Hyperbaric Oxygen Therapy Adjuvant Chemotherapy and Radiotherapy through Inhibiting Stemness in Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and deadliest primary brain tumor in adults. Despite the advances in GBM treatment, outcomes remain poor, with a 2-year survival rate of less than 5%. Hyperbaric oxygen (HBO) therapy is an intermittent, high-concentration, short-term oxygen therapy used to increase cellular oxygen content. In this study, we evaluated the effects of HBO therapy, alone or combined with other treatment modalities, on GBM in vitro and in vivo. In the in vitro analysis, we used a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess the effects of HBO therapy alone, a colony formation assay to analyze the effects of HBO therapy combined with radiotherapy and with temozolomide (TMZ), and a neurosphere assay to assess GBM stemness. In the in vivo analysis, we used immunohistochemical staining and in vivo bioluminescence imaging to assess GBM stemness and the therapeutic effect of HBO therapy alone or combined with TMZ or radiotherapy, respectively. HBO therapy did not affect GBM cell viability, but it did reduce the analyzed tumors' ability to form cancer stem cells. In addition, HBO therapy increased GBM sensitivity to TMZ and radiotherapy both in vitro and in vivo. HBO therapy did not enhance tumor growth and exhibited adjuvant effects to chemotherapy and radiotherapy through inhibiting GBM stemness. In conclusion, HBO therapy shows promise as an adjuvant treatment for GBM by reducing cancer stem cell formation and enhancing sensitivity to chemotherapy and radiotherapy.

Measurement of Interhemispheric Correlation Coefficient in Rodent Model of Middle Cerebral Artery Occlusion Using Near Infrared Spectroscopy.

Near-infrared spectroscopy (NIRS) is a noninvasive brain imaging technique that measures hemodynamics by determining the optical properties of tissue. Clinical potential of NIRS for monitoring cerebral hemodynamics in cerebrovascular diseases, such as stroke, has been studied. However, inconsistencies in measurements among studies, which are believed to be partly due to anatomical variance and diversity in disease presentation, limit the clinical feasibility of NIRS for stroke monitoring. In the present study, bihemispheric frequency-domain NIRS measurements on middle cerebral artery occlusion rats were performed. The discrepancy in interhemispheric synchronicity in hemodynamic oscillation appeared during the early reperfusion stage is related to the size of infarct that developed three days later. These NIRS parameters may have the potential to be early prognostic biomarkers for long-term stroke monitoring in the future translational investigation.

Melatonin attenuated brain death tissue extract-induced cardiac damage by suppressing DAMP signaling.

We tested the hypothesis that melatonin prevents brain death (BD) tissue extract (BDEX)-induced cardiac damage by suppressing inflammatory damage-associated molecular pattern (DAMP) signaling in rats. Six hours after BD induction, levels of a DAMP component (HMGB1) and inflammatory markers (TLR-2, TLR-4, MYD88, IκB, NF-κB, IL-1ß, IFN-γ, TNF-α and IL-6) were higher in brain tissue from BD animals than controls. Levels of HMGB1 and inflammatory markers were higher in BDEX-treated H9C2 cardiac myoblasts than in cells treated with healthy brain tissue extract. These increases were attenuated by melatonin but re-induced with luzindole (all P < 0.001). Additional male rats (n = 30) were divided into groups 1 (negative control), 2 (healthy brain tissue extract implanted in the left ventricular myocardium [LVM]), 3 (BDEX-LVM), 4 (BDEX-LVM + melatonin), and 5 (BDEX-LVM + melatonin + luzindole). Collagen deposition/fibrosis and LVM levels of MTR2, HMGB1, inflammatory markers, oxidative stress, apoptosis, mitochondrial damage and DNA damage were highest in group 3, lowest in groups 1 and 2, and higher in group 5 than in group 4. Heart function and LVM levels of MTR1 and anti-inflammatory, mitochondrial-integrity and anti-oxidative markers exhibited a pattern opposite that of the inflammatory markers in the five groups (all P < 0.0001). These results indicate melatonin inhibits BDEX-induced cardiac damage by suppressing the DAMP inflammatory axis.

Extracorporeal shock wave therapy effectively protects brain against chronic cerebral hypo-perfusion-induced neuropathological changes.

