Mouse Choroid Research Articles

Tissue-Specific Immune Transcriptional Signatures in the Bordering Tissues of the Mouse Retina and Brain.

Bordering the central nervous system (CNS) parenchyma are the choroid (underlying the retina) and the leptomeninges (the inner layers of the meninges enveloping the brain). Although near the neural parenchyma, the choroid and leptomeninges are external to the immune privileged environment of the retina and brain and thus are distinct immune compartments. This study aimed to characterize the transcriptomic signatures of immune cells within the choroid and leptomeninges bordering the healthy adult mouse CNS. Eyes and brains were obtained from 7-week-old C57Bl/6J mice. Choroid and leptomeninges were processed for isolation of CD45+ immune cells and single cell RNA-sequencing. Additionally, single cell RNA-sequencing was performed on immune cells isolated from choroid obtained from human donor eye tissue. Immunostaining and confocal microscopy of wholemount tissue were used to validate selected immune cell populations in situ. A total of 3606 cells were sequenced from mouse tissues, including 2125 CD45+ cells from choroid and 1481 CD45+ cells from leptomeninges. Clustering and differential gene expression analysis revealed heterogeneous subtypes of monocytes/macrophages, dendritic cells, T cells, and B cells. Whereas some clusters were common to both choroid and leptomeninges, others exhibited tissue-specific gene expression profiles and potential functional specializations. Analysis of 6501 CD45+ cells sequenced from human choroid identified similar immune cell populations to mouse choroid. This study provides a detailed characterization of the molecular signatures of immune cells within the vascular connective tissues bordering the healthy retina and brain, and their potential roles in immune protection.

In vivo volumetric analysis of retinal vascular hemodynamics in mice with spatio-temporal optical coherence tomography.

Microcirculation and neurovascular coupling are important parameters to study in neurological and neuro-ophthalmic conditions. As the retina shares many similarities with the cerebral cortex and is optically accessible, a special focus is directed to assessing the chorioretinal structure, microvasculature, and hemodynamics of mice, a vital animal model for vision and neuroscience research. We aim to introduce an optical imaging tool enabling in vivo volumetric mouse retinal monitoring of vascular hemodynamics with high temporal resolution. We translated the spatio-temporal optical coherence tomography (STOC-T) technique into the field of small animal imaging by designing a new optical system that could compensate for the mouse eye refractive error. We also developed post-processing algorithms, notably for the assessment of (i) localized hemodynamics from the analysis of pulse wave-induced Doppler artifact modulation and (ii) retinal tissue displacement from phase-sensitive measurements. We acquired high-quality, in vivo volumetric mouse retina images at a rate of 113Hz over a lateral field of view of . We presented high-resolution en face images of the retinal and choroidal structure and microvasculature from various layers, after digital aberration correction. We were able to measure the pulse wave velocity in capillaries of the outer plexiform layer with a mean speed of 0.35mm/s and identified venous and arterial pulsation frequency and phase delay. We quantified the modulation amplitudes of tissue displacement near major vessels (with peaks of 150nm), potentially carrying information about the biomechanical properties of the retinal layers involved. Last, we identified the delays between retinal displacements due to the passing of venous and arterial pulse waves. The developed STOC-T system provides insights into the hemodynamics of the mouse retina and choroid that could be beneficial in the study of neurovascular coupling and vasculature and flow speed anomalies in neurological and neuro-ophthalmic conditions.

Aging alters the expression of trophic factors and tight junction proteins in the mouse choroid plexus

