Injection Of Substance P Research Articles

Deep electroacupuncture of neurogenic spots attenuates immobilization stress-induced acute hypertension in rats

BackgroundOur previous studies proved that neurogenic inflammatory spots (or neurogenic spots) have the same physiological features as acupuncture points and that neurogenic spot stimulation generates therapeutic effects in various animal models. However, it is unclear how deeply the neurogenic spots should be stimulated to generate therapeutic effects. MethodsThe effects of acupuncture at various needle depths below the neurogenic spot were examined in a rat immobilization stress-induced hypertension (IMH) model. Electroacupuncture was applied to a neurogenic spot at depths of 1, 2, or 3 mm using a concentric bipolar electrode. ResultsElectrical stimulation of the neurogenic spot at a 3-mm depth most effectively lowered blood pressure compared with controls and stimulation at 1- and 2-mm depths, which was inhibited by pretreatment with a local anesthetic lidocaine. Electrical stimulation of the neurogenic spot or injection of substance P (SP) at a 3-mm depth significantly excited the rostral ventrolateral medulla (rVLM) compared with superficial stimulation. Electrical stimulation applied at a 3-mm depth on neurogenic spots dominantly caused c-fos expression from rVLM and ventrolateral periaqueductal gray (vlPAG) in IMH rats. Pretreatment with resiniferatoxin (RTX) injection into the neurogenic spot to ablate SP or calcitonin gene-related peptide (CGRP) prevented the effects of 3-mm neurogenic spot stimulation on blood pressure in IMH rats. Conversely, artificial injection of SP or CGRP generated anti-hypertensive effects in IMH rats. ConclusionOur data suggest that neurogenic spot stimulation at a 3-mm depth generated anti-hypertensive effects through the local release of SP and CGRP and activation of rVLM and vlPAG.

Substance P aggravates ligature-induced periodontitis in mice.

Periodontitis is one of the most common oral diseases in humans, affecting over 40% of adult Americans. Pain-sensing nerves, or nociceptors, sense local environmental changes and often contain neuropeptides. Recent studies have suggested that nociceptors magnify host response and regulate bone loss in the periodontium. A subset of nociceptors projected to periodontium contains neuropeptides, such as calcitonin gene-related peptide (CGRP) or substance P (SP). However, the specific roles of neuropeptides from nociceptive neural terminals in periodontitis remain to be determined. In this study, we investigated the roles of neuropeptides on host responses and bone loss in ligature-induced periodontitis. Deletion of tachykinin precursor 1 (Tac1), a gene that encodes SP, or treatment of gingiva with SP antagonist significantly reduced bone loss in ligature-induced periodontitis, whereas deletion of calcitonin related polypeptide alpha (Calca), a gene that encodes CGRP, showed a marginal role on bone loss. Ligature-induced recruitment of leukocytes, including neutrophils, and increase in cytokines leading to bone loss in periodontium was significantly less in Tac1 knockout mice. Furthermore, intra-gingival injection of SP, but not neurokinin A, induced a vigorous inflammatory response and osteoclast activation in alveolar bone and facilitated bone loss in ligature-induced periodontitis. Altogether, our data suggest that SP plays significant roles in regulating host responses and bone resorption in ligature-induced periodontitis.

Substance P Reduces Infarct Size and Mortality After Ischemic Stroke, Possibly Through the M2 Polarization of Microglia/Macrophages and Neuroprotection in the Ischemic Rat Brain.

