Systemic and Intravitreal Antagonism of the TNFR1 Signaling Pathway Delays Axotomy-Induced Retinal Ganglion Cell Loss

Here, we have blocked the signaling pathway of tumor necrosis factor α (TNFα) in a mouse model of traumatic neuropathy using a small cell permeable molecule (R7050) that inhibits TNFα/TNF receptor 1 (TNFR1) complex internalization. Adult pigmented mice were subjected to intraorbital optic nerve crush (ONC). Animals received daily intraperitoneal injections of R7050, and/or a single intravitreal administration the day of the surgery. Some animals received a combinatorial treatment with R7050 (systemic or local) and a single intravitreal injection of brain derived neurotrophic factor (BDNF). As controls, untreated animals were used. Retinas were analyzed for RGC survival 5 and 14 days after the lesion i.e., during the quick and slow phase of axotomy-induced RGC death. qPCR analyses were done to verify that Tnfr1 and TNFα were up-regulated after ONC. At 5 days post-lesion, R7050 intravitreal or systemic treatment neuroprotected RGCs as much as BDNF alone. At 14 days, RGC rescue by systemic or intravitreal administration of R7050 was similar. At this time point, intravitreal treatment with BDNF was significantly better than intravitreal R7050. Combinatory treatment was not better than BDNF alone, although at both time points, the mean number of surviving RGCs was higher. In conclusion, antagonism of the extrinsic pathway of apoptosis rescues axotomized RGCs as it does the activation of survival pathways by BDNF. However, manipulation of both pathways at the same time, does not improve RGC survival.

TNFα induced cell death does not occur in healthy cells, but in stressed or metabolically imbalanced ones (reviewed in Sedger and McDermott, 2014). Thus, TNFα/TNFR1 signaling depends on the cellular context and can lead to proliferation, apoptosis, or necroptosis. TNFα binding to TNFR1 causes the formation of two multiprotein complexes. Complex I prevents apoptosis, while complex II induces cell death. Complex I is membrane bound, and complex II is activated after being endocytosed and released from the cytoplasmic membrane (reviewed in Cabal-Hierro and Lazo, 2012).
In pre-clinical models of ocular hypertension, the main risk factor of glaucoma, or of traumatic optic nerve injury (optic nerve crush or transection) both, the blockade of TNFα using decoy-receptors (Roh et al., 2012;Tse et al., 2018) or natural anti-TNFα compounds (Kyung et al., 2015), and the deletion of TNFR1 (Tezel et al., 2004) have neuroprotective properties on the injured RGCs.
Anti-TNFα therapies based on decoy-receptors or anti-TNF antibodies have some intrinsic problems such as availability, potential antigenicity, and tissue distribution.
R7050 is a small cell permeable triazoloquinoxaline that blocks TNFα signaling through the inhibition of the ligand-induced TNFR1 endocytosis (Gururaja et al., 2007), impairing the association of TNFR1 with the intracellular adaptor molecules that form the multiprotein complexes. Thus, R7050 is a selective antagonist of the TNFα/TNFR1 pathway. Importantly, R7050 crosses the blood brain barrier and it has been shown that attenuates neurovascular injury and improves neurological behavior in an animal model of intra-cerebral hemorrhage (King et al., 2013).
We have used the well-characterized model of optic nerve crush (ONC) in mice (Galindo-Romero et al., 2011, 2013bSanchez-Migallon et al., 2016. The course of RGC death induced by ONC in mice occurs in two phases, a quick one that lasts 7-9 days during which ∼85% of RGCs die, and a second slower one thereafter. In this model, 50% of RGCs are lost at day 5 post-lesion (reviewed in Vidal-Sanz et al., 2017). Here we have administered R7050 intravitreally and/or intraperitoneally, and BDNF intravitreally, and analyzed RGC survival at 5 or 14 days post-ONC, i.e., during the quick and the slow phase of death.

