Glass Micropipettes Research Articles

Metal-Organic Framework Sub-Nanochannels within the Confined Micropipettes: Precise Construction Makes It a Universal Aptamer-Based Sensing Platform.

It is crucial to precisely construct metal-organic framework (MOF) sub-nanochannels at the tip of micro/nanopipettes for fundamental research and sensing applications. The quality of the MOF modification plays a significant role in influencing subsequent research, particularly in sensing applications. In this work, we present a precise method of constructing MOF sub-nanochannels at the tip of glass micropipettes, which serve as a universal aptamer-based sensing platform for the selective detection of proteins. In situ scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS) mapping, and fluorescence microscopy results demonstrate that the synthesized MOF (UiO-66) nanocrystals fully block the orifice of glass micropipettes (UiO-66-GMs) without forming any nanometer-scale cracks and remain confined within the geometric boundaries of the orifice. The terminal phosphate-modified aptamer readily binds to the surface of UiO-66-GMs through metal (Zr)-phosphate coordination, ultimately forming the aptamer sensor (Apt-UiO-66-GMs). The selective quantification of proteins is achieved via a decrease in current resulting from protein binding to the aptamer. Our results indicate that the precisely constructed Apt-UiO-66-GMs sensor enables highly selective and sensitive detection of SARS-CoV-2 nucleocapsid protein and holds potential for real sample detection. Furthermore, given the sharp tip of the micropipets and the external sensing interface we have constructed, our aptamer-based sensing platform also opens avenues for single-cell analysis and in vivo sensing.

Different taste map for amiloride sensitivity, response frequency, and threshold to NaCl in the rostral nucleus of the solitary tract in rats.

Studies on taste bud cells and brain stem relay nuclei suggest that alternative pathways convey information regarding different taste qualities. Building on the hypothesis that amiloride (epithelial Na channel antagonist)-sensitive neurons respond to palatable salt (low-concentration) and amiloride-insensitive neurons respond to aversive salt (high-concentration), we investigated the histological distribution of taste-sensitive neurons in the rostral nucleus of the solitary tract in rats and their NaCl and amiloride sensitivities. We recorded neuronal activity in extracellular single units using multi-barrel glass micropipettes and reconstructed their locations on the rostrocaudal and mediolateral axes. Seventy-three taste-sensitive neurons were categorized into the best-taste category. The amiloride sensitivities of the 31 neurons were examined for 0.1, 0.2, 0.4, and 0.8 M NaCl. The neuronal distribution of amiloride-sensitive neurons was located in the lateral region, while amiloride-insensitive neurons were located in the medial region. The amiloride-sensitive neurons responded to low salt concentrations, signaling the NaCl levels required by body fluids. Amiloride-insensitive neurons were silent at low salt concentrations but may function as warning signals for high salt concentrations. Low-threshold and/or high-response neurons were located in the rostrolateral region. In contrast, high-threshold and/or low-response neurons were located in the caudal-medial region.

Neuronal rhythmicity and cortical arousal in a mouse model of absence epilepsy

Objectives: Absence seizures impair psychosocial function, yet their detailed neuronal basis remains unknown. Recent work in a rat model suggests that cortical arousal state changes prior to seizures and that single neurons show diverse firing patterns during seizures. Our aim was to extend these investigations to a mouse model with studies of neuronal activity and arousal state to facilitate future fundamental investigations of absence epilepsy.Methods: We performed in vivo extracellular single unit recordings on awake head-fixed C3H/HeJ mice. Mice were implanted with tripolar electrodes for cortical electroencephalography (EEG). Extracellular single unit recordings were obtained with glass micropipettes in the somatosensory barrel cortex, while animals ambulated freely on a running wheel. Signals were digitized and analyzed during seizures and at baseline.Results: Neuronal activity was recorded from 36 cortical neurons in 19 mice while EEG showed characteristic 7–8 Hz spike-wave discharges. Different single neurons showed distinct firing patterns during seizures, but the overall mean population neuronal firing rate during seizures was no different from pre-seizure baseline. However, the rhythmicity of neuronal firing during seizures was significantly increased (p < 0.001). In addition, beginning 10s prior to seizure initiation, we observed a progressive decrease in cortical high frequency (>40 Hz) EEG and an increase in lower frequency (1–39 Hz) activity suggesting decreased arousal state.Significance: We found that the awake head-fixed C3H/HeJ mouse model demonstrated rhythmic neuronal firing during seizures, and a decreased cortical arousal state prior to seizure onset. Unlike the rat model we did not observe an overall decrease in neuronal firing during seizures. Similarities and differences across species strengthen the ability to investigate fundamental key mechanisms. Future work in the mouse model will identify the molecular basis of neurons with different firing patterns, their role in seizure initiation and behavioral deficits, with ultimate translation to human absence epilepsy.

