Embryonic Rat Research Articles

Beating heart cells: Using cultured cardiomyocytes to study cellular structure and contractility in laboratory exercises.

Heart muscle cells, or cardiomyocytes, exhibit intrinsic contractility in vitro. We found that commercially-available mammalian cardiomyocytes serve as an excellent model system for studying the cytoskeleton and cellular contractility, fundamental topics in undergraduate cell and molecular biology courses. Embryonic rat cardiomyocytes were plated on cell culture dishes or glass coverslips and visualized using an inverted phase-contrast microscope. The cardiomyocytes began contracting within 1-2 days after plating and continued to contract for many weeks, allowing their use in multiple laboratory sessions. Following background reading and instruction, students fixed and triple-stained the cardiomyocytes to examine the relative distributions of actin filaments and microtubules and the position of nuclei. Analysis and image capture with fluorescence microscopy provided striking examples of highly organized cytoskeletal elements. Students then designed experiments in which cardiomyocyte intrinsic contractility was explored. Changes in contraction rates were examined after treatment with signaling molecules, such as epinephrine. The addition of epinephrine to the culture medium, within a usable concentration window, increased the rate of contraction. These adaptable exercises provide undergraduate cell and molecular biology students with the exciting opportunity to study cardiomyocytes using standard cell culture and microscopy techniques.

Gamma secretase activity modulates BMP-7-induced dendritic growth in primary rat sympathetic neurons.

Autonomic dysfunction has been observed in Alzheimer's disease (AD); however, the effects of genes involved in AD on the peripheral nervous system are not well understood. Previous studies have shown that presenilin-1 (PSEN1), the catalytic subunit of the gamma secretase (γ-secretase) complex, mutations in which are associated with familial AD function, regulates dendritic growth in hippocampal neurons. In this study, we examined whether the γ-secretase pathway also influences dendritic growth in primary sympathetic neurons. Using immunoblotting and immunocytochemistry, molecules of the γ-secretase complex, PSEN1, PSEN2, PEN2, nicastrin and APH1a, were detected in sympathetic neurons dissociated from embryonic (E20/21) rat sympathetic ganglia. Addition of bone morphogenetic protein-7 (BMP-7), which induces dendrites in these neurons, did not alter expression or localization of γ-secretase complex proteins. BMP-7-induced dendritic growth was inhibited by siRNA knockdown of PSEN1 and by three γ-secretase inhibitors, γ-secretase inhibitor IX (DAPT), LY-411575 and BMS-299897. These effects were specific to dendrites and concentration-dependent and did not alter early downstream pathways of BMP signaling. In summary, our results indicate that γ-secretase activity enhances BMP-7 induced dendritic growth in sympathetic neurons. These findings provide insight into the normal cellular role of the γ-secretase complex in sympathetic neurons.

Age-appropriate potassium clearance from perinatal cerebrospinal fluid depends on choroid plexus NKCC1

Regulation of the volume and electrolyte composition of the cerebrospinal fluid (CSF) is vital for brain development and function. The Na-K-Cl co-transporter NKCC1 in the choroid plexus (ChP) plays key roles in regulating CSF volume by co-transporting ions and mediating same-direction water movements. Our previous study showed ChP NKCC1 is highly phosphorylated in neonatal mice as the CSF K+ level drastically decreases and that overexpression of NKCC1 in the ChP accelerates CSF K+ clearance and reduces ventricle size [1]. These data suggest that NKCC1 mediates CSF K+ clearance following birth in mice. In this current study, we used CRISPR technology to create a conditional NKCC1 knockout mouse line and evaluated CSF K+ by Inductively Coupled Plasma Optical Emission spectroscopy (ICP-OES). We demonstrated ChP-specific reduction of total and phosphorylated NKCC1 in neonatal mice following embryonic intraventricular delivery of Cre recombinase using AAV2/5. ChP-NKCC1 knockdown was accompanied by a delayed perinatal clearance of CSF K+. No gross morphological disruptions were observed in the cerebral cortex. We extended our previous results by showing embryonic and perinatal rats shared key characteristics with mice, including decreased ChP NKCC1 expression level, increased ChP NKCC1 phosphorylation state, and increased CSF K+ levels compared to adult. Collectively, these follow up data support ChP NKCC1’s role in age-appropriate CSF K+ clearance during neonatal development.

