Intranasal Administration of Human MSC for Ischemic Brain Injury in the Mouse: In Vitro and In Vivo Neuroregenerative Functions

Intranasal treatment with C57BL/6 MSCs reduces lesion volume and improves motor and cognitive behavior in the neonatal hypoxic-ischemic (HI) mouse model. In this study, we investigated the potential of human MSCs (hMSCs) to treat HI brain injury in the neonatal mouse. Assessing the regenerative capacity of hMSCs is crucial for translation of our knowledge to the clinic. We determined the neuroregenerative potential of hMSCs in vitro and in vivo by intranasal administration 10 d post-HI in neonatal mice. HI was induced in P9 mouse pups. 1×10⁶ or 2×10⁶ hMSCs were administered intranasally 10 d post-HI. Motor behavior and lesion volume were measured 28 d post-HI. The in vitro capacity of hMSCs to induce differentiation of mouse neural stem cell (mNSC) was determined using a transwell co-culture differentiation assay. To determine which chemotactic factors may play a role in mediating migration of MSCs to the lesion, we performed a PCR array on 84 chemotactic factors 10 days following sham-operation, and at 10 and 17 days post-HI. Our results show that 2×10⁶ hMSCs decrease lesion volume, improve motor behavior, and reduce scar formation and microglia activity. Moreover, we demonstrate that the differentiation assay reflects the neuroregenerative potential of hMSCs in vivo, as hMSCs induce mNSCs to differentiate into neurons in vitro. We also provide evidence that the chemotactic factor CXCL10 may play an important role in hMSC migration to the lesion site. This is suggested by our finding that CXCL10 is significantly upregulated at 10 days following HI, but not at 17 days after HI, a time when MSCs no longer reach the lesion when given intranasally. The results described in this work also tempt us to contemplate hMSCs not only as a potential treatment option for neonatal encephalopathy, but also for a plethora of degenerative and traumatic injuries of the nervous system.     

SAHA Enhances Synaptic Function and Plasticity In Vitro but Has Limited Brain Availability In Vivo and Does Not Impact Cognition

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) used for the treatment of cutaneous T cell lymphoma (CTCL) and under consideration for other indications. In vivo studies suggest reducing HDAC function can enhance synaptic function and memory, raising the possibility that SAHA treatment could have neurological benefits. We first examined the impacts of SAHA on synaptic function in vitro using rat organotypic hippocampal brain slices. Following several days of SAHA treatment, basal excitatory but not inhibitory synaptic function was enhanced. Presynaptic release probability and intrinsic neuronal excitability were unaffected suggesting SAHA treatment selectively enhanced postsynaptic excitatory function. In addition, long-term potentiation (LTP) of excitatory synapses was augmented, while long-term depression (LTD) was impaired in SAHA treated slices. Despite the in vitro synaptic enhancements, in vivo SAHA treatment did not rescue memory deficits in the Tg2576 mouse model of Alzheimer’s disease (AD). Along with the lack of behavioral impact, pharmacokinetic analysis indicated poor brain availability of SAHA. Broader assessment of in vivo SAHA treatment using high-content phenotypic characterization of C57Bl6 mice failed to demonstrate significant behavioral effects of up to 150 mg/kg SAHA following either acute or chronic injections. Potentially explaining the low brain exposure and lack of behavioral impacts, SAHA was found to be a substrate of the blood brain barrier (BBB) efflux transporters Pgp and Bcrp1. Thus while our in vitro data show that HDAC inhibition can enhance excitatory synaptic strength and potentiation, our in vivo data suggests limited brain availability may contribute to the lack of behavioral impact of SAHA following peripheral delivery. These results do not predict CNS effects of SAHA during clinical use and also emphasize the importance of analyzing brain drug levels when interpreting preclinical behavioral pharmacology.     

