Measurement of postmortem outflow facility using iPerfusion.

The key risk factor for glaucoma is elevation of intraocular pressure (IOP) and alleviating it is the only effective therapeutic approach to inhibit further vision loss. IOP is regulated by the flow of aqueous humour across resistive tissues, and a reduction in outflow facility, is responsible for the IOP elevation in glaucoma. Measurement of outflow facility is therefore important when investigating the pathophysiology of glaucoma and testing candidate treatments for lowering IOP. Due to similar anatomy and response to pharmacological treatments, mouse eyes are a common model of human aqueous humour dynamics. The ex vivo preparation, in which an enucleated mouse eye is mounted in a temperature controlled bath and cannulated, has been well characterised and is widely used. The postmortem in situ model, in which the eyes are perfused within the cadaver, has received relatively little attention. In this study, we investigate the postmortem in situ model using the iPerfusion system, with a particular focus on i) the presence or absence of pressure-independent flow, ii) the effect of evaporation on measured flow rates and iii) the magnitude and pressure dependence of outflow facility and how these properties are affected by postmortem changes. Measurements immediately after cannulation and following multi-pressure facility measurement demonstrated negligible pressure-independent flow in postmortem eyes, in contrast to assumptions made in previous studies. Using a humidity chamber, we investigated whether the humidity of the surrounding air would influence measured flow rates. We found that at room levels of humidity, evaporation of saline droplets on the eye resulted in artefactual flow rates with a magnitude comparable to outflow, which were eliminated by a high relative humidity (>85%) environment. Average postmortem outflow facility was ∼4 nl/min/mmHg, similar to values observed ex vivo, irrespective of whether a postmortem delay was introduced prior to cannulation. The intra-animal variability of measured outflow facility values was also reduced relative to previous ex vivo data. The pressure-dependence of outflow facility was reduced in the postmortem relative to ex vivo model, and practically eliminated when eyes were cannulated >40 min after euthanisation. Overall, our results indicate that the moderately increased technical complexity associated with postmortem perfusion provides reduced variability and reduced pressure-dependence in outflow facility, when experimental conditions are properly controlled.

The ocular pulse decreases aqueous humor outflow resistance by stimulating nitric oxide production.

Intraocular pressure (IOP) is not static, but rather oscillates by 2-3 mmHg because of cardiac pulsations in ocular blood volume known as the ocular pulse. The ocular pulse induces pulsatile shear stress in Schlemm's canal (SC). We hypothesize that the ocular pulse modulates outflow facility by stimulating shear-induced nitric oxide (NO) production by SC cells. We confirmed that living mice exhibit an ocular pulse with a peak-to-peak (pk-pk) amplitude of 0.5 mmHg under anesthesia. Using iPerfusion, we measured outflow facility (flow/pressure) during alternating periods of steady or pulsatile IOP in both eyes of 16 cadaveric C57BL/6J mice (13-14 weeks). Eyes were retained in situ, with an applied mean pressure of 8 mmHg and 1.0 mmHg pk-pk pressure amplitude at 10 Hz to mimic the murine heart rate. One eye of each cadaver was perfused with 100 µM L-NAME to inhibit NO synthase, whereas the contralateral eye was perfused with vehicle. During the pulsatile period in the vehicle-treated eye, outflow facility increased by 16 [12, 20] % (P < 0.001) relative to the facility measured during the preceding and subsequent steady periods. This effect was partly inhibited by L-NAME, where pressure pulsations increased outflow facility by 8% [4, 12] (P < 0.001). Thus, the ocular pulse causes an immediate increase in outflow facility in mice, with roughly one-half of the facility increase attributable to NO production. These studies reveal a dynamic component to outflow function that responds instantly to the ocular pulse and may be important for outflow regulation and IOP homeostasis.

A Novel Ventilator Design for COVID-19 and Resource-Limited Settings.

There has existed a severe ventilator deficit in much of the world for many years, due in part to the high cost and complexity of traditional ICU ventilators. This was highlighted and exacerbated by the emergence of the COVID-19 pandemic, during which the increase in ventilator production rapidly overran the global supply chains for components. In response, we propose a new approach to ventilator design that meets the performance requirements for COVID-19 patients, while using components that minimise interference with the existing ventilator supply chains. The majority of current ventilator designs use proportional valves and flow sensors, which remain in short supply over a year into the pandemic. In the proposed design, the core components are on-off valves. Unlike proportional valves, on-off valves are widely available, but accurate control of ventilation using on-off valves is not straightforward. Our proposed solution combines four on-off valves, a two-litre reservoir, an oxygen sensor and two pressure sensors. Benchtop testing of a prototype was performed with a commercially available flow analyser and test lungs. We investigated the accuracy and precision of the prototype using both compressed gas supplies and a portable oxygen concentrator, and demonstrated the long-term durability over 15 days. The precision and accuracy of ventilation parameters were within the ranges specified in international guidelines in all tests. A numerical model of the system was developed and validated against experimental data. The model was used to determine usable ranges of valve flow coefficients to increase supply chain flexibility. This new design provides the performance necessary for the majority of patients that require ventilation. Applications include COVID-19 as well as pneumonia, influenza, and tuberculosis, which remain major causes of mortality in low and middle income countries. The robustness, energy efficiency, ease of maintenance, price and availability of on-off valves are all advantageous over proportional valves. As a result, the proposed ventilator design will cost significantly less to manufacture and maintain than current market designs and has the potential to increase global ventilator availability.