Micromechanical Loading Studies in Ex Vivo Cultured Embryonic Rat Bones Enabled by a Newly Developed Portable Loading Device.

Mechanical loading has been described as having the potential to affect bone growth. In order to experimentally study the potential clinical applications of mechanical loading as a novel treatment to locally modulate bone growth, there is a need to develop a portable mechanical loading device enabling studies in small bones. Existing devices are bulky and challenging to transfer within and between laboratories and animal facilities, and they do not offer user-friendly mechanical testing across both ex vivo cultured small bones and in vivo animal models. To address this, we developed a portable loading device comprised of a linear actuator fixed within a stainless-steel frame equipped with suitable structures and interfaces. The actuator, along with the supplied control system, can achieve high-precision force control within the desired force and frequency range, allowing various load application scenarios. To validate the functionality of this new device, proof-of-concept studies were performed in ex vivo cultured rat bones of varying sizes. First, very small fetal metatarsal bones were microdissected and exposed to 0.4 N loading applied at 0.77Hz for 30s. When bone lengths were measured after 5days in culture, loaded bones had grown less than unloaded controls (p < 0.05). Next, fetal rat femur bones were periodically exposed to 0.4 N loading at 0.77Hz while being cultured ex vivo for 12days. Interestingly, this loading regimen had the opposite effect on bone growth, i.e., loaded femur bones grew significantly more than unloaded controls (p < 0.001). These findings suggest that complex relationships between longitudinal bone growth and mechanical loading can be determined using this device. We conclude that our new portable mechanical loading device allows experimental studies in small bones of varying sizes, which may facilitate further preclinical studies exploring the potential clinical applications of mechanical loading.

Influence of Hypoglycemia on Subcellular Distribution of Phosphorylated Tau Threonine 181 and Phosphorylated Serine 199/202 within Hippocampal astrocytes

AbstractBackgroundThe pathogenesis of Alzheimer’s disease (AD) has a substantial focus on the buildup of beta‐amyloid plaques (Aβ) and phosphorylated Tau protein tangles, in which astrocytes are involved in the clearance of these plaques and tangles. In addition, a large prospective study of type II diabetic patients that experienced hypoglycemia showed a significantly higher incidence of dementia. Furthermore, there was a significant linear association between increased frequency of hypoglycemia and an increased risk of dementia. Recently, studies have shown a correlation between early onset of type II diabetes and subsequent development of dementia.MethodIn the current experiments, embryonic hippocampal rat astrocytes were cultured in complete media (high‐glucose, electrolytes, and 10% fetal bovine serum (FBS). The incubator for cell cultures was maintained at 37 C, 95% humidity, until 85% confluent. The astrocytes were then resuspended in the above media or the same media with the glucose concentration reduced to 30%. Pterostilbene and sulforaphane was dissolved with Captisol® to increase their entry into the astrocytes. Visualizing tau microtubules in vitro was accomplished via utilization of antibodies specific to the tau protein and a Confocal Microscope. Experiments were performed to determine the possible altered distribution of phosphorylated tau at threonine 181(ptau181) and Ser199/Ser202 within the astrocytes.ResultThe levels of phosphorylation of tau were higher in the cells containing lower concentrations of glucose. In addition, astrocytes exposed to low glucose showed higher levels of phosphorylated tau at threonine 181(ptau181) in subcellular fluid, while cells pretreated with low glucose showed higher levels of phosphorylated tau at Ser199/Ser202 in mitochondria and/or nuclei.ConclusionThe subcellular distribution of phosphorylated tau at threonine 181(ptau181) and Ser199/Ser202 within the astrocytes exposed to low glucose media was different.

Enzyme-Mediated Proximity Labeling Identifies Small RNAs in the Endoplasmic Reticulum Lumen.

