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Keywords:

  • interleukin-6;
  • lesion;
  • mouse;
  • organotypic hippocampal slice culture;
  • regeneration

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Interleukin (IL)-6 is a pro-inflammatory cytokine now widely recognized to contribute to the molecular events that follow CNS injury. Little is known, however, about its action on axonal sprouting and regeneration in the brain. We addressed this issue using the model of transection of Schaffer collaterals in mice organotypic hippocampal slice cultures. Transection of slice cultures was associated with a marked release of IL-6 that could be neutralized by an IL-6 blocking antibody. We monitored functional recovery across the lesion by recording synaptic responses using a multi-electrode array. We found that application of IL-6 antibodies to the cultures after lesioning significantly reduced functional recovery across the lesion. Furthermore, the level of expression of the 43-kDa growth-associated protein (GAP-43) was lower in slices treated with the IL-6 neutralizing antibody than in those treated with a control IgG. Conversely, addition of exogenous IL-6 to the culture medium resulted in a dose-dependent enhancement of functional recovery across the lesion and a higher level of expression of GAP-43. Co-culture of CA3 hemi-slices from thy1-YFP mice with CA1 hemi-slices from wild-type animals confirmed that IL-6-treated co-cultures exhibited an increased number of growing fluorescent fibres across the lesion site. Taken together these data indicate that IL-6 plays an important role in CNS repair mechanisms by promoting regrowth and axon regeneration.

Abbreviations used:
Ctrl

control

DIV

days in vitro

EPSP

excitatory postsynaptic potential

GAP-43

growth-associated protein-43

IL

interleukin

L

lesioned

NL

non-lesioned

MAG

myelin-associated glycoprotein

MAPK

mitogen-activated protein kinase

MEA

multi-electrode array

NeuN

neuronal nuclei

NGF

nerve growth factor

SDS

sodium dodecyl sulphate

STAT

signal transducer and activator of transcription

TNF

tumour necrosis factor

YFP

yellow fluorescent protein

Injury to the adult CNS very often leads to irreversible damage owing to the limited capacity of neuronal networks to regenerate and repair. This is in contrast with the PNS where axon regeneration is more readily achieved. The mechanisms underlying the limited ability of the CNS to regenerate have been under extensive study in recent decades and have been shown to primarily involve neuronal growth inhibitors such as myelin-associated glycoprotein (MAG) and Nogo-A (Caroni et al. 1988; Schwab 1990b; Buchli and Schwab 2005). There are also a number of other mechanisms or released factors, particularly those involved in the inflammatory astroglial response associated with injury, that can affect or modulate the capacity for repair. The role of inflammation and scar formation remains, however, controversial (Mutlu et al. 2004), probably because of the complex mechanisms involved and the variety of factors likely to affect recovery.

Among these, interleukin (IL)-6 is probably an important mediator contributing to the response to injury (Gadient and Otten 1997). IL-6 and its receptor are constitutively expressed at a low concentration in many brain regions such as hippocampus, cerebellum and neocortex (Gadient and Otten 1994a,1994b). Its cellular sources are astrocytes, microglial cells (Benveniste et al. 1990; Woodroofe and Cuzner 1993) and also neurones (Ringheim et al. 1995). Importantly, IL-6 levels have been found to be markedly increased in various conditions of brain injury, including ischaemia (Mogi et al. 1994), trauma (Kossmann et al. 1995; Mutlu et al. 2004), but also in degenerative diseases such as Alzheimer's disease (Zhang et al. 2004), Parkinson's disease (Mogi et al. 1994) and multiple sclerosis (Kerschensteiner et al. 2004).

The precise role of IL-6 in response to injury, however, remains unclear (Gadient and Otten 1997). In some instances IL-6 appears to exert neuroprotective effects (Penkowa and Hidalgo 2000; Penkowa et al. 2000; Yamashita et al. 2005), but in other studies it has been shown to promote degenerative mechanisms (Yamada and Hatanaka 1994; Quintanilla et al. 2004). Furthermore, the role of IL-6 in the astroglial reaction, in functional synaptic activity and, more importantly, in the regenerative response of injured axons is little characterized. Two studies of the PNS have suggested a positive action of IL-6 on the capacity to regenerate (Hirota et al. 1996) as well as a correlation between IL-6 mRNA expression and axon regeneration (Streit et al. 2000). In addition, IL-6 knockout mice show defects in regeneration of sensory axons. However, the role of IL-6 in regenerative mechanisms in the CNS is not known.

