Various cross-linking methods inhibit the collagenase I degradation of rabbit scleral tissue.

BACKGROUND: Collagen cross-linking of the sclera is a promising approach to strengthen scleral rigidity and thus to inhibit eye growth in progressive myopia. Additionally, cross-linking might inhibit degrading processes in idiopathic melting or in ocular inflammatory diseases of the sclera. Different cross-linking treatments were tested to increase resistance to enzymatic degradation of the rabbit sclera. METHODS: Scleral patches from rabbit eyes were cross-linked using paraformaldehyde, glutaraldehyde or riboflavin combined with UV-A-light or with blue light. The patches were incubated with collagenase I (MMP1) for various durations up to 24 h to elucidate differences in scleral resistance to enzymatic degradation. Degraded protein components in the supernatant were detected and quantified using measurements of Fluoraldehyde o-Phthaldialdehyde (OPA) fluorescence. RESULTS: All cross-linking methods reduced the enzymatic degradation of rabbit scleral tissue by MMP1. Incubation with glutaraldehyde (1%) and paraformaldehyde (4%) caused nearly a complete inhibition of enzymatic degradation (down to 7% ± 2.8 of digested protein compared to control). Cross-linking with riboflavin/UV-A-light reduced the degradation by MMP1 to 62% ± 12.7 after 24 h. Cross-linking with riboflavin/blue light reduced the degradation by MMP1 to 77% ± 13.5 after 24 h. No significant differences could be detected comparing different light intensities, light exposure times or riboflavin concentrations. CONCLUSIONS: The application of all cross-linking methods increased the resistance of rabbit scleral tissue to MMP1-degradation. Especially, gentle cross-linking with riboflavin and UV-A or blue light might be a clinical approach in future.

Comparison of cellular localisation of the Ca2+ -binding proteins calbindin, calretinin and parvalbumin in the retina of four different Macaca species.

Ca2+ -binding proteins are differentially expressed in the nervous system; their functional role often remains unclear. This immunohistochemical study aimed at characterising and comparing the expression pattern of the Ca2+ -binding proteins calbindin (Calb), calretinin (Calr) and parvalbumin (Parv) in the retina of four species of macaque monkeys: Macaca fascicularis (cynomolgus macaque), M. mulatta (rhesus macaque), M. thibetana (Tibetan macaque) and M. fuscata (Japanese macaque). Calb was found in cone photoreceptors and in a subset of bipolar cells. Calr was expressed in a subpopulation of amacrine cells. Parv was present in horizontal and ganglion cells. In addition, Müller cells were stained using antibodies against the specific marker cellular retinaldehyde-binding protein (CRALBP). Immunostainings were used for calculation of the density of different cell populations. The expression pattern was similar between the examined species and between retinal regions.

The ultrastructure of rabbit sclera after scleral crosslinking with riboflavin and blue light of different intensities.

PURPOSE: We aimed to determine the ultrastructural changes of collagen fibrils and cells in the rabbit sclera after scleral crosslinking using riboflavin and blue light of different intensities. Scleral crosslinking is known to increase scleral stiffness and may inhibit the axial elongation of progressive myopic eyes. METHODS: The equatorial parts of the sclera of one eye of six adult albino rabbits were treated with topical riboflavin solution (0.5 %) followed by irradiation with blue light (200, 400, 650 mW/cm(2)) for 20 min. After 3 weeks, the ultrastructure of scleral cells and the abundance of small- (10-100 nm) and large-diameter (>100 nm) collagen fibrils in fibril bundles of different scleral layers were examined with electron microscopy. RESULTS: In the scleral stroma of control eyes, the thickness of collagen fibrils showed a bimodal distribution. The abundance of small-diameter collagen fibrils decreased from the inner towards the outer sclera, while the amount of large-diameter fibrils and the scleral collagen content did not differ between different stroma layers. Treatment with riboflavin and blue light at 200 mW/cm(2) did not induce ultrastructural changes of cells and collagen fibrils in the scleral stroma. Treatment with blue light of higher intensities induced scleral cell activation in a scleral layer-dependent manner. In addition, outer scleral layers contained phagocytes that engulfed collagen fibrils and erythrocytes. Blue light of the highest intensity induced a reduction of the scleral collagen content, a decreased abundance of large-diameter collagen fibrils, and an increased amount of small-diameter fibrils in the whole scleral stroma. CONCLUSIONS: The data indicate that in rabbits, scleral crosslinking with riboflavin and blue light of 200 mW/cm(2) for 20 min is relatively safe and does not induce ultrastructural alterations of scleral cells and of the collagen composition of the scleral stroma. Irradiation with blue light of intensities between 200 and 400 mW/cm(2) induces scleral cell activation, which may contribute to scleral scarring and stiffening. Higher intensities cause scleritis.