In eukaryotic cells, the subcellular targeting of RNA controls many fundamental aspects of cellular physiology. Despite broad distributions throughout the cytoplasm, RNA molecules are conventionally believed to be excluded from the secretory pathway compartments including the endoplasmic reticulum (ER). Recent discovery of RNA N-glycan modification (glycoRNAs) has challenged this view, but direct evidence of RNA localization in the ER lumen has been lacking. In this study, we applied enzyme-mediated proximity labeling to profile the ER lumen-localized RNAs in human embryonic kidney 293T cells and rat cortical neurons. Our data set reveals the presence of small non-coding RNAs in the ER lumen, including U RNAs and Y RNAs, which raises interesting questions regarding their transport mechanism and biological functions in the ER.

Steady-state intracellular pH and recovery from NH3/NH4+-induced intracellular acid loads in primary cultures of rat hippocampal neurons and astrocytes

Intracellular pH (pH i ) homeostasis is of vital importance because pH i fluctuations alter the net charge on intracellular weak acids and bases (e.g., amino-acid residues of proteins, metabolic intermediates, solutes like inorganic phosphate), and can result in cellular dysfunction. The rate of pH i change (dpH i /dt) is determined by the balance between acid loading (e.g., metabolic production of H + , influx of H + , efflux of HCO 3 − via transmembrane proteins of the SLC4 family) and acid extrusion (e.g., efflux of H + via Na-H exchangers in the SLC9 family, influx of HCO 3 − by members of the SLC4 family). A powerful approach for studying pH i regulation is to impose an acute intracellular acid load using the NH 4 + /NH 3 prepulse technique, and then assess dpH i /dt during the subsequent pH i recovery. Using two such prepulses and recoveries (twin pulses), one can compare 2 experimental conditions. The goal of the present study is to develop a data-analysis approach that facilitates a meaningful presentation of all twin-pulse experiments in a data set. Using primary cultures (~2 weeks) of embryonic (E18–E20) rat hippocampal neurons and astrocytes on coverslips, we loaded cells with the ratiometric pH-sensitive fluorescent dye BCECF, subjected the cells to twin NH 4 + /NH 3 pulses, and calibrated BCECF at the end of each experiment with nigericin at pH=7.0. A standard 12-point nigericin calibration curve allowed us to compute pH i vs. time. Because we studied cells in 21% O 2 but the nominal absence of CO 2 /HCO 3 − , the pH i recoveries mainly reflect Na-H exchange. We fitted the pH i -recovery time course with a double exponential (DExp) to reduce bias. Nevertheless, in both neurons and astrocytes plots of dpH i /dt vs. pH i appeared rather linear, indicating that the pH i recoveries were “quasi” single exponential (SExp). To exploit the near-linearity, for each pH i recovery, we fitted the plot of dpH i /dt vs pH i with a line, the slope of which is the quasi-single exponential rate constant (kqSExp), and the x-intercept of which is the asymptotic pH i of the recovery. We summarize the twin pH i recoveries from individual cells as miniature plots (“thumbnails”) in which we display, for each recovery, both the dpH i /dt values (from the DExp fit of pH i vs. time) vs. pH i and the line of best fit for these dpH i /dt values vs. pH i . A single figure can replicate such twin-pulse experiments from many dozens of cells, so that a reader, at a glance, can surmise the essentials of each pH i recovery. The present work provides background data for additional work in which we studied pH i recoveries at a reduced [O 2 ], with or without CO 2 /HCO 3 − . R01 NS018400, R01 HL160857, R01 DK128315 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acetaminophen causes mitochondrial dysfunction in heart cardiomyocytes