Substance-P (SP) is an 11 amino acid neuropeptide that is known to stimulate the peripheral mobilization of bone marrow mesenchymal stem cells and M2 polarization in monocytes/macrophages in a variety of acute and chronic tissue injuries. To examine the role of SP in protection and recovery from acute ischemic brain injury, experimental ischemic stroke was induced by transient middle cerebral artery occlusion (tMCAo) in rats for 1h with subsequent reperfusion. Two injections of SP, immediately and one day post-tMCAo, resulted in approximately threefold lower mortality and 40% less infarct volume than those of saline-treated rats at seven days post-tMCAo. At 4.5h, SP markedly increased CD11b/c+CD163+/CD 206+ cells in the blood, which were concomitantly decreased in the bone marrow, suggesting that SP preferentially mobilized M2-polarized monocytes. After two days, SP increased the expression of neuroprotective and anti-inflammatory genes in the ischemic brain and induced neuronal survival in the brain penumbra. Additionally, SP markedly increased CD68+CD163+ and CD68+CD206+ M2 microglia/macrophages in the ischemic brain during seven days post-tMCAo. Furthermore, SP preserved the blood‒brain barrier in the ischemic brain, which was confirmed by the abundant levels of SMI71+ brain endothelial cells that colocalized with α-SMA+ pericytes. The beneficial effects of SP on functional recovery and tissue preservation were maintained for six weeks. Collectively, SP treatment in the early phase of ischemic stroke markedly suppressed the destructive inflammatory response and improved the microenvironment for tissue protection and repair.

Effects of Substance P for Liver Regeneration in Rat Hepatectomy Models: A Preliminary Experimental Animal Study.

BackgroundThis experiment was designed to investigate the effect of substance P (SP) on liver regeneration after partial hepatectomy in a rat model.Material/MethodsMale Sprague-Dawley rats were used in this experiment and were divided into an untreated sham group (n=3); a saline group (control group) given a saline injection after hepatectomy (n=4); and an SP group (experimental group) given SP injection after hepatectomy (n=4). The experiments were repeated 3 times in the sham, saline, and SP groups (days 1, 2, and 3). Liver function tests were performed on the serum. Immunohistochemistry was performed for proliferating cell nuclear antigen (PCNA), Ki-67, and CD133, and western blotting was performed for PI3KC, Akt, p38, ERK, and mTOR.ResultsLiver function test results indicated higher levels in the saline group than in the SP group at day 1. However, the difference gradually disappeared at days 2 and 3. PCNA and Ki-67 were more highly expressed in the SP group than in the saline and sham groups at day 1. CD133 was strongly expressed in the SP group at day 2 compared to that in the sham and saline groups. The changes in mTOR/Akt/PI3KC pathway protein and ERK/p38 signaling protein expression levels were not significantly different among groups.ConclusionsSP accelerated the regeneration stage after hepatectomy in a rat model.

Angiotensin (1-7) Attenuates the Nociceptive Behavior Induced by Substance P and NMDA via Spinal MAS1.

The intrathecal (i.t.) injection of substance P (SP) and N-methyl-D-aspartate (NMDA) induce transient nociceptive response by activating neurokinin (NK) 1 and NMDA receptors, respectively. We have recently reported that angiotensin (Ang) (1-7), an N-terminal fragment of Ang II, could alleviate several types of pain including neuropathic and inflammatory pain by activating spinal MAS1. Here, we investigated whether Ang (1-7) can inhibit the SP- and NMDA-induced nociceptive response. The nociceptive response induced by an i.t. injection of SP or NMDA was assessed by measuring the duration of hindlimb scratching directed toward the flank, biting and/or licking of the hindpaw or the tail for 5 min. Localization of MAS1 and either NK1 or NMDA receptors in the lumbar superficial dorsal horn was determined by immunohistochemical observation. The nociceptive response induced by SP and NMDA was attenuated by the i.t. co-administration of Ang (1-7) (0.03-3 pmol) in a dose-dependent manner. The inhibitory effects of Ang (1-7) (3 pmol) were attenuated by A779 (100 pmol), a MAS1 antagonist. Moreover, immunohistochemical analysis showed that spinal MAS1 co-localized with NK1 receptors and NMDA receptors on cells in the dorsal horn. Taken together, the i.t. injection of Ang (1-7) attenuated the nociceptive response induced by SP and NMDA via spinal MAS1, which co-localized with NK1 and NMDA receptors. Thus, the spinal Ang (1-7)/MAS1 pathway could represent a therapeutic target to effectively attenuate spinal pain transmission caused by the activation of NK1 or NMDA receptors.

