Embryonic Rat Research Articles

Remyelination-Promoting DNA Aptamer Conjugate Myaptavin-3064 Binds to Adult Oligodendrocytes In Vitro.

We previously applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology to identify myelin-specific DNA aptamers, using crude mouse central nervous system myelin as bait. This selection identified a 40-nucleotide aptamer (LJM-3064). Multiple biotinylated LJM-3064 molecules were conjugated to a streptavidin core to mimic a multimeric immunoglobulin M (IgM) antibody, generating 3064-BS-streptavidin (Myaptavin-3064). We previously showed that Myaptavin-3064 induces remyelination in the Theiler’s murine encephalomyelitis virus (TMEV) model of chronic spinal cord demyelination. While details of target binding and the mechanism of action remain unclear, we hypothesized that Myaptavin-3064 induces remyelination by binding to oligodendrocytes (OLs). We now report the results of binding assays using the human oligodendroglioma (HOG) cell line, applying both flow cytometry and immunocytochemistry (IC) to assay aptamer conjugate binding to cells. IC assays were applied to compare aptamer conjugate binding to primary embryonic mouse mixed cortical cultures and primary adult rat mixed glial cultures. We show that Myaptavin-3064 binds to HOG cells, with increased binding upon differentiation. In contrast, a negative control aptamer conjugate, 3060-BS, which did not promote central nervous system (CNS) remyelination, does not bind to HOG cells. Myaptavin-3064 did not bind to lung (L2) or kidney (BHK) cell lines. Total internal reflection fluorescence (TIRF) imaging indicates that Myaptavin-3064 binds at the cell membrane of live cells. In addition to HOG cells, Myaptavin-3064 binds to adult rat OLs, but not to embryonic mouse mixed cortical cultures. These data support the hypothesis that Myaptavin-3064 binds to a surface molecule on both rodent and human OLs in a manner that triggers a remyelination signal pathway.

Neither Excessive Nitric Oxide Accumulation nor Acute Hyperglycemia Affects the N-Acetylaspartate Network in Wistar Rat Brain Cells.

The N-acetylaspartate network begins in neurons with N-acetylaspartate production catalyzed by aspartate N-acetyltransferase from acetyl-CoA and aspartate. Clinical studies reported a significant depletion in N-acetylaspartate brain level in type 1 diabetic patients. The main goal of this study was to establish the impact of either hyperglycemia or oxidative stress on the N-acetylaspartate network. For the in vitro part of the study, embryonic rat primary neurons were treated by using a nitric oxide generator for 24 h followed by 6 days of post-treatment culture, while the neural stem cells were cultured in media with 25–75 mM glucose. For the in vivo part, male adult Wistar rats were injected with streptozotocin (65 mg/kg body weight, ip) to induce hyperglycemia (diabetes model) and euthanized 2 or 8 weeks later. Finally, the biochemical profile, NAT8L protein/Nat8l mRNA levels and enzymatic activity were analyzed. Ongoing oxidative stress processes significantly affected energy metabolism and cholinergic neurotransmission. However, the applied factors did not affect the N-acetylaspartate network. This study shows that reduced N-acetylaspartate level in type 1 diabetes is not related to oxidative stress and that does not trigger N-acetylaspartate network fragility. To reveal why N-acetylaspartate is reduced in this pathology, other processes should be considered.

Disease-associated HCN4 V759I variant is not sufficient to impair cardiac pacemaking

The hyperpolarization-activated cation current If is a key determinant for cardiac pacemaker activity. It is conducted by subunits of the hyperpolarization-activated cyclic nucleotide–gated (HCN) channel family, of which HCN4 is predominant in mammalian heart. Both loss-of-function and gain-of-function mutations of the HCN4 gene are associated with sinus node dysfunction in humans; however, their functional impact is not fully understood yet. Here, we sought to characterize a HCN4 V759I variant detected in a patient with a family history of sick sinus syndrome. The genomic analysis yielded a mono-allelic HCN4 V759I variant in a 49-year-old woman presenting with a family history of sick sinus syndrome. This HCN4 variant was previously classified as putatively pathogenic because genetically linked to sudden infant death syndrome and malignant epilepsy. However, detailed electrophysiological and cell biological characterization of HCN4 V759I in Xenopus laevis oocytes and embryonic rat cardiomyocytes, respectively, did not reveal any obvious abnormality. Voltage dependence and kinetics of mutant channel activation, modulation of cAMP-gating by the neuronal HCN channel auxiliary subunit PEX5R, and cell surface expression were indistinguishable from wild-type HCN4. In good agreement, the clinically likewise affected mother of the patient does not exhibit the reported HCN4 variance. HCN4 V759I resembles an innocuous genetic HCN channel variant, which is not sufficient to disturb cardiac pacemaking. Once more, our work emphasizes the importance of careful functional interpretation of genetic findings not only in the context of hereditary cardiac arrhythmias.

