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. 2017 Apr 6;12(4):e0175451.
doi: 10.1371/journal.pone.0175451. eCollection 2017.

Intravitreal injection of β-crystallin B2 improves retinal ganglion cell survival in an experimental animal model of glaucoma

Fabian Anders  1 , Julia Teister  1 , Aiwei Liu  1 , Sebastian Funke  1 , Franz H Grus  1 , Solon Thanos  2 , Harald D von Pein  3 , Norbert Pfeiffer  1 , Verena Prokosch  1
Affiliations

Intravitreal injection of β-crystallin B2 improves retinal ganglion cell survival in an experimental animal model of glaucoma

Fabian Anders et al. PLoS One. .

Abstract

Purpose of this study was to investigate firstly specific proteomic changes within the retina in the course of an animal glaucoma model and to identify secondly new approaches for neuroprotective, therapeutic options in glaucoma by addressing those specific changes. Intraocular pressure was elevated through cauterization of episcleral veins in adult Sprague Dawley rats. Molecular and morphological changes were surveyed using mass spectrometry, optical coherence tomography as well as immunohistochemical cross section- and flat mount stainings. By quantifying more than 1500 retinal proteins, it was found that the HspB5 protein and numerous beta-crystallins showed a uniform and unique shifting expression pattern as a result of different periods of elevated IOP exposure. Crystallins showed a significant downregulation (p<0.05) after 3 weeks of elevated IOP and an upregulation after 7 weeks. Counteracting those typical changes, an intravitreal injection of β-crystallin B2 at the time of IOP elevation was found to reduce retinal ganglion cell loss (p<0.05), decrease of the retinal nerve fiber layer (p<0.05) and impairment of the optic nerve. Ultimately, proteomic data revealed that β-crystallin B2 might influence calcium-depended cell signaling pathways with severe effect on apoptosis and gene regulation. In this context especially annexin A5, calcium-transporting ATPase 1 and various histone proteins seem to play a major role.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. IOP monitoring after episcleral vein cauterization.
IOP comparison from eyes treated by thermic cauterization in four different experimental groups (cf. individual n in Table 1) and their combined contralateral controls (n = 23). IOP elevation can be observed in about 3 weeks after surgery and resulted in an average increase up to 17.40±0.78 mmHg, while the contralateral eye remains at baseline level (p<0.0001, parametric t-test, ±SEM).
Fig 2
Fig 2. Effect of elevated intraocular pressure on retinal morphology.
Brn3A immunohistological staining showed a decrease of RGC due to elevated IOP (A). Compared to the combined control retina (n = 10), which was not exposed to elevated IOP, the 3 weeks (n = 5) and 7 weeks elevated IOP group (n = 5) show a RGC loss of 8% respectively 21% (*p<0.05, parametric t-test, ±SEM). The RNFLT was measured with a 12° diameter circular B-scan, decrease of RNFLT (B) was comparable to RGC loss (*p<0.05, **p<0.01, parametric t-test, ±SEM, n = 5). Grading of the optic nerve (C1) allowed a rough classification of IOP exposed cross-sections. Evaluation of the data showed clear impairment of the optic nerve with progressional period of elevated IOP (n = 7) (C2).
Fig 3
Fig 3. Regulatory changes of crystallin proteins on elevated IOP.
Analyzed members of the different crystallin subclasses showed a unique expression profile at both investigated timepoints of IOP elevation (t1 n = 8, t2 n = 7). (*p<0.05, **p<0.01, Mann-Whitney U test, ±SEM). Fold-changes are plotted using a logarithmic scale with a basis of 2.
Fig 4
Fig 4. β-crystallin B2 staining in retinal cross-sections.
IHC staining could validate the results from mass-spectrometric featured proteomics analyses. After 3 weeks of IOP elevation, almost no β-crystallin B2 could be detected in the retina cross section (n = 3), while a strong increase of protein level could be verified after 7 weeks of elevated IOP (n = 3). Injection of β-crystallin B2 seems to change the protein level in retinal cells after 3 weeks of IOP elevation considerably due to cellular uptake of the crystallin, predominantly by the RGC layer (n = 4).
Fig 5
Fig 5. Morphological effect of β-crystallin B2 injection prior to IOP elevation.
Injection of β-crystallin B2 results in a 12% higher RGC survival rate (n = 4), in case of IOP elevation, compared to the 7 weeks IOP group (n = 7) (A, *p<0.05, parametric t-test, ±SEM). Similar results could be observed for the decrease of the RNFLT, which was found to be 11% lower (n = 4) than in the IOP group, respectivelly (n = 5) (B, *p<0.05, parametric t-test, ±SEM). Neuroprotective effects could also be analyzed in the optic nerve, where cross-sections of crystallin injected animals (n = 4) showed similar results with respect to the untreated control group (n = 10) (C).
Fig 6
Fig 6. Alterations of protein expression by β-crystallin B2 injection.
Crystallin injected animals show proteomic changes in specific calcium signaling key proteins as well as gene regulation proteins (±SEM, n = 4). Fold-changes are plotted using a logarithmic scale with a basis of 2.
Fig 7
Fig 7. Molecular interaction network by Ingenuity Pathway Analysis.
Down-regulation in green, up-regulation marked in red. A = Activation, LO = Localization, E = Expression, PD = Protein/DNA interaction, L = molecular cleavage, P = Phosphorylation. ANXA5 = Annexin V, CYCS = Cytochrome c, somatic, ATPB1 = Calcium-transporting ATPase 1, Hist1h1e = Histone H1, H3F3B = Histone H3, H2AFZ = Histone H2A.Z, MYC = myc proto-oncogen
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Grants and funding

The work was supported by the Deutsche Forschungsgemeinschaft (DFG, grant PR1569/1-1 to VP).