Age estimation based on aspartic acid racemization in human sclera.

Age estimation based on racemization of aspartic acid residues (AAR) in permanent proteins has been established in forensic medicine for years. While dentine is the tissue of choice for this molecular method of age estimation, teeth are not always available which leads to the need to identify other suitable tissues. We examined the suitability of total tissue samples of human sclera for the estimation of age at death. Sixty-five samples of scleral tissue were analyzed. The samples were hydrolyzed and after derivatization, the extent of aspartic acid racemization was determined by gas chromatography. The degree of AAR increased with age. In samples from younger individuals, the correlation of age and D-aspartic acid content was closer than in samples from older individuals. The age-dependent racemization in total tissue samples proves that permanent or at least long-living proteins are present in scleral tissue. The correlation of AAR in human sclera and age at death is close enough to serve as basis for age estimation. However, the precision of age estimation by this method is lower than that of age estimation based on the analysis of dentine which is due to molecular inhomogeneities of total tissue samples of sclera. Nevertheless, the approach may serve as a valuable alternative or addition in exceptional cases.

Effect of flaxseed on choroid-sclera complex thickness and on LDL oxidation in the sclera, choroid and retina of diet-induced hypercholesterolaemic rabbits.

The aim of the present study was to evaluate the effect of flaxseed on choroid-sclera complex thickness and on LDL oxidation in the sclera, choroid and retina of diet-induced hypercholesterolaemic rabbits. New Zealand male albino rabbits (n 21) were divided into two groups: group 1 (G1; n 11), fed a hypercholesterolaemic diet, and group 2 (G2; n 10), fed a hypercholesterolaemic diet enriched with flaxseed flour. The serum concentrations of total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol, TAG and fasting blood glucose were determined at the start of the experiment and on the day of killing (8th week). Choroid and sclera samples were subjected to haematoxylin-eosin (HE) staining and histomorphometric and immunohistochemical analyses with the anti-oxidised LDL antibody. Sensory retina samples were subjected to an immunohistochemical analysis with the primary monoclonal nitrotyrosine antibody. At the end of the experiment, a significant increase was observed in TC and LDL-C concentrations in G1 rabbits when compared with G2 rabbits (P= 0·008 and P= 0·02, respectively). HE staining revealed a significant increase in choroid-sclera complex thickness in G1 rabbits when compared with G2 rabbits (P< 0·001). Immunohistochemical analysis of choroid and sclera samples with the anti-oxidised LDL marker revealed a significant increase in immunoreactivity in G1 rabbits when compared with G2 rabbits (P< 0·001). Immunohistochemical analysis of sensory retina samples with the anti-nitrotyrosine marker revealed a significant increase in immunoreactivity in G1 rabbits when compared with G2 rabbits (P= 0·002). Flaxseed reduced the choroid-sclera complex thickness of diet-induced hypercholesterolaemic rabbits and the expression of oxidised LDL in the choroid-sclera complex as well as the expression of nitrotyrosine in the sensory retina.

Structural and biomechanical effects of photooxidative collagen cross-linking with photosensitizer riboflavin and 370 nm UVA light on human corneoscleral tissues.

This study quantitatively investigated the immediate effects of a photooxidative collagen cross-linking treatment with photosensitizer riboflavin (RF) and 370 nm UVA light in in vitro human corneoscleral collagen fibrils using histology, thickness, scanning electron microscopy, and atomic force microscopy analyses. Twenty 8 x 2 mm corneoscleral strips were dissected sagittally from donor tissue using a scalpel. Four parameters were investigated, including the density, thickness, adhesion force, and stiffness of corneoscleral tissues before and after the collagen cross-linking treatment. The RFUVA-catalyzed collagen cross-linking treatment led to an increase in the density of both corneal (8%) and scleral (23%) stromal collagens. However, there was no difference in corneoscleral thickness. Furthermore, RFUVA-catalyzed collagen cross-linking treatment led to an increased biomechanical response of corneosclera: 25 and 8% increases in corneoscleral stiffness, and 24 and 22% increases in corneoscleral adhesion force. The collagen cross-linking treatment through RF-sensitized photoreaction may cause structural and biomechanical changes in the collagen fibril network of the cornea and the sclera. This is due to narrowing of the interfibrillar spacing and the stromal edema.