SP prevents T2DM complications by immunomodulation

Type 2 diabetic mellitus (T2DM) is characterized by systemic inflammation and insulin resistance due to obesity, and this leads to critical complications, including retinopathy and nephropathy. This study explored the therapeutic effect of substance-p (SP), a neuropeptide, on T2DM progression and its complications. To examine whether SP affects glucose metabolism, lipid metabolism, systemic inflammation, and retinopathy, Otsuka Long-Evans Tokushima Fatty rats (OLETF, 27 weeks old) with chronic inflammation, obesity, and impaired bone marrow stem cell pool was selected. SP was intravenously injected and its effect was evaluated at 2 and 4 weeks after the SP injection. OLETF had typical symptoms of T2DM, including obesity, chronic inflammation, and poor glycemic control. However, SP treatment inhibited the body-weight gain and reduced circulating levels of free fatty acid, cholesterol, and triglyceride, ameliorating the obese environment. SP could suppress inflammation and rejuvenate bone marrow stem cell in OLETF rats. SP-mediated metabolic/immunological change could resolve hyperglycemia and insulin resistance. Histopathological analysis confirmed that SP treatment alleviated the dysfunction of target tissue with insulin resistance. OLETF rats have retinal damage from 27 weeks of age, which was reliably aggravated at 31 weeks. However, SP treatment could restore the damaged retina, sustaining its structure similarly to that of non-diabetic rats. In conclusion, systemic application of SP is capable contribute to the inhibition of the progression of T2DM and diabetic retinopathy.

Rfamide-related peptide-3 suppresses the substance P-induced promotion of the reproductive performance in female rats modulating hypothalamic Kisspeptin expression.

RFamide-related peptide-3 (RFRP-3) has been postulated as the suppressor of the reproductive axis at hypothalamic, pituitary and gonadal levels. Considering the hypothalamic level, RFRP-3 can suppress the activity of gonadotropin-releasing hormone (GnRH) neurons and their upstream neuronal stimulator, namely; the kisspeptin neurons. The effects of the RFRP-3 on the other regulators of GnRH neurons, however, are not completely investigated. Furthermore, substance P (SP) has been known as one of the coordinators of GnRH/ luteinizing hormone (LH) and the kisspeptin/G protein-coupled receptor 54 (GPR54) systems. The present study was aimed at investigating the impacts of RFRP-3 on the effects of SP on the reproductive performance in ovariectomized female rats. After intracerebroventricular (ICV) cannulation, the rats were subjected to the ICV injection of either SP or RFRP-3 and simultaneous injection of them and their selective antagonists. Blood and hypothalamic samplings and also sexual behavioral test were carried out on two main groups of rats. The analyses of the results of LH radioimmunoassay, gene expression assay for hypothalamic Gnrh1, Kisspeptin and Gpr54 accompanied by sexual behavioral examination revealed that the SP administration promotes reproductive behavior and GnRH/LH system and upregulates Kisspeptin expression. The RFRP-3 administration suppressed reproductive behavior, GnRH / LH system and Kisspeptin expression; however, the simultaneous injection of SP and RFRP-3 was devoid of significant alterations in the assessed parameters. The results showed that RFRP-3 can modulates the impacts of SP on the reproductive performance in ovariectomized female rats in part through adjusting Kisspeptin expression.

Substance P enhances the local activation of NK1R-expressing c-kit+ cardiac progenitor cells in right atrium of ischemia/reperfusion-injured heart