This study tested the hypothesis that extracorporeal shock wave (ECSW) therapy could protect mouse brain from chronic cerebral hypoperfusion (CHP)-induced neuropathological changes in a bilateral carotid arterial stenosis (CAS) model. Adult-male C57BL/6 (B6) mice (n=36) were randomized into group 1 (sham-control), group 2 (CHP) and group 3 [CHP+ECSW (100 impulses at 0.15 mJ/mm2) on day 5, 10 and 15 after CHP induction]. By day 60 after CHP induction, the white matter lesion, protein expressions of inflammatory (TNF-α/NF-κB/iNOS), oxidative-stress (NOX-1/NOX-2/NOX-4/nitrotyrosine), angiogenesis (eNOS/CD31), apoptotic (Bax/caspase-3/PARP), fibrotic (Smad3/TGF-ß) and mitochondrial-damaged (cytosolic cytochrome-C) biomarkers were significantly higher in group 2 than in groups 1 and 3, and significantly higher in group 3 than in group 1, whereas the protein expressions of anti-apoptotic (Bcl-2), anti-fibrotic (BMP-2/Smad1/5), and mitochondrial-integrity (mitochondrial cytochrome-C) biomarkers showed an opposite pattern to inflammation among the three groups (all P<0.0001). The cellular expressions of inflammatory (Iba-1/GFAP/CD14, F4/80), apoptotic (TUNEL-assay) and brain-damaged (γ-H2AX/AQP4) biomarkers showed an identical pattern to inflammation, whereas the cellular expressions of endothelial-cell (CD31/vWF), neuron/energy-integrity (NeuN/PGC-1α) and small-vessel density exhibited an opposite pattern to inflammation among the three groups (all P<0.0001). Cellular angiogenesis (VEGF/SDF-1α) significantly and progressively increased from groups 1 to 3 (all P<0.0001). In conclusion, ECSW therapy enhanced angiogenesis, inhibited molecular-cellular perturbations, and protected the white matter and neuron from CHP damage.

The mTOR-FAK mechanotransduction signaling axis for focal adhesion maturation and cell proliferation.

BACKGROUND: Mechanotransduction (MTD) is an important physiopathological signalling pathway associated with cardiovascular disease such as hypertension. Phosphorylation of focal adhesion kinase (FAK) is a MTD-sensing protein. This study tested the hypothesis that mTOR-FAK MTD signaling axis was crucial for focal adhesion (FA) maturation and cell proliferation. METHODS: Shock-wave was adopted as a tool for MTD and mTOR-FAK signaling. RESULTS: After demonstrating a failure in FAK phosphorylation after microfilament depolymerization, we attempted to identify the upstream regulator out of three kinases known to be activated in pressure-stimulated MTD [i.e., GSK-3ß, Akt, and mTORC1 (mammalian target of rapamycin complex 1)]. Of the three specific inhibitors, only rapamycin, an inhibitor of mTORC1, was found to inhibit FAK phosphorylation, suggesting that mTORC1 is the upstream regulator in shock-wave-elicited FAK phosphorylation. Moreover, mTOR and its readout protein S6K were found to be activated by shock-wave stimulation. On the other hand, microscopic examination revealed not only MTD-induced increase in the number of actin stress fibers, but also alternative subcellular localization of mTORC1 as vesicle-like inclusions on microfilaments. Besides, rapamycin was found to destruct the granular pattern of mTORC1, while dissociation between F-actin and mTORC1 was noted after cytochalasin D administration. Since mTORC1 and FAK are essential for cell proliferation, we performed proliferation assay for mesenchymal stem cell (MSC) with and without shock-wave administration/rapamycin treatment/FAK depletion. The results demonstrated significant enhancement of cell proliferation after shock-wave stimulation but remarkable suppression after rapamycin and siFAK treatment. CONCLUSION: Our findings suggest not only a co-ordinated regulation of FAK phosphorylation by mTORC1 and microfilaments, but also the participation of mTORC1-FAK signalling in MSC proliferation.

EPO-cyclosporine combination therapy reduced brain infarct area in rat after acute ischemic stroke: role of innate immune-inflammatory response, micro-RNAs and MAPK family signaling pathway.

