iNOS‐inhibitor driven neuroprotection in a porcine retina organ culture model

Abstract Nitrite oxide plays an important role in the pathogenesis of various retinal diseases, especially when hypoxic processes are involved. This degeneration can be simulated by incubating porcine retinal explants with CoCl2. Here, the therapeutic potential of iNOS‐inhibitor 1400W was evaluated. Degeneration through CoCl2 and treatment with the 1400W were applied simultaneously to porcine retinae explants. Three groups were compared: control, CoCl2, and CoCl2 + iNOS‐inhibitor (1400W). At days 4 and 8, retinal ganglion cells (RGCs), bipolar, and amacrine cells were analysed. Furthermore, the influence on the glia cells and different stress markers were evaluated. Treatment with CoCl2 resulted in a significant loss of RGCs already after 4 days, which was counteracted by the iNOS‐inhibitor. Expression of HIF‐1α and its downstream targets confirmed the effective treatment with 1400W. After 8 days, the CoCl2 group displayed a significant loss in amacrine cells and also a drastic reduction in bipolar cells was observed, which was prevented by 1400W. The decrease in microglia could not be prevented by the inhibitor. CoCl2 induces strong degeneration in porcine retinae by mimicking hypoxia, damaging certain retinal cell types. Treatment with the iNOS‐inhibitor counteracted these effects to some extent, by preventing loss of retinal ganglion and bipolar cells. Hence, this inhibitor seems to be a very promising treatment for retinal diseases.

physiology of the porcine retina is very similar to that of the human retina. 6 The retina is known to be extremely sensitive to fluctuations in oxygen levels and hypoxia is known to cause development of retinopathy and retinal degenerative diseases. Common to all retinal degenerative diseases is the deterioration of the retina caused by the progressive degeneration and death of the different retinal cells. For example, there is evidence for a causal link between oxidative stress and age-related macular degeneration (AMD). [7][8][9][10] Several publications indicate that oxidative stress and ischaemia, an early event, which occurs under the high ocular pressure present in many forms of glaucoma, induces retinal ganglion cells (RGC) damage. [11][12][13][14][15] Vascular occlusions of the retina, including arterial and venous obstructions, are among the most frequent causes of vision loss. 16 During ischaemia of the inner retina, a large number of different cell types are affected, out of those the RGC represent the most sensitive population, usually die first. 17 As with all neurons, regeneration is not possible. Even worse, the preservation of damaged cells is very difficult due to the environment and the signals generated by the surrounding tissue (eg glia cells). Hence, there is an urgent need to develop new and more effective therapeutic strategies to combat these devastating diseases. In order to be able to find new treatment approaches for these diseases, models with which pathophysiology can be simulated are necessary. Hence, an ex vivo model for retinal hypoxia in pig retina was developed. 11 The treatment of retinal explants with cobalt chloride (CoCl 2 ) induces degeneration in the target tissue corresponding to the clinical picture of ischaemic retinopathies. In the here presented study, the protective effect of the inducible nitric oxide synthase (iNOS) inhibitor 1400W were investigated. Cytokine-inducible nitric oxide synthase is an immune regulator in the retina and mainly found in Müller cells and in retinal pigment epithelium. 18 iNOS is induced under pathological conditions by endotoxins, inflammation, and cytokines and causes pathophysiological reactions leading to optic nerve and retinal degeneration. 8 It is involved in phagocytosis during infectious and ischaemic processes. Once induced iNOS produces large amounts of nitric oxide (NO). 19 Nitric oxide is an essential signalling molecule, which plays a role in neurotransmission, host cell defence and vasodilation. 20,21 There are three isoforms of the nitric oxide synthase (NOS), the enzyme that produces NO, neuronal, immunologic and endothelial isoform. The first two are present in the retina. 22 The immunologic isoform is not constitutively expressed and requires induction usually by immunologic activation; calcium is not necessary for its activation as it is for the other two forms. 23 Pathophysiological increase of NO through iNOS has major effects in all tissues, but especially in neuronal tissue, like the retina. NO mediates many of the destructive effects of interleukin (IL)-1 in inflamed tissues. NO has been reported to activate matrix metalloproteinases, 24 inhibit collagen synthesis and induce retinal apoptosis. 22,25 The resulting molecules nitrogen dioxide (NO 2 ), nitrite, peroxynitrite and free radicals are responsible for a retinal degeneration that occurs in glaucoma, ischaemic retinopathies, and AMD. 19 The inhibition of iNOS has been researched for years in cancer therapy and has also made its way into ophthalmology. [25][26][27][28] Here, we present a study investigating the effect of the iNOS-inhibitor 1400W on retinal cells in the CoCl 2 degeneration model 1400W.
This iNOS-inhibitor had a neuroprotective effect on neuronal cells in CoCl 2 induced hypoxic degeneration model.
For this study, a cultivation time of 4 and 8 days was chosen.
Retina explants were exposed to CoCl 2 from day 1 to day 3, for 48 hours (300 mmol/L; Figure 1). At the beginning of degeneration, treatment with 500 µmol/L iNOS-inhibitor 1400W (Merck Millipore) was started simultaneously and lasted 72 hours until day 4. In preliminary studies, shorter time periods were also investigated, but they did not achieve the desired effects. Control retinas were cultivated continuously at 37°C without any treatment. The medium was exchanged completely on days 0, 1, 2 and 3 In addition, 50% medium were exchanged at day 6. On days 4 and 8, samples were frozen for subsequent the analyses immunohistochemistry and quantitative real-time PCR (qRT-PCR).

