CN115990167A - Application of adenylate cyclase inhibitor NB001 in preparation of medicine for treating migraine and anxiety - Google Patents

Abstract

The invention provides an application of an adenylate cyclase inhibitor NB001 in preparing medicaments for treating migraine and anxiety. The drug for inhibiting the AC1 is NB001 or pharmaceutically acceptable salt and solvate thereof; NB001 is 5- ((2- (6-amino-9-H-purin-9-yl) ethyl) amino) pentane-1-pentanol, and has a structural formula:

. The invention provides strong evidence that cortical LTP contributes to migraine-associated pain and anxiety, while drugs that inhibit cortical excitability, such as NB001, would be potential new drugs for future treatment of migraine.

Description

Application of adenylate cyclase inhibitor NB001 in preparation of medicine for treating migraine and anxiety

Technical Field

The invention relates to medical application of an adenylate cyclase inhibitor in treating migraine and anxiety, in particular to application of an adenylate cyclase inhibitor NB001 in preparing a medicine for treating migraine and anxiety.

Background

Migraine is a major form of chronic pain that often presents with associated co-diseases such as somatosensory diffuse allodynia and anxiety. The central mechanisms of migraine and its associated allodynia and anxiety are not yet known. Previous studies have shown that by using migraine animal models (trigeminal vascular models caused by dural inflammatory stimuli), dural inflammation triggers a decrease in nociceptive thresholds and an enhancement of neuronal responses to nociceptive and non-nociceptive stimuli. Most of these studies have focused on Trigeminal Ganglion (TG) and thalamus (.e. neurons of the adult rat thalamus and postthalamus are sensitive and exhibit persistent hyperexcitability to harmless and harmful stimuli the possible effects of the Anterior Cingulate Cortex (ACC) are also poorly understood, which is a key cortex for pain perception and chronic pain recent human brain imaging studies have shown that migraine patients have a reduced anterior cingulate cortex gray matter volume.

ACC synapses have a high degree of plasticity. Changes in anterior cingulate gyrus plasticity (including increased probability of presynaptic neurotransmitter release, increased expression of postsynaptic AMPA receptors) can be triggered by different types of external Zhou Sunshang and further lead to chronic pain and anxiety. A variety of signaling molecules are involved in the intracellular pathways of ACC plasticity, such as NMDA receptors, adenylate cyclase 1 (AC 1), protein kinase mζ (pkmζ), and AMPA receptors. Among these targets, AC1 is considered as a selective target for the treatment of chronic pain. Unlike ataxia and sedation caused by ion channel antagonists (e.g., NMDA receptors), AC1 knockdown does not affect key physiological functions such as learning, memory and cognitive functions. These findings strongly suggest that although most previous studies used animal models of somatosensory and visceral pain, AC1 may be a selective target for the treatment of chronic pain.

In this study, we applied inflammatory mediators to the dura mater of rats to establish chronic migraine. We found that the phosphorylation levels of NMDA and AMPA receptors in ACC neurons were increased and similar to other forms of chronic pain. A selective AC1 inhibitor, NB001, has recently proven safe in animals and humans, topically applied to the anterior cingulate cortex or orally administered, produces potent analgesic and anxiolytic effects in chronic migraine rats. Our findings strongly suggest that plasticity of ACC regulates chronic migraine, whereas AC1 may be a potential target for future chronic migraine treatment.

Disclosure of Invention

The object of the present invention is to provide the use of a medicament for inhibiting AC1 in the manufacture of a medicament for migraine and anxiety.

The application of the drug for inhibiting the AC1 in preparing the drug for treating migraine and anxiety is characterized in that the drug for inhibiting the AC1 is NB001 or pharmaceutically acceptable salts and solvates thereof; NB001 is 5- ((2- (6-amino-9-H-purin-9-yl) ethyl) amino) pentane-1-pentanol, and has a structural formula:

。

use of an AC1 inhibiting medicament for the manufacture of a medicament for the treatment of migraine and anxiety, characterized in that the AC1 inhibiting medicament comprises an active ingredient for enteral or parenteral administration and a suitable pharmaceutically inert carrier material, organic or inorganic.

The use of said AC1 inhibiting medicament for the manufacture of a medicament for the treatment of migraine and anxiety characterized in that said pharmaceutically suitable organic or inorganic inert carrier material comprises water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils and/or polyalkylene glycols.

The dosage of the active ingredients of the drug for inhibiting the AC1 is as follows: 1 ng to 500 mg/kg.

The present invention found that the NMDA receptor GluN2B serine 1303 site phosphorylates and the AMPA receptor GluA1 serine 831 and serine 845 site phosphorylates in migraine rat ACC were increased on average. Presynaptic glutamate release and the response of both postsynaptic AMPA receptors and NMDA receptors are enhanced. Furthermore, anxiety behavior and nociceptive response are also increased. Synaptic long-term potentiation (LTP) is masked in the rat migraine model because theta pulse stimulation (TBS) fails to induce LTP. By using the AC1 selective inhibitor NB001 in ACC to inhibit cortical excitability, behavioral sensitization and anxiety are significantly reduced. The invention provides strong evidence for the contribution of cortical LTP to migraine-associated pain and anxiety, while drugs that inhibit cortical excitability, such as NB001, have utility in the preparation of drugs for the treatment of migraine and anxiety, would be potential new drugs for future treatment of migraine.

Drawings

Figure 1 facial hyperalgesia test in rats with different migraine models.

Figure 2 anxiety-like behavior of different migraine models.

FIG. 3 phosphorylation levels of GluN2B and GluA1 were enhanced in ACC of migraine sufferers.

Figure 4. Characterization of different rat anterior cingulate epithelial whole cell patches.

Fig. 5 IS repeated daily IS stimulation blocks tbs-induced ACC LTP.

FIG. 6 TBS induced LTP was chronically occluded in M-8d rat ACC without IS stimulation.

FIG. 7 response in TBS-induced control group and AH rat ACC.

Fig. 8. Effect of AC1 on migraine rat orbital Zhou Tongjiao allergy.

Fig. 9. Influence of AC1 on migraine related anxiety behavior in migraine rats.

Fig. 10. Synaptic model of migraine and migraine-associated anxiety.

Fig. 11. Low concentrations of NB001 did not alleviate the orbit Zhou Tongjiao allergy in migraine rats.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments and drawings. However, these examples are only for explaining the technical scheme of the present invention, and they should not be construed as limiting the scope of the present invention.

Unless otherwise indicated, the following examples were carried out according to conventional methods known to those skilled in the art using reagents of the type commonly used in the art in the corresponding assays and preparations.