O-184 Piezo-ICSI reduces oocyte degeneration, improves ICSI success rates and has shown less dependence on the experience level of embryologists: cumulative data with 65,033 MII oocytes

Abstract Study question Dose piezo-ICSI improve the overall success rates of in ICSI operations compared with the conventional ICSI by the operators with different level of experience? Summary answer Piezo-ICSI showed lower oocyte degeneration rate during or after ICSI operations compared to the conventional ICSI for embryologists with different level of experience. What is known already Intracytoplasmic sperm injection (ICSI) with a glass micropipette can sometimes result in degeneration of the injected oocyte. Piezo-assisted ICSI has been developed as an alternative method to prevent such degeneration occurring during the procedure. It employs gentler and controlled impacts generated from the piezo electric elements attached to the injection micropipette holder to penetrate oolemma, and it does not require reciprocal aspiration of ooplasm required in the conventional ICSI. While there have been numerous study reports demonstrating the improvements in fertilization rates with piezo-ICSI, most of these studies have the limitation in the size study of population. Study design, size, duration We conducted a retrospective analysis of the ICSI results with 65,033 MII oocytes in 15,687 treatment cycles carried out between January 2018 and February 2023. ICSI were performed using the piezo-assisted equipment with flat-tip micropipettes (PIEZO PMM4G, Prime Tech Ltd., [n = 51,484, Piezo-group]) or in the conventional ICSI method with spiked-tip micropipettes (n = 13,549, C-group). Participants/materials, setting, methods Following the insemination with ICSI, the oocyte degeneration rate, fertilization rate per MII oocytes and blastocyst formation rate per 2PN embryos were evaluated in both the Piezo- and C-groups (n = 51,484 and n = 13,549, respectively). We also evaluated the degeneration rates and fertilization rates of ICSI by the operators: i.e., well-experienced embryologists (at least 10 years of experience) and less experienced embryologists (5 years or less of experience). Main results and the role of chance The fertilization and blastocyst formation rates in the Piezo-group were 83.8% (43,140/51,484) and 54.0% (23,300/43,140), respectively, which were significantly higher than the C-group (74.8% [10,132/13,549] and 36.6% [3,674/10,132], respectively, p &amp;lt; 0.01). The oocyte degeneration rate in the Piezo-group was 2.9% (1,493/51,484), which was significantly lower than the C-group (6.9% [933/13,549], p &amp;lt; 0.05). For the patients over 40 years old, the degeneration rate was also significantly lower in the Piezo-group (3.0% [632/20,770]) than the C-group (7.1% [351/4927], p &amp;lt; 0.01), while the fertilization and blastocyst formation rates were higher in the Piezo-group (82.6% [17,164/20,770] and 46.6% [7,997/17,164], respectively) than the C-group (72.8% [3,588/4,927] and 25.8% [926/3,588], respectively, p &amp;lt; 0.01). We found a marked drop in the oocytes degeneration rates in the Piezo group recorded from both well-experienced and less experienced embryologists. The degeneration rates recorded by less experienced embryologists were 2.8% (119/4,242) in Piezo-group and 8.6% (8/93) in the C group (p &amp;lt; 0.01), while the records by well-experienced embryologists were 2.7% (815/29,681) in the Piezo-group and 6.9% (555/8,037) in the C-group, respectively (p &amp;lt; 0.01). The fertilization rates of the Piezo-group by less experienced and well-experienced embryologists were 84.1%(3,569/4,242) and 83.4% (24,757/29,681), respectively, which were significantly higher the C-group (71.0％ [66/93] and 76.7％ [6,167/8,037], respectively, p &amp;lt; 0.01). Limitations, reasons for caution It is a retrospective study, in which the ICSI operations with piezo and the conventional methods were not equally assigned to the embryologists, especially, for those less experienced, thereby the number of conventional ICSI operations was more skewed to well-experienced embryologists than the less experienced colleagues. Wider implications of the findings Our results with piezo-ICSI showed a marked drop in the oocyte degeneration while achieving comparable fertilization rates amongst operators with different levels of experience. We found these as major advantages over the conventional method in standardizing the ICSI operation by filling the gap between embryologists with different length of carrier. Trial registration number Not applicable