Infusion Mechanisms in Brain White Matter and Their Dependence on Microstructure: An Experimental Study of Hydraulic Permeability.

OBJECTIVE: Hydraulic permeability is a topic of deep interest in biological materials because of its important role in a range of drug delivery-based therapies. The strong dependence of permeability on the geometry and topology of pore structure and the lack of detailed knowledge of these parameters in the case of brain tissue makes the study more challenging. Although theoretical models have been developed for hydraulic permeability, there is limited consensus on the validity of existing experimental evidence to complement these models. In the present study, we measure the permeability of white matter (WM) of fresh ovine brain tissue considering the localised heterogeneities in the medium using an infusion-based experimental set up, iPerfusion. We measure the flow across different parts of the WM in response to applied pressures for a sample of specific dimensions and calculate the permeability from directly measured parameters. Furthermore, we directly probe the effect of anisotropy of the tissue on permeability by considering the directionality of tissue on the obtained values. Additionally, we investigate whether WM hydraulic permeability changes with post-mortem time. To our knowledge, this is the first report of experimental measurements of the localised WM permeability, also demonstrating the effect of axon directionality on permeability. This work provides a significant contribution to the successful development of intra-tumoural infusion-based technologies, such as convection-enhanced delivery (CED), which are based on the delivery of drugs directly by injection under positive pressure into the brain.

The vital role for nitric oxide in intraocular pressure homeostasis.

Catalyzed by endothelial nitric oxide (NO) synthase (eNOS) activity, NO is a gaseous signaling molecule maintaining endothelial and cardiovascular homeostasis. Principally, NO regulates the contractility of vascular smooth muscle cells and permeability of endothelial cells in response to either biochemical or biomechanical cues. In the conventional outflow pathway of the eye, the smooth muscle-like trabecular meshwork (TM) cells and Schlemm's canal (SC) endothelium control aqueous humor outflow resistance, and therefore intraocular pressure (IOP). The mechanisms by which outflow resistance is regulated are complicated, but NO appears to be a key player as enhancement or inhibition of NO signaling dramatically affects outflow function; and polymorphisms in NOS3, the gene that encodes eNOS modifies the relation between various environmental exposures and glaucoma. Based upon a comprehensive review of past foundational studies, we present a model whereby NO controls a feedback signaling loop in the conventional outflow pathway that is sensitive to changes in IOP and its oscillations. Thus, upon IOP elevation, the outflow pathway tissues distend, and the SC lumen narrows resulting in increased SC endothelial shear stress and stretch. In response, SC cells upregulate the production of NO, relaxing neighboring TM cells and increasing permeability of SC's inner wall. These IOP-dependent changes in the outflow pathway tissues reduce the resistance to aqueous humor drainage and lower IOP, which, in turn, diminishes the biomechanical signaling on SC. Similar to cardiovascular pathogenesis, dysregulation of the eNOS/NO system leads to dysfunctional outflow regulation and ocular hypertension, eventually resulting in primary open-angle glaucoma.

The Effect of High-Intensity Focused Ultrasound on Aqueous Humor Dynamics in Patients with Glaucoma.

PURPOSE: To investigate the effects of high-intensity focused ultrasound (HiFU) on aqueous humor dynamics in patients with glaucoma. DESIGN: Comparative, nonrandomized, interventional study. PARTICIPANTS: Adult patients with a diagnosis of open-angle glaucoma or ocular hypertension with suboptimal intraocular pressure (IOP) control despite maximum medical treatment who required further IOP optimization. METHODS: All patients underwent comprehensive ophthalmic examination before aqueous humor dynamics study measurements, including fluorophotometry and digital Schiøtz tonography. All patients received 6 seconds of HiFU therapy. Aqueous humor dynamics studies were repeated 3 months after the treatment (patients had 4-week washout from their glaucoma medication before their aqueous humor dynamics study measurements at baseline and the 3-month visit). MAIN OUTCOME MEASURES: Intraocular pressure, facility of topographic outflow, aqueous flow rate, and uveoscleral outflow. RESULTS: Thirty eyes of 30 patients were included in the study. At the 3-month postoperative visit, the mean postwashout IOP was reduced by 16% (31.7±5.3 vs. 26.6±4.8 mmHg, P = 0.004), and aqueous flow rate was decreased by 15% (2.07±0.73 vs. 1.77±0.55 µl/min, P = 0.05) from baseline. Neither the tonographic outflow facility nor the uveoscleral outflow was significantly different from baseline. There is a 20% risk of treatment failure (those who needed further glaucoma surgical intervention) within 1 month after a single HiFU treatment (n = 6). Only 25 patients (80%) were able to undergo post-treatment washout measurements, and in these eyes, only 26.6% of eyes achieved >20% IOP reduction at 3 months compared with baseline. CONCLUSIONS: We investigated the aqueous humor dynamics effects of a cyclodestructive procedure and specifically HiFU in patients with uncontrolled open-angle glaucoma on maximum tolerated medical therapy. High-intensity focused ultrasound reduced IOP 3 months postoperatively by 16% and aqueous flow decreased by 15% without any significant effect on tonographic outflow facility and uveoscleral outflow.