For a detailed description of embodiments of the present invention, the meaning of the corresponding drawings in the drawings of the present invention is collectively described as follows:

figure 1, facial hyperalgesia test of rats with different migraine models. In the figure: a, saline or IS stimulated cannula implantation schematic. B, schematic of different treatment groups. The control group (upper left) was tested 1 hour after physiological saline stimulation. Acute Headache (AH) group (upper right) was detected 1 hour after IS stimulation. The chronic migraine group (below) was tested on day 1 (M-1 d), day 2 (M-2 d), day 4 (M-4 d), day 8 (M-8 d), day 15 (M-15 d), day 22 (M-22 d) and day 29 (M-29 d), respectively. An example shows a mouse drinking milk, while its facial area contacts the stimulation filaments of the mechanical stimulation module. D,4 sample traces show automatic recordings of drinking behavior for 10 min in control rats (upper), AH rats (middle), M-1D rats (middle) and M-8D rats (lower). E. Migraine rats were exposed to significantly less than the control group for about 3 weeks (control: =27 = -1d: =25; = 22; = -4d: =17; = -8d: =17; = -15d: =17; m-22d: =17; f (6, 135) =3.890, p=0.001, one-way analysis of variance). The contact time of AH (n=22; t (47) =0.880, p=0.383, unpaired t-test) and M-29 rats (n=11, t (36) = -0.825, p=0.415, unpaired t-test) was similar to control rats. Comparison of cumulative contact time during 10 min testing for F,4 different groups (F (2, 396) =30.250, p= 5.948E-13, two-way anova). G, total contact numbers between these different groups were not different (F (8, 166) =0.655, p=0.730, one-way anova). H. The periocular threshold of AH rats was not different from the control group (control group: n=20; AH: n=18; t (36) =1.264, p=0.214, unpaired t-test). Migraine rats (= -1d: = 18; = -2 = 18; = -4 = 17; = -8 = 17; = -15 =: = 17; = -22d: = 17; = (6, 117) = 14.308, p= 3.528E-12, one-way anova) showed a 3 weeks drop in periorbital threshold and recovery on day 29 (n=11, t (29) = -0.266, p=0.792, unpaired t-test). * p < 0.05; * P < 0.01 compared to control group.

Anxiety-like behavior of the different migraine models of fig. 2, different B, C, migraine rats showed anxiety-like behavior in field trials (control: n=8; ah: n= 8;M-1D: n= 8;M-2D: n= 8;M-8D rats: n= 7;F (4, 34) =3.867, p=0.011, unidirectional analysis of variance), but there was no difference in total travel distance (F (4, 34).=0.480, p=0.750, unidirectional analysis of variance) D, 5 EPM test samples from different raters, F, migraine rats showed anxiety-like behavior in EPM trials (control: n=8; ah: n= 8;M-1D: n= 8;M-2D: n= 8;M-8D rats: n= 6;F (4, 34) =3.633, p=0.015, unidirectional analysis of variance) but no difference in total entries (F (4, 33) =0.551, p=0.551, unidirectional analysis of variance). * P < value is 0.01, compared to control group.

FIG. 3 phosphorylation levels of GluN2B and GluA1 were enhanced in ACC of migraine sufferers. A, representative Western blot detection of AMPA receptor in control, AH, M-1d and M-8d rat ACC. The total protein levels of= -E, gluA1 (n= 8,F (3, 28) =0.781, p=0.515, one-way anova) and GluA2 (n= 8,F (3, 28) =0.735, p=0.540, one-way anova) were not different between groups, but the phosphorylation levels of GluA1 serine 831 (n= 7,F (3, 24) =3.748, p=0.024, one-way anova) and serine 845 (n=6, f (3, 20) =5.404, p=0.007, one-way anova) were significantly enhanced in AH, M-1d and M-8d rats. Representative western blots of NMDA receptors in control, acute headache, M-1d and M-8d rat ACC. GluN1 levels (8,F (3, 28) =0.847, p=0.480, one-way anova) were not different between groups, gluN2A (8 per group=, F (3, 28) =0.412, p=0.746, one-way anova), gluN2B (8 per group=, F (3, 28) =0.411, p=0.746, one-way anova) and p-GluN 2B-tyrosine 1472 (n=8 per group, = (3, 28) =1.115, p=0.360, one-way anova), but phosphorylation levels of GluN2 serine were significantly increased in AH, M-1d and M-8d rats (n=6, F (3, 20) =3.400, p=0.038, one-way anova). * p < 0.05, < p < 0.01 vs. Control group. Each data point represents a single animal.

FIG. 4 characterization of different rat anterior cingulate epithelial whole cell patches. a-C, statistics of frequencies (30) = -0.138, p=0.891, unpaired t-test; m-1d: t (32) = -2.078, p=0.046, unpaired t test; m-8d: t (43) = -0.916, p=0.365, unpaired t-test) and amplitude (AH: t (30) =0.149, p=0.882, unpaired t test; m-1d: t (32) = -2.195, p=0.036, unpaired t-test; m-8d rats: t (43) mpescs, p=0.049, unpaired t-test (17 neurons/8 control rats, 15/5 AH rats, 17 neurons/8 = -8d rats) = -2.027, p=0.049, unpaired t-test). D, pooled data showed that AMPA receptor mediated EPSCs input-output curves were shifted to the left (n=15 neurons/6 rats, F (1, 145) =49.962, p= 6.105E-11, bi-directional analysis of variance) and M-8D rats (n=15 neurons/6 rats, = (1, 145) =31.257, p= 1.089E-07, bi-directional analysis of variance) compared to control rats (n=16 neurons/5 rats). At the same time, AH (n=15 neurons/5 rats, F (1, 145) =0.136, p= 0.713, two-way anova) and control rats. E, AMPA receptor mediated I-V curves were not different in ACC neurons (n 20 neurons/8 control rats, 15 neurons/5 AH rats, 15 neurons/7M-1 d rats and 17 neurons/7M-8 d rats, F (3, 464) =1.468, p=0.223, two-way anova). The input-output curves of NMDA receptor-mediated EPSCs in F, M-1d were shifted to the left (total of 14 neurons per 8 rats, F (1, 145) =31.594, p= 9.441E-08, two-way anova) and M-8d rats (n=20 neurons per 7 rats, = (1, 175) =24.718, p=1.574E-06, two-way anova), compared to control group (=17 neurons per 5 rats) and= =16 neurons per 5 rats). The I-V curves for NMDA receptors for G, acute headache rats, M-1d and M-8d rats also shifted to the left (n-15 neurons/7 control rats, 16 neurons/5 AH rats, 13 neurons/7M-1 d rats and 17 neurons/7M-8 d rats, F (3, 456) =6.492, p=2.615E-04, two-way anova). * p < 0.05; * P < 0.01 compared to control group.

FIG. 5 IS a daily repetition of IS stimulation blocking tbs-induced ACC LTP. A. 3 samples showed presence of fEPSP network in ACC of control (left), acute headache (middle) and M-1d (right) rats. The feps was induced by electrical stimulation of one channel (labeled's') and recorded from the other 63 channels 30 min (black) before TBS and 3 hours after TBS. Asterisks indicate channels of fepp recruited in ACC. The sample traces superimposed at different time points (30 min before TBS surgery and 3h after surgery) show the following three plasticity: channels (1) showing L-LTP, channels (2) showing E-LTP, and unenhanced channels (3). B-D, time change of the slope of fEPSP from one sample slice of control rats: the 19 channels had L-LTP (B), the 2 channels had E-LTP (C), the 1 channel had no enhancement (D) E, and the control rats had a final average slope (148.95.+ -. 7.89% of baseline) for all 22 activated channels 3 hours after TBS. F-H, time change of fEPSP ramp from one sample slice of AH rat: 14 channels with L-LTP (F), 1 channel with E-LTP (G), 3 channels without enhancement (H). I, control rats, 3 hours after TBS, the final average slope of all 18 activation channels (165.86 ±, 11.56% of baseline). J-L, time change of fEPSP ramp from one slice of M-1d rat: 3L-LTP channels (J), 2E-LTP channels (K), 12 channels (L) without enhancement. M, in M-1d rats, the final average slope of all 17 activation channels (113.85.+ -. 4.59% of baseline) 3 hours after TBS.