This study tested the hypothesis that erythropoietin (EPO) and cyclosporine (CsA) could effectively reduce brain infarct area (BIA) in rat after acute ischemic stroke (AIS) through regulating inflammation, oxidative stress, MAPK family signaling and microRNA (miR-223/miR-30a/miR-383). Adult male Sprague-Dawley rats (n = 48) were equally divided into group 1 (sham control), group 2 (AIS), group 3 [AIS+EPO (5,000 IU/kg at 0.5/24/48 h, subcutaneous)] and group 4 [AIS+CsA (20.0 mg/kg at 0.5/24/48 h, intra-peritoneal)]. By 72 h, histopathology showed that BIA was largest in group 2 and smallest in group 1, and significantly larger in group 4 than group 3 (all P<0.0001). The three microRNAs expressed were higher in group 2 than in the other three groups (all P<0.04); between these three latter groups there were no significant differences. The protein expressions of MAPK family [phosphorylated (p)-ERK1/2, p-p38/p-JNK], inflammatory (iNOS/MMP-9/TNF-α/NF-κB/IL-12/MIP-1α/CD14/CD68/Ly6g), apoptotic (caspase-3/PARP/mitochondrial-Bax), oxidative-stress (NOX-1/NOX-2/oxidized protein) and mitochondrial-damaged (cytosolic cytochrome-C) biomarkers exhibited an identical pattern to BIA findings (all P<0.0001). The cellular expressions of brain edema (AQP4+), inflammation (CD11+/glial-fibrillary-acid protein+), and cellular damage (TUNEL assay/positive Periodic acid-Schiff stain) biomarkers exhibited an identical pattern, whereas the cellular-integrity markers (neuN+/MAP2+/doublecorin+) exhibited an opposite pattern to BIA (all P value <0.001). EPO-CsA therapy markedly reduced BIA mainly by suppressing the innate immune response to inflammation, oxidative stress, microRNAs (miR-223/miR-30a/miR-383) and MAPK family signaling.

Therapeutic effects of adipose-derived mesenchymal stem cells against brain death-induced remote organ damage and post-heart transplant acute rejection.

We tested the hypothesis that allogenic adipose-derived mesenchymal stem cells (ADMSCs) alleviated brain death (BD)-induced remote organ damage and events of post heart-transplant acute rejection. To determine the impact of BD on remote organ damage, adult-male F344 rats (n=24) were categorized sham-control (SC), BD and BDMSC (allogenic ADMSC/1.2 × 106 cells/derived from F344 by intravenous transfusion 3 h after BD procedure). To determine the protective effect of allogenic ADMSCs, animals (n=8/each group in F344/Lewis) were categorized into groups BD-T(F344 heart transplanted into Lewis by 6h after BD), BD-TMSC(D1/3) (BD induction for 6h then heart transplantation, and allogenic ADMSCs transfusion at days 1 and 5 after heart transplantation), BD-TMSC(3h) (BD + ADMSC/1.2 × 106 cells at 3h and heart transplantation at 6h after BD) and BD-TMSC(3h, D1/3) [BD + ADMSC/1.2 × 106 cells at 3h and heart transplantation at 6h after BD, then ADMSC therapy by days 1/3]. At day 5 post procedure, liver, kidney and heart specimens showed higher molecular-cellular levels of inflammation, immune reaction, apoptosis and fibrosis in BD than in SC that were reversed in BDMSC (all P < 0.0001). These molecular-cellular expressions and circulating/splenic levels of innate/adoptive immune cells were higher in BD-T, lowest in BD-TMSC(3h, D1/3) and higher BD-TMSC(3h) in than BD-TMSC(D1/3), whereas heart function showed an opposite pattern among the four groups (all P < 0.001). In conclusion, ADMSCs suppressed BD-caused remote organ damage and heart-transplant rejection.

Intravenous administration of xenogenic adipose-derived mesenchymal stem cells (ADMSC) and ADMSC-derived exosomes markedly reduced brain infarct volume and preserved neurological function in rat after acute ischemic stroke.

