Brachial artery blood flow by vascular ultrasound in education.

There is extensive and increasing use of ultrasound in medical care and scientific research, so it is important that the technique, indication, and interpretation of ultrasound investigations are included in medical and biological education. Applications of ultrasound in medical care and education employ not only noninvasive imaging of structure but also the evaluation of organ function. Vascular ultrasound is one such application that has been hitherto relatively neglected in physiology education. The techniques of vascular ultrasound and the physiological regulation of human limb blood flow are reviewed to inform students and curriculum designers. Emphasis is placed on the value of converting velocity measurement by ultrasound to volumetric flow and on the mechanisms involved in rapidly changing flows with interventions. Live collection of real data by ultrasound can show macrovascular and microvascular features of vascular physiology. Macrovascular features include imaging and flow velocity profiles. Microvascular perfusion studies show conductance changes with interventions such as exercise and ischemia. Vascular ultrasound offers exciting opportunities for undergraduate research projects using human subjects. The literature is interesting and, though complex, offers excellent educational experience, with scope for the development of critical thinking and meaningful original research.NEW & NOTEWORTHY Ultrasound imaging has emergent prominence in clinical investigation and education. Vascular ultrasound also evaluates function. Simple methods are described that enable the application of basic ultrasound principles to the measurement of velocity and, importantly, to calculate absolute volumetric blood flow. These methods should be useful in undergraduate and graduate education, with application in clinical practice and research.

Ultrasound imaging in teaching cardiovascular physiology: disruption and challenge to foster learning.

This work extends previously described applications of ultrasound technology in illustrating cardiovascular phenomena to investigation of learning effectiveness. To this end, learning in ultrasound-enhanced classes was assessed by quantifying the improvement in single best answer (SBA) exams conducted before and after an ultrasound practical class. This improvement was then compared to that seen in the same SBA exams conducted in matched groups that undertook similar classes conducted without ultrasound equipment. The SBA exams were designed to include questions that directly related to the phenomena being investigated in the practical classes as well as other "filler" questions to disguise the intent of the exam and ensure that standards of physiology knowledge were similar between the two groups. Any small statistically significant gains in performance observed between the two groups were confounded by differences in baseline (pretest) performance between the groups. These results contradict our previous work, which showed that self-reported measures of learning increased after ultrasound classes. It may be optimistic to expect improvements in deep learning and test performance immediately after even the most effective educational intervention. Direct measurement of the phenomena that bring about deep, long-term learning in classes remains problematic. Notwithstanding this, there is much evidence for the value of enhancing physiology learning by providing varying contexts in the physical, semantic, and cognitive domains. Ultrasound technology is a cheap and effective means of providing such context in physiology practical classes.NEW & NOTEWORTHY This work compares learning in cardiovascular physiology classes enhanced by using cheap ultrasound equipment with learning in comparable control classes without ultrasound. Performance improvement in single best answer tests between pre- and postclass tests were compared for the ultrasound and control classes, with little difference shown between the two classes. We question whether it is appropriate to measure deep learning after 2-h classes or whether other, phenomenological, qualitative measures of educational effectiveness would be better.

Snapshot of ultrasound imaging in basic anatomy and physiology teaching in the United Kingdom and Republic of Ireland: perceptions, obstacles, and solutions.

We have used ultrasound imaging and technology as a tool for nonclinical teaching of basic physiological concepts for several years and are aware anecdotally that only a few others in the United Kingdom and Republic of Ireland (UK/ROI) are also using ultrasound with this intention in physiology and anatomy teaching. To better understand what areas ultrasound is used for by others, along with what barriers might exist to its use, we reached out to colleagues in UK/ROI institutions instructing on anatomy and physiology courses by asking them to complete a survey regarding their experiences. Relatively few institutions (9%) reported using the technology in this way but covered physiology and anatomy teaching in most major body systems. The perception of responding educators overall is that, overwhelmingly, ultrasound offers a useful addition to the teaching of physiology and anatomy and is very popular with students. Barriers to its implementation were identified, including unfamiliarity with equipment and potential uses. Lack of funding for equipment and staff, issues with class sizes, and lack of curriculum time were also identified. Despite these potential impediments, most nonusers were interested in finding out about the uses of ultrasound as a teaching tool. We conclude that the teaching community would benefit from wider dissemination of local practices.NEW & NOTEWORTHY We surveyed UK and Republic of Ireland institutions to establish the extent of ultrasound use in teaching undergraduate modules with significant anatomy or physiology content. Responses indicate that although ultrasound is used for a wide variety of systems, only a small proportion of courses use ultrasound for teaching. There is widespread interest in its use, with the main barriers being unfamiliarity with potential uses and the technology. We endorse further dissemination of this teaching practice.

