Retinal vessel dynamic functionality in the eyes of asymptomatic patients with significant internal carotid artery stenosis.

BACKGROUND: The goal of this study was to assess the retinal microvascular function in asymptomatic patients with hemodynamically significant internal carotid artery stenosis (ICAS) and to assess the potential efficacy of carotid endarterectomy (CEA) for the improvement of vessel functionality. METHODS: Retinal vessel caliber and reactions to flicker stimulation were assessed in both eyes of 65 asymptomatic patients with unilateral hemodynamically significant ICAS and 34 healthy subjects. Subsequently, the recruited ICAS patients were referred for standard unilateral CEA procedure. The full ophthalmologic examination of both eyes and vessel analysis were performed 1 day before and 3 months after CEA. RESULTS: The venous responses to flicker stimulation were significantly lower in the EIS (eyes ipsilateral to stenosis) and ECS (eyes contralateral to stenosis) compared with those in the controls (P<0.0001 and P<0.0001, respectively). No changes were identified in retinal vascular flicker responses after CEA in both groups of eyes compared with the baseline values. We observed a decrease in CRVE (central retinal venular equivalent) after the CEA both in eyes ipsilateral (P=0.01) and contralateral (P=0.04) to CEA. Likewise, a decrease in CRAE (central retinal arteriolar equivalent) was identified in the eyes ipsilateral to CEA (P<0.001). CONCLUSIONS: This outcome strongly indicates that microvascular dysfunction is long-lasting despite the recovery of the flow in the carotid artery.

Preclinical Evaluation of Long-Term Neuroprotective Effects of BDNF-Engineered Mesenchymal Stromal Cells as Intravitreal Therapy for Chronic Retinal Degeneration in Rd6 Mutant Mice.

This study aimed to investigate whether the transplantation of genetically engineered bone marrow-derived mesenchymal stromal cells (MSCs) to overexpress brain-derived neurotrophic factor (BDNF) could rescue the chronic degenerative process of slow retinal degeneration in the rd6 (retinal degeneration 6) mouse model and sought to identify the potential underlying mechanisms. Rd6 mice were subjected to the intravitreal injection of lentivirally modified MSC-BDNF or unmodified MSC or saline. In vivo morphology, electrophysiological retinal function (ERG), and the expression of apoptosis-related genes, as well as BDNF and its receptor (TrkB), were assessed in retinas collected at 28 days and three months after transplantation. We observed that cells survived for at least three months after transplantation. MSC-BDNF preferentially integrated into the outer retinal layers and considerably rescued damaged retinal cells, as evaluated by ERG and immunofluorescence staining. Additionally, compared with controls, the therapy with MSC-BDNF was associated with the induction of molecular changes related to anti-apoptotic signaling. In conclusion, BDNF overexpression observed in retinas after MSC-BDNF treatment could enhance the neuroprotective properties of transplanted autologous MSCs alone in the chronically degenerated retina. This research provides evidence for the long-term efficacy of genetically-modified MSC and may represent a strategy for treating various forms of degenerative retinopathies in the future.

Depletion of the Third Complement Component Ameliorates Age-Dependent Oxidative Stress and Positively Modulates Autophagic Activity in Aged Retinas in a Mouse Model.

The aim of the study was to investigate the influence of complement component C3 global depletion on the biological structure and function of the aged retina. In vivo morphology (OCT), electrophysiological function (ERG), and the expression of selected oxidative stress-, apoptosis-, and autophagy-related proteins were assessed in retinas of 12-month-old C3-deficient and WT mice. Moreover, global gene expression in retinas was analyzed by RNA arrays. We found that the absence of active C3 was associated with (1) alleviation of the age-dependent decrease in retinal thickness and gradual deterioration of retinal bioelectrical function, (2) significantly higher levels of antioxidant enzymes (catalase and glutathione reductase) and the antiapoptotic survivin and Mcl-1/Bak dimer, (3) lower expression of the cellular oxidative stress marker-4HNE-and decreased activity of proapoptotic caspase-3, (4) ameliorated retinal autophagic activity with localization of ubiquitinated protein conjugates commonly along the retinal pigment epithelium (RPE) layer, and (5) significantly increased expression of several gene sets associated with maintenance of the physiological functions of the neural retina. Our findings shed light on mechanisms of age-related retinal alterations by identifying C3 as a potential therapeutic target for retinal aging.

Neuroprotective and antiapoptotic activity of lineage-negative bone marrow cells after intravitreal injection in a mouse model of acute retinal injury.

