Methylene blue promotes survival and GAP-43 expression of retinal ganglion cells after optic nerve transection.

AIMS: Neurodegeneration of the optic nerve and retinal ganglion cells (RGCs) leads to progressive vision loss. As part of the central nervous system, RGCs show limited ability to regenerate and there is extensive search for neuroprotective agents for optic nerve damage. Methylene blue (MB) exhibits beneficial effects against various neurodegenerative diseases of the central nervous system. However, the mechanisms associated with its putative protection on neuronal survival and regeneration remain obscure. This study used the optic nerve transection model to examine the effects of MB on RGC survival, the expression of regenerative marker GAP-43 in RGCs and microglial activation. MAIN METHODS: Axons of RGCs were injured by cutting the optic nerve. MB was injected intravitreally either immediately post-injury or delayed to 3 days post-injury. Using immunohistochemical staining, surviving RGCs, GAP-43-positive RGCs and microglial cells were quantified in wholemount retinas 7 days post-injury. KEY FINDINGS: Both immediate and delayed (a more clinically realistic situation) intravitreal injection of MB promoted RGC survival. MB also increased the number of GAP-43-positive RGCs, suggesting an enhanced ability of RGCs to regenerate. This was exemplified by the regenerative sprouting of axon-like processes from injured RGCs after MB treatment. The increase in RGC survival and GAP-43 expression correlated with an increase in the number of microglial cells. SIGNIFICANCE: These results reveal that MB has survival-promoting and growth-promoting effects on RGCs after optic nerve injury. Together with the established safety profile of MB in humans, MB is a promising treatment for neurodegeneration and injury of the optic nerve.

Protection of retinal ganglion cells against optic nerve injury by induction of ischemic preconditioning.

AIM: To explore if ischemic preconditioning (IPC) can enhance the survival of retinal ganglion cells (RGCs) after optic nerve axotomy. METHODS: Twenty-four hours prior to retinal ischemia 60min or axotomy, IPC was applied for ten minutes in groups of (n=72) animals. The survival of RGCs, the cellular expression of heat shock protein 27 (HSP27) and heat shock protein 70 (HSP70) and the numbers of retinal microglia in the different groups were quantified at 7 and 14d post-injury. The cellular expression of HSP27 and HSP70 and changes in the numbers of retinal microglia were quantified to detect the possible mechanism of the protection of the IPC. RESULTS: Ten minutes of IPC promoted RGC survival in both the optic nerve injury (IPC-ONT) and the retinal ischemia 60min (IPC-IR60) groups, examined at 7d and 14d post-injury. Microglial proliferation showed little correlation with the extent of benefit effects of IPC on the rescue of RGCs. The number of HSP27-positive RGCs was significantly higher in the IPC-ONT group than in the sham IPC-ONT group, although the percentage of HSP27-positive RGCs did not significantly differ between groups. For the IPC-IR60 group, neither the number nor the percentage of the HSP27-positive RGCs differed significantly between the IPC and the sham-operated groups. The number of HSP70-positive RGCs was significantly higher for both the IPC-ONT and the IPC-IR60 experimental groups, but the percentages did not differ. CONCLUSION: The induction of IPC enhances the survival of RGCs against both axotomy and retinal ischemia.

The Origin, Expression, Function and Future Research Focus of a G Protein-coupled Receptor, Mas-related Gene X2 (MrgX2).

Mas-related genes (Mrgs) belong to a large family of G protein-coupled receptor genes found in rodents. Human MRGX proteins are G protein-coupled 7-transmembrane proteins sharing 41-52% amino acid identity with each other, but have no orthologs in rodents. MrgX2 is a member of the MrgX family. MRGX2 is expressed in the small neurons of sensory ganglia and mast cells. It can interact with a series of factors and genes such as the peptides substance P, vasoactive intestinal peptide, cortistatin (CST), proadrenomedullin N-terminal peptide (PAMP), LL-37, PMX-53 and ß-defensins. MRGX2 is related to nociception, adrenal gland secretion and mast cell degranulation. Recent research on MrgX2 provides insights into its role in nociception and anti-microbial activities. This article reviewed the origin, expression and function of MrgX2, and discussed possible future research focus.

Remote ischemic postconditioning promotes the survival of retinal ganglion cells after optic nerve injury.

