Background
Optic neuropathies, of which glaucoma is the most common, are characterized by axonal degeneration in the optic nerve and apoptotic death of retinal ganglion cell (RGC) somas, leading to irreversible vision loss [
1]-[
3]. While the exact mechanisms that initiate RGC injury have not been clearly established, animal models of elevated intraocular pressure, axotomy, and optic nerve crush mimic the apoptotic pathways observed during glaucomatous neurodegeneration [
4]-[
9]. Although direct axonal injury ultimately leads to RGC somatic death, resident innate immune cells have long been suspected of playing a critical role during glaucoma [
10]-[
15]. Dendritic cells have been shown to infiltrate the damaged retina after crush injury [
16]; however, it is the retinal glial cells, specifically macroglia (astrocytes and Müller cells) and microglia that comprise the principal populations of resident immune cells in the retina. Under normal circumstances these cells maintain retinal health, but after an injury they undergo an activation response to behave as innate immune cells by presenting antigens and releasing cytokines and other small molecules into the retinal tissue [
17]-[
19]. These signals initiate damage repair and remove critically injured neurons [
15],[
20]; however, the effect of prolonged glial activation on RGC survival continues to be debated. Some research suggests that the innate immune response is critical for RGC protection after injury [
21],[
22], while research in stroke and ischemia models demonstrate greater neuronal loss from activated glia [
14],[
23],[
24]. More specifically in the latter paradigm, glial activation is thought to cause a second wave of RGC loss, termed secondary degeneration [
7],[
11],[
25].
The model of secondary degeneration proposes that ganglion cell death during glaucoma occurs in two waves: first, that axonal injury culminates in the death of a subset of RGCs and the activation of retinal glia; and second, that the activated glia then produce cytotoxic molecules, such as inflammatory cytokines, that critically damage surviving RGCs [
7],[
11],[
25]-[
27]. It has been hypothesized that these cytokines are generated from either macroglia, principally Müller cells [
28], or microglia [
20], or both. Supporting evidence for this model comes from studies showing that minocycline, a broad spectrum anti-inflammatory drug, protects RGCs against optic nerve axotomy, experimental glaucoma, and optic nerve crush [
7],[
29]-[
31], implicating a damaging role for the immune response after injury. While many inflammatory cytokines have been linked to RGC degeneration [
21],[
32],[
33], TNFα has been consistently associated with glaucomatous neuropathy [
10],[
11],[
23],[
34]-[
37].
TNFα is a pro-inflammatory cytokine that is elevated in several neurological diseases including multiple sclerosis [
33], Alzheimer’s disease [
38] and ischemia [
24]. It is generated in the retinas of human glaucoma patients [
35] as well as animal models of retinal injury [
11],[
23],[
28],[
36],[
39]-[
42]. Additionally, the receptors through which TNFα signals, TNFα receptor 1 (TNFR1) and TNFα receptor 2 (TNFR2), are also upregulated after retinal injury [
35],[
43],[
44]. Isolating TNFα from the complex degenerative signaling pathways activated by RGC injury has yielded conflicting results about the role of this cytokine in RGC damage. TNFα is thought to contribute to RGC pathology following NMDA injection and optic nerve crush, which respectively cause RGC death within hours to days [
5],[
7],[
45]; yet an intraocular injection of TNFα requires 2 weeks to cause axonal injury and 8 weeks before RGC somatic loss is significant [
10],[
36],[
46]. Although TNFα injection does ultimately result in RGC loss, the disconnect in the timing of RGC damage suggests that TNFα may not simply flip a switch initiating degeneration, but may instead trigger a cascade of signaling networks that indirectly culminate in neuronal damage over time.