Structural response of human corneal and scleral tissues to collagen cross-linking treatment with riboflavin and ultraviolet A light.

High success rates in clinical trials on keratoconic corneas suggest the possibility of efficient treatment against myopic progression. This study quantitatively investigated the in vitro ultrastructural effects of a photooxidative collagen cross-linking treatment with photosensitizer riboflavin and UVA light in human corneo-scleral collagen fibrils. A total of 30.8 × 2 mm corneo-scleral strips from donor tissue were sagittally dissected using a scalpel. The five analytic parameters namely fibril density, fibril area, corneo-scleral thickness, fibril diameter, and fibril arrangement were investigated before and after riboflavin-UVA-catalyzed collagen cross-linking treatment. Collagen cross-linking effects were measured at the corneo-scleral stroma and were based on clinical corneal cross-linking procedures. The structural response levels were assessed by histology, digital mechanical caliper measurement, scanning electron microscopy, and atomic force microscopy. Riboflavin-UVA-catalyzed collagen cross-linking treatment led to an increase in the area, density, and diameters of both corneal (110, 112, and 103 %) and scleral (133, 133, and 127 %) stromal collagens. It also led to increases in corneal (107 %) and scleral (105 %) thickness. Collagen cross-linking treatment through riboflavin-sensitized photoreaction may cause structural property changes in the collagen fibril network of the cornea and sclera due to stromal edema and interfibrillar spacing narrowing. These changes were particularly prominent in the sclera. This technique can be used to treat progressive keratoconus in the cornea as well as progressive myopia in the sclera. Long-term collagen cross-linking treatment of keratoconic and myopic progression dramatically improves weakened corneo-scleral tissues.

Intraocular distribution of melanin in human, monkey, rabbit, minipig and dog eyes.

The purpose of this study was to quantify the melanin pigment content in sclera, choroid-RPE, and retina, three tissues encountered during transscleral drug delivery to the vitreous, in human, rabbit, monkey, minipig, and dog models. Strain differences were assessed in NZW × NZR F1 and Dutch belted rabbits and Yucatan and Gottingen minipigs. The choroid-RPE and retina tissues were divided into central (posterior pole area) and peripheral (away from posterior pole) regions while the sclera was analyzed without such division. Melanin content in the tissues was analyzed using a colorimetric assay. In all species the rank order for pigment content was: choroid-RPE >retina ≥ sclera, except in humans, where scleral melanin levels were higher than retina and central choroid. The melanin content in a given tissue differed between species. Further, while the peripheral tissue pigment levels tended to be generally higher compared to the central regions, these differences were significant in human in the case of choroid-RPE and in human, monkey, and dogs in the case of retina. Strain difference was observed only in the central choroid-RPE region of rabbits (NZW × NZR F1 >Dutch Belted). Species, strain, and regional differences exist in the melanin pigment content in the tissues of the posterior segment of the eye, with Gottingen minipig being closest to humans among the animals assessed. These differences in melanin content might contribute to differences in drug binding, delivery, and toxicity.

Biomechanical and morphological differences between the sclera canal ring and a peripheral sclera ring in the porcine eye.

AIM: To investigate a possible association between the biomechanical load and unload behaviour and the elastin content of the sclera canal ring (SCR) and a superiorly localized sclera ring (SPS) in the porcine eye. METHODS: Two sclera rings were trephined from each of 40 porcine eyes, one containing the SCR and the other an SPS. The load and the unload curves were measured in the extension range of 0-2.0 mm by a biomaterial tester. Hysteresis was determined from the area enclosed by the loading and unloading curve. Histochemical staining with resorcin-fuchsin and morphometric analysis of paraffin-embedded sections of both rings were performed to detect the area occupied by elastin fibres. RESULTS: At 1 mm extension, the mean load of the SCR was 0.89 ± 0.22 N and that of the SPS 1.13 ± 0.19 N, which was not significantly different between both rings (p > 0.05). Mean hysteresis in the SCR was 1.55 ± 0.30 N × mm and 1.90 ± 0.18 N × mm in the SPS, which was significantly different between both rings (p = 0.01). Mean sclera thickness was 986 µm in the SCR (range: 900-1,060 µm) and 971 µm in the SPS (range: 800-1,200 µm) without a statistically significant difference between both sclera rings (p = 0.78). The area occupied by elastin fibres was 15.5 ± 3.4% in the SCR and 4.5 ± 1.5% in the SPS, which was significantly different between both rings (p = 0.0001). CONCLUSION: Hysteresis in the SCR was significantly lower than in the SPS, indicating a higher elasticity of the SCR in the porcine eye. This effect could be explained by a higher content of elastin in the surrounding ring of the peripapillary optic nerve head providing reversible contraction in cases of intra-ocular pressure variations.

