Mast Cell Degranulation Rates Research Articles

Effects of catgut embedding and PGLA embedding at "Zusanli" (ST 36) on skin mast cells, substance P and histamine in healthy rats

To observe the effects of catgut embedding and polyglycolic acid/poly-lactic acid (PGLA) embedding at "Zusanli" (ST 36) on the activation of local skin mast cells (MC), and expression of substance P (SP) and histamine (HA), and to explore the mechanism of the temporal stimulation effect of acupoint catgut embedding and provide a foundation for further research on the initiation mechanism of acupoint catgut embedding. One hundred and sixty male SPF-grade SD rats were randomly divided into a blank group (10 rats), a sham-embedding group (50 rats), a catgut group (50 rats), and a PGLA group (50 rats). Each intervention group was further randomly divided into five subgroups according to the time points after intervention: 8 hours, 3 days, 7 days, 14 days, and 21 days, with 10 rats in each subgroup. One-time sham-embedding, catgut embedding and PGLA embedding was given at left "Zusanli" (ST 36) in each intervention group, respectively. The skin and subcutaneous connective tissue of the left "Zusanli" (ST 36) were collected at the corresponding time points after intervention, except for the blank group (only one day before intervention). Toluidine blue staining was used to detect MC count and degranulation, and immunohistochemical staining was used to detect the expression of SP and HA positive cells. There was no significant difference in MC count between the subgroups of each intervention group and the blank group (P>0.05). There was no significant difference in MC count between the subgroups of the catgut group and the PGLA group (P>0.05). The MC count in the 8-hour subgroup of PGLA group was higher than that in the 8-hour subgroup of catgut group (P<0.05), while the MC count in the 21-day subgroup of PGLA group was lower than that in the 21-day subgroup of catgut group (P<0.05). Compared with the blank group, the degranulation rates of MC were increased in the 8-hour and 3-day subgroups of sham-embedding group, 8-hour, 3-day, and 7-day subgroups of catgut group, and 8-hour, 3-day, 7-day, and 14-day subgroups of PGLA group (P<0.01, P<0.05, P<0.001). There was no significant difference in the degranulation rate of MC between the subgroups of the catgut group and the PGLA group (P>0.05), and no significant difference in the degranulation rate of MC between the two embedding groups at the same time point (P>0.05). Compared with the blank group, the expression of SP positive cells was increased in the 8-hour subgroup of sham-embedding group, 8-hour, 3-day, 7-day, and 14-day subgroups of catgut group, and 3-day, 7-day, and 14-day subgroups of PGLA group (P<0.001, P<0.05). The expression of SP positive cells in the 7-day subgroup of catgut group was higher than that in the 8-hour subgroup of catgut group (P<0.05), while the expression of SP positive cells in the 14-day subgroup of catgut group was lower than that in the 7-day subgroup of catgut group (P<0.001). The expression of SP positive cells in the 7-day subgroup of PGLA group was higher than that in the 3-day subgroup of PGLA group (P<0.05), while the expression of SP positive cells in the 14-day subgroup of PGLA group was lower than that in the 7-day subgroup of PGLA group (P<0.01). There was no significant difference in the expression of SP positive cells between the subgroups of the two embedding groups at the same time point (P>0.05). Compared with the blank group, the expression of HA positive cells was increased in the 8-hour, 3-day subgroups of sham-embedding group, 8-hour, 3-day, 7-day, and 14-day subgroups of catgut group, and 8-hour, 3-day, 7-day, 14-day, and 21-day subgroups of PGLA group (P<0.001, P<0.01, P<0.05). The expression of HA positive cells in the 14-day subgroup of catgut group was lower than that in the 7-day subgroup of catgut group (P<0.05), while the expression of HA positive cells in the 3-day subgroup of PGLA group was higher than that in the 8-hour subgroup of PGLA group (P<0.05), and the expression of HA positive cells in the 14-day subgroup of PGLA group was lower than that in the 7-day subgroup of PGLA group (P<0.05). The expression of HA positive cells in the 3-day subgroup of PGLA group was higher than that in the 3-day subgroup of catgut group (P<0.05). Catgut and PGLA embedding at "Zusanli" (ST 36) in healthy rats could induce changes in local skin MC, SP, and HA, which may be one of the mechanisms of the temporal stimulation effect after acupoint embedding. There are certain differences between different suture materials. A moderate inflammatory response in the acupoint area, mediated by MC and involving SP and HA, may be one of the initiating factors for the effect of acupoint catgut embedding.