FIG. 6 TBS induced LTP was chronically occluded in M-8d rat ACC without IS stimulation. A, one sample showed the presence of fEPSP network in the M-8d rat ACC. B-D, time change of the fEPSP ramp starting from one slice of M-8D rat: 1 channel with L-LTP (B), 4 channels with E-LTP (C), 16 channels without enhancement (D), E, M-8D rats at 3 hours after TBS, the final average slope of all 21 activation channels (99.12.+ -. 5.03% of baseline). F, average slope of fEPSP for all recorded channels in control group (11 pieces/5 rats), AH (15 pieces/7 rats), M-1d (16 pieces/6 rats) and M-8d (28 pieces/9 rats). The average slope of fEPSP from control channel, AH (1 h: t (24) =0.869, p=0.393, unpaired t test, 2 h: t (24) =0.872, p=0.392, unpaired t test, 3 h: t (24) =0.231, p=0.819, unpaired t test, M-1d (1 h: t (25) =4.548, p=1.201E-04, unpaired t test, 2 h: t (25) =4.685, p= 8.429E-05, unpaired t test, 3 h: t (25) =4.428, p= 1.641E-04, unpaired t test), and M-8d (1: h: t (37) =4.699, p= 3.558E-05, unpaired t test, p=5452E-05, unpaired t test, 2: h: t (37) =4.699, p= 3.558E-05, unpaired t test, p= 8.429E-05, unpaired t test, t (37) and t-8.37, t test). H. M-1d and M-8d rats had fewer L-LTP channels (M-1 d: t (25) = 4.796, p= 6.327E-05, unpaired t test; M-8d: t (37) = 4.424, p= 8.222E-05, unpaired t test) and more N-LTP channels (M-1 d: t (25) = -6.000, p= 2.885E-06, unpaired t test; M-8d: t (37) = -3.419, p=0.002, unpaired t test) were compared to control and AH rats 3h after TBS. * P < value is 0.01, compared to control group.

Figure 7 TBS-induced recruitment response in control and AH rat ACC. a-D, sample sections show the distribution of basal activation channels (blue) and TBS recruitment channels (red) in control rats (a) and AH rats (B), but no distribution E in M-1D rats (C) and M-8D rats (D). The superimposed samples show TBS-induced recruitment response. F. Time variation of the number of recruited feps in G, control and AH rats (n=7 pieces/4 rats/group, t (12) = -3.500, p=0.004, unpaired t-test) H, I, time variation of the amplitude of the recruited channels for control and AH rats (n=7 pieces/4 rats/group, t (12) = -2.570, p=0.025, unpaired t-test) p <0.05; * P < 0.01.

Figure 8 effect of AC1 on migraine allergy to the orbit Zhou Tongjiao of rats. A, control group, AH and migraine rats showed no change in AC1 total protein level (n=8, = (3, 28) = -0.512, p=0.677, one-way analysis of variance) B-D, inhibiting AC1 effectively reduced M-8D rat periocular hyperalgesia by microinjection (12, = (11) = -3.526, p=0.005, paired t-test), intraperitoneal injection (n=12 cases, t (11) = -4.682, p=0.001, paired t-test) or intragastric administration (n= 7,t (6) = -2.558, p=0.043, paired t-test) without affecting contact time (microinjection to ACC: t (11) = -2.088, p=0.061, paired t-test; intraperitoneal injection: t (11) = -1.382, p=0.194, paired t-test; intragastric administration: t (6) = 0.834, p=0.436, paired t-test). * p <0.05, < p < 0.01 vs. Control group.

Figure 9 effect of AC1 on migraine related anxiety behavior in migraine rats. A, 4 EPM test samples from M-8d rats receiving saline or NB001 injection. B-D, inhibition of AC1 was effective to alleviate migraine-related anxiety behavior in ACC microinjection M-8D rats (each group = 6, t (10) = -2.543, p = 0.029, unpaired t test), intraperitoneal injection (M-8d+ saline: n = 7;M-8d+ NB001: n = 6;t (11) = -3.858, p = 0.003, unpaired t test) or intragastric administration (each group n = 6, t (10) = -2.529, p = 0.030, unpaired t test), but did not affect the total input number (ACC microinjection: t (10) = 0.765, p = 0.462, unpaired t test; intraperitoneal injection: t (11) = -1.132, p = 0.282, unpaired t test; intragastric administration: t (10) = 1.081, p = 0.081, paired t test)/(p <0.05, < p < 0.01, p <0. 0.01 vs.

Fig. 10 synaptic models of migraine and migraine-associated anxiety are input from TG neurons that are active around (e.g., periorbital) and projected further into the cortex (including ACC and IC) through PBN and thalamus, resulting in increased excitability of ACC. Increased ACC excitability includes increased presynaptic glutamate release, and enhancement of postsynaptic responses at AMPA and NMDA receptors. Furthermore, FMRP, which was previously shown to be a component of visceral pain, may also contribute to enhancing AMPA and NMDA receptor responses. Subsequently, changes in presynaptic and postsynaptic plasticity of the anterior cingulate cortex further promote anxiety and skin sensitization in chronic migraine rats by a decreased sensory pathway (green arrow). ACC, front cingulum cortex; AC1, adenylate cyclase 1; AMPAR, AMPA receptor; caM, calmodulin, cAMP, cyclic adenosine monophosphate; CREB, cAMP response element binding protein; FMRP, fragile X mental retardation protein; a red acid receptor; hydrocyanic acid, hyperpolarizing activating cyclic nucleotide gating; L-VGCC, L-type voltage-gated calcium channel; NMDAR, NMDA receptor; PAG, ash around the water conduit; PBN, brachial side core; PKA, cyclic adenylate dependent protein kinase; RVM, coracoid; TG, trigeminal ganglion.

Figure 11 low concentrations of NB001 did not alleviate the orbit Zhou Tongjiao allergy in migraine rats. A, two sample traces show the automatic recording of drinking behavior of M-8d rats 10 min prior to injection of (up) and (down) NB 001. B, total contact time before NB001 injection and after NB001 injection during the trial was not different (n=12, t (11) = -0.458, p=0.656, paired t-test) C, total contact number of M-8d rats before NB001 injection was not different (n=12, t (11) = -1.865, p=0.089, paired t-test).

Examples:

materials and methods:

study design:

in this study, we applied inflammatory soup to rat dura mater, established acute headache or chronic migraine animal models, as previously described, and examined changes in pain behavior and anxiety behavior we found that migraine rats had significant periorbital mechanical hyperalgesia and anxiety behavior in view of the known contributing chronic pain in the different treatment groups, and we examined neuronal excitability and postsynaptic related receptors (NMDA and AMPA receptors) in whole cell patch clamp electrophysiology and immunoblot analysis. Subsequently, we induced LTP in migraine rat ACC by extracellular field potential recordings to detect sensitization of migraine rat ACC. Finally, we examined the expression of AC1 in ACC and examined the therapeutic effect of NB001 (AC 1 inhibitor) on migraine and related anxiety by ACC microinjection or systemic administration. The sample size is estimated in consideration of the variation and average of the samples before the experiment, and the number of biological samples is reported in the relevant legend and results. Animals were randomly assigned to groups for all studies, and NB001 treatment and behavioral experiments were assigned to groups and evaluated for results. No animals or samples were excluded from any analysis except for few rats that died after surgery. Data acquisition and analysis was obtained in an unbiased manner.

Animals:

adult male Spragali rats (body weight 200-240 g) were used for the experiment. All rats used in this experiment were purchased from the western traffic university laboratory animal center and randomly fed with human 12 hours light/dark cycle (9 am to 9 pm light) to provide free food and water. The study protocol has been approved by the ethics committee of the university of western traffic.