We investigated effects of bone marrow-derived, lineage-negative cell (Lin(-)BMC) transplantation in acute retinal injury. Lin(-)BMCs were intravitreally injected into murine eyes at 24 h after NaIO3-induced injury. Morphology, function, and expression of apoptosis-related genes, including brain-derived neurotrophic factor (BDNF) and its receptor, were assessed in retinas at 7 days, 28 days, and 3 months after transplantation. Moreover, global gene expression at day 7 was analyzed by RNA arrays. We observed that Lin(-)BMCs integrated into outer retinal layers improving morphological retinal structure and induced molecular changes such as downregulation of proapoptotic caspase-3 gene, a decrease in BAX/BCL-2 gene ratio, and significant elevation of BDNF expression. Furthermore, transplanted Lin(-)BMCs differentiated locally into cells with a macrophage-like phenotype. Finally, Lin(-)BMCs treatment was associated with generation of two distinct transcriptomic patterns. The first relates to downregulated genes associated with regulation of neuron cell death and apoptosis, response to oxidative stress/hypoxia and external stimuli, and negative regulation of cell proliferation. The second relates to upregulated genes associated with neurological system processes and sensory perception. Collectively, our data demonstrate that transplanted Lin(-)BMCs exert neuroprotective function against acute retinal injury and this effect may be associated with their antiapoptotic properties and ability to express neurotrophic factors.

Mixed chimerism and transplant tolerance are not effectively induced in C3a-deficient mice.

Mixed chimerism, a phenomenon involved in the development of specific alloantigen tolerance, could be achieved through the transplantation of hematopoietic stem cells into properly prepared recipients. Because the C3a complement component modulates hematopoietic cell trafficking after transplantation, in the present study, we investigated the influence of the C3a deficiency on mixed chimerism and alloantigen tolerance induction. To induce mixed chimerism, C57BL/6J (wild-type strain; H-2K(b); I-E(-)) and B6.129S4-C3(tm1Crr)/J (C3a-deficient) mice were exposed to 3 G total body irradiation (day -1). Subsequently, these mice were treated with CD8-blocking (day -2) and CD40L-blocking (days 0 and 4) antibodies, followed by transplantation with 20 × 10(6) Balb/c (H-2K(d); I-E(+)) bone marrow cells (day 0). The degree of mixed chimerism in peripheral blood leukocytes was measured several times during the 20-week experiment. The tolerance to Balb/c mouse antigens was assessed based on the number of lymphocytes expressing Vß5 and Vß11 T-cell receptor and on skin-graft (day 0) acceptance. Applying our experimental model, mixed chimerism and alloantigen tolerance were effectively induced in C57BL/6J (wild-type) mice, but not in C3a(-/-) animals. The present study is, to our knowledge, the first to demonstrate that C3a is vital for achieving stable mixed chimerism and related to this induction of transplant tolerance.

In vivo imaging of the mouse retina using high-resolution optical coherence tomography.

PURPOSE: In this study, we demonstrate the advantages of high-resolution optical coherence tomography for the non-invasive, in vivo, three-dimensional imaging of the mouse retina. METHODS: High-resolution optical coherence tomography images of the mouse retina were acquired using the Bioptigen Envisu R2200-HR SD-OCT system. We measured the retinal thickness and compared the measurements to those obtained using conventional histology techniques. RESULTS: High-resolution spectral-domain optical coherence tomography enables high-quality in vivo visualization of retinal structures in mice, providing an accurate quantitative description of retinal layers. Additionally, the ultra-high-speed system offers many advantages over histology, e.g., it permits the visualization of retinal microvasculature and pulsatile flow dynamics. CONCLUSIONS: Spectral domain optical coherence tomography is a new important tool for the in vivo analysis of mouse eyes.

Dose-dependent retinal changes following sodium iodate administration: application of spectral-domain optical coherence tomography for monitoring of retinal injury and endogenous regeneration.

BACKGROUND: The purpose of this study was to demonstrate the progression of acute retinal injury by correlating histological sections with in vivo spectral-domain optical coherence tomography (SD-OCT) images. METHODS: Male C57BL/6 mice were treated intravenously with two different sodium iodate (NaIO3) doses (35 mg/kg or 15 mg/kg). In vivo SD-OCT was performed up to 3 months post-injury. Ex vivo retinal histology, TUNEL and IsolectinB4 immunostaining were also conducted. Quantitative comparison of histopathological images and SD-OCT images was performed. RESULTS: SD-OCT examination revealed that administration of 35 mg/kg NaIO3 was associated with progressive and irreversible retinal degeneration. On day 3 post-injury, we found numerous apoptotic cells in the outer nuclear layer (ONL) that strongly corresponded to hyper-reflective areas in the SD-OCT images. At 7 d post-injury, SD-OCT images showed irregular-shaped patterns of hyper-reflectivity in the retinal pigment epithelium (RPE) that corresponded with the accumulation of macrophages phagocytosing melanin granules and cell debris. Additionally, we documented hyper-reflective opacities in the vitreous that were most numerous at 7 d. At 3 months post-injury, the neurosensory retina was significantly thinner, predominantly due to progressive photoreceptor (PR) loss. In contrast, administration of 15 mg/kg NaIO3 did not induce hyper-reflectivity of ONL in SD-OCT images, which indicates a lack of massive PR cell death. At 3 months post-injury, SD-OCT images showed the complete restoration of outer retina lamination and restoration of hyper-reflective structural bands. Histological assessment of retinas acquired after the last SD-OCT imaging session revealed complete regeneration of the RPE and considerable improvement of PR architecture. CONCLUSIONS: Our findings showed the high level of effectiveness of SD-OCT imaging for monitoring dynamic changes in retinal morphology following acute retinal injury. Moreover, we demonstrated for the first time that SD-OCT can be used to non-invasively detect regeneration in the damaged retina.