Imperial College London

ProfessorDarrylOverby

Faculty of EngineeringDepartment of Bioengineering

Professor of Mechanobiology
 
 
 
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Contact

 

+44 (0)20 7594 6376d.overby

 
 
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Location

 

3.07Bessemer BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

150 results found

Reina-Torres E, De Ieso ML, Pasquale LR, Madekurozwa M, Van Batenburg-Sherwood J, Overby DR, Stamer WDet al., 2021, The vital role for nitric oxide in intraocular pressure homeostasis, PROGRESS IN RETINAL AND EYE RESEARCH, Vol: 83, ISSN: 1350-9462

Journal article

Jamal A, Mongelli M, Vidotto M, Madekurozwa M, Bernardini A, Overby D, De Momi E, Rodriguez y Baena F, Sherwood J, Dini Det al., 2021, Infusion mechanisms in brain white matter and its dependence of microstructure: an experimental study of hydraulic permeability, IEEE Transactions on Biomedical Engineering, Vol: 68, Pages: 1229-1237, ISSN: 0018-9294

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, showing 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.

Journal article

Madekurozwa M, Stamer WD, Reina-Torres E, Sherwood JM, Overby DRet al., 2021, The ocular pulse decreases aqueous humor outflow resistance by stimulating nitric oxide production., Am J Physiol Cell Physiol, Vol: 320, Pages: C652-C665

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.

Journal article

Cassidy PS, Kelly RA, Reina-Torres E, Sherwood JM, Humphries MM, Kiang A-S, Farrar GJ, OBrien C, Campbell M, Stamer WD, Overby DR, Humphries P, OCallaghan Jet al., 2021, siRNA targeting Schlemm’s canal endothelial tight junctions enhances outflow facility and reduces IOP in a steroid-induced OHT rodent model, Molecular Therapy - Methods & Clinical Development, Vol: 20, Pages: 86-94, ISSN: 2329-0501

Systemic or localized application of glucocorticoids (GCs) can lead to iatrogenic ocular hypertension, which is a leading cause of secondary open-angle glaucoma and visual impairment. Previous work has shown that dexamethasone increases zonula occludens-1 (ZO-1) protein expression in trabecular meshwork (TM) cells, and that an antisense oligonucleotide inhibitor of ZO-1 can abolish the dexamethasone-induced increase in trans-endothelial flow resistance in cultured Schlemm’s canal (SC) endothelial and TM cells. We have previously shown that intracameral inoculation of small interfering RNA (siRNA) targeting SC endothelial cell tight junction components, ZO-1 and tricellulin, increases aqueous humor outflow facility ex vivo in normotensive mice by reversibly opening SC endothelial paracellular pores. In this study, we show that targeted siRNA downregulation of these SC endothelial tight junctions reduces intraocular pressure (IOP) in vivo, with a concomitant increase in conventional outflow facility in a well-characterized chronic steroid-induced mouse model of ocular hypertension, thus representing a potential focused clinical application for this therapy in a sight-threatening scenario.

Journal article

Zhu W, Hou F, Fang J, Fard MRB, Liu Y, Ren S, Wu S, Qi Y, Sui S, Read AT, Sherwood JM, Zou W, Yu H, Zhang J, Overby DR, Wang N, Ethier CR, Wang Ket al., 2021, The role of Piezo1 in conventional aqueous humor outflow dynamics, ISCIENCE, Vol: 24

Journal article

Boazak EM, King R, Wang J, Chu CM, Toporek AM, Sherwood JM, Overby DR, Geisert EE, Ethier CRet al., 2021, Smarce1 and Tensin 4 are putative modulators of corneoscleral stiffness, Frontiers in Bioengineering and Biotechnology, Vol: 9, Pages: 1-13, ISSN: 2296-4185

The biomechanical properties of the cornea and sclera are important in the onset and progression of multiple ocular pathologies and vary substantially between individuals, yet the source of this variation remains unknown. Here we identify genes putatively regulating corneoscleral biomechanical tissue properties by conducting high-fidelity ocular compliance measurements across the BXD recombinant inbred mouse set and performing quantitative trait analysis. We find seven cis-eQTLs and non-synonymous SNPs associating with ocular compliance, and show by RT-qPCR and immunolabeling that only two of the candidate genes, Smarce1 and Tns4, showed significant expression in corneal and scleral tissues. Both have mechanistic potential to influence the development and/or regulation of tissue material properties. This work motivates further study of Smarce1 and Tns4 for their role(s) in ocular pathology involving the corneoscleral envelope as well as the development of novel mouse models of ocular pathophysiology, such as myopia and glaucoma.

