In the present study we show for the first time that IL-1β exerts detrimental effects on the plasticity of CNS axons after SCI. These data are in striking contrast to our recent in vitro study, which revealed that IL-1β acts as a potent inducer of axon outgrowth from organotypic brain slices in vitro. IL-1β is one of the most extensively studied proinflammatory cytokines; however, controversial debate continues as to its function in the CNS. In vivo, IL-1β may exert detrimental effects on damaged nervous tissue
, but some evidence exists for a beneficial impact on myelination
 and on peripheral nerve regeneration following sciatic nerve injury
[16, 17]. After SCI, IL-1β and its receptor are upregulated in rodents and humans
[13, 30, 31] in all resident cells of the CNS (including neurons, but mainly astrocytes and microglia), with a peak of mRNA expression 12 hours after lesion
. A functional consequence of increased IL-1β expression may be apoptosis induction, suggested by a threefold increase in caspase-3 activity that can be reversed by administration of the IL-1 receptor antagonist for 72 hours following lesion
. Direct application of IL-1β has also been shown to affect the behavioral outcome after glutamate-induced experimental spinal cord injury
. Even if the precise mechanism of action of IL-1β is still not perfectly clear, an extensive literature describes the pathways that are influenced by this cytokine. Of particular interest is the observation that IL-1β injections stimulate macrophage activation and myelin clearance in spinal cord white matter, while an absence leads to an increased number of intact myelin sheaths
. Furthermore, IL-1β application abrogates neurotrophin-induced neuronal cell survival in vitro[35, 36]. Moreover, administration of IL-1β intrathecally activates p38 mitogen-activated protein kinase, and leads to high levels of inducible nitric oxide synthase and release of nitric oxide
. However, none of these studies investigated the influence of IL-1β on CNS plasticity. The present study demonstrates in vivo that the sum of all these negative effects in the CNS appears to abrogate potential IL-1-dependent axon elongation expected from our recent in vitro study
Here, we provide the first in vivo evidence for a substantial IL-1β effect on plasticity and lesion development. We analyzed the effect of locally applied rIL-1β and its constitutive deficiency on functional recovery, CST fibers and astrogliosis in a mouse model of SCI, which mimics the most common type of spinal cord injury in humans
. Based on previous in vitro findings demonstrating that a high therapeutic dosage of rIL-1β increases axonal outgrowth in an organotypic slice culture model
 we administered perilesionally a high dose of rIL-1β after SCI (20 μg rIL-1β in Gelfoam). Unfortunately, this local application was lethal. However, a lower, nonlethal dosage of rIL-1β (1 μg rIL-1β in Gelfoam) led to a significantly impaired functional recovery according to the BMS. We used a mild lesion to ensure that any possible negative effect of the application of the cytokine could be revealed. This treatment also resulted in a highly reduced number of BDA-positive CST fibers caudal to the lesion compared with PBS-treated mice. Consistently, the analysis of the injured spinal cords of IL-1β-deficient mice revealed a close to fivefold increase in the number of CST fibers caudal to the lesion compared with WT mice.
These mice also displayed a significantly improved neurological outcome. Based on morphological criteria
, BDA-positive fibers counted caudal to the lesion appeared to present newly formed fibers, but due to the nature of the lesion we cannot exclude a small percentage of (undetected) sparing. Counted fibers could thus be a mixture of newly established fibers derived from the site of the lesion and of those sprouting from uninjured fibers.
It is reasonable to assume that multiple spinal motor systems are positively affected by the absence of IL-1β (and the consecutively reduced astrogliosis as discussed hereafter). The improvement in paw placement as an indicator of CST function
[27, 28] and increased numbers of anterogradely labeled CST nerve fibers in the IL-1β-deficient injured spinal cord support the concept that enhanced CST plasticity may significant contribute to the improved clinical outcome demonstrated in our study.
Substantial differences between the in vivo and in vitro model may explain why IL-1β stimulates neurite outgrowth in vitro but has a negative impact on axon plasticity in the present in vivo study. The in vitro study was performed using organotypic slice cultures from postnatal brains as previously described
[22–25]. Acute brain slices as used in our study, should be considered a model for the early, highly acute phase of CNS trauma since they are acutely excised from of the living brain; most neurons are axotomized, the blood–brain barrier is heavily damaged, high levels of neuronal death appear, and astrocytes as well as many immune cells are activated
[39, 40]. Contrastingly, in the in vivo model used here, the SCI is followed by at least three different inflammatory phases (acute, subacute and chronic) that are characterized by dramatic differences, for example, in terms of cytokine levels as well as immune cell activation and migration patterns
. The in vivo effects of IL-1β on functional parameters become clearly detectable about 2 weeks after the highly acute phase. IL-1β may therefore exert direct and/or indirect effects mainly in the subacute or early chronic phase. In a landmark paper, the Kapfhammer group demonstrated that neurite outgrowth can only be reliably studied in embryonic and postnatal brain slices until 4 days after birth
. Brain slice experiments are therefore performed with embryonic or early postnatal brains. In later developmental phases, neurite outgrowth from slices is substantially reduced or absent. A further difference between the models is the absence of systemic neuroendocrinological influences in the brain slice model. The contradicting results in our brain slice study in vitro and the present in vivo study may thus be due to differences in the CNS region (cortex versus spinal cord), the inflammatory phase, the developmental stage (postnatal versus adult) or the presence or absence of systemic neuroendocrinological influences.
In the present study we also show significantly reduced astrogliosis in the perilesional white matter in IL-1βKO mice. This is in line with the role of IL-1β as an astroglial growth factor in the mammalian brain (promoting proliferation)
 and with the IL-1β-dependent astrocyte activation following CNS injury, demonstrated in a murine corticectomy model
, leading to exacerbated astrogliosis. However, in contrast to these studies, which describe early stages after brain injury, we here demonstrate that reduced astrogliosis in the absence of IL-1β is still present 2 weeks after injury. Consistently, we also find a greatly reduced lesion size (and consequently more spared spinal cord tissue).
While the results of the present study are encouraging to further investigate IL-1β modulation for spinal cord repair, they should be interpreted with care keeping in mind the complexity of the plethora of potential mechanisms as outlined in the introduction. Further studies are needed to elucidate the intricate network of IL-1β mechanisms of action after SCI, which is likely to be multifaceted and not limited to demyelination, cell death and cytotoxic neuroinflammation.