In this study we addressed this issue by investigating the role of IL-6 in the mechanisms of axonal sprouting and functional recovery following a lesion in an in vitro mouse model of regeneration in organotypic hippocampal slice cultures. We found that blockade of lesion-induced release of IL-6 prevents functional recovery, whereas treatment of slice cultures with exogenously applied IL-6 promotes the regenerative response, assessed using functional, biochemical and morphological parameters. Taken together, these data provide direct evidence that IL-6 is able to regulate and promote sprouting and postlesion recovery in the CNS, thus opening new and interesting therapeutic perspectives for its action on brain repair.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Organotypic hippocampal slice cultures

Transverse hippocampal slices (400 µm thick) were prepared from 5–6-day-old wild-type C57/Bl6 or Thy-1/yellow fluorescent protein (YFP) (Jackson laboratory; Feng et al. 2000) mice and maintained 17–21 days in culture on porous membranes (confetti, 6 mm diameter) as described previously for rats (Stoppini et al. 1991). The lesions were produced by sectioning the slice culture in two hemi-slices at the level of the CA3–CA1 junction (Fig. 1) using a razor blade. The lesion was produced at room temperature (22°C) with the slice maintained in regular culture medium. The two hemi-slices were then maintained in culture for the next 6–10 days. In many slices, electrical activity was monitored before and immediately after lesioning to test for the effects of sectioning; no evidence of spontaneous epileptiform activity or spreading depression was noted in the individual hemi-slices. To investigate the role of IL-6, slices were treated immediately after the lesion either with a purified rat anti-mouse IL-6 monoclonal antibody (anti-IL-6) or a rat isotype-matched control IgG or, in another series of experiments, with recombinant mouse IL-6 (all products obtained from BD PharMingen, Heidelberg, Germany). The antibodies and IL-6 were added to the culture medium and the treatment was repeated daily. All these experiments were carried out on slice cultures at 17–21 days in vitro (DIV).

image

Figure 1.  Age-dependent functional recovery in mouse hippocampal organotypic slice cultures. (a) Illustration of the arrangement of slice cultures on a MEA for the analysis of synaptic activity evoked across the lesion or within the CA3 region. Slices were cut with a razor blade between CA3 and CA1 (dotted line). Synaptic field potentials elicited in CA3 and recorded in CA1 (across the lesion; before) showed a complete elimination of responses 1 h after the lesion and a significant recovery 6 days after the lesion. In contrast, field responses recorded within the CA3 hemi-slice (CA3), although slightly reduced by the lesion, were still present 1 h and 6 days later. (b) Synaptic field potentials recorded across the lesion or within the CA3 area in young (7 DIV) slice cultures. Data are mean ± SEM of the amplitude of the responses evoked in the two regions as a function of time and expressed as percentage of the values measured before the lesion (time 0). (c) As in (b), but for older (21 DIV) slice cultures. Note the reduced recovery of field responses recorded across the lesion compared with responses obtained within the CA3 hemi-slice. *p < 0.05 versus CA3 hemi-slice (n = 19). (d) Propidium iodide staining experiment showing an absence of damaged neurones in the lesioned slice culture except for a few cells present within the lesioned area (arrows indicate the zone of transection). (e) Western blot indicating the absence of difference in the levels of NeuN in control, non-lesioned (NL) and lesioned (L) cultures of 7 and 21 DIV, 10 days after lesioning.

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Electrophysiological recordings using a multi-electrode array (MEA)

Slice cultures were recorded on sterilized and disposable cartridges that included a MEA consisting of 40 electrodes (30 µm thick) made of pure gold by plasma evaporation (Biocell Interface, SA, La Chaux-de-Fond, Switzerland). The MEA is built on a porous and transparent membrane that is maintained in a sandwich between two chambers: an upper chamber, which is the gas perfusion chamber for maintaining the tissue at the interface, and a lower one, which is the perfusion chamber that contains sterile culture medium under perfusion. The chambers and the array are assembled and inserted into a console unit (Biocell Interface) where the temperature (33°) and the perfusion parameters (10 μl/min) can be monitored precisely. The position of the slices on the electrodes can be visualized directly through the camera integrated within the connector. Each electrode can be assigned as stimulating or recording.

Three to four recording sessions were usually performed: one before lesioning, a second 1 h after creating the lesion, a third on day 6 after lesioning and in some experiments a fourth 10 days after lesioning. In all sessions, four electrodes located in the CA1 area were selected for the recordings of field responses evoked by two stimulating electrodes located in the CA3 area. This stimulation paradigm yielded the activity across the lesion. In addition to this, we also monitored activity in the CA3 hemi-slice, using two stimulating electrodes located in the dentate hilus and four other electrodes located in the CA3 area close to the site of lesion. This stimulation paradigm was used to assess the viability of the tissue. Stimulation parameters were kept constant in all sessions (pulses of 4 V, 0.5 ms, applied every 15 s) and the amplitude of responses recorded during a 10-min period were averaged. Control experiments showed that the field responses evoked and recorded in these slice cultures were abolished by antagonists of glutamate receptors, indicating their mainly synaptic origin. To produce the lesion, the slice cultures were sectioned as described above and replaced on the array in the same position as before the lesion, using a video image (Fig. 1a) for the exact localization. Data are expressed as mean ± SEM of the number of slice cultures analysed and statistical analyses were carried using the unpaired t-test.