Animal Handling
Adult pigmented C57Bl/6 male mice (25 g) were obtained from the University of Murcia breeding colony. All animals were treated in compliance with the European Union guidelines for Animal Care and Use for Scientific Purpose (Directive 2010/63/EU) and the guidelines from the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures were approved by the Ethical and Animal Studies Committee of the University of Murcia, Spain (number: A1320140704).

Animal Groups
Animal groups are detailed in Figure 1. The number of retinas per group and time point was 4-6 for anatomical analysis (see Table 1 for a detailed n), and 4 for qPCR.

Surgery
The left optic nerve was crushed at 0.5 mm from the optic disc following previously described methods (Galindo-Romero et al., 2011;Sanchez-Migallon et al., 2016). In brief, to access the optic nerve at the back of the eye, an incision was made in the skin overlying the superior orbital rim, the supero-external orbital contents were dissected, and the superior and external rectus muscles were sectioned. Then, the optic nerve was crushed at 0.5 mm from the optic disc for 10 s using watchmaker's forceps. Before and after the procedure, the eye fundus was observed through the operating microscope to assess the integrity of the retinal blood flow.
Systemic treatment with R7050 (12 mg/kg i.p.) was done daily for 7 or 14 days. Some animals received a combinatorial therapy of intravitreal and intraperitoneal treatments. All groups are detailed in Figure 1.
FIGURE 1 | Experimental design and animal groups. R7050 toxicity was studied at 0.2 mM in intact retinas, which were analyzed 5 days after administration. As vehicle control groups, we assayed R7050 1 mM vehicle (5% DMSO in saline) since it has been published that at this concentration is toxic for neurons (Galvao et al., 2014). Two groups were done, intact + i.v. of 5% DMSO and ONC + i.v. of DMSO analyzed 5 days later. To reduce the number of animals, control groups for 1% DMSO (R7050 0.2 mM vehicle) were not done since in our conditions neither the population of RGCs nor their loss after axotomy was affected by an i.v. injection of 5% DMSO. Likewise, intraperitoneal vehicle of R7050 and intravitreal vehicle of BDNF groups were not done because we have already shown that those vehicle formulations and administration routes do not have a negative effect in the retina (Galindo-Romero et al., 2013b;Di Pierdomenico et al., 2018). i.v, intravitreal injection; i.p., intraperitoneal injection.

Immunodetection
Animals were perfused transcardially with 0.9% saline solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Retinas were prepared as flat mounts, or eyes were cryo-sectioned. In all experimental groups, RGCs were immunodetected in whole-mounts as reported (Galindo-Romero et al., 2011;Sanchez-Migallon et al., 2016) using mouse α-Brn3a primary antibody (1:500; MAB1585, Merck Millipore; Madrid, Spain). Secondary detection was carried out with donkey α-mouse IgG1-Alexa fluor 594 (1:500; Molecular Probes; Thermo Fisher Scientific, Madrid, Spain). In brief, flat mounted retinas were permeabilized in PBS 0.5% Triton by freezing them for 15 min at −70 • C, rinsed in new PBS 0.5% Triton, and incubated overnight at 4 • C with the primary antibody diluted in blocking buffer (phosphate buffer saline (PBS) with 2% donkey normal serum and 2% Triton). Then, the retinas were washed three times in PBS and incubated for 2 h at room temperature with the secondary antibody. Finally, they were thoroughly washed in PBS, mounted vitreous side up and covered with antifading solution (Vectashield, Vector laboratories, Palex Medical, Barcelona, Spain).

