Glass Micropipettes Research Articles

Automated End-Effector Alignment in Robotic Micromanipulation

Proper alignment of the end-effector is a critical procedure that determines the success of micromanipulation, such as robotic cell manipulation. Presently, end-effector alignment is performed manually and suffers from large misalignment error and inconsistency. Manual alignment often undesirably moves the end-effector (e.g., a glass micropipette) out of the limited field of view under microscopy and risks breaking the fragile end-effector. This article presents automated end-effector alignment in robotic micromanipulation. A rotational degree of freedom was added to a standard micromanipulator with translational degrees of freedom. The kinematic model of end-effector’s rotation was established, and the unknown model parameters were calibrated. To accommodate model uncertainty and parameter variations, a sliding mode controller was designed to achieve end-effector alignment. Experimental results demonstrate that the robotic alignment technique achieved an accuracy of 0.5 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$\pm 0.3^{\circ }$</tex-math></inline-formula> and a time cost of 17.9 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$\pm$</tex-math></inline-formula> 7.3 s, both significantly less than manual alignment. The developed controller based on kinematic modeling and sliding mode control achieved a higher success rate and significantly less time cost for end-effector alignment than the PID controller. Standard micropipettes were used as the end-effectors for sperm immobilization and oocyte penetration, important procedures in cell surgeries. The success rate of sperm immobilization was 98% by robotic micropipette alignment, higher than the success rate of 90% by manual alignment. Oocyte deformation before penetration was 28.1 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$\pm$</tex-math></inline-formula> 7.5 <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 by robotic end-effector alignment, significantly less than the deformation of 54.5 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$\pm$</tex-math></inline-formula> 13.2 <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 by manual alignment.

Robotic Manipulation of Sperm as a Deformable Linear Object

The robotic manipulation of deformable linear objects is a classic and challenging topic. Apart from synthetic objects, such as wires and cables, linear objects are also commonly found in biological cells and organisms. Biomanipulation of such objects is hampered by difficulties, such as limited degrees of freedom of micromanipulators and varied mechanical properties of the biological entities to manipulate. This article presents a robotic manipulation of human sperm, which are deformable cells with a linear shape. The shape and movement of the cell are recapitulated by our developed geometric and kinematic models. Under unfixed constraints between the end-effector and the cell, path planning is designed to update the manipulation point to control cell deformation. A state transition function is formulated in path planning to handle the stiffness variations of sperm without force sensing. A model-predictive controller is designed to minimize the orientation error and manipulation path length. To detect sperm tail for visual feedback, an accuracy of 98% was achieved via deep neural networks. The robotic manipulation of human sperm was performed using a standard clinical setup of a glass micropipette to rotate a sperm to the target orientation. Experimental results showed that robotic sperm rotation achieved an orientation error of 0.8 <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> , a tail curvedness of 0.14 <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 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$^{-1}$</tex-math></inline-formula> , and an operation time of 5.6 s, all significantly less than those of the manual approach. The less orientation error and tail curvedness after robotic rotation led to a significantly lower speed of sperm entering the micropipette during sperm aspiration, resulting in a higher success rate of 97% (versus 76% after manual rotation) for aspiration control.

Abstract 105: Mitotempol Restores Endothelial Dysfunction In Placental Arterioles In Preeclampsia

