The choroidal nervous system: a link between mineralocorticoid receptor and pachychoroid

Abstract

Central serous chorioretinopathy (CSCR) belongs to the pachychoroid spectrum, a pathological phenotype of the choroidal vasculature, in which blood flow is under the choroidal nervous system (ChNS) regulation. The pathogenesis of CSCR is multifactorial, with the most recognised risk factor being intake of glucocorticoids, which activate both the gluco- and the mineralocorticoid (MR) receptors. As MR over-activation is pathogenic in the retina and choroid, it could mediate the pathogenic effects of glucocorticoids in CSCR. But the role of MR signalling in pachychoroid is unknown and whether it affects the ChNS has not been explored. Using anatomo-neurochemical characterisation of the ChNS in rodents and humans, we discovered that beside innervation of arteries, choroidal veins and choriocapillaris are also innervated, suggesting that the entire choroidal vasculature is under neural control. The numerous synapses together with calcitonin gene-related peptide (CGRP) vesicles juxtaposed to choroidal macrophages indicate a neuro-immune crosstalk. Using ultrastructural approaches, we show that transgenic mice overexpressing human MR, display a pachychoroid-like phenotype, with signs of choroidal neuropathy including myelin abnormalities, accumulation and enlargement of mitochondria and nerves vacuolization. Transcriptomic analysis of the RPE/choroid complex in the transgenic mice reveals regulation of corticoids target genes, known to intervene in nerve pathophysiology, such as Lcn2, rdas1/dexras1, S100a8 and S100a9, rabphilin 3a (Rph3a), secretogranin (Scg2) and Kinesin Family Member 5A (Kif5a). Genes belonging to pathways related to vasculature development, hypoxia, epithelial cell apoptosis, epithelial mesenchymal transition, and inflammation, support the pachychoroid phenotype and highlight downstream molecular targets. Hypotheses on the imaging phenotype of pachychoroid in humans are put forward in the light of these new data. Our results provide evidence that MR overactivation causes a choroidal neuropathy that could explain the pachychoroid phenotype found in transgenic mice overexpressing human MR. In patients with pachychoroid and CSCR in which systemic dysautonomia has been demonstrated, MR-induced choroidal neuropathy could be the missing link between corticoids and pachychoroid.

Keywords: CSCR; Choroid; Innervation; Mineralocorticoid; Neuropathy; Pachychoroid.

Fig. 1

Visualisation and general organization of the choroidal nervous system in rats. a Example of a rat choroid stained with TUBB3 after negative filtering using Image J, revealing the general organisation of the choroidal nervous system (ChNS). The two pictures respectively represent a zoomed view of the optic nerve (ON) zone and the transition zone between the choroid and the limbus. The arrows and stars indicate large nerves entering the choroid. Two large nerves are entering in nasal and superior parts of the choroid (stars) whilst two others seem to enter in the nasal and temporal inferior part (arrows). The inferior temporal nerve is out of focus on the long arrow trajectory, remerging just under the LPCA. The zoomed view of the peripheral choroid is showing that the large nerves are responsible for the limbus/corneal innervation. These large fibres are probably corresponding to the human short and long ciliary nerves. Schematic representation of the ChNS (in green) organisation around the vascular system (in pink and blue). Pictures b–e represent the organisation of the ChNS surrounding the IF and the temporal LPCA. b, c Architecture of the IF innervation, the fibres originating in the superior IF bifurcates progressively following the arterial bifurcations. The large nerve (coming from the nasal inferior zone around the optic nerve) is passing under the IF, making collaterals. d, e Architecture of the temporal LPCA innervation, the fibres are surrounding the LPCA and bifurcate progressively at arterial-arteriole bifurcations. Large nerves follow the trajectory of the LPCA toward the peripheral choroid and innervate the limbus/cornea. f, g TUBB3 immunostaining (in green) and phalloidin-rhodamine staining (in red) reveal the ChNS organisation on rat eyes transversal sections. The nerve fibres are strongly organised around the arteries (strongly stained with the phalloidin) and numerous fibres can be observed in the choriocapillaris, juxtaposed to the RPE (white arrows). ON: Optic nerve; LPCA: Long posterior ciliary arteries; IF: Inferior branch