Surgery:

under isoflurane anesthesia, rats were fixed on a stereotactic frame and the scalp covering the back surface thereof was cut to expose the skull. Craniotomy in the right frontal bone (1.5. 1.5mm lateral to midline, 1.5mm posterior to Lei Gema) exposes the dura mater, and an inner cannula with stainless steel is implanted into the plastic cannula without touching the dura mater. The cannula is sealed with a matching obturator cap with a tip just beyond the total length of the cannula to prevent scar tissue from forming on the bore of the inner cannula. Sterile dental bone cement was applied around the cannula, and the cannula was protected with two small screws and fixed to the skull. The skin was sutured with 4-0 nylon, leaving only the obturator cap outside the skin. Penicillin is topically applied post-operatively to prevent infection of the surgical field, and all rats are given a prophylactic antibiotic injection (penicillin, 10 ten thousand IU/100 g) for at least 2 days during recovery. When the mice are fully awake, they are returned to a clean individual cage. All rats were allowed to recover for 7 days prior to the experiment. Periorbital sensation thresholds were measured during the convalescence phase to ensure that they returned to the preoperative baseline.

Treatment groups and repeated chemical stimulation:

after postoperative recovery, rats were randomized into the following three groups, with daily repeated dural injections of 10 μl saline or IS for different days: (a) saline stimulation for 1 day (control, top left of fig. 1B); (b) IS stimulation for 1 day (acute headache (AH) group, top right of fig. 1B); and (c) IS stimulated for 7 days (migraine (M) group, fig. 1B down). To observe migraine group rats on day 1 (M-1 d), day 2 (M-2 d), day 4 (M-4 d), day 8 (M-8 d), day 15 (M-15 d), day 22 (22 d) and day 29 (M-29 d) of migraine, respectively (FIG. 1C). IS a mixture of inflammatory mediators consisting of 2 mM histamine, 2 mM serotonin, 2 mM bradykinin and 0.2 mM prostaglandin E2 in physiological saline at pH 7.4. When rats freely move, physiological saline or IS spreads around the dura mater. Each group was tested 1 hour after injection.

Oral surface operation test:

two standard rat and companion cages were tested using the orofacial stimulus test system (31300, ugo basic). Rats were first placed in companion cages for 10 min to become familiar with the environment. Subsequently, the rats were transferred to a test cage, placed at regular intervals for 10 min, and their orofacial operation behavior was recorded. In the front of the test cage, there is a device with a drinking window, allowing the mouse's head to enter and get rewarded (milk) on the opposite side of the drinking window. The device also includes an infrared beam and a removable mechanical module containing 12 stimulating filaments. Depending on the type of experiment, a detachable mechanical module was used to determine if the rat was subjected to no or mechanical stimulus when attempting to pass the head through the drinking window. The infrared beam is positioned outside the drinking window and connected to a computer, and the duration and the contact times of the milk drinking are automatically recorded. Preoperative adaptation training of rats without mechanical stimulation for two weeks preoperative adaptation training in order to reduce statistical errors, rats that could not meet the criteria were removed with drinking times exceeding 300 seconds as a criterion. After 1 week post-operative recovery, rats were again subjected to 2 weeks post-operative adaptation training without mechanical stimulation to ensure recovery of alcohol intake time to pre-operative levels. Similar to the adaptation training, all experiments in the different groups used mechanical modules, but all experiments were fasted for 12 hours before, rats were familiarized with the test environment for 10 min, and then the evaluation of the facial operation behavior was performed for 10 min.

Periorbital nociception threshold test:

periorbital nociception thresholds were detected using von Frey monofilament method (Ugo basic). Von Frey monofilaments were perpendicular to the periorbital region until slightly flexed and held for 3-6s or until a positive response was observed. The threshold is determined by a "top-down" approach. Positive responses were recorded when the face of the mouse was withdrawn from Fengfu Lei Shansi. Rats that were nonreactive to maximum fiber strength (26 g) were assigned 26 g as their maximum periorbital nociceptive threshold for analysis.

Open air test:

the field test is intended to analyze anxiety-related behaviors and motor activities. The device consisted of a round black base (120 cm diameter) surrounded by black walls (40 cm). The inner space is a circle with the diameter of 90 cm; the outer space is annular and the inner space is 30 cm wide. Illumination is provided by a 40 watt bulb. The animals were placed in the instrument and allowed to explore freely for 5 min. The total distance and the inner zone distance were recorded by an animal behavior trace analysis system (super maze, shanghai Xinruan). The instrument was wiped with 70% alcohol between each test to remove olfactory cues.

Elevated Plus Maze (EPM) test:

EPM was used to measure anxiety-like responses and was performed in a four-arm maze (10 cm x 50 cm) 100 cm above the ground. The two closed arms had a dark wall 40 cm in height and the two open arms had edges 0.5 cm in height. The angle between the two arms is 90 degrees. Illumination is provided by a 40 watt bulb. Rats were placed in the center of the instrument, facing one closed arm, and allowed them to freely explore the 5 minutes spent on the open arm, the total number of arm entries was recorded by an animal behavioral tracking analysis system (super maze, shanghai Xinruan). The instrument was wiped with 70% alcohol between each test to remove olfactory cues.

NB001 microinjection ACC:

to determine the effect of ACC synaptic plasticity in migraine, we locally microinject NB001 in ACC, as reported previously. Briefly, rats were placed in a stereotactic instrument under isoflurane anesthesia. The guide cannula was implanted over the ACC (anterior 2.0mm, 0.8 mm from the outside of the midline, 0.7 mm from below the skull surface). Rats were given a recovery time of at least 2 weeks after cannula implantation. On day 8 of migraine attacks (M-8 d), intra-acc injection was performed using an injection cannula at 3 mm below the skull surface. The microinjection device consisted of a Gaoge glass syringe (1 μl). NB001 (10 mg/ml physiological saline) was injected into each side of ACC at a rate of 0.05. Mu.l/min for 10 min; an equivalent amount of physiological saline was used as a control. After each injection, the microinjection needle was placed for at least 2 min to prevent any solution from flowing outwards, and the behavioral test was started 30 min after the injection.

NB001 intraperitoneal injection and intragastric administration:

NB001 was dissolved in normal saline and either 5 or 20 mg/kg body weight was injected intraperitoneally, or 60 mg/kg body weight was injected intraperitoneally on day 8 (M-8 d) of the migraine attack; saline of equivalent concentration was used as a control. The effect of the drug was examined 30 min after injection.

Slice preparation:

acute coronal sections (300 μm) were prepared from SD rats and the rats were decapitated under 1-2% isoflurane anesthesia as described previously. The whole brain was rapidly removed from the skull of the anesthetized rats and immersed in ice-cold oxygenates (95% O) ₂ And 5% carbon dioxide) cleavage solution (containing mM): 252 sucrose, 2.5 potassium chloride, 6 MgSO ₄ , 0.5 CaCl ₂ , 25 NaHCO ₃ , 1.2 NaH ₂ PO ₄ The number of glucose values is 10, and the pH value is 7.3-7.4. After brief cooling, coronal brain sections (300 μm) containing ACC were cut in the above ice-cold oxygen-containing solution with a shaking group (VT 1200S, leica). Then, in a recovery chamber, artificial cerebrospinal fluid (ACSF) is soaked, which contains (in millimeters): 124 sodium chloride, 4.4 potassium chloride, 2 CaCl ₂ , 1 MgSO ₄ , 25 NaHCO ₃ , 1 NaH ₂ PO _4, And 10 glucose, coronal brain sections were incubated at room temperature for at least 2 hours.

