January 2010
Volume 51, Issue 1
Free
Retina  |   January 2010
Stimulation of Axon Regeneration in the Mature Optic Nerve by Intravitreal Application of the Toll-like Receptor 2 Agonist Pam3Cys
Author Affiliations & Notes
  • Thomas G. Hauk
    From the Departments of Experimental Neurology and
  • Marco Leibinger
    From the Departments of Experimental Neurology and
  • Adrienne Müller
    From the Departments of Experimental Neurology and
  • Anastasia Andreadaki
    From the Departments of Experimental Neurology and
  • Uwe Knippschild
    General, Visceral, and Transplantation Surgery, University of Ulm, Ulm, Germany.
  • Dietmar Fischer
    From the Departments of Experimental Neurology and
  • Corresponding author: Dietmar Fischer, Department of Experimental Neurology, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany; dietmar.fischer@uni-ulm.de
Investigative Ophthalmology & Visual Science January 2010, Vol.51, 459-464. doi:10.1167/iovs.09-4203
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Thomas G. Hauk, Marco Leibinger, Adrienne Müller, Anastasia Andreadaki, Uwe Knippschild, Dietmar Fischer; Stimulation of Axon Regeneration in the Mature Optic Nerve by Intravitreal Application of the Toll-like Receptor 2 Agonist Pam3Cys. Invest. Ophthalmol. Vis. Sci. 2010;51(1):459-464. doi: 10.1167/iovs.09-4203.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: After injury of the optic nerve, mature retinal ganglion cells (RGCs) are normally unable to regenerate axons and undergo apoptosis. However, inflammatory stimulation in the eye induced by the release of β/γ-crystallins from the injured lens or intravitreal zymosan injection transforms RGCs into an active regenerative state, protecting these neurons from cell death and allowing them to regenerate axons back into the optic nerve.

Methods.: The authors tested whether intravitreal application of the selective, water-soluble, toll-like receptor 2 agonist Pam3Cys can delay axotomized RGC cell death and stimulate the regeneration of axons using an in vitro and in vivo paradigm.

Results.: Intravitreal injection of Pam3Cys, as lens injury (LI), induced the upregulation of ciliary neurotrophic factor and glial fibrillary acidic protein expression in retinal glia accompanied by the activation of the JAK/STAT3 pathway in RGCs. As a consequence, RGCs switched to a regenerative state, indicated by a significant upregulation of GAP43 expression and increased neurite outgrowth of RGCs in culture. Repeated intravitreal Pam3Cys application in vivo induced neuroprotective effects and caused stronger axon regeneration in the injured optic nerve than observed after LI.

Conclusions.: Pam3Cys may be a suitable agent for stimulating CNS regeneration.

