Novel rodent models for macular research

Background

Many disabling human retinal disorders involve the central retina, particularly the macula. However, the commonly used rodent models in research, mouse and rat, do not possess a macula. The purpose of this study was to identify small laboratory rodents with a significant central region as potential new models for macular research.

Methodology/Principal Findings

Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli, laboratory rodents less commonly used in retinal research, were subjected to confocal scanning laser ophthalmoscopy (cSLO), fluorescein and indocyanine green angiography, and spectral-domain optical coherence tomography (SD-OCT) using standard equipment (Heidelberg Engineering HRA1 and Spectralis™) adapted to small rodent eyes. The existence of a visual streak-like pattern was assessed on the basis of vascular topography, retinal thickness, and the topography of retinal ganglion cells and cone photoreceptors. All three species examined showed evidence of a significant horizontal streak-like specialization. cSLO angiography and retinal wholemounts revealed that superficial retinal blood vessels typically ramify and narrow into a sparse capillary net at the border of the respective area located dorsal to the optic nerve. Similar to the macular region, there was an absence of larger blood vessels in the streak region. Furthermore, the thickness of the photoreceptor layer and the population density of neurons in the ganglion cell layer were markedly increased in the visual streak region.

Conclusions/Significance

The retinal specializations of Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli resemble features of the primate macula. Hence, the rodents reported here may serve to study aspects of macular development and diseases like age-related macular degeneration and diabetic macular edema, and the preclinical assessment of therapeutic strategies.     

Factors Affecting Reading Speed in Patients with Diabetic Macular Edema Treated with Laser Photocoagulation

Purpose

To study the factors that may affect reading speed in patients with diabetic macular edema previously treated with laser photocoagulation.

Methods

Consecutive patients with type II diabetes treated with laser photocoagulation for diabetic macular edema (DME) at least twelve months previously, with best corrected visual acuity of better than 65 letters (approximately 20/40) measured with Early Treatment Diabetic Retinopathy Study (ETDRS) charts were included in this study. Patients previously treated with pan-retinal photocoagulation, vitrectomy, intravitreal steroid or anti-VEGF therapy were excluded. Any other ocular co-morbidities that may influence reading ability such as cataract, glaucoma or macular degeneration were also excluded. All patients were refracted by a certified examiner, the following measurements were collected: best corrected visual acuity (BCVA), contrast sensitivity with Pelli-Robson chart, reading speed with MNREAD chart, microperimetry with Nidek MP1, and central subfield thickness with Zeiss spectral domain optical coherent topography.

Results

The slow reading group had poorer contrast sensitivity (p = 0.001), reduced retinal sensitivity (p = 0.027) and less stable fixation (p = 0.013). Most interestingly the reduced retinal sensitivity findings were driven by the microperimetry value on the right subfield (p = 0.033), (nasal to the fovea in the right eye and temporal to the fovea in the left eye). Multiple linear regression analysis showed that contrast sensitivity is probably the most important factor that affects reading speed (p = 0.001).

Conclusion

Reduced retinal sensitivity after laser treatment is associated with reduced reading speed in patients with diabetic macular edema.     

Effects of combined ketamine/xylazine anesthesia on light induced retinal degeneration in rats

Objectives

To explore the effect of ketamine-xylazine anesthesia on light-induced retinal degeneration in rats.

Methods

Rats were anesthetized with ketamine and xylazine (100 and 5 mg, respectively) for 1 h, followed by a recovery phase of 2 h before exposure to 16,000 lux of environmental illumination for 2 h. Functional assessment by electroretinography (ERG) and morphological assessment by in vivo imaging (optical coherence tomography), histology (hematoxylin/eosin staining, TUNEL assay) and immunohistochemistry (GFAP and rhodopsin staining) were performed at baseline (ERG), 36 h, 7 d and 14 d post-treatment. Non-anesthetized animals treated with light damage served as controls.