Preparation of a multi-electrode array:

the MED64 detector (P530A, loose) was used for extracellular field potentiometric experiments with a set of 64 planar microelectrodes, each arranged in 8 x 8 mode, with an inter-electrode distance of 300 μm. To ensure that the slice adheres well to the MED64 probe during recording, the new MED64 probe requires hydrophilic treatment. Prior to use, the surface of the MED64 probe was treated with 0.1% polyethylenimine (P-3143, sigma-Aldrich) in 25 mM borate buffer (pH 8.4) at room temperature overnight. Before using the probe in the experiment, the probe surface was rinsed at least three times with sterile distilled water to remove harmful substances affecting brain slice activity.

Extracellular field potential recordings:

after incubation in the recovery chamber, a coronal section is transformedMove onto the prepared MED64 probe, ensuring that the entire microelectrode array covers different layers of ACC. In the recording chamber, the slice is perfused with oxygen-containing oxygen (95% O) ₂ And 5% carbon dioxide) ACSF at 28-30deg.C and maintained at a flow rate of 2 ml/min. After 1 hour incubation of the recording arc table, one channel located in the deep layer of ACC was selected as the stimulation site, and biphasic constant current pulse stimulation (0.2 ms) was applied to the stimulation site to induce field excitatory postsynaptic potentials (feppsps). LTP was induced prior to pulse stimulation (TBS, 5 pulses, 100hz,200 ms intervals; 5 replicates every 10 s), a stable baseline response was recorded for 30 min, and feps responses were recorded 3h after LTP induction.

Whole cell patch clamp electrophysiology:

whole cell patch clamp recordings were performed using an Axon 200B amplifier (molecular device) and bipolar tungsten stimulation electrodes were placed in the ACC deep layers to deliver stimulation. To record micro EPSCs (mEPSCs), the neuron voltage was fixed at-70 mV in the presence of TTX (1. Mu.M), and the pipette (3-5M Ω) was filled with a solution containing (mM) 145 k-gluconic acid, 5 sodium chloride, 1 MgCl ₂ 0.2 EGTA,10 HEPES,2 Mg-ATP,0.1 Na ³ GTP (adjusted to pH 7.2 with potassium hydroxide, 290 mmol). At the same time, the pipette was filled with a sample containing (mM) 112 Cs-gluconate, 5 TEA-Cl, 3.7 sodium chloride, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, 0.1 Na ³ GTP and 5 QX-314 (adjusted to pH 7.2 with CsOH, 290 mmol)Er) For NMDA and AMPA receptor mediated EPSCs recordings. Repeated 0.05 Hz stimulation was applied to induced AMPA receptor mediated EPSCs in the presence of AP-5 (50. Mu.M) when neurons were held at-70 mV by voltage. NMDA receptor mediated EPSCs were recorded at-30 mV by CNQX (20 μm) bathing. Kupi fruit toxin (100. Mu.M) is always present to block GABA _A Receptor-mediated inhibitory synaptic current was used in all experiments. Data were collected and analyzed using software (molecular devices) from clammex 10.3 and clampfit 10.2.

Western blot analysis:

total protein assay briefly, ACC samples were dissected on ice in ice PBS (-) and homogenized in lysis buffer (10 mM Tris-HCl (pH 7.4), 2 mM EDTA, 1% SDS including protease inhibitor cocktail) as previously described (24). The supernatant was then centrifuged (15000 g,20 min,4 ℃). Western blot detection was as described above. Sample protein concentration was quantified using BCA assay (Beyotime) and an equal amount of protein (40 μg) was run on a 7.5% sds-polyacrylamide gel. Isolated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, then blocked with 5% skim milk TBS-T (Triton X-100 triple buffered saline) for 1 hour, then diluted with primary antibodies ((Millipore, US): anti-GluN 1 (1:500), anti-GluN 2A (1:1000), anti-GluN 2B (1:500), anti-p-GluN 2B-serine 1303 (1:1000), anti-p-GluN 2B-tyrosine 1472 (1:1000). Anti-GluA 1 (1:500), anti-pGluA 1 serine 831 (1:1000), anti-pGluA 1 serine 845 (1:1000), anti-GluA 2 (1:1000), anti-AC 1 (1:1000) and anti-tubulin (1:4000; sigma, US)), incubated at 1:5000 (Millipore) overnight at 4℃overnight, the membranes were coupled with horseradish peroxidase anti-rabbit/mouse lgG secondary antibodies, and then immunoblotted with enhanced chemiluminescence (ECL (GE Healthcare)) by means of enhanced chemiluminescence, and immunoblotting (pH) with another dilution of the anti-Glu 1 (1:4000) buffer (1:1000), and immunoblotting was performed by another national assay for immunoblotting with a different mM buffer (pH-6).

Statistical analysis:

data are presented as mean ± scanning electron microscope. Statistical comparisons between the two groups employed either a two-tailed paired or unpaired t-test. The differences between the groups used either single-or two-factor analysis of variance (analysis of variance, IBM SPSS 18). In all cases p < 0.05 was considered statistically significant.

Results:

mechanical withdrawal hyperalgesia in migraine model rats:

hyperalgesia and allodynia are two important symptoms of chronic pain. Previous studies showed that the periorbital nociceptive pain threshold was reduced in migraine animals. To investigate the variation in the orbit Zhou Tongmin of different headache rats, we performed a modified orofacial stimulation experimental system test and von Frey test on the periorbital area of control, acute headache, migraine rats. The animal model building process is shown in fig. 1B. Rats in the control group or in the Acute Headache (AH) group in this study received only one sterile saline or inflammatory decoction (IS) stimulus. Migraine groups were then given IS stimulation 7 times (once daily) consecutively to build a migraine model. Furthermore, to observe the duration of the migraine status, rats of the migraine group were subjected to migraine experiments on day 1 (M-1 d), day 2 (M-2 d), day 4 (M-4 d), day 8 (M-8 d), day 15 (M-15 d), day 22 (M-22 d) and day 29 (M-29 d), respectively, of the migraine attacks (FIG. 1B).

Figures 1C-D show the test procedure of the orofacial stimulus test system, similar to previous studies, when rats drink milk, the stimulus wire will adhere to the periorbital area of the rats and the drinking time will be recorded by infrared means until the rats leave the drinking window. To avoid potential effects of postoperative pain, rats were tested with the stimulation module two weeks after surgery. The cumulative contact time of the control group and acute headache rats was similar over a 10 minute test period (fig. 1F). There was no significant difference in total contact time between the control group and the acute headache group (control group, 353.87 ±19.55 s, n=27; acute headache rats, 323.00 ±30.61 s, n=22; t (47) =0.880, p=0.383, unpaired t-test; fig. 1E). Furthermore, we also recorded the total contact time of migraine rats at different time points. The total contact time of migraine rats on day 1 was significantly lower than that of control group rats and lasted around 3 weeks (M-1 d rats, 255.17 + -26.68 s, n=25; M-2d rats, 214.63 + -24.81 s, n=22; M-4d rats, 218.64 + -29.93 s, n=17; M-8d rats, 263.26 + -27.36 s, n=17; M-15d rats, 252.22 + -22.91 s, n=17; M-22d rats, 277.05 + -29.73 s, n=17; F (6, 135) =3.890, p=0.001; one-way variance analysis; FIG. 1F). In addition, the cumulative contact time at each time point was significantly lower for migraine rats at day 1 and day 8 than for control group (F (2, 396) =30.250, p= 5.948E-13, two-factor analysis of variance; fig. 1E). However, the total contact time at day 29 of the migraine rats was restored to the level of the control group on the fourth week after IS stimulation for 7 consecutive days (M-29 d rats, 381.33 + -19.81 s, n=11; t (36) = -0.825, p=0.415, unpaired t-test; FIG. 1G). Meanwhile, the total number of contacts in the 10-minute test interval was not significantly different in rats in the control group, AH group and migraine group (F (8, 166) =0.655, p=0.730, one-way analysis of variance; fig. 1H). This suggests that the significant reduction in total contact time for migraine rats to drink milk is not due to the reduced number of attempts by the animals to drink milk.

