Development of Novel Techniques for Measuring Bulbar Conjunctival Red Blood Cell Velocity, Oximetry and Redness

Introduction The ocular surface provides a unique opportunity to study hemodynamics since the vessels can be visualized directly, without treatment and non-invasively. The availability of instruments to measure various hemodynamic parameters on the ocular surface in an objective manner are lacking. The quantification of red blood cell velocity, blood oxygen saturation and conjunctival redness on the ocular surface using novel, validated techniques has the potential of providing useful information about vascular physiology. The specific aims of each chapter are as follows: Chapter 3: The objective was to design, develop and validate a system that would non-invasively quantify the red blood cell velocity in the conjunctival vessels. A tool was developed to automatically analyze video sequences of conjunctival vessels, digitally imaged with high enough magnification to resolve movement of the blood within the vessel. Chapter 4: The objective was to: a) design and develop a method in order to non-invasively quantify the changes in blood oxygen saturation (SO2) in the conjunctival vessels and demonstrate reliability of the measures and, b) demonstrate the application of the method by showing a response to an isocapnic hyperoxic provocation and compare those values to the results from a valid instrument. Chapter 5: The aim of this experiment was to examine variations in ocular redness levels, red blood cell velocities and oxygen saturation levels over time in clinically healthy participants and also to compare differences between two age groups. Chapter 6: The aim of this experiment was to examine the ocular redness levels, red blood cell velocities and oxygen saturation levels in clinically healthy participants when a topical ophthalmic decongestant was instilled onto the eye and to demonstrate the validity of the use of two novel techniques. Chapter 7: The aim of this experiment was to examine ocular redness, red blood cell velocity and oxygen saturation in participants who were habitual soft contact lens wearers (study) compared to those that did not (control) and also to compare differences in silicone (SH) and non-silicone hydrogel wearers. Methods Chapter 3: Simulations representing moving RBCs within a vessel and the random variation of each cell in terms of speed, shape and intensity were created in order to evaluate the performance of the algorithm. For each vessel, a signal that correlated to blood cell position was extracted from each frame, and the inter-frame displacement was estimated through a modified dynamic time warping (DTW) algorithm. This provided the red blood cell velocity over time in each point of the vessels. Thus, from these estimates, the mean red blood cell velocity for each vessel was easily evaluated. The true mean velocity from the simulation with the one estimated by the algorithm was compared and the system accuracy was determined. Chapter 4: a) Conjunctival vessels were imaged with two narrow-band interference filters with O2-sensitive and O2-insensitive peak transmissions using a Zeiss slit lamp at 32x magnification. Optical densities were calculated from vascular segments using the average reflected intensities inside and outside the vessels. Optical density ratios were used to calculate relative oxygen saturation values. Video images of the bulbar conjunctiva were recorded at three times of the day. Measurement repeatability was assessed over location at each time and across consecutive frames. b) Subjects initially breathed air for 10 minutes followed by pure oxygen (O2) for 20 minutes, and then air for a final 10 minute period using a sequential re-breathing circuit. Simultaneously, SO2 values measured with a pulse oximeter ear clip and finger clip were recorded. The validity of the dual wavelength method was demonstrated by comparing the values to those from the ear clip pulse oximeter. Chapter 5: Participants attended eight separate visits over the course of a day. Levels of bulbar conjunctival redness, red blood cell velocity and blood oxygen saturation were measured on a vessel of interest. Chapter 6: Participants attended three separate visits during an allotted 60 minute session. Bulbar conjunctival redness, red blood cell velocity and blood oxygen saturation were measured on a vessel of interest, pre-insertion, just after insertion and, 10 minutes after insertion of a topical ocular decongestant. Significant differences between the three measures were assessed and correlations between the three parameters were reported. Chapter 7: Participants were measured 8 times over the course of a day with their contact lenses in place. Bulbar conjunctival redness, red blood cell velocity and blood oxygen saturation were measured. Results Chapter 3: Results for the simulated videos demonstrated a very good concordance between the estimated and actual velocities supporting its validity. The mean relative error for the modified Dynamic Time Warping (DTW) method is 6%. Chapter 4: The intraclass correlations (ICCs) between the three locations at each time point were 0.93, 0.56 and 0.86 respectively. Measurements across 5 consecutive frames showed no significant difference for all subjects (ICC = 0.96). The ICCs between the two methods at each time point were 0.45, 0.10 and 0.11 respectively. a) There was no significant difference in SO2 between the three locations measured using the dual wavelength method for all subjects. There was also no significant difference between the three locations at any of the time points for the dual wavelength method. b) In response to isocapnic hyperoxic provocation using the dual wavelength method, blood oxygen saturation was increased from control values and subsequently recovered after withdrawal of hyperoxia. Blood oxygen saturation values recorded from the ear clip and finger clip of the pulse oximeter also showed an increase from control values and subsequently recovered after withdrawal of hyperoxia. SO2 comparison between the dual wavelength method and the ear-clip pulse oximeter method did not show a significant difference. The interaction between the two methods and time on SO2 was not significant. Chapter 5: From baseline, the group mean redness and oxygen saturation did not change significantly over time. There was a significant difference in the group mean red blood cell velocity values over time. There was no significant difference between age strata for all three measures. Chapter 6: After drop instillation redness values decreased significantly. There was no change in red blood cell velocity and oxygen saturation over time. There was a moderate significant correlation between SO2 and red blood cell velocity just after drop insertion. Chapter 7: When comparing the study and control groups, no significant difference in redness or SO2 over time was found. RBC velocity over time was found to be significantly different between groups. When comparing the two study groups (SH vs. hydrogel) no significant difference across either measure over time was found. Conclusions Chapter 3: Signal displacement estimation through the DTW algorithm can be used to estimate mean red blood cell velocity. Successful application of the algorithm in the estimation of RBC velocity in conjunctival vessels was demonstrated. Chapter 4: The application of the dual wavelength method was demonstrated and optical density ratios can be used in a reliable manner for relative oxygen saturation measurements. This valid method promises to enable the study of conjunctival O2 saturation under various experimental and physiological conditions. Chapter 5: The results of this study support the theory of metabolic regulation. The lack of any significant change across time for redness and oxygen saturation along with significant changes in red blood cell velocity substantiates this notion. Chapter 6: This study supports the literature regarding metabolic regulation of the microvasculature during the use of various stimuli. The results demonstrated that oxygen saturation levels remain stable even when a significant decrease in ocular redness is measured. The novel techniques used in this experiment demonstrated the expected action of the decongestant further contributing to their application and validity. Chapter 7: In summary, the participants in the study group were habitual contact lens wearers that had lower RBC velocities when compared to the control group supporting the notion that contact lenses initiate a hypoxic response. The lack of change in SO2 in either group supports the theory of metabolic regulation