Results

Ketamine-xylazine pre-treatment preserved retinal function and protected against light-induced retinal degeneration. In vivo retinal imaging demonstrated a significant increase of outer nuclear layer (ONL) thickness in the non-anesthetized group at 36 h (p<0.01) and significant reduction one week (p<0.01) after light damage. In contrast, ketamine-xylazine pre-treated animals showed no significant alteration of total retinal or ONL thickness at either time point (p>0.05), indicating a stabilizing and/or protective effect with regard to phototoxicity. Histology confirmed light-induced photoreceptor cell death and Müller cells gliosis in non-anesthetized rats, especially in the superior hemiretina, while ketamine-xylazine treated rats showed reduced photoreceptor cell death (TUNEL staining: p<0.001 after 7 d), thicker ONL and longer IS/OS. Fourteen days after light damage, a reduction of standard flash induced a-wave amplitudes and a-wave slopes (p = 0.01) and significant alterations in parameters of the scotopic sensitivity function (e.g. Vmax of the Naka Rushton fit p = 0.03) were observed in non-treated vs. ketamine-xylazine treated animals.

Conclusions

Our results suggest that pre-treatment with ketamine-xylazine anesthesia protects retinas against light damage, reducing photoreceptor cell death. These data support the notion that anesthesia with ketamine-xylazine provides neuroprotective effects in light-induced cell damage.     

PKCζ Mediates Breakdown of Outer Blood-Retinal Barriers in Diabetic Retinopathy

Aims/hypothesis

Diabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia.

Methods

Studies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor.

Results

PKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death.

Conclusions

In the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology.     

Noninvasive,In Vivo Assessment of Mouse Retinal Structure Using Optical Coherence Tomography

Background

Optical coherence tomography (OCT) is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration.

Methodology/Principal Findings

We achieved to adapt a commercial 3^rd generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Leber''s congenital amaurosis), retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE) due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified.

Conclusions/Significance

We found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical assessment of therapeutic strategies.     

Placental growth factor contributes to micro-vascular abnormalization and blood-retinal barrier breakdown in diabetic retinopathy

Objective

There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1).

Materials and Methods

pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis.

Results

After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation.

Conclusion

This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease.     

Mechanical ventilation with high tidal volumes attenuates myocardial dysfunction by decreasing cardiac edema in a rat model of LPS-induced peritonitis

Background

Injurious mechanical ventilation (MV) may augment organ injury remote from the lungs. During sepsis, myocardial dysfunction is common and increased endothelial activation and permeability can cause myocardial edema, which may, among other factors, hamper myocardial function. We investigated the effects of MV with injuriously high tidal volumes on the myocardium in an animal model of sepsis.

Methods

Normal rats and intraperitoneal (i.p.) lipopolysaccharide (LPS)-treated rats were ventilated with low (6 ml/kg) and high (19 ml/kg) tidal volumes (Vt) under general anesthesia. Non-ventilated animals served as controls. Mean arterial pressure (MAP), central venous pressure (CVP), cardiac output (CO) and pulmonary plateau pressure (P_plat) were measured. Ex vivo myocardial function was measured in isolated Langendorff-perfused hearts. Cardiac expression of endothelial vascular cell adhesion molecule (VCAM)-1 and edema were measured to evaluate endothelial inflammation and leakage.

Results

MAP decreased after LPS-treatment and Vt-dependently, both independent of each other and with interaction. MV Vt-dependently increased CVP and Pplat and decreased CO. LPS-induced peritonitis decreased myocardial function ex vivo but MV attenuated systolic dysfunction Vt-dependently. Cardiac endothelial VCAM-1 expression was increased by LPS treatment independent of MV. Cardiac edema was lowered Vt-dependently by MV, particularly after LPS, and correlated inversely with systolic myocardial function parameters ex vivo.

Conclusion

MV attenuated LPS-induced systolic myocardial dysfunction in a Vt-dependent manner. This was associated with a reduction in cardiac edema following a lower transmural coronary venous outflow pressure during LPS-induced coronary inflammation.     

Ocular application of the kinin B1 receptor antagonist LF22-0542 inhibits retinal inflammation and oxidative stress in streptozotocin-diabetic rats

Purpose

Kinin B₁ receptor (B₁R) is upregulated in retina of Streptozotocin (STZ)-diabetic rats and contributes to vasodilation of retinal microvessels and breakdown of the blood-retinal barrier. Systemic treatment with B₁R antagonists reversed the increased retinal plasma extravasation in STZ rats. The present study aims at determining whether ocular application of a water soluble B₁R antagonist could reverse diabetes-induced retinal inflammation and oxidative stress.