Journal article

Bertrand JA, Woodward DF, Sherwood JM, Spenlehauer A, Silvestri C, Piscitelli F, Marzo VD, Yamazaki M, Sakimura K, Inoue Y, Watanabe K, Overby DRet al., 2021, Deletion of the gene encoding prostamide/prostaglandin F synthase reveals an important role in regulating intraocular pressure, Prostaglandins, Leukotrienes and Essential Fatty Acids, Vol: 165, Pages: 102235-102235, ISSN: 0952-3278

Prostamide/prostaglandin F synthase (PM/PGFS) is an enzyme with very narrow substrate specificity and is dedicated to the biosynthesis of prostamide F2α and prostaglandin F2α (PGF2α.). The importance of this enzyme, relative to the aldo-keto reductase (AKR) series, in providing functional tissue prostamide F2α levels was determined by creating a line of PM/PGFS gene deleted mice. Deletion of the gene encoding PM/PGFS (Fam213b / Prxl2b) was accomplished by a two exon disruption. Prostamide F2α levels in wild type (WT) and PM/PGFS knock-out (KO) mice were determined by LC/MS/MS. Deletion of Fam213b (Prxl2b) had no observed effect on behavior, appetite, or fertility. In contrast, tonometrically measured intraocular pressure was significantly elevated by approximately 4 mmHg in PM/PGFS KO mice compared to littermate WT mice. Outflow facility was measured in enucleated mouse eyes using the iPerfusion system. No effect on pressure dependent outflow facility occurred, which is consistent with the effects of prostamide F2α and PGF2α increasing outflow through the unconventional pathway. The elevation of intraocular pressure caused by deletion of the gene encoding the PM/PGFS enzyme likely results from a diversion of the endoperoxide precursor pathway to provide increased levels of those prostanoids known to raise intraocular pressure, namely prostaglandin D2 (PGD2) and thromboxane A2 (TxA2). It follows that PM/PGFS may serve an important regulatory role in the eye by providing PGF2α and prostamide F2α to constrain the influence of those prostanoids that raise intraocular pressure.

Journal article

Bertrand JA, Woodward DF, Sherwood JM, Wang JW, Overby DRet al., 2020, The role of EP2 receptors in mediating the ultra-long-lasting intraocular pressure reduction by JV-GL1., Br J Ophthalmol

BACKGROUND: A single application of JV-GL1 substantially lowers non-human primate intraocular pressure (IOP) for about a week, independent of dose. This highly protracted effect does not correlate with its ocular biodisposition or correlate with the once-daily dosing regimen for other prostanoid EP2 receptor agonists such as trapenepag or omidenepag. The underlying pharmacological mechanism for the multiday extended activity of JV-GL1 is highly intriguing. The present studies were intended to determine EP2 receptor involvement in mediating the long-term ocular hypotensive activity of JV-GL1 by using mice genetically deficient in EP2 receptors. METHODS: The protracted IOP reduction produced by JV-GL1 was investigated in C57BL/6J and EP2 receptor knock-out mice (B6.129-Ptger2tm1Brey /J; EP2KO). Both ocular normotensive and steroid-induced ocular hypertensive (SI-OHT) mice were studied. IOP was measured tonometrically under general anaesthesia. Aqueous humour outflow facility was measured ex vivo using iPerfusion in normotensive C57BL/6J mouse eyes perfused with 100 nM de-esterified JV-GL1 and in SI-OHT C57BL/6J mouse eyes that had received topical JV-GL1 (0.01%) 3 days prior. RESULTS: Both the initial 1-day and the protracted multiday effects of JV-GL1 in the SI-OHT model for glaucoma were abolished by deletion of the gene encoding the EP2 receptor. Thus, JV-GL1 did not lower IOP in SI-OHT EP2KO mice, but in littermate SI-OHT EP2WT control mice, JV-GL1 statistically significantly lowered IOP for 4-6 days. CONCLUSIONS: Both the 1-day and the long-term effects of JV-GL1 on IOP are entirely EP2 receptor dependent.

