The CORM ALF-186 Mediates Anti-Apoptotic Signaling via an Activation of the p38 MAPK after Ischemia and Reperfusion Injury in Retinal Ganglion Cells

Purpose Ischemia and reperfusion injury may induce apoptosis and lead to sustained tissue damage and loss of function, especially in neuronal organs. While carbon monoxide is known to exert protective effects after various harmful events, the mechanism of carbon monoxide releasing molecules in neuronal tissue has not been investigated yet. We hypothesize that the carbon monoxide releasing molecule (CORM) ALF-186, administered after neuronal ischemia-reperfusion injury (IRI), counteracts retinal apoptosis and its involved signaling pathways and consecutively reduces neuronal tissue damage. Methods IRI was performed in rat´s retinae for 1 hour. The water-soluble CORM ALF-186 (10 mg/kg) was administered intravenously via a tail vein after reperfusion. After 24 and 48 hours, retinal tissue was harvested to analyze mRNA and protein expression of Bcl-2, Bax, Caspase-3, ERK1/2, p38 and JNK. Densities of fluorogold pre-labeled retinal ganglion cells (RGC) were analyzed 7 days after IRI. Immunohistochemistry was performed on retinal cross sections. Results ALF-186 significantly reduced IRI mediated loss of RGC. ALF-186 treatment differentially affected mitogen-activated protein kinases (MAPK) phosphorylation: ALF-186 activated p38 and suppressed ERK1/2 phosphorylation, while JNK remained unchanged. Furthermore, ALF-186 treatment affected mitochondrial apoptosis, decreasing pro-apoptotic Bax and Caspase-3-cleavage, but increasing anti-apoptotic Bcl-2. Inhibition of p38-MAPK using SB203580 reduced ALF-186 mediated anti-apoptotic effects. Conclusion In this study, ALF-186 mediated substantial neuroprotection, affecting intracellular apoptotic signaling, mainly via MAPK p38. CORMs may thus represent a promising therapeutic alternative treating neuronal IRI.


Introduction
As an organ with high demand of nutrients and oxygen, the human brain is very sensitive to hypoperfusion. In patients suffering from stroke, irreversible neuronal cell death may occur within minutes leading to substantial neuronal deficit and increased mortality. [1][2][3][4] Similarly, retinal ischemia and reperfusion injury leads to neuronal cell death and plays an important role in the pathophysiology of several eye diseases such as diabetic retinopathy and glaucoma. [5,6] In the last years a multitude of substances have been tested pre-clinically regarding their neuroprotective potential. [7] However, all of them showed either a weak effect only, or failed to prove relevance in clinical practice at all.
In human organism, carbon monoxide (CO) is produced endogenously as one elimination product of heme oxygenase (HO), an enzyme that facilitates heme catabolism and catalyzes the degradation of heme to biliverdin, CO and ferrous ions. [8,9] It has been shown that even exogenously applied CO may mediate protection in the context of ischemia and reperfusion. Although poisonous at high concentration, inhaled carbon monoxide has attracted attention as a potential therapeutic agent. Being administered after ischemia-reperfusion injury (IRI) and transplantation, CO appears to protect vital organs including heart, lung, kidney and liver. [10][11][12][13] To do so, CO interacts with a variety of physiological processes, among which some of them are responsible for apoptosis. As far as known, targets of CO action are the binding to soluble guanylate cyclase (sGC), stimulation of cGMP production, activation of ca 2+ -dependent potassium channels and stimulation of mitogen-activated protein kinases. [14] Due to the fact of difficulties in handling a gaseous-and dose-dependently toxic-agent and the negative effects of inhaled CO on oxygen transport, molecules containing a heavy-metal framework were created as carriers of covalently-bound CO, defined as carbon monoxide releasing molecules (CORM). These substances, administered either orally or intravenously ensure a constant liberation of CO and delivered a predictable and controllable amount of CO to organs and tissue. [15] The carbon monoxide releasing molecule ALF-186 presents a special group of CORMs, which is water-soluble and releases its bound CO molecules in a slower manner than other CORMs do. [16] Although described in some organ systems, therapeutic effects of CORMs on neuronal structures following neuronal damage have not been investigated so far. In this in-vivo study we analyzed the impact of the CORM ALF-186 after neuronal IRI and the underlying mechanism. We hypothesized that ALF-186 treatment mediates anti-apoptotic effects and protects retinal ganglion cells after IRI via the p38 MAPK. In future, CO might be a promising therapeutic option reducing neuronal damage.

