Retinal Ganglion Cell Survival and Axon Regeneration After Optic Nerve Injury in Naked Mole-Rats

Abstract

In the adult mammalian central nervous system (CNS), axonal damage often triggers neuronal cell death and glial activation, with very limited spontaneous axon regeneration. In this study, we performed optic nerve injury in adult naked mole-rats, the longest living rodent, with a maximum life span exceeding 30 years, and found that injury responses in this species are quite distinct from those in other mammalian species. In contrast to what is seen in other mammals, the majority of injured retinal ganglion cells (RGCs) survive with relatively high spontaneous axon regeneration. Furthermore, injured RGCs display activated signal transducer and activator of transcription-3 (STAT3), whereas astrocytes in the optic nerve robustly occupy and fill the lesion area days after injury. These neuron-intrinsic and -extrinsic injury responses are reminiscent of those in “cold-blooded” animals, such as fish and amphibians, suggesting that the naked mole-rat is a powerful model for exploring the mechanisms of neuronal injury responses and axon regeneration in mammals.

Dependent on species, different responses occur following central nervous system (CNS) injury (Clemente, 1964; Larner et al., 1995; Tanaka and Ferretti, 2009). This might be best documented with intraorbital optic nerve injury (Sperry, 1948; Scalia et al., 1985; Beazley et al., 1986; Dunlop et al., 2000, 2004; Goldberg and Barres, 2000). For example, in anamniotes such as fish, almost all retinal ganglion cells (RGCs) survive, and most injured axons are able to regenerate (Murray, 1982; Becker and Becker, 2014). In amphibians and reptiles, approximately 50-70% of RGCs survive, with different extents of axon regeneration (Stelzner et al., 1986; Dunlop et al., 2004; Tanaka and Ferretti, 2009). However, markedly different outcomes are seen in mammals; massive neuronal death occurs, with only 10-20% of RGCs surviving at 2-4 weeks postinjury, and very limited spontaneous axon regeneration takes place (Berkelaar et al., 1994).

The naked mole-rat (Heterocephalus glaber) is an extraordinary mammalian species (Buffenstein and Jarvis, 2002). They are eusocial mammals that live in large subterranean colonies with a very strict social and reproductive hierarchy. Although naked mole-rats are approximately the size of mice, their maximum life span exceeds 30 years (compared with 2-3 years for traditional laboratory rodents), and they display negligible senescence (Buffenstein, 2008). They are well adapted for the limited availability of oxygen in their environment and thus possess unusual hypoxia resistance (Larson and Park, 2009). In addition, unlike other mammals, their body temperature matches ambient temperatures, thus meeting criteria for classification as a poikilothermic mammal (Buffenstein and Yahav, 1991). These unusual physiological properties make naked mole-rats an interesting model for exploring their injury responses in CNS neurons. In this study, we performed optic nerve injury in young adult naked mole-rats and analyzed both RGC loss and axon regeneration.

MATERIALS AND METHODS

Animals, optic nerve crush, and intravitreal injection

All experimental procedures were performed in compliance with animal protocols approved by the University Animal Care Committee at University of Toronto and IACUC at University of Miami. For all surgical procedures, young adult naked mole-rats (6-12 months old) were anesthetized with Avertin (30 mg/100 g; Sigma, St. Louis, MO). Adult C57BL/6J mice (IMSR catalog No. JAX:000664; RRID:IMSR_JAX:000664; 6-8 weeks old) were anesthetized with a ketamine and xylazine mixture. Buprenorphine (0.05 mg/kg, Bedford Laboratories, Bedford, MA) was administered as postoperative analgesic. For optic nerve crush injury, the left optic nerve was exposed intraorbitally and crushed with forceps for 10 seconds at 1 mm behind the optic disc. For intravitreal injection, a glass micropipette was inserted into the peripheral retina, just behind the ora serrata, and was deliberately angled to avoid damage to the lens. Two days before dissection, approximately 0.5 μl (for naked mole-rat) or 2-3 μl (for mouse) of cholera toxin β subunit (CTB)-Alexa 555 (2 μg/μl, Invitrogen, Carlsbad, CA) was injected into the vitreous with a Hamilton syringe (Hamilton) to label regenerating RGC axons anterogradely.