We tested the hypothesis that combined xenogenic (from mini-pig) adipose-derived mesenchymal stem cell (ADMSC) and ADMSC-derived exosome therapy could reduce brain-infarct zone (BIZ) and enhance neurological recovery in rat after acute ischemic stroke (AIS) induced by 50-min left middle cerebral artery occlusion. Adult-male Sprague-Dawley rats (n = 60) were divided equally into group 1 (sham-control), group 2 (AIS), group 3 [AIS-ADMSC (1.2×106 cells)], group 4 [AIS-exosome (100µg)], and group 5 (AIS-exosome-ADMSC). All therapies were provided intravenously at 3h after AIS procedure. BIZ determined by histopathology (by day-60) and brain MRI (by day-28) were highest in group 2, lowest in group 1, higher in groups 3 and 4 than in group 5, but they showed no difference between groups 3 and 4 (all p < 0.0001). By day-28, sensorimotor functional results exhibited an opposite pattern to BIZ among the five groups (p < 0.005). Protein expressions of inflammatory (inducible nitric oxide synthase/tumor necrosis factor-α/nuclear factor-κB/interleukin-1ß/matrix metalloproteinase-9/plasminogen activator inhibitor-1/RANTES), oxidative-stress (NOX-1/NOX-2/oxidized protein), apoptotic (caspase-3/ Poly-ADP-ribose polymerase), and fibrotic (Smad3/transforming growth factor-ß) biomarkers, and cellular expressions of brain-damaged (γ-H2AX+/ XRCC1-CD90+/p53BP1-CD90+), inflammatory (CD11+/CD68+/glial fibrillary acid protein+) and brain-edema (aquaporin-4+) markers showed a similar pattern of BIZ among the groups (all n < 0.0001). In conclusion, xenogenic ADMSC/ADMSC-derived exosome therapy was safe and offered the additional benefit of reducing BIZ and improving neurological function in rat AIS.

Tissue plasminogen activator deficiency preserves neurological function and protects against murine acute ischemic stroke.

BACKGROUND: We tested the hypothesis that tissue plasminogen activator (tPA) deficiency protected against acute ischemic stroke (AIS)-induced brain injury. METHODS AND RESULTS: Wild-type mice (n=54) were categorized into group 1 (sham control, n=18) and group 3 [AIS by permanent ligation of left common carotid artery (CCA) and cramping right CCA for 1h and then reperfusion followed by hypoxia (11% of oxygen supply for 2h), n=36]. Similarly, tPA knockout (tPA(-/-)) mice (n=54) were randomized into group 2 (sham control, n=18) and group 4 (AIS, n=36). By day 28 after AIS procedure, mortality rate was higher in group 3 (77.8%) than in group 4 (38.9%) and lowest in groups 1 (0%) and 2 (0%) (p<0.001). By days 3 and 28, MRI demonstrated a pattern of changes in brain-infarct volume identical to that of mortality among four groups (p<0.001). By day 28, protein expressions of inflammatory (MMP-9, TNF-α, NF-κB, iNOS, PAI-1, RANTES), oxidative (NOX-1, NOX-2, oxidized protein), apoptotic (cleaved caspase-3 & PARP, Bax), and fibrotic (Smad3, TGF-ß) biomarkers and cellular expressions of inflammation (CD11, F4/80, GFAP), DNA-damage (γ-H2AX) and brain-edema (AQP4) markers exhibited an identical pattern compared to that of mortality (all p<0.001), whereas protein expressions of endothelial (eNOS, CD31), anti-fibrotic (Smad1/5, BMP-2) biomarkers, and number of small vessels displayed an opposite pattern (all p<0.001) among four groups. Expressions of protein and cellular angiogenesis markers (VEGF, SDF-1α, CXCR4) were progressively increased from groups 1 and 2 to group 4 (all p<0.0001). CONCLUSION: tPA deficiency protected the brain from AIS injury.

Intra-carotid arterial administration of autologous peripheral blood-derived endothelial progenitor cells improves acute ischemic stroke neurological outcomes in rats.