Preeclampsia (PreE) is complication of pregnancy, affecting 5% of pregnancies in the United States, and presenting a significant burden to our healthcare systems. Oxidative damage and dysfunction of the placental microcirculation has been implicated. Clinical trials of antioxidant therapies to treat PreE have failed. However, none of these studies have used targeted antioxidants. Given that vascular compromise in preeclampsia may be caused by mitochondrial dysfunction, we propose that MitoTEMPOL (MT), a mitochondrial-targeted superoxide dismutase mimetic, will restore endothelium-dependent vasodilation in human placental arterioles. Arterioles from the maternal portion of the placenta of average diameter 161.1 ± 11.1 μm were cannulated on glass micropipettes and constricted with 0.1-1.5 nM endothelin-1 and flow was generated with a pressure gradient across vessel reservoirs. Flow-mediated dilation (FMD) was measured by video-microscopy presented as % maximal diameter from baseline. Smooth muscle function was evaluated by exposure to 100 μM papaverine. Vessels from normal placentas dilated to flow by 70.2% ± 3.8% (n=11), whereas vessels from PreE placentas dilated to only 47.9% ± 5.6% (n=8, p=0.005). Exposure to 10 μM of MT for 30 minutes in the organ bath restored Pre-E vessel dilation to 80.0% ± 3.9% (n=6, p=0.0003 vs PreE). FMD in normal vessels was not affected by exposure to MT (75.9% ± 7.2%, n=4, p=0.84). Significance was evaluated by 2-way ANOVA, α = 0.05. MT restores endothelium-dependent vasodilation in PreE vessels. Further evaluation of other mitochondrial-targeted antioxidants could lead to novel therapeutic options to improve vascular function in PreE.

Vision-Based 40-nm-Accuracy Liquid Level Detection Compliant With Micromanipulation

Recent efforts in single-cell research call for precise liquid level detection (LLD) for end-effectors to manipulate small volume of liquid samples such as nano- to pico-liter droplets. Existing LLD methods, however, are yet satisfactory for such delicate micromanipulation tasks. This article presents a vision-based contact type LLD method, which adopts the hardware settings normally used in typical micromanipulation scenarios. The detection principle is based on the generation of patterned ripples when the end-effector (e.g., glass micropipette), that is being vibrated, moves to get contact with the liquid surface. Through the microscopic imaging system in micromanipulation, the image frames of patterned ripples such as concentric circles are recorded, from which the wavelength is extracted to determine the contact moment. The vision-based LLD method was cross-validated by the electrical impedance-based method. Experiments revealed that the best detection accuracy was 40 nm, only limited by the micromanipulator's positioning resolution. Moreover, the method was robust to various micromanipulation settings, such as illumination intensity, microscopy magnification, exposure time, and focal plane. As a demonstration, this method was successful in detecting the surface of droplets with a volume as low as 450 pL for different liquids. Given its generality, this method is expected to suit many scenarios involving liquid handling tasks in micromanipulation and industry.

The Role of Angiotensin 1‐7 in Isolated Human Arterioles with SARS‐CoV‐2

IntroductionThe vascular endothelium plays a crucial role to regulate vascular tone. Recently, our laboratory reported severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection significantly reduced endothelial‐dependent vasodilation months after exposed to SARS‐CoV‐2 in human microvessels. SARS‐CoV‐2 enters host cells through angiotensin‐converting enzyme (ACE)2 as a cell surface receptor. Downregulation of ACE2 protein expression by the virus leads to imbalance between angiotensin (ANG) II and ANG 1‐7, causing vasoconstriction, inflammation and increase oxidative stress. With this background, we intend to test our new hypothesis that administration of ANG 1‐7 will improve vasodilation in human arterioles from previously SARS‐CoV‐2 positive subjects.MethodsFresh human tissues were obtained as de‐identified surgical discarded specimens. Control subjects (n=9) were SARS‐CoV‐2 negative and without known virus exposure. We had 11 previously SARS‐CoV‐2 positive subjects or post‐COVID. For all post‐COVID patients, a negative COVID test was obtained prior to collection of surgical specimens. We isolated arterioles (100‐200 µm) and cannulated onto glass micropipettes under 60 mmHg and examined for diameter changes to the endothelial‐dependent vasodilator acetylcholine (ACh; 10−9 to 10−5 M) using videomicroscopy. Flow‐mediated dilation (FMD) was recorded at steady‐state during varying intraluminal pressure gradients (5‐100 cm H2O). All post‐COVID vessels were incubated in a culture media at 37°C for 12–15 hours with or without 1 nM of ANG 1‐7.ResultsDilation to ACh and FMD in arterioles from post‐COVID was significantly reduced (ACh max. dilation at 10−5 M: 74±6% vs. 93±4% in control, n=7‐9, P<0.001; FMD max. dilation at 100 cm H2O: 49±3% vs. 86±2% in control, n=6‐11, P<0.001) while overnight incubation of 1 nM ANG 1‐7 significantly improved the dilation (ACh max. dilation at 10−5 M: 74±6% in post‐COVID vs. 88±5% in post‐COVID + AND 1‐7, n=11, P<0.001; FMD max. dilation at 100 cm H2O: 49±3% in post‐COVID vs. 76±3% in post‐COVID + ANG 1‐7, n=11, P<0.001).ConclusionAdministration of ANG 1‐7 significantly improved vasodilation in isolated arterioles from post‐COVID subjects.