Tissue preparation

Animals were perfused transcardially with PBS, followed by 4% paraformaldehyde (PFA) at 5 ml/minute. The optic nerve and retinas were dissected and post-fixed with 4% PFA for approximately 2 hours. For histological sectioning, samples were cryoprotected by incubating in 30% sucrose overnight. For whole-mount staining, postfixed retinas were kept in 0.1 M PBS at 4°C until processed for staining.

Immunohistochemical staining of viable RGCs

Whole retinas were immunostained with an antibody to neuronal class βIII tubulin (TUJ1; BioLegend, San Diego, CA; catalog No. 801202; RRID:AB_10063408). TUJ1 staining in retinal whole mounts was shown to be adult RGC specific and was efficient for RGC identification (Cui et al., 2003; Robinson and Madison, 2004). Retinas were dissected in 0.1 M PBS solution. After three 10-minute PBS washes, retinas were blocked in 5% normal goat serum and 0.2% Triton X-100 (Sigma) for 1 hour and then incubated in the same medium with TUJ1 antibody (1:1,000) overnight at 4°C. After additional washes, retinas were incubated with goat anti-rabbit antibody (Thermo Fisher Scientific, Fair Lawn, NJ; catalog No. A-11008; RRID:AB_143165, 1:500 dilution;) for 1 hour at room temperature. Retinas were again flat mounted onto slides and coverslipped in Fluoromount-G. For RGC counting, 20-25 fields (mouse) or 10-12 fields (naked mole-rats) were randomly sampled from central, intermediate, and peripheral regions per retina to estimate RGC survival.

Immunohistochemical staining of retinal and optic nerve sections

Retinal cross-sections (10-16 μm in thickness) or longitudinal optic nerve sections (10 μm) were blocked in 5% normal goat serum and 0.2% Triton X-100 for 1 hour. Sections were incubated with the following primary antibodies: rabbit caspase-3 (Cell Signaling Technology, Bedford, MA; catalog No. 9662; RRID:AB_10694681, 1:1,000 dilution); mouse or rabbit anti-TUJ1 (Covance, Berkeley, CA; 1:1,000 dilution); rat antiglial fibrillary acidic protein (GFAP; Invitrogen; catalog No. 13-0300; RRID:AB_2532994, 1:2,000 dilution); phospho-STAT3 (Y705; CST; catalog No. 9145; RRID:AB_561305; 1:100 dilution) overnight at 4°C and washed three times for 15 minutes each with PBS. Secondary antibodies (Jackson Immunoresearch, West Grove, PA; catalog No. A-11006; RRID:AB_2534074; catalog No. A-11008; RRID:AB_143165, catalog No. A-21434; RRID:AB_2535855, 1:500 dilution) were then applied and incubated for 1 hour at room temperature. Sections were again washed three times for 15 minutes each with PBS before a coverslip was attached with Fluoromount-G.

Caspase-3 and p-STAT3 quantification

TUJ1⁺ cells in the ganglion cell layer were counted under a fluorescent microscope in four retina crosssections from each animal. In the same sections, TUJ1⁺ cells with nuclear staining of caspase-3 or phospho-STAT3 (Y705) were identified and counted with the criterion that caspase-3 or p-STAT3 signal was completely surrounded by TUJ1⁺ signal, and confirmed with DAPI nuclear staining signal when necessary.

