Inflammatory responses in three different retinal regeneration paradigms
To assess the inflammatory responses within each paradigm, we first characterized the dynamics of inflammatory cells in the retina. For this purpose, retinal microglia/macrophage was labeled by an intravitreous injection of the isolectin IB4 [20, 28]. In the retina of Tg(mpeg1:EGFP) transgenic zebrafish in which GFP was specifically expressed by microglia/macrophage, the majority of IB4 signals co-localized with GFP (Figure 1A), further confirming the specificity of this staining. In the uninjured retina, a small number of IB4+ cells could be seen scattered in the retina and vitreous (Figure 1B, left panel, Figure 1E). Following stab injury, a large number of IB4+ cells accumulated at the injury site at 2 days post injury (dpi) (Figure 1B,C; p < 0.001), indicating a robust microglia/macrophage response in the retina as shown previously. Intravitreal PBS injection into the uninjured eye did not significantly increase the total number of IB4+ cells in the retina and vitreous (Figure 1B,D,E; p = 0.356), though a small increase in the vitreous was observed at 2 d (Figure 1E, p < 0.001), indicating that some of the IB4+ cells in the retina migrated to the vitreous after PBS injection. Two days after the NMDA-injury or insulin-treatment, significantly increased numbers of IB4+ cells compared with the PBS control could be seen in the retina and vitreous (Figure 1D,E;[retina], NMDA, p < 0.001, insulin, p = 0.009; [vitreous], NMDA, p < 0.001, insulin, p = 0.004). The accumulation of IB4+ cells was most evident in the inner plexiform layer (IPL) in both paradigms (Figure 1D, lower panels). We have previously shown in the stab-injury model that the accumulated IB4+ cells could be seen as early as 6 hours post injury (hpi), and they peaked at 2 dpi before declining at 4 dpi. Interestingly, similar time courses were observed following NMDA-injury or insulin-treatment, though the initial rise in the number of IB4+ cells occurred slightly later than following stab-injury (12 hpi vs 6 hpi, Figure 1F). These results indicate that an acute microglia/macrophage response with similar time courses occurred in the retina and vitreous in the three paradigms.
It has been previously shown that neutrophil infiltrates into the retina after stab injury, but not after sterile retinal cell loss in zebrafish larvae. To characterize the potential neutrophil response in the three paradigms, we took advantage of the Tg(mpx:GFP) transgenic zebrafish in which GFP was selectively expressed in neutrophils but not microglia/macrophages. 3 hours after stab injury, a large number of GFP+ cells were observed infiltrating the injured retina of the Tg(mpx:GFP) zebrafish (Figure 2A,B). The number of neutrophils at the injury site rapidly decreased at 6 hpi (Figure 2A-C), and by 12 and 24 hpi, only a few neutrophils could be seen in this region (Figure 2B,C and data not shown). In contrast to the early neutrophil response, an increased presence of microglia/macrophage within the injured retina was not observed until 6 hpi (Figure 2B). Interestingly, numerous neutrophils were also observed accumulating in the space between the peripheral retina and iris (Figure 2A, white boxes) after stab injury at 3 hpi, and some appeared to migrate along the inner retinal surface toward the injury region (Figure 2A, upper left panel). After 6 hpi, neutrophil accumulation in the peripheral region decreased rapidly in a manner similar to that of the injury site (Figure 2A and data not shown). In contrast to the stab-injury paradigm, no obvious neutrophil infiltration into the retina or vitreous was found after NMDA injury or insulin injection at 3 and 6 hpi, though some neutrophils were seen in the iris (Figure 2A, arrowheads). These findings indicate that the early neutrophil response differs significantly between the stab-injury and other two models.
To investigate if the inflammation levels are comparable in the three models, RNA samples were extracted from the whole retina at different time points (0 h, 6 h, 12 h, 1 d, 2 d and 4 d), and qPCR was performed to determine the expression of typical inflammatory cytokines including il1b, il6, il11a and il11b. In the control retina, there was a relatively low induction of il1b and il6 but not il11a/11b after PBS injection (Figure 2D-G), suggesting that the injection assay itself may cause a weak inflammatory response. qPCR showed that inflammatory cytokines were strongly induced in the retinas of all three models, but their highest levels were observed following NMDA-injury (Figure 2D-G). In insulin-treated retinas, the expression levels of il1b and il11a were higher than those of the stab injury, while their levels of il6 and il11b were largely comparable (Figure 2D-G). Interestingly, qPCR also revealed that the expression of inflammatory cytokines peaked significantly earlier than the number of IB4+ cells (Figure 1F, Figure 2). Specifically, the expression of inflammatory cytokines peaked at 6 hours (NMDA and insulin groups) or 12 hours (stab-injury group). In contrast, the number of IB4+ cells peaked at 2 days in all three paradigms. This inconsistency indicates that cell subtypes or expression patterns of microglia/macrophage may vary between early and later time-points following injury or growth factor treatment.