Aging Alters Integrin‐mediated Vascular Smooth Muscle Function in Soleus Feed Arteries

IntroductionPrevious data have shown that aging impairs vasomotor function and alters vascular smooth muscle cell (VSMC) properties. Integrins play an important role in regulating many cellular functions including arterial contractility. However, how aging affects integrin function, and their contribution to impaired constriction in aged arteries has not yet been elucidated. Thus, the purpose of this study was to investigate the age‐induced alteration of integrin function in isolated VSMC and in intact soleus muscle resistance arteries.MethodsSoleus muscle feed arteries (SFA) were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats, cannulated with glass micropipettes and pressurized at 90cm H2O. Endothelial cells were removed (denuded) by passing 5 ml of air through the artery lumen. Vasoconstrictor responses were assessed by adding of norepinephrine (NE; 10‐9‐10‐4M), angiotensin II (Ang II; 10‐11‐10‐7 M), or phenylephrine (PE; 10‐9‐10‐4 M) in the presence or absence of RGD, an integrin inhibitory peptide. RGE, a non‐inhibitory peptide was used as control. To investigate changes in integrin expression and recruitment at focal adhesions with age, RT‐qPCR, cell adhesion assays, and total internal reflection fluorescence (TIRF) imaging were performed using VSMC isolated from young and old SFA.ResultsOld denuded SFA had reduced vasoconstrictor responses to NE, PE, and Ang II. In the presence of the RGD‐inhibitory peptide, constrictor responses to Ang II were significantly inhibited in denuded old vs. young SFA. Constrictor responses to NE and PE were not significantly altered by RGD. Constrictor responses to NE, PE, and Ang II were also not affected by RGE. Gene expression of specific integrins expressed by VSMC were downregulated in old VSMC compared to young. In addition, RGD‐treated VSMC showed decreased adhesion to fibronectin in both young and old VSMC compared to RGE non‐inhibitory peptide. Quantitative analysis of TIRF images showed that recruitment of integrin α5 and β3 was diminished in old VSMC while β1 was not altered with age.ConclusionsTaken together, these data suggest that aging leads to integrin dysfunction, which contributes to decreased contractile properties of vascular smooth muscle in SFA.

Activation of Glutamate Transport Increases Arteriole Diameter in v ivo: Implications for Neurovascular Coupling.