Optic nerve damage caused by glaucoma, tumors, or traumatic injury is normally associated with an irreversible loss of vision, which is attributed to the failure of mature retinal ganglion cells (RGCs) to regenerate injured axons and to subsequent cell death. However, when exposed to inflammatory stimuli, RGCs switch to an active regenerative state, which enables these neurons to survive injury and to regenerate lengthy axons through the damaged optic nerve. 13 These regenerative conditions can be induced either by transplanting sciatic nerve pieces into the vitreous body, applying β/γ-crystallins into the vitreous body, or releasing these proteins from the injured lens. 1,38 Alternatively, intravitreal injection of the yeast wall extract zymosan, which reportedly binds to the toll-like receptor 2 (TLR2) or dectin-1, 9 mimics these effects. 2,10,11 Both zymosan and β/γ-crystallin injections are associated with the activation of retinal glia and an influx of macrophages into the eye. 8,10,12,13 Activated macrophages, as well as astrocytes/Müller cells, have been proposed to be sources of potent factors mediating these effects. However, depletion of macrophages from the eye did not measurably reduce the axon growth-promoting effects of lens injury (LI), whereas a reduction in the number of activated retinal glia significantly compromised the beneficial effects of zymosan, 10,11,14 suggesting that glial-derived factors are the major mediators of these regenerative effects. Consistently, astrocyte-derived ciliary neurotrophic factor (CNTF) and ApoE have been identified as significant contributors in mediating the beneficial effects induced by LI and zymosan. 11,1417 Furthermore, LI effects are significantly reduced in CNTF-deficient mice and completely abrogated in CNTF/leukemia inhibitory factor (LIF) double-knockout mice (Leibinger M, et al., manuscript submitted), demonstrating that in addition to CNTF, LIF may be another contributing factor mediating the beneficial effects of LI. 
Although intravitreal injections of β/γ-crystallins and zymosan can partially mimic the effects of LI, the number of regenerating axons in the optic nerve and the neuroprotective effects were less robust than those seen after LI. 2,8,10 Moreover, zymosan compromises the passage of light through the eye resulting from a deposition of water-insoluble yeast wall particles in the vitreous body, precluding zymosan as a suitable drug for optic nerve regeneration. Contrary to zymosan, Pam3Cys is a water-soluble bisacyl-lipopeptide and a selective TLR2 agonist. 1821 Here we report that intravitreal application of Pam3Cys is sufficient to induce glial activation, to transform mature RGCs into an active regenerative state, and to significantly stimulate axon regeneration into the injured optic nerve. Thus, intravitreal application of Pam3Cys may be a potential therapeutic strategy for optic nerve repair. 
Materials and Methods
Animals, Optic Nerve Surgery, and Intravitreal Administration
Surgical procedures were approved by the local authorities (Regierungspräsidium, Tübingen, Germany) and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Adult female Sprague-Dawley rats (weight range, 200–230 g) were anesthetized by intraperitoneal injections of ketamine (60–80 mg/kg) and xylazine (10–15 mg/kg). A 1- to 1.5-cm incision was made in the skin above the right orbit. The optic nerve was surgically exposed under an operating microscope, the dural sheath was longitudinally opened, and the nerve was either completely cut or was crushed 1 mm behind the eye by means of jewelers forceps, avoiding injury to the retinal artery. The vascular integrity of the retina was verified by funduscopic examination after surgery. Rats received intravitreal injection of either 10 μL PBS or 10 μg (S)-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH (Pam3Cys) (EMC Microcolections, Tübingen, Germany) solved in 10 μL PBS simultaneously to optic nerve cut (immunohistochemical and Western blot analysis, evaluation of neurite outgrowth in vitro) or 3 days after optic nerve crush (regeneration into the optic nerve). For the latter, intravitreal injections were repeated 7 days after optic nerve surgery in half the animals. If no LI was induced, the integrity of the lens was carefully verified for each animal. For each group, at least five animals were used. 
Dissociated Retinal Cell Cultures
Tissue culture plates (four-well plates; Nunc, Wiesbaden, Germany) were coated with poly-d-lysine (0.1 mg/mL; MWt < 300,000 Da; Sigma, St. Louis, MO), rinsed with distilled water, and air-dried. Wells were then coated with laminin (20 μg/mL; Sigma). To prepare retinal cell cultures, untreated or in vivo-pretreated retinas of rats were killed by overdose of chloral hydrate solution (14%). Retinas were rapidly dissected from the eyecups and incubated at 37°C for 30 minutes in a digestion solution containing papain (16.4 U/mL; Worthington, Katarinen, Germany) and l-cysteine (0.3 μg/mL; Sigma) in Dulbecco's modified Eagle medium (DMEM; Invitrogen). Retinas were then rinsed with DMEM and triturated in 2 mL DMEM. Cells were centrifuged for 5 minutes, and the pellet was carefully resuspended in 5 mL DMEM containing B27-supplement (1:50; Gibco, Grand Island, NY) and 1:50 penicillin/streptomycin (Biochrom, Holliston, MA). Dissociated cells were then passed through a cell strainer (40 μm; Falcon; BD Biosciences, Franklin Lakes, NJ), and 500 μL cell suspension was added to each well. Cultures were arranged in a pseudorandomized manner on the plates so that the investigator would not be aware of their identity. Retinal cells of animals that had received pretreatment in vivo were cultured for 24 hours and fixed with a paraformaldehyde solution (4%) and methanol (both Sigma). They were then processed for immunocytochemical staining using an antibody against βIII-tubulin (TUJ-1; Babco, Richmond, VA) at a dilution of 1:2000. All RGCs with regenerated axons were photographed under a fluorescence microscope (200×). The axon length of RGCs was determined using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). Furthermore, the total number of βIII-tubulin-positive RGCs with an intact nucleus (DAPI) per well was quantified to test for potential neurotoxic or neuroprotective effects after each treatment. Values for axon outgrowth were determined by the sum of the neurite length per well divided by the total number of RGCs per well, resulting in the average axon length per RGC. Values were then normalized to control groups as indicated. The data are given as the mean ± SEM of four replicate wells. Statistical significance was analyzed by a two-tailed Student's t-test (assuming equal variances). The results from individual experiments were averaged within each experimental group. Each experiment was repeated at least three times. 
Immunohistochemistry
Animals were anesthetized and perfused with 4% paraformaldehyde (PFA; Sigma). Eyes were postfixed for several hours, transferred to 30% sucrose overnight (4°C), and embedded (Tissue-Tek; Sakura Finetek, Torrance, CA). Frozen sections of the retinas and optic nerves were longitudinally cut thaw-mounted onto coated glass slides (Superfrost Plus, Fisher, Pittsburgh, PA), and stored at −80°C until further use. Monoclonal antibodies against βIII-tubulin (1:2000; Babco, Richmond, CA) and glial fibrillary acidic protein (GFAP; 1:50; Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal antibodies against rat phospho-STAT3 (1:500; Tyr705; Cell Signaling), and a polyclonal custom-made antibody against GAP43 (1:1000; Invitrogen, Carlsbad, CA) and rat CNTF (1:5000; Serotec, Raleigh, NC) were used. Secondary antibodies included anti-mouse IgG and anti-rabbit IgG antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 (1:1000; Molecular Probes, Eugene, OR). To stain nuclei, sections were incubated in a solution containing 4′, 6-diamidino-2-phenylindol (DAPI) for 1 minute. Fluorescent sections were covered mounting solution (Mowiol; Calbiochem, San Diego, CA) and were analyzed using a microscope. 
Western Blot Assays
For retinal lysate preparation, rats were killed, and their eyeballs were enucleated and dissected. Isolated retinas were collected in lysis buffer (20 mM Tris/HCl, pH 7.5, 10 mM KCl, 250 mM sucrose, 10 mM NaF, 1 mM DTT, 0.1 mM Na3VO4, 1% Triton X-100, 0.1% SDS) with 1/100 protease inhibitor (Calbiochem). Alternatively, retinal explants that were cultured for 2 days were prepared accordingly. Retinas were homogenized and centrifuged at 5000 rpm for 10 minutes at 4°C. The supernatants were analyzed by Western blot assay. Separation of proteins was performed by 10 SDS-PAGE according to standard protocols (Bio-Rad, Hercules, CA). After SDS-PAGE, proteins were transferred to nitrocellulose membranes (Amersham, Buckinghamshire, UK). The blots were blocked either in 5% dried milk or in 2% blocking agent (enhanced chemiluminescence [ECL]; Advance; Amersham) in Tris-buffered saline-Tween-20. They were then processed for immunostaining with either an antiserum against rat phospho-STAT3 (1:5000; Tyr705; Cell Signaling, Beverly, MA), a monoclonal antibody against rat β-actin (1:7500; Sigma), a polyclonal custom-made antibody against GAP43 (1:1000; Invitrogen), an anti-βIII-tubulin (1:1000; Babco; Covance, Princeton, NJ), or a polyclonal antibody against rat CNTF (1:5000; Serotec) at 4°C overnight. Bound antibodies were visualized with anti-rabbit, anti-mouse immunoglobulin G (IgG; 1:80,000) or anti-sheep (1:20,000) secondary antibodies conjugated with horseradish peroxidase (all from Sigma) after incubation for 2 hours at room temperature. The antigen-antibody complexes were detected by ECL (Amersham). 
Retinal Explant Cultures
Untreated retinas were isolated and cultured in a solution containing DMEM (Gibco, Auckland, New Zealand), B27 supplement (1:50; Invitrogen), and penicillin/streptomycin (100 U/mL; Invitrogen) in a humidified atmosphere containing 5% CO2 for 2 days. Isolated retinas were cultured in the presence of BSA (500 μg/mL; Sigma), freshly prepared lens homogenate (500 μg/mL), or increasing concentrations of Pam3Cys (EMC Microcollections) at concentrations of 10, 100, and 250 μg/mL. 
Quantification of Axon Regeneration in the Optic Nerve
Axonal growth in optic nerve sections was quantified using a method described previously. 16,22 In brief, under 400 × magnification, the number of Gap43-positive axons was counted in each of the four sections at 0.25, 0.5, and 1 mm distal to the crush site of the optic nerve. 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 the nerve width. The number of axons per millimeter was then averaged over all sections and was used to calculate the total number of axons regenerating in the optic nerve, as reported previously. 22,23 Statistical significance was analyzed by a two-tailed Student's t-test (assuming equal variances). 
Results
Induction of CNTF and GFAP Expression in Retinal Glia and STAT3 Activation in RGCs by Intravitreal Injection of Pam3Cys
LI, intravitreal injection of β/γ-crystallins, and zymosan induce strong activation of retinal glia, which is indicated by the upregulation of GFAP and CNTF. 2,8,16 To test whether Pam3Cys can mimic the cellular responses to lental proteins or zymosan, we isolated the retina 5 days after optic nerve cut (ONC) + intravitreal injection of PBS, ONC + LI, and ONC + intravitreal injection of Pam3Cys. In contrast to LI, which caused next to the loss of the integrity of the lens a deposition of lens fragments on the surface of the retinal fiber layer, the vitreous body of Pam3Cys-treated animals remained clear, when tested immediately, 5 days, or 14 days after the initial intravitreal injection (data not shown). Nevertheless, LI and Pam3Cys treatment similarly induced a strong upregulation of GFAP and CNTF expression in retinal astrocytes and Müller cells, whereas PBS treatment exhibited only a slight upregulation of CNTF and GFAP expression (Figs. 1A, 1C). Moreover, Western blot and immunohistochemical analyses revealed that Pam3Cys and LI treatments increased phospho-STAT3 (pSTAT3) levels in the nuclei of axotomized RGCs and cells of the inner nuclear layer (most likely Müller cells), whereas retinas treated with intravitreal PBS revealed no or very little activation compared with untreated controls (Figs. 1B, 1C). Next to glial activation, LI and Pam3Cys injection were also associated with an infiltration of macrophages into the eye (Fig. 1A). To investigate whether Pam3Cys is capable of increasing CNTF expression and STAT3 activation in the retina independent of infiltrating macrophages or other peripherally circulating cells, we cultured untreated retinas for 2 days in the presence of BSA, lens homogenate (LH), or increasing concentrations (10, 100, 250 ng/mL) of Pam3Cys. As reported previously for LH and zymosan, 8,16 Pam3Cys induced CNTF expression and activated STAT3 in the retina in a concentration-dependent fashion (Fig. 1D), suggesting that Pam3Cys does induce glial CNTF expression independently of macrophages or other peripherally circulating cells. 
Figure 1.
 