Inflammation is essential for MGPC formation in all three paradigms
To investigate whether inflammation is required for the formation of MGPCs in each paradigm, glucocorticoid dexamethasone (Dex, 15 mg/L) was used to suppress retinal inflammation and previous studies have validated its efficacy in adult zebrafish retina[20, 22]. As it has been shown that 98% of the BrdU+ cells in the inner nuclear layer (INL) at 4 dpi are MGPCs, we quantified the number of INL BrdU+ cells in the retina with or without Dex-treatment at this time point. BrdU immunofluorescence showed that immune suppression significantly reduced the number of INL BrdU+ cells in all three paradigms (Figure 3A-F; p < 0.001). The suppression of MGPC formation after Dex treatment was not a secondary result of decreased cell death, as Dex treatment did not significantly change the number of TUNEL+ cells compared with the control (Figure S1). These results indicate that inflammation is essential for MGPC formation in all three models.
We next ask if inflammation is required for the cell cycle re-entry of MG. Since almost all of the INL BrdU+ cells are proliferating MG at 2 dpi in the stab-injury paradigm, we labeled these cells by BrdU and PCNA immunostaining at this time point. Immunofluorescence microscopy showed that Dex-treatment significantly reduced the number of INL BrdU+ or PCNA+ cells at 2 dpi (Figure 4A-C; [BrdU], p = 0.034; [PCNA], p = 0.032), suggesting that inflammation is necessary for MG proliferation. Furthermore, qPCR was performed to examine the expression of regeneration-associated genes (RAGs) and cell cycle-related genes in control and Dex-treated retina. Our results showed that Dex-treatment significantly reduced the expression of RAGs such as ascl1a, lin28a, hbegfa, igf1, socs3a and socs3b, as well as several cell cycle-related genes including ccna2, ccnb1, ccnd1, ccne1 and cdk2 (Figure 4D). Together these results suggest that inflammation regulates the expression of RAGs and cell cycle-related genes and is necessary for MG proliferation and MGPC formation.
Inflammation alone is insufficient to elicit a regenerative response in the intact retina
The above data shows an essential role of inflammation in the regenerative response in zebrafish retina in different paradigms. To investigate whether inflammation alone is sufficient to induce the reprogramming and proliferation of MG, fish received intravitreal injection of zymosan A (Zym) to activate inflammation in the intact retina. The efficacy of Zym to induce inflammation in the zebrafish CNS has been reported and our previous study also validated its capability to enhance inflammation in stab-injured zebrafish retinas. To better understand the effect of Zym-treatment on retinal cell survival and inflammation, different doses of Zym were tested, and TUNEL experiment showed that 20 and 40 µg/eye of Zym caused significant cell death in the intact retina (Figure 5A; [ONL, 20 µg], p = 0.001; [INL, 20 and 40 µg], p < 0.001 and p =0.048, respectively; [GCL, 20 and 40 µg], p = 0.004 and = 0.022, respectively). Therefore Zym doses at 10 µg/eye or lower (0.1 or 1 µg/eye) were used in the following experiments of this study. We first characterized the inflammatory time course after Zym injection. qPCR of typical inflammatory cytokines showed that intravitreal injection of 10 µg of Zym stimulated an acute inflammatory response which peaked at 12 hours in the uninjured retina (Figure 5B). Importantly, Zym injection significantly increased the number of IB4+ cells in the vitreous (Figure 5C; [0.1, 1, 10 µg], p = 0.005, < 0.001, and < 0.001, respectively), as well as the expression of inflammatory cytokines in the retina in a largely dose-dependent manner (Figure 5D). These findings suggest that Zym injection could dose-dependently induce the inflammatory response in the zebrafish retina.
We next investigated the effect of Zym injection on MG proliferation and MGPC formation in the intact retina. BrdU immunofluorescence showed that all three doses of Zym failed to induce MG proliferation or MGPC formation at 2 days and 4 days after injection (Figure 5E,F; [2 d; 0.1, 1 and 10 µg], p = 0.944, 0.959 and 0.933, respectively; [4 d; 0.1, 1 and 10 µg], p = 0.961, 0.908, and 0.716, respectively). Importantly, qPCR showed that Zym injection failed to induce the expression of key RAGs such as ascl1a, lin28a and hbegfa (Figure 5G), many of the cell cycle-related genes (Figure 5H), and critical cytokines required for MG reprogramming (Figure 5I). Zym injection did cause a significant induction of the Jak-Stat reporter socs3a (Figure 5G), suggesting that inflammation is sufficient to activate this signaling in the retina. Together these results indicate that inflammation alone is insufficient to drive MG into reprogramming and proliferation in the intact retina.
