Refining animal models in fracture research: Seeking consensus in optimising both animal welfare and scientific validity for appropriate biomedical use

Background In an attempt to establish some consensus on the proper use and design of experimental animal models in musculoskeletal research, AOVET (the veterinary specialty group of the AO Foundation) in concert with the AO Research Institute (ARI), and the European Academy for the Study of Scientific and Technological Advance, convened a group of musculoskeletal researchers, veterinarians, legal experts, and ethicists to discuss, in a frank and open forum, the use of animals in musculoskeletal research. Methods The group narrowed the field to fracture research. The consensus opinion resulting from this workshop can be summarized as follows: Results & Conclusion Anaesthesia and pain management protocols for research animals should follow standard protocols applied in clinical work for the species involved. This will improve morbidity and mortality outcomes. A database should be established to facilitate selection of anaesthesia and pain management protocols for specific experimental surgical procedures and adopted as an International Standard (IS) according to animal species selected. A list of 10 golden rules and requirements for conduction of animal experiments in musculoskeletal research was drawn up comprising 1) Intelligent study designs to receive appropriate answers; 2) Minimal complication rates (5 to max. 10%); 3) Defined end-points for both welfare and scientific outputs analogous to quality assessment (QA) audit of protocols in GLP studies; 4) Sufficient details for materials and methods applied; 5) Potentially confounding variables (genetic background, seasonal, hormonal, size, histological, and biomechanical differences); 6) Post-operative management with emphasis on analgesia and follow-up examinations; 7) Study protocols to satisfy criteria established for a "justified animal study"; 8) Surgical expertise to conduct surgery on animals; 9) Pilot studies as a critical part of model validation and powering of the definitive study design; 10) Criteria for funding agencies to include requirements related to animal experiments as part of the overall scientific proposal review protocols. Such agencies are also encouraged to seriously consider and adopt the recommendations described here when awarding funds for specific projects. Specific new requirements and mandates related both to improving the welfare and scientific rigour of animal-based research models are urgently needed as part of international harmonization of standards.

Ocular aberrations in the peripheral visual field

We modified a commercial Hartmann-Shack aberrometer and used it to measure ocular aberrations across the central 42º horizontal x 32º vertical visual fields of five young emmetropic subjects. Some Zernike aberration coefficients show coefficient field distributions that were similar to the field dependence predicted by Seidel theory (astigmatism, oblique astigmatism, horizontal coma, vertical coma), but defocus did not demonstrate such similarity.