Pam3Cys induces GFAP and CNTF expression in retinal glia and activates the JAK/STA3 pathway in RGCs. (A) Immunohistochemical staining of the retina of untreated controls (con), 5 days after ONC + intravitreal injection of PBS, 5 days after ONC + LI, or ONC + intravitreal injection of Pam3Cys (P3C). Expression levels of CNTF (red) and GFAP (green) in retinal astrocytes and Müller cells were markedly increased in the retina after LI and Pam3Cys treatment. Both LI and Pam3Cys treatment were associated with an infiltration of CD68 (ED1)-positive macrophages in the vitreous body. GCL, ganglion cell layer, INL, inner nuclear layer, vitr, vitreous body. Scale bar, 50 μm. (B) Immunohistochemical staining of the retina treated as described in (A) but stained for phospho-STAT3 (red), βIII-tubulin (green), and DAPI (blue). Both LI and Pam3Cys treatment activated the JAK/STAT3 pathway in βIII-tubulin positive RGCs and, to a minor extent, in cells of the inner nuclear layer (most likely Müller cells). Scale bar, 50 μm. (C) Western blot analysis of retinal lysates prepared from animals treated as described in (A) and (B) confirmed the upregulation of CNTF expression and the activation of STAT3 after LI or intravitreal application of Pam3Cys. Bands of β-actin verified that the same amounts of protein were loaded per lane. (D) Western blot analysis of lysates derived from retinal explants cultured and exposed to either BSA (BSA, 200 μg/mL), increasing concentrations of Pam3Cys (P3C) (10–250 μg/mL as indicated), or freshly prepared lens homogenate (LH) for 2 days. Contrary to BSA, LH and Pam3Cys increased CNTF expression and STAT3 activation in a concentration-dependent manner. Bands of β-actin verified that the same amounts of protein were loaded per lane.
Figure 1.
 