Antibody characterization

See Table 1 for a list of all antibodies used. TUJ1 antibody recognized a single band of 50 kD m.w. on Western blots of rat brain (manufacturer’s data sheet) and stained a pattern of cellular morphology and distribution in the mouse retina identical to that in previous reports (Cui et al., 2003; Park et al., 2008; Yungher et al., 2015). GFAP antibody stained a pattern of cellular morphology and distribution in the optic nerve identical to that in previous reports (Park et al., 2008). STAT3 antibody recognized only the two expected isoform bands on Western blots of mouse brain, and no immunohistochemical signal was seen in STAT3 knockout animal tissues (unpublished observation). Caspase-3 antibody shows expected uncleaved and cleaved bands on Western blot analysis of extracts from Jurkat cells, untreated or etoposide-treated (25 μM, 5 hours), and NIH/3T3 cells, untreated or staurosporine-treated (manufacturer’s data sheet).

TABLE 1.

Antibodies Used

Antigen	Description of immunogen	Source, host species, catalog No., RRID	Concentration used (μg/ml)
Tubulin III 3 (TUJ1)	Recognizes an epitope located within the last 15 C-terminal residues	BioLegend, mouse monocloncal, 801202, RRID:AB_2564640	1
Stat3, phospho- (Tyr705)	Synthetic phosphopeptide corresponding to residues surrounding Tyr705 of mouse Stat3	Cell Signaling Technology, rabbit monoclonal, RRID:AB_561305	2
GFAP	Enriched bovine glial filaments	Thermo Fisher Scientific, rat monoclonal, RRID:AB_2532994	0.25
Caspase-3	Synthetic peptide corresponding to residues surrounding the cleavage site of human caspase-3	Cell Signaling Technology, rabbit polyclonal, RRID:AB_10694681	0.1

Optic nerve processing and axon regeneration index

Optic nerves from perfused naked mole-rats were carefully dissected out and postfixed in 4% PFA for 1 hour at room temperature. For mole-rats, anterograde CTB labeling along the optic nerve was observed in whole mounts. Care was taken in the mounting procedure to avoid drying of the tissue. Preparations were observed with an epifluorescence microscope (Olympus). Because a mole-rat optic nerve is extremely thin, this procedure allowed all CTB labeled axons to be visualized in whole mounts. Individual axons that regenerated to 0.2 mm were manually counted in the whole-mount optic nerve. For mouse optic nerves, the total number of CTB labeled axons per optic nerve was calculated from axon counts in sections. Specifically, for each animal, the number of CTB labeled axons was estimated by counting the number of CTB-labeled fibers extending 0.2 mm from the beginning of the crush site in four or five sections per animal. The cross-sectional width of the nerve was measured at the point at which the counts were taken and was used to calculate the number of axons per millimeter of nerve width. The number of axons per millimeter was then averaged over all sections. Σad, the total number of axons extending distance d in a nerve having a radius of r, was estimated by summing over all sections having a thickness t (8 μm): Σad = πr2 × [average axons/mm]/t. Axon regeneration index is the ratio of the total number of axons at 0.2 mm from the lesion start/the total number of surviving RGCs in the same animal (i.e., TUJ1⁺ cells in the retina) at 4 weeks after optic nerve crush.

Statistical analysis

Data are presented as means ± SD. We used ANOVA and Bonferroni post hoc test for multiple comparisons, and Student t-test for two group comparisons.

RESULTS

Naked mole-rats are only slightly larger than mice, but they exhibit remarkable physiological features. Here we sought to compare injury responses in naked mole-rats and mice, the latter regularly serving as a control species in naked mole-rat studies (Kim et al., 2011; Yu et al., 2011; Azpurua et al., 2013). To this end, we sought to compare injury responses in mice and naked mole-rats after optic nerve injury. Consistently with previous results (Berkelaar et al., 1994; Park et al., 2008), at 2 and 4 weeks postaxotomy, approximately 20% and 15% of RGCs survive in young adult mice, respectively (Fig. 1A,B). Remarkably, however, in young adult naked mole-rats, approximately 70% and 60% of RGCs survived at 2 and 4 weeks postinjury, respectively (Fig. 1A,B). Notably, we observe that the RGCs in both mice and naked mole-rats show noticeable reduction in soma size after injury. Although we have not analyzed the RGC survival at later time points, high survival rates seen at 2 and 4 weeks after injury suggested that, unlike those in other mammals, the RGCs in naked mole-rats possess an unusual ability to deal with stress conditions such as axotomy.