In order to meet the energetic demands of cell-to-cell signaling, increases in local neuronal signaling are matched by a coordinated increase in local blood flow, termed neurovascular coupling. Multiple different signals from neurons, astrocytes, and pericytes contribute to this control of blood flow. Previously, several groups demonstrated that inhibition/ablation of glutamate transporters attenuates the neurovascular response. However, it was not determined if glutamate transporter activation was sufficient to increase blood flow. Here, we used multiphoton imaging to monitor the diameter of fluorescently labeled cortical arterioles in anesthetized C57/B6J mice. We delivered vehicle, glutamate transporter substrates, or a combination of a glutamate transporter substrate with various pharmacologic agents via a glass micropipette while simultaneously visualizing changes in arteriole diameter. We developed a novel image analysis method to automate the measurement of arteriole diameter in these time-lapse analyses. Using this workflow, we first conducted pilot experiments in which we focally applied L-glutamate, D-aspartate, or L-threo-hydroxyaspartate (L-THA) and measured arteriole responses as proof of concept. We subsequently applied the selective glutamate transport substrate L-THA (applied at concentrations that do not activate glutamate receptors). We found that L-THA evoked a significantly larger dilation than that observed with focal saline application. This response was blocked by co-application of the potent glutamate transport inhibitor, L-(2S,3S)-3-[3-[4-(trifluoromethyl)-benzoylamino]benzyloxy]-aspartate (TFB-TBOA). Conversely, we were unable to demonstrate a reduction of this effect through co-application of a cocktail of glutamate and GABA receptor antagonists. These studies provide the first direct evidence that activation of glutamate transport is sufficient to increase arteriole diameter. We explored potential downstream mechanisms mediating this transporter-mediated dilation by using a Ca2+ chelator or inhibitors of reversed-mode Na+/Ca2+ exchange, nitric oxide synthetase, or cyclo-oxygenase. The estimated effects and confidence intervals suggested some form of inhibition for a number of these inhibitors. Limitations to our study design prevented definitive conclusions with respect to these downstream inhibitors; these limitations are discussed along with possible next steps. Understanding the mechanisms that control blood flow are important because changes in blood flow/energy supply are implicated in several neurodegenerative disorders and are used as a surrogate measure of neuronal activity in widely used techniques such as functional magnetic resonance imaging (fMRI).

Automatic Cell Rotation Based on Real-Time Detection and Tracking

Cell rotation has great significance for cell manipulation, which is applied to intracytoplasmic sperm injection, preimplantation genetic screening and diagnosis, somatic cell nuclear transfer, etc. In this letter, an automatic cell rotation method is proposed based on real-time detection and tracking of the polar body. Image segmentation and visual tracking are combined to detect the polar body accurately and robustly during cell rotation. Both out-of-plane and in-plane rotation are realized by utilizing a pair of standard glass micropipettes. In experiments, two kinds of oocytes, which are quite different in appearance, are used to validate the system. For mouse oocytes, the robotic system achieved a 98% success rate of out-of-plane rotation and a 93% success rate of in-plane rotation. For porcine oocytes, the robotic system achieved an 80% success rate of out-of-plane rotation and a 93% success rate of in-plane rotation. The average time of cell rotation is approximately 9.1s for mouse oocytes and 12.8s for porcine oocytes. The experiment results demonstrate that the proposed method is effective, stable and high speed for automatic cell rotation in 3D.

A Carbon-Based Ultramicrothermocouple

Abstract Micropipette-based thermocouples provide the advantage of a high tip diameter-to-length aspect ratio allowing the maintenance of a reference temperature crucial for accurate thermal sensing in microdomains. The research efforts in this field strive to achieve high thermoelectric power (voltage change per unit temperature change) while minimizing the sensing area, a pair of tasks that is by nature contradictory and thus, challenging. Herein, the design and fabrication of a carbon-based micropipette thermal sensor are described. A novel manufacturing method and set of materials are used to overcome the reduction in thermoelectric performance associated with small sensor sizes. A glass micropipette is utilized as a template in a chemical vapor deposition process to form a carbon layer in the lumen of the pipette. This carbon micropipette then serves as a scaffold on which gold and nickel are deposited, enabling the device to function as a thermocouple. This low-cost fabrication process results in a thermocouple with a sub-500 nm tip. The response of the thermocouple was characterized and demonstrated good repeatability in a temperature range of 0 to 60 °C. The unique material selection provided a thermoelectric power of 14.9 μV·K−1, a significant improvement (68%) relative to other micropipette-based thermocouples.

Automated microscope-independent fluorescence-guided micropipette

Glass micropipette electrodes are commonly used to provide high resolution recordings of neurons. Although it is the gold standard for single cell recordings, it is highly dependent on the skill of the electrophysiologist. Here, we demonstrate a method of guiding micropipette electrodes to neurons by collecting fluorescence at the aperture, using an intra-electrode tapered optical fiber. The use of a tapered fiber for excitation and collection of fluorescence at the micropipette tip couples the feedback mechanism directly to the distance between the target and electrode. In this study, intra-electrode tapered optical fibers provide a targeted robotic approach to labeled neurons that is independent of microscopy.