The Expression of Mouse Tyrosinase in Chick Cells In Vitro and In Vivo When Controlled by a Constitutive Promoter

Virally introduced mouse tyrosinase expression was checked both in vitro and in vivo in chicken cells and tissues. The results indicate that a constitutive promoter is able to express mouse tyrosinase in a variety of cells and tissues both in vitro and in vivo. Tyrosinase expression is marked by pigment production in situ, which is visible at macroscopic as well as microscopic levels without the use of substrates. It is concluded that tyrosinase can be a valuable marker for tracking gene insertion since it is spontaneously expressed. The expression of tyrosinase in some cells and tissues has a detrimental effect, however, and should be controlled by tissue-specific promoters.     

Modulation in Acetylcholinesterase of Rat Brain by Pyrethroids In Vivo and an In Vitro Kinetic Study

Abstract: The modulation in acetylcholinesterase (AChE) of rat brain by two pyrethroids—permethrin (PM) and cypermethrin (CPM)—was studied both in vivo and in vitro. PM inhibited AChE activity in all regions of the rat brain (cerebral cortex, cerebellum, corpora striata, brainstem, hippocampus, and hypothalamus) at 4, 8, and 12 h after gastric intubation, whereas CPM elevated the enzyme activity in vivo. Substrate-dependent enzyme kinetic studies have shown that PM and CPM behave as mixed-type inhibitors, as evidenced by alterations in both Michaelis-Menten constant ( K _m) and maximal velocity ( V _max) values. This indicates that both PM and CPM and substrate acetylcholine interact at hydrophobic subsites and may be able to bind simultaneously to the enzyme.     

The Expression of CD90/Thy-1 in Hepatocellular Carcinoma: An In Vivo and In Vitro Study

Although the CD90 (Thy-1) was proposed as biomarker of several tumors and cancer stem cells, the involvement of this molecule in the progression of hepatocellular carcinoma (HCC) and other less frequent hepatic neoplasms is still undefined. The distribution of CD90 was investigated both in in vivo (human tissues samples) and in vitro (human HCC cell line JHH-6). A total of 67 liver tumors were analyzed: 51 HCC, 6 cholangiocarcinoma and 10 hepatoblastoma. In all cases, paired tissue sample of both the tumor and cirrhotic liver was available. Hepatic tissue obtained in 12 healthy livers was used as control. CD90 gene expression was studied by RT-qPCR, protein expression was assessed by quantitative Western Blot, immunofluorescence and flow cytometry. The CD90 expression analysis showed a significant increment in tumor compared to both its paired cirrhotic tissue and normal liver (p<0.05 and p<0.001, respectively). This increase was accompanied by the up-regulation of stromal component in the cancer, as demonstrated by alpha smooth muscle actin staining. In vitro analysis of JHH-6 cell line showed a higher proliferation capacity of CD90⁺ compared to CD90^- cells (p<0.001), also noticed in 3D clonogenic assay (p<0.05), associated by a significant higher expression of the promoting factors (hepatocyte growth factor, fibroblast associated protein and alpha smooth muscle actin 2). A higher expression of the breast cancer resistance protein was found in CD90⁺ subpopulation while the multidrug resistance protein 1 showed an opposite behavior. Collectively, these results point to the importance of CD90 in the HCC.     

False In Vitro and In Vivo Elevations of Uric Acid Levels in Mouse Blood

Uric acid (UA) levels in mouse blood have been reported to range widely from 0.1 μM to 760 μM. The aim of this study was to demonstrate false in vitro and in vivo elevations of UA levels in mouse blood. Male ICR mice were anesthetized with pentobarbital (breathing mice) or sacrificed with overdose ether (non-breathing mice). Collected blood was dispensed into MiniCollect® tubes and incubated in vitro for 0 or 30 min at room temperature. After separation of plasma or serum, the levels of UA and hypoxanthine were determined using HPLC. From the non-incubated plasma of breathing mice, the true value of UA level in vivo was 13.5 ± 1.4 μM. However, UA levels in mouse blood increased by a factor of 3.9 following incubation in vitro. This “false in vitro elevation” of UA levels in mouse blood after blood sampling was inhibited by allopurinol, a xanthine oxidase inhibitor. Xanthine oxidase was converted to UA in mouse serum from hypoxanthine which was released from blood cells during incubation. Plasma UA levels from non-breathing mice were 19 times higher than those from breathing mice. This “false in vivo elevation” of UA levels before blood sampling was inhibited by pre-treatment with phentolamine, an α-antagonist. Over-anesthesia with ether might induce α-vasoconstriction and ischemia and thus degrade intracellular ATP to UA. For the accurate measurement of UA levels in mouse blood, the false in vitro and in vivo elevations of UA level must be avoided by immediate separation of plasma after blood sampling from anesthetized breathing mice.     

