Cytokine cascades induced by mechanical trauma injury alter voltage-gated sodium channel activity in intact cortical neurons

Pregnant C57 BL/6J mice were ordered from the Shantou University Medical College Experimental Animal Center, Shantou, China. Animals were conducted according to the NIH Guide for Care and Use of Laboratory Animals.

Primary cultures of mouse cortical neurons were established, as previously described [29, 30], by using post-natal day 1 (P1) mice. In brief, cerebral cortices (without hippocampus) were trypsinized for 2 min with 4 mL 0.25% trypsin (Invitrogen) at 37 °C and 0.5 mL fetal bovine serum (Invitrogen). Cells were collected by centrifugation at 900g for 10 min, resuspended in minimum essential medium (Invitrogen), and seeded onto 12 mm × 12 mm glass cover slips (2 × 10³ cells/mm²) pretreated with 12.5 μg/mL poly-d-lysine (Sigma). Glutamine (2 mM, Sigma) and 2% B-27 supplement (Invitrogen) were added to neurobasal medium immediately before use. Cultures were incubated in 2 mL culture medium at 37 °C in 5% CO₂ atmosphere. Half of the culture medium was changed every 3 days. At day 3, cultures were exposed for 24 h to selective replication inhibitor arabinosylcytosine C at a final concentration of 4 μM to eliminate glial cells.

Neurons and astrocytes were cultured together as previously described [31]. Briefly, cortical tissues from P1 mice were isolated and dissociated, and individual cells (2 × 10³ cells/mm²) were seeded on previously prepared confluent 2-week-old astrocyte cultures with Dulbecco’s Modified Eagle Medium (DMEM)/F12 medium containing 10% fetal calf serum (Invitrogen). After 10–12 days, cocultures were subjected to mechanical injury (trauma) to mimic TBI in vitro [22, 31]. A sterile 21-gauge needle was used to make parallel scratches across circular wells of culture plates (9 × 9 scratches in six-well plates and 6 × 6 scratches in 12-well plates). Control wells were left uninjured. Medium was replaced with serum-free DMEM/F12 medium, and cultures were incubated at 37 °C.

At 6 or 24 h after injury, the medium conditioned by cells subjected to injury was collected and added to primary uninjured cortical neurons. Uninjured (intact) neurons were plated in 12-well plates (1 × 10⁵ cells per well), incubated for 24 h, and washed thrice with prewarmed neurobasal medium. Intact cultures were exposed for 6 or 24 h in mixed medium containing 500 μL of trauma-conditioned medium [with or without BDNF (30 ng/mL, CST)] and 500 μL of fresh neurobasal medium. Control cells were incubated in a mixed medium containing 500 μL of medium conditioned by uninjured cells and 500 μL of fresh neurobasal medium. Cells were incubated for 6 or 24 h in mixed medium for electrophysiological recording.

Electrophysiological recording of cortical cultures was performed as previously described [29, 30]. The bath solution during whole-cell recording of voltage-gated Na⁺ currents contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, 4 mM TEA-Cl, 0.1 mM CdCl₂, and 10 mM glucose. The pH was adjusted to 7.3 using NaOH. The pipette solution contained 145 mM CsCl, 1 mM MgCl₂, 1 mM CaCl₂, 1 mM EGTA, 10 mM HEPES, and 5 mM Na₂ATP. The pH was adjusted to 7.3 using CsOH.

Patch pipettes were pulled from borosilicate glass capillaries to a tip resistance of 2–5 MΩ using a P-97 micropipette puller (Sutter Instruments). Voltage-clamp recording was performed using an EPC-10 amplifier (HEKA), with series resistance compensated by 70–90%, and data frequency was recorded at 200 kHz. To examine the activation of VGSCs, neurons were held at −100 mV and depolarized to 100 mV in 5 mV steps, each of which lasted 20 ms and occurred at a frequency of 0.5 Hz. To examine the inactivation of VGSCs, Na⁺ currents were recorded at 0 mV following a pre-pulse from −70 to 50 mV for 40 ms with 5 mV steps.