Journal article

Reina-Torres E, Boussommier-Calleja A, Sherwood JM, Overby DRet al., 2020, Aqueous humor outflow requires active cellular metabolism in mice., Investigative Ophthalmology and Visual Science, Vol: 61, Pages: 45-45, ISSN: 0146-0404

Purpose: Conventional wisdom posits that aqueous humor leaves the eye by passive bulk flow without involving energy-dependent processes. However, recent studies have shown that active processes, such as cell contractility, contribute to outflow regulation. Here, we examine whether inhibiting cellular metabolism affects outflow facility in mice. Methods: We measured outflow facility in paired enucleated eyes from C57BL/6J mice using iPerfusion. We had three Experimental Sets: ES1, perfused at 35°C versus 22°C; ES2, perfused with metabolic inhibitors versus vehicle at 35°C; and ES3, perfused at 35°C versus 22°C in the presence of metabolic inhibitors. Inhibitors targeted glycolysis and oxidative phosphorylation (2-deoxy-D-glucose, 3PO and sodium azide). We also measured adenosine triphosphate (ATP) levels in separate murine anterior segments treated like ES1 and ES2. Results: Reducing temperature decreased facility by 63% [38%, 78%] (mean [95% confidence interval (CI)], n = 10 pairs; P = 0.002) in ES1 after correcting for changes in viscosity. Metabolic inhibitors reduced facility by 21% [9%, 31%] (n = 9, P = 0.006) in ES2. In the presence of inhibitors, temperature reduction decreased facility by 44% [29%, 56%] (n = 8, P < 0.001) in ES3. Metabolic inhibitors reduced anterior segment adenosine triphosphate (ATP) levels by 90% [83%, 97%] (n = 5, P<0.001), but reducing temperature did not affect ATP. Conclusions: Inhibiting cellular metabolism decreases outflow facility within minutes. This implies that outflow is not entirely passive, but depends partly on energy-dependent cellular processes, at least in mice. This study also suggests that there is a yet unidentified mechanism, which is strongly temperature-dependent but metabolism-independent, that is necessary for nearly half of normal outflow function in mice.

Journal article

Reina-Torres E, Overby D, 2020, DYNAMIC CHANGES IN SEGMENTAL OUTFLOW ARE LOST WITH AGE IN MICE, Annual Meeting of the Association-for-Research-in-Vision-and-Ophthalmology (ARVO), Publisher: ASSOC RESEARCH VISION OPHTHALMOLOGY INC, ISSN: 0146-0404

Conference paper

McDonnell F, Schmitt H, Huang A, Sherwood J, Overby D, Stamer Det al., 2020, Visualization of Vasoregulator Effects on Distal Outflow Vessels in Human Anterior Segments, Annual Meeting of the Association-for-Research-in-Vision-and-Ophthalmology (ARVO), Publisher: ASSOC RESEARCH VISION OPHTHALMOLOGY INC, ISSN: 0146-0404

Conference paper

Feola AJ, Sherwood JM, Pardue MT, Overby DR, Ethier CRet al., 2020, Age and menopause effects on ocular compliance and aqueous outflow., Investigative Ophthalmology and Visual Science, Vol: 61, Pages: 1-7, ISSN: 0146-0404

Purpose: Glaucoma is the second leading cause of blindness worldwide. Recent work suggests that estrogen and the timing of menopause play a role in modulating the risk of developing glaucoma. Menopause is known to cause modest changes in intraocular pressure; yet, whether this change is mediated through the outflow pathway remains unknown. Menopause also affects tissue biomechanical properties throughout the body; however, the impact of menopause on ocular biomechanical properties is not well characterized. Methods: Here, we simultaneously assessed the impact of menopause on aqueous outflow facility and ocular compliance, as a measure of corneoscleral shell biomechanics. We used young (3-4 months old) and middle-aged (9-10 months old) Brown Norway rats. Menopause was induced by ovariectomy (OVX), and control animals underwent sham surgery, resulting in the following groups: young sham (n = 5), young OVX (n = 6), middle-aged sham (n = 5), and middle-aged OVX (n = 5). Eight weeks postoperatively, we measured outflow facility and ocular compliance. Results: Menopause resulted in a 34% decrease in outflow facility and a 19% increase in ocular compliance (P = 0.011) in OVX animals compared with sham controls (P = 0.019). Conclusions: These observations reveal that menopause affects several key physiological factors known to be associated with glaucoma, suggesting that menopause may contribute to an increased risk of glaucoma in women.