Pam3Cys induces GFAP and CNTF expression in retinal glia and activates the JAK/STA3 pathway in RGCs. (A) Immunohistochemical staining of the retina of untreated controls (con), 5 days after ONC + intravitreal injection of PBS, 5 days after ONC + LI, or ONC + intravitreal injection of Pam3Cys (P3C). Expression levels of CNTF (red) and GFAP (green) in retinal astrocytes and Müller cells were markedly increased in the retina after LI and Pam3Cys treatment. Both LI and Pam3Cys treatment were associated with an infiltration of CD68 (ED1)-positive macrophages in the vitreous body. GCL, ganglion cell layer, INL, inner nuclear layer, vitr, vitreous body. Scale bar, 50 μm. (B) Immunohistochemical staining of the retina treated as described in (A) but stained for phospho-STAT3 (red), βIII-tubulin (green), and DAPI (blue). Both LI and Pam3Cys treatment activated the JAK/STAT3 pathway in βIII-tubulin positive RGCs and, to a minor extent, in cells of the inner nuclear layer (most likely Müller cells). Scale bar, 50 μm. (C) Western blot analysis of retinal lysates prepared from animals treated as described in (A) and (B) confirmed the upregulation of CNTF expression and the activation of STAT3 after LI or intravitreal application of Pam3Cys. Bands of β-actin verified that the same amounts of protein were loaded per lane. (D) Western blot analysis of lysates derived from retinal explants cultured and exposed to either BSA (BSA, 200 μg/mL), increasing concentrations of Pam3Cys (P3C) (10–250 μg/mL as indicated), or freshly prepared lens homogenate (LH) for 2 days. Contrary to BSA, LH and Pam3Cys increased CNTF expression and STAT3 activation in a concentration-dependent manner. Bands of β-actin verified that the same amounts of protein were loaded per lane.
Transformation of Mature RGCs into a Regenerative State by Intravitreal Injection of Pam3Cys
To test whether intravitreal injection of Pam3Cys is also sufficient to transform RGCs into a regenerative state, we examined retinal GAP43 expression levels after various treatments in vivo. Western blot analysis revealed a robust induction of GAP43 5 days after ONC + Pam3Cys injection, which was similar to LI, whereas ONC + PBS injections only slightly elevated retinal GAP43 levels compared with naive, untreated retinas (Fig. 1C). The regenerative state of RGCs was functionally assessed in retinal cell cultures. The average length of spontaneous regenerating RGC neurites cultured for 24 hours, 5 days after ONC + intravitreal injection of PBS, ONC + LI, or ONC + Pam3Cys treatment in vivo, are presented in Figures 2A–D. RGCs of rats that had been subjected to ONC + PBS displayed little neurite outgrowth, whereas untreated retinas showed no spontaneous outgrowth (Figs. 2A, 2D). In contrast, LI or Pam3Cys injection induced robust neurite outgrowth (Figs. 2B–D). Pam3Cys treatment induced even significantly more neurite outgrowth compared with LI (Figs. 2C, 2D). All cultures contained similar numbers of RGCs per well, verifying that under these conditions (5 days after ONC and 1 day in culture) cell death of RGCs has not yet occurred and that the differences in neurite outgrowth were not the consequence of neuroprotective or toxic effects (Fig. 2E). 
Figure 2.
 
Effects of Pam3Cys on RGC neurite outgrowth in dissociated retinal cell cultures in vitro. (AC) Representative βIII-tubulin-positive RGCs in dissociated retinal cell cultures 5 days after ONC + LI, ONC + Pam3Cys (P3C) injection, or no previous treatment and 24 hours in culture. Scale bar, 50 μm. (D) Quantification of neurite outgrowth of RGCs of groups as described in (A) and animals that had received ONC + intravitreal injection of saline at the time of optic nerve injury. Data are normalized to the average neurite length of RGCs from animals that were subjected to ONC + LI. **P < 0.001. (E) Quantitation of RGCs of dissociated retinal cultures of groups described in (D). All groups contained a similar number of RGCs per well.
Figure 2.
 