Ocular characteristics of anisometropia

Animal models of refractive error development have demonstrated that visual experience influences ocular growth. In a variety of species, axial anisometropia (i.e. a difference in the length of the two eyes) can be induced through unilateral occlusion, image degradation or optical manipulation. In humans, anisometropia may occur in isolation or in association with amblyopia, strabismus or unilateral pathology. Non-amblyopic myopic anisometropia represents an interesting anomaly of ocular growth, since the two eyes within one visual system have grown to different endpoints. These experiments have investigated a range of biometric, optical and mechanical properties of anisometropic eyes (with and without amblyopia) with the aim of improving our current understanding of asymmetric refractive error development. In the first experiment, the interocular symmetry in 34 non-amblyopic myopic anisometropes (31 Asian, 3 Caucasian) was examined during relaxed accommodation. A high degree of symmetry was observed between the fellow eyes for a range of optical, biometric and biomechanical measurements. When the magnitude of anisometropia exceeded 1.75 D, the more myopic eye was almost always the sighting dominant eye. Further analysis of the optical and biometric properties of the dominant and non-dominant eyes was conducted to determine any related factors but no significant interocular differences were observed with respect to best-corrected visual acuity, corneal or total ocular aberrations during relaxed accommodation. Given the high degree of symmetry observed between the fellow eyes during distance viewing in the first experiment and the strong association previously reported between near work and myopia development, the aim of the second experiment was to investigate the symmetry between the fellow eyes of the same 34 myopic anisometropes following a period of near work. Symmetrical changes in corneal and total ocular aberrations were observed following a short reading task (10 minutes, 2.5 D accommodation demand) which was attributed to the high degree of interocular symmetry for measures of anterior eye morphology, and corneal biomechanics. These changes were related to eyelid shape and position during downward gaze, but gave no clear indication of factors associated with near work that might cause asymmetric eye growth within an individual. Since the influence of near work on eye growth is likely to be most obvious during, rather than following near tasks, in the third experiment the interocular symmetry of the optical and biometric changes was examined during accommodation for 11 myopic anisometropes. The changes in anterior eye biometrics associated with accommodation were again similar between the eyes, resulting in symmetrical changes in the optical characteristics. However, the more myopic eyes exhibited slightly greater amounts of axial elongation during accommodation which may be related to the force exerted by the ciliary muscle. This small asymmetry in axial elongation we observed between the eyes may be due to interocular differences in posterior eye structure, given that the accommodative response was equal between eyes. Using ocular coherence tomography a reduced average choroidal thickness was observed in the more myopic eyes compared to the less myopic eyes of these subjects. The interocular difference in choroidal thickness was correlated with the magnitude of spherical equivalent and axial anisometropia. The symmetry in optics and biometrics between fellow eyes which have undergone significantly different visual development (i.e. anisometropic subjects with amblyopia) is also of interest with respect to refractive error development. In the final experiment the influence of altered visual experience upon corneal and ocular higher-order aberrations was investigated in 21 amblyopic subjects (8 refractive, 11 strabismic and 2 form deprivation). Significant differences in aberrations were observed between the fellow eyes, which varied according to the type of amblyopia. Refractive amblyopes displayed significantly higher levels of 4th order corneal aberrations (spherical aberration and secondary astigmatism) in the amblyopic eye compared to the fellow non-amblyopic eye. Strabismic amblyopes exhibited significantly higher levels of trefoil, a third order aberration, in the amblyopic eye for both corneal and total ocular aberrations. The results of this experiment suggest that asymmetric visual experience during development is associated with asymmetries in higher-order aberrations, proportional to the magnitude of anisometropia and dependent upon the amblyogenic factor. This suggests a direct link between the development of higher-order optical characteristics of the human eye and visual feedback. The results from these experiments have shown that a high degree of symmetry exists between the fellow eyes of non-amblyopic myopic anisometropes for a range of biomechanical, biometric and optical parameters for different levels of accommodation and following near work. While a single specific optical or biomechanical factor that is consistently associated with asymmetric refractive error development has not been identified, the findings from these studies suggest that further research into the association between ocular dominance, choroidal thickness and higher-order aberrations with anisometropia may improve our understanding of refractive error development.

Eye shape and retinal shape, and their relation to peripheral refraction

Purpose: We provide an account of the relationships between eye shape, retinal shape and peripheral refraction. Recent findings: We discuss how eye and retinal shapes may be described as conicoids, and we describe an axis and section reference system for determining shapes. Explanations are given of how patterns of retinal expansion during the development of myopia may contribute to changing patterns of peripheral refraction, and how pre-existing retinal shape might contribute to the development of myopia. Direct and indirect techniques for determining eye and retinal shape are described, and results are discussed. There is reasonable consistency in the literature of eye length increasing at a greater rate than height and width as the degree of myopia increases, so that eyes may be described as changing from oblate/spherical shapes to prolate shapes. However, one study indicates that the retina itself, while showing the same trend, remains oblate in shape for most eyes (discounting high myopia). Eye shape and retinal shape are not the same and merely describing an eye shape as being prolate or oblate is insufficient without some understanding of the parameters contributing to this; in myopia a prolate eye shape is likely to involve both a steepening retina near the posterior pole combined with a flattening (or a reduction in steepening compared with an emmetrope) away from the pole.