Methods

Wistar rats were made diabetic with STZ (65 mg/kg, i.p.) and 7 days later, they received one eye drop application of LF22-0542 (1% in saline) twice a day for a 7 day-period. The impact was determined on retinal vascular permeability (Evans blue exudation), leukostasis (leukocyte infiltration using Fluorescein-isothiocyanate (FITC)-coupled Concanavalin A lectin), retinal mRNA levels (by qRT-PCR) of inflammatory (B₁R, iNOS, COX-2, ICAM-1, VEGF-A, VEGF receptor type 2, IL-1β and HIF-1α) and anti-inflammatory (B₂R, eNOS) markers and retinal level of superoxide anion (dihydroethidium staining).

Results

Retinal plasma extravasation, leukostasis and mRNA levels of B₁R, iNOS, COX-2, VEGF receptor type 2, IL-1β and HIF-1α were significantly increased in diabetic retinae compared to control rats. All these abnormalities were reversed to control values in diabetic rats treated with LF22-0542. B₁R antagonist also significantly inhibited the increased production of superoxide anion in diabetic retinae.

Conclusion

B₁R displays a pathological role in the early stage of diabetes by increasing oxidative stress and pro-inflammatory mediators involved in retinal vascular alterations. Hence, topical application of kinin B₁R antagonist appears a highly promising novel approach for the treatment of diabetic retinopathy.     

Retinal Thickness Measurement Obtained with Spectral Domain Optical Coherence Tomography Assisted Optical Biopsy Accurately Correlates with Ex Vivo Histology

Background

This study determines ‘correlation constants’ between the gold standard histological measurement of retinal thickness and the newer spectral-domain optical coherence tomography (SD-OCT) technology in adult C57BL/6 mice.

Methods

Forty-eight eyes from adult mice underwent SD-OCT imaging and then were histologically prepared for frozen sectioning with H&E staining. Retinal thickness was measured via 10x light microscopy. SD-OCT images and histological sections were standardized to three anatomical sites relative to the optic nerve head (ONH) location. The ratios between SD-OCT to histological thickness for total retinal thickness (TRT) and six sublayers were defined as ‘correlation constants’.

Results

Mean (± SE) TRT for SD-OCT and histological sections was 210.95 µm (±1.09) and 219.58 µm (±2.67), respectively. The mean ‘correlation constant’ for TRT between the SD-OCT and histological sections was 0.96. The retinal thickness for all sublayers measured by SD-OCT vs. histology were also similar, the ‘correlation constant’ values ranged from 0.70 to 1.17. All SD-OCT and histological measurements demonstrated highly significant (p<0.01) strong positive correlations.

Conclusion

This study establishes conversion factors for the translation of ex vivo data into in vivo information; thus enhancing the applicability of SD-OCT in translational research.     

Betacellulin induces increased retinal vascular permeability in mice

Background

Diabetic maculopathy, the leading cause of vision loss in patients with type 2 diabetes, is characterized by hyper-permeability of retinal blood vessels with subsequent formation of macular edema and hard exudates. The degree of hyperglycemia and duration of diabetes have been suggested to be good predictors of retinal complications. Intervention studies have determined that while intensive treatment of diabetes reduced the development of proliferative diabetic retinopathy it was associated with a two to three-fold increased risk of severe hypoglycemia. Thus we hypothesized the need to identify downstream glycemic targets, which induce retinal vascular permeability that could be targeted therapeutically without the additional risks associated with intensive treatment of the hyperglycemia. Betacellulin is a 32 kD member of the epidermal growth factor family with mitogenic properties for the retinal pigment epithelial cells. This led us to hypothesize a role for betacellulin in the retinal vascular complications associated with diabetes.

Methods and Findings

In this study, using a mouse model of diabetes, we demonstrate that diabetic mice have accentuated retinal vascular permeability with a concomitant increased expression of a cleaved soluble form of betacellulin (s-Btc) in the retina. Intravitreal injection of soluble betacellulin induced retinal vascular permeability in normoglycemic and hyperglycemic mice. Western blot analysis of retinas from patients with diabetic retinopathy showed an increase in the active soluble form of betacellulin. In addition, an increase in the levels of A disintegrin and metalloproteinase (ADAM)-10 which plays a role in the cleavage of betacellulin was seen in the retinas of diabetic mice and humans.