Temperature sensitive self-assembling hydroxybutyl chitosan nanoparticles with cationic enhancement effect for multi-functional applications

Hydroxybutyl chitosan (HBC) with different degree of substitution (DS) were prepared using a homogeneous reaction system (KOH/urea), which could achieve temperature-dependent reversible morphological transition in aqueous solution. During hydrophobic assembly, amino groups on HBC chains exposed on the surface of nanoparticles formed a poly-cationic structure. The structure of HBCs was characterized by FTIR, 13C NMR, XRD, TGA and rheology. The morphology and assembly mechanism of HBC nanoparticles were studied by TEM, AFM and DLS. Also, the results of coagulation, bacteriostatic, superoxide anion clearance and anionic contaminant removal tests suggested that HBC nanoparticles had excellent flocculation and removal effect of anionic composites. Moreover, the cytocompatibility test indicated that HBC could effectively promote proliferation and division of mouse fibroblast, mouse embryonic fibroblast and rat bone marrow mesenchymal stem cells. These cationic HBC nanoparticles exhibited great potential in multi-functional applications.

Inhibition of eNOS by L-NAME resulting in rat hind limb developmental defects through PFKFB3 mediated angiogenetic pathway

l-arginine/NOS/NO signaling pathway plays a critical role in controlling variety of vascular diseases. However, whether NOS inhibition by L-NAME suppresses late embryonic development is undefined. The aim of this study is to determine whether NOS inhibition by L-NAME is critical for late embryonic rat hind limb development. The pregnant rat at E13.5 administrated L-NAME by consecutive intraperitoneal injection. The embryos been harvested from E16.5 to E 20.5. Hematoxylin and Eosin Staining, Immunofluorescence and Immunohistochemistry performed to determine hind limb Vasculogenesis, HUVEC culture, Adenoviral PFKFB3 infection, Real time PCR and western blot were performed to determine whether l-arginine/NOS/NO pathway controlling late embryonic hind limb development through PFKFB3 mediated angiogenetic pathway. NOS inhibition by L-NAME resulting in late embryonic hind limb developmental defects characterized by severe hemorrhage. The in vivo studies showed that NOS inhibition strongly suppressed hind limb angiogenetic remodeling by impairing differentiation of endothelial cells and smooth muscle cells, and extracellular matrix synthesis. For underlie mechanism, our studies indicated that L-NAME treatment dramatically suppresses PFKFB3 expression in hematopoietic progenitor cells, tubulogenetic endothelial cells and smooth muscle cells. Knockdown of PFKFB3 dramatically inhibits the expression of angiogenetic genes, as well as tubulogenesis and extracellular matrix related genes. Taken together, our data in this study demonstrated that l-arginine-eNOS-NO pathway is important for rat hind limb development during late embryonic stage. This could be both a useful animal model and a promising therapeutic treatment for defects of late embryonic developmental hind limbs.

Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis.

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study axonal growth, axonal trafficking, synaptogenesis, dendritic growth, dendritic plasticity and nerve-target interactions in co-culture systems. This protocol describes the isolation and dissociation of neurons from the superior cervical ganglia of E21 rat embryos, followed by the preparation and maintenance of pure neuronal cultures in serum-free medium. Since neurons do not adhere to uncoated plastic, neurons will be cultured on either 12 mm glass coverslips or 6-well plates coated with poly-D-lysine. Following treatment with an antimitotic agent (Ara-C, cytosine β-D-arabinofuranoside), this protocol generates healthy neuronal cultures with less than 5% non-neuronal cells, which can be maintained for over a month in vitro. Although embryonic rat SCG neurons are multipolar with 5-8 dendrites in vivo; under serum-free conditions, these neurons extend only a single axon in culture and continue to be unipolar for the duration of the culture. However, these neurons can be induced to extend dendrites in the presence of basement membrane extract, bone morphogenetic proteins (BMPs), or 10% fetal calf serum. These homogenous neuronal cultures can be used for immunocytochemical staining and for biochemical studies. This paper also describes optimized protocol for immunocytochemical staining for microtubule associated protein-2 (MAP-2) in these neurons and for the preparation of neuronal extracts for mass spectrometry.