Scleral cross-linking by riboflavin and blue light application in young rabbits: damage threshold and eye growth inhibition.

BACKGROUND: Scleral cross-linking (SXL) by riboflavin and light application has been introduced as a possible treatment to increase scleral tissue stiffness and to inhibit excessive axial elongation of highly myopic eyes. We evaluated an ocular tissue damage threshold for blue light irradiation, and used SXL treatment to induce eye growth inhibition. METHODS: The sclera of 3-week-old rabbits (39 pigmented and 15 albino rabbits) were treated with different blue light intensities (450 ± 50 nm) and riboflavin. Alterations and a damage threshold were detected in ocular tissues by means of light microscopy and immunohistochemistry. The influence of SXL on the eye growth was examined in 21 young rabbits and was measured by using A-scan ultrasonography, micrometer caliper, and for selected eyes additionally by MR imaging. RESULTS: Light microscopic examinations demonstrated degenerative changes in ocular tissue after irradiation with blue light intensities above 400 mW/cm(2) (with and without riboflavin application). Therefore, that light intensity was defined as the damage threshold. Tissue alteration in retina, choroid, and sclera and activation of retinal microglia cells and Müller cells could be earlier observed at blue light intensities of 150 and 200 mW/cm(2). Albino rabbits were less sensitive to this SXL treatment. A significant reduction of the eye growth could be detected by SXL treatment with the minimal efficient blue light intensity of 15 mW/cm(2) and maintained stable for 24 weeks. CONCLUSIONS: SXL with riboflavin and blue light intensities below a defined damage threshold can induce a long lasting growth inhibitory effect on young rabbit eyes. Therefore, SXL might be a realistic approach to inhibit eye elongation in highly myopic eyes.

Damage threshold in adult rabbit eyes after scleral cross-linking by riboflavin/blue light application.

Several scleral cross-linking (SXL) methods were suggested to increase the biomechanical stiffness of scleral tissue and therefore, to inhibit axial eye elongation in progressive myopia. In addition to scleral cross-linking and biomechanical effects caused by riboflavin and light irradiation such a treatment might induce tissue damage, dependent on the light intensity used. Therefore, we characterized the damage threshold and mechanical stiffening effect in rabbit eyes after application of riboflavin combined with various blue light intensities. Adult pigmented and albino rabbits were treated with riboflavin (0.5 %) and varying blue light (450 ± 50 nm) dosages from 18 to 780 J/cm(2) (15 to 650 mW/cm(2) for 20 min). Scleral, choroidal and retinal tissue alterations were detected by means of light microscopy, electron microscopy and immunohistochemistry. Biomechanical changes were measured by shear rheology. Blue light dosages of 480 J/cm(2) (400 mW/cm(2)) and beyond induced pathological changes in ocular tissues; the damage threshold was defined by the light intensities which induced cellular degeneration and/or massive collagen structure changes. At such high dosages, we observed alterations of the collagen structure in scleral tissue, as well as pigment aggregation, internal hemorrhages, and collapsed blood vessels. Additionally, photoreceptor degenerations associated with microglia activation and macroglia cell reactivity in the retina were detected. These pathological alterations were locally restricted to the treated areas. Pigmentation of rabbit eyes did not change the damage threshold after a treatment with riboflavin and blue light but seems to influence the vulnerability for blue light irradiations. Increased biomechanical stiffness of scleral tissue could be achieved with blue light intensities below the characterized damage threshold. We conclude that riboflavin and blue light application increased the biomechanical stiffness of scleral tissue at blue light energy levels below the damage threshold. Therefore, applied blue light intensities below the characterized damage threshold might define a therapeutic blue light tolerance range.