BackgroundLocalization of neurokinin 1 receptor (NK1R), the endogenous receptor for neuropeptide substance P (SP), has already been described for the right atrium (RA) of the heart. However, the biological role of SP/NK1R signal pathways in the RA remains unclear.Sprague-Dawley rats were randomly divided into 4 groups (n = 22 each); subjected to sham, ischemia/reperfusion-injury (I/R), I/R with 5 nmole/kg SP injection (SP + I/R), and SP + I/R with 1 mg/kg RP67580 injection (RP, a selective non-peptide tachykinin NK1R antagonist) (RP/SP + I/R). The left anterior descending coronary artery was occluded for 40 min followed by 1 day reperfusion with SP or SP + RP or without either. After 1 day, both atria and ventricles as well as the heart apexes were collected.ResultsSP promoted the expression of c-Kit, GATA4, Oct4, Nanog, and Sox2 in only the RA of the SP + I/R rats via NK1R activation. In agreement with these observations, NK1R-expressing c-Kit+ Nkx2.5+GATA4+ cardiac progenitor cells (CPCs) in the ex vivo RA explant outgrowth assay markedly migrated out from RA1 day SP + I/R approximately 2-fold increase more than RA1 day I/R. Treatment of SP promoted proliferation, migration, cardiosphere formation, and potential to differentiate into cardiomyocytes. Using RP inhibitor, NK1R antagonist not only inhibited cell proliferation and migration but also reduced the formation of cardiosphere and differentiation of c-Kit+ CPCs.ConclusionSP/NK1R might play a role as a key mediator involved in the cellular response to c-Kit+ CPC expansion in RA of the heart within 24 h after I/R.

Substance P Administered after Myocardial Infarction Upregulates Microphthalmia-Associated Transcription Factor, GATA4, and the Expansion of c-Kit+ Cells.

Microphthalmia-associated transcription factor (MITF), a basic helix-loop-helix leucine zipper transcription factor, can govern gene expression by binding to E box elements in the promoter region of its target gene. Although high levels of MITF have been observed in cardiomyocytes and the heart, the role of MITF after myocardial infarction (MI) remains unclear. We investigated the association between substance P (SP)/neurokinin-1 receptor (NK1R) signaling and MITF expression after MI. Male Sprague-Dawley rats (8 weeks) were randomly divided in two groups: ischemia/reperfusion injury (I/R) and SP injection (5 nmol/kg, SP+I/R). At the end of 7 days, the left ventricle (LV; LV7daysI/R, LV7daysSP+I/R) and infarct-related areas (IA; IA7daysI/R, IA7daysSP+I/R) from the hearts were collected. Immunofluorescence staining demonstrated that the LV7daysSP+I/R had a larger population of c-Kit+ GATA4high cells, which markedly upregulated MITF, c-Kit, and GATA4. c-Kit+ cells in the explant-derived cells (EDCs) derived from IA7daysSP+I/R migrated more widely than EDCs IA7daysI/R. Immunofluorescence staining, western blot analysis, and qRT-PCR assay showed that SP-treated c-Kit+ cells exhibited a high expression of c-Kit, GATA4, and MITF. FTY720 (a MITF inhibitor), RP67580 (NK1R inhibitor), or both inhibited the migration and proliferation of c-Kit+ cells increased by SP and blocked the upregulation of c-Kit, GATA4, and MITF. Overall, we suggest that MITF might be a potential regulator in SP-mediated c-Kit+ cell expansion post-MI via c-Kit and GATA4.

Effect of systemic delivery of Substance P on experimental tooth movement in rats.

The purpose of this study was to investigate the effect of systemic delivery of Substance P (SP) on experimental tooth movement. Forty-eight adult Sprague-Dawley rats were randomly divided into 2 groups and their maxillary first molars were mesially moved with the use of closed-coil springs. The experiment group received systemic injection of SP and the control group received phosphate-buffered saline solution. Transportation distances of first molars were measured. Hematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, and immunohistochemistry staining were performed to evaluate alveolar bone remodeling. Then the interferon (IFN) γ and tumor necrosis factor (TNF) α concentrations in peripheral blood and local periodontal tissue were measured. Finally, the effects of SP on bone marrow-derived stem cell (BMSC) proliferation and migration were tested invitro. Systemic delivery of SP significantly increased the distance of tooth movement and stimulated both osteoclast and osteoblast activities. The concentrations of IFN-γ and TNF-α increased in peripheral blood at early phases of the experiment and decreased in periodontal tissue at late phases. Invitro, the proliferation and migration of BMSCs were promoted by SP. Systemic delivery of SP can accelerate orthodontic tooth movement and promote alveolar bone remodeling potentially through immunomodulation and mobilizing endogenous mesenchymal stem cells.