F11-Mediated Inhibition of RhoA Signalling Enhances the Spread of Vaccinia Virus In Vitro and In Vivo in an Intranasal Mouse Model of Infection

The cortical actin cytoskeleton beneath the plasma membrane represents a physical barrier that vaccinia virus has to overcome during its exit from an infected cell. Previous observations using overexpression and pharmacological approaches suggest that vaccinia enhances its release by modulating the cortical actin cytoskeleton by inhibiting RhoA signalling using the viral protein F11. We have now examined the role of F11 and its ability to interact with RhoA to inhibit its downstream signalling in the spread of vaccinia infection both in vitro and in vivo. Live cell imaging over 48 hours reveals that loss of F11 or its ability to bind RhoA dramatically reduces the rate of cell-to-cell spread of the virus in a cell monolayer. Cells infected with the ΔF11L virus also maintained their cell-to-cell contacts, and did not undergo virus-induced motility as observed during wild-type infections. The ΔF11L virus is also attenuated in intranasal mouse models of infection, as it is impaired in its ability to spread from the initial sites of infection to the lungs and spleen. Loss of the ability of F11 to bind RhoA also reduces viral spread in vivo. Our results clearly establish that viral-mediated inibition of RhoA signalling can enhance the spread of infection not only in cell monolayers, but also in vivo.     

Merkel Cells as Putative Regulatory Cells in Skin Disorders: An In Vitro Study

Merkel cells (MCs) are involved in mechanoreception, but several lines of evidence suggest that they may also participate in skin disorders through the release of neuropeptides and hormones. In addition, MC hyperplasias have been reported in inflammatory skin diseases. However, neither proliferation nor reactions to the epidermal environment have been demonstrated. We established a culture model enriched in swine MCs to analyze their proliferative capability and to discover MC survival factors and modulators of MC neuroendocrine properties. In culture, MCs reacted to bFGF by extending outgrowths. Conversely, neurotrophins failed to induce cell spreading, suggesting that they do not act as a growth factor for MCs. For the first time, we provide evidence of proliferation in culture through Ki-67 immunoreactivity. We also found that MCs reacted to histamine or activation of the proton gated/osmoreceptor TRPV4 by releasing vasoactive intestinal peptide (VIP). Since VIP is involved in many pathophysiological processes, its release suggests a putative regulatory role for MCs in skin disorders. Moreover, in contrast to mechanotransduction, neuropeptide exocytosis was Ca²⁺-independent, as inhibition of Ca²⁺ channels or culture in the absence of Ca²⁺ failed to decrease the amount of VIP released. We conclude that neuropeptide release and neurotransmitter exocytosis may be two distinct pathways that are differentially regulated.     

Combining mTOR Inhibition with Radiation Improves Antitumor Activity in Bladder Cancer Cells In Vitro and In Vivo: A Novel Strategy for Treatment

Purpose

Radiation therapy for invasive bladder cancer allows for organ preservation but toxicity and local control remain problematic. As such, improving efficacy of treatment requires radiosensitization of tumor cells. The aim of study is to investigate if the mammalian Target of Rapamycin (mTOR), a downstream kinase of the phosphatidylinositol 3-kinase (PI3K)/AKT survival pathway, may be a target for radiation sensitization.

Experimental Design

Clonogenic assays were performed using 6 bladder cancer cell lines (UM-UC3, UM-UC5, UM-UC6, KU7, 253J-BV, and 253-JP) in order to examine the effects of ionizing radiation (IR) alone and in combination with RAD001, an mTOR inhibitor. Cell cycle analysis was performed using flow cytometry. In vivo, athymic mice were subcutaneously injected with 2 bladder cancer cell lines. Treatment response with RAD001 (1.5 mg/kg, daily), fractionated IR (total 9Gy = 3Gy×3), and combination of RAD001 and IR was followed over 4 weeks. Tumor weight was measured at experimental endpoint.