Dose-dependent collagen cross-linking of rabbit scleral tissue by blue light and riboflavin treatment probed by dynamic shear rheology.

PURPOSE: To determine the visco-elastic properties of isolated rabbit scleral tissue and dose-dependent biomechanical and morphological changes after collagen cross-linking by riboflavin/blue light treatment. MATERIAL: Scleral patches from 87 adult albino rabbit eyes were examined by dynamic shear rheology. Scleral patches were treated by riboflavin and different intensities of blue light (450 nm), and the impact on the visco-elastic properties was determined by various rheological test regimes. The relative elastic modulus was calculated from non-treated and corresponding treated scleral patches, and treatments with different blue light intensities were compared. RESULTS: Shear rheology enables us to study the material properties of scleral tissue within physiological relevant parameters. Cross-linking treatment increased the viscous as well as the elastic modulus and changed the ratio of the elastic versus viscous proportion in scleral tissue. Constant riboflavin application combined with different blue light intensities from 12 mW/cm(2) up to 100 mW/cm(2) increased the relative elastic modulus of scleral tissue by factors up to 1.8. Further enhancement of the applied light intensity caused a decline of the relative elastic modulus. This might be due to destructive changes of the collagen bundle structure at larger light intensities, as observed by histological examination. CONCLUSION: Collagen cross-linking by riboflavin/blue light application increases the biomechanical stiffness of the sclera in a dose-dependent manner up to certain light intensities. Therefore, this treatment might be a suitable therapeutic approach to stabilize the biomechanical properties of scleral tissue in cases of pathological eye expansion.

Optical coherence tomography as a diagnostic tool for retinal pathologies in avian ophthalmology.

PURPOSE: Optical coherence tomography (OCT) is an established diagnostic tool for retinal pathologies in human eyes and has been adapted to small animal models. However, there have been only a few attempts to use OCT for examination of avian eyes, and little is known about structural details of healthy or pathologically affected retinas in living birds. METHODS: We used SD-OCT (high-resolution spectral domain OCT) to investigate eyes of various avian species including birds of prey. The birds were anesthetized by isoflurane application during OCT examination. Eyes of a common buzzard (Buteo buteo) could be used for a comparative analysis of OCT images and histologic/immunohistochemical examinations. RESULTS: We investigated 45 wild and domestic birds (25 different species, 40 g-7.7 kg body mass) without and with diverse pathologic indications (e.g., body or head trauma). Animals were generally and ophthalmologically examined, and the diagnostic findings of direct ophthalmoscopy and OCT were compared. The OCT examination revealed an increased number of animals with clinical findings and allowed a detailed assessment of structural changes in retinal and choroidal tissue compared to simple direct ophthalmoscopy. Common findings were retinal and choroidal degeneration, retinal detachment, choroidal schisis, drusen, and drusenoid changes. Histologic and immunohistochemical analysis of retinal tissue confirmed the findings of the OCT examination. CONCLUSIONS: Spectral domain OCT of eyes in living birds is applicable and useful as a diagnostic tool in veterinary clinical practices and for vision research in general. Optical coherence tomography improves the quality of the common assessment methods in avian ophthalmology, and expands the diagnostic possibilities with respect to identification and prognosis of diseases. This will be particularly important for hereditary retinal defects, especially of precious breeding individuals, or estimation of treatment success in traumatized wild birds with the aim of release back into the wild.

Reactive glial cells: increased stiffness correlates with increased intermediate filament expression.