Effects of Pam3Cys on RGC neurite outgrowth in dissociated retinal cell cultures in vitro. (AC) Representative βIII-tubulin-positive RGCs in dissociated retinal cell cultures 5 days after ONC + LI, ONC + Pam3Cys (P3C) injection, or no previous treatment and 24 hours in culture. Scale bar, 50 μm. (D) Quantification of neurite outgrowth of RGCs of groups as described in (A) and animals that had received ONC + intravitreal injection of saline at the time of optic nerve injury. Data are normalized to the average neurite length of RGCs from animals that were subjected to ONC + LI. **P < 0.001. (E) Quantitation of RGCs of dissociated retinal cultures of groups described in (D). All groups contained a similar number of RGCs per well.
Stimulation of Axon Regeneration in the Injured Optic Nerve by Intravitreal Injection of Pam3Cys
To investigate whether Pam3Cys treatment is sufficient to stimulate axon regeneration in the crushed optic nerve or can delay cell death of axotomized RGCs, Pam3Cys injections were performed 3 days after the optic nerve was crushed. In another group, Pam3Cys was applied twice on day 3 and again on day 7 after optic nerve crush. Further experimental groups included animals that received two intravitreal injections of PBS at days 3 and 7 after optic nerve crush or were subjected to LI simultaneously with optic nerve surgery. Animals treated with PBS showed very few, short, GAP43-positive axons beyond the lesion site 14 days after the optic nerve injury (Figs. 3A, 3B), whereas a single injection of Pam3Cys was sufficient to increase the number of axons regenerating beyond the injury site of the optic nerve (Fig. 3B). However, these effects were less pronounced than those measured in response to LI. Two injections of Pam3Cys were more efficient than a single injection, and the number of GAP-43-positive axons growing ≥0.5 mm and ≥1 mm beyond the lesion site of the optic nerve was significantly higher than the number growing beyond the injury site after LI (Figs. 3A, 3B). Two intravitreal injections of Pam3Cys showed significant neuroprotective effects, but they were less robust than the neuroprotection conferred by LI. A single injection of Pam3Cys resulted in no measurable neuroprotective effects of axotomized RGCs compared with animals subjected to optic nerve crush alone (Fig. 3C). 
Figure 3.
 
Effects of Pam3Cys on axonal regeneration in the crushed optic nerve and survival of axotomized RGCs. (A) Longitudinal sections through the optic nerve were stained with an antibody against Gap43 2 weeks after ONC + two intravitreal injections of PBS, ONC + LI, or ONC + two intravitreal injections of Pam3Cys (10 μg). Asterisks: indicate injury site. Scale bar, 200 μm. (B) Quantitative analysis of axon regeneration into the optic nerve at 0.25, 0.5, and 1 mm past the lesion site after ONC + PBS treatment, ONC + LI, ONC + one intravitreal injection of Pam3Cys (P3C I), or two intravitreal injections of Pam3Cys (P3C II). (C) Quantification of surviving βIII-tubulin-positive RGCs in sections of the same eyes as described in (B). con, number of RGCs in naive retina. **P < 0.01, and ***P < 0.001 compared with group treated with ONC + PBS.
Figure 3.
 