Effect of acupuncture pretreatment of "Quchi"(LI11) and "Xuehai" (SP10) on mast cells and IL-33/ST2 in rats with urticaria

To observe the effect of electroacupuncture (EA) pretreatment at "Quchi "(LI11) and "Xuehai "(SP10) on expression of interleukin (IL)-33, suppression of tumorigenicity 2 (ST2) and mast cell degranulation in sensitive area of skin tissue in rats with urticaria, so as to explore its mechanisms underlying prevention of urticaria. A total of 32 male SD rats were randomly divided into blank control, model, EA preconditioning and medication groups, with 8 rats in each group. The urticaria model was established by topical injection of the prepared anti-ovalbumin serum (foreign serum, 0.1 mL/spot) along the bilateral sides of the spinal column on the back, followed by injection of mixture solution of ovalbumin, 0.5% evans blue and normal saline via the tail vein 48 h later. EA intervention (2 Hz/15 Hz, 1 mA) was applied to bilateral LI11 and SP10 for 20 min, once daily for 7 d before modeling.Back sensitization was started from the 5th day on. Rats of the medication group received gavage of loratadine, and those of the model group received gavage of the same volume of normal saline. The diameter of evans blue spots at the back skin tissue was measured; the histopathological changes of the blue spot tissues were observed by light microscope after H.E. staining. The state of degranulation of mast cells in the subcutaneous loose connective tissue was observed by using toluidine blue staining. Serum IgE and histamine contents were detected by ELISA, and the immunoactivity of IL-33 and ST2 in the skin and subcutaneous tissues of the sensitized spots (evans blue exudation spots) was observed by immunohistochemistry. Compared with the blank control group, the diameter of evans blue spot, degranulation rate of mast cells, serum IgE and histamine contents, and the immunoactivity of IL-33 and ST2 in the evans blue exudation spot tissues were significantly increased in the model group (P<0.01). In comparison with the model group, the increase of the above-mentioned indexes was reversed in both EA and medication groups (P<0.01，P<0.05). No significant differences were found between the EA and medication groups in down-regulating the levels of the 6 indexes. H.E. staining of the blue spot tissues of rats in the model group showed incomplete structure of the epidermal layer of the skin, unclear interface of tissues, incomplete keratinization, chaotic epidermal cells, disorderly arrangement of fibers in the dermis, and infiltration of inflammatory cells and edema, which was relatively milder in the EA and medication groups. EA preconditioning can prevent urticaria (reduce size and sensitive reactions) in rats, which may be associated with its functions in lowering the level of IgE through inhibiting IL-33 and ST2.

Voluntary Wheel Running Partially Attenuates Early Life Stress-Induced Neuroimmune Measures in the Dura and Evoked Migraine-Like Behaviors in Female Mice.

Migraine is a complex neurological disorder that affects three times more women than men and can be triggered by endogenous and exogenous factors. Stress is a common migraine trigger and exposure to early life stress increases the likelihood of developing chronic pain disorders later in life. Here, we used our neonatal maternal separation (NMS) model of early life stress to investigate whether female NMS mice have an increased susceptibility to evoked migraine-like behaviors and the potential therapeutic effect of voluntary wheel running. NMS was performed for 3 h/day during the first 3 weeks of life and initial observations were made at 12 weeks of age after voluntary wheel running (Exercise, -Ex) or sedentary behavior (-Sed) for 4 weeks. Mast cell degranulation rates were significantly higher in dura mater from NMS-Sed mice, compared to either naïve-Sed or NMS-Ex mice. Protease activated receptor 2 (PAR2) protein levels in the dura were significantly increased in NMS mice and a significant interaction of NMS and exercise was observed for transient receptor potential ankyrin 1 (TRPA1) protein levels in the dura. Behavioral assessments were performed on adult (>8 weeks of age) naïve and NMS mice that received free access to a running wheel beginning at 4 weeks of age. Facial grimace, paw mechanical withdrawal threshold, and light aversion were measured following direct application of inflammatory soup (IS) onto the dura or intraperitoneal (IP) nitroglycerin (NTG) injection. Dural IS resulted in a significant decrease in forepaw withdrawal threshold in all groups of mice, while exercise significantly increased grimace score across all groups. NTG significantly increased grimace score, particularly in exercised mice. A significant effect of NMS and a significant interaction effect of exercise and NMS were observed on hindpaw sensitivity following NTG injection. Significant light aversion was observed in NMS mice, regardless of exercise, following NTG. Finally, exercise significantly reduced calcitonin gene-related peptide (CGRP) protein level in the dura of NMS and naïve mice. Taken together, these findings suggest that while voluntary wheel running improved some measures in NMS mice that have been associated with increased migraine susceptibility, behavioral outcomes were not impacted or even worsened by exercise.

