Sergio Ortiz

Induction of COX-2 and PGE2 biosynthesis by IL-1β is mediated by PKC and mitogen-activated protein kinases in murine astrocytes

Interleukin‐1 (IL‐1) is an important mediator of immunoinflammatory responses in the brain. In the present study, we examined whether prostaglandin E2 (PGE2) production after IL‐1β stimulation is dependent upon activation of protein kinases in astroglial cells. Astrocyte cultures stimulated with IL‐1β or the phorbol ester, PMA significantly increased PGE2 secretion. The stimulatory action of IL‐1β on PGE2 production was totally abolished by NS‐398, a specific inhibitor of cyclo‐oxygenase‐2 activity, as well as by the protein synthesis inhibitor cycloheximide, and the glucocorticoid dexamethasone. Furthermore, IL‐1β induced the expression of COX‐2 mRNA. This occurred early at 2 h, with a maximum at 4 h and declined at 12 h. IL‐1 β treatment also induced the expression of COX‐2 protein as determined by immunoblot analysis. In that case the expression of the protein remained high at least up to 12 h. Treatment of cells with protein kinase C inhibitors (H‐7, bisindolylmaleimide and calphostin C) inhibited IL‐1β stimulation of PGE2. In addition, PKC‐depleted astrocyte cultures by overnight treatment with PMA no longer responded to PMA or IL‐1. The ablation of the effects of PMA and IL‐1β on PGE2 production, likely results from down‐regulation of phorbol ester sensitive‐PKC isoenzymes. Immunoblot analysis demonstrated the translocation of the conventional isoform cPKC‐α from cytosol to membrane following treatment with IL‐1β. In addition, IL‐1β treatment led to activation of extracellular signal‐regulated kinase (ERK1/2) and p38 subgroups of MAP kinases in astroglial cells. Interestingly, the inhibition of ERK kinase with PD 98059, as well as the inhibition of p38 MAPK with SB 203580, prevented IL‐1β‐induced PGE2 release. ERK1/2 activation by IL‐1β was sensitive to inhibition by the PKC inhibitor bisindolylmaleimide suggesting that ERK phosphorylation is a downstream signal of PKC activation. These results suggest key roles for PKC as well as for ERK1/2 and p38 MAP kinase cascades in the biosynthesis of PGE2, likely by regulating the induction of cyclo‐oxygenase‐2, in IL‐1β‐stimulated astroglial cells.

Optical distortion correction in Optical Coherence Tomography for quantitative ocular anterior segment by three-dimensional imaging

A method for three-dimensional 3-D optical distortion (refraction) correction on anterior segment Optical Coherence Tomography (OCT) images has been developed. The method consists of 3-D ray tracing through the different surfaces, following denoising, segmentation of the surfaces, Delaunay representation of the surfaces, and application of fan distortion correction. The correction has been applied theoretically to realistic computer eye models, and experimentally to OCT images of: an artificial eye with a Polymethyl Methacrylate (PMMA) cornea and an intraocular lens (IOL), an enucleated porcine eye, and a human eye in vivo obtained from two OCT laboratory set-ups (time domain and spectral). Data are analyzed in terms of surface radii of curvature and asphericity. Comparisons are established between the reference values for the surfaces (nominal values in the computer model; non-contact profilometric measurements for the artificial eye; Scheimpflug imaging for the real eyes in vivo and vitro). The results from the OCT data were analyzed following the conventional approach of dividing the optical path by the refractive index, after application of 2-D optical correction, and 3-D optical correction (in all cases after fan distortion correction). The application of 3-D optical distortion correction increased significantly both the accuracy of the radius of curvature estimates and particularly asphericity of the surfaces, with respect to conventional methods of OCT image analysis. We found that the discrepancies of the radii of curvature estimates from 3-D optical distortion corrected OCT images are less than 1% with respect to nominal values. Optical distortion correction in 3-D is critical for quantitative analysis of OCT anterior segment imaging, and allows accurate topography of the internal surfaces of the eye.

Optical coherence tomography for quantitative surface topography

We address the measurement and correction of fan distortion in optical coherence tomography (OCT). This effect arises from the scanning system configuration and prevents one, in general, from obtaining quantitative topographic data from OCT. Computer simulations allowed us to quantify the effect and evaluate its dependence on the scanning mirror separation and design of the collimating lens, as well as to estimate the optimal axial position of that lens to minimize the fan distortion. We also developed a numerical algorithm based on 3-D ray propagation for the correction of the residual fan distortion. The effect was studied experimentally using a custom developed time-domain OCT in a Michelson configuration provided with a confocal channel, and the accuracy of the fan distortion correction algorithm tested on samples of known dimensions (flat surfaces and spherical lenses). With a proper calibration of the system with use of an onfocal channel, this algorithm makes it possible for time-domain OCT devices to be used as topographers.