Materials and Methods Animals
Adult male and female Sprague-Dawley rats (1:1, 280-350g bodyweight, Charles River, Sulzfeld, Germany) were used in these experiments. Animals were fed with a standard diet ad libitum, being kept on a 12-h light/12-h dark cycle. All procedures involving the animals concurred with the statement of The Association for Research in Vision and Ophthalmology for the use of animals in research and were approved a priori by the Committee of Animal Care of the University of Freiburg in accordance with the ARRIVE guidelines (Permit No: .81/G-11/81). All types of surgery and manipulations were performed under general anesthesia with isoflurane/O 2 for retrograde labeling with fluorogold (FG) or with a mixture of intraperitoneally administered ketamine 50 mg/kg (Ceva-Sanofi, Duesseldorf, Germany) and xylazine 2 mg/kg (Ceva-Sanofi) for the ischemia-reperfusion experiment. Body temperature was maintained at 37°C ± 0.5°C with a heating pad. After surgery, buprenorphine (Temgesic 1 0.5 mg/kg; Essex Pharma, Solingen, Germany) was applied intraperitoneally to prevent pain. During recovery from anesthesia, the animals were placed in separate cages. The number of animals used for RGC quantification and molecular analysis was n = 8 per group. For analysis of mRNA-and protein-expression retinal tissue was harvested at t = 24 h and 48 h after reperfusion. (see flow-chart in Fig 1).

Retrograde labeling of RGC
Sprague Dawley rats were anesthetized, placed in a stereotactic apparatus (Stoelting, Kiel, Germany) and retrograde RGC-labeling was done as described previously, briefly summarized as follows: The skin overlying the skull was cut open und retracted. The lambda and bregma sutures served as landmarks for drilling three holes on each site of the bregma sutures. A total amount of 7.8 μl fluorogold (FG) (Fluorochrome, Denver, CO, USA) dissolved in DMSO/PBS was injected into both superior colliculi through the drilling holes. To ensure adequate retrograde transport of FG into the RGC´s perikarya, further experimental interventions were done 7 days after retrograde labeling.

Retinal ischemia/reperfusion injury and treatment with ALF-186
Following randomization, rats were sedated as previously described [17][18][19] and the anterior chamber of the left eye was cannulated with a 30-gauge needle connected to a reservoir containing 0.9% NaCl. Intraocular pressure was increased to 120 mmHg for 60 minutes and ocular ischemia was confirmed microscopically by interruption of the retinal circulation. Reperfusion was initiated by removing the needle tip promptly. Rats without immediate recovery of retinal perfusion at the end of the ischemic period or those with lens injuries were excluded from the study, since the latter prevents RGC death and promotes axonal regeneration. To evaluate possible neuroprotective effects of the treatment with carbon monoxide, released from ALF-186, animals were randomly assigned to receive either treatment with ALF-186 (10 mg/kg body weight i.v., dissolved in PBS) or PBS (vehicle control) alone. Both treatment options were injected intravenously, either immediately or with a delay of 3 hours after IRI. To exclude that the molybdenum-containing metal backbone of ALF-186 exerts protective or crucial effects itself, inactivated ALF-186 (iALF-186) was injected 24 hours after dissolving. In a later set of experiments, rats received the p38 MAPK inhibitor SB203580 (1 mg/kg bodyweight i.v., Sigma-Aldrich, #S8307) sixty minutes prior to IRI and ALF-186 treatment.

