Intravitreal Neuroglobin Mitigates Primate Experimental Glaucomatous Structural Damage in Association with Reduced Optic Nerve Microglial and Complement 3-Astrocyte Activation.

Current management of glaucomatous optic neuropathy is limited to intraocular pressure control. Neuroglobin (Ngb) is an endogenous neuroprotectant expressed in neurons and astrocytes. We recently showed that exogenous intravitreal Ngb reduced inflammatory cytokines and microglial activation in a rodent model of hypoxia. We thus hypothesised that IVT-Ngb may also be neuroprotective in experimental glaucoma (EG) by mitigating optic nerve (ON) astrogliosis and microgliosis as well as structural damage. In this study using a microbead-induced model of EG in six Cynomolgus primates, optical coherence imaging showed that Ngb-treated EG eyes had significantly less thinning of the peripapillary minimum rim width, retinal nerve fibre layer thickness, and ON head cupping than untreated EG eyes. Immunohistochemistry confirmed that ON astrocytes overexpressed Ngb following Ngb treatment. A reduction in complement 3 and cleaved-caspase 3 activated microglia and astrocytes was also noted. Our findings in higher-order primates recapitulate the effects of neuroprotection by Ngb treatment in rodent EG studies and suggest that Ngb may be a potential candidate for glaucoma neuroprotection in humans.

Effects of Exogenous Neuroglobin (Ngb) on retinal inflammatory chemokines and microglia in a rat model of transient hypoxia.

Neuroglobin is an endogenous neuroprotective protein. We determined the safety of direct delivery of Neuroglobin in the rat retina and its effects on retinal inflammatory chemokines and microglial during transient hypoxia. Exogenous Neuroglobin protein was delivered to one eye and a sham injection to the contralateral eye of six rats intravitreally. Fundus photography, Optical Coherence Topography, electroretinogram, histology and Neuroglobin, chemokines level were determined on days 7 and 30. Another 12 rats were subjected to transient hypoxia to assess the effect of Neuroglobin in hypoxia exposed retina by immunohistochemistry, retinal Neuroglobin concentration and inflammatory chemokines. Intravitreal injection of Neuroglobin did not incite morphology or functional changes in the retina. Retinal Neuroglobin protein was reduced by 30% at day 7 post hypoxia. It was restored to normoxic control levels with intravitreal exogenous Neuroglobin injections and sustained up to 30 days. IL-6, TNFα, IL-1B, RANTES, MCP-1 and VEGF were significantly decreased in Neuroglobin treated hypoxic retinae compared to non-treated hypoxic controls. This was associated with decreased microglial activation in the retina. Our findings provide proof of concept suggesting intravitreal Neuroglobin injection is non-toxic to the retina and can achieve the functional level to abrogate microglial and inflammatory chemokines responses during transient hypoxia.

Recombinant neuroglobin ameliorates early brain injury after subarachnoid hemorrhage via inhibiting the activation of mitochondria apoptotic pathway.

Neuroglobin (Ngb) overexpression is considered as an intrinsic neuroprotective response. Therefore, exogenous Ngb increased in brain tissues has become a promising therapeutic strategy for neurological diseases. Previous studies demonstrated that transactivator of transcription (TAT) protein transduction domain was able to mediate synthetic Ngb entrance into neurons, and then protected brain from hypoxia-ischemic injury. However, the role of recombinant Ngb on early brain injury following subarachnoid hemorrhage (SAH) has not been elucidated. The objectives of this study were to investigate the expression of endogenous Ngb in brain using a rabbit model of SAH, and to verify whether TAT-Ngb fusion protein could be delivered into brain parenchyma, as well as to explore the neuroprotective effect of Ngb and its possible mechanisms. We found that Ngb expressions were up regulated in the transcript and protein levels in a similar time dependent manner after SAH as compared to the sham group. Moreover, TAT-Ngb fusion protein was successfully generated and transferred into brain neurons. Compared with the saline- and Ngb-treated group, neuronal viabilities and neurological outcomes were significantly improved 72 h post-SAH in the TAT-Ngb-treated group. Likewise, anti-apoptotic Bcl-2 protein was also elevated obviously. Conversely, pro-apoptotic factors including caspase 3, caspase 9 and Bax were greatly decreased after TAT-Ngb treatment. Our results suggest that Ngb plays a neuroprotective effect in rabbits suffering from SAH possibly through inhibiting the SAH-induced activation of mitochondria apoptotic pathway. Furthermore, TAT-mediated Ngb delivery into brain may be a promising therapeutic approach.