Three-dimensional reconstruction of the crystalline lens gradient index distribution from OCT imaging

We present an optimization method to retrieve the gradient index (GRIN) distribution of the in-vitro crystalline lens from optical path difference data extracted from OCT images. Three-dimensional OCT images of the crystalline lens are obtained in two orientations (with the anterior surface up and posterior surface up), allowing to obtain the lens geometry. The GRIN reconstruction method is based on a genetic algorithm that searches for the parameters of a 4-variable GRIN model that best fits the distorted posterior surface of the lens. Computer simulations showed that, for noise of 5 μm in the surface elevations, the GRIN is recovered with an accuracy of 0.003 and 0.010 in the refractive indices of the nucleus and surface of the lens, respectively. The method was applied to retrieve three-dimensionally the GRIN of a porcine crystalline lens in vitro. We found a refractive index ranging from 1.362 in the surface to 1.443 in the nucleus of the lens, an axial exponential decay of the GRIN profile of 2.62 and a meridional exponential decay ranging from 3.56 to 5.18. The effect of GRIN on the aberrations of the lens also studied. The estimated spherical aberration of the measured porcine lens was 2.87 μm assuming a homogenous equivalent refractive index, and the presence of GRIN shifted the spherical aberration toward negative values (-0.97 μm), for a 6-mm pupil.

Corneal topography from spectral optical coherence tomography (sOCT)

We present a method to obtain accurate corneal topography from a spectral optical coherence tomography (sOCT) system. The method includes calibration of the device, compensation of the fan (or field) distortion introduced by the scanning architecture, and image processing analysis for volumetric data extraction, segmentation and fitting. We present examples of three-dimensional (3-D) surface topography measurements on spherical and aspheric lenses, as well as on 10 human corneas in vivo. Results of sOCT surface topography (with and without fan-distortion correction) were compared with non-contact profilometry (taken as reference) on a spherical lens, and with non-contact profilometry and state-of-the art commercial corneal topography instruments on aspheric lenses and on subjects. Corneal elevation maps from all instruments were fitted by quadric surfaces (as well as by tenth-order Zernike polynomials) using custom routines. We found that the discrepancy in the estimated radius of curvature from nominal values in artificial corneas decreased from 4.6% (without fan distortion correction) to 1.6% (after fan distortion correction), and the difference in the asphericity decreased from 130% to 5%. In human corneas, the estimated corneal radius of curvature was not statistically significantly different across instruments. However, a Bland-Altman analysis showed consistent differences in the estimated asphericity and corneal shape between sOCT topographies without fan distortion correction and the rest of the measurements.

In vivo human crystalline lens topography

Custom high-resolution high-speed anterior segment spectral domain optical coherence tomography (OCT) was used to characterize three-dimensionally (3-D) the human crystalline lens in vivo. The system was provided with custom algorithms for denoising and segmentation of the images, as well as for fan (scanning) and optical (refraction) distortion correction, to provide fully quantitative images of the anterior and posterior crystalline lens surfaces. The method was tested on an artificial eye with known surfaces geometry and on a human lens in vitro, and demonstrated on three human lenses in vivo. Not correcting for distortion overestimated the anterior lens radius by 25% and the posterior lens radius by more than 65%. In vivo lens surfaces were fitted by biconicoids and Zernike polynomials after distortion correction. The anterior lens radii of curvature ranged from 10.27 to 14.14 mm, and the posterior lens radii of curvature ranged from 6.12 to 7.54 mm. Surface asphericities ranged from −0.04 to −1.96. The lens surfaces were well fitted by quadrics (with variation smaller than 2%, for 5-mm pupils), with low amounts of high order terms. Surface lens astigmatism was significant, with the anterior lens typically showing horizontal astigmatism (Z22 ranging from −11 to −1 µm) and the posterior lens showing vertical astigmatism (Z22 ranging from 6 to 10 µm).

Full OCT anterior segment biometry: an application in cataract surgery.