Effects of Aging on Integrin‐Mediated Vascular Smooth Muscle Contractility in Soleus Muscle Feed Arteries

Introduction Vasoconstrictor responses decline with age in soleus muscle feed arteries (SFA). Previous data from our laboratory revealed that alterations in integrin signaling contributed to the impaired constrictor responses. Whether the impaired constrictor responses resulted from integrin signaling in endothelial cells or vascular smooth muscle (VSM) cells is unknown. Thus, the purpose of the study was to test the hypothesis that aging alters VSM contractility due to impaired VSM integrin signaling in SFA. Method Soleus muscle feed arteries (SFA) were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats. SFA were cannulated with glass micropipettes and pressurized to 90 cm H2O for assessment of vasoconstrictor function. To isolate the role of smooth muscle in constrictor responses, endothelial cells were removed (denuded) by passing 5 ml of air through the lumen of the artery. Vasoconstrictor responses were assessed using increasing whole log doses of norepinephrine (NE; 10-9-10-4 M), angiotensin II (Ang II; 10-11-10-7 M), and phenylephrine (PE; 10-9-10-4 M) in the presence or absence of RGD, an integrin inhibitory peptide, and RGE, as control. Results Vasoconstrictor responses to NE, PE, and Ang II were less in old denuded SFA when compared to young SFA. In the presence of RGD, the constrictor response to Ang II (not NE or PE) was significantly reduced in both young and old SFA. RGE did not alter the constrictor responses to any of the agonists. Conclusion The results of this study indicate that VSM contractility declines with age in SFA. In addition, our results suggest that RGD binding integrin-mediated constrictor function declines with age in denuded SFA.

Endogenous Opioid Receptor Activation in the Caudal Medullary Raphe Depresses Respiratory Rate in Decerebrate Rabbits

Background The caudal medullary raphe (CMR) is an essential structure in controlling pain, cardiovascular, and respiratory function (1). Partial reversal of systemic opioid-induced respiratory depression was achieved with naloxone injection into the CMR (2). This study investigated the presence of endogenous opioid activity in the CMR. Methods The study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3-4kg) were anesthetized, tracheotomized, ventilated (FiO2 0.6, PCO2 45-55 mmHg), decerebrated and vagotomized. Phrenic nerve activity was recorded from the c5 rootlet, time averaged and used to calculate inspiratory (TI) and expiratory (TE) duration, breaths per minute (BPM), and peak phrenic activity (PPA). Using a multibarrel glass micropipette, we performed the following injection protocols into the CMR in the midline between 2 mm caudal and 1.5 mm rostral to the functionally identified preBötzinger Complex (preBC, (3)) and into the middle medullary raphe (middle MR (2), 2.5-3.5mm rostral to the preBC) in separate sets of animals: i) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 50 μM, 70 nl), stepsize 0.5mm rostro-caudal, 0.5mm dorso-ventral. ii) The non-selective opioid antagonist naloxone (1-5 mM, 700 nl), stepsize 0.5mm rostro-caudal, 1st injection at the ventral edge of neuronal activity, 2nd injection 1.5-2mm dorsal; and iii) the µ-opioid antagonist CTAP and δ-opioid antagonist naltrindole (each 1 mM, 350-700 nl), stepsize same as ii). Statistical comparison with Wilcoxon signed rank test. Median (25-75% range). Results i) In 6 animals, AMPA injection into the CMR caudal to and at the level of the preBC increased respiratory rate by up to 59% and PPA by up to 38% at multiple dorso-ventral levels. This was due to a decrease in TI and TE. In the middle MR, AMPA injection depressed respiratory rate, at times to apnea, through an increase in TI and TE, and also depressed PPA. ii) Naloxone injection into the CMR increased respiratory rate from 30 (23-34) to 37 (30-6) BPM (p=0.031, n=6) through a decrease in TI from 0.9 (0.8-1.0) to 0.7 (0.6-0.8) sec (p=0.031) and TE from 1.2 (1.0-1.6) to 0.9 (0.7-1.2) sec (p=0.031). We observed effects with injections caudal to, at the level of and rostral to the preBC. iii) CTAP injection increased respiratory rate from 26 (22-27) to 34 (29-34) BPM, and subsequent naltrindole injection increased respiratory rate to 43 (37-44) BPM (n=3). In the middle MR, CTAP + naltrindole increased respiratory rate between 2-5 BPM. Conclusion Excitation of the CMR increased respiratory rate over an area of several millimeters. During quiet breathing under moderate hyperoxic hypercapnia in our decerebrate rabbit preparation, respiratory raphe neurons were depressed by endogenous opioid activity. It will be important to further investigate the role of the CMR in opioid-induced respiratory depression. (1) Holtman et al., J Neurosci 1986, 6: 1185-93 (2) Zhang et al., Anesthesiology 2007, 107: 288-97 (3) Cook-Snyder et al., Resp Physiol Neurobiol 2019, 260: 37-52