Comparative Analysis of the Expression of Chondroitin Sulfate Subtypes and Their Inhibitory Effect on Axonal Growth in the Embryonic, Adult, and Injured Rat Brains.

Chondroitin sulfate glycosaminoglycans (CS-GAGs) are the primary inhibitory GAGs for neuronal growth after central nervous system (CNS) injury. However, the inhibitory or permissive activity of CS-GAG subtypes is controversial and depends on the physiological needs of CNS tissues. In this study, we investigated the characteristics and effects of CS-GAGs on axonal growth, which was isolated from the brain cortices of normal rat embryo at E18, normal adult rat brain and injured adult rat brain. Isolated CS-GAGs from embryo, normal adult, and injured adult rat brains were used for analyzing their effect on attachment and axonal growth using modified spot assay with dorsal root ganglion (DRG) explants and cerebellar granule neurons (CGNs). CS-GAGs were separated using high performance liquid chromatography (HPLC), and the subtypes of CS-GAGs were analyzed. CS-GAGs of all three groups inhibited CGN attachment and axonal growth of DRGs. However, CS-GAGs of normal adult rat brain exhibited higher inhibitory activity than those of the other groups in both assays. When subtypes of CS-GAGs were analyzed using HPLC, CS-A (4S) was the most abundant in all three groups and found in largest amount in normal adult rat brain. In contrast, unsulfated CS (CS0) and CS-C (6S) were more abundant by 3-4-folds in E18 group than in the two adult groups. When compared with the normal adult rat brain, injured rat brain showed relatively similar patterns to that of embryonic rat brain at E18 in the expression of CS subtypes and their inhibitory effect on axonal growth. This phenomenon could be due to differential expression of CS-GAGs subtypes causing decrease in the amount of CS-A and mature-type CS proteoglycan core proteins.

The Epilepsy-Related Protein PCDH19 Regulates Tonic Inhibition, GABAAR Kinetics, and the Intrinsic Excitability of Hippocampal Neurons

PCDH19 encodes for protocadherin-19 (PCDH19), a cell-adhesion molecule of the cadherin superfamily preferentially expressed in the brain. PCDH19 mutations cause a neurodevelopmental syndrome named epileptic encephalopathy, early infantile, 9 (EIEE9) characterized by seizures associated with cognitive and behavioral deficits. We recently reported that PCDH19 binds the alpha subunits of GABAA receptors (GABAARs), modulating their surface availability and miniature inhibitory postsynaptic currents (mIPSCs). Here, we investigated whether PCDH19 regulatory function on GABAARs extends to the extrasynaptic receptor pool that mediates tonic current. In fact, the latter shapes neuronal excitability and network properties at the base of information processing. By combining patch-clamp recordings in whole-cell and cell-attached configurations, we provided a functional characterization of primary hippocampal neurons from embryonic rats of either sex expressing a specific PCDH19 short hairpin (sh)RNA. We first demonstrated that PCDH19 downregulation reduces GABAAR-mediated tonic current, evaluated by current shift and baseline noise analysis. Next, by single-channel recordings, we showed that PCDH19 regulates GABAARs kinetics without altering their conductance. In particular, GABAARs of shRNA-expressing neurons preferentially exhibit brief openings at the expense of long ones, thus displaying a flickering behavior. Finally, we showed that PCDH19 downregulation reduces the rheobase and increases the frequency of action potential firing, thus indicating neuronal hyperexcitability. These findings establish PCDH19 as a critical determinant of GABAAR-mediated tonic transmission and GABAARs gating, and provide the first mechanistic insights into PCDH19-related hyperexcitability and comorbidities.