Figure 1.

RGCs are highly resistant to axotomy-induced cell death in naked mole-rats. A: Representative images of uninjured (intact) retinas and retinas of mouse and naked mole-rat (NMR) at 14 or 28 days postcrush (dpc) after whole-mount TUJ1 staining. B: Quantification of RGC survival as shown in A. Quantification of surviving RGCs is represented as percentage of TUJ1⁺ RGCs compared with that in the uninjured contralateral retinas. In comparison with RGC survival in adult mice, axotomized RGCs in naked mole-rats show significantly higher RGC survival. N = 4 per group. *P < 0.05, two-way repeated measures ANOVA followed by Bonferroni posttest. Error bars, SD. C–E: Levels of caspase-3 immunoreactivity in intact retina and retina at 3 days following axotomy in mice (C) and naked mole-rats (E). D: Quantification of caspase-3⁺ RGCs. N = 3 per group.*P < 0.01, two-way repeated measures ANOVA followed by Bonferroni posttest. Error bars, SD. Scale bars = 20 μm.

In most mammals, death of RGCs following axonal injury is mediated predominantly by apoptosis, as indicated by activation of caspase-3 (Berkelaar et al., 1994; Cui et al., 2003). Given the high survival rates of RGCs in naked mole-rats, we performed immunohistochemistry to examine the levels of active caspase-3 in RGCs at 3 days postinjury. Although axotomy led to caspase-3 activation in many mouse RGCs (i.e., 11% of RGCs), less than 2% of RGCs showed caspase-3 activation in naked mole-rats (Fig. 1D,E). These data suggest that, at least at acute postinjury stages, the apoptotic pathways are only modestly activated in the majority of RGCs in the naked mole-rats, consistent with the limited postinjury RGC loss in these animals.

Because failure of axon regeneration after injury has been considered a hallmark of the adult mammalian CNS, we examined spontaneous axon regeneration responses in naked mole-rats. Naked mole-rats underwent intraorbital crush injury. Two days prior to death, animals received intravitreal CTB injection to anterogradely label regenerating axons. At 2 weeks postinjury, some CTB-labeled regenerating axons are found in the optic nerves distal to the lesion (Fig. 2A), and some CTB-labeled axons had enlarged growing tips, indicative of actively extending growth cones (Fig. 2B). At 4 weeks postinjury, the number of axons that regenerated 0.1 mm and 0.2 mm from the lesion continued to increase but was not significantly higher than that at 2 weeks postinjury (Fig. 2C). Some axons formed many branches in the optic nerve, another key feature of regenerating axons (Fig. 2B; Luo et al., 2013; Pernet et al., 2013). However, although mice have approximately 25-fold more RGCs compared with naked mole-rats (Nemec et al., 2008), spontaneous optic nerve regeneration after crush injury is rare in mice (Fig. 2D). As for comparison, the percentage of surviving RGCs that regenerate axons in mole-rats is approximately threefold higher than that of mice (Fig. 2D,E).

Figure 2.

Axon regeneration in naked mole-rats. Representative optic nerves from naked mole-rats showing CTB-labeled axons at 14 and 28 days postcrush (dpc) in A and B, respectively. A′,B′: Higher magnification of the boxed areas in A,B. Arrow indicates axonal tip reminiscent of a growth cone; arrowhead indicates signs of axonal branching. C: Quantification of axon regeneration represented as total number of CTB-labeled axons seen per whole-mount optic nerve at approximately 0.1 and 0.2 mm from the lesion site. N = 4 per group. D: Representative optic nerve section taken from a mouse at 28 days following an optic nerve crush. E: Axon regeneration index. This index is the ratio of the total number of axons at 0.2 mm from the lesion start/the total number of surviving RGCs in the same animal (i.e., TUJ1⁺ cells in the retina) at 4 weeks after optic nerve crush. There was a significantly higher portion of surviving RGCs regenerate axons in mole-rats compared with mice. Asterisks indicate lesion sites. N = 4 per group. *P < 0.05, unpaired Student t-test. Error bars, SD. Scale bars = 50 μm.