RGC quantification
Animals were sacrificed by CO 2 -inhalation 7 days after ischemia. Retinal tissue was immediately harvested, placed in ice-cold Hank´s balanced salt solution and further processed for whole mount preparation. Retinae were carefully placed on a nitrocellulose membrane with the ganglion cell layer (GCL) on top. After removing the vitreous body, retinae were fixed in 4% paraformaldehyde for 1 h and then embedded in mounting media (Vectashield; Axxora, Loerrach, Germany). The densities of FG-positive RGC were determined in blinded fashion using a fluorescence microscope (AxioImager; Carl Zeiss, Jena, Germany) and the appropriate bandpass emission filter (FG: excitation/emission, 331/418 nm), as previously described. Briefly, we photographed 3 standard rectangular areas (0.200 mm x 0.200 mm = 0.04 mm 2 ) at 1, 2 and 3 mm from the optic disc in the central regions of each retinal quadrant. Hence, we evaluated an area of 0.48 mm 2 per retina. To calculate the average RGC density in cells/mm 2 , we multiplied the number of analyzed cells/0.04 mm 2 with 25. Secondary FG stained activated microglia cells (AMC) after RGC phagocytosis were identified by morphologic criteria and excluded from calculation. All data are presented as mean RGC densities [cells/mm 2 ] ± SD. First row: Fluorogold (FG) labeling seven days prior to IRI, subsequent treatment with or without p38 inhibitor (SB203580) and/or iALF-186/ALF-186 and enucleation another seven days later, giving FG the chance to promote into RGC via axonal transport. Second row: IRI with or without p38 inhibitor (SB203580) and/or iALF-186/ALF-186 treatment and enucleation 24 or 48 hours after IRI for molecular analysis. Third row: IRI with or without p38 inhibitor (SB203580) and/or iALF-186/ALF-186 treatment and enucleation 24 or 48 hours after IRI for immunohistochemical analysis. doi:10.1371/journal.pone.0165182.g001

Real time polymerase chain reaction (RT-PCR)
From retinal tissue harvested 24 h and 48 h after ischemia, total RNA from ¼ of retina was extracted using a column-purification based kit (RNeasy Micro Kit, Qiagen, Hilden, Germany) according to the manufacturer´s instructions. Reverse transcription was performed with 50 ng of total RNA using random primers (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Darmstadt, Germany). Real time polymerase chain reactions (RT-PCR) were done with TaqMan 1 probe-based detection kit (TaqMan 1 PCR universal mastermix, Applied Biosystems, Darmstadt, Germany). Following primers were used: Caspase-3 #Rn00563902_m1, Bax #Rn02532082_g1 and Bcl-2 #Rn99999125_m1 (all from Applied Biosystems, Darmstadt, Germany). The PCR assays were then performed on a RT-PCR System (ABI Prism 7000, Applied Biosystems, Darmstadt, Germany) with the following cycling conditions: 95°C for 10 min, 40 cycles of 95°C for 10 sec and 60°C for 1 min. Reaction specificity was confirmed by running appropriate negative controls. Cycle threshold (CT) values for each gene of interest were normalized to the corresponding CT values for GAPDH (ΔCT). Relative gene expression in IR injured retinal tissue either with injection of ALF-186 or PBS was calculated in relation to the corresponding gene expression in the non-injured retinal tissue of each individual animal (ΔΔCT).

ALF-186 induced p38 expression in the ganglion cell layer and inner nuclear layer
Retinal cross sections were evaluated for p38 immunoreactivity to answer the question, which cells were responsible for p38 upregulation after IRI+ALF. In untreated eyes and after IRI, there was only a weak p38 expression pronounced in the area of the ganglion cell layer (GCL) (Fig 7A, 7D and 7G). Co-staining with Brn3a (B, E, H) revealed a low degree of double-immunoreactivity with RGCs, the p38 signal was seen in a low level in the area of the nerve fibers on top of the GCL (C, F, I). ALF treatment directly after IRI induced upregulation of p38 (J) in the GCL and inner nuclear layer (INL). Double-immunoreactivity with Brn3a (L and Fig 8) was partially seen. Scale bar in L is 100 μm.