Journal article

McDonnell F, Perkumas KM, Ashpole NE, Kalnitsky J, Sherwood JM, Overby DR, Stamer WDet al., 2020, Shear stress in Schlemm's canal as a sensor of intraocular pressure, Scientific Reports, Vol: 10, ISSN: 2045-2322

Elevated intraocular pressure (IOP) narrows Schlemm's canal (SC), theoretically increasing luminal shear stress. Using engineered adenoviruses containing a functional fragment of the shear-responsive endothelial nitric oxide synthase (eNOS) promoter, we tested effects of shear stress and elevated flow rate on reporter expression in vitro and ex vivo. Cultured human umbilical vein endothelial cells (HUVECs) and SC cells were transduced with adenovirus containing eNOS promoter driving secreted alkaline phosphatase (SEAP) or green fluorescent protein (GFP) and subjected to shear stress. In parallel, human anterior segments were perfused under controlled flow. After delivering adenoviruses to the SC lumen by retroperfusion, the flow rate in one anterior segment of pair was increased to double pressure. In response to high shear stress, HUVECs and SC cells expressed more SEAP and GFP than control. Similarly, human anterior segments perfused at higher flow rates released significantly more nitrites and SEAP into perfusion effluent, and SC cells expressed increased GFP near collector channel ostia compared to control. These data establish that engineered adenoviruses have the capacity to quantify and localize shear stress experienced by endothelial cells. This is the first in situ demonstration of shear-mediated SC mechanobiology as a key IOP-sensing mechanism necessary for IOP homeostasis.

Journal article

Bertrand JA, Schicht M, Stamer WD, Baker D, Sherwood JM, Luetjen-Drecoll E, Selwood DL, Overby DRet al., 2020, The beta(4)-subunit of the large-conductance potassium ion channel K(Ca)1.1 regulates outflow facility in mice, Investigative Ophthalmology and Visual Science, Vol: 61, ISSN: 0146-0404

Purpose: The large-conductance calcium-activated potassium channel KCa1.1 (BKCa, maxi-K) influences aqueous humor outflow facility, but the contribution of auxiliary β-subunits to KCa1.1 activity in the outflow pathway is unknown.Methods: Using quantitative polymerase chain reaction, we measured expression of β-subunit genes in anterior segments of C57BL/6J mice (Kcnmb1-4) and in cultured human trabecular meshwork (TM) and Schlemm's canal (SC) cells (KCNMB1-4). We also measured expression of Kcnma1/KCNMA1 that encodes the pore-forming α-subunit. Using confocal immunofluorescence, we visualized the distribution of β4 in the conventional outflow pathway of mice. Using iPerfusion, we measured outflow facility in enucleated mouse eyes in response to 100 or 500 nM iberiotoxin (IbTX; N = 9) or 100 nM martentoxin (MarTX; N = 12). MarTX selectively blocks β4-containing KCa1.1 channels, whereas IbTX blocks KCa1.1 channels that lack β4.Results: Kcnmb4 was the most highly expressed β-subunit in mouse conventional outflow tissues, expressed at a level comparable to Kcnma1. β4 was present within the juxtacanalicular TM, appearing to label cellular processes connecting to SC cells. Accordingly, KCNMB4 was the most highly expressed β-subunit in human TM cells, and the sole β-subunit in human SC cells. To dissect functional contribution, MarTX decreased outflow facility by 35% (27%, 42%; mean, 95% confidence interval) relative to vehicle-treated contralateral eyes, whereas IbTX reduced outflow facility by 16% (6%, 25%).Conclusions: The β4-subunit regulates KCa1.1 activity in the conventional outflow pathway, significantly influencing outflow function. Targeting β4-containing KCa1.1 channels may be a promising approach to lower intraocular pressure to treat glaucoma.