Acetaminophen (APAP) is a common over-the-counter medication used to treat pain and fever. It can be taken orally, topically, or intravenously and is considered a safe and effective drug when taken at therapeutic doses. However, recent research suggests that taking APAP regularly can result in increased blood pressure and cardiovascular risk. Previous publications found that Non-Steroidal-Anti-Inflammatory-Drugs (NSAIDs) increased Reactive Oxygen Species (ROS) in cardiomyocytes, resulting in cardiac dysfunction. Although APAP is not a NSAID, APAP likely has similar effects. We hypothesized that regular APAP usage resulted in increased ROS levels, leading to potential dysfunction in hearts and other organs. We used the embryonic rat heart cell line H9C2 to test our hypothesis. We conducted a ROS Assay using H9C2 cells treated with dichlorodihydrofluorescein diacetate (DFCDA). The cells were then treated with vehicle, various concentrations of APAP (25μM, 50μM, 100μM, and 200μM), or 100μM hydrogen peroxide for 1.5 hours before measuring the ROS levels of the cells. We also measured the cell viability of H9C2 cells. Cells were treated with 10μM Alamar Blue, then treated with vehicle, various concentrations of APAP (25μM, 50μM, 100μM, and 200μM), or 100μM or 200μM hydrogen peroxide for 48 hours before detecting the percentage of viable cells. The mitochondrial membrane potential (MMP) of cells treated with APAP was also measured. Cells were treated with vehicle, various concentrations of APAP (25μM, 50μM, 100μM, and 200μM), or 250μM p-triflouromethoxyphenylhydrazone (FCCP) for 24 hours before being incubated with JC-10 for one hour. The cells were measured using a fluorescent reader at excitation/emission 490/525 nm and 540/590 nm. For the ROS assay, the cells treated with 100μM APAP, 200μM APAP, and 100μM Hydrogen Peroxide significantly increased total ROS compared to the control. The cell viability assay resulted in a significant decrease in percent viability in cells treated with 200μM APAP, 100μM hydrogen peroxide, and 200μM hydrogen peroxide compared to the control. The MMP Assay resulted in a significant decrease in MMP at all concentrations of APAP and 250μM FCCP compared to the control. These results suggest that treatment of H9C2 cardiac cells with physiological concentrations of APAP causes an increase in intracellular ROS levels, decreased cell viability, and decreased MMP. Overall, APAP causes mitochondrial dysfunction, which is likely to cause cardiomyocyte dysfunction. This research is funded by the NIEHS/Superfund Research Program (P42 ES004699). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells.

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

Circulating Extracellular Vesicle-Propagated microRNA Signature as a Vascular Calcification Factor in Chronic Kidney Disease.

Chronic kidney disease (CKD) accelerates vascular calcification via phenotypic switching of vascular smooth muscle cells (VSMCs). We investigated the roles of circulating small extracellular vesicles (sEVs) between the kidneys and VSMCs and uncovered relevant sEV-propagated microRNAs (miRNAs) and their biological signaling pathways. We established CKD models in rats and mice by adenine-induced tubulointerstitial fibrosis. Cultures of A10 embryonic rat VSMCs showed increased calcification and transcription of osterix (Sp7), osteocalcin (Bglap), and osteopontin (Spp1) when treated with rat CKD serum. sEVs, but not sEV-depleted serum, accelerated calcification in VSMCs. Intraperitoneal administration of a neutral sphingomyelinase and biogenesis/release inhibitor of sEVs, GW4869 (2.5 mg/kg per 2 days), inhibited thoracic aortic calcification in CKD mice under a high-phosphorus diet. GW4869 induced a nearly full recovery of calcification and transcription of osteogenic marker genes. In CKD, the miRNA transcriptome of sEVs revealed a depletion of 4 miRNAs, miR-16-5p, miR-17~92 cluster-originated miR-17-5p/miR-20a-5p, and miR-106b-5p. Their expression decreased in sEVs from CKD patients as kidney function deteriorated. Transfection of VSMCs with each miRNA-mimic mitigated calcification. In silico analyses revealed VEGFA (vascular endothelial growth factor A) as a convergent target of these miRNAs. We found a 16-fold increase in VEGFA transcription in the thoracic aorta of CKD mice under a high-phosphorus diet, which GW4869 reversed. Inhibition of VEGFA-VEGFR2 signaling with sorafenib, fruquintinib, sunitinib, or VEGFR2-targeted siRNA mitigated calcification in VSMCs. Orally administered fruquintinib (2.5 mg/kg per day) for 4 weeks suppressed the transcription of osteogenic marker genes in the mouse aorta. The area under the curve of miR-16-5p, miR-17-5p, 20a-5p, and miR-106b-5p for the prediction of abdominal aortic calcification was 0.7630, 0.7704, 0.7407, and 0.7704, respectively. The miRNA transcriptomic signature of circulating sEVs uncovered their pathologic role, devoid of the calcification-protective miRNAs that target VEGFA signaling in CKD-driven vascular calcification. These sEV-propagated miRNAs are potential biomarkers and therapeutic targets for vascular calcification.