Substance-P Prevents Cardiac Ischemia-Reperfusion Injury by Modulating Stem Cell Mobilization and Causing Early Suppression of Injury-Mediated Inflammation.

Therapies using stem/progenitor cells have been experimentally and clinically investigated to regenerate damaged hearts. Substance-P (SP) induces bone marrow (BM) stem cell mobilization and suppresses inflammation in ischemic injuries. This study investigated the role of SP in BM stem cell mobilization and immune responses for tissue repair after ischemic-reperfusion injury (IRI), in comparison with that of granulocyte colony-stimulating factor (GCSF). SP was intravenously injected into IRI rats and its affect was evaluated by determining colony forming efficiency, immune cell/ cytokine profiles, histological changes, and heart function through echocardiography. In the rat cardiac IRI model, SP suppressed IRI-mediated tumor necrosis factor-α induction, but increased the levels of interleukin-10, CD206+ monocytes, and regulatory T cells in the blood; reduced myocardial apoptosis at day 1 post-IRI; and markedly stimulated colony forming unit (CFU)-e and (CFU)-f cell mobilization. Efficacy of SP in the recovery of cardiac function after IRI was demonstrated by increased cardiac contractility, accompanied by reduced infarction sizes and fibrosis, and increased revascularization of vessels covered with alpha smooth muscle actin. These effects of SP were confirmed in an acute myocardial infarction (AMI) model. All effects mediated by SP were superior to those mediated by GCSF. Systemic injection of SP decreased early inflammatory responses and promoted stem cell mobilization, leading to a compact vasculature and improved cardiac function in cardiac IRI and AMI.

Los neuropéptidos SP y CGRP subyacen en las propiedades eléctricas de los puntos de acupuntura

Electrical skin measurements at acupuncture points (acupoints) have been utilized as a diagnostic and therapeutic aid for more than 50 years. Although acupoints are described as having distinct electrical properties, such as high conductance and low impedance, the underlying mechanisms are currently unknown. The present study investigated in a rat model of hypertension whether the high conductance at acupoints is a result of the release of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) during neurogenic inflammation in the referred pain area. When plasma extravasation from neurogenic inflammation was examined by exploring the leakage of intravenously injected Evans blue dye (EBD) to the skin, extravasated EBD was found most frequently in acupoints on the wrist. The increased conductance and temperature at these acupoints occurred during the development of hypertension. The increase in conductance and plasma extravasation at acupoints in hypertensive rats was ablated by cutting median and ulnar nerves, blocking small diameter afferent fibers with resiniferatoxin (RTX) injection into median and ulnar nerves, or antagonizing SP or CGRP receptors in acupoints. In turn, intradermal injection of SP or CGRP resulted in increased conductance and plasma extravasation in naïve rats. Elevated levels of SP and CGRP were found in the acupoints of hypertensive rats. These findings suggest that the high conductance at acupoints is due to vascular leakage following local release of SP and CGRP during neurogenic inflammation.

Neuropeptides SP and CGRP Underlie the Electrical Properties of Acupoints.

Electrical skin measurements at acupuncture points (acupoints) have been utilized as a diagnostic and therapeutic aid for more than 50 years. Although acupoints are described as having distinct electrical properties, such as high conductance and low impedance, the underlying mechanisms are currently unknown. The present study investigated in a rat model of hypertension whether the high conductance at acupoints is a result of the release of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) during neurogenic inflammation in the referred pain area. When plasma extravasation from neurogenic inflammation was examined by exploring the leakage of intravenously injected Evans blue dye (EBD) to the skin, extravasated EBD was found most frequently in acupoints on the wrist. The increased conductance and temperature at these acupoints occurred during the development of hypertension. The increase in conductance and plasma extravasation at acupoints in hypertensive rats was ablated by cutting median and ulnar nerves, blocking small diameter afferent fibers with resiniferatoxin (RTX) injection into median and ulnar nerves, or antagonizing SP or CGRP receptors in acupoints. In turn, intradermal injection of SP or CGRP resulted in increased conductance and plasma extravasation in naïve rats. Elevated levels of SP and CGRP were found in the acupoints of hypertensive rats. These findings suggest that the high conductance at acupoints is due to vascular leakage following local release of SP and CGRP during neurogenic inflammation.