Subsequently, we applied the traditional method, von Frey test, to further verify the variation of periorbital pain threshold for rats in different treatment groups (fig. 1H). The von Frey assay results for periorbital areas of rats of different treatment groups were similar to those of the orofacial stimulation test system. There was no significant difference in periorbital pain threshold between control and acute headache rats (control, 17.55±1.54 g, n=20; acute headache rats, 14.14±2.27 g, n=18; t (36) =1.264, p=0.214, unpaired t-test; fig. 1H). The periorbital pain threshold of migraine rats was significantly lower than that of control rats (M-1 d rats, 6.66+ -1.42 g, n=18; M-2d rats, 4.81+ -1.02 g, n=18; M-4d rats, 3.69+ -0.71 g, n=17; M-8d rats, 5.52+ -1.10 g, n=17; M-15d rats, 5.69+ -1.47 g, n=17; M-22d rats, 10.24+ -1.60 g, n=17; F (6, 117) =14.308, p= 3.528E-12; one-way variance analysis; FIG. 1H) and returned to the level of control rats on day 29 (M-29 d rats, 18.27+ -2.37 g, n=11; t (29) = -0.266, p=0.792; unpaired t-test; FIG. 1H). These results suggest that the periorbital area of rats in the migraine group is exposed to mechanical hyperalgesia of longer duration.

Migraine model migraine related anxiety behavior in rats:

chronic pain is reported to cause anxiety and other negative emotions associated with pain. To test anxiety-related behavior in rats of the different treatment groups in this study, we performed open field and EPM tests on these rats. We found that rats in the migraine group had anxiety-like behavior associated with their headache. In open field experiments, the total movement distances of control, AH, M-1d, M-2d, and M-8d rats were not different (35.28±2.28M, n=8 for control, AH rats, 33.16 ±2.98M, n= 8;M-1d rats, 30.59 ±1.80M, n= 8;M-2d rats, 31.55±3.63M, n= 8;M-8d rats, 31.88±1.85M, n= 7;F (4, 34) =0.480, p=0.750, one-way analysis of variance; fig. 2B), but the movement distances of migraine rats in the central region were significantly smaller than those of control (control, 5.81±0.75M, n=8 for AH rats, 3.89±0.68M, n= 8;M-1d rats, 2.56±0.40M, n=95-2 d rats, 3.39±0.68M, n=69±8-8 d rats, p=3.68, p=3.35M, p=0.75M, p=3.35M, p=8.35, p=3.40). In the EPM experiments, there was no significant difference in total number of entries in the control, AH, M-1d, M-2d, and M-8d groups (F (4, 33) =0.772, p=0.551, one-way analysis of variance; fig. 2E), but migraine rats were significantly less time in the open arm than control rats (control, 28.49±4.83%, n=8 AH, 27.90±6.02%, n= 8;M-1d, 13.48±2.21%, n= 8;M-2d, 11.78±3.34%, n= 8;M-8d, 10.93±6.39%, n= 6;F (4, 33) =3.633, p=0.015, one-way analysis of variance; fig. 2F). Furthermore, acute headache has no effect on the anxiety behavior associated with headache in rats. These results indicate that chronic migraine attacks induce anxiety-related behavior in rats, rather than a single acute headache.

Increased levels of phosphorylation of GluA1 and GluN2B in ACC in migraine patients:

both AMPA receptors and NMDA receptors are tetramers consisting of four subunits. Among them, gluN2B and GluA1 play a key role in sensitization of ACC in chronic pain. To determine whether these two receptor subtypes are involved in migraine-induced ACC sensitization, we examined the levels of these two receptor subtypes in different treatment groups of ACC. We found that the total protein levels of GluA1 (F (3, 28) =0.781, p=0.515, single factor anova; fig. 3 AB) and Glu N2B (F (3, 28) =0.411, p=0.746, single factor anova; fig. 3I) did not show differences between the control, acute headache, M-1d and M-8d rats. Furthermore, we examined the levels of the other several major AMPAR subtypes and NMDAR subtypes in the different treatment groups ACC. We found that the total protein levels of GluA2 (F (3, 28) =0.735, p=0.540, single factor anova; fig. 3E), gluN1 (F (3, 28) =0.847, p=0.480, single factor anova; fig. 3G) and GluN2A (F (3, 28) =0.412, p=0.746, single factor anova; fig. 3H) did not vary between the control, acute headache, M-1d and M-8d rats. Subsequently, the study further compares the changes in phosphorylation sites of GluA1 and GluN2B in the rat ACC of the different treatment groups. We found that the degree of phosphorylation of GluA 1-serine 831 site (F (3, 24) =3.748, p=0.024, one-way anova; FIG. 3 AC) and serine 845 site (F (3, 20) =5.404, p=0.007, one-way anova; FIG. 3D) were significantly increased in acute headache, M-1D and M-8D rats compared to control rats. Furthermore, we found that the degree of phosphorylation of GluN 2B-serine 1303 site (F (3, 20) =3.400, p=0.038, one-way anova; fig. 3 FJ) was also significantly increased in acute headache, M-1d and M-8d rats, but that the level of phosphorylation of GluN 2B-tyrosine 1472 site was not significantly changed (F (3, 28) =1.115, p=0.360, one-way anova; fig. 3K). These results indicate that NMDAR and AMPAR are involved in migraine induced ACC sensitization primarily through the phosphorylation sites of GluN2B and GluA 1.

Long-term presynaptic and postsynaptic expansion in ACC in migraine rats:

to determine whether synaptic transmission of ACC was enhanced in migraine rats, we recorded miniature EPSCs (mpescs) using whole cell patch clamp. We found that the frequency difference between control and test M-1d rats was significant, but there was no difference between control and test M-8d rats (FIG. 4B). In addition, the amplitudes of both the test group M-1d and M-8d rats were significantly greater than the control group (FIG. 4C). Meanwhile, there was no change in frequency and amplitude of the mEPSCs in both control and test rats (FIGS. 4A-C). These results indicate that pre-and post-synaptic factors are involved in the enhancement of migraine rat ACC transmission.

Postsynaptic AMPA and NMDA receptor mediated current play a key role in the induction and maintenance of ACC LTP in chronic pain. To determine which receptors are associated with enhanced delivery of the anterior cingulate cortex in migraine rats, we recorded the input (stimulus intensity) -output (EPSC amplitude) efficiency and I-V relationship of AMPA and NMDA receptor-mediated synaptic responses. The AMPA receptor mediated input-output (I-O) curves for M-1D rats were shifted to the left compared to control rats, which was similar to that for M-8D rats, whereas the I-O curves for control and AH rats were not different (fig. 4D). These results indicate that AMPA receptor mediated excitatory responses are enhanced in migraine rats. However, there was no significant difference in the I-V curves (-70 to +50 mV) between the two groups (fig. 4E).