Conclusions

These results suggest that excessive amounts of betacellulin in the retina may contribute to the pathogenesis of diabetic macular edema.     

SDOCT imaging to identify macular pathology in patients diagnosed with diabetic maculopathy by a digital photographic retinal screening programme

Introduction

Diabetic macular edema (DME) is an important cause of vision loss. England has a national systematic photographic retinal screening programme to identify patients with diabetic eye disease. Grading retinal photographs according to this national protocol identifies surrogate markers for DME. We audited a care pathway using a spectral-domain optical coherence tomography (SDOCT) clinic to identify macular pathology in this subset of patients.

Methods

A prospective audit was performed of patients referred from screening with mild to moderate non-proliferative diabetic retinopathy (R1) and surrogate markers for diabetic macular edema (M1) attending an SDOCT clinic. The SDOCT images were graded by an ophthalmologist as SDOCT positive, borderline or negative. SDOCT positive patients were referred to the medical retina clinic. SDOCT negative and borderline patients were further reviewed in the SDOCT clinic in 6 months.

Results

From a registered screening population of 17 551 patients with diabetes mellitus, 311 patients met the inclusion criteria between (March 2008 and September 2009). We analyzed images from 311 patients’ SDOCT clinic episodes. There were 131 SDOCT negative and 12 borderline patients booked for revisit in the OCT clinic. Twenty-four were referred back to photographic screening for a variety of reasons. A total of 144 were referred to ophthalmology with OCT evidence of definite macular pathology requiring review by an ophthalmologist.

Discussion

This analysis shows that patients with diabetes, mild to moderate non-proliferative diabetic retinopathy (R1) and evidence of diabetic maculopathy on non-stereoscopic retinal photographs (M1) have a 42.1% chance of having no macular edema on SDOCT imaging as defined by standard OCT definitions of DME when graded by a retinal specialist. SDOCT imaging is a useful adjunct to colour fundus photography in screening for referable diabetic maculopathy in our screening population.     

Association of Preoperative Optical Coherence Tomography Markers with Residual Inner Limiting Membrane in Epiretinal Membrane Peeling

Purpose

To identify preoperative markers on spectral domain optical coherence tomography (SD-OCT) for residual inner limiting membrane (ILM) in epiretinal membrane (ERM) peeling.

Methods

In this retrospective case series the preoperative SD-OCTs from 119 eyes of 119 consecutive patients who underwent surgery for idiopathic ERM by a single surgeon were evaluated for markers predisposing for ILM persistence after ERM removal. ILM persistence was determined via intraoperative indocyanine green staining. The main outcome measures were correlation of central foveal thickness (CFT), ERM thickness, extent of elevated ERM and retinal folding, intraretinal cysts, and discontinuation of the ERM, with ILM persistence after ERM peeling.

Results

The persistence of the ILM was found in 50.4% (n = 60). After Bonferroni correction for multiple testing, a greater extent of elevated ERM and thicker ERMs were associated with persistence of the ILM (p<0.005). The other parameters showed no statistically significant correlations with the persistence of the ILM (p≥0.005).

Conclusion

Residual ILM can be found in nearly half of the eyes after ERM peeling. A loose connection between the ERM and the retinal surface predisposes for ILM persistence. Preoperative SD-OCT is helpful in identifying risk markers for the persistence of the ILM in ERM surgery.     

Comparison of Longitudinal In Vivo Measurements of Retinal Nerve Fiber Layer Thickness and Retinal Ganglion Cell Density after Optic Nerve Transection in Rat

Purpose

To determine the relationship between longitudinal in vivo measurements of retinal nerve fiber layer thickness (RNFLT) and retinal ganglion cell (RGC) density after unilateral optic nerve transection (ONT).