Acute in vitro effects on embryonic rat dorsal root ganglion (DRG) cultures by in silico predicted neurotoxic chemicals: Evaluations on cytotoxicity, neurite length, and neurophysiology

The Hard-Soft Acid and Base hypothesis can be used to predict the potential bio-reactivity (electrophilicity) of a chemical with intracellular proteins, resulting in neurotoxicity. Twelve chemicals predicted to be neurotoxic were evaluated in vitro in rat dorsal root ganglia (DRG) for effects on cytotoxicity (%LDH), neuronal structure (total neurite length/neuron, NLPN), and neurophysiology (mean firing rate, MFR). DRGs were treated acutely on days in vitro (DIV) 7 (1–100 μM) with test chemical; %LDH and NLPN were measured after 48 h. 4-cyclohexylhexanone (4-C) increased %LDH release at 50 (29%) and 100 μM (56%), citronellal (Cit) and 1-bromopropane increased %LDH at 100 μM (22% and 26%). 4-C, Cit, 2,5 Hexanedione (2,5Hex), phenylacetylaldehyde (PAA) and 2-ethylhexanal decreased mean NLPN at 48 h; 50 and 100 μM for 4-C (28% and 60%), 100 μM Cit (52%), 100 μM 2,5- Hex (37%) 100 μM PAA (41%) and 100 μM for 2-ethylhexanal (23%). Separate DRG cultures were treated on DIV 14 and changes in MFR measured. Four compounds decreased MFR at 50 or 100 μM: Acrylamide (−83%), 3,4-dichloro-1-butene (−93%), 4-C (−89%) and hexane (−79%, 50 μM). Changes in MFR and NLPN occurred in absence of cytotoxicity. While the current study showed little cytotoxicity, it gave insight to initial changes in MFR. Results provide insight for future chronic exposure experiments to evaluate neurotoxicity.

Isoliquiritigenin attenuates diabetic cardiomyopathy via inhibition of hyperglycemia-induced inflammatory response and oxidative stress

Inflammation and oxidative stress play essential roles in the occurrence and progression of diabetic cardiomyopathy (DCM). Isoliquiritigenin (ISL), a natural chalcone, exhibits strong anti-inflammatory and antioxidant activities. In this study, we aimed to investigate the protective effects of ISL on DCM using high glucose (HG)-challenged cultured cardiomyocytes and streptozotocin (STZ)-induced diabetic mice. Embryonic rat heart-derived H9c2 cells challenged with a high concentration of glucose were used to evaluate the anti-inflammatory and antioxidant effects of ISL. STZ-induced diabetic mice were used to study the effects of ISL in DCM in vivo. Furthermore, cardiac fibrosis, hypertrophy, and apoptosis were explored both in vitro and in vivo. ISL effectively inhibited HG-induced hypertrophy, fibrosis, and apoptosis probably by alleviating the inflammatory response and oxidative stress in H9c2 cells. Results from in vivo experiments showed that ISL exhibited anti-inflammatory and antioxidant stress activities that were characterized by the attenuation of cardiac hypertrophy, fibrosis, and apoptosis, which resulted in the maintenance of cardiac function. The protective effects of ISL against inflammation and oxidative stress were mediated by the inhibition of mitogen-activated protein kinases (MAPKs) and induction of nuclear factor-erythroid 2 related factor 2 (Nrf2) signaling pathway, respectively. Our results provided compelling evidence that ISL, by virtue of neutralizing excessive inflammatory response and oxidative stress, could be a promising agent in the treatment of DCM. Targeting the MAPKs and Nrf2 signaling pathway might be an effective therapeutic strategy for the prevention and treatment of DCM.

Central Cholinergic Synapse Formation in Optimized Primary Septal-Hippocampal Co-cultures.

Septal innervation of basal forebrain cholinergic neurons to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer's disease. To understand the molecular events underlying physiological cholinergic synaptogenesis and remodeling, as well as pathological loss, we developed an optimized primary septal-hippocampal co-culture system. Hippocampal and septal tissue were harvested from embryonic Sprague-Dawley rat brain and cultured together at varying densities, cell ratios, and in the presence of different growth factors. We identified conditions that produced robust septal-hippocampal synapse formation. We used confocal microscopy with primary antibodies and fluorescent ligands to validate that this system was capable of generating developmentally mature cholinergic synapses. Such synapses were comprised of physiological synaptic partners and mimicked the molecular composition of in vivo counterparts. This co-culture system will facilitate the study of the formation, plasticity, and dysfunction of central mammalian cholinergic synapses.