Action potentials were recorded in current-clamp mode to measure spike thresholds and firing rates. Cells were held at 0 pA, and potentials were elicited using 120 ms depolarizing currents that were varied stepwise from −50 to 70 pA in 10 pA steps or a ramp current of 0–500 pA lasting 100 ms. The external solution contained 140 mM NaCl, 3 mM KCl, 2 mM MgCl₂, 0.1 mM CdCl₂, 1 mM CaCl₂, and 10 mM HEPES. The pH was adjusted to 7.3 using NaOH. The pipette solution contained 140 mM KCl, 10 mM EGTA, 5 mM Mg-ATP, and 5 mM HEPES. The pH was adjusted to 7.3 using KOH. All experiments were performed at 23–25 °C.

Total RNA was extracted from mouse primary mixed neuron–astrocyte cultures after mechanical injury as described above. An aliquot of total RNA (5 μg) was reverse-transcribed using oligo (dT) primers and Super-Script II reverse transcriptase (Invitrogen) in a total volume of 20 μl. Levels of messenger RNAs (mRNAs) encoding pro-inflammatory cytokines were quantified by quantitative real-time PCR (qPCR) using the primers in Table 1 and the Power SYBR Green PCR Master Mix Kit (Invitrogen) in a 7500 real-time PCR system (Applied Biosystems) according to the manufacturer’s instructions. Specificity of the SYBR Green PCR signal was confirmed by melting curve analysis. In each experiment, mouse GAPDH mRNA was amplified as an internal control.

To complement these mRNA analyses, cytokine expression was measured in the medium of mixed cultures using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems).

Membrane fractions were prepared from mouse cortical neurons treated by the conditioned medium using discontinuous sucrose gradient centrifugation. Briefly, lysate in 0.32 M sucrose/5 mM Tris (pH 7.4) was layered onto 1.2 M sucrose/5 mM Tris (pH 7.4) and centrifuged at 10,000g for 30 min. The layer at 0.8–1.2 M sucrose was collected, diluted twofold with 0.8 M sucrose/5 mM Tris (pH 7.4), and centrifuged at 20,000g for 20 min. The resulting pellet was re-suspended in a RIPA buffer containing 25 mM Tris, 150 mM NaCl, 1 mM EDTA, and 2% Triton X-100 (pH 7.4) and then centrifuged at 20,000g for 20 min to yield the final membrane preparation (supernatant). Complete protease inhibitor (Roche) was included throughout the procedure.

The membrane protein preparations were fractionated by SDS-PAGE (100 μg protein per well) and the gel band with pan-Nav and β-actin was cut horizontally and transferred onto a Hydrophobic PVDF membrane (Millipore) separately. The membrane was blocked with 5% non-fat milk. The pan-Nav and β-actin band were separately probed with a mouse anti-pan-Nav antibody (1:1000; Sigma) and rabbit anti-β-actin antibody (1:1000; Sigma) overnight at 4 °C. The membrane was washed with TBS/Tween-20, incubated in horseradish peroxidase-conjugated secondary antibody (goat anti-mouse 1:2000 or goat anti-mouse 1:2000 Sigma), washed again with TBS/Tween-20, and visualized by standard chemiluminescence. Ensure the signal images of pan-Nav or β-actin in every independent repeat experiments have a similar but not saturated exposure. Quantification of western blots was obtained from three separate experiments.

Data were expressed as means ± SEM and analyzed using Origin (Origin Lab Corporation, Northampton) and SPSS 15.0 (IBM, Chicago, IL, USA) software. One-way ANOVA and subsequent Tukey’s post hoc test were used to evaluate differences in voltage of half-maximal activation or inactivation (V _1/2), slope (k) of activation and inactivation, peak current density, standard cell viability, and threshold and firing rate for action potential. Differences in cytokine secretion tested by ELISA and normative protein expression levels were analyzed using two-way ANOVA analysis. Treatment and exposure time were considered independent variables. Student’s t test was performed to compare differences in mRNA relative expression of BDNF receptors of the two groups. Efficiency of target amplification and normalization of products was within 0.9–1.1 in real-time PCR. Results were calculated using the 2^−(ΔΔCT) method. p value of <0.05 was considered statistically significant.