Release profile and transscleral permeation of triamcinolone acetonide loaded nanostructured lipid carriers (TA-NLC): in vitro and ex vivo studies.

Nanostructured lipid carriers (NLC) have been developed for sustained release of triamcinolone acetonide (TA), a corticosteroid commonly indicated for macular edema, neovascularization, and other ocular inflammatory disorders. TA-NLC were prepared by high-pressure homogenization and characterized for in vitro release by dialysis bag. Ex vivo permeation profile was assessed using rabbit sclera isolated and mounted in Franz diffusion cells. TA-NLC were placed in episcleral donor compartment and choroidal side was perfused with HEPES buffer. Tissue sections underwent drug wash-out, following analysis by validated RP-HPLC of drug content and perfused fractions collected over 24 hours. Drug release followed one-order kinetics and permeability studies confirmed that TA is able to diffuse across rabbit sclera in sustained profile, following zero-order kinetics. Strong tissue binding was observed, providing a drug depot. These findings are of potential use when designing future TA therapy strategies for ocular diseases of posterior segment.

Validating a Sclera-Based Smartphone Application for Screening Jaundiced Newborns in Ghana.

OBJECTIVES: Reducing the burden of bilirubin-induced neurologic complications in low-resource countries requires reliable and accessible screening tools. We sought to optimize and validate a sclera-based smartphone application, Neonatal Scleral-Conjunctival Bilirubin (neoSCB), for screening neonatal jaundice. METHODS: Using a cross-sectional design, consecutive eligible infants (aged 0-28 days, in the hospital, not critically ill) were enrolled in Ghana from March 2019 to April 2020. Jaundice screening was performed with neoSCB (Samsung Galaxy S8) to quantify SCB and JM-105 (Dräger) for transcutaneous bilirubin (TcB). Screening values were compared with total serum bilirubin (TSB) measured at the point of care. RESULTS: Overall, 724 infants participated in the optimization and validation phases of the study. The analysis for validation included 336 infants with no previous treatment of jaundice. Single neoSCB image captures identified infants with TSB >14.62 mg/dL (250 µmol/L) with reasonably high sensitivity, specificity, and receiver operating characteristic area under the curve at 0.94 (95% confidence interval [CI], 0.91 to 0.97), 0.73 (95% CI, 0.68 to 0.78), and 0.90, respectively. These findings were comparable to the sensitivity and specificity of JM-105 (0.96 [95% CI, 0.90 to 0.99] and 0.81 [95% CI, 0.76 to 0.86], respectively). The TcB/TSB had a larger correlation coefficient (r = 0.93; P < .01) than SCB/TSB (r = 0.78; P < .01). Performance of both devices was lower in infants with previous phototherapy (n = 231). CONCLUSIONS: The diagnostic performance of neoSCB was comparable to JM-105 and is a potential, affordable, contact-free screening tool for neonatal jaundice.

Pharmacokinetics of pirfenidone after topical administration in rabbit eye.

PURPOSE: Pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) is a new, broad-spectrum agent that has an inhibition effect on the proliferation, migration, and collagen contraction of human Tenon's fibroblasts, and thus modulating the wound healing process of glaucoma filtering surgical site. This study investigated the pharmacokinetics of topically administered pirfenidone (0.5%) in rabbit eyes. METHODS: Pirfenidone solution (50 µl) was instilled into the rabbit's conjunctival sac. The rabbits were quickly sacrificed at 2, 5, 8, 10, 15, 20, 30, 60, 90, and 120 min after the administration and ocular tissues were obtained. The concentrations of pirfenidone in conjunctiva, sclera, cornea, aqueous humor, and vitreous were determined by high performance liquid chromatography. RESULTS: After topical administration, there was wide distribution and fast clearance of pirfenidone among the various ocular tissues. The mean maximum concentrations (C(max)) of pirfenidone in cornea, conjunctiva, sclera, aqueous humor, and vitreous were 9.64 mg/g, 9.62 mg/g, 2.13 mg/g, 34.88 mg/l and 0.52 mg/l, respectively. The half-life for these tissues was 18.26, 34.16, 15.71, 70.91, and 39.48 min, respectively. CONCLUSIONS: Measurable concentrations of pirfenidone are achieved in ocular tissues after topical application in rabbit model. Topical administration of pirfenidone may be an effective approach for modulation of wound healing responses in glaucoma filtration surgical site.