Using two-dimensional ultrasound imaging to examine venous pressure.

Ultrasound imaging is being used increasingly to aid in the teaching of human physiology and anatomy. Here we describe how its use can be integrated into the teaching of concepts surrounding venous circulation, specifically 1) venous valves and the muscle pump, 2) the effects of hydrostatic pressure on venous pressure, and 3) central venous pressure. The imaging procedures described are relatively simple but add a dimension that helps deliver the teaching points clearly and is enjoyable for students. They also aid in the link of basic physiology to clinical aspects of venous circulatory physiology.

Increased SOX9 Expression in Premalignant and Malignant Pancreatic Neoplasms.

BACKGROUND: SOX9, a progenitor cell marker, is important for pancreatic ductal development. Our goal was to examine SOX9 expression differences in intraductal papillary mucinous neoplasms (IPMNs) and ductal adenocarcinoma (PDAC) compared with benign pancreatic duct (BP). METHODS: SOX9 expression was evaluated by immunohistochemistry performed on 93 specimens: 37 BP, 24 low grade (LG) IPMN, 12 high grade (HG) IPMN, and 20 PDAC. A linear mixed-effects model was used to compare the percentage of cells expressing SOX9 by specimen type. A separate linear mixed-effects model evaluated differences in SOX9 expression by staining intensity in pancreatic epithelial cells. RESULTS: Nuclear SOX9 expression was detected in the epithelial cells of 98% HG IPMN, 93% LG IPMN, 83% PDAC, and 60% BP. Compared with BP, SOX9 was expressed from a significantly greater percentage of cells in LG IMPN, HG IMPN, and PDAC (p < 0.001 for each). BP and PDAC showed greater variability in SOX9 expression in epithelial cells compared with IPMNs which showed strong, homogenous SOX9 expression in almost all cells. Compared with BP, both LG and HG IPMN showed significantly greater SOX9 expression (p < 0.001 for each), but there was no significant difference in SOX9 expression between LG and HG IPMN (p > 0.05). PDAC had significantly higher expression of SOX9 compared with BP but significantly lower SOX9 expression compared with LG or HG IPMN (p < 0.001 for each). CONCLUSIONS: IPMNs demonstrated the highest expression levels of SOX9. SOX9 expression in BP and PDAC demonstrated much more heterogeneity compared with the strong, uniform expression in IPMN.

Muscle hypotrophy, not inhibition, is responsible for quadriceps weakness during rehabilitation after anterior cruciate ligament reconstruction.