Recapitulation of Neural Crest Specification and EMT via Induction from Neural Plate Border-like Cells

SummaryNeural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

THE CHARACTERISTICS OF RAT EMBRYONIC FIBROBLASTS (REFS) AND RAT BONE MARROW DERIVED MESENCHYMAL STEM CELLS (RAT-BMMSCS) FROM THE WISTAR RAT: A COMPARATIVE STUDY

Objective: Rat embryonic fibroblasts (REFs) and rat bone marrow-derived mesenchymal stem cells (rat-BMMSCs) an be used as in vitro models for a variety of studies, including for degenerative diseases such as arterial ischemia, tissue engineering and development of induced pluripotent stem cells (iPSCs). Therefore, the further developments of the use of these two cells of great importance.
 Methods: The experiments were performed with Wistar rat, those with 15-17 d gestation (aged 32 w) as a REFs source and those aged 12 w as a BMMSCs source. Dulbecco's modified eagle medium (DMEM) was used for both cell cultures but with different media supplements. Proliferation ability was determined for both by calculating population doubling time (PDT). Characterization was performed by differentiation testing into osteocyte, chondrocyte and adipocyte cells by staining with Alizarin Red, Alcian Blue and Oil Red O and by an investigation of specific antigen characteristics using flow cytometry with positive CD90 and CD29 and negative CD34 markers.
 Results: Morphologically, the REFs and rat-BMMSCs had the same fibroblasts like shape. PDT was higher for the REFs than the BMMSCs (p<0.05), and both could differentiate into osteocytes, chondrocytes and adipocyte. The characteristics of the positive markers (CD29 and CD90) were higher in rat-BMMSCs than in REFs.
 Conclusion: In this study demonstrated that the explant method for REFs isolation and flushing method for rat-BMMSC isolation are both effective. It also showed that rat-BMMSC grow faster than REFs, and that both cells have the same differentiation ability as rat-BMMSCs but with different specific surface antigen characteristics.

THE VIABILITY TEST OF SAPPAN WOOD (CAESALPINIA SAPPAN L.) ETHANOL EXTRACT IN THE H9C2 CELL LINE

Objective: In this study, the embryonic rat cardiomyocyte cell line H9C2 was used to investigate the cardiotoxicity effect of sappan wood ethanol extract (SWEE).
 Methods: Sappan wood was extracted in 96% ethanol and divided into dose concentrations of 2.5, 5, 10, 50, 100, and 150 μg/ml, with deferiprone used as a control. Cell viability was assessed using the PrestoBlue Cell Viability Reagent, according to manufacturer protocols.
 Results: Microscopic examination showed that the cell viability of H9C2 was preserved by SWEE treatments at a dose of 10 μg/ml and suggested dose concentrations of 50 μg/ml of SWEE. The percentage of viable cells was greater than 95% with a dose concentration of 10 μg/ml of SWEE, but it was significantly reduced with a dose concentration of 50 μg/ml of SWEE (p<0.05).
 Conclusion: The optimal dose concentration of SWEE to reach 95% cell viability was 10 μg/ml.

Reg-1α Promotes Differentiation of Cortical Progenitors via Its N-Terminal Active Domain.

Reg-1α belongs to the Reg family of small, secreted proteins expressed in both pancreas and nervous system. Reg-1α is composed of two domains, an insoluble C-type lectin domain and a short soluble N-terminal peptide, which is released from the molecule upon proteolytic N-terminal processing, although the biological significance of this proteolysis remains unclear. We have previously shown that binding of Reg-1α to its receptor Extl3 stimulates axonal outgrowth. Reg-1α and Extl3 genes are expressed in the developing cortex but their expression decreases in adulthood, pointing to a possible function of this signaling system at the early developmental stages. Here, we demonstrate that recombinant Reg-1α increases migration and differentiation of cultured embryonic rat telencephalic progenitors via the activation of GSK-3β activity. In vivo overexpression of Reg-1α by in utero electroporation, has a similar effect, favoring premature differentiation of cortical progenitors. Notably, the N-terminal soluble domain, but not the C-type lectin domain, is largely responsible for Reg-1α effects on cortical neuronal differentiation. We thus conclude that Reg-1α via its proteolytically generated N-terminal domain is required for basic development processes.

Innervation and electrical pulse stimulation - in vitro effects on human skeletal muscle cells.