Methods

Nineteen adult Brown-Norway rats were studied; N = 10 ONT plus RGC label, N = 3 ONT plus vehicle only (sans label), N = 6 sham ONT plus RGC label. RNFLT was measured by spectral domain optical coherence tomography (SD-OCT) at baseline then weekly for 1 month. RGCs were labeled by retrograde transport of fluorescently conjugated cholera toxin B (CTB) from the superior colliculus 48 hours prior to ONT or sham surgery. RGC density measurements were obtained by confocal scanning laser ophthalmoscopy (CSLO) at baseline and weekly for 1 month. RGC density and reactivity of microglia (anti-Iba1) and astrocytes (anti-GFAP) were determined from post mortem fluorescence microscopy of whole-mount retinae.

Results

RNFLT decreased after ONT by 17% (p<0.05), 30% (p<0.0001) and 36% (p<0.0001) at weeks 2, 3 and 4. RGC density decreased after ONT by 18%, 69%, 85% and 92% at weeks 1, 2, 3 and 4 (p<0.0001 each). RGC density measured in vivo at week 4 and post mortem by microscopy were strongly correlated (R = 0.91, p<0.0001). In vivo measures of RNFLT and RGC density were strongly correlated (R = 0.81, p<0.0001). In ONT- CTB labeled fellow eyes, RNFLT increased by 18%, 52% and 36% at weeks 2, 3 and 4 (p<0.0001), but did not change in fellow ONT-eyes sans CTB. Microgliosis was evident in the RNFL of the ONT-CTB fellow eyes, exceeding that observed in other fellow eyes.

Conclusions

In vivo measurements of RNFLT and RGC density are strongly correlated and can be used to monitor longitudinal changes after optic nerve injury. The strong fellow eye effect observed in eyes contralateral to ONT, only in the presence of CTB label, consisted of a dramatic increase in RNFLT associated with retinal microgliosis.     

α-Melanocyte-Stimulating Hormone Protects Retinal Vascular Endothelial Cells from Oxidative Stress and Apoptosis in a Rat Model of Diabetes

Aims

Oxidative stress and apoptosis are among the earliest lesions of diabetic retinopathy. This study sought to examine the anti-oxidative and anti-apoptotic effects of α-melanocyte-stimulating hormone (α-MSH) in early diabetic retinas and to explore the underlying mechanisms in retinal vascular endothelial cells.

Methods

Sprague-Dawley rats were injected intravenously with streptozocin to induce diabetes. The diabetic rats were injected intravitreally with α-MSH or saline. At week 5 after diabetes, the retinas were analyzed for reactive oxygen species (ROS) and gene expression. One week later, the retinas were processed for terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and transmission electron microscopy. Retinal vascular endothelial cells were stimulated by high glucose (HG) with or without α-MSH. The expression of Forkhead box O genes (Foxos) was examined through real-time PCR. The Foxo4 gene was overexpressed in endothelial cells by transient transfection prior to α-MSH or HG treatment, and oxidative stress and apoptosis were analyzed through CM-H2DCFDA and annexin-V assays, respectively.

Results

In diabetic retinas, the levels of H₂O₂ and ROS and the total anti-oxidant capacity were normalized, the apoptotic cell number was reduced, and the ultrastructural injuries were ameliorated by α-MSH. Treatment with α-MSH also corrected the aberrant changes in eNOS, iNOS, ICAM-1, and TNF-α expression levels in diabetic retinas. Furthermore, α-MSH inhibited Foxo4 up-regulation in diabetic retinas and in endothelial cells exposed to HG, whereas Foxo4 overexpression abrogated the anti-oxidative and anti-apoptotic effects of α-MSH in HG-stimulated retinal vascular endothelial cells.

Conclusions

α-MSH normalized oxidative stress, reduced apoptosis and ultrastructural injuries, and corrected gene expression levels in early diabetic retinas. The protective effects of α-MSH in retinal vascular endothelial cells may be mediated through the inhibition of Foxo4 up-regulation induced by HG. This study suggests an α-MSH-mediated potential intervention approach to early diabetic retinopathy and a novel regulatory mechanism involving Foxo4.     

Activation of c-Src tyrosine kinase mediated the degradation of occludin in ventilator-induced lung injury

Background

Ventilator-induced lung injury (VILI) is characterized by increased alveolar permeability, pulmonary edema. The tyrosine kinase, c-Src, is involved in VILI but its role has not been fully elucidated. This study examined the relationship between c-Src activation and occludin levels in VILI both in vitro and in vivo.