Results

Clonogenic assays revealed that in all bladder cell lines tested, an additive effect was observed in the combined treatment when compared to either treatment alone. Our data indicates that this effect is due to arrest in both G1 and G2 phases of cell cycle when treatments are combined. Furthermore, our data show that this arrest is primarily regulated by changes in levels of cyclin D1, p27 and p21 following treatments. In vivo, a significant decrease in tumor weight was observed in the combined treatment compared to either treatment alone or control.

Conclusions

Altering cell cycle by inhibiting the mTOR signaling pathway in combination with radiation have favorable outcomes and is a promising therapeutic modality for bladder cancer.     

Synaptic Transfer between Rod and Cone Pathways Mediated by AII Amacrine Cells in the Mouse Retina

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Ethanol Impairs Intestinal Barrier Function in Humans through Mitogen Activated Protein Kinase Signaling: A Combined In Vivo and In Vitro Approach

Background

Ethanol-induced gut barrier disruption is associated with several gastrointestinal and liver disorders.

Aim

Since human data on effects of moderate ethanol consumption on intestinal barrier integrity and involved mechanisms are limited, the objectives of this study were to investigate effects of a single moderate ethanol dose on small and large intestinal permeability and to explore the role of mitogen activated protein kinase (MAPK) pathway as a primary signaling mechanism.

Methods

Intestinal permeability was assessed in 12 healthy volunteers after intraduodenal administration of either placebo or 20 g ethanol in a randomised cross-over trial. Localization of the tight junction (TJ) and gene expression, phosphorylation of the MAPK isoforms p38, ERK and JNK as indicative of activation were analyzed in duodenal biopsies. The role of MAPK was further examined in vitro using Caco-2 monolayers.

Results

Ethanol increased small and large intestinal permeability, paralleled by redistribution of ZO-1 and occludin, down-regulation of ZO-1 and up-regulation of myosin light chain kinase (MLCK) mRNA expression, and increased MAPK isoforms phosphorylation. In Caco-2 monolayers, ethanol increased permeability, induced redistribution of the junctional proteins and F-actin, and MAPK and MLCK activation, as indicated by phosphorylation of MAPK isoforms and myosin light chain (MLC), respectively, which could be reversed by pretreatment with either MAPK inhibitors or the anti-oxidant L-cysteine.

Conclusions

Administration of moderate ethanol dosage can increase both small and colon permeability. Furthermore, the data indicate a pivotal role for MAPK and its crosstalk with MLCK in ethanol-induced intestinal barrier disruption.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00928733","term_id":"NCT00928733"}}NCT00928733     

Expansion of Cord Blood CD34+ Cells in Presence of zVADfmk and zLLYfmk Improved Their In Vitro Functionality and In Vivo Engraftment in NOD/SCID Mouse

Background

Cord blood (CB) is a promising source for hematopoietic stem cell transplantations. The limitation of cell dose associated with this source has prompted the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). However, the expansion procedure is known to exhaust the stem cell pool causing cellular defects that promote apoptosis and disrupt homing to the bone marrow. The role of apoptotic machinery in the regulation of stem cell compartment has been speculated in mouse hematopoietic and embryonic systems. We have consistently observed an increase in apoptosis in the cord blood derived CD34⁺ cells cultured with cytokines compared to their freshly isolated counterpart. The present study was undertaken to assess whether pharmacological inhibition of apoptosis could improve the outcome of expansion.

Methodology/Principal Findings

CB CD34⁺ cells were expanded with cytokines in the presence or absence of cell permeable inhibitors of caspases and calpains; zVADfmk and zLLYfmk respectively. A novel role of apoptotic protease inhibitors was observed in increasing the CD34⁺ cell content of the graft during ex vivo expansion. This was further reflected in improved in vitro functional aspects of the HSPCs; a higher clonogenicity and long term culture initiating potential. These cells sustained superior long term engraftment and an efficient regeneration of major lympho-myeloid lineages in the bone marrow of NOD/SCID mouse compared to the cells expanded with growth factors alone.