Automated Piezo-Assisted Sperm Immobilization

Sperm immobilization is a crucial procedure in clinical cell surgery for infertility treatment. Current immobilization is implemented by tapping the sperm tail with a glass micropipette, but its effectiveness is restricted by sperm orientation and ineffective membrane ablation. Ineffective ablation leads to limited release of oocyte activating factors and lowers fertilization rate; and sperm swim in small angles relative to the micropipette tip cannot be tapped due to the risk of damaging the sperm’s genetic materials contained in the sperm head. This paper reports automated piezo-assisted sperm immobilization with enhanced efficacy of cell membrane ablation and sperm orientation control. The designed piezo drill consists of two orthogonal vibration modules to generate controlled micropipette vibration along axial and lateral axes. Through stiffness modeling, the flexure joints guide the motion of the central beam of each vibration module. To achieve sperm orientation control, whirl flow is induced by both axial and lateral vibration of the micropipette tip. To immobilize sperm, only micropipette’s axial vibration is generated to prevent lateral vibration from damaging sperm head. A visual servoing scheme is developed by decoupling sperm wiggling from positioning error for immobilization. Experimental results showed that sperm orientation control by the piezo drill achieved an error of 1.4 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$^{\circ}$</tex-math> </inline-formula> and a time cost of 2.5 s. Visual servoing with sperm wiggling decoupling achieved a positioning error of 1.7 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mu$</tex-math> </inline-formula> m. Furthermore, the piezo-assisted sperm immobilization technique led to effective membrane ablation. With membrane-impermeable stains, it took 5.6 s for the immobilized sperm to be stained after piezo-assisted immobilization, significantly less than 49.2 s by conventional micropipette tapping. <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Note to Practitioners</i> —This work tackled the challenge of ineffective membrane ablation and orientation limit in clinical cell surgeries. Conventional manual immobilization suffers from low membrane ablation efficacy, which leads to limited release of oocyte activating factors and lowers fertilization rate. Moreover, sperm swim in small angles relative to the micropipette tip cannot be tapped due to the risk of damaging the sperm’s genetic materials contained in the sperm head. In this paper, we propose automation techniques for effective membrane ablation and orientation control of sperm. A clinically compatible piezo drill is developed to generate controllable micropipette motion along both axial and lateral directions. The whirl flow generated by micropipette vibration is employed to rotate sperm, which greatly increased the number of available sperm for immobilization. A visual servoing controller is developed to keep the sperm at the center of field of view for immobilization by decoupling sperm wiggling from positioning error. The developed methods can be generalized to the manipulation of other types of cells. The piezo drill can be used for effective membrane ablation of oocyte, embryo, yeast cell and so on. The orientation control strategy leveraging piezo-induced whirl flow is applicable to non-contact rotation of a variety of microorganism.

The Role of NO and H2O2 in Ach-Induced Dilation of Human Arterioles in the Absence and Presence of Coronary Artery Disease