PURPOSE: Quadriceps weakness is common after anterior cruciate ligament reconstruction (ACLR). Limited neuromuscular activation may have a role in the weakness. The purpose of this study was to use peripheral magnetic stimulation to measure changes in quadriceps inhibition in patients during rehabilitation from ACLR. METHODS: Ten patients (7M/3F; age 35 ± 8 years; BMI 26.0 ± 4.8 kg/m2) who had ACLR with patellar tendon autograft were recruited. At 3 and 6 months postoperatively, patients' knee extension peak torque was measured during maximum voluntary isometric contraction (MVIC), magnetic stimulation-evoked contraction, and MVIC augmented with superimposed burst magnetic stimulation to the femoral nerve. All tests were done bilaterally at 30° and 65° of knee flexion on a dynamometer. Central activation ratio was calculated by dividing the peak torque before stimulation by peak torque after stimulation. RESULTS: Patients had marked deficits in MVIC, with improvement from 3 to 6 months that was more apparent at 65° versus 30° (P < 0.05). There was significant deficit in stimulation-evoked torque on the involved side that diminished over time, and this change occurred differently between the two angles (P < 0.05). Central activation ratio was lower on the involved side versus the noninvolved side and this effect was more prominent at 3 versus 6 months: combining the angles, mean central activation ratio on the involved and noninvolved sides, respectively, was 91.4 ± 7.6% and 97.5 ± 5.3% at 3 months, and 93.0 ± 7.8% and 95.8 ± 6.8% at 6 months. CONCLUSIONS: At 3 and 6 months after ACLR, there were significant deficits in quadriceps strength and activation. Quadriceps activation levels were high (> 90%) for both sides at both time points. The substantial strength deficits at this postoperative period may be largely due to muscle atrophy with limited contribution from central inhibition. Rehabilitation interventions to normalize quadriceps strength should emphasize hypertrophic stimuli as opposed to neuromuscular activation strategies. LEVEL OF EVIDENCE: II, prospective cohort study.

Understanding basic vein physiology and venous blood pressure through simple physical assessments.

An understanding of the complexity of the cardiovascular system is incomplete without a knowledge of the venous system. It is important for students to understand that, in a closed system, like the circulatory system, changes to the venous side of the circulation have a knock-on effect on heart function and the arterial system and vice versa. Veins are capacitance vessels feeding blood to the right side of the heart. Changes in venous compliance have large effects on the volume of blood entering the heart and hence cardiac output by the Frank-Starling Law. In healthy steady-state conditions, venous return has to equal cardiac output, i.e., the heart cannot pump more blood than is delivered to it. A sound understanding of the venous system is essential in understanding how changes in cardiac output occur with changes in right atrial pressure or central venous pressure, and the effect these changes have on arterial blood pressure regulation. The aim of this paper is to detail simple hands-on physiological assessments that can be easily undertaken in the practical laboratory setting and that illustrate some key functions of veins. Specifically, we illustrate that venous valves prevent the backflow of blood, that venous blood pressure increases from the heart to the feet, that the skeletal muscle pump facilitates venous return, and we investigate the physiological and clinical significance of central venous pressure and how it may be assessed.

Reducing low birth weight: prioritizing action to address modifiable risk factors.

Background: Low birth weight (LBW) affects 6.9% of all UK births and has remained largely unchanged for many years. The United Nations and the World Health Assembly have set targets to substantially reduce global incidence. Understanding the contribution of modifiable risk factors to the burden of LBW is required to ensure appropriate interventions are in place to achieve this reduction. Methods: Data from published studies on the risks from key modifiable factors were used alongside prevalence data from the Welsh population to calculate the population attributable risk for each factor individually and in combination. Results: Fourteen risk factors accounted for nearly half of LBW births, and 60% of those to younger mothers (<25 years). Tobacco smoke exposure was the largest contributor. We estimated that smoking in pregnancy was a factor in one in eight LBW births, increasing to one in five for women aged under 25. Conclusions: Risk factors are interrelated and inequitably distributed within the population. Exposure to one factor increases the likelihood of exposure to a constellation of factors further increasing risk. Action to address LBW must consider groups where the risk factors are most prevalent and address these risk factors together using multi-component interventions.

Ion channel mechanisms of rat tail artery contraction-relaxation by menthol involving, respectively, TRPM8 activation and L-type Ca2+ channel inhibition.