Journal article

Alaghband P, Galvis E, Ramirez A, Madekurozwa M, Chu B, Overby D, Lim KSet al., 2020, The Effect of High-Intensity Focused Ultrasound on Aqueous Humor Dynamics in Patients with Glaucoma, OPHTHALMOLOGY GLAUCOMA, Vol: 3, Pages: 122-129, ISSN: 2589-4234

Journal article

Chatzidimitriou F, Soon Seng NG, Tamir Rashid S, Sherwood JM, Overby DRet al., 2020, A liver-in-chip platform for preserving ex vivo tissue viability, Pages: 981-982

Organ-on-a-chip technologies provide new approaches to study biological systems in controllable microenvironments1. However, most of these platforms use isolated cells, a setting where fundamental features of tissue microenvironment, such as native extracellular matrix, vascularity and multiple cell types are lacking2. In this context, we develop a novel liver-in-chip model that preserves the viability of native tissue specimens using perfusion to deliver nutrients. This approach provides a powerful new tool to study human disease.

Conference paper

Alaghband P, Baneke AJ, Galvis E, Madekurozwa M, Chu B, Stanford M, Overby D, Lim KSet al., 2019, Aqueous Humor Dynamics in Uveitic Eyes, American Journal of Ophthalmology, Vol: 208, Pages: 347-355, ISSN: 0002-9394

PURPOSE: To investigate aqueous humor dynamics in uveitic eyes DESIGN: A cross-sectional study PARTICIPANTS: Patients with recurrent(≥3 attacks) anterior uveitis(now quiescent) and being treated for glaucoma or OHT(group-1), previous recurrent anterior uveitis(≥3 attacks) without glaucoma or OHT(group-2), and normal subjects with no ocular problems and IOP<21mmHg at screening, formed the control group(group-3). METHODS: Patients had one-off measurements. Group-1 patients who were on anti-hypertensives, were washed out for a 4-week period, prior to their study measurements. MAIN OUTCOME MEASURE: Tonographic outflow facility, aqueous 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 IOP was higher in the group-1 compared to the others(25±10.2(group-1) vs 16±2.7(group-2) vs 16±2.2mmHg(group-3), p<0.001). The tonographic outflow facility was lower in group-1 compared to the others(0.18±0.1(group-1) vs 0.25±0.1(group-2) vs 0.27±0.1μl/min/mmHg(group-3), p=0.005). However, aqueous flow rate was not statistically different(2.47±0.9(group-1) vs 2.13±0.9(group-2) vs 2.25±0.7μl/min(group-3), p=0.3). There was also no significant difference in calculated uveoscleral outflow. CONCLUSION: This is the first aqueous humor dynamic study in patients with uveitic glaucoma/OHT and recurrent anterior uveitis compared with age-matched controls. We have demonstrated that the elevated IOP 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 demonstrated any discernible difference. However, the exact mechanism remains to be elucidated.

Journal article

Sherwood JM, Boazak EM, Feola AJ, Parker K, Ethier CR, Overby DRet al., 2019, Measurement of ocular compliance using iPerfusion, Frontiers in Bioengineering and Biotechnology, Vol: 7, Pages: 1-15, ISSN: 2296-4185

The pressure-volume relationship of the eye is determined by the biomechanical properties of the corneoscleral shell and is classically characterised by Friedenwald's coefficient of ocular rigidity or, alternatively, by the ocular compliance (OC), defined as dV/dP. OC is important in any situation where the volume (V) or pressure (P) of the eye is perturbed, as occurs during several physiological and pathological processes. However, accurately measuring OC is challenging, particularly in rodents. We measured OC in 24 untreated enucleated eyes from 12 C57BL/6 mice using the iPerfusion system to apply controlled pressure steps, whilst measuring the time-varying flow rate into the eye. Pressure and flow data were analysed by a “Discrete Volume” (integrating the flow trace) and “Step Response” method (fitting an analytical solution to the pressure trace). OC evaluated at 13 mmHg was similar between the two methods (Step Response, 41 [37, 46] vs. Discrete Volume, 42 [37, 48] nl/mmHg; mean [95% CI]), although the Step Response Method yielded tighter confidence bounds on individual eyes. OC was tightly correlated between contralateral eyes (R2 = 0.75, p = 0.0003). Following treatment with the cross-linking agent genipin, OC decreased by 40 [33, 47]% (p = 0.0001; N = 6, Step Response Method). Measuring OC provides a powerful tool to assess corneoscleral biomechanics in mice and other species.

