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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.
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.
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.
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- Materials and methods
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.