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold-sensing role in sensory neurons, it is expressed and functional in several nonneuronal tissues, including vasculature. We previously demonstrated that menthol causes variable mechanical responses (vasoconstriction, vasodilatation, or biphasic reactions) in isolated arteries, depending on vascular tone. Here we aimed to dissect the specific ion channel mechanisms and corresponding Ca2+ signaling pathways underlying such complex responses to menthol and other TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology, confocal Ca2+ imaging, and ratiometric Ca2+ recording. Menthol (300 µM, a concentration typically used to induce TRPM8 currents) strongly inhibited L-type Ca2+ channel current (L-ICa) in isolated myocytes, especially its sustained component, most relevant for depolarization-induced vasoconstriction. In contraction studies, with nifedipine present (10 µM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol, similar to resting (i.e., without α-adrenoceptor stimulation by PE) conditions, when L-type channels were mostly deactivated. Menthol-induced increases in PE-induced vasoconstrictions could be inhibited both by the TRPM8 antagonist AMTB (thus confirming the specific role of TRPM8) and by cyclopiazonic acid treatment to deplete Ca2+ stores, pointing to a major contribution of Ca2+ release from the sarcoplasmic reticulum in these contractile responses. Immunocytochemical analysis has indeed revealed colocalization of TRPM8 and InsP3 receptors. Moreover, menthol Ca2+ responses, which were somewhat reduced under Ca2+-free conditions, were strongly reduced by cyclopiazonic acid treatment to deplete Ca2+ store, whereas caffeine-induced Ca2+ responses were blunted in the presence of menthol. Finally, two other common TRPM8 agonists, WS-12 and icilin, also inhibited L-ICa With respect to L-ICa inhibition, WS-12 is the most selective agonist. It augmented PE-induced contractions, whereas any secondary phase of vasorelaxation (as with menthol) was completely lacking. Thus TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels and largely obscure TRPM8-mediated vasoconstriction. These findings will promote our understanding of the vascular TRPM8 role, especially the well-known hypotensive effect of menthol, and may also have certain translational implications (e.g., in cardiovascular surgery, organ storage, transplantation, and Raynaud's phenomenon).

Electrospun Fibers for Drug Delivery after Spinal Cord Injury and the Effects of Drug Incorporation on Fiber Properties.

There is currently no cure for individuals with spinal cord injury (SCI). While many promising approaches are being tested in clinical trials, the complexity of SCI limits several of these approaches from aiding complete functional recovery. Several different categories of biomaterials are investigated for their ability to guide axonal regeneration, to deliver proteins or small molecules locally, or to improve the viability of transplanted stem cells. The purpose of this study is to provide a brief overview of SCI, present the different categories of biomaterial scaffolds that direct and guide axonal regeneration, and then focus specifically on electrospun fiber guidance scaffolds. Much like other polymer guidance approaches, electrospun fibers can retain and deliver therapeutic drugs. The experimental section presents new data showing the incorporation of two therapeutic drugs into electrospun poly-L-lactic acid fibers. Two different concentrations of either riluzole or neurotrophin-3 were loaded into the electrospun fibers to examine the effect of drug concentration on the physical characteristics of the fibers (fiber alignment and fiber diameter). Overall, the drugs were successfully incorporated into the fibers and the release was related to the loading concentration. The fiber diameter decreased with the inclusion of the drug, and the decreased diameter was correlated with a decrease in fiber alignment. Subsequently, the study includes considerations for successful incorporation of a therapeutic drug without changing the physical properties of the fibers.

Ultrasound imaging in teaching cardiac physiology.

This laboratory session provides hands-on experience for students to visualize the beating human heart with ultrasound imaging. Simple views are obtained from which students can directly measure important cardiac dimensions in systole and diastole. This allows students to derive, from first principles, important measures of cardiac function, such as stroke volume, ejection fraction, and cardiac output. By repeating the measurements from a subject after a brief exercise period, an increase in stroke volume and ejection fraction are easily demonstrable, potentially with or without an increase in left ventricular end-diastolic volume (which indicates preload). Thus, factors that affect cardiac performance can readily be discussed. This activity may be performed as a practical demonstration and visualized using an overhead projector or networked computers, concentrating on using the ultrasound images to teach basic physiological principles. This has proved to be highly popular with students, who reported a significant improvement in their understanding of Frank-Starling's law of the heart with ultrasound imaging.

Recent advances in thermoregulation.