Prior to testing the effect of inflammatory cytokines on VGSCs and cortical neuron excitability, we examined changes in expressions of multiple cytokines after mechanical trauma injury in neuron–astrocyte mixed cultures in vitro. As shown in Fig. 1a, mRNA expression of proinflammatory cytokines (IL-1β, IL-6, and TNF-α), chemokines (monocyte chemoattractant protein (MCP)-1, C-X-C motif chemokine ligand 1 (CXCL1), and chemokine (C-C motif) ligand 5) (CCL5)), anti-inflammatory cytokines (IL-10, TGF-β1, and OX-2 membrane glycoprotein (CD200)), and neurotrophic factors (nerve growth factor (NGF) and BDNF) increased in different degrees 6 h after trauma. IL-1β, IL-6, TNF-α, MCP-1, and TGF-β1 showed significantly higher upregulations than those of controls. ELISA was performed to determine protein levels of IL-1β, IL-6, TNF-α, and TGF-β1. Two-way ANOVA examined the effects of mechanically injured mixture and exposure duration on secretion of these proinflammatory cytokines. Mechanical injury exerted statistically significant enhancement effect on secretion concentration of IL-β1 (F (1, 8) = 20.27; p = 0.002), IL-6 (F (1, 8) = 16.89; p = 0.003), TNF-α (F (1, 8) = 10.33; p = 0.012), and TGF-β1 (F (1, 12) = 9.773; p = 0.009) in mixed glia. Exposure time significantly affected TGF-1β concentration (F (1, 12) = 8.126; p = 0.031). Tukey’s post hoc test revealed that mechanical injury significantly induced upregulation of IL-β1 (p = 0.008), IL-6 (p = 0.005), TNF-α (p = 0.014), and TGF-β1 (p = 0.041) compared with that of respective control group at 24 h (Fig. 1c–f, respectively). IL-6 and TGF-β1 increased at 6 h (Figs. 1b, d, respectively). We also examined BDNF/TrkB/p75NTR signal because BDNF/TrkB performs a neuroprotective role in various central nervous system diseases. In acute mechanical trauma injury, BDNF high-affinity receptors, namely, TrkB full-length (TrkB.FL) (p = 0.141) and TrkB.truncated type 1 (TrkB.T1) (p = 0.077), were not altered, but low-affinity receptor p75NTR (p = 0.036) significantly increased by fivefold at mRNA level (Fig. 1b). These results demonstrated that acute mechanical trauma injury in mixed astrocyte–neuron cultures upregulated multiple inflammatory mediators, mainly cytokines, but not neurotrophic factors in in vitro cell model of TBI.

An inward current with fast activation and inactivation was elicited by depolarization steps at −100 mV of holding potential. Such current was recorded at 200 kHz of sampling frequency in whole-cell patch-clamp recording and blocked completely using tetrodotoxin (TTX, 100 nM) (Fig. 2a). This result indicated that the currents were TTX-sensitive Na⁺ currents through VGSCs. Afterward, we examined the effect of trauma-conditioned medium containing multiple cytokines, which were secreted by mixed cultures, on voltage-gated Na⁺ current densities at 6 and 24 h. A statistically significant difference existed between groups, as determined by one-way ANOVA (F (2, 17) = 4.230; p = 0.032). Tukey’s post hoc test revealed that voltage-gated Na⁺ current densities significantly increased by 37.6% ± 4.5% in pure neuronal cultures exposed to conditioned medium harvested at 6 h after mechanical injury compared with those in control medium(p = 0.034) (Figs. 2b, c, e). Similarly, one-way ANOVA also showed statistically significant difference between groups at 24 h (F (2, 19) = 19.81; p <0.001). Tukey’s post hoc test also revealed that Na⁺ current densities significantly increased by 82.2% ± 5.4% in cultures exposed to trauma-conditioned medium for 24 h compared with those of controls (p < 0.001) (Fig. 2f). Adding BDNF (30 ng/mL) to conditioned medium almost completely abolished increased density of Na⁺ currents compared with those of controls (p < 0.001) (Figs. 2b–g). BDNF also showed an apparent dose-dependent effect on Na⁺ current density reduction of Na⁺ currents induced by the trauma-conditioned mediums (Fig. 2h, i). BDNF concentration appeared to correlate negatively with Na⁺ current density induced by trauma-conditioned medium in primary cortical neurons.