Photobiomodulation improves the synapses and cognitive function and ameliorates epileptic seizure by inhibiting downregulation of Nlgn3

BackgroundTemporal lobe epilepsy (TLE) remains one of the most drug-resistant focal epilepsies. Glutamate excitotoxicity and neuroinflammation which leads to loss of synaptic proteins and neuronal death appear to represent a pathogen that characterizes the neurobiology of TLE. Photobiomodulation (PBM) is a rapidly growing therapy for the attenuation of neuronal degeneration harboring non-invasiveness benefits. However, the detailed effects of PBM on excitotoxicity or neuroinflammation remain unclear. We investigated whether tPBM exerts neuroprotective effects on hippocampal neurons in epilepsy mouse model by regulating synapse and synapse-related genes.MethodsIn an in vitro study, we performed imaging analysis and western blot in primary hippocampal neurons from embryonic (E17) rat pups. In an in vivo study, RNA sequencing was performed to identify the gene regulatory by PBM. Histological stain and immunohistochemistry analyses were used to assess synaptic connections, neuroinflammation and neuronal survival. Behavioral tests were used to evaluate the effects of PBM on cognitive functions.ResultsPBM was upregulated synaptic connections in an in vitro. In addition, it was confirmed that transcranial PBM reduced synaptic degeneration, neuronal apoptosis, and neuroinflammation in an in vivo. These effects of PBM were supported by RNA sequencing results showing the relation of PBM with gene regulatory networks of neuronal functions. Specifically, Nlgn3 showed increase after PBM and silencing the Nlgn3 reversed the positive effect of PBM in in vitro. Lastly, behavioral alterations including hypoactivity, anxiety and impaired memory were recovered along with the reduction of seizure score in PBM-treated mice.ConclusionsOur findings demonstrate that PBM attenuates epileptic excitotoxicity, neurodegeneration and cognitive decline induced by TLE through inhibition of the Nlgn3 gene decrease induced by excitotoxicity.Graphical

Superficial groove structure in the size of focal adhesion can clarify cell-type-specific differences in force-dependent substrate mechanosensing

Mechanosensing-based cell orientation and migration in microenvironments, such as microgrooved surfaces, are essential in biological tissue growth and repair. These responses might be cell type-dependent as they are deeply involved in cellular functions. Despite their orientation and migration responses are depended on cell-substrate adhesion, which is deeply involved with focal adhesions (FAs) of cells, we have limited information on the cell behavior on FA-sized microgrooved surfaces. Here, we systematically investigated the cell orientation and migration behavior of primary porcine aortic smooth muscle cells (PASMs), embryonic rat aortic smooth muscle cells with fibroblast-like phenotype (A7r5), and human cervical cancer cells (HeLa) on a substrate comprised of superficial grooves with three widths (1 μm, 5 μm, and 10 μm) and 150-nm depth, which are the same order of magnitude of the three-dimensional size of FAs. PASM and A7r5 cells including thick bundles of actin fibers with elongated FAs showed pronounced cell polarization and directional migration on the 1 and 5 μm wide grooves. PASMs were more sensitive to the superficial grooves than A7r5 cells. HeLa cells adhering to the grooves had non-oriented actin fibers with smaller FAs, and they did not show specific orientation nor directional migration in any groove type. Atomic force microscopy elucidated that the mechanical tension of the actin fibers in live cells had a significant positive correlation with the length/width ratio of FAs, reflecting the cell alignment and directional migration on the grooves. The increase or decrease in the mechanical tension of the actin fibers improved or diminished the mechanosensing for the grooves, respectively. The results strongly suggested that the differences in cell type-specific alignment and migration become much more pronounced on the microgrooves which are similar in three-dimensional size of FA, and that is useful for clarifying slight differences in force-dependent cellular mechanosensing.