Fig. 2

ChNS neurochemical profiles, vascular targets, and contact with resident macrophages. TUBB3/Synaptophysin (neuronal and synaptic marker) VIP/ChAT (parasympathetic) NPY (sympathetic), CGRP (sensory) phalloidin/α-SMA (vascular markers) were used to visualise the neurochemical profile and targets of the choroidal innervation. a–c General organisation of the ChNS showing important vascular targeting, both arterial and venous structure are innervated. d–f Parasympathetic (VIP/ChAT) component is the major component of the ChNS. Parasympathetic fibres are found in the ciliary nerves (e), forming sometimes bifurcations (arrow) which innervate the choroid. VIP-positive fibres are targeting the vessels and are also highly represented in the intervascular space. g–i Sympathetic nervous system is the second major component of the ChNS. It is present in the ciliary nerves (h) and is almost exclusively organised around the choroidal vessels. j–l Sensory nervous system is the less represented component of the ChNS. It is highly present in the ciliary nerves, which form multiple bifurcations innervating the choroid. The CGRP-positive fibres are targeting the choroidal vessels but are also found in the intervascular space. m–o IBA1 and Synaptophysin immunostaining reveals the anatomical proximity between the choroidal macrophages and the choroidal nervous system. Numerous immune cells are organised around the vessels, in different layers, and close to the surrounding synapses. Numerous synaptophysin-positive zones are found in contact with IBA1-positive cells. p–r CGRP-positive fibres are found close to immune cells, sometimes displaying apparent contacts, which suggest an interaction between the immune system and the sensory nervous system within the choroid

Fig. 3

Ultrastructure of mouse choroidal innervation by SBF-SEM and TEM. a A single section of the mouse choroid from a volume acquired by SBF-SEM, showing a large choroidal nerve (purple), choroidal nerve fibres (magenta) and the choriocapillaris (CC, in red). Other small fibres are visible (arrows) as well as pigments from melanocytes (white) and large vessels (dark). b 3D reconstruction of the SBF-SEM volume presented in A, with the myelin of the large nerve (purple), the choroidal nerve fibre (magenta) and the choriocapillaris (red) segmented. The reconstruction reveals the ultrastructural organisation of the choroidal innervation, with a clear innervation of large vessels (arrow) and CC/RPE (star). c, d Transmission electron microscopy (TEM) analysis of a large choroidal nerve after osmium contrasting. The large nerve is composed by large, myelinated fibres (mf) nearby myelinating Schwann cells (mSC) and group of non-myelinated fibres (nmf) where non-myelinating Schwann cells (nmSC) are visible. N: myelinated Schwann cell nucleus

Fig. 4

Immunohistochemical and ultrastructural analysis of the human choroidal nervous system. a General organisation of the human choroidal nervous system (TUBB3) and its proximity with the choroidal vessels (Lectin), iChNS are clearly noticeable. b large choroidal nerve corresponding to human ciliary nerve. c NF200 is found in the inter-capillary space (white arrows), showing innervation of the choriocapillaris (CC) (Collagen IV). (d) TUBB3 staining allows the visualisation of IChNS and their neurites. e–h Parasympathetic fibres and VIP-positive IChNS (g-h) are present in the human choroid, both in large nerve (e) and around choroidal vessels (f). i–l Sympathetic fibres are numerous in the human choroid, both around large vessels (I) but also at the level of the CC, just under the RPE (k). Some IChNS were also found positive for NPY (l). m–p Sensory fibres are rare and are noticeable in large nerve fibres (m) and around choroidal vessels (n–o). Few IChNS carry CGRP-vesicles, indicating sensory properties (p). q–s Transversal Semithin sections of healthy aged human eye. Large choroidal nerves are easily noticeable (r, s) with myelinated (s, white star) and unmyelinated fibres (s, white arrow). t, u TEM observation of transversal section of human choroid. t Large choroidal nerve containing myelinated fibres (mf) and their myelinating Schwann cell (mSC) as well as non-myelinated fibres (nmf). u Non-myelinated choroidal fibres nearby a choroidal vessel. v Non-myelinated choroidal fibres contacting the choriocapillaris