Mesopic rod and S-cone interactions revealed by modulation thresholds

We analyzed mesopic rod and S-cone interactions in terms of their contributions to the blue-yellow opponent pathway. Stimuli were generated using a 4-primary colorimeter. Mixed rod and S-cone modulation thresholds (constant L-, M-cone excitation) were measured as a function of their phase difference. Modulation amplitude was equated using threshold units and contrast ratios. This study identified three interaction types: (1) A linear and antagonistic rod:S-cone interaction, (2) probability summation (3) and a previously unidentified mutual nonlinear reinforcement. Linear rod:S-cone interactions occur within the blue-yellow opponent pathway. Probability summation involves signaling by different post-receptoral pathways. The origin of the nonlinear reinforcement is possibly at the photoreceptors.

Corneal changes following near work in myopic anisometropia

Purpose: To examine the symmetry of corneal changes following near work in the fellow eyes of non-amblyopic myopic anisometropes. Methods: Thirty-four non-amblyopic, myopic anisometropes (minimum 1 D spherical equivalent anisometropia) had corneal topography measured before and after a controlled near work task. Subjects were positioned in a headrest to minimise head movements and read continuous text on a computer monitor for 10 minutes at an angle of 25 degrees downward gaze and an accommodation demand of 2.5 D. Measures of the morphology of the palpebral aperture during primary and downward gaze were also obtained. Results: The more and less myopic eyes exhibited a high degree of interocular symmetry for measures of palpebral aperture morphology during both primary and downward gaze. Following the near work task, fellow eyes also displayed a symmetrical change in superior corneal topography (hyperopic defocus) which correlated with the position of the upper eyelid during downward gaze. Greater changes in the spherical corneal power vector (M) following reading were associated with narrower palpebral aperture during downward gaze (p = 0.07 for more myopic and p = 0.03 for less myopic eyes). A significantly greater change in J0 (an increase in against the rule astigmatism) was observed in the more myopic eyes (-0.04 ± 0.04 D) compared to the less myopic eyes (-0.02 ± 0.06 D) over a 6 mm corneal diameter (p = 0.01). Conclusions: Changes in corneal topography following near work are highly symmetrical between the fellow eyes of myopic anisometropes due to the interocular symmetry of the palpebral aperture. However, the more myopic eye exhibits changes in corneal astigmatism of greater magnitude compared to the less myopic eye.

Assessment of neuroretinal function in a group of functional amblyopes with documented LGN deficits

Purpose In this study we examine neuroretinal function in five amblyopes, who had been shown in previous functional MRI (fMRI) studies to have compromised function of the lateral geniculate nucleus (LGN), to determine if the fMRI deficit in amblyopia may have its origin at the retinal level. Methods We used slow flash multifocal ERG (mfERG) and compared averaged five ring responses of the amblyopic and fellow eyes across a 35 deg field. Central responses were also assessed over a field which was about 6.3 deg in diameter. We measured central retinal thickness using optical coherence tomography. Central fields were measured using the MP1-Microperimeter which also assesses ocular fixation during perimetry. MfERG data were compared with fMRI results from a previous study. Results Amblyopic eyes had reduced response density amplitudes (first major negative to first positive (N1-P1) responses) for the central and paracentral retina (up to 18 deg diameter) but not for the mid-periphery (from 18 to 35 deg). Retinal thickness was within normal limits for all eyes, and not different between amblyopic and fellow eyes. Fixation was maintained within the central 4° more than 80% of the time by four of the five participants; fixation assessed using bivariate contour ellipse areas (BCEA) gave rankings similar to those of the MP-1 system. There was no significant relationship between BCEA and mfERG response for either amblyopic or fellow eye. There was no significant relationship between the central mfERG eye response difference and the selective blood oxygen level dependent (BOLD) LGN eye response difference previously seen in these participants. Conclusions Retinal responses in amblyopes can be reduced within the central field without an obvious anatomical basis. Additionally, this retinal deficit may not be the reason why the LGN BOLD (blood oxygen level dependent) responses are reduced for amblyopic eye stimulation.

