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.