Journal article

Koudouna E, Young RD, Overby DR, Ueno M, Kinoshita S, Knupp C, Quantock AJet al., 2019, Ultrastructural variability of the juxtacanalicular tissue along the inner wall of Schlemm's canal., Molecular Vision, Vol: 25, Pages: 517-526, ISSN: 1090-0535

Purpose: Increased resistance of aqueous humor drainage from the eye through Schlemm's canal (SC) is the basis for elevated intraocular pressure in glaucoma. Experimental evidence suggests that the bulk of outflow resistance lies in the vicinity of the inner wall endothelial lining of SC and the adjacent juxtacanalicular tissue (JCT). However, there is little understanding of how this resistance is generated, and a detailed understanding of the structure-function relationship of the outflow pathway has not been established yet. In the present study, regional variations in the ultrastructure of the JCT and the inner wall of SC were investigated in three dimensions. Methods: With the use of serial block face scanning electron microscopy (SBF-SEM), the volume occupied by the electron lucent spaces of the JCT compared to that occupied by the cellular and extracellular matrix was investigated and quantified. The distribution of giant vacuoles (GVs) and pores in the inner wall endothelium of SC was further examined. Results: With increasing distance from the inner wall of SC, the volume of the electron lucent spaces increased above 30%. In contrast, the volume of these spaces in immediate contact with the inner wall endothelium was minimal (<10%). Circumferential variability in the type and distribution of GVs was observed, and the percentage of GVs with pores varied between 3% and 27%. Conclusions: These studies provide a detailed quantitative analysis of the ultrastructure of JCT and the distribution of GVs along the circumference of SC in three dimensions, supporting the non-uniform or segmental aqueous outflow.

Journal article

Reina-Torres E, Bertrand JA, O'Callaghan J, Sherwood JM, Humphries P, Overby DRet al., 2019, Reduced humidity experienced by mice in vivo coincides with reduced outflow facility measured ex vivo, Experimental Eye Research, Vol: 186, Pages: 1-5, ISSN: 0014-4835

Mice are routinely used to study aqueous humour dynamics. However, physical factors such as temperature and hydration affect outflow facility in enucleated eyes. This retrospective study examined whether differences in temperature and relative humidity experienced by living mice within their housing environment in vivo coincide with differences in outflow facility measured ex vivo. Facility data and environmental records were collected for one enucleated eye from 116 mice (C57BL/6J males, 9–15 weeks old) at two institutions. Outflow facility was reduced when relative humidity was below the lower limit of 45% recommended by the UK Code of Practice, but there was no detectable effect of temperature on outflow facility. Even when accounting for effects of humidity, there were differences in outflow facility measured between institutions and between individual researchers at the same institution. These data indicate that humidity, as well as additional environmental factors experienced by living mice within their housing environment, may significantly affect outflow facility measured ex vivo.

Journal article

Torres ER, Sherwood JM, Overby DR, 2019, Aqueous humour outflow requires active cellular metabolism, Annual Meeting of the Association-for-Research-in-Vision-and-Ophthalmology (ARVO), Publisher: ASSOC RESEARCH VISION OPHTHALMOLOGY INC, ISSN: 0146-0404

Conference paper

Overby DR, Spenlehauer A, Cairoli A, Sherwood JM, Vahabikashi A, Stamer WD, Lee CFet al., 2019, Actomyosin contractility and the vimentin cytoskeleton influence giant vacuole life-cycle in Schlemm's canal endothelial cells, Annual Meeting of the Association-for-Research-in-Vision-and-Ophthalmology (ARVO), Publisher: ASSOC RESEARCH VISION OPHTHALMOLOGY INC, ISSN: 0146-0404

Conference paper

Bertrand JA, Sherwood JM, Schicht M, Lutjen-Drecoll E, Selwood D, Stamer WD, Overby DRet al., 2019, Blockade of the BK-a/beta 4 potassium ion channel reduces outflow facility in mice, Annual Meeting of the Association-for-Research-in-Vision-and-Ophthalmology (ARVO), Publisher: ASSOC RESEARCH VISION OPHTHALMOLOGY INC, ISSN: 0146-0404

Conference paper

McDonnell F, Perkumas KM, Ashpole NE, Kalnitsky J, Sherwood JM, Overby DR, Stamer WDet al., 2019, Elevated IOP increases Shear Stress in Schlemm's Canal, Annual Meeting of the Association-for-Research-in-Vision-and-Ophthalmology (ARVO), Publisher: ASSOC RESEARCH VISION OPHTHALMOLOGY INC, ISSN: 0146-0404

