Hypothermia-induced neurite outgrowth is mediated by tumor necrosis factor-alpha

Abstract

Systemic or brain-selective hypothermia is a well-established method for neuroprotection after brain trauma. There is increasing evidence that hypothermia exerts beneficial effects on the brain and may also support regenerative responses after brain damage. Here, we have investigated whether hypothermia influences neurite outgrowth in vitro via modulation of the post-injury cytokine milieu. Organotypic brain slices were incubated: deep hypothermia (2 h at 17 degrees C), rewarming (2 h up to 37 degrees C), normothermia (20 h at 37 degrees C). Neurite density and cytokine release (IL 1beta, IL-6, IL-10, and TNF-alpha) were investigated after 24 h. For functional analysis mice deficient in NT-3/NT-4 and TNF-alpha as well as the TNF-alpha inhibitor etanercept were used. Hypothermia led to a significant increase of neurite outgrowth, which was independent of neurotrophin signaling. In contrast to other cytokines investigated, TNF-alpha secretion by organotypic brain slices was significantly increased after deep hypothermia. Moreover, hypothermia-induced neurite extension was abolished after administration of the TNF-alpha inhibitor and in TNF-alpha knockout mice. We demonstrate that TNF-alpha is responsible for inducing neurite outgrowth in the context of deep hypothermia and rewarming. These data suggest that hypothermia not only exerts protective effects in the CNS but may also support neurite outgrowth as a potential mechanism of regeneration.

Figure 1

Outgrowth model. A. Outgrowth assay (see Material and Methods section for details); the entorhinal cortex (EC) explant was embedded in a three‐dimensional collagen I gel matrix; the density of the outgrowing neurites (arrows) was photo‐documented at higher magnification and quantified by two blinded investigators. B. Neurite outgrowth in control organotypic brain slices. The white box indicates the area shown at higher magnification in 1F. C. Multiple neurites are visible by light‐microscopy (LM) growing out of the concave part of the EC explant. Immunofluorescence staining using specific antibodies reveals immunreactivity against Tau‐1, which is found on neurites but not on glial cells. D. No immunoreactivity of the extensions is detectable using a specific antibody against glial fibrillary acidic protein (GFAP) as a marker for astrocytes and their extensions. E. No immunoreactivity of the extensions is found using the secondary antibody alone as a negative control. F. Higher magnification of the white box in 1B: Tau‐1‐labeled neurites at light microscopy (LM) show characteristic growth cones that are also immunoreactive for Tau‐1 as a marker for neurites.

Figure 3

Hypothermia‐induced neurite growth is independent of neurotrophins and deep hypothermia upregulates the secretion of TNF‐alpha by organotypic brain slices. A. Neurite growth is increased by deep hypothermia and rewarming in wildtype mice as well as in NT‐4 or NT‐3 ± /NT‐4 mice. B. Hypothermia‐induced increase in neurite growth remains present after application of the pan‐neurotrophin receptor antagonist K252a, which is a potent inhibitor of TrkA, TrkB and TrkC in nanomolar concentrations compared with the solvent DMSO alone at the same concentration. C–E. The levels of IL‐1beta, IL‐6 and IL‐10 do not significantly change after deep hypothermia and rewarming. F. Deep hypothermia and rewarming significantly upregulate (nearly fourfold) the secretion of TNF‐alpha by organotypic brain slices ***P < 0.001.

Figure 2

Quantification by image analysis reveals stimulation of neurite outgrowth by hypothermia and rewarming. A, B. Representative photomicrographs of a brain slice with intermediate outgrowth (A) and a brain slice with strong outgrowth (B); the white dotted box in Figure 2A indicates the area shown at higher magnification in 1E. C, D. To quantify the density of the outgrowing neurites (arrowheads) image processing based on the Sobel algorithm was performed to determine the mean intensity in a standardized area parallel to the brain slice edge (indicated as white boxes). E. Higher magnification of the white dotted box in 1A; characteristic growth cones (arrows) are visible at the end of the neurites. F. Control brain slices kept at 37°C during the experiment display average growth of neurites (arrowheads). G. Applying the dynamic time–temperature protocol over 24 h leads to significantly increased neurite density after deep hypothermia and rewarming. H. Analysis of neurite density reveals a significant increase of more than 60% in organotypic brain slice cultures after deep hypothermia. **P < 0.01; arrow heads: single neurites. Abbreviation: EC = entorhinal cortex.

Figure 4

TNF‐alpha is necessary and sufficient for hypothermia‐induced neurite growth. A. Representative photomicrograph of a normothermic organotypic brain slice showing moderate outgrowth of neurites (arrowhead). B. Hypothermia and rewarming induce a substantial increase of the outgrowth of neurites (arrowheads). C. The TNF‐apha inhibitor etanercept does not influence neurite outgrowth of normothermic brain slices compared with controls. D. Etanercept inhibits hypothermia‐induced neurite growth. E. TNF‐alpha absence in brain slices derived from TNF‐alpha‐deficient mice blocks hypothermia‐induced neurite growth. F. TNF‐alpha‐deficient mice fully eliminated the effect of deep hypothermia and rewarming. G. TNF‐alpha significantly increases neurite density similar to hypothermia and rewarming. In the presence of the TNF‐alpha inhibitor etanercept and in the absence of TNF‐alpha in TNF‐alpha‐deficient mice the hypothermia‐induced increase in neurite growth is abolished. **P < 0.01. Abbreviation: EC = entorhinal cortex.