Unravelling the induction and development of intramembranous bones in the chicken eye

x, 96 leaves : ill. ; 29 cm.Includes abstract.Includes bibliographical references (leaves 79-83).Bone development is a complex process, involving multiple tissues and hierarchical inductive interactions. The study of skeletal development has focused on endochondral bones while intramembranous bones (e.g. scleral ossicles) have received less attention. Although, sonic hedgehog was recently demonstrated to be present during induction of scleral ossicles, other signalling molecules are still unknown. Using in situ hybridization and bead implantation I have demonstrated the presence or absence and the involvement of candidate genes and gene families during the development of scleral ossicles. Scleraxis, patched, as well as the bone morphogenetic protein (BMP) family of genes, were found to be involved in the development of scleral ossicles. Conversely, it was determined that indian hedgehog and msx2 are not present. This study has contributed to unravelling the signals involved during the induction and subsequent development of scleral ossicles and has contributed to the current understanding of intramembranous bone development

Measurement of Average Real-Time Red Blood Cell Velocity in Conjunctival Vessels

Measurement of retinal blood flow has been used to provide a microcirculatory assessment of the haemodynamic state of the retina. To date, there have been no studies that have looked measuring real-time blood velocity in bulbar conjunctival vessels. The objective was to design and develop a system to non-invasively quantify the blood velocity in the conjunctival vessels, with the prospect of gaining a comprehensive understanding of the conjunctival circulation in health and disease

Signals from the brain induce variation in avian facial shape

BACKGROUND: How developmental mechanisms generate the phenotypic variation that is the raw material for evolution is largely unknown. Here we explore whether variation in a conserved signaling axis between the brain and face contributes to differences in morphogenesis of the avian upper jaw. In amniotes, including both mice and avians, signals from the brain establish a signaling center in the ectoderm (the Frontonasal ectodermal zone or “FEZ”) that directs outgrowth of the facial primordia. RESULTS: Here we show that the spatial organization of this signaling center differs among avians, and these correspond to Sonic hedgehog (Shh) expression in the basal forebrain and embryonic facial shape. In ducks this basal forebrain domain is present almost the entire width, while in chickens it is restricted to the midline. When the duck forebrain is unilaterally transplanted into stage matched chicken embryos the face on the treated side resembles that of the donor. CONCLUSIONS: Combined with previous findings, these results demonstrate that variation in a highly conserved developmental pathway has the potential to contribute to evolutionary differences in avian upper jaw morphology

Functional slit lamp biomicroscopy for imaging bulbar conjunctival microvasculature in contact lens wearers

