPO-increased VEGF and BDNF may be involved in angiogenesis and neurogenesis, which could contribute to functional recovery.

Sildenafil (Viagra) induces neurogenesis and promotes functional recovery after stroke in rats.

BACKGROUND AND PURPOSE: We tested the hypothesis that sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, promotes functional recovery and neurogenesis after stroke. METHODS: Male Wistar rats were subjected to embolic middle cerebral artery occlusion. Sildenafil (Viagra) was administered orally for 7 consecutive days starting 2 or 24 hours after stroke onset at doses of 2 or 5 mg/kg per day. Ischemic rats administered the same volume of tap water were used as a control group. Functional outcome tests (foot-fault, adhesive removal) were performed. Rats were killed 28 days after stroke for analysis of infarct volume and newly generated cells within the subventricular zone and the dentate gyrus. Brain cGMP levels, expression of PDE5, and localized cerebral blood flow were measured in additional rats. RESULTS: Treatment with sildenafil significantly (P<0.05) enhanced neurological recovery in all tests performed. There was no significant difference of infarct volume among the experimental groups. Treatment with sildenafil significantly (P<0.05) increased numbers of bromodeoxyuridine-immunoreactive cells in the subventricular zone and the dentate gyrus and numbers of immature neurons, as indicated by betaIII-tubulin (TuJ1) immunoreactivity in the ipsilateral subventricular zone and striatum. The cortical levels of cGMP significantly increased after administration of sildenafil, and PDE5 mRNA was present in both nonischemic and ischemic brain. CONCLUSIONS: Sildenafil increases brain levels of cGMP, evokes neurogenesis, and reduces neurological deficits when given to rats 2 or 24 hours after stroke. These data suggest that this drug that is presently in the clinic for sexual dysfunction may have a role in promoting recovery from stroke.

Activated neural stem cells contribute to stroke-induced neurogenesis and neuroblast migration toward the infarct boundary in adult rats.

Stroke increases neurogenesis. The authors investigated whether neural stem cells or progenitor cells in the adult subventricular zone (SVZ) of rats contribute to stroke-induced increase in neurogenesis. After induction of stroke in rats, the numbers of cells immunoreactive to doublecortin, a marker for immature neurons, increased in the ipsilateral SVZ and striatum. Infusion of an antimitotic agent (cytosine-beta-D-arabiofuranoside, Ara-C) onto the ipsilateral cortex eliminated more than 98% of actively proliferating cells in the SVZ and doublecortin-positive cells in the ipsilateral striatum. However, doublecortin-positive cells rapidly replenished after antimitotic agent depletion of actively proliferating cells. Depleting the numbers of actively proliferating cells in vivo had no effect on the numbers of neurospheres formed in vitro, yet the numbers of neurospheres derived from stroke rats significantly (P<0.05) increased. Neurospheres derived from stroke rats self-renewed and differentiated into neurons and glia. In addition, doublecortin-positive cells generated in the SVZ migrated in a chainlike structure toward ischemic striatum. These findings indicate that in the adult stroke brain, increases in recruitment of neural stem cells contribute to stroke-induced neurogenesis, and that newly generated neurons migrate from the SVZ to the ischemic striatum.