Quantification of IL-6 release by ELISA

The culture medium from control and treated slice cultures was collected and the concentrations of IL-6 measured by ELISA using commercial kits (R & D Systems, Minneapolis, MN, USA). Briefly, 100 µL assay diluent was added to each well of a 96-well plate (precoated with an affinity-purified polyclonal antibody specific for the IL-6). Then, 100 µL of either standard, control medium or of slice culture medium was added per well. The 100 µL of slice culture medium was collected from three separate inserts (33 µL each) with four slice cultures per insert. The 96-well plates were incubated for 2 h at room temperature. The solutions of each well were aspirated and washed a total of five times (washed by filling each well with 400 µL wash buffer). The plates were inverted and blotted against clean paper towels. Rat IL-6 conjugate (100 µL) was then added to each well. The 96-well plates were incubated for 2 h at room temperature. The plates were washed and dried as described above. Thereafter, 100 µL of a substrate solution was added to each well. The plates were incubated in the dark for 30 min. Finally, 100 µL stop solution was added to each well. The optical density of each well was determined within 30 min by using a microplate reader set to 450 nm (wavelength correction set to 540 nm).

Electrophoresis and western blotting for growth-associated protein-43 (GAP-43), neuronal nuclei (NeuN) and β-actin

Hippocampal cultures were lysed in buffer containing 1% Nonidet P-40, 1% sodium cholate, 0.05% sodium dodecyl sulphate (SDS), 20 mm Tris (pH 7.5) and 100 mm NaCl, and then sonicated. Protein concentration was determined using the Bradford dye-binding assay (Bio-Rad, Hercules, CA, USA), according to the manufacturer's protocol, with bovine serum albumin as the standard. Fifteen micrograms of total protein was separated by SDS–polyacrylamide gel electrophoresis (10% gels) under reducing conditions and blotted on to pure nitrocellulose membranes (Bio-Rad). Membranes were incubated with either antibodies against GAP-43 (1 : 1000, polyclonal GAP-43; Chemicon, Temecula, CA, USA), β-actin (1 : 5000; Chemicon) or NeuN (1 : 5000; Chemicon), followed by a horseradish peroxidase-conjugated secondary antibody (1 : 5000; Bio-Rad). The immunoreactive proteins were detected with an enhanced chemiluminescence kit (ECL; Amersham Pharmacia, Piscataway, NJ, USA) according to the manufacturer's protocol, by using ECL-Hyperfilm (Amersham Pharmacia). Quantification of signal intensities in western blots was performed with a densitometer using ImageJ software (http://rsb.info.nih.gov/ij). Densitometric values are expressed as mean ± SEM in arbitrary units.

Co-culture experiment

A fluorescent YFP mouse strain [Tg(Thy1-YFP)16Jrs; C57Bl/6 × CBA founders, backcrossed for 1–4 generations in C57Bl/6] was used to visualize regeneration in slice cultures. Wild-type and Thy-1 slice cultures maintained for 17–21 days in vitro were lesioned as described above. The Thy-1/YFP CA3 half-slice was then apposed to the wild-type CA1 hemi-slice and maintained in cultures on a confetti of 10 mm in diameter. Fluorescence analyses were carried out using a confocal microscope and 10 × or 40 × objectives. The level of regrowth of fluorescent YFP fibres within the CA1 region was quantified by measuring the level of fluorescence detected within a given constant field located in the CA2 region apposed to the lesion. Fluorescence intensity, calculated as the sum of the pixel values of the entire image minus background, was quantified using ImageJ software. Data are expressed in arbitrary units ± SEM. Control electrophysiological experiments with lesioned YFP slice cultures showed no differences from control, C57Bl/6 cultures.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Mice organotypic hippocampal slice cultures have an age-dependent capacity to regenerate