Central Injection of Substance P Antagonizes the RF Amide-Related Peptide-3 Impacts on Hypothalamic KISS-1 and GnRH Gene Expressions in Male Wistar Rats

Background: Gonadotropin-inhibitory hormone and its mammalian orthologues (RFRP: RF amid related peptide) are known to inhibit the secretion of gonadotropins. In addition, substance P (SP) a member of tachykinin’s family is demonstrated that can increase the firing rate of kisspeptin/neurokinin B/dynorphin (KNDy) neurons and provokes the of secretion gonadotropins. In this experimental study we investigated the effects of co-administration of RFRP-3 and SP on the expression of KISS-1 and GnRH genes in male rats. Methods: forty-two mature Wistar rats were randomly allocated in 7 groups (n=6 in each group). Animals in each group intracerebroventricularly received either saline+DMSO, 1nmol SP, 5 nmol RFRP-3, 1nmol SP plus 5 nmol RFRP-3, 1 nmol SP plus10 nmol RF9 (RFRP-3 receptor antagonist), 1 nmol SP plus 1 nmol P234 (kisspeptin receptor antagonist) plus 5 nmol RFRP-3 or 1 nmol SP plus 5 nmol CP-96,345 (SP receptor antagonist) plus 5 nmol RFRP-3 in final volume of 3 µl. After two hours following injections hypothalamic samples were collected for evaluating the expression of target genes by real-time PCR technique. Results: Injections in SP and SP plus RF9 groups increase the expression of the both GnRH and KISS-1 genes (P<0.05). Injections in RFRP-3 group and SP plus RFRP-3 plus SP antagonist group significantly decrease the expression of both GnRH and KISS-1 genes (P<0.05). Injections of SP plus RFRP-3 group and SP plus RFRP-3 plus P234 group did not significantly change the expression of GnRH and KISS-1 genes. Conclusion: The results indicate that SP antagonizes the effects of RFRP-3 on the expression of Hypothalamic KISS-1 and GnRH genes.

Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice

The present study evaluates whether the injection of serotonin, acetylcholine, glutamate, bradykinin, histamine, or substance P (SP) into the Zusanli (Stomach 36, ST 36) acupoint can also produce the acupuncture-induced antinociceptive effect on inflammatory or neuropathic pain. In this in vivo experimental study, a total of 450 male Swiss mice were used. Mice were injected with saline or complete Freund's adjuvant (CFA) or subjected to sham or chronic constriction injury (CCI) surgery. After the establishment of the inflammatory (4 hours) or the neuropathic pain (3 days), the animals (n = 6) received manual acupuncture, sham acupuncture, or injection of saline, serotonin, acetylcholine, glutamate, bradykinin, histamine, or SP into the ST 36 and were evaluated for up to 24 hours. Mechanical threshold was evaluated, and the L4-L6 dorsal root ganglion was used for analysis of the transient receptor potential vanilloid type 1 overexpression. The mice from both the CFA and CCI models treated with manual acupuncture had significant increases in the thresholds for more than 24 hours. Sham acupuncture stimulation did not change the thresholds. In the mice injected with each of the mediators, the thresholds were significantly increased for all times in both the CFA and CCI models. Transient receptor potential vanilloid type 1 overexpression in CFA and CCI mice was reduced at all times by injection of serotonin, acetylcholine, or SP but not by injection of glutamate, histamine, or bradykinin. Our data suggest that the neuroactive mediators released by acupuncture-induced tissue injury may contribute to acupuncture-induced analgesia.