The endothelium regulates vascular tone through the release of various vasodilatory factors such as NO, PGI2, and endothelium-derived hyperpolarizing (EDH) signaling that is more important in resistance arteries and arterioles. Using freshly isolated human arterioles, previous studies indicate that hydrogen peroxide (H2O2), an EDH factor, mediates flow-mediated dilation in patients with coronary artery disease (CAD), whereas NO plays a more prominent role in patients without CAD. However, the mechanisms of receptor agonists (e.g., acetylcholine)-induced dilation remains to be clarified in human arterioles. The objective of this study was to compare the role of NO and H2O2 in acetylcholine (Ach-induced dilation in human adipose arterioles (HAA) from non-CAD and CAD patients. HAA (100-200 μm) dissected from discarded surgical samples of non-CAD and CAD patients, were cannulated onto glass micropipettes and pressurized at 60 mmHg for measurement of diameter changes by using videomicroscopy. Microvessels were incubated with L-NAME (100 μM), an inhibitor of NO synthase, Indomethacin (Indo, 10 μM), an inhibitor of PGI2 synthesis and peg-CAT (500 U/ml), a scavenger of H2O2. To study the dose-dependent response to Ach (log 10-9 – 10-5 M), HAA were preconstricted with endothelin-1 (0.1-0.5 nM). In non-CAD tissues Ach induced potent concentration-dependent dilation (maximal dilation at 10−5 M: 95.6±1.8%). After preincubation with L-NAME, this dilation was partially reduced (maximal dilation at 10−5 M: 77.4±3.1%, P&lt;0.05 vs. control), while the combination of L-NAME and Indo reduced it to the same extent as L-NAME by itself (maximal dilation at 10−5 M: 73.1±7.4%, P&lt;0.05 vs. control). To further determine the role of H2O2 in dilatory response to Ach, we treated HAA with peg-CAT. Incubation of peg-CAT significantly reduced the dilation (maximal dilation at 10−5 M: 74.6±8.1% and 51.3±8.7% in the absence and presence of L-NAME and Indo, P&lt;0.05 vs. control). In HAA obtained from CAD patients, the dilation response to Ach was similar to non-CAD (maximal dilation at 10−5 M: 93.9± 1.8%) and the combination of L-NAME and Indo reduced this dilation to a lesser extent (maximal dilation at 10−5 M: 83.1±3%, P=0.055), compared to non-CAD patients (maximal dilation at 10−5 M: 73.1±7.4%, P&lt;0.05 vs. control). The preincubation with peg-CAT attenuated the dilation only at log -7 M (maximal dilation at 10−7 M: 33.8±9.5%, P&lt;0.05 vs. 54.6±7.5% in control). The large portion of the dilation remaining after the combination of L-NAME and Indo was almost completely abolished in the presence of high K+ concentration (maximal dilation at 10−5 M: 12.9±5.1% P&lt;0.05 vs. control). Together, these results provide new evidence that in non-CAD arterioles both NO and H2O2 play an important role in Ach-induced dilation whereas PGI2 is likely not involved. In CAD arterioles, NO, PGI2 and H2O2 have much less contribution to Ach-induced dilation, while a H2O2-independent EDH signaling plays the major role in the dilatory response. Further studies are required to determine the specific mechanism or factor involved in Ach-induced EDH-dependent dilation in HAA. This work was supported by the National Heart, Lung, and Blood Institute [R01HL096647]. This is the full abstract presented at the American Physiology Summit 2024 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.

First Babies Conceived With Automated Intracytoplasmic Sperm Injection

(Abstracted from Reprod Biomed Online 2023;47:103237 Intracytoplasmic sperm injection (ICSI) is a common procedure used in approximately 70% of in vitro fertilization (IVF) cycles. The procedure involves using glass micropipettes to penetrate the oolemma and injection of sperm into the oocyte.

Implantation of Flexible Electrodes for Simultaneous in-vivo Extracellular Recording and Two-Photon Imaging

Introduction: Rigid silicon electrodes like Utah array grids and Neuropixel probes have been used in human and animal brain models to understand the dynamics of neural computation, treat neurodegenerative disorders, and act as brain-machine-interfaces. However, when implanted chronically, glial proliferation can rapidly disrupt the interaction between neurons and electrodes, drastically reducing recording fidelity. The development of flexible electrodes has the potential to minimize tissue damage and inflammation, which allows for long-term recordings over several months. In line with this objective, the Nano-neurotechnology Lab at Purdue University has developed a 6-µm thick, flexible, and biocompatible Parylene probe to facilitate chronic recordings in awake mice. However, flexible electrodes present a unique engineering challenge as the force required to insert into the brain causes the probe to buckle and fail during insertion. &#x0D; Methods and Results: Here, I designed a micropipette shuttle using a glass micropipette and custom insertion system which provided reproducible probe implantation into the cortex. The implantation device was designed in CAD software and 3D-printed for rapid prototyping. The procedure was developed on brain phantoms made of 0.6% agarose with a comparable Young’s modulus to mouse brain tissue. Utilizing 3D-printed pieces and the surface tension of diluted poly-vinyl-acrylate adhesive to align the probe to a micropipette, insertion of the electrode and retraction of the shuttle was accomplished in awake mice. &#x0D; Conclusion: The implications of flexible recording electrodes are extensive. Long-term implantation opens the door for understanding behavioral and learning dynamics over time. Moreover, the flexibility of these probes allows for the combination of 2-photon optical microscopy, thus enabling multi-modal investigation of neuronal physiology. A low-cost, consistent procedure is the first step in the implementation of these flexible probes for further advancements in fundamental neuroscience research and its potential applications in human and animal studies.