To investigate the role of IL-6 in regeneration, we used the model of Schaffer collateral lesion in mouse hippocampal organotypic slice cultures (Stoppini et al. 1993, 1997; McKinney et al. 1997, 1999). Slices were sectioned into two pieces using a razor blade and regeneration was assessed by monitoring synaptic activity across the lesion using a MEA (Fig. 1). For each recording session, we measured and averaged the amplitude of field responses recorded across the lesion through four separate CA1 recording electrodes. We also analysed, using the same approach, the responses elicited within the CA3 hemi-slice in order to evaluate the viability of the tissue. Measurements were carried out before, 1 h after and then 6 and 10 days after production of the lesion. For each slice, data were expressed as a percentage of values recorded before production of the lesion. As illustrated in Fig. 1(a), responses evoked across the lesion were fully abolished when recordings were carried out 1 h after lesioning, indicating complete sectioning of the connections between CA3 and CA1 neurones. Field potentials then progressively recovered within the next 6–10 days. In contrast, field responses evoked in the hilus or dentate gyrus and recorded in the CA3 area were not abolished by the lesion and could be continuously recorded throughout the experiment. Their amplitude was, however, usually reduced after hemi-section of the slice (Fig. 1b), probably reflecting some cell loss or damage produced to CA3 neurones close to the lesion. Cell loss was, however, probably minor, as propidium iodide staining carried out after lesioning only indicated the presence of a few dying cells in the vicinity of the lesion site (Fig. 1d). Furthermore, the NeuN content measured by western blotting showed no differences between control and lesioned slices (Fig. 1e).

These CA3 responses were therefore used as controls to monitor cell survival in the tissue compared with recovery across the lesion. Figures 1(b) and (c) show the level of amplitude of responses evoked across the lesion when transection was carried out in 1- and 3-week-old slice cultures. In agreement with previous studies, we found that functional recovery was almost complete 6 and 10 days after the lesion when transection was carried out in very young, 1-week-old slice cultures (Fig. 1b). The mean amplitude of responses across the lesion recovered to 71.2 ± 9 and 70.7 ± 11.5% of the initial activity after 6 and 10 days (n = 19) respectively, which was close to the values obtained in the non-lesioned CA3 area (75.1 ± 10% and 76.6 ± 9.9; n = 19). In contrast, recovery was considerably reduced and incomplete when transection was carried out in older, 3-week-old tissue. The amplitude of responses across the lesion recovered to only 28.5 ± 11.6 and 32.4 ± 11.7% after 6 and 10 days respectively (n = 21), and was much lower than values in the non-lesioned CA3 area (74.7 ± 5.6 and 76.2 ± 14%; n = 19, p < 0.01). These results indicated that the sprouting and recovery response that followed hemi-section of a slice culture showed similar properties in mice to those previously reported in rat (Stoppini et al. 1993, 1997).

Lesion-induced release of IL-6

IL-6 and its receptor have been shown to be expressed at low concentrations in various brain regions including the hippocampus (Schobitz et al. 1993; Gadient and Otten 1994a, b) and this capacity to release IL-6 is also retained in organotypic slice cultures (Huuskonen et al. 2005). We tested whether production of a lesion to Schaffer collaterals affected the release of IL-6 in our mouse model. For this, we used 21 DIV slice cultures, collected the supernatant from intact and lesioned cultures 1 and 2 days after lesioning, and assessed the IL-6 content using an ELISA. As shown in Fig. 2, the level of IL-6 in the supernatant from intact cultures was rather low, varying in the two sets of cultures used as unlesioned controls between 2 and 5 pg/mL. However, when the slice culture was sectioned and maintained in culture for an additional 1 or 2 days, the level of IL-6 recovered in the supernatant increased markedly (by about an order of magnitude) 1 day after lesioning (80 ± 6 pg/mL; n = 9, p < 0.001). This level then progressively decreased to basal values over the next day, and was not significantly higher than that in control slices 2 days after creating the lesion (8 ± 2 pg/mL; n = 9, p < 0.05). Thus production of a lesion in slice cultures resulted in a marked release of IL-6 that peaked within the day after lesioning.

image

Figure 2.  Release of IL-6 by transection of hippocampal slice cultures. The amount of IL-6 measured by ELISA and recovered in 100 µL of culture medium from hippocampal slice cultures (21 DIV) collected from control, non-lesioned slices (white bars), from slices 1 and 2 days after lesioning (black bars), or from slices 1 and 2 days after lesioning but treated with different concentrations of IL-6 antibody (10 , 1 and 0.1 µg/mL; hatched bars). Data are mean ± SEM values obtained from three wells in three different series of experiments. Note the marked increase in IL-6 detected in the culture medium at 1 day, but not 2 days after the lesion. This IL-6 release could be fully neutralized by treatment of slice cultures with an IL-6 antibody. *p < 0.001 versus non-lesioned control(n = 9). Ctrl, control.