A possible explanation for this disconnect may be the opposing roles for TNFR1 and TNFR2 [
43],[
47],[
48]. In human glaucoma, TNFR1 has been linked with the upregulation of pro-apoptotic proteins including BAX and CASP1 [
37], and TNFR1 deficiency protected neuronal cell cultures from glutamate excitotoxicity [
47], and increased RGC survival in a mouse model of optic nerve crush [
11],[
37]. Conversely, TNFR2 deficiency increased neuronal susceptibility to glutamate [
47], and caused greater RGC loss in a mouse model of ischemia/reperfusion [
43]. Given that TNFα appears to play an important role during retinal injury, there is a clear need to better understand through which pathway(s) this cytokine is signaling. The present study investigates further the role of TNFα in the pathology of RGCs after optic nerve damage in mice. After optic nerve crush we detected a modest increase in
Tnfα gene expression. Experimental evidence suggests that this inflammatory cytokine may have a protective role early in the RGC death process.
Materials and methods
Animals
Adult C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, USA) were handled in accordance with the Association for Research in Vision and Ophthalmology statement on the use of animals in research. All experimental protocols and the ethical care of the mice were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Wisconsin. Mice were housed in microisolator cages and kept on a 12-hour light/dark cycle and maintained on a 4% fat diet (8604 M/R; Harland Teklad, Madison, WI, USA).
Bax-deficient mice were generated from breeding
Bax
+/-
animals on a C57BL/6J background.
Tnfα
-/-
mice were obtained from the Jackson Laboratory and as a gift from Dr Matyas Sandor at the Univeristy of Wisconsin. NFΚB expression was monitored with
cis-NFΚB
EGFP reporter mice [
49] that were obtained from Dr Christian Jobin at the University of North Carolina. All genotypes were on the C57BL/6J background.
Optic nerve crush surgery
Prior to surgery, mice were anesthetized with ketamine (120 mg/kg) and xylazine (11.3 mg/kg) and the eye numbed with a drop of 0.5% proparacaine hydrochloride (Akorn, Lake Forest, IL, USA). Optic nerve crush surgery was performed as previously described [
5],[
9]. Briefly, a lateral canthotomy was performed followed by an incision through the conjunctiva at the limbal junction. The sclera was cleared of excess tissue before the optic nerve was exposed using self-closing N7 forceps (Fine Science Tools, Foster City, CA, USA), and clamped for 3 seconds. After surgery, the eye was covered with triple antibiotic ointment, and a subcutaneous injection of buprenex (0.2 mg/kg) was delivered to alleviate pain. Surgery was not performed on the right eye of each mouse, as previous studies have shown that mock surgery does not affect ganglion cell morphology or number [
50],[
51].
Intraocular injections
Mice were anesthetized with ketamine/xylazine and a drop of proparacaine was applied to numb the eye. A small hole was made through the conjunctiva and scleral tissue with a 30G needle, and then a 30G beveled Nanofil needle attached to a Nanofil syringe (World Precision Instruments, Inc., Sarasota, FL, USA) was inserted through the hole and a 2 μl volume of either 50 ng or 100 ng TNFα (Sigma, St Louis, MO, USA) was slowly delivered to the vitreous over 60 seconds. Care was taken not to damage the lens. After delivery, the needle was held in the eye for an additional 30 seconds before being retracted. A subcutaneous injection of buprenex was delivered to alleviate pain and the mouse was allowed to recover.
RNA isolation and quantitative analysis of mRNA expression by quantitative PCR
Mice were euthanized with a lethal overdose of pentobarbital sodium prior to tissue harvest. Retinal tissue was collected and flash frozen on dry ice. At least three retinas were analyzed for each condition tested. Total RNA was isolated from the tissue using a solution of 50% phenol containing 1.67 M guanidine thiocyanate, 14.3 mM sodium acetate, 10.4 mM sodium citrate, 0.3% β-mercaptoethanol, and 0.005% Sarkosyl. Retinal tissue was sonicated in 1 ml of the phenol solution with 10 pulses at 50% power using a Branson Sonifier SLPe Energy Cell Disruptor (All-Spec Industries, Willmington, NC, USA). The RNA was then extracted with chloroform and precipitated with isopropanol. The pellet was washed in 70% ethanol and dried before being resuspended in DEPC-treated water (Fisher Scientific, Waltham, MA, USA). The total RNA concentration was determined using a BioPhotometer (Eppendorf, Hamburg, Germany). A DNase treatment with DNase I (Promega, Madison, WI, USA) was then performed on 4 μg of RNA to eliminate contaminating genomic DNA. The DNase-treated RNA samples were extracted with phenol and chloroform, and precipitated with ethanol. The pellet was washed with 70% ethanol and dried before being resuspended in DEPC-treated water (Fisher Scientific). Finally the RNA was converted to cDNA with oligo(dT) 15 primers and Moloney murine leukemia virus reverse transcriptase (Promega).