PVN ‐ projecting MnPO neurons are involved in blood pressure and sympathetic nerve activity regulation in CIH hypertension

Sleep apnea (SA) is characterized by repeated interruptions in breathing resulting in hypoxia, hypercapnia, and increased thoracic pressure. Chronic intermittent hypoxia (CIH) in rats mimics the repetitive hypoxemias that occur with SA leading to a sustained increase in blood pressure associated with increased sympathetic activity and chemoreceptor sensitivity. CIH increases the peripheral renin-angiotensin system (RAS) activating the subfornical organ (SFO) which in turn projects to the hypothalamic paraventricular nucleus (PVN) and the median preoptic nucleus (MnPO). Angiotensin receptor knockdown in SFO or MnPO neurons and virally –mediated caspase lesions PVN-projecting MnPO neurons prevents CIH hypertension. These results suggest that PVN-projecting MnPO neurons play a significant role in blood pressure regulation in CIH hypertension. However, the contribution of MnPO to increased sympathetic nerve activity in CIH hypertension has not been directly tested. Our hypothesis is that the chronic activation of PVN by SFO via MnPO contributes to increased sympathetic outflow sustaining CIH hypertension through the diurnal cycle. In order to selectively inhibit PVN-projecting MnPO neurons, a retrograde Cre-dependent virus was microinjected bilaterally into PVN (AAV9.hSyn.HI.eGFP-Cre.WPRE.SV40) and a Cre-dependent inhibitory DREADD (PAAV.Syn.DIO.hM4D(Gi)-mCherry) was injected into MnPO. Two weeks later, rats were submitted to a 7-day CIH protocol (3 min of 10% oxygen followed by 3 min of 21% oxygen, 8 h/day) or normoxia. Rats were anesthetized (urethane 800 mg/kg and α-chloralose 80 mg/kg ip) and the trachea was cannulated for artificial ventilation. A femoral vein catheter (PE-50 tubing) was implanted for continuous infusion of gallamine triethiodide (20 mg/mL, 0.25 mL/h iv) for neuromuscular blockade. Blood pressure was measured by a Millar catheter system in the femoral artery. The rats were placed in a stereotaxic frame and a craniotomy was performed for intracerebroventricular (ICV) injections. Renal and splanchnic nerves were isolated and placed in a bipolar electrode. Noise levels were determined after a bolus injection of hexamethonium (30 mg/kg iv). Using a pneumatic picopump (WPI) connected to a single-barreled glass micropipette, DREADD agonist CNO (1µg/1µl) was injected ICV and responses were collected after 30 min of the injection. In rats exposed to normoxia, chemogenetic inhibition of PVN-projecting MnPO neurons slightly increased splanchnic SNA (4.9±21, n=3), renal SNA (17± 17, n=3) and increased blood pressure (Normoxia: 4.3±4, n=3; CIH; -0.3± 4, n=3). In rats exposed to CIH, CNO reduced splanchnic SNA (-42.9±37, n=4) and renal SNA (-88±49, n=3). These preliminary results suggest that CIH triggers a neuroadaptation that changes the relationship between the SFO-MnPO-PVN pathway and SNA.