Fig. 5

Semithin and ultrastructural analysis of P1.hMR and WT littermate mice choroid and choroidal nerves. a–c Semithin transversal eye section of WT littermate mice. The photoreceptor outer segments are aligned and the choriocapillaris are clearly noticeable between large choroidal artery and RPE (a). RPE pigments are concentrated and well spread toward the apical pole of the cells (a inset and b). Choroidal nerve displays healthy structure with normal myelin organisation (c). (d–g) Semithin transversal eye section of transgenic P1.hMR mice where several features of pachychoroid are observed. Dilated vessels in direct contact with Bruch membrane and effacement of the overlying choriocapillaris (“pachyvessel”) (d, left inset) associated with irregular pigment distribution in the RPE (d, right inset). Dilated veins (e, black double arrows), with focal area of elongated undigested photoreceptor outer segments (f, black arrows) and subretinal deposits associated with abnormal RPE/photoreceptor outer segment interface (f, white arrows). These are accompanied by irregular pigment distribution in the RPE and subretinal migration of RPE cells (f, inset). These features resemble pachychoroid epitheliopathy described in humans. In P1.hMR, enlarged nerves with irregular myelin shedding (white arrows) and disorganisation were observed (g). These neural abnormalities were more obvious in electron microscopy observations. TEM of WT littermate (h, i) and transgenic P1.hMR mice (k, l) choroidal nerves. Transgenic mice display signs of neuropathy, including myelin abnormalities (k, white arrows), increased number of large mitochondria (l, white arrows) and loss of organisation and vacuolization of the nerve. SBF-SEM sections of a choroidal nerve in a WT littermate (j, purple area) or in a transgenic mouse (m, green area), and the corresponding 3D segmentation of the myelin (n). Myelin abnormalities in transgenic animals are clearly apparent, including large variation of the G-ratio

Fig. 6

Differential transcriptomic analysis and gene concept network for differentially expressed genes in P1.hMR mice versus WT mice. Volcano plot (a) and heat map (b) display 45 significantly downregulated and 56 upregulated genes in P1.hMR mice compared to their age/sex-matched WT littermates. c Significantly regulated pathways in Gene Ontology gene sets (C5:GO) enrichment analysis. d Significantly regulated pathways in hallmark gene sets (H)

Fig. 7

Foveal SD-OCT in healthy and CSCR conditions. a Foveal SD-OCT of a healthy retina, showing a thickened choroid with dilated choroidal vessels. In the enlarged quadrant, vessels are visualised as round hyporeflective structures with a homogenous hyperreflective border, more pronounced towards the RPE. The choriocapillaris remains visible despite choroidal enlargement. b In this chronic resolving CSCR, pachychoroid is observed, as reported on SD-OCT of the left eye. An enlarged choroidal vessel in the perifoveal zone is visualised underneath the RPE and prevents correct visualisation of the choriocapillaris (white arrows), associated with discrete RPE elevation and outer retina displacement. In the enlarged quadrant, several hyperreflective dots are observed, both surrounding large choroidal vessels and within the choriocapillary space, with a granular irregular appearance

Fig. 8

Clinical features on multimodal imaging of a 55-year-old man with chronic CSCR and widespread pigment epitheliopathy. The RPE atrophy is highlighted by the extensive window defect shown on early-phase fluorescein angiography (a), which allowed a more detailed visualisation of choroidal vasculature in the early-phase IGC cliché at 1 min (b), showing the dye in the intravascular space. On infrared en-face imaging (c), choroidal vessels were visualised as dark areas (yellow star). Adjacent to choroidal vasculature dark reflectance, white linear structures with increased IR reflectance were observed (blue arrowhead). These were hypofluorescent on ICG (b, corresponding yellow stars and arrowheads). d SD-OCT EDI scan showing photoreceptor disorganisation and RPE loss, pachychoroid and enlarged choroidal vessels. The green vertical line shows that the B-scan is placed over the white linear structure in the en-face IR image (e, white arrow). The corresponding location on SD-OCT (f, enlarged image) shows a hyperreflective area (yellow circle) very close to enlarged choroidal vessels (white star), underneath the choriocapillaris