We next attempted to explore possible mechanisms underlying such high neuronal survival and spontaneous axon regeneration in naked mole-rats. Previous studies suggested that neuronal mTOR activity is an important indicator of intrinsic regenerative ability in mice (Park et al., 2008). However, available antibodies failed to detect signals of the mTOR activity marker p-S6 and other signaling molecules in the mTOR pathway in sectioned retinae from naked mole-rats. Another intracellular pathway important for cell survival and axon regeneration is the JAK/STAT pathway (Smith et al., 2009; Sun et al., 2011; Elsaeidi et al., 2014). In particular, phosphorylation and activation of STAT3 leading to nuclear translocation has been shown to be a key event for neural repair in different model systems, including mammals and zebrafish (Bareyre et al., 2011; Sun et al., 2011; Leibinger et al., 2013; Elsaeidi et al., 2014). Immunohistochemistry with an antibody against p-STAT3 shows virtually no p-STAT3 immunoreactivity in RGC nuclei in intact mice and at 3 days following crush injury. On the other hand, many RGCs expressed nuclear p-STAT3 after injury in naked mole-rats (Fig. 3A,B), suggesting that the JAK/STAT pathway might be differently regulated in injured RGCs in mice and naked mole-rats.

Figure 3.

STAT3 activation in injured RGCs of naked mole-rats. A: Signals detected with TUJ1 or anti-p-STAT3 antibodies in the retinal sections from mice and naked mole-rats at 3 days postcrush. B: Percentage of RGCs with nuclear phospho-STAT3 signals. N = 4 per group. *P < 0.001, **P < 0.0001, two-way repeated measures ANOVA followed by Bonferroni posttest. Error bars, SD. Scale bar = 50 μm.

In addition to neuron-intrinsic factors, extrinsic factors might also play an important role in axon regeneration after injury (Shearer and Fawcett, 2001; Silver and Miller, 2004). We thus compared temporal and spatial glial responses in the optic nerve of naked mole-rats and mice by performing immunohistochemistry with an antibody against GFAP, a marker for reactive astrocytes. At 3 days after crush, GFAP immunoreactivity pattern is similar in mice and mole-rats (intense around the lesion site) but is markedly less pronounced in the lesion epicenter (data not shown), indicating death and loss of astrocytes and their processes within the lesion at this stage of injury. However, at 14 days postinjury, distinct patterns are seen in these two species. In contrast to the large absence of GFAP immunoreactivity in the lesion site of mice, intense GFAP immunoreactivity could be detected in the lesion of mole-rats (Fig. 4A,B). These results suggest that glial cells in the CNS of mole-rats possess injury responses that are quite distinct from those of other mammalian species; unlike the case in mice, astrocytes and their processes in naked mole-rat optic nerve heavily occupy the lesion epicenter, which might be one contributing factor to the observed incident of spontaneous axon regeneration.

Figure 4.

Astrocytes fill the lesion in naked mole-rats. A: Optic nerve sections taken from mouse at 14 days postcrush, showing CTB labeling and GFAP immunostaining. B: Optic nerve sections taken from naked mole-rat at 14 days postcrush showing CTB labeling and GFAP immunostaining. Asterisks, lesion core. Scale bars = 50 μm.