Aqueous Humor Dynamics in Uveitic Eyes.

PURPOSE: To investigate aqueous humor dynamics in uveitic eyes. DESIGN: Cross-sectional study. PARTICIPANTS: Patients with recurrent (≥3 attacks) anterior uveitis (now quiescent) and being treated for glaucoma or ocular hypertension (OHT) (Group 1), previous recurrent anterior uveitis (≥3 attacks) without glaucoma or OHT (Group 2), and normal subjects with no ocular problems and IOP < 21 mm Hg at screening (control group; Group 3). METHODS: Patients had one-off measurements. Group 1 patients who were on antihypertensives were washed out for a 4-week period, prior to their study measurements. Main outcome measures were tonographic outflow facility, aqueous humor flow rate, and uveoscleral outflow. RESULTS: One hundred and one patients were screened between February 2014 and February 2017. Nine patients did not meet the inclusion criteria. Groups 1 and 3 each included 30 patients, and Group 2 included 32 patients. The mean intraocular pressure was higher in Group 1 compared to the others (25 ± 10.2 mm Hg in Group 1 vs 16 ± 2.7 mm Hg in Group 2 vs 16 ± 2.2 mm Hg in Group 3, P < .001). The tonographic outflow facility was lower in Group 1 compared to the others (0.18 ± 0.1 µL/min/mm Hg in Group 1 vs 0.25 ± 0.1 µL/min/mm Hg in Group 2 vs 0.27 ± 0.1 µL/min/mm Hg in Group 3, P = .005). However, aqueous humor flow rate was not statistically different (2.47 ± 0.9 µL/min in Group 1 vs 2.13 ± 0.9 µL/min in Group 2 vs 2.25 ± 0.7 µL/min in Group 3, P = .3). There was also no significant difference in calculated uveoscleral outflow. CONCLUSION: This is the first aqueous humor dynamics study in patients with uveitic glaucoma/OHT and recurrent anterior uveitis compared with age-matched controls. We have demonstrated that the elevated intraocular pressure seen in the uveitic glaucoma/OHT eyes (3-6 attacks) was due to reduced tonographic outflow facility. The aqueous humor flow rate was not detectibly different, nor did the calculated uveoscleral outflow demonstrate any discernible difference. However, the exact mechanism remains to be elucidated.

Direct measurement of pressure-independent aqueous humour flow using iPerfusion.

Reduction of intraocular pressure is the sole therapeutic target for glaucoma. Intraocular pressure is determined by the dynamics of aqueous humour secretion and outflow, which comprise several pressure-dependent and pressure-independent mechanisms. Accurately quantifying the components of aqueous humour dynamics is essential in understanding the pathology of glaucoma and the development of new treatments. To better characterise aqueous humour dynamics, we propose a method to directly measure pressure-independent aqueous humour flow. Using the iPerfusion system, we directly measure the flow into the eye when the pressure drop across the pressure-dependent pathways is eliminated. Using this approach we address i) the magnitude of pressure-independent flow in ex vivo eyes, ii) whether we can accurately measure an artificially imposed pressure-independent flow, and iii) whether the presence of a pressure-independent flow affects our ability to measure outflow facility. These studies are conducted in mice, which are a common animal model for aqueous humour dynamics. In eyes perfused with a single cannula, the average pressure-independent flow was 1 [-3, 5] nl/min (mean [95% confidence interval]) (N = 6). Paired ex vivo eyes were then cannulated with two needles, connecting the eye to both iPerfusion and a syringe pump, which was used to impose a known pressure-independent flow of 120 nl/min into the experimental eye only. The measured pressure-independent flow was then 121 [117, 125] nl/min (N = 7), indicating that the method could measure pressure-independent flow with high accuracy. Finally, we showed that the artificially imposed pressure-independent flow did not affect our ability to measure facility, provided that the pressure-dependence of facility and the true pressure-independent flow were accounted for. The present study provides a robust method for measurement of pressure-independent flow, and demonstrates the importance of accurately quantifying this parameter when investigating pressure-dependent flow or outflow facility.