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Intravitreal NGF Administration Counteracts Retina Degeneration After Permanent Carotid Artery Occlusion in Rat

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Intravitreal NGF Administration Counteracts Retina Degeneration After Permanent Carotid Artery Occlusion in Rat

Sandra Sivilia et al. BMC Neurosci.

Abstract

Background: The neurotrophin nerve growth factor (NGF) is produced by different cell types in the anterior and posterior eye, exerting a neuroprotective role in the adult life. The visual system is highly sensitive to NGF and the retina and optic nerve provides suitable subjects for the study of central nervous system degeneration. The model of bilateral carotid occlusion (two-vessel occlusion, 2VO) is a well-established model for chronic brain hypoperfusion leading to brain capillary pathology, to retina and optic nerve degeneration. In order to study if a single intravitreal injection of NGF protects the retina and the optic nerve from degeneration during systemic circulatory diseases, we investigated morphological and molecular changes occurring in the retina and optic nerve of adult rats at different time-points (8, 30 and 75 days) after bilateral carotid occlusion.

Results: We demonstrated that a single intravitreal injection of NGF (5 microg/3 microl performed 24 hours after 2VO ligation) has a long-lasting protective effect on retina and optic nerve degeneration. NGF counteracts retinal ganglion cells degeneration by early affecting Bax/Bcl-2 balance- and c-jun- expression (at 8 days after 2VO). A single intravitreal NGF injection regulates the demyelination/remyelination balance after ischemic injury in the optic nerve toward remyelination (at 75 days after 2VO), as indicated by the MBP expression regulation, thus preventing optic nerve atrophy and ganglion cells degeneration. At 8 days, NGF does not modify 2VO-induced alteration in VEFG and related receptors mRNA expression.

Conclusion: The protective effect of exogenous NGF during this systemic circulatory disease seems to occur also by strengthening the effect of endogenous NGF, the synthesis of which is increased by vascular defect and also by the mechanical lesion associated with NGF or even vehicle intraocular delivery.

Figures

Figure 1
Figure 1
A, B: Histological staining (Hematoxylin-Eosin) of the retina of sham-operated (A) and 2VO ligated 8 days after surgery (B). C, D: Histological staining (Masson Trichromic staining) of the optic nerve of sham (C) and 2VO animals 8 days after surgery (D). E-R: Optic nerve staining of sham- (E-L) and 2VO- operated animals 8 days after surgery (M-R) with the markers listed in the figure. Images are obtained by conventional fluorescence microscopy. Bars: 100 μm (A, B); 200 μm (C-R). Abbreviations: ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer;GCL: ganglion cell layer.
Figure 2
Figure 2
Morphometric analysis of the retina and optic nerve in the experimental groups. In the retina, the OPL layer almost disappears in 2VO animal 8 days after surgery (A; Student-t test: ***p < 0.0001). A reduction in the number of ganglion cells 75 days after surgery was also found (B, Student's t test: *p < 0.05). The atrophic effect of ligation is shown as reduction of the optic nerve diameter (C; one-way ANOVA and post hoc Tukey's test: ##p < 0.001). The semiquantitative analysis of relative immunoreactive area for the betaIII-Tubulin (D) shows a strong reduction at 8, 30 and 75 days after ligation (one-way ANOVA and post hoc Tukey's test: #p < 0.05, ##p < 0.001), such as the analysis of MBP (E, one-way ANOVA and post hoc Tukey's test: #p < 0.05; ##p < 0.001). Abbreviations: ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer;GCL: ganglion cell layer.
Figure 3
Figure 3
Morphometric analysis of NGF effect on optic nerve diameter (A), ganglion cells (B), betaIII-Tubulin (C) and MBP (D) immunostaining. NGF administration partially prevents 2VO-induced optic nerve atrophy (A) and ganglion cells degeneration at 75 days after surgery. NGF also induces a partially recovery of MBP- immunoreactivity (D), whereas betaIII-tubuline immunostaining is not modified (C). Statistical analysis: one-way ANOVA and post hoc Tukey's test: #p < 0.05; ##p < 0.01 and Student-t test: *p < 0.05.).
Figure 4
Figure 4
Real time PCR of Bax, Bcl-2 and c-jun genes. The induction of hypoperfusion induces an increase in the pro-apoptotic gene Bax mRNA level (2VO-saline versus sham-saline) in the retina (B) which then decreases after NGF treatment. The relative expression of the anti-apoptotic gene Bcl-2 conversely increases in the 2VO-NGF group compared with the sham-saline group of animals (A). The vascular lesion also causes the induction of c-jun mRNA expression (2VO-saline versus sham-saline) (D), which is counteracted by NGF intravitreal injection (E). Experiments were performed in triplicate. Results are presented as mean values ± SEM. Statistical analyses performed: one-way ANOVA with multiple comparison post hoc Tukey's test (#p < 0.05) and Student's t test (*p < 0.05; **p < 0.01).
Figure 5
Figure 5
Relative mRNA expression of NGF and its receptors p75 and TrkA in the retina at 8 days after vessel ligation. NGF mRNA increased in all the experimental groups when compared with the sham-operated group of animals (A) and also in the 2VO-NGF group when compared with 2VO-saline (NGF effect on vascular lesion) (C). The opposite effect was found for the high affinity NGF receptor, TrKA, with a decrease observed in the mRNA level in all the experimental groups compared with the sham-operated group of animals (G). The mRNA level of the low affinity NGF receptor p75 is not significantly altered in the experimental groups studied except in the case of NGF injected sham group of animals (F). Results are presented as mean values ± SEM of experiments performed in duplicate. Statistical analysis was performed by one-way ANOVA and post hoc Tukey's multiple comparison test (#p < 0.05; ###p < 0.001) or Student's t test (*p < 0.05).
Figure 6
Figure 6
Relative mRNA expression of VEGF-A, Flt-1 and Flk-1 in the retina at 8 days after vessel ligation. The relative expression of VEGF (A) and its Flt-1 receptor (F) is increased in the 2VO groups with respect to the sham group of animals while the relative expression of Flk-1 decreases in 2VO groups compared with sham-group (G). NGF injection reduced the damage-induced VEGF mRNA expression. Results were considered statistically significant when one-way ANOVA and post hoc Tukey's test, # p < 0.01, ### p < 0.001; Student's t test: *p < 0.05.

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