BackgroundThe choroid plexus (CP) is an understudied tissue in the central nervous system and is primarily implicated in cerebrospinal fluid (CSF) production. CP also produces numerous neurotrophic factors (NTF) which circulate to different brain regions. Regulation of NTFs in the CP during natural aging is largely unknown. Here, we investigated the age and gender-specific transcription of NTFs along with the changes in the tight junctional proteins (TJPs) and the water channel protein Aquaporin (AQP1).MethodsMale and female mice were used for our study. Age-related transcriptional changes were analyzed using quantitative PCR at three different time points: mature adult, middle-aged, and aged. Transcriptional changes during aging were further confirmed with digital droplet PCR. Additionally, we used immunohistochemical analysis (IHC) for the evaluation of in vivo protein expression. We further investigated the cellular phenotype of these NTFS, TJP, and water channel proteins in the mouse CP by co-labeling them with the classical vascular marker, Isolectin B4, and epithelial cell marker, Plectin.ResultsAging significantly altered NTF gene expression in the CP. Brain-derived neurotrophic factor (BDNF), Midkine (MDK), VGF, Insulin-like growth factor (IGF1), IGF2, Klotho (KL), Erythropoietin (EPO), and its receptor (EPOR) were reduced in the aged CP of males and females. Vascular endothelial growth factor (VEGF) transcription was gender-specific; in males, gene expression was unchanged in the aged CP, while females showed an age-dependent reduction. Age-dependent changes in VEGF localization were evident, from vasculature to epithelial cells. IGF2 and klotho localized in the basolateral membrane of the CP and showed an age-dependent reduction in epithelial cells. Water channel protein AQP1 localized in the tip of epithelial cells and showed an age-related reduction in mRNA and protein levels. TJP’s JAM, CLAUDIN1, CLAUDIN2 and CLAUDIN5 were reduced in aged mice.ConclusionsOur study highlights transcriptional level changes in the CP during aging. The age-related transcriptional changes exhibit similarities as well as gene-specific differences in the CP of males and females. Altered transcription of the water channel protein AQP1 and TJPs could be involved in reduced CSF production during aging. Importantly, reduction in the neurotrophic factors and longevity factor Klotho can play a role in regulating brain aging.Graphical

Isolating Immune Cells from Mouse Choroid Plexuses

Isolating Immune Cells from Mouse Choroid Plexuses

Non-endothelial expression of endomucin in the mouse and human choroid

Endomucin (EMCN) is a 261 amino acid transmembrane glycoprotein that is highly expressed by venous and capillary endothelial cells where it plays a role in VEGF-mediated angiogenesis and regulation of immune cell recruitment. However, it is better known as a histological marker, where it has become widespread due to the commercial availability of high-quality antibodies that work under a wide range of conditions and in many tissues. The specificity of EMCN staining has been well-validated in retinal vessels, but while it has been used extensively as a marker in other tissues of the eye, including the choroid, the pattern of expression has not been described in detail. Here, in addition to endothelial expression in the choriocapillaris and deeper vascular layers, we characterize a population of EMCN-positive perivascular cells in the mouse choroid that did not co-localize with cells expressing other endothelial markers such as PECAM1 or PODXL. To confirm that these cells represented a new population of EMCN-expressing stromal cells, we then performed single cell RNA sequencing in choroids from adult wild-type mice. Analysis of this new dataset confirmed that, in addition to endothelial cells, Emcn mRNA expression was present in choroidal pericytes and a subset of fibroblasts, but not vascular smooth muscle cells. Besides Emcn, no known endothelial gene expression was detected in these cell populations, confirming that they did not represent endothelial-stromal doublets, a common technical artifact in single cell RNA seq datasets. Instead, choroidal Emcn-expressing fibroblasts exhibited high levels of chemokine and interferon signaling genes, while Emcn-negative fibroblasts were enriched in genes encoding extracellular matrix proteins. Emcn expressing fibroblasts were also detected in published datasets from mouse brain and human choroid, suggesting that stromal Emcn expression was not unique to the choroid and was evolutionarily conserved. Together, these findings highlight unique fibroblast and pericyte populations in the choroid and provide new context for the role of EMCN in the eye.

In vivo multi-contrast depth-resolved choroidal imaging of a mouse using polarization-diversity optical coherence tomography.

The results of depth-resolved multi-contrast in vivo mouse choroidal imaging using a polarization-diversity optical coherence tomography (PD-OCT) system are presented. A selectively chosen depth of focus that was fine-tuned with a sensorless adaptive optics technique and a simple segmentation based on the degree of polarization uniformity signal visualizes the detailed features of a mouse choroid from the OCT angiography images. A comprehensive image analysis of the choroid revealed the distinctive pathological characteristics of the laser-induced choroidal neovascularization mouse.