Elevation of Intra-Cellular Calcium in Nucleus Pulposus Cells with Micro-Pipette-Guided Ultrasound

Modulation of intra-cellular calcium by ultrasound offers a possible means for therapeutic applications. One such possibility is the modulation of nucleus pulposus cells as a preventive measure for inter-vertebral disc degeneration. We report a cellular stimulation device (micro-pipette ultrasound) using a glass micro-pipette as a waveguide to deliver ultrasound through the pipette tip and to elevate intra-cellular calcium in nucleus pulposus cells. The device generates two relevant stimuli at the cellular level: ultrasound propagation throughout the cell and acoustic streaming on the apical side. Ultrasound is radiated from a tip of a few microns, and its amplitude is proportional to the input voltage; acoustic streaming can be controlled by the duty factor. The novelty of the device is to impose a unique cellular loading: shear stress on cell apical surfaces combined with compressional waves propagating through the cells. G protein–coupled receptors and acid-sensing ion channel 3 were shown to play a role in calcium elevation by micro-pipette ultrasound in nucleus pulposus cells. Our results demonstrate that micro-pipette ultrasound can be an effective tool to elevate intra-cellular calcium levels in different cells, facilitating the identification of different mechanoreceptors in action.

Intracytoplasmic sperm injection versus conventional IVF

Intracytoplasmic sperm injection is a widely used fertilisation method that involves injecting a single spermatozoon into oocyte cytoplasm using a glass micropipette.1 Although it was introduced as a modification of conventional in-vitro fertilisation (IVF) to overcome severe male factor infertility,2 intracytoplasmic sperm injection has become the most common laboratory technique in assisted reproductive technology, accounting for almost 70% of the around 2·8 million annual assisted reproductive technology cycles, according to global estimates.

An integrated set-up for ex vivo characterisation of biaxial murine artery biomechanics under pulsatile conditions

Ex vivo characterisation of arterial biomechanics enables detailed discrimination of the various cellular and extracellular contributions to arterial stiffness. However, ex vivo biomechanical studies are commonly performed under quasi-static conditions, whereas dynamic biomechanical behaviour (as relevant in vivo) may differ substantially. Hence, we aim to (1) develop an integrated set-up for quasi-static and dynamic biaxial biomechanical testing, (2) quantify set-up reproducibility, and (3) illustrate the differences in measured arterial stiffness between quasi-static and dynamic conditions. Twenty-two mouse carotid arteries were mounted between glass micropipettes and kept fully vasodilated. While recording pressure, axial force (F), and inner diameter, arteries were exposed to (1) quasi-static pressure inflation from 0 to 200 mmHg; (2) 300 bpm dynamic pressure inflation (peaking at 80/120/160 mmHg); and (3) axial stretch (λz) variation at constant pressures of 10/60/100/140/200 mmHg. Measurements were performed in duplicate. Single-point pulse wave velocities (PWV; Bramwell-Hill) and axial stiffness coefficients (cax = dF/dλz) were calculated at the in vivo value of λz. Within-subject coefficients of variation were ~ 20%. Dynamic PWVs were consistently higher than quasi-static PWVs (p < 0.001); cax increased with increasing pressure. We demonstrated the feasibility of ex vivo biomechanical characterisation of biaxially-loaded murine carotid arteries under pulsatile conditions, and quantified reproducibility allowing for well-powered future study design.

Gold nanoparticles produce transient reactive gliosis in the adult brain

Gold nanoparticles (GNPs) have unique physical and chemical properties that allow them to function as a drug-delivery system for several tissues: skin, eye, liver, and others. However, information about the biological response of brain tissue against GNPs is limited. Astrocytes and microglia cells are the first line of defense against brain insults and proper indicators of the level of brain damage. This study was aimed to evaluate the astrocytic and microglia response after an intracerebral injection of polyethylene-glycol-coupled GNPs (PEGylated GNPs). We injected spherical PEGylated GNPs (85 × 106 nanoparticles /nl) with a glass micropipette (inner diameter =35 μm) into the striatum of P60 CD1 mice. We evaluated the cellular response of astrocytes and microglia on days 3, 7, 14, 30, and 90 after intracerebral injection. For both astrocytes and microglia cells, our findings indicated that the glial response was transient and mainly circumscribed to the injection site. This evidence suggests that PEGylated GNPs are well-tolerated by the neural tissue. Understanding the effects of GNPs in the adult brain is a crucial step to design proper pharmacological vehicles to deliver long-lasting drugs.