Conference paper

Wu P-J, Masouleh MI, Paterson C, Dini D, Török P, Overby DR, Kabakova IVet al., 2019, Detection of proteoglycan loss from articular cartilage using Brillouin microscopy, with applications to osteoarthritis, Biomedical Optics Express, Vol: 10, Pages: 2457-2466, ISSN: 2156-7085

The degeneration of articular cartilage (AC) occurs in osteoarthritis (OA), which is a leading cause of pain and disability in middle-aged and older people. The early disease-related changes in cartilage extra-cellular matrix (ECM) start with depletion of proteoglycan (PG), leading to an increase in tissue hydration and permeability. These early compositional changes are small (<10%) and hence difficult to register with conventional non-invasive imaging technologies (magnetic resonance and ultrasound imaging). Here we apply Brillouin microscopy for detecting changes in the mechanical properties and composition of porcine AC. OA-like degradation is mimicked by enzymatic tissue digestion, and we compare Brillouin microscopy measurements against histological staining of PG depletion over varying digestion times and enzyme concentrations. The non-destructive nature of Brillouin imaging technology opens new avenues for creating minimally invasive arthroscopic devices for OA diagnostics and therapeutic monitoring.

Journal article

Madekurozwa M, 2019, Aqueous Humour Outflow Dynamics in Mice

Glaucoma is the leading cause of irreversible blindness worldwide. The major risk factor of glaucoma is sustained elevation of intraocular pressure (IOP), and lowering IOP is the only proven method for halting the progression of glaucomatous blindness. IOP is determined by the balance between aqueous humour (AH) production and drainage through pressure-dependent and pressure-independent outflow pathways. Elevated IOP is caused by increased hydraulic resistance through the pressure-dependent outflow pathway. Most glaucoma therapies aimed at lowering IOP do not effectively target pressure-dependent outflow due to an incomplete understanding of its regulation. We aim to use mice to study outflow regulation in the context of glaucoma.Mice are commonly used to study IOP regulation due to their resemblance to human ocular anatomy, genetics and pharmacology. However, while the bulk of AH drainage passes through the pressure-dependent pathway in humans, it has been reported to predominantly flow through the pressure-independent pathway in mice, which if true would invalidate the mouse as a model for studying outflow as occurs in humans. Here we present the first direct measurement of pressure-independent outflow in mice, showing it to be indistinguishable from zero which supports the mouse being a good model for pressure-dependent outflow as occurs in humans.We also investigated the role of the ocular pulse in outflow facility regulation, which arises due to cardiac pulsations in ocular blood volume. To do this we designed an apparatus to apply a sinusoidal pressure waveform superimposed onto a steady pressure whilst simultaneously measuring outflow resistance. We show that the ocular pulse leads to immediate decrease in outflow resistance in mice, and the effect was partly mediated through nitric oxide synthase.Finally, we developed a new apparatus and method to measure outflow resistance in living mice accounting for the influence of anaesthesia that introduces time-depen

Thesis dissertation

Sherwood JM, Stamer WD, Overby DR, 2019, A model of the oscillatory mechanical forces in the conventional outflow pathway, Journal of the Royal Society Interface, Vol: 16, ISSN: 1742-5662

Intraocular pressure is regulated by mechanosensitive cells within the conventional outflow pathway, the primary route of aqueous humour drainage from the eye. However, the characteristics of the forces acting on those cells are poorly understood. We develop a model that describes flow through the conventional outflow pathway, including the trabecular meshwork (TM) and Schlemm’s canal (SC). Accounting for the ocular pulse, we estimate the time-varying shear stress on SC endothelium and strain on the TM. We consider a range of outflow resistances spanning normotensive to hypertensive conditions. Over this range, the SC shear stress increases significantly and becomes highly oscillatory. TM strain also increases, but with negligible oscillations. Interestingly, TM strain responds more to changes in outflow resistance around physiological values, while SC shear stress responds more to elevated levels of resistance. A modest increase in TM stiffness, as observed in glaucoma, suppresses TM strain and practically eliminates the influence of outflow resistance on SC shear stress. As SC and TM cells respond to mechanical stimulation by secreting factors that modulate outflow resistance, our model provides insight regarding the potential role of SC shear and TM strain as mechanosensory cues for homeostatic regulation of outflow resistance and hence intraocular pressure.