AFM study for morphological and mechanical properties of human scleral surface.

This study examined the structures and the elastic and viscous properties of human scleral collagen fibrils by atomic force microscopy (AFM). Sample preparation was performed to minimize the sources of artifacts for further imaging. To observe the morphological and property characteristics of human scleral surfaces, AFM was used as a microscopic tool. The AFM topography, phase shift and deflection images of the dehydrated scleral collagen fibrils were obtained. The visco-elasticity of collagen fibrils was determined from the force-distance curves of the AFM. Inspection of the fibril surface in high resolution showed that the D-period spacing along the collagen fibrils was clearly evident. The fibril diameter over a scan size of 5 x 5 microm2 was 145.22 +/- 17.78 nm (n = 178) ranging from 98 to 220 nm, and the D-periodicity was 69.14 +/- 14.15 nm (n = 189), which is similar to the normal 67 nm D-periodicity. Force-distance analysis indicated that human scleral collagen had comparatively high adhesion force and elasticity, to protect the eye from external trauma and to withstand the expansive force made by the intraocular pressure.

[Sclera of the glaucomatous eye: physicochemical analysis].

It has been shown that the collagen content and denaturation temperature in scleral tissue increase during the development of glaucoma whereas the fluorescence intensity does not change. These effects may be related to the disturbance of collagen catabolism.

[Biochemical, structural and biomechanical features of human scleral matrix in primary open-angle glaucoma].

The goal of the investigation was to study the changes of scleral matrix collagen in progressing primary open-angle glaucoma (POAG). In scleral samples of 16 patients aged 52-81 years with different stages of POAG amino acid composition, collagen content and cross-linking ratio characterizing tissue rigidity were estimated. Collagen I was found to be the dominant type in sclera of glaucomatous and normal eyes, though as POAG progressing the content and cross-linking increase, that underlies the scleral rigidity increase and permeability decrease. Similar ratio of collagen cross-linking with age and glaucoma progression give evidence of specific pathologic changes of the dominant matrix component, that may be one of significant pathogenic factor of POAG.

Two-photon microscopy of deep intravital tissues and its merits in clinical research.

Multiphoton excitation laser scanning microscopy, relying on the simultaneous absorption of two or more photons by a molecule, is one of the most exciting recent developments in biomedical imaging. Thanks to its superior imaging capability of deeper tissue penetration and efficient light detection, this system becomes more and more an inspiring tool for intravital bulk tissue imaging. Two-photon excitation microscopy including 2-photon fluorescence and second harmonic generated signal microscopy is the most common multiphoton microscopic application. In the present review we take diverse ocular tissues as intravital samples to demonstrate the advantages of this approach. Experiments with registration of intracellular 2-photon fluorescence and extracellular collagen second harmonic generated signal microscopy in native ocular tissues are focused. Data show that the in-tandem combination of 2-photon fluorescence and second harmonic generated signal microscopy as two-modality microscopy allows for in situ co-localization imaging of various microstructural components in the whole-mount deep intravital tissues. New applications and recent developments of this high technology in clinical studies such as 2-photon-controlled drug release, in vivo drug screening and administration in skin and kidney, as well as its uses in tumourous tissues such as melanoma and glioma, in diseased lung, brain and heart are additionally reviewed. Intrinsic emission two-modal 2-photon microscopy/tomography, acting as an efficient and sensitive non-injurious imaging approach featured by high contrast and subcellular spatial resolution, has been proved to be a promising tool for intravital deep tissue imaging and clinical studies. Given the level of its performance, we believe that the non-linear optical imaging technique has tremendous potentials to find more applications in biomedical fundamental and clinical research in the near future.

Scleral Lens Sensor for Ocular Electrolyte Analysis.