Methods

For the in vivo study, Wistar rats were randomly divided into five groups: control (group C); normal tidal volume (group M); normal tidal volume + c-Src inhibitor (PP2) (group M + P); high tidal volume (group H); and high tidal volume + c-Src inhibitor (PP2) (group H + P). Rats in all groups but group C underwent mechanical ventilation for 4 h. For the in vitro study, MLE-12 cells pretreated with PP2 and siRNA underwent cyclic stretching at 8% or 20% for 0, 1, 2 and 4 h. The expressions of occludin, c-Src, and p-c-Src were analyzed by western blotting, hematoxylin and eosin (HE) staining, and immunofluorescence.

Results

For the in vivo study, rats in group H showed decreased occludin expression and activated c-Src compared with group C. HE staining and lung injury score showed more severe lung injury and alveolar edema in group H compared with group M and group C. Group H + P had less pulmonary edema induced by the high tidal volume ventilation. For the in vitro study, occludin expression decreased and c-Src activation increased as indicated by the phosphorylation of c-Src over time. Consistently, PP2 could restore occludin levels.

Conclusions

Mechanical ventilation can activate c-Src by phosphorylation and increase the degradation of occludin. c-Src inhibitor can ameliorate barrier function and lung injury by up-regulating occludin.     

FeCo/graphite nanocrystals for multi-modality imaging of experimental vascular inflammation

Background

FeCo/graphitic-carbon nanocrystals (FeCo/GC) are biocompatible, high-relaxivity, multi-functional nanoparticles. Macrophages represent important cellular imaging targets for assessing vascular inflammation. We evaluated FeCo/GC for vascular macrophage uptake and imaging in vivo using fluorescence and MRI.

Methods and Results

Hyperlipidemic and diabetic mice underwent carotid ligation to produce a macrophage-rich vascular lesion. In situ and ex vivo fluorescence imaging were performed at 48 hours after intravenous injection of FeCo/GC conjugated to Cy5.5 (n = 8, 8 nmol of Cy5.5/mouse). Significant fluorescence signal from FeCo/GC-Cy5.5 was present in the ligated left carotid arteries, but not in the control (non-ligated) right carotid arteries or sham-operated carotid arteries (p = 0.03 for ligated vs. non-ligated). Serial in vivo 3T MRI was performed at 48 and 72 hours after intravenous FeCo/GC (n = 6, 270 µg Fe/mouse). Significant T2* signal loss from FeCo/GC was seen in ligated left carotid arteries, not in non-ligated controls (p = 0.03). Immunofluorescence staining showed colocalization of FeCo/GC and macrophages in ligated carotid arteries.

Conclusions

FeCo/GC accumulates in vascular macrophages in vivo, allowing fluorescence and MR imaging. This multi-functional high-relaxivity nanoparticle platform provides a promising approach for cellular imaging of vascular inflammation.     

Atrophic cardiac remodeling induced by taurine deficiency in wistar rats

Introduction

Micronutrient deficiency is observed in heart failure patients. Taurine, for example, represents 50% of total free amino acids in the heart, and in vivo studies have linked taurine deficiency with cardiomyopathy.

Methods

Thirty-four male Wistar rats (body weight = 100 g) were weighed and randomly assigned to one of two groups: Control (C) or taurine-deficient (T (-)). Beta-alanine at a concentration of 3% was added to the animals’ water to induce taurine deficiency in the T (-) group. On day 30, the rats were individually submitted to echocardiography; morphometrical and histopathological evaluation and metalloproteinase activity, oxidative stress and inflammation evaluation were performed. Tissue samples were collected to determine the taurine concentration in the heart.

Results

Taurine deficiency led to decreases in: ventricular wall thickness, left ventricle dry weight, myocyte sectional area, left ventricle posterior wall thickness and ventricular geometry. With regard to heart function, the velocity of the A wave, the ratio between the E and A wave, the ejection fraction, fractional shortening and cardiac output values were decreased in T (-) rats, suggesting abnormal diastolic and systolic function. Increased fibrosis, inflammation and increased activation of metalloproteinases were not observed. Oxidative stress was increased in deficient animals.