Image Acquisition and Analysis
Images were acquired using an epifluorescence microscope (Axioscop 2 Plus; Zeiss Mikroskopie, Jena, Germany) equipped with a computer-driven motorized stage (ProScan H128 Series; Prior Scientific Instruments, Cambridge, UK) controlled by image analysis software (Image-Pro Plus, IPP 5.1 for Windows; Media Cybernetics, Silver Spring, MD).
Retinal photomontages of Brn3a + RGCs were reconstructed from 154 (11 × 14) individual images (Galindo-Romero et al., 2011). The whole population of Brn3a + RGCs was quantified automatically and their distribution assessed by neighbor maps using previously reported methods (Galindo-Romero et al., 2013a). Briefly for each RGC its center mass position coordinates (x, y) were measured using the IPP macro language and the data were exported to a spreadsheet (Office Excel 2000; Microsoft Corp., Redmond, WA). Next, the Java (Oracle Corporation, Redwood Shores, California, USA) application described previously (Galindo-Romero et al., 2013a) was used to calculate the number of neighbors around each cell by measuring their euclidean distance to the rest of cells. Those cells closer than the fixed radius (0.22 mm) were counted. Finally, spatial study was used to spatially plot every RGC, and the number of neighbors served to color each one with a color scale representing the number of neighbors for each one. All maps were plotted using SigmaPlot (SigmaPlot 9.0 for Windows; Systat Software, Inc., Richmond, CA, USA). qPCR Fresh dissected retinas were immediately frozen on dry ice (n = 4 per group and time point). Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific) and the RNA samples were dissolved in 20 µL Milli-Q water. Total RNA concentration was determined using SimpliNanoTM (GE Healthcare Life Sciences, Madrid, Spain). Complementary DNA amplification was performed according to the instructions provided by the manufacturer SuperScript TM IV VILO TM Master Mix, Thermo Fisher Scientific) using 1 µg total RNA.
Mouse pre-designed SYBR green primers (pair 1) for Tnfrs1, Tnfα, and Hrpt (housekeeping) were purchased from Sigma Aldrich. SYBR Premix Ex Taq II (Tli RNaseH Plus, TaKara; Thermo Fisher Scientific) based qPCR was carried out by the Genomic Platform at the IMIB-Arrixaca in a final volume of 5 µl with a primer concentration of 450 nM using the QuantStudio 5 (Applied Biosystems; Thermo Fisher Scientific). Technical triplicates were done for each sample. The Ct values were converted to relative quantification using the 2 Ct method (Livak and Schmittgen, 2001).

Statistics
Data were analyzed and plotted with GraphPad Prism v.7 (GraphPad San Diego, USA). Anatomical data are presented as mean ± standard deviation (SD), and qPCR data as mean ± standard error of the mean (SEM). Differences were considered significant when p < 0.05. Tests are detailed in results.

TNFR1 Expression in Intact and Injured Retinas
In intact retinas, TNFR1 is expressed in the inner nuclear layer (INL) and in the ganglion cell layer (GCL) (Figure 2A). TNFR1 + cells in the GCL do not express Brn3a, and most possibly are displaced amacrine cells, since they are 50% of the cells in the GCL (Pérez De Sevilla Müller et al., 2007;Nadal-Nicolás et al., 2015). Five days after ONC, TNFR1 is expressed more brightly and by more cells than in intact retinas. In the GCL many RGCs are TNFR1 positive (Figure 2B, yellow arrows). Interestingly, TNFR1 expression is observed in those RGCs with a lower signal of Brn3a, as we observed before for those RGCs expressing the cleaved form of caspase 3 (Sanchez-Migallon et al., 2016).
qPCR analyses support the anatomical data, and at 3, 5, and 9 days after ONC there is a 2-3-fold increase of Tnfr1 mRNA compared to intact retinas ( Figure 2C, left graph). Furthermore, after ONC there is as well a significant increase of the Tnfr1 ligand, TNFα ( Figure 2C, right graph), in agreement with previous reports (Tse et al., 2018).