Optically Controlled Dissolution Kinetics of Vaterite Microcapsules: Toward Novel Crystal Growth Strategies.

Controllable continuous release of functional materials from capsules is one of the unmet functions of theragnosis particles; on this way, understanding cargo-fluid interactions in vitro is an essential milestone. We develop a flexible platform to investigate single particle-fluid interactions utilizing a glass micropipette as a highly localized flow source around an optically trapped particle. In proof-of-concept experiments, this microparticle is sensitive to local microflow distribution, thus serving as a probe. The very same flows are capable of the particle rotating (i.e., vaterite drug cargo) at frequencies dependent on the mutual particle-pipette position. Platform flexibility comes from different interactions of a tweezer (optical forces) and a pipette (mechanical/hydrodynamical) with a microparticle, which makes this arrangement an ideal microtool. We studied the vaterite dissolution kinetics and demonstrated that it can be controlled on demand, providing a wide cargo release dynamic rate. Our results promote the use of inorganic mesoporous nanoparticles as a nanomedicine platform.

Automated Orientation Control of Motile Deformable Cells

Automated manipulation of deformable objects is challenging due to the object&#x2019;s deformation behavior. Different from still deformable objects such as wires and cloth, biological organisms such as sperm and worms are both deformable and motile, requiring the control of both deformation and motion. This paper reports automated orientation control of live sperm, as an example of motile deformable cells. Robotic manipulation of human sperm was performed by using a glass micropipette, which is a standard clinical tool, to rotate individual motile sperm. Sperm rotation must be performed before immobilization, as required in clinical cell surgery for infertility treatment. To control tail deformation during sperm rotation, a path planner was designed based on kinematic analysis and manipulation point update. To deal with the intrinsic motion of a motile sperm, a motorized stage was controlled to compensate for sperm swimming motion, and an observer was designed to decouple sperm orientation from its wiggling motion. A sliding mode controller was designed to cope with stiffness variances along the sperm tail and among different sperm. Deep neural networks were developed for robust sperm tail detection, and Kalman filter was used to predict tail motion. Experimental results demonstrated that automated sperm manipulation achieved an orientation error of 0.8<inline-formula> <tex-math notation="LaTeX">$^{\circ}$</tex-math> </inline-formula> and operation time of 6.8 s, both significantly less than those of manual operation. The designed observer was effective to reduce sperm orientation error by reducing the disturbance from sperm wiggling motion. The developed sliding mode controller outperformed the PID controller in operation time, reducing the time of oocyte exposure to the ambient environment. <i>Note to Practitioners</i>&#x2014;This work tackled the challenge of rotating a fast-swimming and deformable sperm in clinical cell surgeries. Automated manipulation of deformable objects has wide applications in industrial and service settings such as manipulating wires and folding cloth. However, the intrinsic motion of a motile sperm and the lack of a rotational degree of freedom in standard micromanipulators pose difficulties to automated sperm manipulation. In this paper, we propose automation techniques for sperm orientation control. For sperm tail detection, deep learning was used to handle the variances of shape and length among different sperm. A path planning strategy and a controller were designed to achieve automated rotation of motile sperm, with its deformation and motion both controlled. The developed methods can be generalized to the manipulation of other deformable objects such as wires, cables and cloth. These objects exhibit significant variance of mechanical properties, and calibration is often time-consuming. The designed controller can be used to manipulate deformable objects with robustness to varied mechanical parameters. Path planning was designed by updating the manipulation point based on the object&#x2019;s deformation behavior, and is suitable in manipulation where constraints are imposed such as the object&#x2019;s strain.

Sensitive Current Sensor Based on a Lanthanide Framework with Lewis Basic Bipyridyl Sites for Cu2+ Detection.

A new Yb-based three-dimensional metal-organic framework with free Lewis basic sites, [Yb2(ddbpdc)3(CH3OH)2] (referred to as ACBP-6), from YbCl3 and (6R,8R)-6,8-dimethyl-7,8-dihydro-6H-[1,5]dioxonino[7,6-b:8,9-b']dipyridine-3,11-dicarboxylic acid (H2ddbpdc) was synthesized by a conventional solvothermal method. Two Yb3+ are connected by three carboxyl groups to form the [Yb2(CO2)5] binuclear unit, which is further bridged by two carboxyl moieties to produce a tetranuclear secondary building unit. With further ligation of the ligand ddbpdc2-, a 3-D MOF with helical channels is constructed. In the MOF, Yb3+ only coordinates with O atoms, leaving the bipyridyl N atoms of ddbpdc2- unoccupied. The unsaturated Lewis basic sites make this framework possible to coordinate with other metal ions. After growing the ACBP-6 in situ into a glass micropipette, a novel current sensor is formed. This sensor shows high selectivity and a high signal-to-noise ratio toward Cu2+ detection with a detection limit of 1 μM, due to the stronger coordination ability between the Cu2+ and the bipyridyl N atoms.