Thermoregulation is the maintenance of a relatively constant core body temperature. Humans normally maintain a body temperature at 37°C, and maintenance of this relatively high temperature is critical to human survival. This concept is so important that control of thermoregulation is often the principal example cited when teaching physiological homeostasis. A basic understanding of the processes underpinning temperature regulation is necessary for all undergraduate students studying biology and biology-related disciplines, and a thorough understanding is necessary for those students in clinical training. Our aim in this review is to broadly present the thermoregulatory process taking into account current advances in this area. First, we summarize the basic concepts of thermoregulation and subsequently assess the physiological responses to heat and cold stress, including vasodilation and vasoconstriction, sweating, nonshivering thermogenesis, piloerection, shivering, and altered behavior. Current research is presented concerning the body's detection of thermal challenge, peripheral and central thermoregulatory control mechanisms, including brown adipose tissue in adult humans and temperature transduction by the relatively recently discovered transient receptor potential channels. Finally, we present an updated understanding of the neuroanatomic circuitry supporting thermoregulation.

Investigation of physiological properties of nerves and muscles using electromyography.

The measurement and representation of the electrical activity of muscles [electromyography (EMG)] have a long history from the Victorian Era until today. Currently, EMG has uses both as a research tool, in noninvasively recording muscle activation, and clinically in the diagnosis and assessment of nerve and muscle disease and injury as well as in assessing the recovery of neuromuscular function after nerve damage. In the present report, we describe the use of a basic EMG setup in our teaching laboratories to demonstrate some of these current applications. Our practical also illustrates some fundamental physiological and structural properties of nerves and muscles. Learning activities include 1) displaying the recruitment of muscle fibers with increasing force development; 2) the measurement of conduction velocity of motor nerves; 3) the assessment of reflex delay and demonstration of Jendrassik's maneuver; and 4) a Hoffman reflex experiment that illustrates the composition of mixed nerves and the differential excitability thresholds of fibers within the same nerve, thus aiding an understanding of the reflex nature of muscle control. We can set up the classes at various levels of inquiry depending on the needs/professional requirements of the class. The results can then provide an ideal platform for a discovery learning session/tutorial on how the central nervous system controls muscles, giving insights on how supraspinal control interacts with reflexes to give smooth, precise muscular activation.

Investigating autonomic control of the cardiovascular system: a battery of simple tests.

Sympathetic and parasympathetic divisions of the autonomic nervous system constantly control the heart (sympathetic and parasympathetic divisions) and blood vessels (predominantly the sympathetic division) to maintain appropriate blood pressure and organ blood flow over sometimes widely varying conditions. This can be adversely affected by pathological conditions that can damage one or both branches of autonomic control. The set of teaching laboratory activities outlined here uses various interventions, namely, 1) the heart rate response to deep breathing, 2) the heart rate response to a Valsalva maneuver, 3) the heart rate response to standing, and 4) the blood pressure response to standing, that cause fairly predictable disturbances in cardiovascular parameters in normal circumstances, which serve to demonstrate the dynamic control of the cardiovascular system by autonomic nerves. These tests are also used clinically to help investigate potential damage to this control.

The role of neural tension in stretch-induced strength loss.

The purpose of this study was to determine if neural tension during passive stretching affected subsequent strength loss. Eleven healthy subjects (10 men, 1 woman; age 34 ± 12 years) performed maximal isometric hamstring contractions at 100°, 80°, 60°, and 40° knee flexion before and after five 1-minute hamstring stretches performed in either a spinal neutral position or a neural tension position. One leg was stretched in the neutral position and the other in the neural tension position. Hamstring electromyography (EMG) activity was recorded during all contractions and stretches. Passive resistance to stretch was reduced by 11% after stretching (p < 0.01; no difference between neutral or neural tension stretches p = 0.41). Stretch-induced strength loss was apparent after neural tension stretches (12%, p < 0.01) but not after neutral stretches (5%, p = 0.09). There was a rightward shift in the angle-torque curve after neutral stretches (strength loss on ascending limb, strength gain on descending limb, p < 0.01). This effect was not apparent after neural tension stretches (p = 0.43). Stretching did not affect EMG activity during isometric contractions (<2% decline p = 0.58; no difference between neutral and neural tension, p = 0.86). Hamstring stretching with the spine in a neutral position did not result in a significant strength loss but shifted the length-tension relationship such that strength was decreased at short muscle lengths and increased at long muscle lengths. Hamstring stretching with increased neural tension resulted in strength loss with no associated shift in the length-tension relationship.