Effects of Pam3Cys on axonal regeneration in the crushed optic nerve and survival of axotomized RGCs. (A) Longitudinal sections through the optic nerve were stained with an antibody against Gap43 2 weeks after ONC + two intravitreal injections of PBS, ONC + LI, or ONC + two intravitreal injections of Pam3Cys (10 μg). Asterisks: indicate injury site. Scale bar, 200 μm. (B) Quantitative analysis of axon regeneration into the optic nerve at 0.25, 0.5, and 1 mm past the lesion site after ONC + PBS treatment, ONC + LI, ONC + one intravitreal injection of Pam3Cys (P3C I), or two intravitreal injections of Pam3Cys (P3C II). (C) Quantification of surviving βIII-tubulin-positive RGCs in sections of the same eyes as described in (B). con, number of RGCs in naive retina. **P < 0.01, and ***P < 0.001 compared with group treated with ONC + PBS.
Discussion
Intravitreal injections of β/γ-crystallins, the slow release of β/γ-crystallins from the injured lens, or zymosan injection transform RGCs into an active regenerative state, enabling these neurons to regenerate axons in the injured optic nerve. 1,4,8,10,12,22 Astrocyte/Müller cell-derived and macrophage-derived factors have been proposed to be sources of factors mediating these beneficial effects. 2,15,16 Although all treatments evoke glial and macrophage activation, the receptors through which β/γ-crystallins or zymosan stimulate the cellular responses and thereby promote axon regeneration are still unknown. It is further unclear whether crystallins, LI, or zymosan induce their beneficial effects through the same or different receptors. However, the identification of these receptors may open the avenue for the development and use of specific drugs to protect RGCs from cell death and to stimulate axon regeneration. It is well known that zymosan binds to and stimulates several cell types via dectin-1 or TLR2, 9,24,25 pointing to the possibility that specific agonists of these receptors may be suitable drugs to induce the beneficial effects evoked by LI or zymosan more specifically. In the present study, we tested this possibility and found that intravitreal application of the specific TLR2 agonist Pam3Cys induced, as LI, the expression of CNTF and GFAP in retinal astrocytes/Müller cells. Furthermore, Pam3Cys application was also associated with an infiltration of macrophages, which is consistent with previous studies showing an activation of macrophages by Pam3Cys through the TLR2. 26,27 Most important, repeated intravitreal injections of Pam3Cys stimulated more axons to regenerate into the injured optic nerve than was seen for LI. Given that Pam3Cys is a selective TLR2 agonist and that TLR2 receptor is expressed in macrophages and astrocytes, 28,29 it is possible that the cascade of events leading to these beneficial effects on RGCs were mediated through TLR2 signaling. Further experiments are necessary to test this possibility. The observation that Pam3Cys induced retinal CNTF expression associated with an activation of the JAK/STAT3 pathway, also in the absence of blood-borne macrophages in culture, strongly suggests that Pam3Cys induced glial CNTF expression independently of peripherally circulating cells. Furthermore, we have previously shown that macrophages infiltrating the inner eye do not express CNTF 16 and, therefore, do not contribute to the increased CNTF expression levels measured in the retina. However, further research is necessary to test whether CNTF expression in astrocytes/Müller cells is directly increased by TLR2 signaling or indirectly via other resident TLR2-expressing cells in the retina, such as microglia. 
The advantage of intravitreal treatment with Pam3Cys compared with LI or intravitreal zymosan injection is, in addition to the stronger stimulatory effect on axon regeneration, that water-soluble Pam3Cys treatment does not compromise the passage of light in the eye. This argument is particularly important from a clinical point of view. Compared with intravitreal injection of β/γ-crystallins, which did also not affect the transparency of the vitreous body, 8 repeated intravitreal application of Pam3Cys stimulated significantly more axonal regeneration in the crushed optic nerve than did LI. However, our study also demonstrates that Pam3Cys must be applied repeatedly to achieve stronger regeneration than can be achieved with LI. The most likely explanation for this may be a rapid clearance of Pam3Cys from the vitreous body because of its water solubility, whereas LI causes a continuous release of crystallins over several days. Therefore, the resultant induction and release of beneficial factors, such as CNTF, may be more restricted over time in Pam3Cys treatment than after LI. Consistent with this idea, a single intravitreal injection of Pam3Cys stimulated axon regeneration of RGCs stronger than LI when evaluated in culture 5 days after surgery, whereas a single injection caused little regeneration of axons in the optic nerve. This time-restriction effect for Pam3Cys seems to become more evident when regeneration or neuroprotection is measured after longer time periods. 
In summary, we show here that intravitreal application of the selective TLR2 agonist Pam3Cys transforms mature RGCs into an active regenerative state and allows these neurons to regenerate more axons into the lesioned optic nerve than LI. Next to optic nerve regeneration, zymosan treatment of sensory ganglia, combined with a modification of chondroitin sulfate proteoglycans, results in robust and functional regeneration of sensory axons through the dorsal root entry zone after root injury. 30 Therefore, Pam3Cys may be also a suitable and superior agent to stimulate axonal regeneration not only in the optic nerve but in other regions of the CNS as well. Thus, Pam3Cys may be a useful compound for the development of future strategies for optic nerve repair and to improve recovery after CNS injuries in general. 
Footnotes
 Supported by the state of Baden-Württemberg and the German Research Foundation.
Footnotes
 Disclosure: T.G. Hauk, None; M. Leibinger, None; A. Müller, None; N. Andreadaki, None; U. Knippschild, None; D. Fischer, None
References
Fischer D Pavlidis M Thanos S . Cataractogenic lens injury prevents traumatic ganglion cell death and promotes axonal regeneration both in vivo and in culture. Invest Ophthalmol Vis Sci. 2000; 41: 3943–3954. [PubMed]
Yin Y Cui Q Li Y . Macrophage-derived factors stimulate optic nerve regeneration. J Neurosci. 2003; 23: 2284–2293. [PubMed]
Fischer D Heiduschka P Thanos S . Lens-injury-stimulated axonal regeneration throughout the optic pathway of adult rats. Exp Neurol. 2001; 172: 257–272. [CrossRef] [PubMed]
Lorber B Berry M Logan A . Lens injury stimulates adult mouse retinal ganglion cell axon regeneration via both macrophage- and lens-derived factors. Eur J Neurosci. 2005; 21: 2029–2034. [CrossRef] [PubMed]
Berry M Ahmed Z Lorber B Douglas M Logan A . Regeneration of axons in the visual system. Restorative Neurol Neurosci. 2008; 26: 147–174.
Berry M Carlile J Hunter A . Peripheral nerve explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve. J Neurocytol. 1996; 25: 147–170. [CrossRef] [PubMed]
Berry M Carlile J Hunter A Tsang W Rosenstiel P Sievers J . Optic nerve regeneration after intravitreal peripheral nerve implants: trajectories of axons regrowing through the optic chiasm into the optic tracts. J Neurocytol. 1999; 28: 721–741. [CrossRef] [PubMed]
Fischer D Hauk TG Muller A Thanos S . Crystallins of the beta/gamma-superfamily mimic the effects of lens injury and promote axon regeneration. Mol Cell Neurosci. 2008; 37: 471–479. [CrossRef] [PubMed]
Dillon S Agrawal S Banerjee K . Yeast zymosan, a stimulus for TLR2 and dectin-1, induces regulatory antigen-presenting cells and immunological tolerance. J Clin Invest. 2006; 116: 916–928. [CrossRef] [PubMed]
Hauk TG Muller A Lee J Schwendener R Fischer D . Neuroprotective and axon growth promoting effects of intraocular inflammation do not depend on oncomodulin or the presence of large numbers of activated macrophages. Exp Neurol. 2008; 209: 469–482. [CrossRef] [PubMed]
Lorber B Berry M Douglas MR Nakazawa T Logan A . Activated retinal glia promote neurite outgrowth of retinal ganglion cells via apolipoprotein E. J Neurosci Res. 2009; 87: 2645–2652. [CrossRef] [PubMed]
Pernet V Di Polo A . Synergistic action of brain-derived neurotrophic factor and lens injury promotes retinal ganglion cell survival, but leads to optic nerve dystrophy in vivo. Brain. 2006; 129: 1014–1026. [CrossRef] [PubMed]
Lorber B Berry M Logan A Tonge D . Effect of lens lesion on neurite outgrowth of retinal ganglion cells in vitro. Mol Cell Neurosci. 2002; 21: 301–311. [CrossRef] [PubMed]
Lorber B Berry M Logan A . Different factors promote axonal regeneration of adult rat retinal ganglion cells after lens injury and intravitreal peripheral nerve grafting. J Neurosci Res. 2008; 86: 894–903. [CrossRef] [PubMed]
Fischer D . CNTF, a key factor mediating the beneficial effects of inflammatory reactions in the eye. Brain. 2008; 131(6): e97. [CrossRef]
Muller A Hauk TG Fischer D . Astrocyte-derived CNTF switches mature RGCs to a regenerative state following inflammatory stimulation. Brain. 2007; 130: 3308–3320. [CrossRef] [PubMed]
Muller A Hauk TG Leibinger M Marienfeld R Fischer D . Exogenous CNTF stimulates axon regeneration of retinal ganglion cells partially via endogenous CNTF. Mol Cell Neurosci. 2009; 41: 233–246. [CrossRef] [PubMed]
Takeuchi O Kawai T Muhlradt PF . Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol. 2001; 13: 933–940. [CrossRef] [PubMed]
Dobrovolskaia MA Medvedev AE Thomas KE . Induction of in vitro reprogramming by Toll-like receptor (TLR)2 and TLR4 agonists in murine macrophages: effects of TLR “homotolerance” versus “heterotolerance” on NF-kappa B signaling pathway components. J Immunol. 2003; 170: 508–519. [CrossRef] [PubMed]
Takeuchi O Sato S Horiuchi T . Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol. 2002; 169: 10–14. [CrossRef] [PubMed]
Alexopoulou L Thomas V Schnare M . Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice. Nat Med. 2002; 8: 878–884. [PubMed]
Fischer D Petkova V Thanos S Benowitz LI . Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation. J Neurosci. 2004; 24: 8726–8740. [CrossRef] [PubMed]
Fischer D He Z Benowitz LI . Counteracting the Nogo receptor enhances optic nerve regeneration if retinal ganglion cells are in an active growth state. J Neurosci. 2004; 24: 1646–1651. [CrossRef] [PubMed]
Shah VB Huang Y Keshwara R Ozment-Skelton T Williams DL Keshvara L . Beta-glucan activates microglia without inducing cytokine production in Dectin-1-dependent manner. J Immunol. 2008; 180: 2777–2785. [CrossRef] [PubMed]
Bonfim CV Mamoni RL Souza MH Blotta L . TLR-2, TLR-4 and dectin-1 expression in human monocytes and neutrophils stimulated by Paracoccidioides brasiliensis . Med Mycol. 2009; 1–12.
Hauschildt S Hoffmann P Beuscher HU . Activation of bone marrow-derived mouse macrophages by bacterial lipopeptide: cytokine production, phagocytosis and Ia expression. Eur J Immunol. 1990; 20: 63–68. [CrossRef] [PubMed]
Hoffmann P Wiesmuller KH Metzger J Jung G Bessler WG . Induction of tumor cytotoxicity in murine bone marrow-derived macrophages by two synthetic lipopeptide analogues. Biol Chem Hoppe Seyler. 1989; 370: 575–582. [CrossRef] [PubMed]
Phulwani NK Esen N Syed MM Kielian T . TLR2 expression in astrocytes is induced by TNF-alpha- and NF-kappa B-dependent pathways. J Immunol. 2008; 181: 3841–3849. [CrossRef] [PubMed]
Esen N Tanga FY DeLeo JA Kielian T . Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus . J Neurochem. 2004; 88: 746–758. [CrossRef] [PubMed]
Steinmetz MP Horn KP Tom VJ . Chronic enhancement of the intrinsic growth capacity of sensory neurons combined with the degradation of inhibitory proteoglycans allows functional regeneration of sensory axons through the dorsal root entry zone in the mammalian spinal cord. J Neurosci. 2005; 25: 8066–8076. [CrossRef] [PubMed]
Figure 1.
 