3,3',5-Triiodothyroacetic Acid Transporters.

Introduction: Thyroid hormone transporters are essential for thyroid hormones to enter target cells. Monocarboxylate transporter (MCT) 8 is a key transporter and is expressed at the blood-brain barrier (BBB), in neural cells and many other tissues. Patients with MCT8 deficiency have severe neurodevelopmental delays because of cerebral hypothyroidism and chronic sequelae of peripheral thyrotoxicosis. The T3 analog 3,3',5-triiodothyroacetic acid (TRIAC) rescued neurodevelopmental features in animal models mimicking MCT8 deficiency and improved key metabolic features in patients with MCT8 deficiency. However, the identity of the transporter(s) that facilitate TRIAC transport are unknown. Here, we screened candidate transporters that are expressed at the human BBB and/or brain-cerebrospinal fluid barrier and known thyroid hormone transporters for TRIAC transport. Materials and Methods: Plasma membrane expression was determined by cell surface biotinylation assays. Intracellular accumulation of 1 nM TRIAC was assessed in COS-1 cells expressing candidate transporters in Dulbecco's phosphate-buffered saline (DPBS)/0.1% glucose or Dulbecco's modified Eagle's medium (DMEM) with or without 0.1% bovine serum albumin (BSA). Expression of Slc22a8 was determined by fluorescent in situ hybridization in brain sections from wild-type and Mct8/Oatp1c1 knockout mice at postnatal days 12, 21, and 120. Results: In total, 59 plasma membrane transporters were selected for screening of TRIAC accumulation (n = 40 based on expression at the human BBB and/or brain-cerebrospinal fluid barrier and having small organic molecules as substrates; n = 19 known thyroid hormone transporters). Screening of the selected transporter panel showed that 18 transporters facilitated significant intracellular accumulation of TRIAC in DPBS/0.1% glucose or DMEM in the absence of BSA. In the presence of BSA, substantial transport was noted for SLCO1B1 and SLC22A8 (in DPBS/0.1% glucose and DMEM) and SLC10A1, SLC22A6, and SLC22A24 (in DMEM). The zebrafish and mouse orthologs of these transporters similarly facilitated intracellular accumulation of TRIAC. Highest Slc22a8 mRNA expression was detected in mouse brain capillary endothelial cells and choroid plexus epithelial cells at early postnatal time points, but was reduced at P120. Conclusions: Human SLC10A1, SLCO1B1, SLC22A6, SLC22A8, and SLC22A24 as well as their mouse and zebrafish orthologs are efficient TRIAC transporters. These findings contribute to the understanding of TRIAC treatment in patients with MCT8 deficiency and animal models thereof.

The Pattern of GH Action in the Mouse Brain.

GH acts in numerous organs expressing the GH receptor (GHR), including the brain. However, the mechanisms behind the brain's permeability to GH and how this hormone accesses different brain regions remain unclear. It is well-known that an acute GH administration induces phosphorylation of the signal transducer and activator of transcription 5 (pSTAT5) in the mouse brain. Thus, the pattern of pSTAT5 immunoreactive cells was analyzed at different time points after IP or intracerebroventricular GH injections. After a systemic GH injection, the first cells expressing pSTAT5 were those near circumventricular organs, such as arcuate nucleus neurons adjacent to the median eminence. Both systemic and central GH injections induced a medial-to-lateral pattern of pSTAT5 immunoreactivity over time because GH-responsive cells were initially observed in periventricular areas and were progressively detected in lateral brain structures. Very few choroid plexus cells exhibited GH-induced pSTAT5. Additionally, Ghr mRNA was poorly expressed in the mouse choroid plexus. In contrast, some tanycytes lining the floor of the third ventricle expressed Ghr mRNA and exhibited GH-induced pSTAT5. The transport of radiolabeled GH into the hypothalamus did not differ between wild-type and dwarf Ghr knockout mice, indicating that GH transport into the mouse brain is GHR independent. Also, single-photon emission computed tomography confirmed that radiolabeled GH rapidly reaches the ventral part of the tuberal hypothalamus. In conclusion, our study provides novel and valuable information about the pattern and mechanisms behind GH transport into the mouse brain.

