Estudo das alterações estruturais e ultra-estruturais da camada de fotorreceptores da retina de ratos diabéticos aloxânicos

Pós-graduação em Bases Gerais da Cirurgia - FMB

Association of retinal arteriolar dilatation with lower verbal memory::The Edinburgh Type 2 Diabetes Study

Abstract


AIMS/HYPOTHESIS:

Retinal vascular calibre changes may reflect early subclinical microvascular disease in diabetes. Because of the considerable homology between retinal and cerebral microcirculation, we examined whether retinal vascular calibre, as a proxy of cerebral microvascular disease, was associated with cognitive function in older people with type 2 diabetes.

METHODS:

A cross-sectional analysis of 954 people aged 60-75 years with type 2 diabetes from the population-based Edinburgh Type 2 Diabetes Study was performed. Participants underwent standard seven-field binocular digital retinal photography and a battery of seven cognitive function tests. The Mill Hill Vocabulary Scale was used to estimate pre-morbid cognitive ability. Retinal vascular calibre was measured from an image field with the optic disc in the centre using a validated computer-based program.

RESULTS:

After age and sex adjustment, larger retinal arteriolar and venular calibres were significantly associated with lower scores for the Wechsler Logical Memory test, with standardised regression coefficients -0.119 and -0.084, respectively (p?<?0.01), but not with other cognitive tests. There was a significant interaction between sex and retinal vascular calibre for logical memory. In male participants, the association of increased retinal arteriolar calibre with logical memory persisted (p?<?0.05) when further adjusted for vocabulary, venular calibre, depression, cardiovascular risk factors and macrovascular disease. In female participants, this association was weaker and not significant.

CONCLUSIONS/INTERPRETATION:

Retinal arteriolar dilatation was associated with poorer memory, independent of estimated prior cognitive ability in older men with type 2 diabetes. The sex interaction with stronger findings in men requires confirmation. Nevertheless, these data suggest that impaired cerebral arteriolar autoregulation in smooth muscle cells, leading to arteriolar dilatation, may be a possible pathogenic mechanism in verbal declarative memory decrements in people with diabetes.

Assessment of retinal structure and visual function in association with diabetic peripheral neuropathy

Diabetes is an increasingly prevalent disease worldwide. Providing early management of the complications can prevent morbidity and mortality in this population. Peripheral neuropathy, a significant complication of diabetes, is the major cause of foot ulceration and amputation in diabetes. Delay in attending to complication of the disease contributes to significant medical expenses for diabetic patients and the community. Early structural changes to the neural components of the retina have been demonstrated to occur prior to the clinically visible retinal vasculature complication of diabetic retinopathy. Additionally visual functionloss has been shown to exist before the ophthalmoscopic manifestations of vasculature damage. The purpose of this thesis was to evaluate the relationship between diabetic peripheral neuropathy and both retinal structure and visual function. The key question was whether diabetic peripheral neuropathy is the potential underlying factor responsible for retinal anatomical change and visual functional loss in people with diabetes. This study was conducted on a cohort with type 2 diabetes. Retinal nerve fibre layer thickness was assessed by means of Optical Coherence Tomography (OCT). Visual function was assessed using two different methods; Standard Automated Perimetry (SAP) and flicker perimetry were performed within the central 30 degrees of fixation. The level of diabetic peripheral neuropathy (DPN) was assessed using two techniques - Quantitative Sensory Testing and Neuropathy Disability Score (NDS). These techniques are known to be capable of detecting DPN at very early stages. NDS has also been shown as a gold standard for detecting 'risk of foot ulceration'. Findings reported in this thesis showed that RNFL thickness, particularly in the inferior quadrant, has a significant association with severity of DPN when the condition has been assessed using NDS. More specifically it was observed that inferior RNFL thickness has the ability to differentiate individuals who are at higher risk of foot ulceration from those who are at lower risk, indicating that RNFL thickness can predict late-staged DPN. Investigating the association between RNFL and QST did not show any meaningful interaction, which indicates that RNFL thickness for this cohort was not as predictive of neuropathy status as NDS. In both of these studies, control participants did not have different results from the type 2 cohort who did not DPN suggesting that RNFL thickness is not a marker for diagnosing DPN at early stages. The latter finding also indicated that diabetes per se, is unlikely to affect the RNFL thickness. Visual function as measured by SAP and flicker perimetry was found to be associated with severity of peripheral neuropathy as measured by NDS. These findings were also capable of differentiating individuals at higher risk of foot ulceration; however, visual function also proved not to be a maker for early diagnosis of DPN. It was found that neither SAP, nor flicker sensitivity have meaningful associations with DPN when neuropathy status was measured using QST. Importantly diabetic retinopathy did not explain any of the findings in these experiments. The work described here is valuable as no other research to date has investigated the association between diabetic peripheral neuropathy and either retinal structure or visual function.