The quantitative analysis of tear analytes in point-of-care settings can enable early diagnosis of ocular diseases. Here, a fluorescent scleral lens sensor is developed to quantitatively measure physiological levels of pH, Na+ , K+ , Ca2+ , Mg2+ , and Zn2+ ions. Benzenedicarboxylic acid, a pH probe, displays a sensitivity of 0.12 pH units within pH 7.0-8.0. Crown ether derivatives exhibit selectivity to Na+ and K+ ions within detection ranges of 0-100 and 0-50 mmol L-1 , and selectivities of 15.6 and 8.1 mmol L-1 , respectively. A 1,2 bis(o-aminophenoxy)ethane-N,N,-N',N'-tetraacetic-acid-based probe allows Ca2+ ion sensing with 0.02-0.05 mmol L-1 sensitivity within 0.50-1.25 mmol L-1 detection range. 5-Oxazolecarboxylic acid senses Mg2+ ions, exhibiting a sensitivity of 0.10-0.44 mmol L-1 within the range of 0.5-0.8 mmol L-1 . The N-(2-methoxyphenyl)iminodiacetate Zn2+ ion sensor has a sensitivity of 1 µmol L-1 within the range of 10-20 µmol L-1 . The fluorescent sensors are subsequently multiplexed in the concavities of an engraved scleral lens. A handheld ophthalmic readout device comprising light-emitting diodes (LEDs) and bandpass filters is fabricated to excite as well as read the scleral sensor. A smartphone camera application and an user interface are developed to deliver quantitative measurements with data deconvolution. The ophthalmic system enables the assessment of dry eye severity stages and the differentiation of its subtypes.

Ocular swabs on exhumed bodies: An alternative to the collection of "classical" tissue samples in forensic genetics.

We present the results obtained on DNA extracted from ocular (scleral/corneal) swabs collected from exhumed bodies at different times of burial. To our knowledge, there are no publications in the scientific forensic literature dealing with sclera/cornea as a source of DNA in the forensic laboratory. The obtained results demonstrate that cornea/sclera swabbing might be a promising alternative to the sampling of other tissues for DNA extraction even in highly putrefied bodies.

Cytosine-functionalized bioinspired hydrogels for ocular delivery of antioxidant transferulic acid.

Contact lenses (CLs) are being pointed out as feasible platforms for controlled delivery of ophthalmic drugs. Bioinspired strategies may endow CLs with affinity for a given drug by mimicking its physiological receptor using adequate functional monomers and tuning their conformation in the space through the molecular imprinting technology. However, there are some active substances, such as efficient antioxidant agents, that cannot be used as templates because they degrade during polymerization or even hinder the polymerization itself. Therefore, the development of CLs able to sustain the release of antioxidants for the prevention and/or treatment of several age-related and light-induced eye diseases has not been explored yet. Searching for an alternative bioinspired strategy, the present work relies on the fact that some drugs owe their therapeutic action to their ability to interact with nucleotides that build up DNA and RNA. Thus, the aim of this work was to design hydrogels functionalized with the nitrogenous base cytosine for the controlled uptake and release of transferulic acid (TA) having a complementary chemical structure in terms of hydrogen bonding and π-π stacking ability. Hydrogels were prepared from mixtures of 2-hydroxyethyl methacrylate (HEMA), glycidyl methacrylate (GMA) and ethyleneglycolphenylether methacrylate (EGPEM). GMA was used as a bridge to immobilize cytosine after hydrogel synthesis, while EGPEM was added to reinforce hydrophobic interactions with TA. The hydrogels were characterized in terms of suitability to be used as CLs (swelling, light transmission, mechanical properties, biocompatibility) and capability to host TA and sustain its release in lachrymal fluid while maintaining the antioxidant activity. Relevantly, the bioinspired CLs favored TA accumulation in cornea and sclera tissues.

Stabilization of scleral collagen by glycerol aldehyde cross-linking.

The paper aims at the evaluation of prospects for using glyceraldehyde as a cross-linking agent for the scleral tissue. Stability parameters (denaturation temperature, Young's modulus, ultimate tensile stress, proteolytic resistance) and analytical parameter (fluorescence intensity) were determined during the glycation process of isolated rabbit sclera. The analysis of fluorescence spectral characteristic provided information about some glycation products. The glyceraldehyde treatment was resulted in a significant increase in thermal stability, proteolytic resistance and improvement of biomechanical characteristics (Young's modulus, ultimate tensile stress). Unique properties of the reaction between scleral collagen and glyceraldehyde are observed at short cross-linking times. The appearance of intermediate collagen fraction with lowest thermal and proteolytic stability was detected.

