Anterior insular cortex mediates hyperalgesia induced by chronic pancreatitis in rats

Abstract

Central sensitization plays a pivotal role in the maintenance of chronic pain induced by chronic pancreatitis (CP), but cortical modulation of painful CP remains elusive. This study was designed to examine the role of anterior insular cortex (aIC) in the pathogenesis of hyperalgesia in a rat model of CP. CP was induced by intraductal administration of trinitrobenzene sulfonic acid (TNBS). Abdomen hyperalgesia and anxiety were assessed by von Frey filament and open field tests, respectively. Two weeks after surgery, the activation of aIC was indicated by FOS immunohistochemical staining and electrophysiological recordings. Expressions of VGluT1, NMDAR subunit NR2B and AMPAR subunit GluR1 were analyzed by immunoblottings. The regulatory roles of aIC in hyperalgesia and pain-related anxiety were detected via pharmacological approach and chemogenetics in CP rats. Our results showed that TNBS treatment resulted in long-term hyperalgesia and anxiety-like behavior in rats. CP rats exhibited increased FOS expression and potentiated excitatory synaptic transmission within aIC. CP rats also showed up-regulated expression of VGluT1, and increased membrane trafficking and phosphorylation of NR2B and GluR1 within aIC. Blocking excitatory synaptic transmission significantly attenuated abdomen mechanical hyperalgesia. Specifically inhibiting the excitability of insular pyramidal cells reduced both abdomen hyperalgesia and pain-related anxiety. In conclusion, our findings emphasize a key role for aIC in hyperalgesia and anxiety of painful CP, providing a novel insight into cortical modulation of painful CP and shedding light on aIC as a potential target for neuromodulation interventions in the treatment of CP.

Keywords: Anterior insular cortex; Anxiety; Chronic pancreatitis; Excitatory synaptic transmission; Hyperalgesia; Long-term potentiation; Rat.

Fig. 1

TNBS-treated rats exhibit abdomen hyperalgesia and anxiety-like behaviors. a-c: Representative pancreas histology of sham group (a), TNBS-treated group on POD 14 (b) and POD 28 (C). Bar = 100 μm. d-f: TNBS treatment increased serum content of amylase (d), lipase (e) and total bilirubin (f) compared to sham rats along the course of CP, one-way ANOVA in (d, e) and Kruskal-Wallis test in (f). g: TNBS-treated rats showed decreased abdomen withdrawal threshold along the course of CP, while rats with sham surgery exhibited transient abdomen mechanical hypersensitivity which returned to baseline on POD 14, one-way repeated ANOVA. h-i: Rats with CP traveled less distance in the open field (h) as well as in the center of the open field (i) than sham rats from POD 7 to 28, unpaired t-test. Numbers within the bars denote the numbers of rats in each group, * P < 0.05, ** P < 0.01, *** P < 0.001, TNBS vs sham; ^### P < 0.001, sham vs naïve

Fig. 2

The number of FOS-expressing neurons is up-regulated within IC in CP rats. a-c: Immunochemical staining of FOS within different coronal sections of IC in sham (left) and CP rats on POD 14 (right). Bar = 200 μm. d: Histogram showing the qualification of FOS-expressing neurons within different parts of IC in saline or TNBS-treated rats. n = 3 slices from 3 rats in each group, unpaired t-test, * P < 0.05, ** P < 0.01, TNBS vs sham

Fig. 3

Post-LTP is occluded within aIC in rats with CP. a: Light microscopy photograph showing relative location of aIC within the probe. b: Schematic diagram showing the recording array arrangement. c: The input-output curve of the number of activated channels in slices of sham and CP rats, one-way repeated ANOVA. d, e: A sample of an overview of multisite synaptic responses recorded at baseline (black) and 2 h after TBS (red) in sham and CP groups, respectively. The flash denotes the stimulated channel. Red and black filled circles mark all activated channels undergoing and not undergoing LTP, respectively, while the black rectangle represents a typical channel not exhibiting any response in the baseline. These example traces are shown in an enlarged scale below. Vertical lines demarcate different layers. f, g: Time course of averaged fEPSP slope and amplitude of all active channels in sham and TNBS groups. The arrow indicates the time of TBS application. Dashed line indicates the baseline of 100% slope or amplitude. h: The average slope and amplitude of fEPSPs of all active channels within the last 20 min of 140 min recording in sham and TNBS groups, unpaired t-test. i, j: The polygonal diagram of activated (blue) and LTP-occurring (red) channels within aIC after TBS in sham (i) and TNBS (j) groups. The red dots indicate the stimulation sites. n = 6 slices from 6 rats in each group. * P < 0.05, ** P < 0.01, *** P < 0.001, TNBS vs sham