Paraquat-Induced Retinal Degeneration Is Exaggerated in CX3CR1-Deficient Mice and Is Associated with Increased Retinal Inflammation

PURPOSE:
To investigate the role of the Fractalkine receptor CX3CR1 pathway in oxidative insults-mediated retinal degeneration and immune activation.
METHODS:
A prooxidant, paraquat (0.75 µM) was injected into the vitreous of C57BL/6J, CX3CR1(gpf/+), and CX3CR1(gfp/gfp) mice. Retinal lesions were investigated clinically by topic endoscopic fundus imaging and fluorescence angiography, and pathologically by light- and electron microscopy. Retinal immune gene expression was determined by real-time RT-PCR. Microglial activation and immune cell infiltration were examined by confocal microscopy of retinal flatmounts.
RESULTS:
Intravitreal injection of paraquat (0.75 µM) resulted in acute retinal capillary nonperfusion within 2 days, which improved from 4 days to 4 weeks postinjection (p.i.). Panretinal degeneration was observed at 4 days p.i. and progressed further at 4 weeks p.i. In the absence of CX3CR1, retinal degeneration was exaggerated and was accompanied by increased TNF-a, iNOS, IL-1ß, Ccl2, and Casp-1 gene expression. Confocal microscopy of retinal flatmounts revealed microglial activation and CD44(+)MHC-II(+) monocyte and GR1(+) neutrophil infiltration in paraquat-injected eyes. The number of activated microglia and infiltrating leukocytes was significantly higher in CX3CR1(gfp/gfp) mice than in CX3CR1(gfp/+) mice.
CONCLUSIONS:
Our results suggest that the CX3CR1 signaling pathway may play an important role in controlling retinal inflammation under oxidative and ischemia/reperfusion conditions. In the absence of CX3CR1, uncontrolled retinal inflammation results in exaggerated retinal degeneration.

Intravitreal Injection of Normal Saline Induces Retinal Degeneration in the C57BL/6J Mouse

Purpose: To investigate the adverse effect of intravitreal injection of normal saline (NS) and phosphate buffered saline (PBS) in mouse eyes.

Methods: NS or PBS was injected intravitreally into C57BL/6J mouse eyes. Retinal lesions were monitored by fundus imaging, spectral-domain optical coherence tomography (SD-OCT), and histological investigations. Retinal immune gene expression was determined by real-time polymerase chain reaction (PCR). The toxic effect of NS and PBS or retinal protein from NS- or PBS-injected eyes on retinal pigment epithelium (RPE) was tested in B6-RPE-07 mouse RPE cell cultures.

Results: Intravitreal injection of NS dose-dependently induced localized retinal lesion in mice. Histological investigations revealed multiple vacuoles in photoreceptor outer segments and RPE cells. The lesions recovered over time and by 3 weeks post injection the majority of lesions vanished in eyes receiving 1 μl NS. Inflammatory genes, including TNF-α, IL-1β, IL-6, iNOS, and VEGF were upregulated in NS injected eyes. Intravitreal injection of PBS did not cause any pathology. The treatment of B6-RPE07 cells with 30% PBS or 30% NS did not affect RPE viability. However, incubation of 1-μg/ml retinal protein from NS-injected eyes, but not PBS-injected eyes induced RPE cell death.

Conclusion: NS is toxic to the C57BL/6J mouse retina and should not be used as a vehicle for intraocular injection. PBS is not toxic to the retina and is a preferred vehicle.

Translational Relevance: NS is not a physiological solution for intraocular injection in the C57BL/6J mice and questions its suitability for intraocular injection in other species, including human.