The cDNA samples were then diluted and 100 ng was analyzed by quantitative PCR (qPCR) for changes in gene expression of
Aif1,
Gfap, Nrn1,
Sncg,
Tnfα and
S16 ribosomal protein mRNA. The cDNA was added to diluted SYBR Green PCR master mix (Applied Biosystems, Grand Island, NY, USA) with 0.25 μm of each primer in a 20 μl reaction volume. Each cDNA sample was run in triplicate on an ABI 7300 Real Time PCR system (Applied Biosystems), superimposed on a standard curve to determine absolute transcript quantities, and normalized to
S16. Cycling conditions were 95°C (15 seconds) and 60°C (60 seconds) for 40 cycles with a dissociation step. Primer sequences are listed in Table
1.
Table 1
Quantitative PCR primer sequences
Aif1
| Forward: AGAGAGGTGTCCAGTGGC | 200 |
Reverse: CCCCACCGTGTGACCTCC |
Gfap
| Forward: CAAACTGGCTGATGTCTACC | 269 |
Reverse: AGAACTGGATCTCCTCATCC |
Nrn1
| Forward: TTCACTGATCCTCGCGGTGC | 238 |
Reverse: TACTTTCGCCCCTTCCTGGC |
Sncg
| Forward: GACCAAGCAGGGAGTAACGG | 240 |
Reverse: TCCAAGTCCTCCTTGCGCAC |
Tnfα
| Forward: CGCGACGTGGAACTGGCAGAA | 276 |
Reverse: GTGGTTTGCTACGACGTGGGCT |
S16
| Forward: CACTGCAAACGGGGAAATGG | 198 |
Reverse: TGAGATGGACTGTCGGATGG |
Cell counts from retinal whole mounts
After euthanasia the superior portion of the eye was marked with a cautery, and then the whole eye was enucleated and fixed in 4% paraformaldehyde. After 50 minutes, the eye was rinsed in PBS and the anterior segment removed to create an eye cup. The retina was removed from the eye cup and placed with the ganglion cell layer (GCL) facing up onto a Superfrost Plus slide (Fisher Scientific); three additional relaxing cuts were made to allow the retina to lay flat. The whole mounts were stained with 300 ng/ml 4',6-diamidino-2-phenylindole (DAPI; Fisher Scientific) and then thoroughly rinsed in PBS before being covered with Immu-mount (Fisher Scientific), coverslipped and stored at 4°C in the dark. Images were captured at 400× from all around the periphery of the retina, and nuclear counts were obtained from 24 distinct fields (120 μm
2) for each retina and averaged together. Only rounded nuclei with at least one nucleolus, typical of both RGCs and amacrine cells in this layer, were included in the counts. Endothelial cells exhibiting elongated nuclei and no nucleolus, and densely staining astrocytes were excluded [
52]. The GCL cell counts for each experimental retina were compared to the cell counts for the corresponding contralateral retina using the following formula to yield a percent change: [(cell count experimental) - (cell count control)]/(cell count control) × 100. Retinal ganglion cells represent about 50% of the GCL population [
53]. It should be noted that while
Bax -/- mice have twice as many neurons as wild-type mice, the RGCs still represent about 50% of the GCL population [
54].
