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, 20 (5), 700-707

C1 Neurons Mediate a Stress-Induced Anti-Inflammatory Reflex in Mice

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C1 Neurons Mediate a Stress-Induced Anti-Inflammatory Reflex in Mice

Chikara Abe et al. Nat Neurosci.

Abstract

C1 neurons, located in the medulla oblongata, mediate adaptive autonomic responses to physical stressors (for example, hypotension, hemorrhage and presence of lipopolysaccharides). We describe here a powerful anti-inflammatory effect of restraint stress, mediated by C1 neurons: protection against renal ischemia-reperfusion injury. Restraint stress or optogenetic C1 neuron (C1) stimulation (10 min) protected mice from ischemia-reperfusion injury (IRI). The protection was reproduced by injecting splenic T cells that had been preincubated with noradrenaline or splenocytes harvested from stressed mice. Stress-induced IRI protection was absent in Chrna7 knockout (a7nAChR-/-) mice and greatly reduced by destroying or transiently inhibiting C1. The protection conferred by C1 stimulation was eliminated by splenectomy, ganglionic-blocker administration or β2-adrenergic receptor blockade. Although C1 stimulation elevated plasma corticosterone and increased both vagal and sympathetic nerve activity, C1-mediated IRI protection persisted after subdiaphragmatic vagotomy or corticosterone receptor blockade. Overall, acute stress attenuated IRI by activating a cholinergic, predominantly sympathetic, anti-inflammatory pathway. C1s were necessary and sufficient to mediate this effect.

Conflict of interest statement

Competing financial interests. The authors have stated that they have no financial interest.