Fabrication of Glass Micropipette Device Using Reflow Processes and Its Integration With Microfluidic Channels for Patch Clamp Recording of Cell

This paper presents a borosilicate glass micropipette device that has a high-aspect-ratio pore for patch clamp recording applications. The device was fabricated by carrying out glass reflow processes on a silicon substrate. The shape of the micropipette was determined by the width and thickness of the reflowed glass structure, which matches well with the rounded tip of a conventional glass pipette. The diameter of the micropipette pore was first defined by deep reactive ion etching (DRIE) of a silicon pillar, which was positioned at the center of the glass structure. Then, the silicon pillar was removed, and the pore diameter was additionally reduced to the submicron scale by depositing a parylene-C film. The fabricated device was integrated with microfluidic channels for patch clamp recording experiments: microfluidic PDMS layers were assembled on the top and bottom surfaces of the micropipette chip for the medium exchange and the application of negative pressure, respectively. The quality of seal between a cerebral cortex cell of a rat and the glass micropipette was characterized by the amplitude of the current produced by an applied voltage pulse. A negative pressure applied through the bottom microfluidic channel pulled down the cell and increased the seal resistance from 9.82 $\text{M}\Omega $ to 2.06 $\text{G}\Omega $ . This indicates that the gigaseal was successfully formed around the micropipette pore. In addition, the action potentials of the patched cortical neuron were recorded using the fabricated device.

Eukaryotic initiation factor 4 gamma 2 contributes to neuropathic pain through down-regulation of Kv1.2 and the mu opioid receptor in mouse primary sensory neurones

BackgroundNerve injury-induced changes in gene expression in the dorsal root ganglion (DRG) contribute to neuropathic pain genesis. Eukaryotic initiation factor 4 gamma 2 (eIF4G2) is a general repressor of cap-dependent mRNA translation. Whether DRG eIF4G2 participates in nerve injury-induced alternations in gene expression and nociceptive hypersensitivity is unknown. MethodsThe expression and distribution of eIF4G2 mRNA and protein in mouse DRG after spinal nerve ligation (SNL) were assessed. Effects of eIF4G2 siRNA microinjected through a glass micropipette into the injured DRG on the SNL-induced DRG mu opioid receptor (MOR) and Kv1.2 downregulation and nociceptive hypersensitivity were examined. In addition, effects of DRG microinjection of adeno-associated virus 5-expressing eIF4G2 (AAV5-eIF4G2) on basal DRG MOR and Kv1.2 expression and nociceptive thresholds were analysed. ResultseIF4G2 protein co-expressed with Kv1.2 and MOR in DRG neurones. Levels of eIF4G2 mRNA (1.7 [0.24] to 2.3 [0.14]-fold of sham, P<0.01) and protein (1.6 [0.14] to 2.5 [0.22]-fold of sham, P<0.01) in injured DRG were time-dependently increased on days 3–14 after SNL. Blocking increased eIF4G2 through microinjection of eIF4G2 siRNA into the injured DRG attenuated SNL-induced downregulation of DRG MOR and Kv1.2 and development and maintenance of nociceptive hypersensitivities. DRG microinjection of AAV5-eIF4G2 reduced DRG MOR and Kv1.2 expression and elicited hypersensitivities to mechanical, heat and cold stimuli in naïve mice. ConclusionseIF4G2 contributes to neuropathic pain through participation in downregulation of Kv1.2 and MOR in injured DRG and is a potential target for treatment of this disorder.

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 °C and positioned in a stereotactic frame with a cervical bend of about 30°. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 µL of blood was injected in the cisterna magna followed by a 30° downward tilt of the animal for 2 minutes. The second infusion of 30 µL of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.