Cassette analysis of eight beta-blockers in bovine eye sclera, choroid-RPE, retina, and vitreous by liquid chromatography-tandem mass spectrometry.

A simple, selective, and sensitive LC-MS/MS method was developed for the simultaneous extraction and determination of eight beta-blockers (atenolol, sotalol, nadolol, pindolol, timolol, metoprolol, betaxolol and propranolol) in various bovine eye tissues including sclera, choroid-RPE, retina, and vitreous. The analytes were extracted by liquid-liquid extraction after samples were alkalinized with 2% NaOH solution in water. The chromatographic separation was performed on a Hypersil-ODS C18 column (100 mm x 2.1 mm, 3.9 microm) using a gradient mixture of (A) 5 mM ammonium formate in water (pH 3.5 adjusted with formic acid) and (B) acetonitrile:methanol (75:25) containing 0.02% triethyl amine (pH 4.0; adjusted with formic acid) as mobile phase at a flow rate of 0.4 ml/min. The compounds were ionized in the positive electrospray ionization (ESI) mode and detected in the multiple reaction monitoring (MRM) mode. The average recoveries in all four eye tissues for all beta-blockers were >82%, except for sotalol (>51%). The matrix effect for beta-blockers ranged from 81 to 110% in the four eye tissues. This analytical method was validated and applied successfully for simultaneous quantification of the beta-blockers in sclera after tissue exposure using cassette dosing method. The calibration curve was linear in the range of 10-2000 ng/ml for all analytes, with the correlation coefficient >0.996. Intra-day and inter-day precision (% CV) was less than 15%, and accuracy ranged from 85 to 110% for all analytes. Scleral uptake was the lowest for sotalol and atenolol, two hydrophilic beta-blockers, and the highest for propranolol, a lipophilic beta-blocker.

[Investigation of scleral fibronectin in primary open-angle glaucoma: immunohistochemical study].

The present investigation was first to study scleral fibronectin (FN) levels in primary open-angle glaucoma, by applying an immunohistochemical study. The enhanced accumulation of this glycoprotein was found in the deep and inner scleral layers. Active FN expression was ascertained to correspond to the scleral morphological changes characteristic for glaucoma (garneting of collagenous fibers, accumulation of free glycosamine glycans) and to be of focal nature. The findings show the role of FN in the mechanisms of scleral destruction in primary open-angle glaucoma.

Microstructure-based numerical simulation of the mechanical behaviour of ocular tissue.

This paper aims to present a novel full-eye biomechanical material model that incorporates the characteristics of ocular tissues at microstructural level, and use the model to analyse the age-related stiffening in tissue behaviour. The collagen content in ocular tissues, as obtained using X-ray scattering measurements, was represented by sets of Zernike polynomials that covered both the cornea and sclera, then used to reconstruct maps of collagen fibril magnitude and orientation on the three-dimensional geometry of the eye globe. Fine-mesh finite-element (FE) models with eye-specific geometry were built and supported by a user-defined material model (UMAT), which considered the regional variation of fibril density and orientation. The models were then used in an iterative inverse modelling study to derive the material parameters that represent the experimental behaviour of ocular tissues from donors aged between 50 and 90 years obtained in earlier ex vivo studies. Sensitivity analysis showed that reducing the number of directions that represented the anisotropy of collagen fibril orientation at each X-ray scattering measurement point from 180 to 16 would have limited and insignificant effect on the FE solution (0.08%). Inverse analysis resulted in material parameters that provided a close match with experimental intraocular pressure-deformation behaviour with a root mean square of error between 3.6% and 4.3%. The results also demonstrated a steady increase in mechanical stiffness in all ocular regions with age. A constitutive material model based on distributions of collagen fibril density and orientation has been developed to enable the accurate representation of the biomechanical behaviour of ocular tissues. The model offers a high level of control of stiffness and anisotropy across ocular globe, and therefore has the potential for use in planning surgical and medical procedures.