OBJECTIVE: We tested the hypothesis that transfusion of autologous peripheral blood-derived endothelial progenitor cells (PBDEPC) via the internal carotid artery could reduce brain-infarct zone (BIZ) and neurological deficit in rats following acute ischemic stroke (IS) induced by 50-min left middle cerebral artery occlusion. DESIGN: Adult male Sprague-Dawley rats (n=60) were equally divided into group 1 [sham control (SC)], group 2 [SC-PBDEPC (5.7 × 10(6)/kg)], group 3 (IS), group 4 [IS-low-dose PBDEPC (1.7 × 10(6)/kg)], group 5 [IS-high-dose PBDEPC (5.7×10(6)/kg)]. Groups 2 to 5 received G-CSF (35 µg/kg subcutaneously) for 4 days before drawing blood for PBDEPC culture. MEASUREMENTS AND MAIN RESULTS: By day 90, BIZ determined by histopathology (area) and brain MRI (volume) were highest in group 3, lowest in groups 1 and 2, higher in group 4 than in group 5 (all p<0.0001), and not significantly different between groups 1 and 2. Sensorimotor functional results exhibited an opposite pattern of BIZ among groups 3 to 5 (p<0.005). Angiogenesis biomarkers (SDF-1α, CXCR4, VEGF, angiopoietin-1) significantly increased progressively from groups 1 and 2 to group 5 (all p<0.0001). Oxidative-stress (NOX-1, NOX-2, oxidized protein), apoptotic (cleaved caspase 3 and PARP, mitochondrial Bax), inflammatory (MMP-9, TNF-α, AQP-4, GFAP, iNOS), and brain-damaged (cytosolic cytochrome-C) biomarkers showed an identical pattern, whereas anti-inflammatory (Bcl-2), mitochondrial preservation (mitochondrial cytochrome-C, PGC-1α), and endothelial function (CD31+, vWF+, eNOS) biomarkers, and vessel density showed an opposite pattern of BIZ among these five groups (all p<0.001). CONCLUSION: Higher-dose was superior to lower-dose EPC treatment for reducing BIZ and improving neurological functional outcome.

Extracorporeal shock wave effectively attenuates brain infarct volume and improves neurological function in rat after acute ischemic stroke.

BACKGROUND: To investigate the effect of shock wave (SW) on brain-infarction volume (BIV) and neurological function in acute ischemic stroke (AIS) by left internal carotid artery occlusion in rats. METHODS AND RESULTS: SD rats (n=48) were divided into group 1 [sham-control (SC)], group 2 [SC-ECSW (energy dosage of 0.15 mJ/mm(2)/300 impulses)], group 3 (AIS), and group 4 (AIS-ECSW) and sacrificed by day 28 after IS induction. In normal rats, caspase-3, Bax and TNF-α biomarkers did not differ between animals with and without ECSW therapy, whereas Hsp70 was activated post-ECSW treatment. By day 21 after AIS, Sensorimotor-functional test identified a higher frequency of turning movement to left in group 3 than that in group 4 (P<0.05). By day 28, brain MRI demonstrated lager BIV in group 3 than that in group 4 (P<0.001). Angiogenesis biomarkers at cellular (CD31, α-SMA+) and protein (eNOS) levels and number of neuN+ cells were higher in groups 1 and 2 than those in groups 3 and 4, and higher in group 4 than those in group 3, whereas VEGF and Hsp70 levels were progressively increased from groups 1 and 2 to group 4 (all P<0.001). Protein expressions of apoptosis (Bax, caspase 3, PARP), inflammation (MMP-9, TNF-α), oxidative stress (NOX-1, NOX-2, oxidized protein) and DNA-damage marker (γ-H2AX), and expressions of γ-H2AX+, GFAP+, AQP-4+ cells showed an opposite pattern compared to that of angiogenesis among the four groups (all P<0.001). CONCLUSION: ECSW therapy was safe and effective in reducing BIV and improved neurological function.

Combined therapy with shock wave and autologous bone marrow-derived mesenchymal stem cells alleviates left ventricular dysfunction and remodeling through inhibiting inflammatory stimuli, oxidative stress & enhancing angiogenesis in a swine myocardial infarction model.

BACKGROUND: We hypothesized that combined therapy with shock wave (SW) and autologous bone marrow-derived mesenchymal stem cells (BMDMSCs) is superior to either therapy alone for alleviating left ventricular (LV) dysfunction. METHODS AND RESULTS: Male mini-pigs (n=30) equally divided into group 1 (sham control), group 2 [acute myocardial infarction (AMI) by left coronary artery ligation], group 3 (AMI-SW), group 4 (AMI-BMDMSC), and group 5 (AMI-SW-BMDMSC) were sacrificed by day 60 and the hearts were collected for studies. Baseline LV injection fraction [LVEF (%)] and LV chamber size did not differ among the five groups (p>0.5). By day 60, LVEF was highest in group 1 and lowest in group 2, significantly higher in group 5 than that in groups 3 and 4, and significantly higher in group 4 than that in group 3 (p<0.001). Cellular and protein levels of VEGF, CXCR4, and SDF-1α were significantly increased progressively from groups 1 to 5 (all p<0.05). Small vessel number and protein expressions of CD31 and eNOS were highest in groups 1 and 5, lowest in group 2, and significantly higher in group 4 than those in group 3 (p<0.001). Protein (MMP-9, TNF-1α and NF-κB) and cellular (CD14+, CD40+) levels of inflammatory biomarkers, protein expressions of oxidative stress (oxidized protein, NOX-1, NOX-2), apoptosis (Bax, caspase-3, PARP), infarct size, and LV dimensions showed a pattern opposite to that of LVEF among all groups (all p<0.001). CONCLUSIONS: Combined SW-BMDMSC therapy is superior to either therapy alone for improving LVEF, reducing infarct size, and inhibiting LV remodeling.