Pyramidal neuron morphogenesis requires a septin network that stabilizes filopodia and suppresses lamellipodia during neurite initiation

Pyramidal neurons are a major cell type of the forebrain, consisting of a pyramidally shaped soma with axonal and apicobasal dendritic processes. It is poorly understood how the neuronal soma develops its pyramidal morphology, while generating neurites of the proper shape and orientation. Here, we discovered that the spherical somata of immature neurite-less neurons possess a circumferential wreath-like network of septin filaments, which promotes neuritogenesis by balancing the protrusive activity of lamellipodia and filopodia. In embryonic rat hippocampal and mouse cortical neurons, the septin wreath network consists of curvilinear filaments that contain septins 5, 7, and 11 (Sept5/7/11). The Sept5/7/11 wreath network demarcates a zone of myosin II enrichment and Arp2/3 diminution at the base of filopodial actin bundles. In Sept7-depleted neurons, cell bodies are enlarged with hyperextended lamellae and abnormally shaped neurites that originate from lamellipodia. This phenotype is accompanied by diminished myosin II and filopodia lifetimes and increased Arp2/3 and lamellipodial activity. Inhibition of Arp2/3 rescues soma and neurite phenotypes, indicating that the septin wreath network suppresses the extension of lamellipodia, facilitating the formation of neurites from the filopodia of a consolidated soma. We show that this septin function is critical for developing a pyramidally shaped soma with properly distributed and oriented dendrites in cultured rat hippocampal neurons and invivo in mouse perinatal cortical neurons. Therefore, the somatic septin cytoskeleton provides a key morphogenetic mechanism for neuritogenesis and the development of pyramidal neurons.

Accelerating Neurite Growth and Directing Neuronal Connections Constrained by 3D Porous Microtubes.

Investigation of neural growth and connection is crucial in the field of neural tissue engineering. Here, using a femtosecond laser direct writing (fs-DLW) technique, we propose a directionally aligned porous microtube array as a culture system for accelerating the growth of neurons and directing the connection of neurites. These microtubes exhibited an unprecedented guidance effect toward the outgrowth of primary embryonic rat hippocampal neurons, with a wrap resembling the myelin sheaths of neurons. The speed of neurite growth inside these microtubes was significantly faster than that outside these microtubes. We also achieved selective/directing connection of neural networks inside the magnetic microtubes via precise microtube delivery to a gap between two neural clusters. This work not only proposes a powerful microtube platform for accelerated growth of neurons but also offers a new idea for constructing biological neural circuits by arranging the size, location, and pattern of microtubes.

Semi-Automated Quantitative Evaluation of Neuron Developmental Morphology In Vitro Using the Change-Point Test.

Neuron morphology gives rise to distinct axons and dendrites and plays an essential role in neuronal functionality and circuit dynamics. In rat hippocampal neurons, morphological development occurs over roughly one week in vitro. This development has been qualitatively described as occurring in 5 stages. Still, there is a need to quantify cell growth to monitor cell culture health, understand cell responses to sensory cues, and compare experimental results and computational growth model predictions. To address this need, embryonic rat hippocampal neurons were observed in vitro over six days, and their processes were quantified using both standard morphometrics (degree, number of neurites, total length, and tortuosity) and new metrics (distance between change points, relative turning angle, and the number of change points) based on the Change-Point Test to track changes in path trajectories. Of the standard morphometrics, the total length of neurites per cell and the number of endpoints were significantly different between 0.5, 1.5, and 4 days in vitro, which are typically associated with Stages 2-4. Using the Change-Point Test, the number of change points and the average distance between change points per cell were also significantly different between those key time points. This work highlights key quantitative characteristics, both among common and novel morphometrics, that can describe neuron development in vitro and provides a foundation for analyzing directional changes in neurite growth for future studies.

Sodium-Calcium Exchanger 2: A Pivotal Role in Oxaliplatin Induced Peripheral Neurotoxicity and Axonal Damage?

Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN) is a frequent adverse event of colorectal cancer treatment. OIPN encompasses a chronic and an acute syndrome. The latter consists of transient axonal hyperexcitability, due to unbalance in Na+ voltage-operated channels (Na+VOC). This leads to sustained depolarisation which can activate the reverse mode of the Na+/Ca2+ exchanger 2 (NCX2), resulting in toxic Ca2+ accumulation and axonal damage (ADa). We explored the role of NCX2 in in vitro and in vivo settings. Embryonic rat Dorsal Root Ganglia (DRG) organotypic cultures treated with SEA0400 (SEA), a NCX inhibitor, were used to assess neuroprotection in a proof-of-concept and pilot study to exploit NCX modulation to prevent ADa. In vivo, OHP treated mice (7 mg/Kg, i.v., once a week for 8 weeks) were compared with a vehicle-treated group (n = 12 each). Neurophysiological and behavioural testing were performed to characterise acute and chronic OIPN, and morphological analyses were performed to detect ADa. Immunohistochemistry, immunofluorescence, and western blotting (WB) analyses were also performed to demonstrate changes in NCX2 immunoreactivity and protein expression. In vitro, NCX inhibition was matched by ADa mitigation. In the in vivo part, after verifyingboth acute and chronic OIPN had ensued, we confirmed via immunohistochemistry, immunofluorescence, and WB that a significant NCX2 alteration had ensued in the OHP group. Our data suggest NCX2 involvement in ADa development, paving the way to a new line of research to prevent OIPN.

Sulforaphane inhibits angiotensin II-induced cardiomyocyte apoptosis by acetylation modification of Nrf2.

Oxidative stress is the central cause of angiotensin II (Ang II)-induced myocardial injury, and nuclear factor erythroid 2-related factor (Nrf2) is the core molecule of the anti-oxidant defense system. We have previously demonstrated that sulforaphane (SFN) can prevent Ang II-induced myocardial injury by activating Nrf2; however, the underlying molecular mechanism is still unclear. This study aimed to evaluate whether SFN prevents Ang II-induced cardiomyocyte apoptosis through acetylation modification of Nrf2. Wild-type and Nrf2 knockdown embryonic rat cardiomyocytes (H9C2) were exposed to Ang II to induce apoptosis, oxidative stress, and inflammatory responses. SFN treatment significantly reduced Ang II-induced cardiomyocyte apoptosis, inflammation and oxidative stress. Activation of Nrf2 played a critical role in preventing cardiomyocyte apoptosis. After Nrf2 was knockdown, the anti-inflammatory, antioxidant stress of SFN were eliminated. Furthermore, Nrf2 activation by SFN was closely related to the decreased activity of histone deacetylases (HDACs) and increased histone-3 (H3) acetylation levels in Nrf2 promoter region. These findings confirm that Nrf2 plays a key role in SFN preventing Ang II-induced cardiomyocyte apoptosis. SFN activates Nrf2 by inhibiting HDACs expression and activation.

SCO-spondin-derived peptide NX210 rescues neurons from cerebral ischemia/reperfusion injury through modulating the Integrin-β1 mediated PI3K/Akt pathway

Ischemic stroke is a common condition with high morbidity and mortality, causing irreversible neuronal damage and seriously affecting neurological function. There has been no ideal effective treatment so far. The NX210 peptide is derived from the thrombospondin type 1 repeat (TSR) sequence of SCO-spondin, and has been reported to exert various neurogenic properties. This study investigated whether NX210 had therapeutic effects and possible underlying mechanisms against cerebral ischemia/reperfusion (I/R). Therefore, primary embryonic rat cortical neurons and Sprague–Dawley (SD) rats that were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion/reperfusion (MCAO/R) injuries, respectively, were treated with or without NX210. We found that NX210 reduced OGD/R-induced cell viability loss and cytotoxicity. NX210 decreased cerebral infarct volume and brain edema, ameliorated neurological dysfunction, attenuated oxidative stress damage, and diminished neuronal apoptosis in MCAO/R rats. Furthermore, western blot analysis shown that treatment with NX210 up-regulated the expression of Integrin-β1, phosphorylated-PI3K (p-PI3K) and phosphorylated-Akt (p-Akt). The Integrin-β1 specific inhibitor, ATN-161, was used to identify pathways involved. The anti-oxidation activities and anti-apoptosis of NX210 was reversed by treatment with ATN-161. Overall, our results indicated that NX210 prevents oxidative stress and neuronal apoptosis in cerebral I/R via upregulation of the Integrin-β1/PI3K/Akt signaling pathway. These results indicated that NX210 may be a promising therapeutic candidate for ischemic stroke.