Increased stiffness of reactive glial cells may impede neurite growth and contribute to the poor regenerative capabilities of the mammalian central nervous system. We induced reactive gliosis in rodent retina by ischemia-reperfusion and assessed intermediate filament (IF) expression and the viscoelastic properties of dissociated single glial cells in wild-type mice, mice lacking glial fibrillary acidic protein and vimentin (GFAP(-/-)Vim(-/-)) in which glial cells are consequently devoid of IFs, and normal Long-Evans rats. In response to ischemia-reperfusion, glial cells stiffened significantly in wild-type mice and rats but were unchanged in GFAP(-/-)Vim(-/-) mice. Cell stiffness (elastic modulus) correlated with the density of IFs. These results support the hypothesis that rigid glial scars impair nerve regeneration and that IFs are important determinants of cellular viscoelasticity in reactive glia. Thus, therapeutic suppression of IF up-regulation in reactive glial cells may facilitate neuroregeneration.

Different profiles of buprenorphine-induced analgesia and antihyperalgesia in a human pain model.

Different mechanisms were proposed for opioid-induced analgesia and antihyperalgesia, which might result in different pharmacodynamics. To address this issue, the time course of analgesic and antihyperalgesic effects of intravenous (i.v.) and sublingual (s.l.) buprenorphine was assessed in an experimental human pain model. Fifteen volunteers were enrolled in this randomized, double-blind, and placebo controlled cross-over study. The magnitude of pain and the area of secondary hyperalgesia following transcutaneous stimulation were repetitively assessed before and up to 150 min after administration of (1) 0.15 mg buprenorphine i.v. and placebo pill s.l., (2) 0.2 mg buprenorphine s.l. and saline 0.9% i.v. or (3) saline 0.9% i.v. and placebo pill s.l. as a control. The sessions were separated by 2 week wash-out periods. For both applications of buprenorphine the antihyperalgesic effects were more pronounced as compared to the analgesic effects (66+/-9 vs. 26+/-5% and 43+/-10 vs. 10+/-6%, for i.v. and s.l. application, respectively). This contrasts the pattern for the intravenous administration of pure mu-receptor agonists in the same model in which the antihyperalgesic effects are weaker. The apparent bioavailability of buprenorphine s.l. as compared to buprenorphine i.v. was 58% with a 15.8 min later onset of antinociceptive effects. The half-life of buprenorphine-induced analgesic and antihyperalgesic effects were 171 and 288 min, respectively. In contrast to pure mu-receptor agonists, buprenorphine exerts a lasting antihyperalgesic effect in our model. It will be of major clinical interest whether this difference will translate into improved treatment of pain states dominated by central sensitization.

The cyclooxygenase isozyme inhibitors parecoxib and paracetamol reduce central hyperalgesia in humans.

Non-steroidal antiinflammatory drugs (NSAIDs) are known to induce analgesia mainly via inhibition of cyclooxygenase (COX). Although the inhibition of COX in the periphery is commonly accepted as the primary mechanism, experimental and clinical data suggest a potential role for spinal COX-inhibition to produce antinociception and reduce hypersensitivity. We used an experimental model of electrically evoked pain and hyperalgesia in human skin to determine the time course of central analgesic and antihyperalgesic effects of intravenous parecoxib and paracetamol (acetaminophen). Fourteen subjects were enrolled in this randomized, double blind, and placebo controlled cross-over study. In three sessions, separated by 2-week wash-out periods, the subjects received intravenous infusions of 40 mg parecoxib, 1000 mg paracetamol, or placebo. The magnitude of pain and areas of pinprick-hyperalgesia and touch evoked allodynia were repeatedly assessed before, and for 150 min after the infusion. While pain ratings were not affected, parecoxib as well as paracetamol significantly reduced the areas of secondary hyperalgesia to pinprick and touch. In conclusion, our results provide clear experimental evidence for the existence of central antihyperalgesia induced by intravenous infusion of two COX inhibitors, parecoxib and paracetamol. Since the electrical current directly stimulated the axons, peripheral effects of the COX inhibitors on nociceptive nerve endings cannot account for the reduction of hyperalgesia. Thus, besides its well-known effects on inflamed peripheral tissues, inhibition of central COX provides an important mechanism of NSAID-mediated antihyperalgesia in humans.