Extracorporeal shock wave therapy effectively prevented diabetic neuropathy.

BACKGROUND: We tested the hypothesis that extracorporeal shock wave (ECSW) therapy can effectively protect sciatic nerve (SN) from diabetes mellitus (DM)-induced neuropathy in leptin-deficient (ob/ob) mice. METHODS AND RESULTS: Eighteen-week C57BL/6 mice (n=8) served as age-matched controls (group 1) and ob/ob mice (n=16) were categorized into DM (group 2) and DM + ECSW (0.12 mJ/mm(2) for 4 times of 200 impulses at 3-week intervals) (group 3). The animals were sacrificed two weeks post-ECSW. In vitro results showed that the protein expressions of oxidative stress (NOX-1, NOX-2, oxidized protein), inflammation (MMP-9, TNF-α, iNOS), apoptosis (Bax, cleaved caspase-3, & PARP), and DNA-damage marker (γ-H2AX) were significantly higher in RT4-D6P2T (schwannoma cell line) treated by menadione (25 µM) compared with control group and were significantly reversed after ECSW (0.12 mJ/mm(2), 200 impulses) (all p<0.001). mRNA expressions of inflammation (MMP-9, TNF-α, iNOS), oxidative stress (NOX-1, NOX-2) and apoptosis (Bax, caspase-3) in SN were significantly higher in group 2 than in group 1 and were significantly reversed in group 3, whereas the mRNA expressions of anti-oxidants (HO-1, NQO1) progressively increased from group 1 to group 3 (all p<0.001). Cellular expressions of F4/80+, CD14+, γ-H2AX+ cells, and number of vacuolar formation in SN showed a pattern identical to that of inflammation markers among all groups (all p<0.001). Microscopic findings of Schwann cells and myelin-sheath scores, and number of eNOS+ cells in SN showed a reversed pattern compared to that of inflammation among all groups (all p<0.001). CONCLUSIONS: ECSW therapy protected SN against DM-induced neuropathy.

The prognostic values of leukocyte Rho kinase activity in acute ischemic stroke.

OBJECTIVE: It has been reported that leukocyte ROCK activity is elevated in patients after ischemic stroke, but it is unclear whether leukocyte ROCK activity is associated with clinical outcomes following acute stroke events. The objective of this study is to investigate if leukocyte ROCK activity can predict the outcomes in patients with acute ischemic stroke. MATERIALS AND METHODS: We enrolled 110 patients of acute ischemic stroke and measured the leukocyte ROCK activity and plasma level of inflammatory cytokines to correlate the clinical outcomes of these patients. RESULTS: The leukocyte ROCK activity at 48 hours after admission in acute ischemic stroke patients was higher as compared to a risk-matched population. The leukocyte ROCK activity significantly correlated with National Institute of Health Stroke Scale (NIHSS) difference between admission and 90 days after stroke event. Kaplan-Meier survival estimates showed lower stroke-free survival during follow-up period in patients with high leukocyte ROCK activity or plasma hsCRP level. Leukocyte ROCK activity independently predicted the recurrent stroke in patients with atherosclerotic stroke. CONCLUSIONS: This study shows elevated leukocyte ROCK activity in patients with ischemic stroke as compared to risk-matched subjects and is an independent predictor for recurrent stroke.

Comparison of acute versus convalescent stage high-sensitivity C-Reactive protein level in predicting clinical outcome after acute ischemic stroke and impact of erythropoietin.