Interleukin-17-mediated protective cytokine signaling against degeneration of the retinal pigment epithelium.

Injuries to the retinal pigment epithelium (RPE) and outer retina often result in the accumulation of retinal microglia within the subretinal space. These subretinal microglia play crucial roles in inflammation and resolution, but the mechanisms governing their functions are still largely unknown. Our previous research highlighted the protective functions of choroidal γδ T cells in response to RPE injury. In the current study, we employed single-cell RNA sequencing approach to characterize the profiles of immune cells in mouse choroid. We found that γδ T cells were the primary producer of interleukin-17 (IL-17) in the choroid. IL-17 signaled through its receptor on the RPE, subsequently triggering the production of interleukin-6. This cascade of cytokines initiated a metabolic reprogramming of subretinal microglia, enhancing their capacity for lipid metabolism. RPE-specific knockout of IL-17 receptor A led to the dysfunction of subretinal microglia and RPE pathology. Collectively, our findings suggest that responding to RPE injury, the choroidal γδ T cells can initiate a protective signaling cascade that ensures the proper functioning of subretinal microglia.

Exploring the Potential of Wild Olive (Acebuche) Oil as a Pharm-Food to Prevent Ocular Hypertension and Fibrotic Events in the Retina of Hypertensive Mice.

Our laboratoryhas previously described the antioxidant and anti-inflammatory potential of a wild olive (acebuche, ACE) oil against hypertension-associated vascular retinopathies. The current study aims to analyze the antifibrotic effect of ACE oil on the retina of hypertensive mice. Mice are rendered hypertensive by administration of NG-nitro-L-arginine-methyl-ester (L-NAME) and simultaneously subjected to dietary supplementation with ACE oil or a reference extra virgin olive oil (EVOO). Intraocular pressure (IOP) is measured by rebound tonometry, and retinal vasculature/layers are analyzed by fundus fluorescein angiography and optical coherence tomography. Different fibrosis-related parameters are analyzed in the retina and choroid of normotensive and hypertensive mice with or without oil supplementation. Besides preventing the alterations found in hypertensive animals, including increased IOP, reduced fluorescein signal, and altered retinal layer thickness, the ACE oil-enriched diet improves collagen metabolism by regulating the expression of major fibrotic process modulators (matrix metalloproteinases, tissue inhibitors of metalloproteinases, connective tissue growth factor, and transforming growth factor beta family). Regular consumption of EVOO and ACE oil (with better outcomes in the latter) might help reduce abnormally high IOP values in the context of hypertension-related retinal damage, with significant reduction in the surrounding fibrotic process.

In vitro investigation of the effect of proinflammatory cytokines on mouse choroid plexus membrane transporters Ncbe and NKCC1

Intraventricular hemorrhage is a potentially life-threatening condition. Approximately 20% of patients develop posthemorrhagic hydrocephalus with increased ventricular volume and intracranial pressure. Hydrocephalus develops partially due to increased secretion of cerebrospinal fluid by the choroid plexus. During hemorrhage a multitude of factors are released into the cerebrospinal fluid. Many of these have been implicated in the hypersecretion. In this study, we have investigated the isolated effect of inflammatory components, on the abundance of two membrane transporters involved in cerebrospinal fluid secretion by the choroid plexus: the Na+-dependent Cl−/HCO3− exchanger, Ncbe, and the Na+, K+, 2Cl− cotransporter, NKCC1. We have established a primary choroid plexus epithelial cell culture from 1 to 7 days old mouse pups. Seven days after seeding, the cells formed a monolayer. The cells were treated with either tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β), or interleukin 6 (IL-6) to mimic inflammation. The data show that treatment with TNFα, and IL-1β only transiently increased NKCC1 abundance whereas the effect on Ncbe abundance was a transient decrease. IL-6 however significantly increased NKCC1 (242%), the phosphorylated NKCC1 (147%), as well as pSPAK (406%) abundance, but had no effect on Ncbe. This study suggests that the inflammatory pathway involved in hypersecretion primarily is mediated by activation of basolateral receptors in the choroid plexus, mainly facilitated by IL-6. This study highlights the complexity of the pathophysiological circumstances occurring during intraventricular hemorrhage.