Creating the Phenotype of Preeclamptic Endothelial Dysfunction in Placental Vessels

Objective: To determine if placental vessel endothelial dysfunction can be produced in control vessels by exposure to preeclamptic plasma, establishing a model to further explore the mechanisms of dysfunction and therapeutics to restore function in preeclampsia. Hypothesis: Preeclamptic plasma administered to unaffected vessels induces endothelial dysfunction. Methods: Placental microvessels were dissected from the maternal side of placentas from patients consented to the Medical College of Wisconsin Maternal Research Placenta and Cord Blood Bank. Control vessels were treated with 10% healthy maternal plasma or 10% preeclamptic (PreE) maternal plasma within the vessel lumen for 12-16 hours[MJ1]. PreE vessels were not treated with plasma. Microvessels were then cannulated on glass micropipettes, pressurized to 60mmHg and constricted with 0.1-1.0 nM Endothelin-1 (ET-1). A 1-100cm H2O pressure gradient was applied and the inner diameter of the vessel was measured by video microscopy. The change in diameter was calculated as a % of maximum diameter as assessed by exposure to 100μM papaverine. Flow mediated dilation (FMD) was evaluated by a two-way ANOVA test with Dunnett’s multiple comparisons. The maximum dilation values were evaluated by a Kruskal-Wallis test with Dunn’s multiple comparative test or t-test as appropriate. FMD is evaluated as mean +/- SEM. Data: Control vessels at maximum flow dilated to 81.1 % ± 7.1 % (n=5), whereas vessel from PreE placentas dilated to 36.9 % ± 8.6% (n=3,). Incubation of control vessels with normal plasma resulted in maximal dilation of 80.6% ± 3.3%, n=3. Control vessels incubated with plasma from PreE patients dilated to 44.7% ± 4.7%. n=3). Summary of Results: Control vessels dilated significantly more than PreE vessels when evaluated by t-test (p=0.05). Incubation of control vessels with normal plasma did not change maximal dilation (p&gt;0.99). Control vessels incubated with PreE plasma demonstrated a near significant drop in dilatory capacity (p=0.05). PreE plasma exposed vessels compared to untreated PreE vessels showed no significant difference (p=0.46[DDG2] ).When comparing overall flow mediated dilatory responses in these vessels there is a significant difference observed between control vessels, control vessels treated with PreE plasma, and PreE vessels at 50cm H2O (p&lt;0.05). This difference is even more significant (p&lt;0.01) at the maximum pressure gradient when analyzed by two-way ANOVA. There is no significant difference at any point between control vessels and control vessels treated with normal plasma as evaluated by two-way ANOVA (p&gt;0.99). Conclusions: The addition of PreE plasma to control vessels demonstrates similar impairment in endothelium-dependent vasodilation as seen in untreated PreE vessels. Future directions will include evaluation of the mechanisms of vasodilation and therapeutic options that could restore endothelial function in PreE vessels. NHLBI 1K08HL150340-01 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.

Argon Gas Flow Through Micro- and Nano-pipettes

Glass micropipettes and nanopipettes are widely used for injection to or aspiration from living cells, and nanopipettes can be also used for nano-fabrication by deposition. Here, we show a non-destructive and contamination-free method of examining the inside of glass micropipettes and nanopipettes by argon gas flow through the pipettes from the tip-top to the tube-end. The exit pressure for each pipette was measured at the entrance pressure of 50 kPa, and the semi-log plot of the vacuum conductance versus pipette inner diameter is compared with the calculation using tapered tube approximation assuming molecular flow. The experimental results were consistent in trend with the calculation, and the difference between the experimental results and the calculation was due to the intermediate gas flow as the pipette inner diameter increases. Therefore, the formula of the calculation was transformed using the Gompertz function to fit the experimental results well. By utilizing the plot of the inner diameter dependence of the exit pressure, the inner diameter of glass pipettes can be estimated from our developed gas flow method.