BACKGROUND AND AIM: Currently, no data on the optimal time point after acute ischemic stroke (IS) at which high-sensitivity C-reactive protein (hs-CRP) level is most predictive of unfavorable outcome. We tested the hypothesis that hs-CRP levels during both acute (48 h after IS) and convalescent (21 days after IS) phases are equally important in predicting 90-day clinical outcome after acute IS. We further evaluated the impact of erythropoietin (EPO), an anti-inflammatory agent, on level of hs-CRP after acute IS. METHODS: Totally 160 patients were prospectively randomized to receive either EPO therapy (group 1, n = 80) (5,000 IU each time, subcutaneously) at 48 h and 72 h after acute IS, or placebo (group 2, n = 80). Serum level of hs-CRP was determined using ELISA at 48 h and on day 21 after IS and once in 60 healthy volunteers. RESULTS: Serum level of hs-CRP was substantially higher in all patients with IS than in healthy controls at 48 h and day 21 after IS (all p < 0.001). Levels of hs-CRP did not differ between group 1 and 2 at 48 h and day 21 after IS (all p > 0.5). Multivariate analysis showed that hs-CRP levels (at 48 h and day 21) were independently predictive of 90-day major adverse neurological event (MANE) (defined as recurrent stroke, NIHSS≥8, or death) (all p < 0.03), whereas EPO therapy was independently predictive of reduced 90-day MANE (all p < 0.02). CONCLUSION: EPO therapy which was independently predictive of freedom from 90-day MANE did not alter the crucial role of hs-CRP levels measured at 48 h and 21-day in predicting unfavorable clinical outcome after IS.

Enhanced protection against pulmonary hypertension with sildenafil and endothelial progenitor cell in rats.

BACKGROUND: Sildenafil and bone marrow-derived endothelial progenitor cells (BMDEPCs) have been shown to ameliorate monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) in the rat. We test whether combined sildenafil and BMDEPC treatment exerts additional protection against MCT-induced PAH in rats. METHODS: Male Sprague-Dawley rats were randomized to receive saline injection only (group 1), MCT (70 mg/kg) only (group 2), MCT plus autologous BMDEPC (2.0×10(6) cells) transplantation (group 3), MCT with sildenafil (30 mg/kg/day) (group 4), and MCT with combined BMDEPCs-sildenafil (30 mg/kg/day) (group 5). Intravenous BMDEPC and oral sildenafil were given on day 3 after MCT administration. Hemodynamics were analyzed using Labchart software, whereas cellular and molecular parameters were measured using flow cytometry, real-time PCR, TUNEL assay, Western blot, and immunohistochemical staining. RESULTS: By day 35 following MCT treatment, lower expression of connexin43, protein kinase C-ε, Bcl-2, and endothelial nitric oxide synthase and higher expression of tumor necrosis factor-α and caspase 3 were found in right ventricle (RV) and lung in group 2 compared with other groups (all p<0.05). The number of alveolar sacs and lung arterioles were also lower in group 2 than in other groups (all p<0.05). Furthermore, RV systolic pressure (RVSP), RV weight, and RV-to-final body weight ratio were substantially increased in group 2 than in other groups, and notably higher in groups 3 and 4 than in groups 1 and 5 (all p<0.0001). CONCLUSIONS: Combined therapy with autologous BMDEPC and sildenafil is superior to either BMDPEC or sildenafil alone for preventing MCT-induced PAH.

Combination of cyclosporine and erythropoietin improves brain infarct size and neurological function in rats after ischemic stroke.

BACKGROUND: This study tested the superiority of combined cyclosporine A (CsA)-erythropoietin (EPO) therapy compared with either one in limiting brain infarction area (BIA) and preserving neurological function in rat after ischemic stroke (IS). METHODS: Fifty adult-male SD rats were equally divided into sham control (group 1), IS plus intra-peritoneal physiological saline (at 0.5/24/48 h after IS) (group 2), IS plus CsA (20.0 mg/kg at 0.5/24h, intra-peritoneal) (group 3), IS plus EPO (5,000IU/kg at 0.5/24/48h, subcutaneous) (group 4), combined CsA and EPO (same route and dosage as groups 3 and 4) treatment (group 5) after occlusion of distal left internal carotid artery. RESULTS: BIA on day 21 after acute IS was higher in group 2 than in other groups and lowest in group 5 (all p < 0.01). The sensorimotor functional test showed higher frequency of left turning in group 2 than in other groups and lowest in group 5 (all p < 0.05). mRNA and protein expressions of apoptotic markers and number of apoptotic nuclei on TUNEL were higher in group 2 than in other groups and lowest in group 1 and 5, whereas the anti-apoptotic markers exhibited an opposite trend (all p < 0.05). The expressions of inflammatory and oxidized protein were higher in group 2 than in other groups and lowest in group 1 and 5, whereas anti-inflammatory markers showed reversed changes in group 1 and other groups (all p < 0.05). The number of aquaporin-4+ and glial fibrillary acid protein+ stained cells were higher in group 2 as compared to other groups and lowest in groups 1 and 5 (all p < 0.01). CONCLUSION: combined treatment with CsA and EPO was superior to either one alone in protecting rat brain from ischemic damage after IS.