Optical and biomechanical factors, associated with near work and myopia

Near work may play an important role in the development of myopia in the younger population. The prevalence of myopia has also been found to be higher in occupations that involve substantial near work tasks, for example in microscopists and textile workers. When nearwork is performed, it typically involves accommodation, convergence and downward gaze. A number of previous studies have examined the effects of accommodation and convergence on changes in the optics and biometrics of the eye in primary gaze. However, little is known about the influence of accommodation on the eye in downward gaze. This thesis is primarily concerned with investigating the changes in the eye during near work in downward gaze under natural viewing conditions. To measure wavefront aberrations in downward gaze under natural viewing conditions, we modified a commercial Shack-Hartmann wavefront sensor by adding a relay lens system to allow on-axis ocular aberration measurements in primary gaze and downward gaze, with binocular fixation. Measurements with the modified wavefront sensor in primary and downward gaze were validated against a conventional aberrometer using both a model eye and in 9 human subjects. We then conducted an experiment to investigate changes in ocular aberrations associated with accommodation in downward gaze over 10 mins in groups of both myopes (n = 14) and emmetropes (n =12) using the modified Shack-Hartmann wavefront sensor. During the distance accommodation task, small but significant changes in refractive power (myopic shift) and higher order aberrations were observed in downward gaze compared to primary gaze. Accommodation caused greater changes in higher order aberrations (in particular coma and spherical aberration) in downward gaze than primary gaze, and there was evidence that the changes in certain aberrations with accommodation over time were different in downward gaze compared to primary gaze. There were no obvious systematic differences in higher order aberrations between refractive error groups during accommodation or downward gaze for fixed pupils. However, myopes exhibited a significantly greater change in higher order aberrations (in particular spherical aberration) than emmetropes for natural pupils after 10 mins of a near task (5 D accommodation) in downward gaze. These findings indicated that ocular aberrations change from primary to downward gaze, particularly with accommodation. To understand the mechanism underlying these changes in greater detail, we then extended this work to examine the characteristics of the corneal optics, internal optics, anterior biometrics and axial length of the eye during a near task, in downward gaze, over 10 mins. Twenty young adult subjects (10 emmetropes and 10 myopes) participated in this study. To measure corneal topography and ocular biometrics in downward gaze, a rotating Scheimpflug camera and an optical biometer were inclined on a custom built, height and tilt adjustable table. We found that both corneal optics and internal optics change with downward gaze, resulting in a myopic shift (~0.10 D) in the spherical power of the eye. The changes in corneal optics appear to be due to eyelid pressure on the anterior surface of the cornea, whereas the changes in the internal optics (an increase in axial length and a decrease in anterior chamber depth) may be associated with movement of the crystalline lens, under the action of gravity, and the influence of altered biomechanical forces from the extraocular muscles on the globe with downward gaze. Changes in axial length with accommodation were significantly greater in downward gaze than primary gaze (p < 0.05), indicating an increased effect of the mechanical forces from the ciliary muscle and extraocular muscles. A subsequent study was conducted to investigate the changes in anterior biometrics, axial length and choroidal thickness in nine cardinal gaze directions under the actions of the extraocular muscles. Ocular biometry measurements were obtained from 30 young adults (10 emmetropes, 10 low myopes and 10 moderate myopes) through a rotating prism with 15° deviation, along the foveal axis, using a non-contact optical biometer in each of nine different cardinal directions of gaze, over 5 mins. There was a significant influence of gaze angle and time on axial length (both p < 0.001), with the greatest axial elongation (+18 ± 8 μm) occurring with infero-nasal gaze (p < 0.001) and a slight decrease in axial length in superior gaze (−12 ± 17 μm) compared with primary gaze (p < 0.001). There was a significant correlation between refractive error (spherical equivalent refraction) and the mean change in axial length in the infero-nasal gaze direction (Pearson's R2 = 0.71, p < 0.001). To further investigate the relative effect of gravity and extraocular muscle force on the axial length, we measured axial length in 15° and 25° downward gaze with the biometer inclined on a tilting table that allowed gaze shifts to occur with either full head turn but no eye turn (reflects the effect of gravity), or full eye turn with no head turn (reflects the effect of extraocular muscle forces). We observed a significant axial elongation in 15° and 25° downward gaze in the full eye turn condition. However, axial length did not change significantly in downward gaze over 5 mins (p > 0.05) in the full head turn condition. The elongation of the axial length in downward gaze appears to be due to the influence of the extraocular muscles, since the effect was not present when head turn was used instead of eye turn. The findings of these experiments collectively show the dynamic characteristics of the optics and biometrics of the eye in downward gaze during a near task, over time. These were small but significant differences between myopic and emmetropic eyes in both the optical and biomechanical changes associated with shifts of gaze direction. These differences between myopes and emmetropes could arise as a consequence of excessive eye growth associated with myopia. However the potentially additive effects of repeated or long lasting near work activities employing infero-nasal gaze could also act to promote elongation of the eye due to optical and/or biomechanical stimuli.