Immunofluorescent labeling
Whole eyes were fixed in 4% paraformaldehyde before the anterior segment was removed to create an eye cup. The eye cups were then rinsed in PBS, post-fixed overnight in 0.4% paraformaldehyde, and equilibrated in 30% sucrose in PBS. The eye cups were embedded in optimal cutting temperature compound (Fisher Scientific) in blocks and frozen on dry ice. Frozen sections were cut at 10 to 14 μm. Slides were rinsed in PBS and then blocked in 0.2% Triton-X, 1% BSA, and 5% donkey serum in PBS for 1 hour at room temperature. Primary antibodies (see Table
2) were incubated overnight at 4°C in PBS containing 1% BSA. Slides were thoroughly rinsed in PBS and incubated in Texas Red-conjugated or FITC-conjugated secondary antibodies (Jackson ImmunoResearch, Inc., West Grove, PA, USA) in the dark for 2 hours at room temperature in PBS containing 1% BSA. Slides were thoroughly rinsed in PBS before being incubated with 300 ng/ml DAPI for 5 minutes at room temperature. Finally, the slides were rinsed in PBS and coverslipped with Immu-Mount and stored at 4°C in the dark.
Table 2
Primary antibodies
Allograft inflammatory factor 1 | AIF1 | Polyclonal rabbit | 1:1000 | WAKOa
| 019-19741 |
BRN3A | BRN3A | Monoclonal mouse | 1:50 | Milliporeb
| MAB1585 |
Caspase 3 | CASP3 | Polyclonal rabbit | 1:1000 | R&Dc
| AF835 |
Glial fibrillary acidic protein | GFAP | Polyclonal rabbit | 1:1000 | DAKOd
| Z0334 |
JUN | JUN | Polyclonal rabbit | 1:1000 | Abcame
| Ab40766 |
Transcription factor SOX-9 | SOX9 | Polyclonal rabbit | 1:1000 | Millipore | AB5535 |
Tumor necrosis factor alpha | TNFα | Polyclonal goat | 1:100 | R&D | AF-410-NA |
Whole mounts labeled with BRN3A were stained as previously described by Nadal-Nicolas and colleagues [
55], with minor modifications. Briefly, following fixation of the globe, the anterior segment was removed and the eye cups were incubated in PBS containing 0.5% Triton-X100 and 2% donkey serum (Jackson ImmunoResearch, Inc.) for 1.5 hours at room temperature. They were then transferred into the same buffer containing primary antibody (see Table
2) overnight at 4°C. After incubation, the eye cups were thoroughly rinsed in PBS with 0.5% Triton-X100, and then fixed for an additional 10 minutes in 4% paraformaldehyde. Eye cups were rinsed in PBS and whole mounted onto Fisher Plus slides, and then incubated in 2% Triton-X100 and 2% donkey serum with 1:500 secondary antibody (Jackson ImmunoResearch) for 2 hours at room temperature. The whole mounts were rinsed in PBS and stained with 300 ng/ml DAPI for 5 minutes at room temperature. After a final wash with PBS, the slides were coverslipped with Immu-Mount and photographed.
Microscopy
All immunofluorescent photographs were acquired using a Zeiss Axioplan 2 Imaging microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY, USA) with a digital black and white camera. Images were analyzed using the Zeiss Axiovision Image Analysis software v4.6 (Carl Zeiss Microimaging, Inc.).
Statistical analyses
Means from qPCR quantification are reported with the standard deviation of the mean, and cell counts are reported with standard error. Statistical significance between two means was determined using a two-sided Student’s t-test. P values were considered significant at a value equal to or less than 0.05.