Molecular Mechanisms of Organic Anion Transporting Polypeptide-Mediated Organic Anion Clearance at the Blood-Cerebrospinal Fluid Barrier.

The blood-cerebrospinal fluid barrier (BCSFB), formed by the choroid plexus epithelial (CPE) cells, plays an active role in removing drugs and metabolic wastes from the brain. Recent functional studies in isolated mouse choroid plexus (CP) tissues suggested the presence of organic anion transporting polypeptides (OATPs, encoded by SLCOs) at the apical membrane of BCSFB, which may clear large organic anions from the cerebrospinal fluid (CSF). However, the specific OATP isoform involved is unclear. Using quantitative fluorescence imaging, we showed that the fluorescent anions sulforhodamine 101 (SR101), fluorescein methotrexate (FL-MTX), and 8-fluorescein-cAMP (fluo-cAMP) are actively transported from the CSF to the subepithelial space in CP tissues isolated from wild-type mice. In contrast, transepithelial transport of these compounds across the CPE cells was abolished in Oatp1a/1b-/- mice due to impaired apical uptake. Using transporter-expressing cell lines, SR101, FL-MTX, and fluo-cAMP were additionally shown to be transported by mouse OATP1A5 and its human counterpart OATP1A2. Kinetic analysis showed that estrone-3-sulfate and SR101 are transported by OATP1A2 and OATP1A5 with similar Michaelis-Menten constants (Km). Immunofluorescence staining further revealed the presence of OATP1A2 protein in human CP tissues. Together, our results suggest that large organic anions in the CSF are actively transported into CPE cells by apical OATP1A2 (OATP1A5 in mice), then subsequently effluxed into the blood by basolateral multidrug resistance-associated proteins (MRPs). As OATP1A2 transports a wide array of endogenous compounds and xenobiotics, the presence of this transporter at the BCSFB may imply a novel clearance route for drugs and neurohormones from the CSF. SIGNIFICANCE STATEMENT: Drug transporters at the blood-cerebrospinal fluid (CSF) barrier play an important but understudied role in brain drug disposition. This study revealed a functional contribution of rodent organic anion transporting polypeptide (OATP) 1A5 towards the CSF clearance of organic anions and suggested a similar role for OATP1A2 in humans. Delineating the molecular mechanisms governing CSF organic anion clearance may help to improve the prediction of central nervous system (CNS) pharmacokinetics and identify drug candidates with favorable CNS pharmacokinetic properties.

Downregulation of VEGF in the retinal pigment epithelium followed by choriocapillaris atrophy after NaIO3 treatment in mice

Sodium iodate (NaIO3) induces retinal pigment epithelium (RPE) dysfunction, which leads to photoreceptor degeneration. Previously, we used electron microscopy to show that the administration of NaIO3 resulted in the accumulation of cell debris in the subretinal space, which was thought to be caused by failed phagocytosis in the outer segment of the photoreceptor due to RPE dysfunction. We further analyzed the pathological changes in the retina and choroid of NaIO3-injected mice, and found that the expression of OTX2, an RPE marker, disappeared from central part of the RPE 1 day after NaIO3 administration. Furthermore, fenestrated capillaries (choriocapillaris, CC) adjacent to the RPE could not be identified only 2 days after NaIO3 administration. An examination of the expression of the CC-specific protein plasmalemma vesicle-associated protein (PLVAP), in sections and flat-mount retina/choroid specimens showed destruction of the CC, and complete disappearance of the PLVAP signal 7 days after NaIO3 administration. In contrast, CD31 flat-mount immunohistochemistry of the retina indicated no difference in retinal vessels between NaIO3-treated mice and controls. Electron microscopy showed that the fenestrated capillaries in the kidney and duodenum were morphologically indistinguishable between control and NaIO3-treated mice. We examined cytokine production in the retina and RPE, and found that the Vegfa transcript level in the RPE decreased starting 1 day after NaIO3 administration. Taken together, these observations show that NaIO3 reduces the CC in the early stages of the pathology, which is accompanied by a rapid decrease in Vegfa expression in the RPE.

