Assessment of Neurotrophins and Inflammatory Mediators in Vitreous of Patients With Diabetic Retinopathy

Abstract

Purpose: To assess vitreous levels of inflammatory cytokines and neurotrophins (NTs) in diabetic retinopathy (DR) and elucidate their potential roles.

Methods: A prospective study was performed on 50 vitreous samples obtained from patients with DR (n = 22) and the nondiabetic controls (n = 28). All patients were candidates for vitrectomy. Inflammatory cytokine and NT levels were determined with ELISA. Potential source and role of NTs was determined by using human retinal Müller glia and mouse photoreceptor cells and challenging them with TNF-α or IL-1β, followed by detection of NTs and cell death.

Results: Vitreous NT levels of all DR patients were significantly higher than those of nondiabetic controls (nerve growth factor [NGF, P = 0.0001], brain-derived neurotrophic factor [BDNF, P = 0.009], neurotrophin-3 [NT-3, P < 0.0001], neurotrophin-4 [NT-4, P = 0.0001], ciliary neurotrophic factor [CNTF, P = 0.0001], and glial cell-derived neurotrophic factor [GDNF, P = 0.008]). Similarly, the levels of inflammatory mediators IL-1β (P < 0.0001), IL-6 (P = 0.0005), IL-8 (P < 0.0001), and TNF-α (P < 0.0001) were also higher in eyes with DR. Interestingly, inflammatory cytokine and NT levels, particularly TNF-α (P < 0.05), IL-8 (P < 0.004), NT-3 (P = 0.012), NGF (P = 0.04), GDNF (P = 0.005), and CNTF (P = 0.002), were higher in eyes with nonproliferative diabetic retinopathy (NPDR) than in eyes with active proliferative diabetic retinopathy (PDR). Cytokine stimulation of Müller glia resulted in production of NTs, and GDNF treatment reduced photoreceptor cell death in response to inflammation and oxidative stress.

Conclusions: Together, our study demonstrated that patients with DR have higher levels of both inflammatory cytokines and NTs in their vitreous. Müller glia could be the potential source of NTs under inflammatory conditions to exert neuroprotection.

Figure 1

Box-and-whisker plot analysis showing comparison of vitreous NGF, BDNF, NT-3, NT-4, CNTE, and GDNF levels in diabetics (n = 22) compared to nondiabetics (n = 28) (A) and diabetic patients with NPDR compared to active PDR (B).

Figure 2

Box-and-whisker plot analysis showing comparison of vitreous IL-6, IL-1β, IL-8, and TNF-α in diabetics (n = 22) compared to nondiabetics (n = 28) (A) and diabetic patients with NPDR compared to PDR (B).

Figure 3

Box-and-whisker plot analysis of vitreous neurotrophin (A) and inflammatory cytokine (B) levels in diabetics who have undergone peripheral retinal ablation with PRP compared to diabetics without a history of PRP.

Figure 4

Inflammatory cytokines induce NT production in Müller glia. Human retinal Müller glia (MIO-M1 cell line) were challenged with recombinant TNF-α or IL-1β (100 ng/mL each) (A, B), IL-4 (100 ng/mL) (C), and dexamethasone (100 μM) (D) for the indicated time points. The secretion of NTs into the conditioned media was detected by dot blot, and the intensities of the dots were quantitated by densitometric analysis, using ImageJ and presented as fold-changes (arbitrary units; AU), using a value of 1 for the control samples (B). Statistical analysis was performed by using a 1-way ANOVA (*P < 0.01; **P = 0.001), for comparisons of control versus stimulated cells. Data points and bars represent mean ± SD of triplicates from three independent experiments.

Figure 5

GDNF protects oxidative stress and inflammation-induced photoreceptor cell death. Mouse cone photoreceptor cells (661W cell line) were challenged with H₂O₂ (100 μM) and TNF-α (100 ng/mL) in the presence and absence of GDNF (100 ng/mL) for 24 hours. The neuroprotective effect of GDNF was measured by measuring the cell death by TUNEL staining (blue, 4′,6-diamidino-2-phenylindole [DAPI] nuclear stain; green, TUNEL+ve cells).

Figure 6

Schematic of the relationship between inflammatory mediators and NTs in diabetic retinopathy. Patients with diabetic retinopathy have increased levels of inflammatory mediators in vitreous. This increased inflammatory milieu triggers the production of NTs in retinal cells, such as Müller glia, which in turn exert neuroprotective and anti-inflammatory effects to prevent neuronal cell death in the retina.