| Histology
Fixation of retinal explants was performed for 15 minutes using 4% paraformaldehyde. Each explant was cryo-protected with 15% sucrose for 15 minutes, 30% sucrose for 30 minutes, and then F I G U R E 1 Procedure of the 1400W treatment in the CoCl 2 degeneration model. CoCl 2 , cobalt chloride; IHC, immunohistochemistry; iNOS-inh., iNOS-inhibitor 1400W frozen in liquid nitrogen. Slides of 10 µm thick slices were cut using a cryostat.

| Immunohistology
Retinal cross-sections were pre-incubated with blocking buffer containing 0.1%-0.2% PBS/TritonX-100 mixture (Merck Millipore) and 10%-20% normal donkey serum (Dianova) for one hour. Primary antibodies ( Table 1) were diluted in the blocking buffer and slices were incubated over night at room temperature. At the next day, retinal cross-sections were incubated with fluorescence-labelled secondary antibodies diluted in the same blocking buffer (Table 1).

| Immunohistological examination
Six retinal slices per explant were used for the evaluation. In the end, 24 masked images were counted for each staining. Cells were counted as positive, when the specific marker (RNA-binding protein with multiple splicing [RBPMS], calretinin and Protein kinase C alpha [PKCα]) was co-localized with DAPI. The total amount of microglia population was evaluated by counting all Ionized calcium-binding adapter molecule 1 (Iba1 + ) and DAPI + cells. Active microglia were counted when Fcy-Rezeptor (Fcy-R + ) signals were additionally seen.
All cell numbers are given in cells/mm.

| Quantitative real-time PCR
The expression of cell specific markers, like parvalbumin (PVALB),

| Statistical analysis
In regard to immunohistological data, ANOVA followed by Tukey's post-hoc test was applied to analyse differences between groups (Statistica, V 12). In accordance, qRT-PCR data were analysed using ANOVA followed by Tukey's post-hoc test to analyse differences between groups (GraphPad Prism 8). For all statistical tests, significance with respect to the control group is indicated using the following symbols and significance levels: *P < .05; **P < .01; ***P < .001.