In vivo three-dimensional (3-D) anterior segment biometry before and after cataract surgery was analyzed by using custom high-resolution high-speed anterior segment spectral domain Optical Coherence Tomography (OCT). The system was provided with custom algorithms for denoising, segmentation, full distortion correction (fan and optical) and merging of the anterior segment volumes (cornea, iris, and crystalline lens or IOL), to provide fully quantitative data of the anterior segment of the eye. The method was tested on an in vitro artificial eye with known surfaces geometry at different orientations and demonstrated on an aging cataract patient in vivo. Biometric parameters CCT, ACD/ILP, CLT/ILT Tilt and decentration are retrieved with a very high degree of accuracy. IOL was placed 400 μm behind the natural crystalline lens, The IOL was aligned with a similar orientation of the natural lens (2.47 deg superiorly), but slightly lower amounts (0.77 deg superiorly). The IOL was decentered superiorly (0.39 mm) and nasally (0.26 mm).

Theiler's virus infection induces the expression of cyclooxygenase-2 in murine astrocytes: Inhibition by the anti-inflammatory cytokines interleukin-4 and interleukin-10

Theilers murine encephalomyelitis virus (TMEV) causes an acute encephalomyelitis followed by a persistent infection of the central nervous system (CNS) resulting in a chronic inflammation and axonal demyelination in susceptible strains of mice. The pathogenesis of TMEV-induced demyelinating disease remains unknown, but infection of brain glial cells is a critical factor for virus persistence in the CNS. In the present study we investigated the effects of the anti-inflammatory cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10) on the production of inflammatory mediators, such as prostaglandins, after infection of primary astroglial SJL/J murine cultures with TMEV. This infection resulted in a time-dependent transcription of the gene encoding cyclooxygenase-2 (COX-2) and an increased production of prostaglandin E2 (PGE(2)). Both, IL-4 but mainly, IL-10 (1 and 10 ng/ml) decreased the TMEV-induced expression of COX-2 as well as the synthesis of PGE(2). Interestingly, treatment with IL-10 completely abrogated COX-2 induction. The molecular mechanisms involved in the regulation of COX-2 expression by TMEV are unknown, but the effects of anti-inflammatory cytokines may involve the inhibition of the transcription factor nuclear factor B activity and lead to strategies capable of interrupting the inflammatory cascade triggered by TMEV in brain glial cells.

Behavioural and gene transcription alterations induced by spontaneous cannabinoid withdrawal in mice.

This study examined behavioural signs that occur during tolerance development to cannabinoid treatment and hormonal and gene expression alterations induced by spontaneous cannabinoid withdrawal in mice. Tolerance to CP‐55,940 treatment developed for hypothermia, ambulatory and exploratory locomotor activity. Cessation of cannabinoid treatment resulted in a behavioural withdrawal syndrome characterized by a pronounced increase in ambulatory activity and rearings. Corticosterone plasma concentrations dramatically increased 24 and 72 h after cessation of cannabinoid treatment. Similarly, an increase (40%) in cannabinoid [35S]GTPγS binding autoradiography was detected on days 1 and 3 of abstinence. Spontaneous cannabinoid withdrawal produced time‐related significant alterations in gene transcription: (i) decreased (20%) tyrosine hydroxylase (TH) mRNA levels in the ventral tegmental area and increased (50%) in substantia nigra; (ii) increased proenkephalin (PENK) gene expression more than 100% in caudate‐putamen, nucleus accumbens, olfactory tubercle and piriform cortex; (iii) increased (20–40%) pro‐opiomelanocortin (POMC) gene expression in the arcuate nucleus of the hypothalamus. These results suggest that spontaneous cannabinoid withdrawal occur after cessation of CP‐55,940 treatment. This ‘syndrome’ includes behavioural, hormonal and gene transcription alterations that seems to be part of the regulation of neuronal plasticity induced by spontaneous cannabinoid withdrawal.

Distortions of the posterior surface in optical coherence tomography images of the isolated crystalline lens: effect of the lens index gradient

We quantify the posterior surface distortions in optical coherence tomography (OCT) images of isolated crystalline lenses. The posterior radius of curvature and asphericity obtained from OCT images acquired with the beam incident first on the anterior, and then the posterior, surface were compared. The results were compared with predictions of a ray-tracing model which includes the index gradient. The results show that the error in the radius of curvature is within the measurement reproducibility and that it can be corrected by assuming a uniform refractive index. However, accurate asphericity values require a correction algorithm that takes into account the gradient.