Impact of Peripheral Nerve Block Technique on Incidence of Phrenic Nerve Palsy in Shoulder Surgery.

Peripheral nerve blocks are an increasingly common method of providing postoperative analgesia for shoulder surgeries. However, the standard technique, the interscalene block (ISB), inevitably causes hemidiaphragmatic paresis (HDP), secondary to phrenic nerve palsy. This can cause morbidity in patients with preexisting respiratory compromise, prompting investigation into alternative "phrenic-sparing" nerve blocks. The aim of this review was to give an overview of these blocks and critically evaluate the current literature to determine if any are suitable replacements for ISB. The incidence of HDP and analgesic efficacy were considered. We queried four electronic databases and one register. Twenty-eight original articles were selected for review. The use of ultrasound guidance, lower volumes of local anaesthetic (LA), and injection 4 mm outside the brachial plexus fascia reduced HDP incidence for the ISB; however, no single modification did so sufficiently. While the anterior suprascapular nerve block (SSNB) showed comparable analgesic effects to the ISB, HDP prevalence was also high. The posterior SSNB produced consistently low HDP incidences but also inferior analgesia to ISB, except when combined with an infraclavicular brachial plexus block. The superior trunk block (STB) provided equivalent analgesia to the ISB while reducing HDP incidence, but not significantly. Lower LA volumes consistently led to lower HDP incidence across all blocks, likely due to a reduced ability to spread to the phrenic nerve. Further investigation into the minimum effective volumes of the extrafascial ISB, anterior SSNB, STB, and combined posterior SSNB with infraclavicular block is warranted to determine if any of these blocks can successfully balance HDP prevention with analgesic efficacy.

Ultrasound skills teaching in UK medical education: A systematic review.

BACKGROUND: Sonography is increasingly integrated into medical curricula to prepare students for clinical practice. In 2022, we conducted a systematic review to explore the degree to which ultrasound skill acquisition is incorporated into undergraduate medial education in the United Kingdom. METHODS: A search of Medline and Embase databases from 2003 to 2022 identified 15 relevant articles. Studies were included if they described ultrasound skills training in UK undergraduate medical education. FINDINGS: A range of teaching methods were reported including didactic demonstrations, hands-on experience and combinations thereof. Portable machines were more common than cart-based machines, and most demonstrators were ultrasound-trained clinicians. Ultrasound teaching is well received, with improvements in confidence using ultrasound, motivation to learn anatomy and retention of knowledge. DISCUSSION: Obstacles to integration were noted including training, cost, curriculum time constraints and the issue of incidental pathology. One study demonstrated that anatomists with appropriate training could provide ultrasound teaching, reducing the need for clinicians or sonographers. Costs may be reduced by renting machines or purchasing portable/hand-held devices. Allowing access to machines during student's free time may address scheduling difficulties. A final recommendation is to pre-scan volunteers prior to the teaching session. CONCLUSION: We have outlined approaches to ultrasound skills teaching and the inherent hurdles to this, as well as potential solutions. This may aid educators wishing to augment their curricula. Although there are relatively few studies from the United Kingdom, there is consensus that students enjoy the incorporation of ultrasound practice and believe it complements existing teaching, especially in a small group setting.

Bidirectional TRP/L Type Ca2+ Channel/RyR/BKCa Molecular and Functional Signaloplex in Vascular Smooth Muscles.