Figures

Figure 1
Figure 1. Restraint stress protects against renal ischemia-reperfusion injury (IRI)
(a) Time line of experiments shown in panels b-e. Effect of prior restraint stress on plasma creatinine in DβHCre/0 (dopamine β-hydroxylase) mice (DBH-cre mice) (b), Kidney Injury Molecule-1 (Kim-1) mRNA (DBH-cre mice) (c), and acute tubular necrosis (DBH-cre mice) (d) (n = 6/group). IR, ischemia-reperfusion. One-way ANOVA with Tukey–Kramer test (b) or Kruskal-Wallis with Steel-Dwass test (c and d) was used for statistical analysis; [F(2, 15) = 35.64, P < 0.0001] (b), [H = 14.36, P < 0.0001] (c), and [H = 14.36, P < 0.0001] (d). * vs. Restraint(−):IR(−); † vs. Restraint(−):IR(+). Single or triple significant symbols indicate P < 0.05 or P < 0.001, respectively. (e) Restraint stress protects wild-type mice (α7WT) against IRI but has no effect in α7nAChR −/− mice (α7KO). Statistics: two-way ANOVA with Tukey–Kramer test; [F(1, 18) = 37.03, P < 0.0001]. *** P < 0.001 vs. Restraint(−):IR(+) in α7WT and α7KO or Restraint(+):IR(+) in α7KO.
Figure 2
Figure 2. Adoptive transfer of splenocytes protects against renal IRI
(a, b) adoptive transfer of splenocytes. Splenocytes were harvested from restraint stress-exposed donor mice. Then these cells (1*10^6, 5*10^6, or 1*10^7) were injected to the recipient mice. Statistics: two-way ANOVA with Tukey–Kramer test; [F(1, 32) = 6.299, P < 0.0173]. * P < 0.05 vs. 5*10^6:Restraint(−):IR(+), †† P < 0.01 vs. 1*10^6:Restraint(−):IR(+), and ‡‡‡P < 0.001 vs. 1*10^6:Restraint(+):IR(+). (c, d) adoptive transfer of noradrenaline-treated CD4 T cells. CD4 T cells were harvested from splenocytes of the donor mice, these cells were treated with noradrenaline. Then these cells were injected to the recipient mice. Statistics: unpaired t test; [t(11) = 7.657, P < 0.0001]. *** P < 0.001 vs. vehicle.
Figure 3
Figure 3. C1 neuron stimulation protects against renal IRI
(a) Time line of experiment. (b) Section through the rostral medulla oblongata of a DBH-cre mouse 5–6 weeks after local injection of AAV2–DIO–EF1α–ChR2–mCherry. mCherry (magenta) and tyrosine-hydroxylase (TH, green) immunoreactivities are colocalized (white). Most catecholaminergic neurons contain mCherry. A small lesion surrounded by green autofluorescence shows the location of the tip of the optical fiber (scale bar: 100 μm). (c–e) Effect of prior C1 neuron stimulation on plasma creatinine, Kidney Injury Molecule-1 (Kim-1) mRNA (Havcr1/Gapdh ratio) in the kidney, and acute tubular necrosis (% of kidney section surface area) in DBH-cre mice after IRI (n = 6/group). Vector used: AAV2–DIO–EF1α–ChR2–mCherry (ChR2) or AAV2–DIO–EF1α–mCherry (mCherry). Laser: 5 Hz, 10 min. IR, ischemia-reperfusion. Statistical analysis (Kruskal-Wallis with Steel-Dwass test): [H = 18.62, P = 0.0003] (c), [H = 19.22, P = 0.0004] (d), [H = 19.77, P < 0.0001] (e), * vs. ChR2:Laser(−):IR(−); † vs. ChR2:Laser(−):IR(+) or mCherry:Laser(+):IR(+). Single, double, or triple significant symbols indicate P < 0.05, P < 0.01, or P < 0.001, respectively.
Figure 4
Figure 4. C1 neurons mediate the protective effect of restraint stress against renal ischemia-reperfusion injury (IRI)
(a) mCherry (magenta) and TH (green) immunoreactivity in the left medulla oblongata of a DBH-cre mouse six weeks after stereotaxic microinjection of AAV2–DIO–hSyn–hm4D(Gi)–mCherry (DREADD) (transverse section; scale bar: 100 μm). (b) Rostrocaudal distribution (mm caudal to bregma) of mCherry and TH immunoreactivities (n = 12). (c) Choline acetyltransferase- (ChAT; magenta) and TH- (green) immunoreactivities in the left medulla oblongata of a DBH-cre mouse 6 weeks after injection of AAV2–DIO–taCasp3–TEVp (AAV2-caspase). After AAV2-caspase treatment, the C1 neurons in the rostral ventrolateral medulla (RVLM) are undetectable (left and lower middle panels) but other catecholaminergic neurons (dorsal medulla, DMM and locus coeruleus, LC) are intact (top middle and top right panels). ChAT+ neurons are unaffected by AAV2-caspase treatment regardless of location (left and bottom middle). Scale bar: 500 μm (left) or 100 μm (four right panels). Amb, nucleus ambiguus. (d) Rostrocaudal distribution of TH-immunoreactive RVLM neurons in control DBH-cre mice (n = 7) vs. caspase-treated DBH-cre mice (n = 10). Lesions were bilateral; cells were counted on one side only. Statistics: two-way ANOVA with Tukey–Kramer test; [F(12, 180) = 25.99, P < 0.0001]. (e) The protective effect of restraint stress against renal IRI was attenuated by inhibiting (DREADD) or lesioning (caspase) the C1 neurons (n = 6 DBH-cre mice/group). CNO: clozapine N-oxide (3 mg/kg); Vehicle: saline. Statistics: one-way ANOVA with Tukey–Kramer test; [F(5, 30) = 14.11, P < 0.0001]. * vs. Restraint(-):IR(+) and † vs. Restraint(+):IR(+) and DREADD:Vehicle:Restraint(+):IR(+). Single, double, or triple significant symbols indicate P < 0.05, P < 0.01, or P < 0.001, respectively.
Figure 5
Figure 5. Corticosterone is released by C1 stimulation and restraint stress but plays no detectable role in protecting kidneys from renal ischemia-reperfusion injury (IRI)