Up-regulated expression of substance P in CD8+ T cells and NK1R on monocytes of atopic dermatitis

BackgroundLarge numbers of CD8+ T cells were observed in atopic dermatitis (AD) skin, and monocytes from AD patients showed increased prostaglandin E2 production. However, little is known about the expression of substance P (SP) and its receptor NK1R in blood leukocytes of patients with AD.ObjectiveTo explore the expression of SP and NK1R in leukocytes of AD and the influence of allergens on SP and NK1R expression.MethodsThe expression levels of SP and NK1R in patients with AD were examined by flow cytometry, ELISA and a mouse AD model.ResultsThe plasma SP level was 4.9-fold higher in patients with AD than in HC subjects. Both the percentage of SP expression in the population and mean fluorescence intensity (MFI) of SP expression were elevated in CD8+ T cells in the blood of AD patients. However, both the CD14+NK1R+ population and MFI of NK1R expression on CD14+ cells were enhanced in the blood of AD patients. Allergens ASWE, HDME and PPE failed to up-regulate SP expression in CD8+ T cells. However, allergens ASWE and HDME both enhanced NK1R expression on CD14+ blood leukocytes regardless of AD or HC subjects. OVA-sensitized AD mice showed an elevated proportion and MFI of SP-expressing CD8+ T cells in the blood, which agrees with the SP expression situation in human AD blood. Injection of SP into mouse skin did not up-regulate NK1R expression on monocytes.ConclusionsAn elevated plasma SP level, up-regulated expression of SP and NK1R indicate that the SP/NK1R complex is important in the development of AD. Therefore, SP and NK1R antagonist or blocker agents may help to treat patients with AD.Trial registration Registration number: ChiCTR-BOC-16010279; Registration date: Dec., 28, 2016; retrospectively registered

Inhibition of substance P-induced defensive behavior via neurokinin-1 receptor antagonism in the central and medial but not basolateral nuclei of the amygdala in male Wistar rats

RationaleThe production of unconditioned defensive behaviors has been related to the amygdala, a key component of the encephalic aversion system. Microinjection of the neuropeptide substance P (SP) in the amygdala elicits defensive behaviors via the activation of type 1 neurokinin (NK-1) receptors. However, no studies have investigated whether intra-amygdala SP/NK-1 mechanisms can elicit other types of defensive responses, such as antinociception and ultrasonic vocalizations (USVs). MethodsThe present study investigated the effects of SP-induced activation of the neurokininergic system in three main nuclei of the amygdala—basolateral (BLA), central (CeA), and medial (MeA) nuclei—in rats that were subjected to the elevated plus maze (EPM), tail-flick test, and USV recording. The effects of SP in these amygdaloid nuclei were challenged with combined injections of the NK-1 receptor antagonist spantide. ResultsThe present study showed that SP injections in the CeA and MeA but not BLA exerted anxiogenic-like effects. In contrast to the CeA, the anxiogenic-like effects of SP in the MeA were not dependent on NK-1 mechanisms. In the tail-flick test, SP microinjections produced antinociceptive effects only in the MeA through NK-1 receptor activation. No USV emissions were detected after the SP microinjections. ConclusionsThe present study showed that NK-1 receptors in the CeA and MeA but not BLA are involved in defensive reactions to conditions of fear. The present results may provide a better understanding of the neurochemical mediation of fear states.

Substance P accelerates wound healing in type 2 diabetic mice through endothelial progenitor cell mobilization and Yes-associated protein activation

Wound healing is delayed in diabetes due to a number of factors, including impaired angiogenesis and poor dermal healing. The present study demonstrated that subcutaneous administration of substance P (SP) accelerates wound healing in db/db type 2 diabetic mice (db/db mice). SP injection (10 nM/kg, subcutaneously) enhanced angiogenesis, induced the mobilization of endothelial progenitor cells (EPCs) and increased the number of EPC-colony forming units (EPC-CFUs) in the bone marrow of db/db mice. Immunohistochemistry was performed to check the effects of SP on the cellular proliferation and the subcellular localization of Yes-associated protein (YAP) in the wound dermis. SP also upregulated cellular proliferation in the injured dermis of db/db mice. Compared with the control group, an increased number of cells in the wound dermis of SP-treated mice exhibited nuclear localization of YAP, which induces cellular proliferation. The results of the current study indicate that subcutaneous administration of SP may be a promising therapeutic strategy to treat diabetic wounds exhibiting impaired angiogenesis and dysfunctional dermal wound healing.