Contraction-induced adaptations in skeletal muscles are well characterized in vivo, but the underlying cellular mechanisms are still not completely understood. Cultured human myotubes represent an essential model system for human skeletal muscle that can be modulated ex vivo, but they are quiescent and do not contract unless being stimulated. Stimulation can be achieved by innervation of human myotubes in vitro by co-culturing with embryonic rat spinal cord, or by replacing motor neuron activation by electrical pulse stimulation (EPS). Effects of these two in vitro approaches, innervation and EPS, were characterized with respects to the expression of myosin heavy chains (MyHCs) and metabolism of glucose and oleic acid in cultured human myotubes. Adherent human myotubes were either innervated with rat spinal cord segments or exposed to EPS. The expression pattern of MyHCs was assessed by quantitative polymerase chain reaction, immunoblotting, and immunofluorescence, while the metabolism of glucose and oleic acid were studied using radiolabelled substrates. Innervation and EPS promoted differentiation towards different fiber types in human myotubes. Expression of the slow MyHC-1 isoform was reduced in innervated myotubes, whereas it remained unaltered in EPS-treated cells. Expression of both fast isoforms (MyHC-2A and MyHC-2X) tended to decrease in EPS-treated cells. Both approaches induced a more oxidative phenotype, reflected in increased CO2 production from both glucose and oleic acid. Novelty: Innervation and EPS favour differentiation into different fiber types in human myotubes. Both innervation and EPS promote a metabolically more oxidative phenotype in human myotubes.

(Z)-7,4'-dimethoxy-6-hydroxy-aurone-4-O-β-glucopyranoside attenuates lipoteichoic acid-induced damage in rat cardiomyoblast cells.

ObjectiveExcessive inflammatory responses in the endocardium are related to progression of infectious endocarditis. This study aimed to investigate whether (Z)-7,4'-dimethoxy-6-hydroxy-aurone-4-O-β-glucopyranoside (DHAG), a compound isolated from the endophytic fungus Penicillium citrinum of Bruguiera gymnorrhiza, could attenuate cell damage caused by lipoteichoic acid (LTA) in embryonic rat heart cells (H9c2).MethodsLTA-induced cell damage occurred in H9c2 cells and the protective effects of DHAG at different concentrations (1–10 µM) were assessed. Indicators of oxidative stress and inflammatory responses in H9c2 cells were measured.ResultsDHAG (1–10 µM) significantly attenuated LTA-induced damage in H9c2 cells, as evidenced by increased cell viability and mitochondrial membrane potential, decreased cytochrome c release and DNA fragmentation, inhibition of caspase-3 and -9 activity, and altered expression of apoptosis-related proteins. DHAG also decreased oxidative stress by increasing protein expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Furthermore, DHAG inhibited inflammatory responses by decreasing protein expression of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs).ConclusionDHAG exerted protective effects against LTA-induced cell damage, at least partially by decreasing oxidative stress and inhibiting inflammatory responses. Our results provide a scientific rational for developing DHAG as a therapy against infectious endocarditis.

Neuroimmunomodulatory and Neuroprotective Effects of the Flavonoid Apigenin in in vitro Models of Neuroinflammation Associated With Alzheimer's Disease.