Discussion
The role of TNFΑ in neurodegeneration has been extensively studied, yet caveats still remain in understanding the mechanism by which it contributes to disease. In the context of RGC injury and death, TNFΑ has been considered by some researchers as detrimental and has been modeled as a secondary inducer of RGC loss [
7],[
11],[
13],[
25]-[
27],[
79]. In accordance with the literature supporting this theory, we found that
Tnfα mRNA is elevated in our model of optic nerve crush, and that the cytokine does lead to a delayed loss of RGCs through an extrinsic apoptotic mechanism. However, a single intraocular injection of TNFΑ does not cause rapid RGC loss as is seen with other ocular injury models to which TNFΑ has been linked. Additional research supports that TNFΑ is beneficial and protective to neurons [
43],[
47],[
48],[
80]. Consistent with this we found that genetic deletion of the
Tnfα gene rendered mice more susceptible to optic nerve injury, and that pre-treatment with exogenous TNFΑ promoted RGC survival after crush. It appears contradictory that TNFΑ is both detrimental and protective to RGCs; however, our data present a potentially critical timing component that has not previously been studied in regards to TNFΑ signaling in the retina. More specifically, it appears that early TNFΑ exposure prior to an injury may be protective, while chronic TNFΑ expression may eventually culminate in neuronal damage and loss.
This phenomenon of neuronal protection from pre-conditioning has been previously observed in stroke patients, in which those with a history of transient ischemia attacks fared better following a cerebral infarction than those without a similar history [
81]. This protection has been mimicked in cell culture models of neuronal insult and animal models of brain ischemia with TNFΑ exposure prior to a damaging stimulus [
47],[
82]. Early treatment with TNFΑ has been positively correlated with survival pathways mediated by a number of proteins, including phosphoinositide-3-kinase [
47], the transcription factor NFΚB, and the histone acetyltransferase CREB binding protein [
82]. The upregulation of CREB binding protein was observed only in neurons, even when co-cultured with astrocytes [
82], suggesting that TNFΑ was acting directly on the neurons and not being mediated through the glia. However, in the retina, astrocytes are only one subpopulation of glial cells - Müller cells and microglia also contribute to retinal health and injury repair, complicating the mechanism by which TNFΑ may be promoting RGC survival.
TNFΑ signals through TNFR1 and TNFR2, and following retinal ischemia and a mouse model of glaucoma, both receptors are upregulated in cells of the inner nuclear layer and GCL [
43],[
44]. TNFR1 is a transmembrane protein with an intracellular death domain that, upon activation, can interact with adaptor proteins and initiate apoptosis through CASP8 [
37],[
83],[
84], and several studies have shown that
Tnfr1 -/- mice exhibit significantly less RGC loss than wild-type mice after injury [
11],[
43]. In comparison, TNFR2 does not contain a death domain and has been linked with sustained NFΚB activation [
43],[
47]. Unlike the RGC protection seen after injury with
Tnfr1 deficiency,
Tnfr2 -/- mice fair worse than wild-type mice following ischemia [
43] and glutamate excitotoxicity [
47]. Interestingly, the
Tnfα -/- mice in our study more closely reflect the enhanced RGC pathology seen in
Tnfr2 -/- mice after injury. This might suggest a preference for TNFΑ to bind TNFR2, possibly explaining the protection afforded when TNFR1 is genetically ablated, restricting TNFΑ to signal through the TNFR2 protective networks. Alternatively,
Tnfr2 deficiency may enhance apoptotic signals through TNFR1, rendering central nervous system tissue more susceptible to injury. In both scenarios, it seems critical to understand the proteins downstream of each receptor that are being affected after injury, and in particular which retinal cell types are responding to this cytokine.