| Effect of 1400W on oxidative stress in retinal organ cultures
First, it was examined whether the iNOS-inhibitor in the retinal organ cultures develops its effect via testing the protein and mRNA expression of the hypoxia marker HIF-1-α. As previously described, induction with CoCl 2 leads to an increase in oxidative stress and activates transcription of HIF-1α. 3,4 This is a specific oxygen-sensitive subunit that regulates the activity of the transcription factor HIF-1, which increases after ischaemia and can either promote or prevent neuronal survival.
Histologically, an HIF-1α signal could be observed after 4 days in the CoCl 2 group, which seemed to be only slightly reduced after 4 days under treatment with iNOS-inhibitor 1400W ( With the 4-day-groups no significant changes of the HIF-1α mRNA level were observable ( Figure 2B). After 8 days of cultivation, mRNA expression of the HIF-1α was increased 1.9-fold (P = .061) in the CoCl 2 group. 1400W significantly reduced mRNA expression (P = .037; Figure 2B).

| Influence of 1400W on iNOS and HSP70 expression
HIF-1 is known to induce transcription of more than 60 genes, including vascular enothelial groth factor (VEGF; data not shown) and iNOS. In order to check whether this is the case in our model, we also analysed the mRNA expression of those markers.
Investigating of the mRNA expression of the iNOS revealed no alterations in the CoCl 2 group and a non-significant twofold reduction of the mRNA in 1400W-treated group after 4 days. In the later time point, a significant (fourfold, P = .0036) mRNA increase by CoCl 2 was observed, which was prevented by 1400W (P = .0085; Figure 3B).
This loss could be counteracted by treatment with 1400W. In the retinae of the treatment group significantly more RGCs were detected compared with the untreated group (CoCl 2 + iNOS-inh.: 33.9 ± 2.2 cells/mm, P = .021) and also no significant difference to the control group was noticed (P = .109). After 8 days of cultivation a significant decline of the RGCs in the CoCl 2 -treated retinae was also observed (control: 31.7 ± 2.0 cells/mm; CoCl 2 : 19.4 ± 0.9 cells/mm, P = .0002).
Treatment with the iNOS-inhibitor significantly reduced this effect, as these retinae contained significantly more RGCs than the untreated ones (CoCl 2 + iNOS-inh.: 28.5 ± 1.8 cells/mm, P = .002). Furthermore, there was no significant difference in the number of RGCs between the treatment and the control group (P = .374; Figure 4B).

| CoCl 2 induced irreversible degeneration of microglia and decreases their activity
Immunohistochemical staining was used to examine the total population of microglia in the retina (anti-Iba1 (red); Figure 5A). Activated microglia exhibited a strongly increased expression of the Fcy-receptor; thus, all Iba1 + and Fcy-R + cells (green) were evaluated as active microglia. As previously observed, 3 Figure 5C).
In addition, the relative mRNA expression of CD11b, another marker of the microglia, and CCL2, a CC chemokine, which regulates the activation and recruitment of macrophages, was tested. Here, a down-regulation of immunocompetent cells was also clearly evident.
After 8 days of cultivation, there was still a reduction of CD11b mRNA, which was not significant (P = .0512) ( Figure 5D). And there were no differences in CCL2 mRNA expression ( Figure 5E).
F I G U R E 3 mRNA expression of the HIF-1α target genes. A, CoCl 2 had a significant effect on iNOS mRNA expression after 4 d and the additional treatment with 1400W only lead to a small decrease in mRNA. After 8 d, CoCl 2 induced a fourfold increase in iNOS mRNA expression, this could be prevented by the 1400W treatment. B, After 4 d, a massive increase in mRNA expression of HSP70 was observed in the CoCl 2 retinas, which was significantly reduced by treatment with 1400W. After 8 d, a significantly increased expression was still observed, but not as pronounced as at the previous time. However, the protective effect of the inhibitor was still detectable after 8 d. All data are shown as mean ± SEM; **P < .01

| No rescue of amacrine cells through iNOSinhibitor treatment
The degenerative influence of CoCl 2 and the potential protective effect of 1400W on amacrine cells were investigated using relative CoCl 2 + iNOS-inh.: 5.1 ± 3.6 calretinin + cells/mm, P = .001). Also, no significant difference in relative PVALB expression could be measured between the groups after 4 days ( Figure 6C). At day 8, a 2.8fold decrease in relative PVALB expression was noted in the CoCl 2 group compared to the controls (P = .0518), which could not be eliminated by treatment with iNOS-inhibitor (4-fold decrease, P = .0265; Figure 6C). of the bipolar cells, which was prevented by the 1400W treatment (45.5 ± 7.1 PKCα + cells/mm; P = .0017; Figure 7B).