We then tested NMDA receptor-mediated currents in these rats. The results of the I-O profile for the NMDA receptor are similar to those for the AMPA receptor. We found that the NMDA receptor mediated I-O curves for M-1d and M-8d rats shifted to the left compared to the control group (FIG. 4F). In addition, the I-V curves for the NMDA receptors of AH, M-1d and M-8d rats also shifted to the left compared to the control group (FIG. 4G). These results indicate that both NMDA and AMPA receptor mediated synaptic transmission is enhanced in migraine model rats.

No LTP induction in migraine rat ACC:

long Term Potentiation (LTP) is a key synaptic mechanism for chronic pain. To determine if acute headache is sufficient to induce LTP in ACC, we recorded network LTP in control and AH rats using MED 64 system. In control rat sample sections, 19 channels were activated for 3h in late LTP phase (L-LTP); 2 channels are activated in the early stage of LTP (E-LTP) for less than 3 hours; 1 activation channel has no LTP (N-LTP). The final average slope for all 22 activation channels was 148.95 ±,7.89% of baseline levels 3 hours after LTP induction (fig. 5B-E). The experimental results of AH rats were similar to the control group. In a typical AH rat sample slice, there are 14 channels active in L-LTP, 1 channel active in E-LTP, and 3 active channels showing N-LTP. The final average slope of all 18 activation channels was 165.86 ±and 11.56% of baseline levels 3 hours after LTP induction (fig. 5F-I).

The 144 activation channels of 11 pieces of 5 control rats were analyzed and the average induction rates of 3 different types of reactions were found to be 66.07.+ -. 10.19% (L-LTP), 14.75.+ -. 5.15% (E-LTP) and 19.18.+ -. 6.37% (N-LTP), respectively (FIG. 6H). At 3 hours, the final average slope for all 144 activated channels was 135.80 ±6.99% of baseline level (fig. 6F-G). Of 15 sections of 7 AH rats, 51.21 + -7.88% of the channels showed L-LTP, 12.94+ -4.68% of the channels showed E-LTP and 35.84+ -7.43% of the channels failed to be enhanced (FIG. 6H). The final average slope of all 204 activation channels in AH rats after LTP induction for 3h was also similar to control rats (fig. 6F-G). These results indicate that acute headache is insufficient to induce LTP in ACC.

To investigate whether the synaptic plasticity of migraine rat ACC was altered, we also recorded the network LTP of M-1d rats. FIG. 5J-M shows typical 17 activation channels for M-1d rats, with only 3 activated channels showing L-LTP, while 2 activation channels show E-LTP and 12 activation channels show N-LTP. The final average slope of 17 activation channels 3h after LTP induction was 113.85 ±4.59% of baseline (fig. 5M). 16 sections of 6M-1 d rats showed that the average inductivity of the different types of channels of the M-1d rats was also affected. M-1d rats showed more N-LTP channels (72.25.+ -. 5.88%); fewer channels (14.16.+ -. 5.65%) were present for L-LTP (FIG. 6H). The final average slope of all 167 activation channels in M-1d rats was significantly lower than that of the control group (103.86.+ -. 3.60% of the M-1d rats baseline; FIGS. 6F-G). All these data indicate enhanced synaptic connection of the anterior cingulate cortex of migraine rats.

Migraine caused TBS-induced long-term occlusion of LTP:

to determine whether LTP can be maintained for long periods in ACC in migraine rats in the absence of IS stimulation, we recorded network LTP in M-8d rats. As shown in FIG. 6, the number of different types of channels in a typical slice with 21 activation channels was similar to that of the M-1d rat. After LTP induction, there were 1L-LTP channel, 4E-LTP channels, and 16 non-enhanced channels in the sample sections. Meanwhile, the final average slope of this representative sample from M-8d rats was 99.12±5.03% of baseline value 3 hours after LTP induction (fig. 6E). Our experimental results on 347 activation channels from 28 sections of 9M-8 d rats showed that the induction rate of L-LTP channel by M-8d rats was significantly lower than that by control group rats (M-8 d rats, 23.86.+ -. 4.48%), while that by N-LTP channel in M-8d rats was significantly higher than that by control group rats (M-8 d rats, 51.99.+ -. 5.45%) (FIG. 6H). In addition, the final average slope of all 347 activation channels in M-8d rats was also significantly lower than in control rats (M-8 d rats, 107.01.+ -. 3.08% of baseline; FIG. 6F-G). These results indicate that migraine headache results in TBS-induced long-term occlusion of LTP in the absence of IS stimulation, which may lead to long-term hyperalgesia in migraine rats.

Response was in ACC of control group and acute headache rats, but not migraine rats.

Previous studies have shown that silent synapses can be responded to by postsynaptic transport of AMPA receptors, and that this response can be observed by a multichannel recording system. To investigate the effect of different types of headaches on response, we analyzed the number and amplitude of response channels in different types of headache rats. During baseline recordings, 12.82±2.14 activation channels were observed per slice for control rats (fig. 7A). Although the response to the responses could not be recorded in all sections, 1.57.+ -. 0.30 was recorded 3 hours after TBS in the control rat sections where the response was generated in 30 response channels (FIGS. 5F-G), which were located at the edge of the basal active zone. (FIGS. 7A-B).

Similar to the control rats, there was no difference in the number of basal activation channels between the control and AH rats, and 2.29±0.61 response channels per day were observed in 3hAH rats after TBS (fig. 7F-G). However, there was an interesting phenomenon-AH rats recorded more recruitment 1 hour after TBS than the control group (fig. 7F). For chronic migraine rats, no recruitment was observed for both M-1D and M-8D rats (FIGS. 7C-D).

Fig. 7H shows the time course of the fEPSP amplitude change for the answered channel. Rats in the control group had a gradual increase in amplitude of the responded channel after TBS, and eventually increased to 7.07±0.77 μv after 3 h. Whereas the amplitude of the post-TBS 3hAH rats was 10.34.+ -. 1.01. Mu.V, which was greater than that of the control rats (FIG. 7H-I). These results indicate that headache stimulus induces postsynaptic transport of AMPA receptors in silent synapses, and that this transport may occur within 1 hour after headache stimulus.

Effect of AC1 on mechanical hyperalgesia in migraine rats

Recent studies have shown that up-regulation of the AC1 total protein level in visceral pain is important for chronic pain. Thus, we examined the level of AC1 in the migraine rat ACC to observe changes in chronic migraine rat AC 1. We found that there was no difference in total protein levels of AC1 between the control group, AH, M-1d and M-8d rats (FIG. 8A). This result suggests that migraine does not affect the total protein level of AC1, unlike neuropathic pain or visceral pain.

Then, we applied the AC1 antagonist NB001 to M-8d rats to determine if AC1 participated in migraine in an activity dependent form. We found that microinjection of NB001 in the ACC of migraine rats effectively prolonged the milk drinking time (upper left corner of fig. 8B), but did not affect the total number of contacts (lower left corner of fig. 8B). We also performed intraperitoneal and intragastric injections of NB001 in migraine rats. We found that NB001 was also effective in improving the hydration time of migraine rats at 20 mg/kg (i.p, upper left corner of FIG. 8C) and 60 mg/kg ((i.g, upper left corner of FIG. 8D), but did not affect the total number of contacts (lower left corner of FIG. 8C and lower left corner of FIG. 8D). However, low doses of NB001 (i.p, 5 mg/kg) had no significant improvement in migraine rat orbital Zhou Tongjiao allergy (FIG. 11B).