Application of near infrared (NIR) spectroscopy for determining the thickness of articular cartilage

The determination of the characteristics of articular cartilage such as thickness, stiffness and swelling, especially in the form that can facilitate real-time decisions and diagnostics is still a matter for research and development. This paper correlates near infrared spectroscopy with mechanically measured cartilage thickness to establish a fast, non-destructive, repeatable and precise protocol for determining this tissue property. Statistical correlation was conducted between the thickness of bovine cartilage specimens (n = 97) and regions of their near infrared spectra. Nine regions were established along the full absorption spectrum of each sample and were correlated with the thickness using partial least squares (PLS) regression multivariate analysis. The coefficient of determination (R2) varied between 53 and 93%, with the most predictive region (R2 = 93.1%, p < 0.0001) for cartilage thickness lying in the region (wavenumber) 5350–8850 cm−1. Our results demonstrate that the thickness of articular cartilage can be measured spectroscopically using NIR light. This protocol is potentially beneficial to clinical practice and surgical procedures in the treatment of joint disease such as osteoarthritis.

Interocular corneal symmetry in refractive error groups

Purpose: To examine between eye differences in corneal higher order aberrations and topographical characteristics in a range of refractive error groups. Methods: One hundred and seventy subjects were recruited including; 50 emmetropic isometropes, 48 myopic isometropes (spherical equivalent anisometropia ≤ 0.75 D), 50 myopic anisometropes (spherical equivalent anisometropia ≥ 1.00 D) and 22 keratoconics. The corneal topography of each eye was captured using the E300 videokeratoscope (Medmont, Victoria, Australia) and analyzed using custom written software. All left eye data were rotated about the vertical midline to account for enantiomorphism. Corneal height data were used to calculate the corneal wavefront error using a ray tracing procedure and fit with Zernike polynomials (up to and including the eighth radial order). The wavefront was centred on the line of sight by using the pupil offset value from the pupil detection function in the videokeratoscope. Refractive power maps were analysed to assess corneal sphero-cylindrical power vectors. Differences between the more myopic (or more advanced eye for keratoconics) and the less myopic (advanced) eye were examined. Results: Over a 6 mm diameter, the cornea of the more myopic eye was significantly steeper (refractive power vector M) compared to the fellow eye in both anisometropes (0.10 ± 0.27 D steeper, p = 0.01) and keratoconics (2.54 ± 2.32 D steeper, p < 0.001) while no significant interocular difference was observed for isometropic emmetropes (-0.03 ± 0.32 D) or isometropic myopes (0.02 ± 0.30 D) (both p > 0.05). In keratoconic eyes, the between eye difference in corneal refractive power was greatest inferiorly (associated with cone location). Similarly, in myopic anisometropes, the more myopic eye displayed a central region of significant inferior corneal steepening (0.15 ± 0.42 D steeper) relative to the fellow eye (p = 0.01). Significant interocular differences in higher order aberrations were only observed in the keratoconic group for; vertical trefoil C(3,-3), horizontal coma C(3,1) secondary astigmatism along 45 C(4, -2) (p < 0.05) and vertical coma C(3,-1) (p < 0.001). The interocular difference in vertical pupil decentration (relative to the corneal vertex normal) increased with between eye asymmetry in refraction (isometropia 0.00 ± 0.09, anisometropia 0.03 ± 0.15 and keratoconus 0.08 ± 0.16 mm) as did the interocular difference in corneal vertical coma C (3,-1) (isometropia -0.006 ± 0.142, anisometropia -0.037 ± 0.195 and keratoconus -1.243 ± 0.936 μm) but only reached statistical significance for pair-wise comparisons between the isometropic and keratoconic groups. Conclusions: There is a high degree of corneal symmetry between the fellow eyes of myopic and emmetropic isometropes. Interocular differences in corneal topography and higher order aberrations are more apparent in myopic anisometropes and keratoconics due to regional (primarily inferior) differences in topography and between eye differences in vertical pupil decentration relative to the corneal vertex normal. Interocular asymmetries in corneal optics appear to be associated with anisometropic refractive development.