Effect of erythropoietin on level of circulating endothelial progenitor cells and outcome in patients after acute ischemic stroke.

INTRODUCTION: Erythropoietin (EPO) enhances the circulating level of endothelial progenitor cells (EPCs), which has been reported to be associated with prognostic outcome in ischemic stroke (IS) patients. The aim of this study was to evaluate the time course of circulating EPC level and the impact of EPO therapy on EPC level and clinical outcome in patients after acute IS. METHODS: In total, 167 patients were prospectively randomized to receive either EPO therapy (group 1) (5,000 IU each time, subcutaneously) at 48 h and 72 h after acute IS, or serve as placebo (group 2). The circulating level of EPCs (double-stained markers: CD31/CD34 (E1), CD62E/CD34 (E2) and KDR/CD34 (E3)) was determined using flow cytometry at 48 h and on days 7 and 21 after IS. EPC level was also evaluated once in 60 healthy volunteers. RESULTS: Circulating EPC (E1 to E3) level at 48 h after IS was remarkably higher in patients than in control subjects (P < 0.02). At 48 h and on Day 7 after IS, EPC (E1 to E3) level did not differ between groups 1 and 2 (all P > 0.1). However, by Day 21, EPC (E1 to E3) level was significantly higher in group 1 than in group 2 (all P < 0.03). Additionally, 90-day recurrent stroke rate was notably lower in group 1 compared with group 2 (P = 0.022). Multivariate analysis demonstrated that EPO therapy (95% confidence interval (CI), 0.153 to 0.730; P = 0.006) and EPC (E3) (95% CI, 0.341 to 0.997; P = 0.049) levels were significantly and independently predictive of a reduced 90-day major adverse neurological event (MANE) (defined as recurrent stroke, National Institutes of Health Stroke scale ≥8, or death). CONCLUSIONS: EPO therapy significantly improved circulating EPC level and 90-day MANE. TRIAL REGISTRATION NUMBER: ISRCTN: ISRCTN96340690.

Bone marrow cells reduce fibrogenesis and enhance regeneration in fibrotic rat liver.

BACKGROUND: This study aimed at investigating the cellular and molecular impacts of bone marrow-derived mononuclear cells (BMCs) on regeneration of fibrotic liver in rats. MATERIALS AND METHODS: Thirty male adult Fisher rats were randomized into three groups: group 1 (normal controls, n = 10); group 2 (carbon tetrachloride-induced liver fibrosis, n = 10), and group 3 (liver fibrosis with portal venous infusion of autologous BMCs, 1 × 10(6), n = 10). After 7-d culturing, BMCs were characterized by flow cytometry. Groups 2 and 3 received BMC aspiration through bilateral femurs 5 d before hepatectomy. All animals received 70% hepatectomy, whereas only group 3 received a bolus of intra-portal BMC infusion 48 h after hepatectomy. Liver-to-body weight ratio, degree of fibrosis (Masson trichrome staining), oxidative stress, peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α), collagen I, tumor necrosis factor-α (TNF-α), and transforming growth factor- ß (TGF-ß) expressions were analyzed 14 d after hepatectomy. Immunohistochemical staining for albumin, α-smooth muscle actin, and CD31 was performed for tracing cellular differentiation. RESULTS: Cellular phenotypes typical of hepatocyte, hepatic stellate cell (HSC), and endothelial cell were identified in the engrafted BMCs. Liver-to-body weight ratio was enhanced with PGC-1α significantly preserved, whereas oxidative index, collagen I, α-SMA, TNF-α, and TGF-ß expressions were all decreased in group 3 compared with group 2 (all P < 0.05). CONCLUSIONS: This study demonstrated a positive impact of intra-portal BMC infusion in enhancing regeneration and reducing fibrosis of the regenerating fibrotic liver in rats through suppressing HSC activation and inflammatory cytokine expressions, preserving mitochondrial function and reducing oxidative stress.