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In order to investigate the role of IL-6 in this process, we incubated the tissue with different concentrations of neutralizing IL6 antibody, with the aim of interfering with activation of the IL-6 receptor. Preincubation of slice cultures with different concentrations of blocking IL-6 antibody (10, 1 and 0.1 µg/mL) did indeed prevent the lesion-induced release of IL-6 recovered in the supernatant (Fig. 2). At the three concentrations tested, the level of IL-6 detected in the supernatant of lesioned cultures 1 and 2 days after the lesion was no longer different from that obtained in intact, non-lesioned tissue. The antibody was therefore able to neutralize the released IL-6 sufficiently effectively to prevent its detection in the supernatant.

Effect of IL-6 blockade on functional recovery and GAP-43 expression

We then used this same condition to investigate the role of IL-6 in the lesion-induced regenerative response. Different concentrations of IL-6 neutralizing antibody were added to the culture medium of 17–21 DIV cultures immediately after the mechanical cut and maintained throughout the next 6 days. Six days after lesioning, functional recovery was assessed using the MEA as described above. Fig. 3(a) illustrates representative field responses obtained across the lesion by one of the four recording electrodes in control (treated with 10 µg/mL rat IgG1) and anti-IL-6 Ig-treated slices before (black trace) and after (grey trace) lesioning. For the three concentrations of anti-IL-6 Ig tested the field potentials elicited across the lesion were significantly smaller [19.9 ± 3.4% (n = 13), 16.7 ± 5.32% (n = 14) and 20.63 ± 3.76% (n = 14) for 10, 1 and 0.1 µg/mL anti-IL-6 Ig respectively] than those recorded under control conditions (10 µg/mL rat IgG: 35.2 ± 5.34%; n = 13, p < 0.05). In contrast. analysis of evoked responses within the CA3 region of transected slice cultures showed no significant differences between the anti-IL-6 Ig-treated groups and control slices (72.4 ± 6.3, 70.9 ± 9.8, 72.4 ± 10.6 vs. 72.9 ± 10 for 10, 1 and 0.1 µg/mL anti-IL-6 Ig respectively vs. 10 µg/mL control rat IgG). This suggests that the anti-IL-6 Ig treatment decreased the regenerative response, without affecting cell survival or synaptic transmission. To test this further, we treated intact slice cultures with a blocking antibody for 6 days (Fig. 3d) and analysed how this affected basal synaptic transmission. Again, no differences in the size of synaptic field responses recorded after 6 days could be detected when compared with the initial synaptic activity (amplitude ratios 85.2 ± 17, 92.6 ± 17, 92 ± 22.7 vs. 89.1 ± 12.5% for 10, 1 and 0.1 µg/mL anti-IL-6 Ig respectively vs. 10 µg/mL control rat IgG; n = 5).

image

Figure 3.  Reduced functional recovery following treatment with a blocking IL-6 antibody. (a) Examples of the evoked synaptic responses recorded across the lesion before (black trace) and 6 days after (grey trace) lesioning in control, IgG1-treated cultures (upper panel) and anti-IL-6 IgG-treated slices (lower panel; slice culture age 17–21 DIV). (b) Amplitude of synaptic field responses recorded across the lesion 6 days after the lesion under control conditions and in the presence of different concentrations of IL-6 antibody (10, 1 and 0.1 µg/mL). Data are mean ± SEM of the results obtained in 13–14 slice cultures. *p < 0.05 versus control. (c) Same as in (b), but for the field responses recorded within the CA3 hemi-slice. (d) Amplitudes of evoked field responses recorded in the presence of control, IgG1 antibody or IL-6 IgG antibody in non-lesioned slice cultures, indicating an absence of effect of the antibodies on synaptic transmission (n = 5). EPSP, excitatory postsynaptic potential.

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To complement these data and obtain further confirmation that the sprouting response might indeed be affected, we also measured the level of GAP-43 expression in control and treated slice cultures using western blotting. GAP-43 is a protein known to be expressed in growing axons of the peripheral and central nervous systems (Meiri et al. 1986), and its expression level has been shown to correlate with the regenerative response of newly formed axons in lesioned slice cultures (McKinney et al. 1997, 1999). We compared the level of expression of GAP-43 6 days after the lesion in control and rat-IgG-treated slices and in cultures treated with the neutralizing IL-6 antibody. Figure 4(a) shows representative blots obtained from one experiment. Although no differences in β-actin level were observed, GAP-43 appeared to be clearly expressed at a higher level in the control group than in the anti-IL-6 condition. Quantification of the optical densities under these conditions showed that, in comparison with the control group, the anti-IL-6-treated groups displayed a markedly reduced content of GAP-43 (70.8 ± 6, 71.3 ± 10 and 63.1 ± 4% percent of control for 10, 1 and 0.1 µg/mL anti-IL-6 Ig respectively; n = 8, p < 0.05) (Fig. 4b). Taken together, these results indicated that interference with the lesion-induced release of IL-6 in transected slice cultures reduced the axonal regrowth response and decreased the capacity for re-establishment of functional synaptic transmission. This suggests a positive action of IL-6 on regeneration.