Intraperitoneal and Intravitreal Antagonism of TNFR1 Rescues RGCs From Optic Nerve Crush
To evaluate if a systemic treatment with R7050 protects axotomized RGCs, we performed ONC and animals were treated daily with an intraperitoneal injection of 12 mg/kg of R7050. This dose was chosen based on a previous work where this antagonist was used to treat the brain (King et al., 2013).
Five and fourteen days after the lesion, when without treatment 50 and 92%, respectively, of the RGCs have died, the number of RGCs is significantly higher in the treated than in the untreated groups (Table 1; Figures 3, 4).
Next, we wondered whether a single intravitreal treatment would improve this outcome. We tested two different concentrations (0.2 and 1 mM) because this is the first time this route is used for this drug. Since R7050 is dissolved in DMSO, which has been reported toxic for RGCs after intravitreal administration (Galvao et al., 2014), we injected 5% DMSO in PBS (the maximum concentration used here) in intact retinas and after ONC, and both groups were analyzed 5 days later. Our data show that in intact retinas 5% DMSO does not cause RGC loss, and it does not increase the loss of RGCs after ONC (Table 1).
Five days after the lesion, R7050 intravitreal treatment increases significantly the number of surviving RGCs compared to untreated retinas. With the 0.2 mM intravitreal injection, the elicited neuroprotection was significantly higher than after intraperitoneal treatment (Table 1; Figure 3). Both intravitreal doses achieved a similar RGC rescue and we chose the lower one for subsequent experiments. To verify that the treatment itself was not toxic for RGCs, 0.2 mM R7050 was intravitreally injected in intact retinas. As observed in Table 1, at this dose there was no RGC loss 5 days post-injection. Because the 1 mM dose was not used further and to save animals, we did not test its toxicity.
At 14 days, the intravitreal treatment still protects RGCs (Table 1; Figure 4), but the mean number of surviving RGCs is smaller than with the intraperitoneal treatment, although this difference does not reach statistical significance.
Finally, we combined both routes and treated animals with a single intravitreal injection and a daily intraperitoneal one. This posology does not improve the neuroprotection observed by intravitreal injection alone at 5 days postlesion, therefore we did not assayed this combination at 14 days (Table 1; Figure 3).
The study of RGC topography by neighbor maps, shows in agreement with previous works, that RGC loss by axotomy affects the whole retina (Galindo-Romero et al., 2011;Nadal-Nicolás et al., 2015;Sanchez-Migallon et al., 2016. In addition, here we show that the intraperitonal and intravitreal administration of R7050 protects RGCs across the whole retina (Figures 3B, 4B).