The role of WNT singling activation and its blockage in the focal retinal lesion of Ccl2-/-/Cx3cr1-/- mice with and without rd8 background

Purpose: The canonical Wnt signaling is activated by retinal injury. Under disease conditions, the Wnt mediates inflammatory responses. Inflammation has been detected in age-related macular degeneration (AMD) retinas and Ccl2-/-/Cx3cr1-/- (DKO) mice with or without rd8 background, a model with progressive AMD-like lesions including focal photoreceptor/RPE degeneration and A2E accumulation. We evaluated the effects of Wnt-β-catenin activation and an antibody against LRP6, the co-receptor of Wnt on these two models.

Methods: anti-LRP6 antibody (2F1, 1 μl of 5 μg/μL) was intravitreally injected into the right eyes in 3 separate experiments (DKOrd8, N=35; DKO, N=10). The left eyes were injected with mouse IgG as controls. Fundoscopy was taken before injection and sequentially monthly after injection. Two months after injection, light-adapted ERG responses were recorded; then the eyes were harvested for histopathology, the determination of retinal A2E, and molecular analysis. The microarray of ocular mRNA of 92 Wnt genes was compared between the treated and the control eyes. The phosphorylated types of LRP6 and β-catenin and endogenous forms of the proteins were assayed by Western blotting.

Results: For DKOrd8 mice, the fundus showed a slower progression or alleviation of retinal lesions in the right eyes as compared to the left eyes. Among 35 pairs of eyes, 26 (74.3%) were improved, 7 (20%) stayed the same and 2 (5.7%) remained progressing. Histology confirmed the clinical observation. Light-adapted ERG of the treated eyes exhibited larger amplitudes compared to control eyes (n=6), with greater improvements under UV light stimulus. There was a significantly lower A2E in the treated eyes compared to controls. Microarray of 92 Wnt genes expression pattern was similar in both eyes. Western blotting indicated local administration of 2F1 antibody to suppress the activation of Wnt pathway in the retina. For DKO mice, the treatment improved ERG but less effect on RPE degeneration.

Conclusions: The canonical Wnt signaling plays a role in the focal retina lesion of both DKOrd8 and DKO mice; and intravitreal anti-LRP6 antibody might be neuroprotective via deactivation of canonical Wnt pathway.

Identification and evaluation of candidate genes for inherited retinal -degenerative diseases

Although more than 100 genes associated with inherited retinal disease have been mapped to chromosomal locations, less than half of these genes have been cloned. This text includes identification and evaluation of candidate genes for three autosomal dominant forms of inherited retinal degeneration: atypical vitelliform macular dystrophy (VMD1), cone-rod dystrophy (CORD), and retinitis pigmentosa (RP). ^ VMD1 is a disorder characterized by complete penetrance but extremely variable expressivity, and includes macular or peripheral retinal lesions and peripappilary abnormalitites. In 1984, linkage was reported between VMD1 and soluble glutamate-pyruvate transaminase GPT); however, placement of GPT to 8q24 on linkage maps had been debated, and VMD1 did not show linkage to microsatellite markers in that region. This study excluded linkage between the loci by cloning GPT, identifying the nucleotide substitution associated with the GPT sozymes, and by assaying VMD1 family samples with an RFLP designed to detect the substitution. In addition, linkage of VMD1 to the known dominant macular degeneration loci was excluded. ^ CORD is characterized by early onset of color-vision deficiency, and decreased visual acuity, However, this retinal degeneration progresses to no light perception, severe macular lesion, and “bone-spicule” accumulations in the peripheral retina. In this study, the disorder in a large Texan family was mapped to the CORD2 locus of 19q13, and a mutation in the retina/pineal-specific cone-rod homeobox gene (CRX) was identified as the disease cause. In addition, mutations in CRX were associated with significantly different retinal disease phenotypes, including retinitis pigmentosa and Leber congenital amaurosis. ^ Many of the mutations leading to inherited retinal disorders have been identified in genes like CRX, which are expressed predominantly in the retina and pineal gland. Therefore, a combination of database analysis and laboratory investigation was used to identify 26 novel retina/pineal-specific expressed sequence tag (EST) clusters as candidate genes for inherited retinal disorders. Eight of these genes were mapped into the candidate regions of inherited retinal degeneration loci. ^ Two of the eight clusters mapped into the retinitis pigmentosa RP13 candidate region of 17p13, and were both determined to represent a single gene that is highly expressed in photoreceptors. This gene, the Ah receptor-interacting like protein-1 (AIPL1), was cloned, characterized, and screened for mutations in RP13 patient DNA samples. ^

Polyamine system in developing rat eye and an animal model of retinopathy of prematurity