Pam3Cys induces GFAP and CNTF expression in retinal glia and activates the JAK/STA3 pathway in RGCs. (A) Immunohistochemical staining of the retina of untreated controls (con), 5 days after ONC + intravitreal injection of PBS, 5 days after ONC + LI, or ONC + intravitreal injection of Pam3Cys (P3C). Expression levels of CNTF (red) and GFAP (green) in retinal astrocytes and Müller cells were markedly increased in the retina after LI and Pam3Cys treatment. Both LI and Pam3Cys treatment were associated with an infiltration of CD68 (ED1)-positive macrophages in the vitreous body. GCL, ganglion cell layer, INL, inner nuclear layer, vitr, vitreous body. Scale bar, 50 μm. (B) Immunohistochemical staining of the retina treated as described in (A) but stained for phospho-STAT3 (red), βIII-tubulin (green), and DAPI (blue). Both LI and Pam3Cys treatment activated the JAK/STAT3 pathway in βIII-tubulin positive RGCs and, to a minor extent, in cells of the inner nuclear layer (most likely Müller cells). Scale bar, 50 μm. (C) Western blot analysis of retinal lysates prepared from animals treated as described in (A) and (B) confirmed the upregulation of CNTF expression and the activation of STAT3 after LI or intravitreal application of Pam3Cys. Bands of β-actin verified that the same amounts of protein were loaded per lane. (D) Western blot analysis of lysates derived from retinal explants cultured and exposed to either BSA (BSA, 200 μg/mL), increasing concentrations of Pam3Cys (P3C) (10–250 μg/mL as indicated), or freshly prepared lens homogenate (LH) for 2 days. Contrary to BSA, LH and Pam3Cys increased CNTF expression and STAT3 activation in a concentration-dependent manner. Bands of β-actin verified that the same amounts of protein were loaded per lane.
Figure 1.
 