Carbon monoxide (CO) exerts a variety of pharmacological effects, mediating among others anti-apoptotic effects in the cardiovascular, immune and nervous systems. [20,21] Due to difficult handling in treatment with the potentially toxic volatile agent CO, carbon monoxide releasing molecules have attracted attention. These molecules contain metal carbonyls like manganese, ruthenium, boron and iron as CO carrier leading to a constant and reliable release  Retinal cross sections were evaluated for p38 immunoreactivity to answer the question, which cells were responsible for p38 upregulation after IRI+ALF. In controls and after IRI+PBS, there was only a weak p38 expression, pronounced in the area of the ganglion cell layer (GCL) (Fig 5, right side, A, D, G). Co-staining with Brn3a (B, E, H) revealed a low degree of double-immunoreactivity, the p38 signal was seen in a low level in the area of the nerve fibers on top of the GCL (C, F, I). ALF treatment directly after IRI induced upregulation of p38 (J) in the GCL and inner nuclear layer (INL), as expected from the results of the p38 mRNA expression. Double-immunoreactivity with Brn3a (K+L) was more frequent and more intense but not exclusively expressed in RGCs. Scale bar in L is 100 μm. doi:10.1371/journal.pone.0165182.g007 Carbon Monoxide Provides Neuroprotection via p38 of CO. 1 , [15] Since water-soluble substances are available, oral or intravenous application simplified CO treatment avoiding environmental impact and potential poisoning.
In this in-vivo study, we have examined the potential protective effect of ALF-186 on neuronal tissue. Hence, we chose a well-established model of retinal IRI in rats. [17][18][19] We found that treatment with ALF-186 reduced IRI mediated loss of retinal ganglion cells and therefore mediated protection. While protective effects of carbon monoxide are well studied in a variety of damage models of non-neuronal organs, studies describing the protective effect of CO and CORM on neuronal tissue are limited. [22][23][24][25][26] Zeynalov and Doré demonstrated that inhalative carbon monoxide reduced infarct size after focal transient brain ischemia in a mouse model. [27] Wang et al. also exposed mice to carbon monoxide after middle cerebral artery occlusion and described a reduced infarct volume. [28] The neuroprotective effects in this study are comparable to our previous findings. [17] Intracellular signaling via MAPK ERK1/2, p38 and JNK plays a significant role in the regulation of neuronal [29][30][31] and retinal [32][33][34] apoptosis. However, the effects of ERK1/2 phosphorylation are considered to be contradictory: both survival and death signals can activate ERK1/2 and therefore activation itself may result in neuronal death or survival. [29] Cellular stress triggers MAPK p38 and JNK activation, for example in the context of cerebral ischemia. [35] It is assumed that p38 and JNK mediated apoptosis is transmitted by mitochondrial proapoptotic proteins like Bax and Bcl-2. [35] In our study we have found that ALF 186 modulated MAPK differently. ALF treatment resulted in a decreased ERK1/2 phosphorylation and, on contrary, increased p38 activation while JNK phosphorylation did not changed. Following cell and tissue damage, the influence of carbon monoxide on MAPK is a topic of controversy in the literature whereby it is important to note that the type of injury differently affected the MAPK. Our findings are in accordance to the results of Sethi et al., who exposed primary pulmonary artery endothelial cells of the rat to carbon monoxide after TNFα stimulation. Injured cells presented increased ERK1/2 and JNK activation in contrast to p38 suppression. Application of CO reversed TNFα mediated effects on ERK1/2 and p38 while CO had no influence on JNK. [36] Li et al. describe in their study the p38 MAPK pathway as a key signaling mechanism of CO´s