Restoring retinal neurovascular health via substance P

Abstract

Regulation of vascular permeability plays a major role in the pathophysiology of visually threatening conditions such as retinal vein occlusion and diabetic retinopathy. Principally, several factors such as vascular endothelial growth factor (VEGF), are up-regulated or induced in response to hypoxia thus adversely affecting the blood-retinal barrier (BRB), resulting in retinal edema and neovascularisation. Furthermore, current evidence supports a dysregulation of the inner retinal neural-vascular integrity as a critical factor driving retinal ganglion cell (RGC) death and visual loss. The principal objective of this study was to interrogate whether Substance P (SP), a constitutive neurotransmitter of amacrine and ganglion cells, may protect against N -methyl- d -aspartate (NMDA)-induced excitotoxic apoptosis of ganglion cells and VEGF-induced vessel leakage in the retina. Tight junctional protein expression and a Vascular Permeability Image Assay were used to determine vascular integrity in vitro . The protective effect of SP on RGC was established in ex vivo retinal explants and in vivo murine models. After NMDA administration, a reduction in TUNEL+ cells and a maintained number of Brn-3a+ cells were found, indicating an inhibition of RGC apoptosis mediated by SP. Additionally, SP maintained endothelial tight junctions and decreased VEGF-induced vascular permeability. In conclusion, administration of SP protects against NMDA apoptosis of RGC and VEGF-induced endothelial barrier breakdown.

Treatment of diabetic retinopathy through neuropeptide Y‐mediated enhancement of neurovascular microenvironment

“…As NPY receptors are expressed in the inner retina and recognition that RGC are perturbed in diabetes, we then wished to evaluate whether NPY played a homeostatic role and exerted a protective effect to preserve RGC. To investigate this further, the NMDA‐induced RGC excitotoxic model was employed in retinal explants …”

“…Permeability visualization experiments were performed on 18 × 18 mm glass coverslips as previously described . For assays, serum‐free culture medium supplemented with the treatment (combinations of NPY, N‐methyl‐D‐aspartate [NMDA], VEGF) was added for a further 24 hours.…”

“…Retinal explant culture was performed as previously described . Healthy Han Wistar rat's retina (20‐old‐day) was dissected into four equal‐sized pieces, and the explants were separately transferred onto 12‐mm‐diameter filters (0.4 μm pore, Millipore) with the RGC side facing up.…”

“…Eyes from 6‐month diabetic mice and cultured rat retinal explants were fixed with 4% PFA and snap‐frozen in OCT (VWR Chemicals). For immunostaining, 8‐μm cryosections were washed in PBS and blocked in 5% normal goat serum (NGS), 2% BSA and 0.3% Triton X‐100 in PBS for 30 minutes at RT followed by incubating with the following primary antibodies: mouse anti‐GFAP (#3670, Cell Signaling Technology, 1:200), mouse anti‐Brn‐3a (sc‐8429, Santa Cruz Biotechnology, Inc, 1:50) and rabbit anti‐Rhodopsin (ab3424, Abcam, 1:100) overnight at 4°C. The secondary antibodies Cy3 conjugated goat antimouse IgG (Merck) and Alexa Fluor 488‐labelled donkey anti‐rabbit IgG (Invitrogen), both with 1:200 dilution in 2% BSA in PBS at RT for 1 hour in the dark.…”

“…NMDA (Sigma‐Aldrich) was used to induce excitotoxicity of RGC in the retina, under experimental conditions described above. A dose‐response was undertaken previously to ascertain the optimal dosing of NPY (5 to 20 nmol); then, mice received the following intravitreal injections (2 µL) with a 33‐gauge needle into one eye: NMDA (10 nmol); NPY (10 nmol); NMDA with NPY together; and PBS vehicle.…”