F I G U R E 4 Rescue of retinal ganglion cells after CoCl 2 -induced degeneration.
A, Representative images of the immunohistological staining. RGCs were stained with an antibody against RBPMS (red) and cell nuclei with DAPI (blue). A significant loss of RGCs in the untreated degeneration groups (CoCl 2 ) was observed over the cultivation period of 4 and 8 d. B, After 4 d, neuroprotection of the RGCs was observed by treatment with the iNOS-inhibitor compared with the CoCl 2 group. Even after 8 d of cultivation, a protection of the RGCs by 1400W could be noticed. The retinae of the treatment groups contained significantly more RGCs than the untreated retinae. GCL, ganglion cell layer; IPL, inner plexiform layer. Scale bar = 20 µm. All data are shown as mean ± SEM; *P < .05; **P < .01; ***P < .001 F I G U R E 5 CoCl 2 degeneration irreversibly reduces the microglia. A, Microglia were stained with anti-Iba1 (red) on day 4 and 8 of cultivation. Fcy-R (green, arrows) in combination with Iba1 served as an activity marker of the microglia. Cell nuclei are shown in blue. B, The addition of CoCl 2 triggered a significant loss of microglia in comparison with controls at both times. In the 1400W-treated group also, significantly fewer microglia were present than in the control. C, In addition, the number of activated microglia was significantly lower in the CoCl 2 group than in the control group after 4 and 8 d. Again, treatment with the iNOS-inhibitor had no protective effect. D, The relative CD11b mRNA expression was also significantly reduced in the CoCl 2 group, after 4 and 8 d of cultivation. The 1400W treatment did not result in an improvement compared with control at both times. E, The analysis of the relative CCL2 mRNA expression showed that it was significantly reduced after 4 d in retinae of the CoCl 2 group. Again, mRNA expression could not be altered by the 1400W. After 8 d of cultivation, no differences between the groups were observed. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar = 20 µm. All data are shown as mean ± SEM; **P < .01; ***P < .001