Defining diurnal fluctuations in mouse choroid plexus and CSF at high molecular, spatial, and temporal resolution

Transmission and secretion of signals via the choroid plexus (ChP) brain barrier can modulate brain states via regulation of cerebrospinal fluid (CSF) composition. Here, we developed a platform to analyze diurnal variations in male mouse ChP and CSF. Ribosome profiling of ChP epithelial cells revealed diurnal translatome differences in metabolic machinery, secreted proteins, and barrier components. Using ChP and CSF metabolomics and blood-CSF barrier analyses, we observed diurnal changes in metabolites and cellular junctions. We then focused on transthyretin (TTR), a diurnally regulated thyroid hormone chaperone secreted by the ChP. Diurnal variation in ChP TTR depended on Bmal1 clock gene expression. We achieved real-time tracking of CSF-TTR in awake TtrmNeonGreen mice via multi-day intracerebroventricular fiber photometry. Diurnal changes in ChP and CSF TTR levels correlated with CSF thyroid hormone levels. These datasets highlight an integrated platform for investigating diurnal control of brain states by the ChP and CSF.

Distribution of the cysteinyl leukotriene system components in the human, rat and mouse eye

The cysteinyl leukotrienes (CysLTs) have important functions in the regulation of inflammation and cellular stress. Blocking the CysLT receptors (CysLTRs) with specific antagonists is beneficial against progression of retinopathies (e.g. diabetic retinopathy, wet AMD). However, the exact cellular localization of the CysLTRs and their endogenous ligands in the eye have not been elucidated in detail yet. It is also not known whether the expression patterns differ between humans and animal models. Therefore, the present study aimed to describe and compare the distribution of two important enzymes in CysLT biosynthesis, 5-lipoxygenase (5-LOX) and 5-lipoxygenase-activating protein (FLAP), and of CysLTR1 and CysLTR2 in healthy human, rat and mouse eyes.Human donor eyes (n = 10) and eyes from adult Sprague Dawley rats (n = 5) and CD1 mice (n = 8) of both sexes were collected. The eyes were fixed in 4% paraformaldehyde and cross-sections were investigated by immunofluorescence with specific antibodies against 5-LOX, FLAP (human tissue only), CysLTR1 and CysLTR2. Flat-mounts of the human choroid were prepared and processed similarly. Expression patterns were assessed and semiquantitatively evaluated using a confocal fluorescence microscope (LSM710, Zeiss).We observed so far unreported expression sites for CysLT system components in various ocular tissues. Overall, we detected expression of 5-LOX, CysLTR1 and CysLTR2 in the human, rat and mouse cornea, conjunctiva, iris, lens, ciliary body, retina and choroid. Importantly, expression profiles of CysLTR1 and CysLTR2 were highly similar between human and rodent eyes. FLAP was expressed in all human ocular tissues except the lens. Largely weak immunoreactivity of FLAP and 5-LOX was observed in a few, yet unidentified, cells of diverse ocular tissues, indicating low levels of CysLT biosynthesis in healthy eyes. CysLTR1 was predominantly detected in ocular epithelial cells, supporting the involvement of CysLTR1 in stress and immune responses. CysLTR2 was predominantly expressed in neuronal structures, suggesting neuromodulatory roles of CysLTR2 in the eye and revealing disparate functions of CysLTRs in ocular tissues.Taken together, we provide a comprehensive protein expression atlas of CysLT system components in the human and rodent eye. While the current study is purely descriptive and therefore does not allow significant functional conclusions yet, it represents an important basis for future studies in diseased ocular tissues in which distribution patterns or expression levels of the CysLT system might be altered. Furthermore, this is the first comprehensive study to elucidate expression patterns of CysLT system components in human and animal models that will help to identify and understand functions of the system as well as mechanisms of action of potential CysLTR ligands in the eye.