Conclusions

These data suggest that taurine deficiency promotes structural and functional cardiac alterations with unique characteristics.     

In Vivo Measurement of Hippocampal GABAA/cBZR Density with [18F]-Flumazenil PET for the Study of Disease Progression in an Animal Model of Temporal Lobe Epilepsy

Purpose

Imbalance of inhibitory GABAergic neurotransmission has been proposed to play a role in the pathogenesis of temporal lobe epilepsy (TLE). This study aimed to investigate whether [¹⁸F]-flumazenil ([¹⁸F]-FMZ) PET could be used to non-invasively characterise GABA_A/central benzodiazepine receptor (GABA_A/cBZR) density and affinity in vivo in the post-kainic acid status epilepticus (SE) model of TLE.

Methods

Dynamic [¹⁸F]-FMZ -PET scans using a multi-injection protocol were acquired in four male wistar rats for validation of the partial saturation model (PSM). SE was induced in eight male Wistar rats (10 weeks of age) by i.p. injection of kainic acid (7.5–25 mg/kg), while control rats (n = 7) received saline injections. Five weeks post-SE, an anatomic MRI scan was acquired and the following week an [¹⁸F]-FMZ PET scan (3.6–4.6 nmol). The PET data was co-registered to the MRI and regions of interest drawn on the MRI for selected structures. A PSM was used to derive receptor density and apparent affinity from the [¹⁸F]-FMZ PET data.

Key Findings

The PSM was found to adequately model [¹⁸F]-FMZ binding in vivo. There was a significant decrease in hippocampal receptor density in the SE group (p<0.01), accompanied by an increase in apparent affinity (p<0.05) compared to controls. No change in cortical receptor binding was observed. Hippocampal volume reduction and cell loss was only seen in a subset of animals. Histological assessment of hippocampal cell loss was significantly correlated with hippocampal volume measured by MRI (p<0.05), but did not correlate with [¹⁸F]-FMZ binding.

Significance

Alterations to hippocampal GABA_A/cBZR density and affinity in the post-kainic acid SE model of TLE are detectable in vivo with [¹⁸F]-FMZ PET and a PSM. These changes are independent from hippocampal cell and volume loss. [¹⁸F]-FMZ PET is useful for investigating the role that changes GABA_A/cBZR density and binding affinity play in the pathogenesis of TLE.     

Axonal transmission in the retina introduces a small dispersion of relative timing in the ganglion cell population response

Background

Visual stimuli elicit action potentials in tens of different retinal ganglion cells. Each ganglion cell type responds with a different latency to a given stimulus, thus transforming the high-dimensional input into a temporal neural code. The timing of the first spikes between different retinal projection neurons cells may further change along axonal transmission. The purpose of this study is to investigate if intraretinal conduction velocity leads to a synchronization or dispersion of the population signal leaving the eye.

Methodology/Principal Findings

We ‘imaged’ the initiation and transmission of light-evoked action potentials along individual axons in the rabbit retina at micron-scale resolution using a high-density multi-transistor array. We measured unimodal conduction velocity distributions (1.3±0.3 m/sec, mean ± SD) for axonal populations at all retinal eccentricities with the exception of the central part that contains myelinated axons. The velocity variance within each piece of retina is caused by ganglion cell types that show narrower and slightly different average velocity tuning. Ganglion cells of the same type respond with similar latency to spatially homogenous stimuli and conduct with similar velocity. For ganglion cells of different type intraretinal conduction velocity and response latency to flashed stimuli are negatively correlated, indicating that differences in first spike timing increase (up to 10 msec). Similarly, the analysis of pair-wise correlated activity in response to white-noise stimuli reveals that conduction velocity and response latency are negatively correlated.

Conclusion/Significance

Intraretinal conduction does not change the relative spike timing between ganglion cells of the same type but increases spike timing differences among ganglion cells of different type. The fastest retinal ganglion cells therefore act as indicators of new stimuli for postsynaptic neurons. The intraretinal dispersion of the population activity will not be compensated by variability in extraretinal conduction times, estimated from data in the literature.