PLoS One. 2015 Oct 15; 10(10): e0140387. doi: 10.1371/journal.pone.0140387. eCollection 2015.

Cerium Oxide Nanoparticles Reduce Microglial Activation and Neurodegenerative Events in Light Damaged Retina.

Fiorani L1, Passacantando M2, Santucci S2, Di Marco S1, Bisti S1, Maccarone R1.

1Department of Applied Clinical Science and Biotechnology, University of L’Aquila, Via Vetoio, Coppito II, 67100 L’Aquila, Italy.

2Department of Physical and Chemical Sciences, University of L’Aquila, Via Vetoio, Coppito I, 67100 L’Aquila, Italy.

 

Abstract

The first target of any therapy for retinal neurodegeneration is to slow down the progression of the disease and to maintain visual function. Cerium oxide or ceria nanoparticles reduce oxidative stress, which is known to play a pivotal role in neurodegeneration. Our aim was to investigate whether cerium oxide nanoparticles were able to mitigate neurodegeneration including microglial activation and related inflammatory processes induced by exposure to high intensity light. Cerium oxide nanoparticles were injected intravitreally or intraveinously in albino Sprague-Dawley rats three weeks before exposing them to light damage of 1000 lux for 24 h. Electroretinographic recordings were performed a week after light damage. The progression of retinal degeneration was evaluated by measuring outer nuclear layer thickness and TUNEL staining to quantify photoreceptors death. Immunohistochemical analysis was used to evaluate retinal stress, neuroinflammatory cytokines and microglial activation. Only intravitreally injected ceria nanoparticles were detected at the level of photoreceptor outer segments 3 weeks after the light damage and electoretinographic recordings showed that ceria nanoparticles maintained visual response. Moreover, this treatment reduced neuronal death and “hot spot” extension preserving the outer nuclear layer morphology. It is noteworthy that in this work we demonstrated, for the first time, the ability of ceria nanoparticles to reduce microglial activation and their migration toward outer nuclear layer. All these evidences support ceria nanoparticles as a powerful therapeutic agent in retinal neurodegenerative processes.

 

Supplement:

Overproduction of free radicals can cause oxidative damage to biomolecules and leading to many neurodegenerative diseases such as Retinal diseases, like Retinitis Pigmentosa, glaucoma, Stargardt and Age related Macular degeneration. The final event in these diseases is the photoreceptor death and consequently loss of vision up to blindness [1, 2].

Albino rats exposed to high intensity light are considered a good and reproducible model of oxidative stress [3]. In retinal diseases like AMD, also in light damaged rats the final event is photoreceptors death and later the gradual loss of vision. For these reasons, the first target in many therapies is the reduction of the oxidative stress in order to maintain visual function as long as possible. Cerium oxide (CeO2) nanoparticles (NPs) are innate antioxidants that possess regenerative radical scavenging activities due to the presence of oxygen vacancies on the surface of these nano-sized particles and to the auto-regenerative cycle between the two-oxidation states, Ce3+ and Ce4+.

Figure 1(A) shows a diagram on the composition of the nanoparticles, in which the crystalline CeO2 (Ce4+) core part and the surface is amorphous Ce2O3 (Ce3+). In fig. 1(B), we report a fluorescence image of the synthesized ceria nanoparticles conjugated with fluorescein isothiocyanate (FITC), that were used to analyze their biodistribution in the retina after injection. The use of fluorescently tagged NPs has allowed their rapid visualization, as well as multiple labeling approaches, in confocal microscopy.

 

 

fig1

Figure 1: A, diagram on the composition of the nanoparticles, in which the crystalline CeO2 (Ce4+) core part and the surface is amorphous Ce2O3 (Ce3+). B, Fluorescent image of ceria nanoparticles conjugated with fluorescein isothiocyanate.

 

Cerium oxide (CeO2) nanoparticles have been successfully proposed as neuroprotectants and drug delivery devices see for ref.[1,4]. However, oxidative stress is not the only player during the progression of neurodegeneration; many other factors, including release of chemokines and neuroinflammation, heavily influence the advancement of the pathology. Microglia play an important role in maintaining the homeostasis of neurotrophic factors due to cross talk with Müller cells.  Oxidative stress can induce microglial activation that, in turn, can be responsible for the progression of degeneration in light damaged rats as well as in many other retinal diseases [3,5,6].

Our aim was to investigate the possible ways of action of Cerium oxide nanoparticles in preventing retinal degeneration.

Our results showed that we were able to develop and select extremely efficient ceria nanoparticles that when injected intravitreally, were able to reduce photoreceptor death induced by bright continuous light (BCL), maintaining morphology and function as already described [7]. (Fig. 2)

 

 fig2

Figure 2: A, Albino rats were anaesthetized with Ketamine/Xylazine (10 mg/100gr-1.2 mg/100 gr) and 2 ml of CeO2 nanoparticles (1 mM) were injected intravitreally by using an Hamilton syringe.

B, The picture shows a detail of electroretinographic equipment used to evaluate retinal function, composed by a stereotaxic apparatus, the ganzfeld dome and gold electrodes loop.

C, representative image of retinal section stained with with propidium iodide, to highlight retinal morphology,. and acquired by using a confocal microscope (Nikon 80i).

 

 

We demonstrated that FITC- CeO2 nanoparticles injected intravitreally were able to cross the entire retina, where they remain stable over a long time.

In addition, we provided a novel observation: the ability of ceria nanoparticles to reduce reactive response of retinal Müller cells and eventually to control microglial activation. Recently, great attention has been devoted to microglia physiology and function and there are increasing evidence of the pivotal role played by microglia in both pathogenesis and progression of neurodegenerative diseases where neuroinflammation is involved including age related macular degeneration see for ref [5,6]. The general role of microglia is very dynamic and critical for neuronal maintenance. In response to stress-injury-related signals, there is a sudden activation of microglia migration and phagocytosis. To visualize the recruitment of microglia/monocytes we used Iba1 marker. Intravitreal injection of CeO2 nanoparticles significantly reduced Iba1-positive cells with respect to non-injected group, mainly in the photoreceptor layer. Moreover activated microglia and apoptotic figures overlap in the same area suggesting a link between neuronal death and microglia.

Furthermore, we demonstrated the ability of CeO2 nanoparticles to reduce Tumor Necrosis Factor alpha that is a secreted pro-inflammatory cytokine enrolled in several cellular events, including apoptosis, cell survival, and proliferation see for ref. [8].

 

Importance of the study

This work has confirmed that the chemical structure of the nanoparticles is a crucial point for their therapeutic efficacy. Cerium oxide nanoparticles injected in the vitreous, maintain a stable localization and, therefore, are able to perform their action for a long time. Based on these results, it is possible to consider non-stoichiometric cerium oxide nanoparticles as a convenient treatment in degenerative retinal processes, where oxidative stress plays a relevant role.

 

Funding statement

PRIN 2011 to SB contributed to the fellowship for LF for a year.

 

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