Conclusion/Significance

Our data show that, use of either zVADfmk or zLLYfmk in the culture medium improves expansion of CD34⁺ cells. The strategy protects stem cell pool and committed progenitors, and improves their in vitro functionality and in vivo engraftment. This observation may complement the existing protocols used in the manipulation of hematopoietic cells for therapeutic purposes. These findings may have an impact in the CB transplant procedures involving a combined infusion of unmanipulated and expanded grafts.     

Transient Hypoxia Alters Striatal Catecholamine Metabolism in Immature Brain: An In Vivo Microdialysis Study

Microdialysis probes were inserted bilaterally into the striatum of 7-day-old rat pups (n = 30) to examine extracellular fluid levels of dopamine, its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA). The dialysis samples were assayed by HPLC with electrochemical detection. Baseline levels, measured after a 2-h stabilization period, were as follows: dopamine, not detected; DOPAC, 617 +/- 33 fmol/min; HVA, 974 +/- 42 fmol/min; and 5-HIAA, 276 +/- 15 fmol/min. After a 40-min baseline sampling period, 12 animals were exposed to 8% oxygen for 120 min. Hypoxia produced marked reductions in the striatal extracellular fluid levels of both dopamine metabolites (p less than 0.001 by analysis of variance) and a more gradual and less prominent reduction in 5-HIAA levels (p less than 0.02 by analysis of variance), compared with controls (n = 12) sampled in room air. In the first hour after hypoxia, DOPAC and HVA levels rose quickly, whereas 5-HIAA levels remained suppressed. The magnitude of depolarization-evoked release of dopamine (elicited by infusion of potassium or veratrine through the microdialysis probes for 20 min) was evaluated in control and hypoxic animals. Depolarization-evoked dopamine efflux was considerably higher in hypoxic pups than in controls: hypoxic (n = 7), 257 +/- 32 fmol/min; control (n = 12), 75 +/- 14 fmol/min (p less than 0.001 by analysis of variance). These data demonstrate that a brief exposure to moderate hypoxia markedly disrupts striatal catecholamine metabolism in the immature rodent brain.     

Deep Sea Water Prevents Balloon Angioplasty-Induced Hyperplasia through MMP-2: An In Vitro and In Vivo Study

Major facts about the development of restenosis include vascular smooth muscle cells (VSMCs) proliferation and migration. A previous study showed that in vitro treatment with magnesium chloride has the potential to affect the proliferation and migration of VSMCs. Magnesium is the major element in deep sea water (DSW) and is a biologically active mineral. It is unclear whether DSW intake can prevent abnormal proliferation and migration of VSMCs as well as balloon angioplasty-induced neointimal hyperplasia. Thus, we attempted to evaluate the anti-restenotic effects of DSW and its possible molecular mechanisms. Several concentrations of DSW, based on the dietary recommendations (RDA) for magnesium, were applied to a model of balloon angioplasty in SD rats. The results showed that DSW intake markedly increased magnesium content within the vascular wall and reduced the development of neointimal hyperplasia. The immunohistochemical analysis also showed that the expression of proteins associated with cell proliferation and migration were decreased in the balloon angioplasty groups with DSW supplement. Furthermore, in vitro treatment with DSW has a dose-dependent inhibitory effect on serum-stimulated proliferation and migration of VSMCs, whose effects might be mediated by modulation of mitogen-activated protein kinase (MAPK) signaling and of the activity of matrix metalloproteinase-2 (MMP-2). Our study suggested that DSW intake can help prevent neointimal hyperplasia (or restenosis), whose effects may be partially regulated by magnesium and other minerals.     