BACKGROUND: In experimental models of retinopathy of prematurity (ROP), a vasoproliferative disorder of the retina, retinal lesions are usually assessed by morphological examination. However, studies suggest that the polyamine system may be useful in monitoring proliferation processes. For this reason, polyamine concentrations in rat erythrocytes (RBC) and the regulation of polyamine system in rat eyes under the conditions relevant to ROP were investigated. METHODS: Newborn Wistar rats were reared in room air (control) or exposed first to hyperoxia (60% or 80% oxygen, 2 weeks) and then to normoxia (relative hypoxia, 1 or 2 weeks). Blood was collected from orbital vessels at 2 weeks of age and before death. Polyamine system-related enzyme activities were measured in retina and lens with radioassays. Polyamines were quantified by fluorometry after extraction, dansylation and HPLC separation. RESULTS: Oxygen (80% only) significantly decreased RBC polyamine concentrations, which then markedly increased after rats were transferred for a week to normal air, suggesting retardation of growth processes and compensatory stimulation, respectively. However, polyamine system changes in the rat eye were not so pronounced. Enzyme activities and polyamine concentrations tended to be lower in retina after hyperoxia and were only slightly higher, with the exception of ornithine decarboxylase, after a subsequent 1 week of normoxia. In litters subjected to normoxia for longer periods no changes were found. CONCLUSION: The transient and short-lived alteration in polyamine metabolism, especially in the eye, suggests that exposure of newborn rats to high oxygen supplementation followed by normoxia does not necessarily result in marked retinopathy.

Parainflammation, chronic inflammation, and age-related macular degeneration

Inflammation is an adaptive response of the immune system to noxious insults to maintain homeostasis and restore functionality. The retina is considered an immune-privileged tissue as a result of its unique anatomic and physiologic properties. During aging, the retina suffers from a low-grade chronic oxidative insult, which sustains for decades and increases in level with advancing age. As a result, the retinal innate-immune system, particularly microglia and the complement system, undergoes low levels of activation (parainflammation). In many cases, this parainflammatory response can maintain homeostasis in the healthy aging eye. However, in patients with age-related macular degeneration, this parainflammatory response becomes dysregulated and contributes to macular damage. Factors contributing to the dysregulation of age-related retinal parainflammation include genetic predisposition, environmental risk factors, and old age. Dysregulated parainflammation (chronic inflammation) in age-related macular degeneration damages the blood retina barrier, resulting in the breach of retinal-immune privilege, leading to the development of retinal lesions. This review discusses the basic principles of retinal innate-immune responses to endogenous chronic insults in normal aging and in age-related macular degeneration and explores the difference between beneficial parainflammation and the detrimental chronic inflammation in the context of age-related macular degeneration.

Wnt signaling in age-related macular degeneration: human macular tissue and mouse model

BACKGROUND: The wingless-type MMTV integration site (Wnt) signaling is a group of signal transduction pathways. In canonical Wnt pathway, Wnt ligands bind to low-density lipoprotein receptor-related protein 5 or 6 (LRP5 or LRP6), resulting in phosphorylation and activation of the receptor. We hypothesize that canonical Wnt pathway plays a role in the retinal lesion of age-related macular degeneration (AMD), a leading cause of irreversible central visual loss in elderly.

METHODS: We examined LRP6 phosphorylation and Wnt signaling cascade in human retinal sections and plasma kallistatin, an endogenous inhibitor of the Wnt pathway in AMD patients and non-AMD subjects. We also used the Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 and Ccl2 (-/-) /Cx3cr1 (gfp/gfp) mouse models with AMD-like retinal degeneration to further explore the involvement of Wnt signaling activation in the retinal lesions in those models and to preclinically evaluate the role of Wnt signaling suppression as a potential therapeutic option for AMD.

RESULTS: We found higher levels of LRP6 (a key Wnt signaling receptor) protein phosphorylation and transcripts of the Wnt pathway-targeted genes, as well as higher beta-catenin protein in AMD macula compared to controls. Kallistatin was decreased in the plasma of AMD patients. Retinal non-phosphorylated-β-catenin and phosphorylated-LRP6 were higher in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 mice than that in wild type. Intravitreal administration of an anti-LRP6 antibody slowed the progression of retinal lesions in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 and Ccl2 (-/-) /Cx3cr1 (gfp/gfp) mice. Electroretinography of treated eyes exhibited larger amplitudes compared to controls in both mouse models. A2E, a retinoid byproduct associated with AMD was lower in the treated eyes of Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 mice. Anti-LRP6 also suppressed the expression of Tnf-α and Icam-1 in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 retinas.