PURPOSE: To develop, test and validate functional slit lamp biomicroscopy (FSLB) for generating non-invasive bulbar conjunctival microvascular perfusion maps (nMPMs) and assessing morphometry and hemodyanmics. METHODS: FSLB was adapted from a traditional slit-lamp microscope by attaching a digital camera to image the bulbar conjunctiva to create nMPMs and measure venular blood flow hemodyanmics. High definition images with a large field of view were obtained on the temporal bulbar conjunctiva for creating nMPMs. A high imaging rate of 60 frame per second and a ~210× high magnification were achieved using the camera inherited high speed setting and movie crop function, for imaging hemodyanmics. Custom software was developed to segment bulbar conjunctival nMPMs for further fractal analysis and quantitatively measure blood vessel diameter, blood flow velocity and flow rate. Six human subjects were imaged before and after 6 hours of wearing contact lenses. Monofractal and multifractal analyses were performed to quantify fractality of the nMPMs. RESULTS: The mean bulbar conjunctival vessel diameter was 18.8 ± 2.7 μm at baseline and increased to 19.6 ± 2.4 μm after 6 hours of lens wear (P = 0.020). The blood flow velocity was increased from 0.60 ± 0.12 mm/s to 0.88 ± 0.21 mm/s (P = 0.001). The blood flow rate was also increased from 129.8 ± 59.9 pl/s to 207.2 ± 81.3 pl/s (P = 0.001). Bulbar conjunctival nMPMs showed the intricate details of the bulbar conjunctival microvascular network. At baseline, fractal dimension was 1.63 ± 0.05 and 1.71 ± 0.03 analyzed by monofractal and multifractal analysis, respectively. Significant increases in fractal dimensions were found after 6 hours of lens wear (P < 0.05). CONCLUSIONS: Microvascular network’s fractality, morphometry and hemodyanmics of the human bulbar conjunctiva can be measured easily and reliably using FSLB. The alternations of the fractal dimensions, morphometry and hemodyanmics during contact lens wear may indicate ocular microvascular responses to contact lens wear

Limbal and Bulbar Hyperaemia in Normal Eyes

Purpose:  To investigate the appearance of limbal and bulbar hyperaemia in normal eyes, their relationship and the inter-observer agreement of clinical grading. Methods:  The right eyes of 120 healthy, non-contact lens-wearing subjects (m = 57, f = 63, median age = 45 years, range 18–77 years) were examined by two trained observers. Limbal and bulbar hyperaemia were scored using the Cornea and Contact Lens Research Unit (CCLRU) redness grading scales interpolated into 0.1 increments. Redness of four quadrants, and overall, were assessed, and quadrant-average redness was calculated. Inter-observer agreement was assessed at the start and end of the study (20 subjects each). Results:  For limbal redness, the overall (1.62 ± 0.46) (mean units ± S.D.) was not significantly different from the quadrant-average (1.61 ± 0.40) score. For bulbar redness, the overall (2.02 ± 0.49) was higher than the quadrant-average (1.82 ± 0.39) score (p < 0.0001). Significant correlations were found between bulbar and limbal quadrants (Pearson: r ≥ 0.43, p < 0.0001). Significant differences in redness were found between quadrants (p < 0.0001), with nasal and temporal redder than superior and inferior quadrants. Small effects of age and gender were found for limbal redness. The inter-observer 95% limits of agreement were similar at the start and end of the study. They were larger for overall (0.57) compared with quadrant-average (0.28) redness. Conclusions:  For similar populations, a limbal redness above 2.5 or a bulbar redness above 2.6 (quadrant-average) or 3.0 (overall) may be considered abnormal. Limbal and bulbar redness were correlated. Quadrant-average scores are recommended instead of overall scores, as inter-observer agreement was better

DNA nanoparticles for ophthalmic drug delivery

Nucleic acids represent very appealing building blocks for the construction of nano-scaled objects with great potential applications in the field of drug delivery where multifunctional nanoparticles (NPs) play a pivotal role. One opportunity for DNA nanotechnology lies in the treatment of ophthalmic diseases as the efficacy of eye drops is impaired by the short survival time of the drug on the eye surface. As a consequence, topical administration of ocular therapeutics requires high drug doses and frequent administration, still rarely providing high bioavailability. To overcome these shortcomings we introduce a novel and general carrier system that is based on DNA nanotechnology. Non-toxic, lipid-modified DNA strands (12mers with 4 lipid modified thymines at the 5' end) form uniform NPs (micelles), which adhere to the corneal surface for extended periods of time. In a single self-assembly step they can be equipped with different drugs by hybridization with an aptamer. The long survival times of DNA NPs can be translated into improved efficacy. Their functionality was demonstrated in several ex-vivo experiments and in an in-vivo animal model. Finally, the NPs were confirmed to be applicable even for human tissue. (C) 2017 Elsevier Ltd. All rights reserved