Slibinin governs high glucose induced autophagy in cardiac myocyte cells via sphingosine kinase 1 pathway

As a disorder of the myocardium caused by diabetes mellitus, DCM has become a key health concern in the world. Autophagy plays an important role in the pathogenesis of DCM. Sphingosine kinase 1 benefits to cell survival and growth, and regulates the pathogenesis of many diseases, including diabetes and cardiovascular disease. Silibinin is widely used as a hepatoprotective and antioxidant agent in Asia and Europe. However, how silibinin governs DCM remains poorly understood. To this aim, the present study is to mine the role of Sphk1 in autophagy induction and cell survival in myocardial cells under high glucose treatment, and how silibinin regulates DCM. Herein, the study used the embryonic rat heart-derived myogenic H9C2 cells and adenovirus based gene manipulation was utilized. It was found that silibinin could induce autophagy in a dose-dependent manner. Overexpression of Sphk1 significantly increased mRNA expression of autophagy gene markers in H9C2 cells. Gain of function of Sphk1 significantly increased protein level of Beclin1 in H9C2 cells under treatments of low (5.5 mM) and high (25 mM) glucose. High glucose inhibited autophagy induction in H9C2 cells. Gain of function of Sphk1 compromised inhibition of on autophagy induction by high glucose. The results of the present study may assist in attaining an improved understanding of the pathogenesis of DCM, and developing novel therapies for treatment of DCM.

Casein kinase 2 attenuates brain injury induced by intracerebral hemorrhage via regulation of NR2B phosphorylation.

ObjectiveIntracerebral hemorrhage (ICH) is a common cerebrovascular disease with high incidence, disability, and mortality. Casein kinase 2 (CK2) is a serine/threonine kinase with hundreds of identified substrates and plays an important role in many diseases. This study aimed to explore whether CK2 plays protective roles in ICH-induced neuronal apoptosis, inflammation, and oxidative stress through regulation NR2B phosphorylation.MethodsCK2 expression level of brain tissues taken from ICH patients was determined by immunoblotting. Neurons from embryonic rat and astrocytes from newborn rats were cultured and treated by Hemoglobin chloride (Hemin). The proliferation of astrocytes, the apoptosis and oxidative stress of neurons and the inflammatory factors of astrocytes were detected. CK2 expression was determined in ICH model rats. The effects of CK2 overexpression plasmid (pc-CK2) on neurobehavioral defects and brain water content in ICH rats were observed.ResultsCK2 expression in ICH patients was down-regulated. Overexpression of CK2 promoted the astrocyte proliferation, inhibited neuronal apoptosis, and reduced astrocyte-mediated inflammation. N-methyl-D-aspartate receptor 2B (NR2B) reversed the effects of pc-CK2 on neurons and astrocytes. CK2 phosphorylated NR2B at the S1480 site, down-regulated the expression of NR2B and interfered with the interaction between NR2B and postsynaptic density protein 95 (PSD95). In vivo experiments showed that the expression of CK2 decreased and the expression of NR2B increased in ICH rats. Furthermore, pc-CK2 attenuated neurobehavioral defects, brain water content and neuronal damage in ICH rats.ConclusionCK2 phosphorylated NR2B, down-regulated the expression of NR2B, interfered with the interaction between NR2B and PSD95, alleviated inflammatory reactions, inhibited neuronal apoptosis and oxidative stress after ICH. CK2 and NR2B may be new potential therapeutic targets for the treatment of ICH. However, the limitation of this study is that we only investigated the regulation of NR2B by CK2.