TRP channels are expressed both in vascular myocytes and endothelial cells, but knowledge of their operational mechanisms in vascular tissue is particularly limited. Here, we show for the first time the biphasic contractile reaction with relaxation followed by a contraction in response to TRPV4 agonist, GSK1016790A, in a rat pulmonary artery preconstricted with phenylephrine. Similar responses were observed both with and without endothelium, and these were abolished by the TRPV4 selective blocker, HC067047, confirming the specific role of TRPV4 in vascular myocytes. Using selective blockers of BKCa and L-type voltage-gated Ca2+ channels (CaL), we found that the relaxation phase was inducted by BKCa activation generating STOCs, while subsequent slowly developing TRPV4-mediated depolarisation activated CaL, producing the second contraction phase. These results are compared to TRPM8 activation using menthol in rat tail artery. Activation of both types of TRP channels produces highly similar changes in membrane potential, namely slow depolarisation with concurrent brief hyperpolarisations due to STOCs. We thus propose a general concept of bidirectional TRP-CaL-RyR-BKCa molecular and functional signaloplex in vascular smooth muscles. Accordingly, both TRPV4 and TRPM8 channels enhance local Ca2+ signals producing STOCs via TRP-RyR-BKCa coupling while simultaneously globally engaging BKCa and CaL channels by altering membrane potential.

Feeder-Cell-Free and Serum-Free Expansion of Natural Killer Cells Using Cloudz Microspheres, G-Rex6M, and Human Platelet Lysate.

The versatility of natural killer cells has ignited growing interest in their therapeutic use for cancer and other immunotherapy treatments. However, NK cells compose a small portion of peripheral blood mononuclear cells (5%-20% of PBMCs) and clinical doses require billions of cells. Manufacturing suitable doses of NK cells remains a major challenge for NK immunotherapy. The current standard for expanding NK cells relies on feeder cells and fetal bovine serum to achieve large expansion, but both encounter regulatory concerns. We developed NK Cloudz, a dissolvable polymer-based microsphere platform, as an alternative to a feeder cell approach to expand NK cells. We demonstrated that a combination of NK Cloudz, a G-Rex6M culture vessel, and GMP Human Platelet Lysate expanded NK cells 387 ± 100-fold in 10 days from a PBMC starting population. The NK purity, viability, and cytotoxicity were similar to both a feeder cell protocol and an FBS-based protocol. Additionally, we found no significant differences between FBS and GMP Human Platelet Lysate and concluded that platelet lysate is a good xeno-free alternative to FBS for NK expansion. Overall, we demonstrated a feeder-cell-free and FBS-free protocol that leverages NK Cloudz as a promising step toward a commercial GMP manufacturing method to expand NK cells for therapeutic use.

Changes in muscle sympathetic nerve activity and vascular responses evoked in the spinotrapezius muscle of the rat by systemic hypoxia.

Responses evoked in muscle sympathetic nerve activity (MSNA) by systemic hypoxia have received relatively little attention. Moreover, MSNA is generally identified from firing characteristics in fibres supplying whole limbs: their actual destination is not determined. We aimed to address these limitations by using a novel preparation of spinotrapezius muscle in anaesthetised rats. By using focal recording electrodes, multi-unit and discriminated single unit activity were recorded from the surface of arterial vessels.This had cardiac- and respiratory-related activities expected of MSNA, and was increased by baroreceptor unloading, decreased by baroreceptor stimulation and abolished by autonomic ganglion blockade. Progressive, graded hypoxia (breathing sequentially 12, 10, 8% O2 for 2min each) evoked graded increases in MSNA.In single units, mean firing frequency increased from 0.2±0.04 in 21% O2 to 0.62 ± 0.14 Hz in8% O2, while instantaneous frequencies ranged from 0.04­6Hz in 21% O2 to 0.09­20 Hz in 8%O2. Concomitantly, arterial pressure (ABP), fell and heart rate (HR) and respiratory frequency(RF) increased progressively, while spinotrapezius vascular resistance (SVR) decreased (Spinotrapezius blood flow/ABP), indicating muscle vasodilatation. During 8% O2 for 10 min, the falls in ABP and SVR were maintained, but RF, HR and MSNA waned towards baselines from the second to the tenth minute. Thus, we directly show that MSNA increases during systemic hypoxia to an extent that is mainly determined by the increases in peripheral chemoreceptor stimulation and respiratory drive, but its vasoconstrictor effects on muscle vasculature are largely blunted by local dilator influences, despite high instantaneous frequencies in single fibres.