Enhancing inflammation influences MGPC formation in a context-dependent manner
As immune suppression inhibited MGPC formation, we predicted that enhancing inflammation would increase MGPC formation. Indeed, intravitreal injection of Zym (10 µg/eye) significantly increased the number of MGPCs in the stab-injury model at 4 dpi (Figure 3A,B, p < 0.001), consistent with the result from immune suppression (Figure 3A,B). Surprisingly, enhancing inflammation by intravitreous injection of the same dose of Zym following NMDA-injury or insulin-treatment significantly inhibited MGPC formation (Figure 3C-F, p < 0.001 for both groups). These results were unlikely caused by a significant change in retinal cell death, as Zym injection did not affect the number of TUNEL+ cells in the NMDA-treated retina (Figure S1), and very few TUNEL+ cells were seen in retinas of the insulin model (Figure S1). The unexpected results prompted us to further examine the influence of inflammation on MGPC formation. Since our previous data showed that the levels of typical inflammatory cytokines following NMDA-injury or insulin-treatment were higher than that of stab-injury in the retina (Figure 2), we hypothesize that Zym’s inhibitory effect on MGPC formation in these two models was caused by an excessive level of inflammation. To answer this question, various doses of Zym were used and their effects on MGPC formation in the three paradigms were examined. In the stab-injury model, Zym injection significantly increased the number of MGPCs at 4 dpi in a dose-dependent manner (Figure 6A,D; [0.1, 1, 10 µg], p = 0.012, p < 0.001 and p < 0.001, respectively). In the NMDA-injury paradigm, low doses of Zym (0.01, 0.05, 0.1 and 1 µg/eye) dose-dependently increased MGPC formation (Figure 6B,E; [0.01, 0.05, 0.1 and 1 µg], p = 0.007, 0.003, 0.006, and 0.002, respectively). However, this promoting effect vanished at the dose of 2.5 µg (Figure 6E; p = 0.415), and was reversed at the dose of 10 µg (Figure 6B,E, p < 0.001). A similar dual effect of Zym on MGPC formation was also observed in the insulin model (Figure 6C,F). These results support our hypothesis and indicate a dual role of inflammation in MGPC formation in the NMDA and insulin models.
To investigate if the effect of enhancing inflammation also depends on the degree of retinal damage, retinas were treated with different concentrations of NMDA (0.5 mM, 5 mM, or 50 mM used in the above experiments) to manipulate the extent of tissue damage. TUNEL experiment showed a dose-dependent increase of cell death in NMDA-treated retinas (Figure 7A,B; [0.5 mM vs PBS], p = 0.008; [5 mM vs 0.5 mM], p = 0.037; [50 mM vs 5 mM], p = 0.002), suggesting the degree of retinal damage is correlated with the NMDA dose. BrdU immunofluorescence showed that enhancing inflammation with 0.1 µg of Zym increased the number of MGPCs in all three NMDA concentrations (Figure 7C,D; [50, 5 and 0.5 mM], p = 0.003, p < 0.001, and p = 0.012, respectively). 10 µg of Zym significantly inhibited MGPC formation in retinas treated with 50 mM of NMDA (Figure 7C,D, p < 0.001), consistent with the data above (Figure 6E). Surprisingly, this inhibitory effect of 10 µg of Zym was completely reversed in retinas treated with 5 mM or 0.5 mM of NMDA (Figure 7C,D; [5 mM and 0.5 mM], p < 0.001 and p = 0.008, respectively). The reversal of 10 µg Zym’s effect was unlikely caused by a marked decrease in the overall background level of inflammation, as qPCR showed no significant difference in the levels of typical inflammatory cytokines between the 5 mM and 50 mM NMDA groups, and 4 out of 6 of these cytokines were also comparable between the 0.5mM and 50 mM NMDA groups (Figure 7E). These results indicate that the influence of inflammation on MGPC formation also depends on the degree of retinal damage. Taken together, these data demonstrate a complex function of inflammation following retinal injury, and indicate the influence of inflammation on MGPC formation is dependent on the injury paradigm, the level of inflammation, as well as the degree of retinal damage.