Effects of AC1 on migraine-associated anxiety:

finally, we also tested the effect of AC1 on migraine-associated anxiety behavior. ACC microinjection NB001 significantly increased the time to arm of M-8d rats (right in fig. 9B), but did not affect total entry (left in fig. 9B). Intraperitoneal and intragastric administration were also similar to the results of microinjection (FIGS. 9G-H). These findings indicate that AC1 is involved in migraine and anxiety associated with migraine by its own activity change, not by protein synthesis. This is different from previous studies on neuropathic pain or visceral pain.

Discussion:

recent studies using animal models of chronic pain have consistently shown that plasticity of the front cingulate cortex is likely to be a novel approach to the treatment of neuropathic pain, cancer pain, visceral pain and inflammatory pain for behavioral sensitization and emotional anxiety. In this study, we demonstrate that AC1 plays a key role in chronic migraine. After stimulation of dural inflammation, both NMDA and AMPA receptors in ACC are phosphorylated, and presynaptic release of glutamate and postsynaptic responses of AMPA and NMDA receptors are enhanced. Our behavioral studies have found that inhibition of ACC excitability by topical administration of the AC1 inhibitor NB001 significantly relieves anxiety and inhibits hyperalgesia. To our knowledge, this is the first demonstration that AC 1-dependent ACC plasticity plays a key role in chronic migraine.

Behavioral hyperalgesia in chronic migraine patients:

skin sensitivity, or ectopic pain, is a typical symptom of migraine. Previous studies have shown that migraine rats present a diffuse ectopic pain (including contralateral head and legs) which we demonstrated in this study by a modified orofacial stimulus test system. We found that periorbital hyperalgesia persisted for about 3 weeks without persistent inflammatory stimuli. Previous studies using animal models of inflammatory or neuropathic pain have found that postsynaptic enhancement of the anterior cingulate skin layer (otherwise known as postltp enhancement) contributes to peripheral sensitization in rodents suffering from chronic pain. In this study, we found a significant enhancement of excitatory postsynaptic transmission of chronic migraine rat ACC. Postsynaptic AMPA receptor mediated increases in current and amplitude of mpescs. Consistent with these findings, we found that post LTP of TBS-induced migraine rat ACC was occluded. Biochemical experiments showed that the phosphorylation level of AMPA GluA1 (including serine 831 and serine 845) was elevated in migraine rat ACC. Our previous studies indicate that an increase in GluA1 phosphorylation level is important after LTP in ACC. Thus, our findings strongly suggest that the cortical mechanism regulated by AMPA receptors plays an important role in chronic migraine and chronic pain induced by somatosensory lesions.

In addition to enhancement of AMPA receptors, we found that NMDA receptor-mediated postsynaptic currents are also enhanced in chronic migraine. In addition, the phosphorylation level of GluN2B was increased. Recent studies have shown that phosphorylation of NMDA GluN2B receptors contributes to cortical excitation and chronic pain of somatic and visceral pain. For example, it has been reported that in the mouse visceral pain model, NMDA receptor function is enhanced in ACC, mainly by increasing GluN2B phosphorylation at serine 1303 and tyrosine 1472 sites. In this study we found that the phosphorylation of GluN2B was only increased at the serine site in chronic migraine rat ACC, whereas the phosphorylation of tyrosine 1472 was not increased. This difference may be due to different signal pathways and different pain models. In addition, gluN2B tyrosine 1472 is phosphorylated in the trigeminal nucleus. These findings indicate that different sites of GluN2B phosphorylation in migraine are associated with different functions of NMDA receptors in different central regions.

As is well known, spinal pain transmission is subject to down regulation from structures on the spinal cord, including the brainstem, the gray matter surrounding the water guide tube and the cortex, in addition to cortical excitation, recent studies report that anterior cingulate gyrus also have a strong down promoting effect on spinal pain transmission. acc-spinal cord predisposition is likely to cause allodynia with chronic migraine. ACC excitation reported in this study may affect the transmission of spinal pain through down-regulation of facilitation. Previous studies on downstream regulation of migraine have focused mainly on downstream inhibitory pathways from the lower levels of the brainstem (such as the medial medullary kissing flank and PAG), and future studies clearly need to reveal downstream facilitated regulation of migraine by ACC.

Emotional anxiety in chronic migraine sufferers:

previous clinical studies have shown that migraine sufferers are anxious and anxiety aggravates migraine. However, the exact neuronal mechanism of interaction between migraine and anxiety is not yet known. In this study we found that chronic migraine rats had long-term anxiety. Previous studies only measured anxiety behavior immediately after inflammatory stimuli, but did not examine long-term anxiety behavior. Here we found that in chronic migraine rats, long-term anxiety behavior can last for at least one week if there is no persistent inflammatory stimulus of the dura mater. Since we detected a pre-synaptic enhancement of glutamate release in chronic migraine, this pre-synaptic enhancement is likely to contribute to behavioral anxiety. It has been previously demonstrated that ac1 dependent pre-LTP in ACC is important for damage induced anxiety. Based on this finding, we found that microinjection of AC1 inhibitors in ACC can alleviate anxiety in migraine rats.

Clinical significance

Triptans and Calcitonin Gene Related Peptide (CGRP) receptor antagonists, two common peripheral drugs used to treat/prevent acute migraine, have been reported to be less effective in skin sensitization and anxiety in humans or rodents suffering from chronic migraine. This may be due to the fact that these inhibitors are ineffective in blocking central sensitization of the cortex of chronic migraine. AC1 is a neuronal form of subtype ACs that has been shown to contribute to both pre-LTP and post-LTP of ACC. In this study, we found that NB001 was topically applied to ACC or orally administered, producing significant analgesic and anxiolytic effects in chronic migraine rats, suggesting that ac 1-dependent ACC plasticity contributes to migraine. This finding is consistent with our previous findings that AC1 is critical to ACC plasticity, and knockout or inhibition of AC1 can alleviate pain and anxiety behavior in different chronic pain animal models. Furthermore, our recent studies report that CGRP enhances excitatory transmission of the anterior cingulate gyrus, and this enhancement also requires AC1 activity. Recently, CGRP antagonists have been approved for the treatment of idiopathic pain and ectopic pain to prevent migraine. However, they cause side effects such as hepatotoxicity unlike CGRP antagonists, our previous animal data and phase I clinical studies have found NB001 to be very safe for both animals and humans. It is believed that AC1 could be a new drug target for the treatment of chronic migraine, while NB001 is a powerful candidate.

Claims (5)

1. Use of a medicament for inhibiting AC1 in the manufacture of a medicament for treating migraine and anxiety.

2. Use of an AC1 inhibiting medicament according to claim 1 for the manufacture of a medicament for the treatment of migraine and anxiety, wherein the AC1 inhibiting medicament is NB001 or a pharmaceutically acceptable salt, solvate thereof; NB001 is 5- ((2- (6-amino-9-H-purin-9-yl) ethyl) amino) pentane-1-pentanol, and has a structural formula:

。

3. use of an AC1 inhibiting medicament according to claim 1 or 2 for the manufacture of a medicament for the treatment of migraine and anxiety, wherein the AC1 inhibiting medicament comprises an active ingredient for enteral or parenteral administration and a suitable pharmaceutically organic or inorganic inert carrier material.

4. Use of a medicament for inhibiting AC1 according to claim 3 for the manufacture of a medicament for the treatment of migraine and anxiety, wherein the suitable pharmaceutically organic or inorganic inert carrier material comprises water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils and/or polyalkylene glycols.

5. Use of an AC1 inhibiting medicament according to claim 1 or 2 for the preparation of a medicament for the treatment of migraine and anxiety, characterized in that the AC1 inhibiting medicament is administered in an active ingredient dose of: 1 ng to 500 mg/kg.

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