Newly-established in vitro inner BRB spheroids to elucidate retinal Ang2-linked substance transfer.

Conjugation of angiopep-2 (Ang2) with drugs/compounds is known to increase plasma membrane permeability across endothelial barriers. The inner blood-retinal barrier (BRB) regulates retinal drug distribution and is formed by retinal capillary endothelial cells, supported by Müller cells and retinal pericytes. To elucidate the potential of Ang2 conjugation in promoting retinal drug distribution after peripheral administration across the inner BRB, an in vivo administration study and in vitro transport experiments using newly developed multicellular inner BRB spheroids were performed. After intravenous administration of Ang2-linked green fluorescence protein (GFP-Ang2) in mice, GFP-derived signals were observed in the neural retina. In contrast, GFP-derived signals were not observed after intravenous GFP administration, suggesting the promotion of the retinal distribution of substances by Ang2 conjugation. To overcome the limitations of in vitro studies using cells cultured on dishes, inner BRB spheroids were established using conditionally immortalized rat retinal capillary endothelial cells, Müller cells, and retinal pericytes. Immunocytochemistry of marker molecules suggests that the central part of the spheroids is occupied by Müller cells, and encapsulated by retinal pericytes and capillary endothelial cells. Studies on the expression and functions of tight junctions suggest that tight junctions are formed on the surface of the inner BRB spheroids by retinal capillary endothelial cells. The functional expression of drug transporters, such as P-glycoprotein, was observed in the spheroids, implying that the inner side of the spheroids reflects the retinal side of the inner BRB. In the inner BRB spheroids, energy-dependent accumulation of GFP-Ang2 and Ang2-linked 5(6)-carboxyfluorescein (FAM-Ang2) was observed. Moreover, an endocytic inhibition study revealed that clathrin-dependent endocytosis/transcytosis was involved in the transcellular transport of Ang2-conjugated drugs/compounds across the inner BRB. Consequently, it is suggested that the Ang2 linkage is useful for promoting retinal drug distribution via clathrin-dependent transcytosis at the inner BRB.

Nitric Oxide Synthase (NOS) Isoform Expression after Peripheral Nerve Transection in Mice.

Localization of the nitric oxide (NO)-producing enzyme, nitric oxide synthase (NOS), and its functions are currently being investigated in several tissues and organs. It has been suggested that NO is involved in nerve cell death and the development of neurodegenerative disease. The purpose of this study was to immunohistochemically investigate expression of NOS to clarify its function in the degeneration and regeneration of transected mouse sciatic nerve. Scattered neuronal NOS (nNOS)-positive Schwann cells observed on the central side of the stump on day 1 after transection showed an increase in number on day 7. None were observed at the stump on day 14, however. Expression of nNOS was observed in axons extending from the stump. The number of nNOS-positive axons increased on day 21. Inducible NOS was expressed in inflammatory cells at the stump on day 1. This positive reaction subsequently weakened by day 7, however. Endothelial NOS was expressed in blood vessels at the stump on day 7, but decreased thereafter. The results of the present study suggest that NO is involved in the proliferation and migration of Schwann cells, as well as in axon regeneration at an early stage following nerve transection.

Anatomical study of phrenic nerve course in relation to neck dissection.

The present study sought to clarify the course of the phrenic nerve and its correlation with anatomical landmarks in the neck region. We examined 17 cadavers (30 sides). In each, the phrenic nerves was dissected from the lateral side of the neck, and its position within the triangle formed by the mastoid process and sternal and acromial ends of the clavicle was determined. The point where the phrenic nerve arises in the posterior triangle was found to be similar to the point where the cutaneous blanches of the cervical plexus emerge at the middle of the posterior border of the sternocleidomastoid muscle. In the supraclavian triangle, the phrenic nerve crosses the anterior border of the anterior scalene muscle near Erb's point where the superficial point is 2-3 cm superior from the clavicle and posterior border of the sternocleidomastoid muscle. The phrenic nerve arises in the posterior triangle near the nerve point, then descends to the anterior surface of the anterior scalene muscle in the supraclavian triangle. It is necessary to be aware of the supraclavian triangle below Erb's point during neck dissection procedures.