(a) Restraint stress increased plasma corticosterone in DBH-cre mice. Statistics: unpaired t test; [t(10) = 4.991, P = 0.0005]. *** P < 0.001 vs. Restraint(−). (b) C1 neuron optogenetic stimulation (AAV2–DIO–EF1α–ChR2–mCherry, ChR2) increased plasma corticosterone in DBH-cre mice whereas laser light alone was ineffective in DBH-cre mice injected with control vector (AAV2–DIO–EF1α–mCherry, mCherry). Laser: laser stimulation (5 Hz for 10 min). Statistics: one-way ANOVA with Tukey–Kramer test; [F(2, 21) = 6.959, P = 0.0048]. * P < 0.05 vs. ChR2:Laser(−) and mCherry:Laser(+). (c) Mifepristone (30 mg/kg) did not alter the protective effect of C1 neuron stimulation against renal IRI in DBH-cre mice (n = 6/group). Statistics: unpaired t test; t(10) = 0.495, P = 0.6313).
Figure 6
Figure 6. C1 neuron stimulation activates both divisions of the autonomic nervous system
(a) C1 stimulation (left side; 10 ms pulses, 10 s trains) activates ipsilateral vagal nerve efferent activity (VNA) in DBH-cre mice. (b) C1 stimulation (left side; 10 ms pulses, 10 s trains) activates contralateral vagal nerve efferent activity (VNA) in DBH-cre mice. Statistics: repeated measure one-way ANOVA with Tukey–Kramer test; [F(4, 25) = 112.9, P < 0.0001] (a) and [F(4, 25) = 14.4, P < 0.0001] (b). * vs. Dummy, † vs. Dummy and 5 Hz, ‡ vs. Dummy, 5 Hz, and 10 Hz, and # vs. Dummy, 5 Hz, 10 Hz, and 15 Hz. Double symbols, P < 0.01; triple symbols, P < 0.001. (c) C1 neuron stimulation (10 ms, 1 Hz) produced a robust activation of renal sympathetic nerve activity (RSNA) in DBH-cre mice. (d) Effect of repetitive C1 stimulation (5-20 Hz; 10 ms pulses, 10 s train) on RSNA. Statistics: repeated measure one-way ANOVA with Tukey–Kramer test; [F(4, 25) = 4.651, P = 0.0061]. * P < 0.05 vs. Dummy and 5 Hz. DMV, dorsal motor nucleus of the vagus; RSN, renal sympathetic nerve; SGN, sympathetic ganglionic neuron; SPGN, sympathetic preganglionic neurons; 7, facial motor nucleus.
Figure 7
Figure 7. Protection against renal IRI by C1 neuron stimulation is mediated via the autonomic nervous system and the spleen
(a) Butoxamine (But), or hexamethonium (Hex) but not vehicle (Veh) attenuated the protective effect of C1 neuron stimulation (Laser 5 Hz, 10 min) against ischemia-reperfusion (IR) injury (n = 6 or 9 DBH-cre mice/group). Statistics: one-way ANOVA with Tukey–Kramer test; [F(3, 26) = 22.17, P < 0.0001]. * vs. Veh:Laser(−):IR(+), † vs. Veh:Laser(+):IR(+). Double symbols, P<0.01; triple symbols, P < 0.001. (b) Splenectomy eliminated the protective effect of C1 neuron stimulation in DBH-cre mice (n = 6/group). Statistics: unpaired t test; t(10) = 0.49, P = 0.63. (c) Subdiaphragmatic vagotomy (sVNX) does not reduce the protective effect of C1 neuron stimulation in DBH-cre mice (n = 6/group). Statistics: one-way ANOVA with Tukey–Kramer test; [F(2, 13) = 44.75, P < 0.0001]. * vs. Sham:Laser(−):IR(+). Triple symbols, P < 0.001. DMV, dorsal motor nucleus of the vagus; NA, noradrenaline; ACh, acetylcholine; SGN, sympathetic ganglionic neuron; SPGN, sympathetic preganglionic neurons; VN, vagal nerve.
Figure 8
Figure 8. Restraint stress and C1 neuron stimulation produce opposite effects on arterial pressure and heart rate
(a) Effect of restraint stress on arterial pressure (AP) and heart rate (HR) in a DBH-cre mouse. (b and c) Mean AP and HR before (Pre), during and after (Post) restraint (each period: 10 min) in DBH-cre mice (n = 4). Statistics: one-way repeated measures ANOVA with Tukey–Kramer test; [F(2, 9) = 29.11, P = 0.0001] (b) and [F(2, 9) = 21.93, P = 0.0003] (c). * vs. Pre. Double symbols, P < 0.01 and triple symbols, P < 0.001. (d) Effect of C1 stimulation (10 ms, 5 Hz, 10 min) on arterial pressure (AP) and heart rate (HR) in a DBH-cre mouse. (e and f) Effect of C1 neuron stimulation (laser 5 Hz) on AP and HR in DBH-cre mice. Mean AP and HR before (Pre), during and after (Post) C1 neuron stimulation (each period: 10 min) (n = 4/group). Statistics: one-way repeated measure ANOVA with Tukey–Kramer test; [F(2, 9) = 12.7, P = 0.0024] (e) and [F(2, 9) = 9.249, P = 0.0066] (f). * vs. Pre. Single symbols, P < 0.05 and double symbols, P < 0.01.

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References

    1. Li L, et al. IL-17 produced by neutrophils regulates IFN-gamma-mediated neutrophil migration in mouse kidney ischemia-reperfusion injury. J Clin Invest. 2010;120:331–342. - PMC - PubMed
    1. Pavlov VA, Tracey KJ. Neural circuitry and immunity. Immunol Res. 2015;63:38–57. - PMC - PubMed
    1. Olofsson PS, Rosas-Ballina M, Levine YA, Tracey KJ. Rethinking inflammation: neural circuits in the regulation of immunity. Immunol Rev. 2012;248:188–204. - PMC - PubMed
    1. Yamakawa K, et al. Electrical vagus nerve stimulation attenuates systemic inflammation and improves survival in a rat heatstroke model. PloS one. 2013;8:e56728. - PMC - PubMed
    1. Jiang Y, et al. Vagus nerve stimulation attenuates cerebral ischemia and reperfusion injury via endogenous cholinergic pathway in rat. PloS one. 2014;9:e102342. - PMC - PubMed

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