Applying the Taguchi Design for Optimized Formulation of Sustained Release Gliclazide Chitosan Beads: An In Vitro/In Vivo Study

Gliclazide is a second generation of hypoglycemic sulfonylurea and acts selectively on pancreatic β cell to control diabetes mellitus. The objective of this study was to produce a controlled release system of gliclazide using chitosan beads. Chitosan beads were produced by dispersion technique using tripolyphosphate (TPP) as gelating agent. The effects of process variables including chitosan molecular weight, concentration of chitosan and TPP, pH of TPP, and cross-linking time after addition of chitosan were evaluated by Taguchi design on the rate of drug release, mean release time (MRT), release efficiency (RE₈%), and particle size of the beads. The blood glucose lowering effect of the beads was studied in normal and streptozotocin-diabetic rats. The optimized formulation CL₂T₅P₂t₁₀ with about 31% drug loading, 2.4 h MRT, and 69.16% RE₈% decreased blood glucose level in normal rats for 24 h compared to pure powder of gliclazide that lasted for just 10 h.     

The Effects of Mechanical Loading on Tendons - An In Vivo and In Vitro Model Study

Mechanical loading constantly acts on tendons, and a better understanding of its effects on the tendons is essential to gain more insights into tendon patho-physiology. This study aims to investigate tendon mechanobiological responses through the use of mouse treadmill running as an in vivo model and mechanical stretching of tendon cells as an in vitro model. In the in vivo study, mice underwent moderate treadmill running (MTR) and intensive treadmill running (ITR) regimens. Treadmill running elevated the expression of mechanical growth factors (MGF) and enhanced the proliferative potential of tendon stem cells (TSCs) in both patellar and Achilles tendons. In both tendons, MTR upregulated tenocyte-related genes: collagen type I (Coll. I ∼10 fold) and tenomodulin (∼3–4 fold), but did not affect non-tenocyte-related genes: LPL (adipocyte), Sox9 (chondrocyte), Runx2 and Osterix (both osteocyte). However, ITR upregulated both tenocyte (Coll. I ∼7–11 fold; tenomodulin ∼4–5 fold) and non-tenocyte-related genes (∼3–8 fold). In the in vitro study, TSCs and tenocytes were stretched to 4% and 8% using a custom made mechanical loading system. Low mechanical stretching (4%) of TSCs from both patellar and Achilles tendons increased the expression of only the tenocyte-related genes (Coll. I ∼5–6 fold; tenomodulin ∼6–13 fold), but high mechanical stretching (8%) increased the expression of both tenocyte (Coll. I ∼28–50 fold; tenomodulin ∼14–48 fold) and non-tenocyte-related genes (2–5-fold). However, in tenocytes, non-tenocyte related gene expression was not altered by the application of either low or high mechanical stretching. These findings indicate that appropriate mechanical loading could be beneficial to tendons because of their potential to induce anabolic changes in tendon cells. However, while excessive mechanical loading caused anabolic changes in tendons, it also induced differentiation of TSCs into non-tenocytes, which may lead to the development of degenerative tendinopathy frequently seen in clinical settings.     

壳聚糖/ 魔芋葡甘露聚糖复合膜体内外促创面愈合研究

目的:制备壳聚糖/魔芋葡甘露聚糖复合膜,研究其促创面愈合作用。方法:壳聚糖溶液和魔芋葡甘露聚糖溶液混合后冷冻干燥制成复合膜。扫描电镜观察膜的形态和孔径,并研究比较膜的吸水率,水蒸气透过率,拉伸强度,断裂伸长率和体外降解率。建立大鼠皮肤损伤模型,敷以复合膜治疗,比较创面愈合率,观察创面组织染色结果,评价复合膜的促创面愈合作用。结果:壳聚糖/魔芋葡甘露聚糖复合膜具有三维网状结构,壳聚糖复合魔芋葡甘露聚糖后,膜的吸水率、拉伸强度和断裂伸长率提高,体外降解加速,水蒸气透过率改善。愈合实验表明壳聚糖/魔芋葡甘露聚糖膜具有促进创面愈合作用。结论:壳聚糖/魔芋葡甘露聚糖复合膜制备工艺简单,能有效促进创面愈合,具有成为创伤敷料的潜力。