Morphological and mitochondrial changes in murine choroid plexus epithelial cells during healthy aging

BackgroundChoroid plexuses (ChPs) are intraventricular structures mainly composed by specialized epithelial cells interconnected by tight junctions that establish the blood-cerebrospinal fluid (CSF) barrier. ChPs are essential to produce CSF and transport solutes from and into the brain. Deterioration of ChP function and morphology has been correlated to worsening of neurodegenerative disorders. We here map morpho-functional changes in the ChP epithelial cells during healthy aging, starting from young adult to 2-years old mice.MethodsWe used a multi-tiered approach, including transmission electron microscopy (TEM), immunohistochemistry, RT-qPCR, Western Blot and 2-photon microscopy (2-PM) at multiple timepoints ranging from young adult to 2-years old mice.ResultsWe identified distinct morpho-functional modifications in epithelial cells of ChP starting from 8 to 12 months of age, which mostly remained stable up to 2 years. These changes include flattening of the epithelium, reduction of microvilli length and an augmentation of interrupted tight junctions. We also found a decrease in mitochondria density together with elongation of mitochondria in older mice. Morphological mitochondrial rearrangements were accompanied by increased superoxide levels, decreased membrane potential and decreased mitochondrial motility in aged mice. Interestingly, most of the age-related changes were not accompanied by modification of protein and/or gene expression levels and aged mitochondria effectively responded to acute pharmacological stressful stimuli.ConclusionsOur study suggests a long-term progression of multiple morpho-functional features of the mouse choroid plexus epithelium during adulthood followed by structural remodeling during the aging process. These findings can lead to a better understanding on how functional and morphological rearrangements of ChP are correlated during aging.

Genetic deficiency and pharmacological modulation of RORα regulate laser-induced choroidal neovascularization.

Choroidal neovascularization (CNV) causes acute vision loss in neovascular age-related macular degeneration (AMD). Genetic variations of the nuclear receptor RAR-related orphan receptor alpha (RORα) have been linked with neovascular AMD, yet its specific role in pathological CNV development is not entirely clear. In this study, we showed that Rora was highly expressed in the mouse choroid compared with the retina, and genetic loss of RORα in Staggerer mice (Rorasg/sg) led to increased expression levels of Vegfr2 and Tnfa in the choroid and retinal pigment epithelium (RPE) complex. In a mouse model of laser-induced CNV, RORα expression was highly increased in the choroidal/RPE complex post-laser, and loss of RORα in Rorasg/sg eyes significantly worsened CNV with increased lesion size and vascular leakage, associated with increased levels of VEGFR2 and TNFα proteins. Pharmacological inhibition of RORα also worsened CNV. In addition, both genetic deficiency and inhibition of RORα substantially increased vascular growth in isolated mouse choroidal explants ex vivo. RORα inhibition also promoted angiogenic function of human choroidal endothelial cell culture. Together, our results suggest that RORα negatively regulates pathological CNV development in part by modulating angiogenic response of the choroidal endothelium and inflammatory environment in the choroid/RPE complex.

Mouse Choroid Proteome Revisited: Focus on Aging.

Age is a major risk factor for age-related macular degeneration (AMD), and age has a role in the disease phenotypes of heritable macular dystrophies. The proteomes of C57Bl6/J mouse choroids at 2 ages were analyzed to identify biochemical processes affected by aging. Proteins of interest were identified as those contributing most to the variance in principal component analysis and those showing the largest significant differences between ages. These proteins implicated altered ECM composition, immune system function, and lipid metabolism.

Identification of CD157-Positive Vascular Endothelial Stem Cells in Mouse Retinal and Choroidal Vessels: Fluorescence-Activated Cell Sorting Analysis.