Conductance–voltage curves were generated and fitted using Boltzmann equation to examine activation and inactivation properties of VGSCs (Fig. 3a, b). Fast inactivation was measured by applying a peak current at 0 mV test voltage and normalizing obtained currents to maximal current (Fig. 3b). Normative currents were plotted against prepulse voltage, and current–voltage curves were fitted using the Boltzmann equation.

V _1/2 of fast inactivation showed statistically significant difference between groups, as determined by one-way ANOVA (F (2, 23) = 8.078; p = 0.002). Post hoc Tukey’s test revealed that exposure of intact cortical neurons to trauma-conditioned medium did not affect VGSC activation (p = 0.086) (Fig. 3a), but a slight shift was observed in V _1/2 of fast inactivation to hyperpolarization (from −52.47 ± 0.47 mV to −54.56 ± 0.32 mV) (p = 0.004) (Fig. 3b and Table 2). Similarly, a statistically significant difference was noted in V _1/2 of activation between groups, as determined by one-way ANOVA [F (2, 23) = 4.138; p = 0.029]. Tukey’s post hoc test revealed that addition of BDNF (30 ng/mL) to trauma-conditioned medium slightly shifted V _1/2 of activation to hyperpolarization (from −32.63 ± 0.57 mV to 34.70 ± 0.44 mV) (p = 0.031) (Fig. 3a and Table 2). Neither trauma-conditioned medium nor BDNF changed recovery properties of VGSCs from fast inactivation (Fig. 3c). These results suggested that both trauma-conditioned medium and BDNF did not apparently alter dynamic properties of VGSCs.

Consistent with our whole-cell patch-clamp findings, two-way ANOVA analysis showed that conditioned medium resulted in statistically significant influence on expression of functional VGSCs in plasma membrane between groups [F (2, 6) = 25.66; p = 0.002] but not on exposure time [F (2, 6) = 2.708; p = 0.151]. Post hoc Tukey’s test revealed significant increase in the number of functional VGSCs in plasma membrane (by 77.54 and 111.96%) after treatment with trauma-conditioned medium for 6 h (p = 0.005) and 24 (p = 0.048) compared with those of controls. Addition of BDNF (30 ng/mL) for 24 h almost completely reversed upregulation of VGSCs by trauma-conditioned medium (p = 0.037) (Fig. 4a, b). Cell viability assays were performed to determine the effect of conditioned medium on survival of intact neurons and neuroprotective effect of BDNF. Conditioned medium significantly affected cell viability of neurons at 6 [F (2, 15) = 15.62; p < 0.001] and 24 h [F (2, 6) = 7.896; p = 0.021], respectively, as determined by one-way ANOVA. Tukey’s post hoc test revealed that treatment with conditioned medium significantly decreased viability of intact neurons about 30% both at 6 (p < 0.001) and 24 h (p = 0.022). BDNF (30 ng/mL) only showed apparent neuroprotective effects on neuronal viability at 6 h (p = 0.006) but not at 24 h (p = 0.053). Altogether, these results demonstrated that upregulation of Na⁺ current density possibly resulted from increased functional VGSCs in intact neurons. Possibly, BDNF partly played its neuroprotective role by maintaining VGSCs at normal levels, thereby maintaining normal influx of Na⁺ into neurons.

Examinations were performed to determine the effects of conditioned medium on spike behavior. Firing threshold of spike was not significantly altered by trauma-conditioned medium (with or without BDNF) after 6 [F (2, 31) = 1.855; p = 0.174] or 24 h [F (2, 28) = 0.282; p = 0.757] compared with respective control group (Fig. 5a–d), as determined by one-way ANOVA. Trauma-conditioned medium (with or without BDNF) significantly altered firing rate of spike at 6 [F (2, 32) = 7.782; p = 0.002] or 24 h [F (2, 20) = 5.826; p = 0.011] compared with each control group (Fig. 5e–h). Tukey’s post hoc test revealed that exposure to trauma-conditioned medium conditioned for 6 (p = 0.003) or 24 h (p = 0.013) significantly increased firing rate (Fig. 5e–h), and BDNF significantly inhibited increase in firing rate by trauma-conditioned medium conditioned for 6 h (p = 0.006) (Fig. 5e, f) but not at 24 h (p = 0.281) (Fig. 5g, h). These results indicate that the enhancement of firing rate is induced in intact neurons by the trauma-conditioned medium and that BDNF may be able to protect against the enhancement of firing rate.