DISCUSSION

The naked mole-rat possesses sets of biological and physical traits that make it extremely unique among mammals. It is a eusocial mammal with very low metabolic and respiratory rates. In addition, it is extremely resistant to cancer and is the longest living rodent. Our results here suggest that high neuronal tolerance to axotomy is an additional unusual biological feature of naked mole-rats. Importantly, such responses are similar to what is seen in certain reptilian and amphibian species, in which approximately 70% of RGCs survive after optic nerve injury (Stelzner et al., 1986; Dunlop et al., 2004; Becker and Becker, 2014). In addition, we observed higher degrees of spontaneous axon regeneration in naked mole-rats compared with mice. An obvious similarity between the naked mole-rat and reptiles as well as amphibians is that these are “cold-blooded” or poikilothermic animals. Thus, it is possible that these animals have evolved unusual stress responses as a consequence of their unique ecological niche.

We observed that RGC survival in naked mole-rat is relatively high at 14 and 28 days after injury. However, one important question that remains is whether RGC survival is maintained at later time points in naked mole-rat. It is possible that most RGCs in naked mole-rats eventually die later, but the fact that they are tolerant initially, with it taking much longer for them to die after injury, is intriguing. Cell death after axon injury is said to be triggered by multiple factors (including lack of retrograde trophic support, mitochondrial defect, reactive oxygen species stress, and endoplasmic reticulum stress, ultimately leading to caspase cascades. The extent and how these and other injury events occur and are managed in the naked mole-rat RGCs remain to be examined.

In our attempts to examine mechanisms underlying tolerance to injury and ability to regenerate axons in this species, we used several antibodies and performed immunohistochemistry on retina and optic nerve sections. Most antibodies, including those against endoplasmic stress markers (i.e., CHOP) and injury-response genes (i.e., DLK1, ATF3, and c-JUN), failed to detect clear signals in the mole-rat RGCs, possibly because of lack of antibody specificity for mole-rats. We have also tested antibodies against RHEB and p-S6, which are associated with activation of mTORC1 and axon regeneration (Park et al., 2008; Wu et al., 2015). However, we did not see obvious p-S6 and RHEB expression in naked mole-rat retinal sections using a standard staining protocol (Park et al., 2008). These data with the pS6 antibody suggest that mTORC1 might not play a role in RGC survival and axon regeneration in naked mole-rat. However, we cannot exclude the possibility that mTORC1 in fact is present in naked mole-rat RGCs but at low levels (i.e., at levels not detected via immunohistochemistry) and contributes to RGC survival and regeneration. Nonetheless, among the markers we examined, we found that the activated form of STAT3 (i.e., phosphorylated at the tyrosine 705 residue) is highly expressed in injured mole-rat RGCs. The level of expression of phosphorylated or activated STAT3 is very low in neurons of mammals after injury (Sun et al., 2011; Leibinger et al., 2013). In contrast, a previous study has demonstrated a considerable increase in p-STAT3 level as well as its potential activator components, including the IL-6 family of cytokines, in zebrafish RGCs following axotomy (Elsaeidi et al., 2014). Thus, it seems that in mole-rats the JAK/STAT3 pathway activation resembles that in zebrafish, which could contribute to enhanced RGC survival and axon regeneration. Future studies will be aimed to analyze other aspects of intrinsic regenerative regulation.

In mammals, damage to the CNS results in the formation of cysts and fibrotic scars that block axon regeneration with limited formation of permissive glia bridges across the lesion (Soderblom et al., 2013; Cregg et al., 2014). In contrast, glial cells in zebrafish and newts robustly infiltrate the injury site and form a permissive environment for axon growth (Zukor et al., 2011; Becker and Becker, 2014). For mole-rat optic nerves, we observed striking differences in astrocyte occupancy in the lesion compared with mice. Taken together, our results suggest that, after axonal injury, mole-rats possess some aspects of neuron-intrinsic and -extrinsic features that are similar to those of cold-blooded animals. Recent efforts to decode the whole genome sequence of the naked mole-rat (Kim et al., 2011; Keane et al., 2014) will allow further characterization of the molecular mechanisms underlying their unusual properties, which should provide important insights for developing strategies for promoting neuronal survival and axon regeneration in mammals.