Pam3Cys induces GFAP and CNTF expression in retinal glia and activates the JAK/STA3 pathway in RGCs. (A) Immunohistochemical staining of the retina of untreated controls (con), 5 days after ONC + intravitreal injection of PBS, 5 days after ONC + LI, or ONC + intravitreal injection of Pam3Cys (P3C). Expression levels of CNTF (red) and GFAP (green) in retinal astrocytes and Müller cells were markedly increased in the retina after LI and Pam3Cys treatment. Both LI and Pam3Cys treatment were associated with an infiltration of CD68 (ED1)-positive macrophages in the vitreous body. GCL, ganglion cell layer, INL, inner nuclear layer, vitr, vitreous body. Scale bar, 50 μm. (B) Immunohistochemical staining of the retina treated as described in (A) but stained for phospho-STAT3 (red), βIII-tubulin (green), and DAPI (blue). Both LI and Pam3Cys treatment activated the JAK/STAT3 pathway in βIII-tubulin positive RGCs and, to a minor extent, in cells of the inner nuclear layer (most likely Müller cells). Scale bar, 50 μm. (C) Western blot analysis of retinal lysates prepared from animals treated as described in (A) and (B) confirmed the upregulation of CNTF expression and the activation of STAT3 after LI or intravitreal application of Pam3Cys. Bands of β-actin verified that the same amounts of protein were loaded per lane. (D) Western blot analysis of lysates derived from retinal explants cultured and exposed to either BSA (BSA, 200 μg/mL), increasing concentrations of Pam3Cys (P3C) (10–250 μg/mL as indicated), or freshly prepared lens homogenate (LH) for 2 days. Contrary to BSA, LH and Pam3Cys increased CNTF expression and STAT3 activation in a concentration-dependent manner. Bands of β-actin verified that the same amounts of protein were loaded per lane.
Figure 2.
 
Effects of Pam3Cys on RGC neurite outgrowth in dissociated retinal cell cultures in vitro. (AC) Representative βIII-tubulin-positive RGCs in dissociated retinal cell cultures 5 days after ONC + LI, ONC + Pam3Cys (P3C) injection, or no previous treatment and 24 hours in culture. Scale bar, 50 μm. (D) Quantification of neurite outgrowth of RGCs of groups as described in (A) and animals that had received ONC + intravitreal injection of saline at the time of optic nerve injury. Data are normalized to the average neurite length of RGCs from animals that were subjected to ONC + LI. **P < 0.001. (E) Quantitation of RGCs of dissociated retinal cultures of groups described in (D). All groups contained a similar number of RGCs per well.
Figure 2.
 
Effects of Pam3Cys on RGC neurite outgrowth in dissociated retinal cell cultures in vitro. (AC) Representative βIII-tubulin-positive RGCs in dissociated retinal cell cultures 5 days after ONC + LI, ONC + Pam3Cys (P3C) injection, or no previous treatment and 24 hours in culture. Scale bar, 50 μm. (D) Quantification of neurite outgrowth of RGCs of groups as described in (A) and animals that had received ONC + intravitreal injection of saline at the time of optic nerve injury. Data are normalized to the average neurite length of RGCs from animals that were subjected to ONC + LI. **P < 0.001. (E) Quantitation of RGCs of dissociated retinal cultures of groups described in (D). All groups contained a similar number of RGCs per well.
Figure 3.
 
Effects of Pam3Cys on axonal regeneration in the crushed optic nerve and survival of axotomized RGCs. (A) Longitudinal sections through the optic nerve were stained with an antibody against Gap43 2 weeks after ONC + two intravitreal injections of PBS, ONC + LI, or ONC + two intravitreal injections of Pam3Cys (10 μg). Asterisks: indicate injury site. Scale bar, 200 μm. (B) Quantitative analysis of axon regeneration into the optic nerve at 0.25, 0.5, and 1 mm past the lesion site after ONC + PBS treatment, ONC + LI, ONC + one intravitreal injection of Pam3Cys (P3C I), or two intravitreal injections of Pam3Cys (P3C II). (C) Quantification of surviving βIII-tubulin-positive RGCs in sections of the same eyes as described in (B). con, number of RGCs in naive retina. **P < 0.01, and ***P < 0.001 compared with group treated with ONC + PBS.
Figure 3.
 
Effects of Pam3Cys on axonal regeneration in the crushed optic nerve and survival of axotomized RGCs. (A) Longitudinal sections through the optic nerve were stained with an antibody against Gap43 2 weeks after ONC + two intravitreal injections of PBS, ONC + LI, or ONC + two intravitreal injections of Pam3Cys (10 μg). Asterisks: indicate injury site. Scale bar, 200 μm. (B) Quantitative analysis of axon regeneration into the optic nerve at 0.25, 0.5, and 1 mm past the lesion site after ONC + PBS treatment, ONC + LI, ONC + one intravitreal injection of Pam3Cys (P3C I), or two intravitreal injections of Pam3Cys (P3C II). (C) Quantification of surviving βIII-tubulin-positive RGCs in sections of the same eyes as described in (B). con, number of RGCs in naive retina. **P < 0.01, and ***P < 0.001 compared with group treated with ONC + PBS.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×