Diffusion-sensitive magnetic resonance spectroscopy and imaging in biomedical sciences

We present a mini-review of the development and contemporary applications of diffusion-sensitive nuclear magnetic resonance (NMR) techniques in biomedical sciences. Molecular diffusion is a fundamental physical phenomenon present in all biological systems. Due to the connection between experimentally measured diffusion metrics and the microscopic environment sensed by the diffusing molecules, diffusion measurements can be used for characterisation of molecular size, molecular binding and association, and the morphology of biological tissues. The emergence of magnetic resonance was instrumental to the development of biomedical applications of diffusion. We discuss the fundamental physical principles of diffusion NMR spectroscopy and diffusion MR imaging. The emphasis is placed on conceptual understanding, historical evolution and practical applications rather than complex technical details. Mathematical description of diffusion is presented to the extent that it is required for the basic understanding of the concepts. We present a wide range of spectroscopic and imaging applications of diffusion magnetic resonance, including colloidal drug delivery vehicles; protein association; characterisation of cell morphology; neural fibre tractography; cardiac imaging; and the imaging of load-bearing connective tissues. This paper is intended as an accessible introduction into the exciting and growing field of diffusion magnetic resonance.

Through-focus performance with multifocal contact lenses : effect of binocularity, pupil diameter and inherent ocular aberrations

Purpose to evaluate the effects of the wearer’s pupil size and spherical aberration on visual performance with centre-near, aspheric multifocal contact lenses (MFCLs). The advantage of binocular over monocular vision was also investigated. Methods Twelve young volunteers, with an average age of 27±5 years, participated in the study. LogMAR Visual Acuity (VA) was measured under cycloplegia for a range of defocus levels (from +3.0 to -3.0D, in 0.5D steps) with no correction and with three aspheric MFCLs (Air Optix Aqua Multifocal, Ciba Vision, Duluth, GA, US) with a centre-near design, providing correction for “Low”, “Med” and “High” near demands. Measurements were performed for all combinations of the following conditions: i) artificial pupils of 6mm and 3mm diameter, ii) binocular and monocular (dominant eye) vision. Depth-of-focus (DOF) was calculated from the VA vs. defocus curves. Ocular aberrations under cycloplegia were measured using iTrace. Results VA at -3.0D defocus (simulating near performance) was statistically higher for the 3mm than for the 6mm pupil (p=0.006), and for binocular rather than for monocular vision (p<0.001). Similarly, DOF was better for the 3mm pupil (p=0.002) and for binocular viewing conditions (p<0.001, ANOVA). Both VA at –3.0D defocus and DOF increased as the “addition” of the MFCL correction increased. Finally, with the centre-near MFCLs a linear correlation was found between VA at –3.0D defocus and the wearer’s ocular spherical aberration (R2=0.20 p<0.001 for 6mm data), with the eyes exhibiting the higher positive spherical aberration experiencing lower VAs. By contrast, no correlation was found between VA and spherical aberration at 0.00D defocus (distance vision). Conclusions Both near VA and depth-of-focus improve with these MFCLs, with the effects being more pronounced for small pupils and binocular than for monocular vision. Coupling of the wearer’s ocular spherical aberration with the aberration profiles provided by MFCLs affects their functionality.