Fig. 4

Enhanced pyramidal neuron excitability within aIC after TNBS treatment. a: Schematic diagram indicating the placement of stimulating and recording electrodes in the aIC. b: The different firing patterns of pyramidal neuron (left, repetitive action potentials with frequency adaptation) and interneuron (right, fast-spike firing) within aIC after positive current injected into the cell under current-clamp mode. c: Sample traces of the spikes recorded in pyramidal neurons within the superficial layer of aIC of sham (left) and CP (right) rats in response to depolarizing current injections, step = 100 pA, duration = 400 ms. d: The spike number input-output curve from CP rats was steeper than that from sham rats, one-way repeated ANOVA. e: Sample traces showing CP rats exhibited decreased rheobase current (42 pA in CP rats vs 194 pA in sham rats) and hyperpolarized RMP (− 63.30 mV in CP rats vs − 71.22 mV in sham rats) in insular pyramidal neurons, step = 2 pA, duration = 400 ms. f: The rheobase current was decreased in CP rats (74.33 ± 8.97 pA) compared to sham rats (120.7 ± 20.85 pA), unpaired t-test. g: The RMP was increased in CP rats (− 70.3 ± 1.57 mV) compared to sham rats (− 65.67 ± 1.48 mV), unpaired t-test. n = 9 in sham group and 12 in TNBS group, * P < 0.05, TNBS vs sham

Fig. 5

Enhanced presynaptic glutamate release within aIC after TNBS treatment. a: Representative sEPSCs recorded in the superficial layer of aIC from sham (top) and TNBS-treated (bottom) rats holding at − 60 mV. b: Cumulative inter-event interval (left) and amplitude (right) histograms of sEPSCs recorded in the same neurons showed in (a). c: Summary plots showing the frequency (left) and the amplitude (right) of sEPSC was increased in CP rats (1.46 ± 0.28 Hz, 18.48 ± 0.84 pA, n = 12) compared to sham rats (0.65 ± 0.26 Hz, 15.06 ± 0.96 pA, n = 11), unpaired t-test. d: Representative traces of PPF with an interval of 35 ms recorded in the superficial layer of aIC (top). PPF at time intervals of 35, 50 and 75 ms was reduced in CP rats (bottom). n = 13 cells in sham group and 15 in TNBS group, one-way repeated ANOVA. e: Representative western blot sample for VGluT1 within aIC obtained on POD 7, 14 and 28 (top). Statistical analyses showing enhanced expression of VGluT1 from POD 7 to 28 after TNBS treatment (bottom). n = 3 rats in each group, one-way ANOVA. f: The immunoreactivities of VGluT1 within aIC were remarkably increased in TNBS-treated rats compared to sham rats. Microphotographs indicating double-immunoflurescence histochemistry for VGluT1 (green) and NeuN (red) within aIC (left). The framed areas in upper images were magnified in lower images. Bars = 200 μm in upper images and 40 μm in lower images. Quantifications and statistical analyses of VGluT1 immunoreactivities presented in the graph (right). * P < 0.05, ** P < 0.01, TNBS vs sham

