Compartmentalized ocular lymphatic system mediates eye-brain immunity.

The eye, an anatomical extension of the central nervous system (CNS), exhibits many molecular and cellular parallels to the brain. Emerging research demonstrates that changes in the brain are often reflected in the eye, particularly in the retina1. Still, the possibility of an immunological nexus between the posterior eye and the rest of the CNS tissues remains unexplored. Here, studying immune responses to herpes simplex virus in the brain, we observed that intravitreal immunization protects mice against intracranial viral challenge. This protection extended to bacteria and even tumours, allowing therapeutic immune responses against glioblastoma through intravitreal immunization. We further show that the anterior and posterior compartments of the eye have distinct lymphatic drainage systems, with the latter draining to the deep cervical lymph nodes through lymphatic vasculature in the optic nerve sheath. This posterior lymphatic drainage, like that of meningeal lymphatics, could be modulated by the lymphatic stimulator VEGFC. Conversely, we show that inhibition of lymphatic signalling on the optic nerve could overcome a major limitation in gene therapy by diminishing the immune response to adeno-associated virus and ensuring continued efficacy after multiple doses. These results reveal a shared lymphatic circuit able to mount a unified immune response between the posterior eye and the brain, highlighting an understudied immunological feature of the eye and opening up the potential for new therapeutic strategies in ocular and CNS diseases.

Identification and Localization of Cell Types in the Mouse Olfactory Bulb Using Slide-SeqV2.

Spatial transcriptomics maps RNA molecules to the location in a tissue where they are expressed. Here we document the use of Slide-SeqV2 to visualize gene expression in the mouse olfactory bulb (OB). This approach relies on spatially identified beads to locate and quantify individual transcripts. The expression profiles associated with the beads are used to identify and localize individual cell types in an unbiased manner. We demonstrate the various cell types and subtypes with distinct spatial locations in the olfactory bulb that are identified using Slide-SeqV2.

A freeze-substitution approach with solvent-based glyoxal fixative to prevent distortion of ocular structures.

Investigating the function of delicate mammalian eyes often requires chemical fixation, histological sectioning, immunohistochemistry (IHC) and in situ hybridization (ISH). One of the long-standing challenges in the ocular histology field is the limited success of maintaining intact morphology via cryo- or paraffin procedures. Although our latest protocol significantly improved the morphology of mouse eyeball sections, the window technique is time-consuming and requires extensive practice to avoid damage while making windows. In this study, we present a novel glyoxal fixative that is suitable for a freeze-substitution approach to improve both morphology and molecular target preservation of mouse eyes. The method prevents morphology distortion in all tested eyeballs. Therefore, it suits a variety of research needs from morphological examination to investigation of single-molecule RNA expression, using hematoxylin and eosin (H&E) stain, IHC, and ISH assays on either frozen (cryo) or paraffin-infiltrated tissue sections. In addition, this method can be easily performed in many histology laboratories.

A review on histotechnology practices in COVID-19 pathology investigations.

COVID-19 disease in humans, caused by the novel SARS-CoV-2 virus, was first reported in the city of Wuhan, China in December 2019. This disease has quickly developed into a global pandemic, resulting in over 350,000 deaths worldwide and over 5 million confirmed infections in a matter of 6 months. Although the genome of this novel viral RNA was sequenced quickly and testing kits were manufactured to assist in combatting COVID-19, the diagnosis and treatment will remain relatively unsuccessful until the pathology of this disease is fully understood. Histotechnology plays an important role in understanding the pathology of many diseases, including COVID-19. The first postmortem biopsy of a COVID-19 patient was collected on 27 January 2020, and the pathology finding was published in mid-February 2020. Since then, more studies have been published in scientific literatures as the global outbreak continues. This mini-review summarizes the published articles in which histotechnology aspects were utilized with the intent to understand the pathology of COVID-19. In addition, it is anticipated there will be more molecular and immunohistochemical studies to further understand the mechanism of the disease in the near future.