Journal article

Alaghband P, Beltran-Agulló L, Galvis EA, Overby DR, Lim KSet al., 2018, Effect of phacoemulsification on facility of outflow, British Journal of Ophthalmology, Vol: 102, Pages: 1520-1526, ISSN: 0007-1161

PURPOSE: Phacoemulsification has been shown to reduce intraocular pressure (IOP). The mechanism of action is thought to be via increased trabecular outflow facility. However, studies on the relationship between phacoemulsification and outflow facility have been inconsistent. This study intended to examine the change in electronic Schiotz tonographic outflow facility (TOF) and IOP measurements following phacoemulsification. METHODS: Patients who were due to undergo a standard clear corneal incision phacoemulsification with intraocular lens (IOL) implantation, at St Thomas' Hospital, were invited to participate in this study. IOP was measured using Goldmann's applanation tonometer, and TOF was measured by electronic Schiotz tonography at baseline and at 3, 6 and 12 months postoperatively. RESULTS: Forty-one patients were recruited. Tonography data for 27 patients were reliable and available at all time points. Eleven cases had primary open angle glaucoma and cataract, while 16 patients had cataract only. Mean IOP reduced at every time point postoperatively significantly compared with baseline. TOF improved significantly after cataract extraction at all time points (baseline of 0.14±0.06 vs 0.18±0.09 at 3 months, P=0.02 and 0.20±0.09 at 6 months, P=0.003, 0.17±0.07 µL/min mmHg at 12 months, P=0.04). Five contralateral eyes of patients with cataracts only who did not have any intraocular surgery during the follow-up period were used as comparison. Their IOP and TOF did not change significantly at any postoperative visits. CONCLUSION: This is the first study using electronic Schiotz tonography with documented anterior chamber depth and gonioscopy after modern cataract surgery (CS) with phacoemulsification and IOL implantation. We demonstrated that phacoemulsification increases TOF and this fully accounts for the IOP reduction following CS. ISTCRN REGISTRATION NUMBER: ISRCTN04247738.

Journal article

Wu P-J, Kabovka I, Ruberti J, Sherwood J, Dunlop IE, Paterson C, Torok P, Overby Det al., 2018, Water content, not stiffness, dominates Brillouin spectroscopy measurements in hydrated materials, Nature Methods, Vol: 15, Pages: 561-562, ISSN: 1548-7091

Journal article

McDonnell F, Dismuke WM, Overby DR, Stamer WDet al., 2018, Pharmacological regulation of outflow resistance distal to Schlemm’s canal, American Journal of Physiology - Cell Physiology, Vol: 315, Pages: C44-C51, ISSN: 0363-6143

The trabecular meshwork (TM) and Schlemm's canal (SC) are responsible for generating the majority of outflow resistance, however the distal regions of the conventional outflow pathway appear to account for 25-50% of total. Sections of these distal vessels are surrounded by α-smooth muscle actin containing cells, indicating that they may be vasoregulated. This study examined the effect of a potent vasodilator, nitric oxide (NO) and its physiological antagonist endothelin-1 (ET-1) on the regulation of outflow resistance in the distal regions of the conventional outflow pathway. Using a physiological model of the conventional outflow pathway, human and porcine anterior segments were perfused in organ culture under constant flow conditions, while intrachamber pressure was continually monitored. For porcine anterior segments, a stable baseline outflow facility with TM intact was first achieved before anterior segments were removed and a trabeculotomy performed. For human anterior segments, a trabeculotomy was immediately performed. In human anterior segments, 100 nM ET-1 significantly decreased distal outflow facility from 0.49{plus minus}0.26 to 0.31{plus minus}0.18 (mean{plus minus}SD) µl/min/mmHg, p<0.01, a decrease of 38{plus minus}16%. Perfusion with 100µM DETA-NO in the presence of 1 nM ET-1 immediately reversed ET-1 effects, significantly increasing distal outflow facility to 0.54{plus minus}0.35 µl/min/mmHg, p=0.01, an escalation of 175{plus minus}49%. Similar results were obtained in porcine anterior segment experiments. In conclusion, data show a dynamic range of resistance generation by distal vessels in both the human and porcine conventional outflow pathways. Interestingly, maximal contraction of vessels in the distal outflow tract generated resistance very near physiological levels for both species.

Journal article

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