Mouse In Vivo Placental Targeted CRISPR Manipulation.

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Single-Cell Quantification of the Mechanical Stability of Cell-Cell Adherens Junction Using Glass Micropipettes.

Micropipette-based methods have been widely used for the manipulation of cells and characterization of the mechanical properties at the cell or tissue level. Here, we introduce the glass micropipette-based mechanical assays for the stability of cell-cell adhesion. A probing microbead coated with specific adhesion ligands, captured by a glass micropipette, is manipulated to form the adhesion complexes with the corresponding receptors on a single cell. Once the cell is moving away from the micropipette, forces are generated from 20 pN to 100 nN to the adhesion complexes, which are quantified in real-time based on the bending of the glass micropipette. We specifically emphasize the principle and method to probe the rupturing forces of the adhesion complexes at controlled force loading rates, the ligand coating on the probe microbeads, the force calibration of the glass micropipette, and the applications of the method to probe the E-cadherin-based cell-cell adhesions. The principles can be broadly applied to other cell adhesions such as cell-matrix adhesions, neuronal synapses, and bacterial-cell adhesions.

Improved Genome Editing via Oviductal Nucleic Acids Delivery (i-GONAD): Protocol Steps and Additional Notes.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice). Such experiments are performed by highly skilled technicians (especially for MI). We recently developed a novelgenome editing method, called "GONAD (Genome-editing via Oviductal Nucleic Acids Delivery)," which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed "improved-GONAD (i-GONAD)." The i-GONAD method involves injection of CRISPR reagents into the oviduct of an anesthetized pregnant female using a mouthpiece-controlled glass micropipette under a dissecting microscope, followed by EP of the entire oviduct allowing the CRISPR reagents to enter into the zygotes present inside the oviduct, in situ. After the i-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. The i-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, the i-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about the i-GONAD method. Additionally, even though the detailed protocols of GONAD and i-GONAD have been published elsewhere (Gurumurthy et al., Curr Protoc Hum Genet 88:15.8.1-15.8.12, 2016 Nat Protoc 14:2452-2482, 2019), we provide all the protocol steps of i-GONAD in this chapter so that the reader can find most of the information, needed for performing i-GONAD experiments, in one place.

Functional Expression of Mechanosensitive Piezo1/TRPV4 Channels in Mouse Osteoblasts.

Mechanical stress is an important regulatory factor in bone homeostasis. Mechanical stimulation of osteoblasts has been shown to elicit an increase in the concentration of intracellular free Ca2+ ([Ca2+]i). The pattern of functional expression of mechanosensitive ion channels remains unclear, however. Therefore, the purpose of this study was to investigate the pharmacological characteristics of [Ca2+]i in response to direct mechanical stimulation in osteoblasts. The morphological expression of mechanosensitive ion channels was also examined. Mouse osteoblast-like cells (MC3T3-E1 cells) were loaded with fura-2-acetoxymethyl ester, after which [Ca2+]i was measured. Increased levels of [Ca2+]i were observed in MC3T3-E1 cells in response to direct mechanical stimulation by means of a glass micropipette, but no desensitization. Application of a hypotonic solution also induced an increase in [Ca2+]i but was accompanied by a desensitizing effect. Extracellular Gd3+, GsMTx4, or RN-1734 reversibly inhibited this mechanical stimulation-induced increase in [Ca2+]i, whereas no inhibitory effect was observed with HC030031 or clemizole. When osteoblasts were stimulated with Yoda1, an increase was observed in [Ca2+]i together with a significant desensitizing effect. Immunoreactivity against Piezo1 and TRPV4 channel antibodies was detected in MC3T3-E1 cells. These results suggest that osteoblasts express Piezo1 and TRPV4 channels, which are involved in mechanosensitive processes during mechanical stress.

Flexible Disk Ultramicroelectrode for High-Resolution and Substrate-Tolerable Scanning Electrochemical Microscopy Imaging.