Ketamine-xylazine anaesthesia and orofacial administration of substance P: A lethal combination in rats

Background and aimsKetamine+xylazine mixture is a widely used anaesthetic in animal experiments. In rats anaesthetized with this mixture, we have shown that injection of carrageenan, a standard proinflammatory stimulus, into the cheek (intra-oral injection) induced oedema. A likely mediator of this oedema is substance P (SP), a major transmitter of sensory nerves in orofacial tissue. We have assessed the effects of intra-oral injection of SP in rats. Experimental approachSP (50–1μg per rat) was injected intra-orally in male adult Holtzman or Wistar rats, anaesthetized with ketamine+xylazine. For comparison, histamine (50μg) and 5-HT (5μg) were similarly injected. Antagonists of SP (SR140333, 2mg/kg), of histamine (pyrilamine, 2mg/kg) or of 5-HT (pizotifen, 2mg/kg) were subcutaneously (s.c.) injected, 30min before the corresponding agonist. Oedema in the cheek was assessed by measuring tissue thickness with calipers. ResultsIntra-oral injection of SP (1–50μg per rat) in Holtzman or Wistar rats anaesthetized with ketamine+xylazine induced, dose-dependently, death within 15min, accompanied by signs of excessive salivation. Rats pretreated with SR140333 were protected against SP-induced lethality and the excessive salivation. However, intra-oral injection of either histamine or 5-HT did not induce death, only a characteristic cheek oedema. These doses of SP injected into the hindpaws of conscious Holtzman and Wistar rats only induced oedema with no deaths. In rats anaesthetized with inhaled isoflurane, intra-oral SP (50μg) induced only cheek oedema, with no deaths or excessive salivation. This oedema was prevented by pre-treating rats with SR140333, pyrilamine and pizotifen. ConclusionIt is likely that the deaths were due to excessive salivation induced by the particular combination of ketamine and SP. Our results are presented as a warning to other experimenters who might use these two otherwise non-toxic conditions and the consequent unexpected and needless loss of experimental animals.

Intravenous Administration of Substance P Attenuates Mechanical Allodynia Following Nerve Injury by Regulating Neuropathic Pain-Related Factors.

This study aimed to investigate the analgesic effect of substance P (SP) in an animal model of neuropathic pain. An experimental model of neuropathic pain, the chronic constriction injury (CCI) model, was established using ICR mice. An intravenous (i.v.) injection of SP (1 nmole/kg) was administered to the mice to examine the analgesic effects of systemic SP on neuropathic pain. Behavioral testing and immunostaining was performed following treatment of the CCI model with SP. SP attenuated mechanical allodynia in a time-dependent manner, beginning at 1 h following administration, peaking at 1 day post-injection, and decaying by 3 days post-injection. The second injection of SP also increased the threshold of mechanical allodynia, with the effects peaking on day 1 and decaying by day 3. A reduction in phospho-ERK and glial fibrillary acidic protein (GFAP) accompanied the attenuation of mechanical allodynia. We have shown for the first time that i.v. administration of substance P attenuated mechanical allodynia in the maintenance phase of neuropathic pain using von Frey’s test, and simultaneously reduced levels of phospho-ERK and GFAP, which are representative biochemical markers of neuropathic pain. Importantly, glial cells in the dorsal horn of the spinal cord (L4–L5) of SP-treated CCI mice, expressed the anti-inflammatory cytokine, IL-10, which was not seen in vehicle saline-treated mice. Thus, i.v. administration of substance P may be beneficial for improving the treatment of patients with neuropathic pain, since it decreases the activity of nociceptive factors and increases the expression of anti-nociceptive factors.