Influence of enhancing inflammation on MG reprogramming and proliferation
To further understand how enhancing inflammation influences MGPC formation, the cell cycle re-entry of MG was examined by BrdU and PCNA immunofluorescence at 2 days post injury. In the stab-injury model, Zym injection had no effect on the number of INL BrdU+ cells at 2 dpi, but caused a significant increase of INL PCNA+ cells (Figure 8A,B; [BrdU], p = 0.589; [PCNA], p < 0.001). Since it has been shown that proliferating cells in the INL at 2 dpi are MG [3, 20], this suggests enhancing inflammation promoted the entry of more MG into the cell cycle as shown by the PCNA staining at 2 dpi, but they hadn’t reached the S phase yet as indicated by the BrdU incorporation. Indeed, BrdU staining at 3 dpi showed a significant increase of INL BrdU+ cells in Zym-treated retinas (Figure 8C,D, p < 0.001), consistent with our notion. Importantly, qPCR analysis showed that Zym injection significantly increased the expression of key RAGs such as ascl1a, lin28a, hbegfa, igf1 and socs3a, as well as multiple cell cycle-related genes in the retina (Figure 4D). These results indicate that in the stab-injury paradigm, enhancing inflammation promotes MG reprograming and drives more MG from quiescence into the cell cycle, which in turn generates more MGPCs at 4 dpi.
We then investigated the effect of enhancing inflammation on MG proliferation by a low or high dose of Zym (0.1 µg or 10 µg) following NMDA injury (50 mM). In line with the aforementioned data, intravitreous injection of 10 µg Zym significantly reduced the number of INL BrdU+ or PCNA+ MG at 2 dpi (Figure 8E,F; [BrdU], p = 0.009; [PCNA], p < 0.001), suggesting a high level of inflammation prevents MG from entering the cell cycle in the NMDA model. Interestingly, no difference in MG proliferation measured by PCNA or BrdU immunofluorescence was observed between the 0.1 µg Zym group and the control at 2 dpi (Figure 8E-F, [BrdU], p = 0.097; [PCNA], p = 0.167; Figure 8G). We speculate there was a delay in MG response to Zym in the NMDA model compared with the stab injury, therefore the MG proliferation was further examined at 3 dpi. Indeed, 0.1 µg of Zym significantly increased the number of INL proliferating MG at 3 dpi (Figure 8H,I; [BrdU], p = 0.027; [PCNA], p = 0.013). To further explore if this phenomenon was caused by an increased proliferation of MG-derived progenitors, or a result of cell cycle entrance from a new population of MG, proliferating MG were labeled by a pulse of BrdU at 2 dpi, and their progeny cells were analyzed by BrdU/PCNA staining at 3 dpi (Figure 8J). In the control retina, most of the INL PCNA+ cells were also BrdU+ at 3 dpi (94.6±0.9%, Figure 8K). In contrast, a significantly lower proportion of INL PCNA+ cells at 3 dpi were BrdU+ in retinas received 0.1 µg of Zym (Figure 8K, 61.9±2.2%, p < 0.001), suggesting the increased proliferation was caused by the entry into the cell cycle of a new MG population between 2-3 dpi. Taken together, our findings indicate that enhancing inflammation following stab or NMDA injury may drive more MG into the cell cycle, but if excessive inflammation is elicited, it may also exhibit an opposite effect in the NMDA model.
Inflammation promotes the regeneration of retinal neurons in the stab-injury model
To investigate the influence of inflammation on the regeneration of retinal neurons in the stab-injury paradigm, MGPCs were labeled with a pulse of BrdU at 4 dpi, and their distribution and differentiation were examined at 30 dpi (Figure 9A). BrdU immunofluorescence showed that Dex-treatment significantly reduced the total number of BrdU+ cells in the retina at this time point, while Zym injection had an opposite effect (Figure 9A,B; [Dex], p = 0.002; [Zym], p = 0.002). Further analysis revealed a similar effect of immune manipulation on the number of BrdU+ cells in different retinal layers, though the difference was only significant in the ONL and INL (Figure 9A,C; [Dex; ONL, INL and GCL], p = 0.004, 0.007 and 0.093, respectively; [Zym; ONL, INL and GCL], p = 0.001, 0.001 and 0.118, respectively). Interestingly, the percentage of BrdU+ cells in each layer in Dex- or Zym-treated retinas was comparable to that of control (Figure 9D; [Dex; ONL, INL and GCL], p = 0.837, 0.575, and 0.389, respectively; [Zym; ONL, INL and GCL], p = 0.465, 0.478 and 0.244, respectively), suggesting modulation of inflammation had no effect on MGPC fates. To examine the effect of inflammation on MGPC differentiation, immunofluorescence of retinal cell markers Zpr1 (photoreceptors) and HuC/D (amacrine cells in the INL and RGCs in the GCL) were performed and their co-localization with BrdU signal was examined. Our results showed that immune suppression decreased, while enhancing inflammation increased the number of BrdU+/Zpr1+ cells in the ONL (Figure 9E,F; [Dex], p = 0.016; [Zym], p < 0.001) and BrdU+/HuC/D+ cells in the INL (Figure 9E,F; [Dex], p = 0.013; [Zym], p = 0.006), respectively. Neither treatment had any effect on the number of BrdU+/HuC/D+ cells in the GCL (Figure 9E,F; [Dex], p = 0.117; [Zym], p = 0.481). These findings indicate inflammation promoted the regeneration of photoreceptors and amacrine cells, but not RGCs. Together these data demonstrated that inflammation increased the MGPC population and neuronal regeneration in the stab-injured retina, without affecting the cell fate decisions of MGPCs.
