Lymphatic and vascular markers in an optic nerve crush model in rat.

Only few tissues lack lymphatic supply, such as the CNS or the inner eye. However, if the scleral border is compromised due to trauma or tumor, lymphatics are detected in the eye. Since the situation in the optic nerve (ON), part of the CNS, is not clear, the aim of this study is to screen for the presence of lymphatic markers in the healthy and lesioned ON. Brown Norway rats received an unilateral optic nerve crush (ONC) with defined force, leaving the dura intact. Lesioned ONs and unlesioned contralateral controls were analyzed 7 days (n = 5) and 14 days (n = 5) after ONC, with the following markers: PDGFRb (pericyte), Iba1 (microglia), CD68 (macrophages), RECA (endothelial cell), GFAP (astrocyte) as well as LYVE-1 and podoplanin (PDPN; lymphatic markers). Rat skin sections served as positive controls and confocal microscopy in single optical section mode was used for documentation. In healthy ONs, PDGFRb is detected in vessel-like structures, which are associated to RECA positive structures. Some of these PDGFRb+/RECA+ structures are closely associated with LYVE-1+ cells. Homogenous PDPN-immunoreactivity (IR) was detected in healthy ON without vascular appearance, showing no co-localization with LYVE-1 or PDGFRb but co-localization with GFAP. However, in rat skin controls PDPN-IR was co-localized with LYVE-1 and further with RECA in vessel-like structures. In lesioned ONs, numerous PDGFRb+ cells were detected with network-like appearance in the lesion core. The majority of these PDGFRb+ cells were not associated with RECA-IR, but were immunopositive for Iba1 and CD68. Further, single LYVE-1+ cells were detected here. These LYVE-1+ cells were Iba1-positive but PDPN-negative. PDPN-IR was also clearly absent within the lesion site, while LYVE-1+ and PDPN+ structures were both unaltered outside the lesion. In the lesioned area, PDGFRb+/Iba1+/CD68+ network-like cells without vascular association might represent a subtype of microglia/macrophages, potentially involved in repair and phagocytosis. PDPN was detected in non-lymphatic structures in the healthy ON, co-localizing with GFAP but lacking LYVE-1, therefore most likely representing astrocytes. Both, PDPN and GFAP positive structures are absent in the lesion core. At both time points investigated, no lymphatic structures can be identified in the lesioned ON. However, single markers used to identify lymphatics, detected non-lymphatic structures, highlighting the importance of using a panel of markers to properly identify lymphatic structures.

Time-dependent retinal ganglion cell loss, microglial activation and blood-retina-barrier tightness in an acute model of ocular hypertension.

Glaucoma is a group of neurodegenerative diseases characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, and is the second leading cause of blindness worldwide. Elevated intraocular pressure is a well known risk factor for the development of glaucomatous optic neuropathy and pharmacological or surgical lowering of intraocular pressure represents a standard procedure in glaucoma treatment. However, the treatment options are limited and although lowering of intraocular pressure impedes disease progression, glaucoma cannot be cured by the currently available therapy concepts. In an acute short-term ocular hypertension model in rat, we characterize RGC loss, but also microglial cell activation and vascular alterations of the retina at certain time points. The combination of these three parameters might facilitate a better evaluation of the disease progression, and could further serve as a new model to test novel treatment strategies at certain time points. Acute ocular hypertension (OHT) was induced by the injection of magnetic microbeads into the rat anterior chamber angle (n = 22) with magnetic position control, leading to constant elevation of IOP. At certain time points post injection (4d, 7d, 10d, 14d and 21d), RGC loss, microglial activation, and microvascular pericyte (PC) coverage was analyzed using immunohistochemistry with corresponding specific markers (Brn3a, Iba1, NG2). Additionally, the tightness of the retinal vasculature was determined via injections of Texas Red labeled dextran (10 kDa) and subsequently analyzed for vascular leakage. For documentation, confocal laser-scanning microscopy was used, followed by cell counts, capillary length measurements and morphological and statistical analysis. The injection of magnetic microbeads led to a progressive loss of RGCs at the five time points investigated (20.07%, 29.52%, 41.80%, 61.40% and 76.57%). Microglial cells increased in number and displayed an activated morphology, as revealed by Iba1-positive cell number (150.23%, 175%, 429.25%,486.72% and 544.78%) and particle size analysis (205.49%, 203.37%, 412.84%, 333.37% and 299.77%) compared to contralateral control eyes. Pericyte coverage (NG2-positive PC/mm) displayed a significant reduction after 7d of OHT in central, and after 7d and 10d in peripheral retina. Despite these alterations, the tightness of the retinal vasculature remained unaltered at 14 and 21 days after OHT induction. While vascular tightness was unchanged in the course of OHT, a progressive loss of RGCs and activation of microglial cells was detected. Since a significant loss in RGCs was observed already at day 4 of experimental glaucoma, and since activated microglia peaked at day 10, we determined a time frame of 7-14 days after MB injection as potential optimum to study glaucoma mechanisms in this model.

Long-QT syndrome-associated caveolin-3 mutations differentially regulate the hyperpolarization-activated cyclic nucleotide gated channel 4.

Background Caveolin-3 (cav-3) mutations are linked to the long-QT syndrome (LQTS) causing distinct clinical symptoms. Hyperpolarization-activated cyclic nucleotide channel 4 (HCN4) underlies the pacemaker current If. It associates with cav-3 and both form a macromolecular complex. Methods To examine the effects of human LQTS-associated cav-3 mutations on HCN4-channel function, HEK293-cells were cotransfected with HCN4 and wild-type (WT) cav-3 or a LQTS-associated cav-3 mutant (T78M, A85T, S141R, or F97C). HCN4 currents were recorded using the whole-cell patch-clamp technique. Results WT cav-3 significantly decreased HCN4 current density and shifted midpoint of activation into negative direction. HCN4 current properties were differentially modulated by LQTS-associated cav-3 mutations. When compared with WT cav-3, A85T, F97C, and T78M did not alter the specific effect of cav-3, but S141R significantly increased HCN4 current density. Compared with WT cav-3, no significant modifications of voltage dependence of steady-state activation curves were observed. However, while WT cav-3 alone had no significant effect on HCN4 current activation, all LQTS-associated cav-3 mutations significantly accelerated HCN4 activation kinetics. Conclusions Our results indicate that HCN4 channel function is modulated by cav-3. LQTS-associated mutations of cav-3 differentially influence pacemaker current properties indicating a pathophysiological role in clinical manifestations.