Fig. 6

Enhanced AMPAR and NMDAR currents within aIC after TNBS treatment. a: The AMPAR-mediated synaptic input-output curve of CP rats (n = 11) was steeper than that from sham rats (n = 7), one-way repeated ANOVA. b: The NMDAR-mediated synaptic input-output curve of CP rats (n = 9) was steeper than that from sham rats (n = 10), one-way repeated ANOVA. c: I-V curves of AMPAR-EPSCs of aIC pyramidal neurons recorded at holding potentials ranging from − 60 to + 50 mV in sham and CP rats, one-way repeated ANOVA. d: Comparison of the rectification index of AMPAR-EPSCs in sham (n = 11) and CP (n = 10) rats, unpaired t-test. e: I-V curves of NMDAR-EPSCs of aIC pyramidal neurons recorded at holding potentials ranging from − 60 to + 50 mV in sham and CP rats, one-way repeated ANOVA. f: Comparison of the rectification index of NMDAR-EPSCs in sham (n = 9) and CP (n = 12) rats, unpaired t-test. * P < 0.05, ** P < 0.01, TNBS vs sham

Fig. 7

TNBS treatment facilitates the phosphorylation and the trafficking of insular glutamate receptor subunits into membrane. a: Representative western blot for NR2B and GluR1 within aIC on POD 7, 14 and 28. b, c: The expressions of NR2B (b) and GluR1 (c) were significantly enhanced from POD 7 to 28 after TNBS treatment. d: Fractionation of insular tissue was probed for N-cadherin and GAPDH to verify the accuracy of subcellular fractionation procedure. e, f: Representative western blot samples for membrane (e) and cytosol (f) NR2B, pNR2B, GluR1 and pGluR1 within aIC obtained on POD 7, 14 and 28 in TNBS-treated rats and sham rats. g, h Membrane NR2B was significantly increased on POD 7, 14 and 28 after TNBS treatment compared to sham group while cytosol NR2B was significantly decreased on POD 14 and 28. i, j Membrane GluR1 was significantly increased on POD 7, 14 and 28 after TNBS treatment compared to sham group while cytosol GluR1 showed no change. k, l Membrane pNR2B was significantly increased on POD 7, 14 and 28 after TNBS treatment compared to sham group while cytosol NR2B showed no change. m, n Membrane pGluR1 was significantly increased on POD 7, 14 and 28 after TNBS treatment compared to sham group while cytosol pGluR1 showed no change. n = 3 rats in each group, one-way ANOVA, * P < 0.05, ** P < 0.01, *** P < 0.001, TNBS vs sham

Fig. 8

Bilateral microinjection of CNQX and AP-5 within aIC alleviates visceral hypersensitivity of CP rats. a: The schematic diagram of the behavioral experiment. b: A representative coronal section showing the sites of cannula implantation within bilateral aIC. Bar = 1 mm. c: Bilateral microinjections of CNQX and AP-5, instead of saline, significantly enhanced the AWT in CP rats on POD 14. n = 5 in saline and CNQX-treated groups and 6 in AP5-treated group, one-way ANOVA, * P < 0.05, ** P < 0.01, CNQX or AP5 vs saline

Fig. 9

Non-selectively chemogenetic inactivation of bilateral insular neurons alleviates abdomen mechanical hyperalgesia and anxiety-like behavior in CP rats. a: The schematic diagram of the behavioral experiment (up) and rAAV2/9-hSyn-hM4Di-mCitrine construct (down). b: A representative coronal section showing virus injection sites within bilateral aIC. Bar = 1 mm. c: Inhibiting bilateral insular neurons via i.p. CNO injection significantly increased the AWT in CP rats on POD 14. d, e: Inhibiting bilateral insular neurons led to a trend toward increased total traveling distance (d) and a significant increase in central traveling distance (e) in CP rats. n = 7 in mCitrine group and 6 in Gi group, unpaired t-test. * P < 0.05, ** P < 0.01, Gi vs mCitrine

Fig. 10

Chemogenetic inactivation of bilateral insular pyramidal neurons alleviates abdomen mechanical hyperalgesia and anxiety-like behavior in CP rats. a: The schematic diagram of the behavioral experiment (up) and rAAV2/9-CaMKIIa-hM4Di-mCherry construct (down). b: A representative coronal section showing virus injection sites within bilateral aIC. Bar = 1 mm. c: Inhibiting bilateral insular pyramidal neurons via i.p. CNO injection significantly increased the AWT in CP rats on POD 14. d, e: Inhibiting bilateral insular neurons led to a significant increase in total traveling distance (d) as well as central traveling distance (e) in CP rats. n = 6 in each group, unpaired t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, Gi vs mCherry