It is important to note that while we have shown an early protective potential of TNFΑ, the long-term consequence of TNFΑ exposure still appears to be detrimental. This dual function of TNFΑ may reflect different responses of individual cell types to this cytokine, which is consistent with a recent publication by Dvoriantchikova and Ivanov [
85]. Their research found that, in response to TNFΑ, RGC cultures exhibited sustained JNK activation and death, while astrocytes upregulated NFΚB and promoted survival [
85]. Therefore, the localization of TNFΑ expression and the cells responding to this cytokine will influence whether TNFΑ has a beneficial or detrimental effect on RGCs. TNFΑ is expressed by a number of innate immune responders, and has been co-localized to optic nerve head, nerve fiber layer, GCL, and the inner nuclear layer of human glaucoma patients [
34],[
35],[
43],[
86], and is upregulated by macroglia and microglia in the optic nerve and optic nerve head [
11],[
34]. Additional studies have shown that dendritic cells also infiltrate the retina following a similar optic nerve crush paradigm described here [
16],[
87], and it is conceivable that they may be the source of TNFΑ. The proximity of TNFΑ production to specific cell types in the retina may generate the differential protective versus detrimental effects. The protective effects may occur through an indirect mechanism, by TNFΑ-induced changes in retinal glia. Based on the rapid induction of NFΚB activity and JUN accumulation after exposure to exogenous TNFΑ, we attribute this rapid response to Müller cells (see below). Conversely, the detrimental effects may result from a direct interaction with the RGCs. Kitaoka and colleagues noted that intravitreal injection of TNFΑ in rabbits resulted in relatively early-onset axonal damage followed by soma death many weeks after TNFΑ exposure [
40].
Unlike the delayed effect of TNFΑ on RGC somas, we have shown that Müller cells respond rapidly to TNFΑ within 1 day of exposure by accumulating JUN and upregulating NFΚB, two known targets downstream of TNFΑ [
85]. It is important to note that JUN is activated by phosphorylation; however, the p-JUN antibody is less reliable than that for JUN due to cross-reactivity [
50]. Therefore, the data presented in this manuscript are documented as nuclear accumulation of JUN rather than activation. Additionally, JUN is known to autoregulate its own expression following its activation [
88], and JUN levels have been used as a surrogate of JUN activity. In response to an intraocular delivery of TNFΑ, both JUN and NFΚB exhibit nuclear activity, and it is interesting that the primary glial cells responding to TNFΑ are the Müller cells. However, while JUN accumulation was present in all of the Müller cells, only a subset exhibited NFΚB activity. This might suggest that JUN is upstream of NFΚB, and that all of the Müller cells have not yet been able to activate the latter gene, although the literature suggests a more complex interplay between these transcription factors [
71],[
73]. It is also unclear if either of these pathways are involved in the protective effect of TNFΑ. However, given the strong association of NFΚB with survival pathways [
47],[
73], it is possible that the cells expressing this transcription factor might be mediating the protective effect seen in our studies involving intravitreal injection of exogenous TNFΑ. A paradox with this interpretation, however, is that neither JUN nor NFΚB were activated after optic nerve crush. While this may have been a function of reduced or more localized levels of TNFΑ production (such as by infiltrating dendritic cells), it remains unclear how the endogenous TNFΑ signaling response provides a protective environment for RGCs. Further studies involving cell-specific ablation of one or both of these transcription factors are needed to decipher whether they play a role in the endogenous TNFΑ protective effect.
The signaling pathways activated in the injured retina are complex, but we have identified a critical timing component in TNFΑ signaling: specifically, that early exposure to this cytokine protects RGCs from subsequent optic nerve damage. A considerable amount of literature has identified a damaging role for TNFΑ, yet this research has indirectly focused on the effect of TNFΑ signaling late after injury. Future studies should consider the advantage of early immune activation in the retina, specifically with an emphasis on Müller cell activation. By bolstering protective pathways early, rather than eliminating cytokine signaling entirely, RGC loss may be minimized following a severe insult and improve the prognosis for patients with optic neuropathies, such as glaucoma.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RWN and RTL conceived the study and participated in its design and coordination. CEM conducted the optic nerve crush and intraocular injections on wild-type, Tnfα -/- mice, and cis-NFΚBEGFP reporter mice, and subsequent immunofluorescence and quantitative PCR. KAF conducted the JUN studies and performed optic nerve crush on Tnfα -/- mice. CLS participated in study design and data analysis. CEM and RWN drafted the manuscript. All authors read and approved the final manuscript.