PurposeCD157 (also known as Bst1) positive vascular endothelial stem cells (VESCs), which contribute to vascular regeneration, have been recently identified in mouse organs, including the retinas, brain, liver, lungs, heart, and skin. However, VESCs have not been identified in the choroid. The purpose of this study was to identify VESCs in choroidal vessels and to establish the protocol to isolate retinal and choroidal VESCs.MethodsWe established an efficient protocol to create single-cell suspensions from freshly isolated mouse retina and choroid by enzymatic digestion using dispase, collagenase, and type II collagenase. CD157-positive VESCs, defined as CD31+CD45−CD157+ cells, were sorted using fluorescence-activated cell sorting (FACS).ResultsIn mouse retina, among CD31+CD45− endothelial cells (ECs), 1.6 ± 0.2% were CD157-positive VESCs, based on FACS analysis. In mouse choroid, among CD31+CD45− ECs, 4.5 ± 0.4% were VESCs. The CD157-positive VESCs generated a higher number of EC networks compared with CD157-negative non-VESCs under vascular endothelial growth factor (VEGF) in vitro cultures. The EC network area, defined as the ratio of the CD31-positive area to the total area in each field, was 4.21 ± 0.39% (retinal VESCs) and 0.27 ± 0.12% (retinal non-VESCs), respectively (P < 0.01). The EC network area was 8.59 ± 0.78% (choroidal VESCs) and 0.14 ± 0.04% (choroidal non-VESCs), respectively (P < 0.01). The VESCs were located in large blood vessels but not in the capillaries.ConclusionsWe confirmed distinct populations of CD157-positive VESCs in both mouse retina and choroid. VESCs are located in large vessels and have the proliferative potential. The current results may open new avenues for the research and treatment of ocular vascular diseases.

Live Tissue Imaging Reveals Distinct Transcellular Pathways for Organic Cations and Anions at the Blood-Cerebrospinal Fluid Barrier.

Formed by the choroid plexus epithelial (CPE) cells, the blood-cerebrospinal fluid barrier (BCSFB) plays an active role in removing drugs, toxins, and metabolic wastes from the brain. Several organic cation and anion transporters are expressed in the CPE cells, but how they functionally mediate transepithelial transport of organic cations and anions remain unclear. In this study, we visualized the transcellular transport of fluorescent organic cation and organic anion probes using live tissue imaging in freshly isolated mouse choroid plexuses (CPs). The cationic probe, 4-[4-(dimethylamino)phenyl]-1-methylpyridinium iodide (IDT307) was transported into CPE cells at the apical membrane and highly accumulated in mitochondria. Consistent with the lack of expression of organic cation efflux transporters, there was little efflux of IDT307 into the blood capillary space. Furthermore, IDT307 uptake and intracellular accumulation was attenuated by approximately 70% in CP tissues from mice with targeted deletion of the plasma membrane monoamine transporter (Pmat). In contrast, the anionic probe fluorescein-methotrexate (FL-MTX) was rapidly transported across the CPE cells into the capillary space with little intracellular accumulation. Rifampicin, an inhibitor of organic anion transporting polypeptides (OATPs), completely blocked FL-MTX uptake into the CPE cells whereas MK-571, a pan-inhibitor of multidrug resistance associated proteins (MRPs), abolished basolateral efflux of FL-MTX. In summary, our results suggest distinct transcellular transport pathways for organic cations and anions at the BCSFB and reveal a pivotal role of PMAT, OATP and MRP transporters in organic cation and anion transport at the blood-cerebrospinal fluid interface. SIGNIFICANCE STATEMENT: Live tissue imaging revealed that while organic cations are transported from the cerebrospinal fluid (CSF) into the choroid plexus epithelial cells by plasma membrane monoamine transporter without efflux into the blood, amphipathic anions in the CSF are efficiently transported across the BCSFB through the collaborated function of apical organic anion transporting polypeptides and basolateral multidrug resistance associated proteins. These findings contribute to a mechanistic understanding of the molecular and cellular pathways for choroid plexus clearance of solutes from the brain.