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Figure 4.  Reduced expression of GAP-43 by a blocking IL-6 antibody in transected cultures. (a) Western blot for GAP-43 and β-actin in transected slice cultures (17–21 DIV) 6 days after treatment with either a control IgG1 antibody (Ctrl) or an anti-IL-6 IgG applied at the indicated concentrations. (b) Relative optical density obtained for GAP-43 immunoreactivity under control conditions and in anti-IL-6 IgG-treated slices. Note the decrease in GAP-43 level in anti-IL-6-treated slices. *p < 0.05 versus control (n = 8).

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PEffect of exogenous IL-6 on functional recovery and axonal sprouting

In order to test the possibility that IL-6 could improve regeneration, we carried out experiments using 17–21 DIV cultures showing only partial regeneration, and applied exogenous, recombinant IL-6 to the culture medium for a period of 6 days. In a first group of studies we analysed synaptic activity across the lesion using the multielectrode array. Figure 5(a) illustrates recordings obtained before (black trace) and 6 days after (grey trace) transection across the lesion in control and IL-6-treated cultures. Interestingly, we found that the mean amplitude of field responses recovered in CA1 following stimulation in CA3 (Fig. 5b) was larger in slices treated with 150 ng/mL IL-6 than in untreated controls (53.2 ± 7.3% vs. 29.1 ± 7.2% of the initial activity; n = 13, p < 0.01). The difference represented an almost two-fold improvement in functional recovery of these older slice cultures, similar to the functional recovery obtained in young, 1-week-old cultures (Fig. 1). Analyses made in the CA3 regions of these hemi-sectioned slices also showed no intergroup differences between control and IL-6-treated cultures (71.8 ± 7.7 and 72.5 ± 10.8% respectively; n = 14). Moreover, measurement of evoked field potentials in intact slices treated for 6 days with IL-6 revealed no differences between the two groups (amplitude ratio 96.6 ± 11.5 and 93.4 ± 8% respectively; n = 6). Together, these results indicated that IL-6 had no detectable effects on basal synaptic transmission under the conditions used and rather pointed to an improvement of the regenerative response.

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Figure 5.  Enhanced functional recovery following exogenous application of IL-6 to transected slice cultures. (a) Examples of the evoked field responses recorded across the lesion before (black trace) and 6 days after (grey trace) lesioniong in control (upper panel) and IL-6-treated (150 ng/mL) slice cultures (lower panel; 21 DIV). (b) Amplitude of synaptic field responses recorded across the lesion after 6 days under control conditions (Ctrl) and following treatment with IL-6 (150 ng/mL). Data are mean ± SEM of the values obtained in 14 slice cultures. *p < 0.05 versus control. (c) Same as in (b), but for the field potentials recorded within the CA3 hemi-slice (n = 14). (d) Treatment of slice cultures with 150 ng/mL IL-6 did not alter synaptic field responses in CA1 (n = 6). EPSP, excitatory postsynaptic potential.

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To examine this further, we investigated GAP-43 expression levels using western blot analysis and the same paradigm. As illustrated in Fig. 6(a), the level of GAP-43 found in IL-6-treated and control slices following a lesion showed a clear increase in signal when IL-6 had been added to the culture medium, thus corroborating the electrophysiological data. Quantitative analyses of the optical density observed in six different experiments with different concentrations of IL-6 confirmed this result. As illustrated in Fig. 6(b), GAP-43 expression after lesioning was significantly increased in a dose-dependent manner when recovery occurred in the presence of 10 and 100 ng/mL IL-6, but not with 1 ng/mL (100 ng: 137 ± 6%n = 20, p < 0.01; 10 ng/mL: 122.2 ± 5%, n = 24, p < 0.05; 1 ng/mL: 107.8 ± 5%, n = 19, p < 0.05). This effect, however, was not due to a non-specific increase in GAP-43 expression produced by IL-6, because no differences in GAP-43 expression level were observed if slices were maintained in the presence of IL-6 but without lesioning (Fig. 6a). Taken together these data clearly supported the idea that the delivery of IL-6 in the culture medium during the recovery period promoted functional recovery and the reactive axonal regrowth response.

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Figure 6.  Increased expression of GAP-43 by exogenous application of IL-6 to transected slice cultures. (a) Western blots for GAP-43 and β-actin in transected slice cultures (21 DIV) 6 days after treatment with 150 ng/mL IL-6 (left panel). Control experiments in non-lesioned (NL) slice cultures showed that IL-6 treatment did not affect levels of GAP-43 expression (right panel). (b) Relative optical density obtained for GAP-43 immunoreactivity under control conditions and in slice cultures treated with different concentrations of IL-6 (1, 10 and 50 ng/mL). Data are mean ± SEM of the results obtained from 20 slices. *p < 0.01 versus control.