CONCLUSIONS: Wnt signaling may be disturbed in AMD patients, which could contribute to the retinal inflammation and increased A2E levels found in AMD. Aberrant activation of canonical Wnt signaling might also contribute to the focal retinal degenerative lesions of mouse models with Ccl2 and Cx3cr1 deficiency, and intravitreal administration of anti-LRP6 antibody could be beneficial by deactivating the canonical Wnt pathway.

Potentiel des liposomes pour l'injection intravitréenne de molécules thérapeutiques [Potential of liposomes for the intravitreal injection of therapeutic molecules].

Intravitreal administration has been widely used since 20 years and has been shown to improve the treatment of diseases of the posterior segment of the eye with infectious origin or in edematous maculopathies. This route of administration allows to achieve high concentration of drug in the vitreous and avoids the problems resulting from systemic administration. However, two basic problems limit the use of intravitreal therapy. Many drugs are rapidly cleared from the vitreous humor; therefore, to reach and to maintain effective therapy repeated injections are necessary. Repeated intravitreal injections increase the risk of endophthalmitis, damage to lens, retinal detachment. Moreover, some drugs provoke a local toxicity at their effective dose inducing side-effects and possible retinal lesions. In this context, the development and the use of new drug delivery systems for intravitreal administration are necessary to treat chronic ocular diseases. Among them, particulate systems such as liposomes have been widely studied. Liposomes are easily injectable and permit to reduce the toxicity and to increase the residence time of several drugs in the eye. They are also able to protect in vivo poorly-stable molecules from degradation such as peptides and nucleic acids. Some promising results have been obtained for the treatment of retinitis induced by cytomegalovirus in human and more recently for the treatment of uveitis in animal. Finally, the fate of liposomes in ocular tissues and fluids after their injection into the vitreous and their elimination routes begin to be more known.

Unusual macular thickness in Alport syndrome: case report

This case report describes the presence of bilateral macular atrophy in a patient with Alport syndrome and compares this finding with literature. At fundoscopy, there was a discrete circumscribed macular thinning showing intense retinal pigment epithelium color and the presence of whitish circular retinal lesions ("dots" and "flecks") at nasal mid periphery of both eyes. Optical coherence tomography showed bilateral partial atrophy of the neurosensory retina in the macula, with a greater extent in the temporal region. This case describes a rare ophthalmological finding in Alport syndrome and important to be recognized for a precise diagnosis as well as for determining visual prognosis.

Vitreous pharmacokinetics and electroretinographic findings after intravitreal injection of acyclovir in rabbits

OBJECTIVES: Acute retinal necrosis is a rapidly progressive and devastating viral retinitis caused by the herpesvirus family. Systemic acyclovir is the treatment of choice; however, the progression of retinal lesions ceases approximately 2 days after treatment initiation. An intravitreal injection of acyclovir may be used an adjuvant therapy during the first 2 days of treatment when systemically administered acyclovir has not reached therapeutic levels in the retina. The aims of this study were to determine the pharmacokinetic profile of acyclovir in the rabbit vitreous after intravitreal injection and the functional effects of acyclovir in the rabbit retina. METHODS: Acyclovir (Acyclovir; Bedford Laboratories, Bedford, OH, USA) 1 mg in 0.1 mL was injected into the right eye vitreous of 32 New Zealand white rabbits, and 0.1 mL sterile saline solution was injected into the left eye as a control. The animals were sacrificed after 2, 9, 14, or 28 days. The eyes were enucleated, and the vitreous was removed. The half-life of acyclovir was determined using high-performance liquid chromatography. Electroretinograms were recorded on days 2, 9, 14, and 28 in the eight animals that were sacrificed 28 days after injection according to a modified protocol of the International Society for Clinical Electrophysiology of Vision. RESULTS: Acyclovir rapidly decayed in the vitreous within the first two days after treatment and remained at low levels from day 9 onward. The eyes that were injected with acyclovir did not present any electroretinographic changes compared with the control eyes. CONCLUSIONS: The vitreous half-life of acyclovir is short, and the electrophysiological findings suggest that the intravitreal delivery of 1 mg acyclovir is safe and well tolerated by the rabbit retina.