Immune suppression results in the best neuronal regeneration in the NMDA-injury model
Similar to the experiments above, we also labeled the proliferating MGPCs by a pulse of BrdU at 4 dpi in the NMDA-injury paradigm, and examined the influence of inflammation on their distribution and differentiation at 30 dpi. Immunofluorescence showed that the majority of BrdU+ cells in the NMDA control group were in the ONL at 30 dpi (Figure 10A,C). As expected, enhancing inflammation by 0.1 µg of Zym resulted in significantly more BrdU+ cells (Figure 10A,B, p < 0.001), with the most obvious increase in the INL (Figure 10A,C; [ONL], p = 0.019; [INL], p < 0.001; [GCL], p = 0.655). Surprisingly, early immune suppression by Dex generated the largest number of BrdU+ cells in the retina at 30 dpi (Figure 10A,B, p < 0.001). Analysis of their distribution showed that Dex treatment significantly increased the number of BrdU+ cells in the ONL and INL, with the most dramatic change observed in the latter layer (Figure 10A,C; [ONL, INL], p < 0.001; [GCL], p = 0.085). Zpr1 and HuC/D immunofluorescence showed that while enhancing inflammation produced more retinal neurons compared with the NMDA control at 30 dpi (Figure 10D,E; [Zpr1+], p = 0.005; [INL HuC/D+], p < 0.001; [GCL HuC/D+], p = 0.047), immune suppression regenerated the highest number of photoreceptors and amacrine cells in the three groups (Figure 10D,E; [Zpr1+, INL HuC/D+], p < 0.001; [GCL HuC/D+], p = 0.095). As Dex treatment reduced the number of MGPCs in the NMDA model at 4 dpi (Figure 3), we asked how immune suppression could produce the best regeneration result. To address this question, MGPCs from the three groups were labeled with BrdU at 4 dpi, and their number and distribution were examined at 6, 14 and 30 dpi (Figure 10F, Figure S2). In the NMDA control and Zym-treated retinas, an initial increase of ONL BrdU+ cells was observed from 6-14 dpi (Figure 10F, left panel; [NMDA], p < 0.001; [NMDA+Zym], p = 0.001; Figure S2), but followed by a dramatic decrease from 14-30 dpi (Figure 10F, left panel; [NMDA], p < 0.001; [NMDA+Zym], p < 0.001; Figure S2). In contrast, there was no significant change in the number of MGPCs in the ONL in Dex-treated retina from 6-30 dpi (Figure 10F, left panel; [14 d], p = 0.132; [30 d], p = 0.129; Figure S2). In the INL, the number of BrdU+ cells in the NMDA control and Zym-treated retinas decreased dramatically from 6-14 dpi, with partial recovery in the latter group from 14-30 dpi (Figure 10F, middle panel, p < 0.001 for both groups; Figure S2). In contrast, there was an initial increase of INL BrdU+ cells from 6-14 dpi, followed by a slight decrease from 14-30 dpi in Dex-treated retinas (Figure 10F, middle panel; [14 d], p = 0.044; [30 d], p = 0.128; Figure S2). In the GCL, a gradual but significant decrease of BrdU+ cells was found in all three groups during this time period (Figure 10F, right panel; [NMDA; 14 d, 30 d], p < 0.001; [NMDA+Zym; 14 d, 30 d], p = 0.004 and p < 0.001, respectively; [NMDA+Dex; 14 d, 30 d], p = 0.135 and 0.013, respectively; Figure S2). Taken together, these results indicate a dramatic loss of MGPCs from 6-30 dpi in the NMDA control and Zym-treated retinas, presumably due to cell death. Importantly, immune suppression prevented MGPC depletion in the ONL and INL, resulting in the best neuronal regeneration in the NMDA paradigm.