Therefore, we investigated whether IL-1 has a comparable negative impact on NT-3-induced neurite growth from organotypic brain slices and transverse spinal cord slices

Therefore, we investigated whether IL-1 has a comparable negative impact on NT-3-induced neurite growth from organotypic brain slices and transverse spinal cord slices. NT-3 significantly increased the number of brain slices displaying maximal neurite growth compared to single treatments. These data show that these two factors synergistically stimulate two unique aspects of neurite outgrowth, namely neurite density and neurite length from acute organotypic brain slices. Keywords:interleukin 1 beta, IL-1, neurotrophin 3, NT-3, NGF, spinal cord, brain slices, neurite growth, axon outgrowth, neuroplasticity == Introduction == Interleukin-1 beta (IL-1) is usually a member of the IL-1 family of cytokines which have potent pro-inflammatory properties. It is produced in the periphery mainly by monocytes and is a strong activator of the host immune response to both injury and contamination [1,2]. In the central nervous system (CNS) IL-1 is usually primarily produced by microglia and invading monocytes/macrophages, but other types of resident cells of the nervous system, including neurons and astrocytes, are also capable of its production [3]. It is generally believed that inflammatory processes stimulated by pro-inflammatory cytokines and particularly by IL-1, are rather detrimental and can aggravate the primary damage caused by infection of the CNS. This has been suggested by variousin vivostudies, in line with its enhanced expression in the brain after damage or in neurodegenerative diseases, including Alzheimer’s disease (AD). Consistently, IL-1 deficient mice display reduced neuronal loss and infarct volumes after ischemic brain damage [4] and direct application of the recombinant cytokine results in an enhanced infarct volume [5]. In traumatic brain injury, antibodies against IL-1 reduce the loss of hippocampal neurons [6]. Consistently, in a mouse model of AD, an inhibitor of pro-inflammatory cytokine production suppressed neuroinflammation leading to a restoration of hippocampal synaptic dysfunction markers [7]. In AD it has also been exhibited that members of the IL-1 family are associated with an increased risk of contracting the disease [8]. The findings in variousin vitromodels suggest a rather elaborated mechanism. In culture, IL-1 exhibited neurotoxic effects towards hippocampal neurons exposed to high concentrations (500 ng/ml) combined with long-term exposure (three days). However, no effect was observed in lower concentrations following short-term exposure (one day) [9]. In otherin vitromodels, IL-1 has even been seen to display beneficial effects towards neuronal survival in the CNS [10,11]. This has also been observed in axonal growth in the peripheral nervous system bothin vivofollowing sciatic nerve injury [12,13] andin vitroin adult dorsal root ganglion (DRG) collagen gel explant cultures [14], but not in dissociated single DRG neuron cultures [15]. Previously, it has been exhibited that IL-1 impairs neurotrophin-induced neuronal cell survival [16,17]. It has long been hypothesized that cytokine effects on neurite growth may be mediated at least in part by modulating neurotrophin signalling accordingly [18]. In addition to their positive effect on cell death, the neurotrophins Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3) and NT-4 have also a well documented impact on axon plasticity and regeneration [19,20]. This is crucial in the context of CNS insult to provide re-innervation and thus consecutive functional recovery. Based on these observations we investigated whether IL-1 is also a modulator of neurotrophin-induced neurite outgrowth in the CNSin vitro, using organotypic brain and spinal cord slice cultures. The present study shows that surprisingly, IL-1 LXR-623 did not abrogate NT-3-induced neurite outgrowth but conversely showed a significant synergistic effect. These data show that IL-1 differentially regulates the effect of NT-3 on neuronal survival and neurite extension. == Materials and methods == == Animals and factors == C57BL/6 wildtype mice and LXR-623 IL-1-deficient mice [21] were housed in a conventional animal facility (Center for Anatomy, Charit-Universittsmedizin, Berlin, Germany). All experiments were performed in accordance with German guidelines on the use of laboratory animals. Recombinant neurotrophins NGF, BDNF, NT-3 and NT-4 were used in a concentration of 500 ng/mL (all Tebu-Bio, Offenbach, Germany). Recombinant IL-1 (Tebu-Bio, Offenbach, Germany) was used in concentrations of 5, 50 and 500 ng/mL. == Acute organotypic brain slice culture == LXR-623 The entorhinal slice cultures were prepared from mouse brains at postnatal day 2 as previously explained [22-25]. In brief, after decapitation, the entorhinal cortex was dissected in ice-cold preparation medium, made up of MEM with L-Glutamine (2 mM) and Trisbase (8 mM). Transverse slices 350 Rabbit polyclonal to ACAD9 m solid were cut using a tissue chopper (Bachhofer, Reutlingen, Germany). Collagen was prepared as previously explained [26]. Each entorhinal slice was LXR-623 embedded in a drop of collagen matrix on glass slides. The recombinant factors (neurotrophins and IL-1) were mixed into the sterile cultivation medium made up of MEM, 25% HBSS, 25% heat-inactivated normal horse serum, 4 mM L-glutamine, 4 g/ml insulin LXR-623 (all from Gibco, Karlsruhe, Germany), 2.4 mg/ml glucose (Braun, Melsungen, Germany), 0.1 mg/ml streptomycin, 100 U/ml penicillin, and 800 g/ml vitamin C (all Sigma-Aldrich, Taufkirchen, Germany)..