Combinatorial Treatment: TNFR1 Antagonism and BDNF
BDNF is, excluding knocking out pro-apoptotic proteins or transfecting anti-apoptotic ones (Malik et al., 2005;Nickells et al., 2008), the best neuroprotectant for axotomized RGCs. Thus, we decided to test whether a combinatory therapy with BDNF and R7050 was better than each one alone.
At 5 days after ONC, a single intravitreal injection of BDNF rescues as many RGCs as the intravitreal or intraperitoneal treatment with R7050 (Table 1; Figure 3). An intravitreal injection of BDNF and 0.2 mM of R7050 increases the mean number of surviving RGCs, but no significantly so compared to either treatment alone. To save animals, we did not combine BDNF with systemic R7050, because R7050 administered intravitreally gives better results at this time point.
At 14 days post-injury, RGC neuroprotection by BDNF alone was significantly higher than R7050 administered intravitreally and better, but no significantly, than intraperitoneal R7050. Combination of BDNF and R7050 administered intravitreally or systemically, does not increase significantly the number of surviving RGCs compared to BDNF treatment alone. Even so, and as it occurs at 5 days, the mean number of RGCs is higher in the combinatory experiment (Table 1; Figure 4).
Again, and as observed for the single therapy with R7050, RGC neuroprotection by the combinatory treatments expands the whole retina (Figures 3B, 4B).
In summary, while none of the treatments rescued the whole population of RGCs (see Table 1, intact retinas and neighbor map from an intact retina in Figure 3B), they elicited significant neuroprotection: at 5 days and compared to ONC alone, the percent of surviving RGCs in the treated retinas is 19% (BDNF), and 28% (BDNF + intravitreal R7050) higher. Again neuroprotection is relatively better at 14 than at 5 days: at 14 days there are 270% (BDNF), 301% (BDNF + intravitreal R7050), and 320% (BDNF + intraperitoneal R7050) more RGCs than in untreated retinas.
Our results confirm and extend those previous reports connecting axonal damage, RGC loss and TNFα. Our strategy differs from previous works targeting TNFα (Roh et al., 2012;Tse et al., 2018;Park et al., 2019) in that instead of blocking TNFα using decoy-receptors, we have used an antagonist of the TNFα/TNFR1 signaling pathway. R7050 inhibits the endocytosis of the TNFα/TNFR1 multiprotein complex (Gururaja et al., 2007), thus blocking the extrinsic pathway of apoptosis. This antagonist is a small cell permeable molecule that crosses the blood brain barrier, allowing a systemic administration to treat the central nervous system (King et al., 2013). TNFα decoy receptors are also administered systemically, however because they are proteins they come with inherent problems such as bioavailability, tissue distribution and most importantly, antigenicity.
Our data show that R7050 administered locally or systemically, rescues axotomized RGCs. Intravitreal treatment elicits a better neuroprotection at 5 days than the intraperitoneal route. At long time post-lesions, intraperitoneal and intravitreal administration render similar results. Nevertheless, we should not forget that here we performed a single intravitreal injection, while the systemic dose was administered daily.
Neuroprotection by R7050 14 days after ONC is higher (54-42%) than that shown in a previous work where TNFα was intercepted with etanercept, a decoy-receptor (Tse et al., 2018). Tse et al. reported an increase of ∼24% of RGC survival. These differences could mean that R7050 is a better inhibitor of the TNFα apoptotic signaling. Alternatively, the differences could be due to the different route (subcutaneous) or methodology, as they sample the retinas and present RGC densities, while here we quantify the total population of RGCs.
Another strategy to study the implication of a given protein in axotomy-induced RGC death is the use of knockout mice. In a time course study using TNFR1 −/− mice, Tezel et al. (2004) analyzed the retinas from 1 to 6 weeks after ONC and observed, in agreement with our data that compared to wild type animals, RGC neuroprotection in the deficient mice was higher at longer times post-lesion. RGC loss by axotomy triggers a myriad of signals, encompassing multiple pro-death, and pro-survival pathways (Agudo et al., 2008Agudo-Barriuso et al., 2013), and this is believed to be one of the reasons why single treatments are not enough (Harvey, 2007). Thus, combinatory therapies are needed to increase neuroprotection not only in number of neurons, but also in time. Here we combined BDNF, whose neuroprotective potential has been widely studied (Mansour-Robaey et al., 1994;Peinado-Ramon et al., 1996;Di Polo et al., 1998;Pernet and Di, 2006;Parrilla-Reverter et al., 2009;Sanchez-Migallon et al., 2011Galindo-Romero et al., 2013b;Valiente-Soriano et al., 2015;Feng et al., 2016Feng et al., , 2017Osborne et al., 2018) with R7050. We hypothesized that activating pro-survival pathways while inhibiting pro-apoptotic ones would increase RGC survival. However, our data show that the combined rescue of R7050 and BDNF is not summative. This suggest that either both pathways share a common denominator, and/or that the activation of the survival pathways by BDNF blocks the apoptotic signals thus effectively overriding the effect of R7050. Further experiments are needed to assess whether a sequential treatment first with BDNF and then with R7050 (or vice versa) would be more effective.
In conclusion, here we show for the first time that local and systemic treatment with the small inhibitor of the TNFR1 signaling, R7050, protects RGCs from axotomy-induced degeneration. While knockout or transgenic animals are very useful to assign specific functions to specific proteins, this strategy cannot (yet) be used in human patients. Given that to date there are no therapies to overcome neuronal death, proof of concept pre-clinical studies are needed to expand the possible therapeutic avenues to treat not only traumatic neuropathies, but also neurodegenerative diseases.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the manuscript/supplementary files.

ETHICS STATEMENT
The animal study was reviewed and approved by the Ethical and Animal Studies Committee of the University of Murcia, Spain (number: A1320140704).

AUTHOR CONTRIBUTIONS
FL-R, CG-R, MS-N, MG-R: methodology, data analysis and representation, and review and editing. MV-S: writing, review and editing, and funding acquisition. MA: conceptualization,