protective effect. In primary hepatocytes of rats´ethanol increased p38 phosphorylation while additional CORM further stimulated p38 phosphorylation. JNK and ERK1/2 were not affected. [37] The cytoprotective effect of ERK1/2 downregulation was described by Choi et al. Glucose deprivation led to an increase of ERK1/2 and p38 phosphorylation in BNL CL.2 cells whereas JNK was not affected. In contrast to our findings, the presence of CORM only suppressed ERK1/2 phosphorylation. [38] Ning et al. showed in their in-vitro study that stimulating lung cells, with IL16 only affected MAPK ERK1/2, but neither p38 nor JNK, and increased levels of phosphorylated ERK1/2. Exposure to CO (250 ppm) mitigated this effect. [39] Similarly, CO treatment suppressed elevated ERK1/2 phosphorylation in liver grafts after cold IRI due to orthotopic liver transplantation in rats. Although CO also decreased p38 phosphorylation, the influence of CO was identified as not significant. [13] Our research group showed that CORM increased p38 phosphorylation in staurosporine treated EBL cells, but not ERK1/2 nor JNK. [40] In another study, postconditioning with inhalative CO (250 ppm) showed similar effects on retinal ganglion cells with an increase of cell count after IRI. Interestingly, an opposite effect on MAPK was demonstrated. [19] Inhalative CO reduced p38 phosphorylation and induced ERK1/2 activation while JNK was not affected. The reason for this might be the different way of administration.
Due to neuronal injury, neurons release molecules interacting cell survival and cell death signaling. In particular, Bcl-2 family proteins like Bax and Bcl-2 are involved in IRI. [41]  Thereby, Bcl-2 promotes neuronal survival modulating Ca 2+ signaling and Bax operates as an apoptotic stimulus. Shifting this balanced Bax/Bcl-2 ratio towards Bax causes mitochondrial damage that activates the caspase cascade. [42] Our study demonstrated, that ALF-186 reduced IRI induced apoptosis by inhibition of Bax expression, activation of Bcl-2 and, consecutively, by suppression of caspase-3 cleavage. In part, these results are in agreement with Schallner et al.: Inhalation of CO prior to IRI caused a decrease of Bax and caspase-3 cleavage while Bcl-2 was not affected. [19] Cheng and Levy also reported a beneficial effect of carbon monoxide on neuronal tissue. In their study, subclinical CO decreased caspase-3 activation in mice pulps following isoflurane exposition. [43] In a study with endothelial lung cells Wang et al. described that CO inhibited hyperoxia induced cell death by decreasing pro-apoptotic Bax. [44] Nakao et al. examined the effect of CO treatment on intestinal apoptosis due to IRI in rats. CO inhalation reduced Bax and upregulated Bcl-2. [45] Pretreatment with p38 inhibitor SB203580 reduced the impact of ALF-186 on retinal apoptosis. We have found that p38 inhibition resulted in an increase of caspase-3 cleavage and Bax expression and also in a decrease of Bcl-2 expression. Furthermore, p38 inhibition reversed ALF-186 mediated p38 phosphorylation, reduced neuronal ganglion cell count and cancelled neuroprotection. We focused on this proposed mechanism as part of the carbon monoxide mediated neuroprotective effect in Fig 9. Although neuroprotective effects are described [46], in our study SB203580 alone did not affect retinal apoptosis. This is also confirmed by Mori et al., who did not notice protection in traumatic brain injury after treatment with SB203580. [47] In summary, we demonstrate that the treatment with ALF-186 as a therapeutic option reduced retinal apoptosis after IRI in-vivo mainly in the first 24 hours after neuronal damage. ALF186 affected and modulated intracellular MAPK and apoptosis signaling and reduced loss of retinal ganglion cells, therefore representing a novel and promising alternative in treating ischemia-related injury of neuronal organs.