Ischemic conditioning, the application of a mild ischemic stimulus to an ischemia-sensitive structure like the heart or brain either before (preconditioning) or after (postconditioning) its exposure to a lethal ischemic insult, is known to switch on endogenous protective mechanisms. However, most studies of its neuroprotective effect in the central nervous system (CNS) have focused on ischemic damage or related conditions like hypoxia, while its potential in treating other neural diseases remains uncertain. In particular, the recent discovery of remote ischemic postconditioning whereby mild ischemia applied to a region remote from the target after the main ischemic insult also confers protection offers an attractive paradigm to study its potential in other types of neural injury. Retinal ganglion cells damaged by optic nerve transection undergo extensive cell death. However, application of a series of mild ischemic/reperfusion cycles to the hind limb (limb remote ischemic postconditioning) at 10 min or 6 h after optic nerve cut was found to promote ganglion cell survival at 7 days post-injury, with the 10 min postconditioning still exerting protection at 14 days post-injury. Concomitant with the increased ganglion cell survival, 51 % more ganglion cells expressed the small heat shock protein HSP27, when remote ischemic postconditioning was performed at 10 min post-injury, as compared to the sham conditioning group. Our results highlight the potential of using remote ischemic postconditioning as a noninvasive neuroprotective strategy in different CNS disorders like spinal cord and traumatic brain injury.

Different in vitro toxicity of ribosome-inactivating proteins (RIPs) on sensory neurons and Schwann cells.

OBJECTIVE: To study the neurotoxicity induced by Ricinus communis agglutinin (RCA), ricin A chain (RTA), and trichosanthin (TCS) in vitro. METHODS: Rat neurons and Schwann cells were cultured and real-time up-take of RIPs was traced. TUNEL, Annexin V and DAPI were employed to study the mechanism. RESULTS: The purity of both primary neuronal and Schwann cell cultures attained 80-90%. In neuritis, transport of FITC-RCA was demonstrated, but RTA and TCS were not detected. RCA elicited the strongest TUNEL and annexin V signals in both cultures. RTA evoked a stronger apoptotic signal than TCS in neurons. In contrast, compared with TCS, RTA elicited an attenuated apoptotic reaction in Schwann cells. All internalized RIPs were concentrated in the cytoplasm of the cells and their nuclei were not stained by DAPI. CONCLUSION: The toxicity of these RIPs on neurons is different from that on Schwann cells. Although they enter cells by different mechanisms they all induce apoptosis. These results may find application in in vivo neural lesioning studies and clinical therapy.

Co-localization of hyperphosphorylated tau and caspases in the brainstem of Alzheimer's disease patients.

Hyperphosphorylation of microtubule associated protein tau had limited studies in Alzheimer's disease (AD) brainstem. We compared the distribution and number of neurons with hyperphosphorylated tau in two age groups of AD brainstems with mean ages of 65.4 +/- 5.7 and 91.1 +/- 6.4 years. The degree of co-localization of hyperphosphorylated tau positive cells with either cleaved caspase-3 or cleaved caspase-6 was also quantified. Results showed hyperphosphorylated tau mainly occurred in hypoglossal, dorsal motor vagal, trigeminal sensory/motor nuclei as well as in dorsal raphe, locus coeruleus and substantia nigra. Older AD brainstem consistently had higher density of hyperphosphorylated tau cells. Up to 70% of tau positive cells also displayed either cleaved caspase-3 or caspase-6, and the number of co-localized tau cells in each caspase subfamily group was always higher in older aged group. Some hyperphosphorylated tau cells with cleaved caspases had TUNEL positive nuclei. These findings suggest that these latter cells went through the apoptotic process or DNA fragmentation.

Lens epithelial cells promote regrowth of retinal ganglion cells in culture and in vivo.

Lens damage has been demonstrated to promote axonal regeneration of retinal ganglion cells. Various mechanisms associated with this enhancement have been proposed, including macrophage recruitment and stimulatory factors from the lesioned lens. Lens epithelial cells, which become activated as a result of injury, are another potential stimulus. A recent study of co-culturing lens epithelial cells adjacent to retinal explants without direct contact showed that neurites were attracted to grow towards them. We explored the ability of lens epithelial cells to act as a favorable substrate for ganglion cell axonal regeneration, by culturing retinal explants on top of a lens epithelial cell layer, as well as in vivo by transplanting freshly isolated lens epithelial cells to the cut optic nerve. Retinal explants cultured on lens epithelial cells regenerated more and longer neurites than those cultured on either an acellular substrate or a substrate of corneal cells, while lens epithelial cells transplanted to the optic nerve stimulated axons to regenerate in close association with them.