A simple and universal strategy for fabricating flexible 25 μm platinum (Pt) disk ultramicroelectrodes (UMEs) was proposed, where a pulled borosilicate glass micropipette acted as a mold for shaping the flexible tip with flexible epoxy resin. The whole preparation procedure was highly efficient, enabling 10 or more probes to be manually fabricated within 10 h. Intriguingly, this technique permits an adjustable RG ratio, tip length, and stiffness, which could be tuned according to varying experimental demands. Besides, the electroactive area of the probe could be exposed and made renewable with a thin blade, allowing its reuse in multiple experiments. The flexibility characterization was then employed to optimize the resin/hardener mass ratio of epoxy resin and the tip position during HF etching in the fabrication process, suggesting that more hardener, a larger RG value, or a longer tip length obtained stronger deformation resistance. Subsequently, the as-prepared probe was examined by optical microscopy, cyclic voltammetry, and SECM approach curves. The results demonstrated the probe possessed good geometry with a small RG ratio of less than 3 and exceptional electrochemical properties, and its insulating sheath remained undeformed after blade cutting. Owing to the tip's flexibility, it could be operated in contactless mode with an extremely low working distance and even in contact mode scanning to achieve high spatial resolution and high sensitivity while guaranteeing that the tip and samples would suffer minimal damage if the tip crashed. Finally, the flexible probe was successfully employed in three scanning scenarios where tilted and 3D structured PDMS microchips, a latent fingerprint deposited on the stiff copper sheet, and soft egg white were included. In all, the flexible probe encompasses the advantages of traditional disk UMEs and circumvents their principal drawbacks of tip crash and causing sample scratches, which is thus more compatible with large specimens of 3D structured, stiff, or even soft topography.

Maintaining Single-Channel Recordings on a Silver Nanoneedle through Probe Design and Feedback Tip Positioning Control.

Ion channel proteins showed great promise in the field of nanopore sensing and molecular flux imaging applications due to the atomic-level precision of the pore size and a high signal-to-noise ratio. More specifically, ion channel probes, where the protein channels are integrated at the end of a solid probe, can achieve highly localized detection. Metal probe materials such as gold and silver have been developed to support lipid bilayers and enable the use of smaller probes, or nanoneedles, compared to more traditional glass micropipette ion channel probes. Silver probes are preferable because they support sustained DC stable channel current due to the AgCl layer formed around the tip during the fabrication process. However, one of the current challenges in ion channel measurements is maintaining a single-channel recording. Multiple protein insertions complicate data analysis and destabilize the bilayer. Herein, we combine the promising probe material (Ag/AgCl) with an approach based on current feedback-controlled tip positioning to maintain long-term single-channel recordings for up to 3 h. We develop a hybrid positioning control system, where the channel current is used as feedback to control the vertical movement of the silver tip and, subsequently, control the number of protein channels inserted in the lipid membrane. Our findings reveal that the area of the lipid bilayer decreases with moving the silver tip up (i.e., decreasing the displacement in the z-direction). By reducing the bilayer area around the fine silver tip, we minimize the probability of multiple insertions and remove unwanted proteins. In addition, we characterize the effect of lipid properties such as fluidity on the lipid membrane area. We believe that the use of silver nanoneedles, which enables DC stable channel current, coupled with the developed tip displacement mechanism will offer more opportunities to employ these probes for chemical imaging and mapping different surfaces.

Impact of Goreisan components on rat mesenteric collecting lymphatic vessel pumping.

Goreisan is a traditional herbal formulation with diuretic properties tested as a clinical therapeutic to alleviate lymphedema in Japan. The present study aimed to determine how Goreisan and its five different components affect lymphatic pump function. Mesenteric collecting lymphatics were isolated from anesthetized Sprague-Dawley rats and mounted on resistance-matched glass micropipettes in a 37°C physiological salt solution bath for studies. Diameter was continuously measured to obtain the following lymphatic pump parameters: contraction frequency (CF), end diastolic diameter (EDD), and end systolic diameter (ESD), contraction amplitude (AMP), ejection fraction (EF), and fractional pump flow (FPF). Goreisan and each of its components (Cinnamomi Cortex, Atractylodis Rhizoma, Alismatis Rhizoma, Polyporus, and Poria) were applied to the bath at concentrations of 1-30 μg/mL. The results show that while Goreisan causes no significant changes to lymphatic pumping, Alismatis Rhizoma and Polyporus each significantly reduce CF and FPF. In addition, rats that received oral administration of Goreisan and Alismatis Rhizoma for 1 week had elevated expression of VEGFR-3 in their mesenteric collecting lymphatics. Collectively, the results suggest that some components of Goreisan have a direct, rapid impact on lymphatic pumping. These findings provide new insights but also raise new questions about the therapeutic potential of Goreisan in patients with secondary lymphedema.