| D ISCUSS I ON
The degeneration processes of many retinal diseases have not been fully investigated yet. In order to understand the pathological changes, reliable models are needed, in which eye diseases can be simulated. In this study, we present a promising option in which not only the degeneration process in retinal tissue could be simulated in a straight-forward and standardized way, but also drug therapy testing could be performed. The advantages of these ex vivo cultures are obvious: The anatomy of pig eyes compared to human eyes is morphologically and physiologically more similar to that of rodents. 6 In addition, the complex structure of the retina is preserved and the reproducibility is much higher as a higher number of samples can be obtained. 2,30 Retinopathy is the main cause of blindness and visual impairment in people of all ages. The pathogenesis of retinopathy is caused by numerous factors. Considering only the 'hypoxic factors', these include changes that contribute to oxidative stress, like increased nitric oxide and superoxide production, changes in the expression of various isoforms of nitric oxide synthase, or the endogenous antioxidant system. 31 Hypoxia is a main trigger of the pathogenic mechanism in retinal diseases. This is a multifactorial, dynamic process involving oxidative stress, inflammation, and cell death as well as the activation of regenerative mechanisms dependent on the hypoxia inducible transcription factor HIF-1α. 32,33 HIF-1α is one of the key regulatory components in the cell's hy- CoCl 2 , similar to hypoxia, prevents the degradation of the α-subunit of hypoxia-inducible factor and thus mediates its stabilization. 40 With our CoCl 2 induced retinal degeneration model, we already proved the neuroprotective effect of hypothermia. 4 Here, we specifically examined the influence of the inhibition of iNOS on the course of degeneration.
CoCl 2 induced hypoxia led to a significant loss of RGCs after 4 and 8 days, which was counteracted by treatment with 1400W.
As described before, incubation with CoCl 2 induces apoptosis, especially in RGCs. 3,41 We have already shown that CoCl 2 not only mimics hypoxia by stabilizing HIF-1α, but also leads to an elevated ROS level by disrupting the mitochondrial respiratory chain. 4,33 The mechanism behind the 1400W mediated protection of RGCs is possibly based on reduced NO production and thus on the prevention of apoptosis. In our model, it could be observed that treatment with the iNOS inhibitor significantly reduced the amount of iNOS and HIF-1α mRNA expression. 42 It is known that hypoxia induces HIF-1α and its target genes, such as VEGF and iNOS, in many tissues. 43 The pathophysiological accumulation of these factors has been associated with neuronal death under hypoxic-ischaemic conditions. Moreover, overproduction leads to increased extracellular accumulation of glutamate and inflammatory cytokines, which damage the neurons. 44 Another marker for cellular stress is HSP70, whose expression can be induced by HIF-1α. 45 In CoCl 2 -stressed retinas, the mRNA expression of HSP70 was strongly elevated. 46 HSPs are chaperones that are up-regulated during cellular stress. Their task is to prevent misfolded proteins and protein aggregation. Thus, HSPs plays an important role for the accumulation and function of HIF-1α. 47  vitro, and they may therefore be a major source of iNOS expression.
Furthermore, other cell types, such as amacrine, horizontal, bipolar and microglial cells, contribute to the NO production during ischaemic proliferative retinopathy. 48 Microglia are an essential mediator of neuroinflammation in many neurological disorders and are susceptible to HIF-1α. 49 Likewise, there are reports that describe that besides oligodendrocytes, the microglia are the glial cell types most susceptible to hypoxia 50 and are extremely sensitive to their microenvironment. 44,51 We observed the same effects in our ex vivo model ( Figure 5). Incubation with CoCl 2 has massive degenerative effects on microglia. 3 The inhibition of iNOS is not an obvious way to increase microglia number in this case. Based on the type of cultivation, with just a piece of the retina, no interaction with the optic nerve and the retinal pigment epithelium, the microglia are more turned to pro-inflammatory M1 subtypes. iNOS is a marker which is only produced by M1 microglia/macrophages. 52 The reduction of iNOS inhibited the microglia. Furthermore, high levels of VEGF could reduce the number of M1 microglia as well, which is already shown in an ischaemic brain rat model. 53 Therefore, the treatment with the iNOS inhibitor had no beneficial effect on the microglia number in contrast to the already published treatment with hypothermia. 4 Other publications describe a balance between harmful and protective factors in the retina after hypoxia. It is therefore conceivable that microglia react early to hypoxic stress but are down-regulated after 4 or 8 days to protect the retina. 44,54,55 Inner layers of the retina are known to be most sensitive to hypoxic challenges, whereas the outer retina is more resistant to hypoxic stress. 56,57 Investigations of other cell types of the inner retina revealed that CoCl 2 led to a loss of calretinin positive amacrine cells and PKCα-positive bipolar cells after 8 days, which was also described before. 3  Furthermore, increased production of NO is believed to mediate neuronal injury caused by glutamate acting on NMDA receptors. 44,61 This might be one mechanism how CoCl 2 induced loss of amacrine cells and why it could not be prevented by the iNOS-inhibitor.

| CON CLUS ION
The iNOS-inhibitor 1400W led to neuroprotective effects in the retina and many but not all cell types responded with an increased survival rate to the therapy. This allowed us to prove the neuroprotective properties of 1400W and at the same time prove that ex vivo organ cultures are very suitable for drug therapy testing.

ACK N OWLED G EM ENTS
This project is supported in part by the SET Stiftung, Germany.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R S ' CO NTR I B UTI O N S
AMM-B, FH and LH cultivated retinal explants and performed the histological examinations of the explants. SK supported the statistical analysis of the data. JH performed the qRT-PCR examination and was a major contributor in writing the manuscript. SS and SCJ revised the manuscript, planed and designed the study. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article.