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Analysis of axonal sprouting using co-cultures of YFP expressing mice

In order to establish the beneficial effect of IL-6 on axonal sprouting in a more direct way, we developed a morphological approach in which growing axons could be directly visualized and assessed. We performed co-culture experiments in which the CA3 hemi-slice obtained from a transgenic mouse expressing the fluorescent protein YFP in the hippocampus (Feng et al. 2000) was apposed to a hemi-slice obtained from a wild-type animal. These YFP/ wild-type co-cultures were maintained in vitro for the next 6 days in the presence or absence of IL-6. The regrowth reaction involving YFP-positive axons from CA3 cells growing into the CA1 could be assessed by analysing the level of fluorescence visualized in the stratum radiatum of the CA1 region adjacent to the lesion (Fig. 7a). Comparison of images obtained from co-cultures treated with IL-6 with those from control co-cultures indicated that a greater number of fluorescent fibres could be seen crossing the lesion site as a result of IL-6 treatment (Figs 7c and d). Quantification of the level of fluorescence by summation of pixel values measured in the same reference frame in four different co-cultures per condition indicated a clear increase in fluorescence levels in IL-6-treated compared with control co-cultures (n = 4, p < 0.05) (Fig. 7b). These results supported the conclusion that exogenously applied IL-6 promoted the regrowth reaction induced by a lesion in hippocampal organotypic slices.

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Figure 7.  Enhanced axonal sprouting promoted by IL-6 treatment of transected slice cultures. (a) Low-magnification views of a co-culture between a YFP-CA3 and a wild-type (wt) CA1 hemi-slice (21 DIV) illustrated with the corresponding bright-field (upper panel) and fluorescence (lower panel) images. The lesion site is indicated by the arrowheads (upper panel) and the dotted line (lower panel). The dotted square indicates the area in which fluorescence intensity was assessed 6 days after lesioning. (b) Fluorescence level (mean ± SEM arbitrary units) measured in the area bounded by the dotted square illustrated in (a) in control, YFP/wt co-cultures and co-cultures treated with 150 ng/mL IL-6. *p < 0.05 versus control (Ctrl) (n = 4). (c) Illustration at higher magnification of the sprouting fluorescent axons observed in the area of analysis (dotted square in a) in a control YFP/wt co-culture. (d) Same as in (c), but following 6 days' treatment with 150 ng/mL IL-6.

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Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study provides strong evidence supporting the conclusion that the pro-inflammatory cytokine IL-6 promotes axonal regrowth and network repair of CNS tissue following a lesion. This conclusion is based on several observations made using an in vitro model of lesioned hippocampal slice cultures. First, we showed that IL-6 was released by hippocampal tissue during the 48 h after production of a lesion. Second, we found that interference with lesion-induced IL-6 release using a neutralizing antibody reduced sprouting and functional recovery, indicating a positive action of the endogenously released IL-6 on the regenerative response. Finally, we provided evidence that exogenous application of IL-6 in the culture medium enhanced sprouting and functional recovery. The regenerative response in these experiments was assessed using a multidisciplinary approach based on electrophysiological recordings, western blot analyses of GAP-43 expression, which is directly related to sprouting, and confocal imaging of fluorescent fibres crossing the lesion site. Overall, the results obtained with these different approaches were consistent and thus clearly point to a modulation of the regenerative capacity of CNS neurones by IL-6. This is in line with work carried out in the PNS (Hirota et al. 1996) and with other studies suggesting a positive action of IL-6 on sprouting in a model of dopaminergic cell toxicity (Parish et al. 2002). Finally, this is also consistent with the report of defects in regeneration of sensory axons in IL-6 knockout mice (Cafferty et al. 2004).