Neurodegenerative disorders (ND) are characterized by the progressive and irreversible loss of neurons. Alzheimer’s Disease (AD) is the most incident age-related ND, in which the presence of a chronic inflammatory compound seems to be related to its pathogenesis. Different stimuli in the central nervous system (CNS) can induce activation, proliferation, and changes in phenotype and glial function, which can be modulated by anti-inflammatory agents. Apigenin (4,5,7–trihydroxyflavone) is a flavonoid found in abundance in many fruits and vegetables, that has shown important effects upon controlling the inflammatory response. This study evaluated the neuroprotective and neuroimmunomodulatory potential of apigenin using in vitro models of neuroinflammation associated with AD. Co-cultures of neurons and glial cells were obtained from the cortex of newborn and embryonic Wistar rats. After 26 days in vitro, cultures were exposed to lipopolysaccharide (LPS; 1 μg/ml), or IL-1β (10 ng/ml) for 24 h, or to Aβ oligomers (500 nM) for 4 h, and then treated with apigenin (1 μM) for further 24 h. It was observed that the treatment with apigenin preserved neurons and astrocytes integrity, determined by Rosenfeld’s staining and immunocytochemistry for β-tubulin III and GFAP, respectively. Moreover, it was observed by Fluoro-Jade-B and caspase-3 immunostaining that apigenin was not neurotoxic and has a neuroprotective effect against inflammatory damage. Additionally, apigenin reduced microglial activation, characterized by inhibition of proliferation (BrdU+ cells) and modulation of microglia morphology (Iba-1 + cells), and decreased the expression of the M1 inflammatory marker CD68. Moreover, as determined by RT-qPCR, inflammatory stimuli induced by IL-1β increased the mRNA expression of IL-6, IL-1β, and CCL5, and decreased the mRNA expression of IL-10. Contrary, after treatment with apigenin in inflammatory stimuli (IL-1β or LPS) there was a modulation of the mRNA expression of inflammatory cytokines, and reduced expression of OX42, IL-6 and gp130. Moreover, apigenin alone and after an inflammatory stimulus with IL-1β also induced the increase in the expression of brain-derived neurotrophic factor (BDNF), an effect that may be associated with anti-inflammatory and neuroprotective effects. Together these data demonstrate that apigenin presents neuroprotective and anti-inflammatory effects in vitro and might represent an important neuroimmunomodulatory agent for the treatment of neurodegenerative conditions.

Preclinical Ultrasound Imaging—A Review of Techniques and Imaging Applications

Ultrasound imaging is a well-established clinical imaging technique providing real-time, quantitative anatomical and physiological information in humans. The lack of ionising radiation and relative low purchase and maintenance costs results in it being one of the most frequently used clinical imaging techniques with increasing use for guiding interventional clinical procedures. Until 20 years ago, translation of clinical ultrasound practices to preclinical applications proved a significant technological challenge due to the smaller size (25g vs 70kg) and rapid conscious heart-rate (500-700bpm vs 60bpm) of the mouse requiring an increase in both spatial and temporal resolution of 10-20 fold in order to achieve diagnostic information comparable to that achieved clinically. Since 2000 (Foster et al 2000), these technological challenges have been overcome and commercial high frequency ultrasound scanners have enabled longitudinal studies of disease progression in small animal models to be undertaken. Adult, neonatal and embryonic rats, mice and zebrafish can now be scanned with resolutions down to 30 microns and with sufficient temporal resolution to enable cardiac abnormalities in all these species to be identified. In mice and rats, quantification of blood flow in cardiac chambers, renal, liver and uterine vessels and intra-mural tissue movements can be measured using the Doppler technique. Ultrasonic contrast microbubbles used routinely for clinical applications are now being further developed to include targeting mechanisms and drug-loading capabilities and the results in animal models bode well for translation for targeted drug delivery in humans.

Cortical cells are altered by factors including bone morphogenetic protein released from a placental barrier model under altered oxygenation.

Episodes of hypoxia and hypoxia/reoxygenation during foetal development have been associated with increased risk of neurodevelopmental conditions presenting in later life.The mechanism for this is not understood; however, several authors have suggested that the placenta plays an important role. Previously we found both placentas from a maternal hypoxia model and pre-eclamptic placentas from patients release factors lead to a loss of dendrite complexity in rodent neurons. Here to further explore the nature and origin of these secretions we exposed a simple in vitro model of the placental barrier, consisting of a barrier of human cytotrophoblasts, to hypoxia or hypoxia/reoxygenation. We then exposed cortical cultures from embryonic rat brains to the conditioned media (CM) from below these exposed barriers and examined changes in cell morphology, number, and receptor presentation.The barriers released factors that reduced dendrite and astrocyte process lengths, decreased GABAB1 staining, and increased astrocyte number. The changes in astrocytes required the presence of neurons and were prevented by inhibition of the SMAD pathway and by neutralising Bone Morphogenetic Proteins (BMPs) 2/4. Barriers exposed to hypoxia/reoxygenation also released factors that reduced dendrite lengths but increased GABAB1 staining. Both oxygen changes caused barriers to release factors that decreased GluN1, GABAAα1 staining and increased GluN3a staining. We find that hypoxia in particular will elicit the release of factors that increase astrocyte number and decrease process length as well as causing changes in the intensity of glutamate and GABA receptor staining. There is some evidence that BMPs are released and contribute to these changes.