The model of lesioning of Schaffer collaterals used here in organotypic slice cultures has been validated in several previous studies using similar parameters to assess sprouting and regeneration (Stoppini et al. 1993; McKinney et al. 1997, 1999). This model has features that are very similar to what occurs in vivo. The slice culture, which remains three-dimensional, shows many properties of in situ hippocampus both in terms of organization, morphology and function (Gahwiler 1988). Hemi-sectioning of the slice is associated with a repair process that involves proliferation of astrocytes and formation of a scar (Stoppini et al. 1997) as happens in the CNS. The regrowth response is developmentally regulated and considerably reduced in older tissue (Stoppini et al. 1997), a process that could be related to the progressive myelination that takes place at this time of development in the hippocampus and the influence of myelin-associated inhibitors on regeneration (Schwab et al. 2006). The effects of IL-6 reported here are thus likely to be relevant and representative of an involvement of IL-6 in brain repair mechanisms. Although these effects were obtained at rather high doses of IL-6, the results in Fig. 6 show that they were dose dependent and already apparent at concentrations below 100 ng/mL. Furthermore, although the various parameters analysed here strongly indicate an effect on axon regrowth and regeneration, another possible aspect of IL-6 action could involve neuroprotection and promotion of cell survival. Such an effect has been reported in the case of cultured neurones or ischaemia (Hama et al. 1989; Zhang et al. 2004). It does not seem, however, that such a neuroprotective effect accounts significantly for the results obtained here. Although sectioning a slice culture did result in some damage to neurones close to the site of the lesion and some decrease in synaptic responses, control experiments showed that IL-6 treatment did not significantly alter field responses and synaptic transmission evoked within each hemi-slice or the level of GAP-43 expression in intact tissue. This leads to the conclusion that the main action of IL-6 in this slice culture model was to promote regrowth and recovery.

The cellular and molecular mechanisms by which IL-6 exerted its beneficial effect on axonal regrowth and functional recovery remain to be understood. Several possibilities can be considered, based either on direct or indirect effects on axonal sprouting. It is likely that the regeneration response to the lesion involves complex regulation of several cell types and, in particular, an astroglial reaction. It is significant that IL-6 and IL-6 receptors are expressed in the hippocampus by astrocytes, microglia and neurones. There is also evidence in other models of lesions, particularly in the model of entorhinal cortex lesion, that proliferation of astrocytes and phagocytic activity by microglial and astrocytic cells plays an important role (Bechmann and Nitsch 1997). It might be assumed, therefore, that IL-6 could promote this reaction, facilitating the removal of debris and proteins such as growth-inhibiting myelin proteins (Schwab 1990a) and consequently enhance sprouting. An involvement of IL-6 in astrocyte proliferation and differentiation has indeed been reported (Marz et al. 1999).

Alternatively, IL-6 might affect or interfere with the release of some of the numerous factors that contribute to the response to injury. Synthesis and release of multiple trophic factors, such as nerve growth factor by astrocytes (Kossmann et al. 1996) or brain-derived neurotrophic factor and glia-derived neurotrophic factor by microglial cells (Batchelor et al. 1999) have been reported following injury. More importantly IL-6 appears to inhibit the synthesis of tumour necrosis factor (TNF)-α produced by astrocytes (Benveniste et al. 1995) and released under various conditions of injury, including in our model of hippocampal slice culture lesion (data not shown). TNF-α has been shown to inhibit neurite outgrowth in cultured neurones and this effect appears to result from activation of the GTPase RhoA (Neumann et al. 2002), known to be involved in the intracellular pathway of signal transduction mediated by Nogo-A and MAG (Niederost et al. 2002; Schwab et al. 2006). Thus, IL-6 might actually promote sprouting by counteracting the release and/or the effects of TNF-α.

Finally, and in addition to this, IL-6 might exert a more direct action on neuronal regrowth itself. IL-6 is a member of the glycoprotein-130 receptor family that activates the Janus-associated kinase/signal transducer and activator of transcription (STAT) and mitogen-activated protein kinase (MAPK) pathways (Heinrich et al. 1998; Schumann et al. 1999), a result also confirmed in organotypic hippocampal slice cultures (Pizzi et al. 2004). These different pathways have been proposed to play important roles in the intracellular signalling mechanisms triggered by injury or associated with synaptic plasticity. Phospho-STAT3 immunoreactivity, for example, has been detected in sprouting cholinergic neurones following a lesion (Xia et al. 2002). Furthermore, the MAPK pathway is associated with cyclic AMP responsive element-binding protein signalling and proposed to regulate plasticity programmes. IL-6 might thus activate these signalling cascades and in this way promote regeneration in lesioned neurones (Teng and Tang 2006). The inability of IL-6 null mice to regenerate could support this interpretation (Cafferty et al. 2004). The possibility of either inhibiting or promoting regeneration by applying IL-6 antibodies or by adding exogenous IL-6 respectively, as shown in this study, provides an experimental model in which some of hypotheses can be investigated. The observation that IL-6 can exert a positive effect on regrowth and regeneration of CNS axons opens new avenues into possible therapeutic applications.

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Special thanks to Rosy Bonfante, Caroline Waltzinger, John Challier, John Donnely, Marlis Moosmayer and Chantal Alliod for their technical support. This work was supported by Serono SA (DH) and by the Swiss National Science Foundation (grant 3100A0-105721 to DM).

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  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
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