Effect of electroacupuncture on expressions of Lyn and Syk in mast cells of subcutaneous loose connective tissue in rats with urticarial

To observe the effect of electroacupuncture (EA) preconditioning on the expressions of tyrosine kinase Lyn and spleen tyrosine kinase (Syk) in mast cells of subcutaneous loose connective tissue in the rats with urticaria and explore the potential biological mechanism of EA in the intervention of urticaria. A total of 32 SD rats were randomized into a blank group, a model group, an EA group and a positive medication group, 8 rats in each one. Except of the blank group, the passive cutaneous anaphylaxis (PCA) was adopted to prepare the model of urticaria in the rats of the rest three groups. In the EA group, EA was applied to bilateral "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36), with disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in current intensity, once daily, for 20 min each time, consecutively for 7 days. In the positive medication group, loratadine (1 mg•kg-1•d-1) was for intragastric administration, once daily, consecutively for 7 days. The samples were collected for index detection 30 min after PCA antigen challenge in the rats of each group. Spectrophotometer was adopted to determine the effusion quantity of Evans blue in the allergized site of skin. HE staining was used to observe the morphological changes in the allergized site of skin. Toluidine blue staining was provided to observe mast cell degranulation in subcutaneous loose connective tissue in the allergized site of skin. Immunohistochemistry was applied to determine the protein expressions of Lyn and Syk during degranulation of mast cells. In the rats of the odel group, the eipdermis of allergized site was thickening, cells were disorganized in hierarchy and inflammatory cells were infiltrated largely in the dermis. In the positive medication group and the EA group, the epidermis was getting thin, cell arrangement was clear and the inflammatory cell infiltration was obviously alleviated as compared with the model group. Compared with the blank group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all increased in the model group (P<0.01). Compared with the model group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all reduced in the EA group and the positive medication group (P<0.01). Compared with the positive medication group, the degranulation rate of mast cells was increased significantly in the EA group (P<0.01). Electroacupuncture at "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36) reduces vascular permeability and gives play to the role of anti-allergy by the way of regulating and controlling the degranulation of mast cells in the rats with urticaria and the effect mechanism of electroacupuncture may be related to the inhibition of protein expressions of Lyn and Syk in mast cells.

Foot shock stress generates persistent widespread hypersensitivity and anhedonic behavior in an anxiety-prone strain of mice.

A significant subset of patients with urologic chronic pelvic pain syndrome suffer from widespread, as well as pelvic, pain and experience mood-related disorders, including anxiety, depression, and panic disorder. Stress is a commonly reported trigger for symptom onset and exacerbation within these patients. The link between stress and pain is believed to arise, in part, from the hypothalamic-pituitary-adrenal axis, which regulates the response to stress and can influence the perception of pain. Previous studies have shown that stress exposure in anxiety-prone rats can induce both pelvic and widespread hypersensitivity. Here, we exposed female A/J mice, an anxiety-prone inbred murine strain, to 10 days of foot shock stress to determine stress-induced effects on sensitivity, anhedonia, and hypothalamic-pituitary-adrenal axis regulation and output. At 1 and 28 days after foot shock, A/J mice displayed significantly increased bladder sensitivity and hind paw mechanical allodynia. They also displayed anhedonic behavior, measured as reduced nest building scores and a decrease in sucrose preference during the 10-day foot shock exposure. Serum corticosterone was significantly increased at 1 day after foot shock, and bladder mast cell degranulation rates were similarly high in both sham- and shock-exposed mice. Bladder cytokine and growth factor mRNA levels indicated a persistent shift toward a proinflammatory environment after foot shock exposure. Together, these data suggest that chronic stress exposure in an anxiety-prone mouse strain may provide a useful translational model for understanding mechanisms that contribute to widespreadness of pain and increased comorbidity in a subset of patients with urologic chronic pelvic pain syndrome.

(242) Stress-Induced Bladder Hypersensitivity, Hind Paw Allodynia, and Depression-Like Behavior in an Anxiety-Prone Strain of Mice

An estimated 80% of Americans suffer from at least one stress-related symptom during a month's time and co-morbidity of chronic pain and mood disorders is very common. Stress activates the Hypothalamic-Pituitary-Adrenal (HPA) axis and subsequently initiates and/or exacerbates symptoms related to both chronic pain and mood disorders. The impact of stress on allodynia and anhedonic behaviors in highly-anxious individuals has not been well studied. Here, we are testing the hypothesis that chronic stress exposure can increase somatic and visceral sensitivity and induce anhedonic behaviors in an anxiety-prone mouse strain. Female, 12-week-old A/J mice were exposed to repeated foot shock stress for 10 continuous days and tested after 1 (acute) or 28 days (persistent) for mechanical hind paw sensitivity, sucrose preference, nest building behavior, visceromotor response (VMR) during urinary bladder distension (UBD), mast cell degranulation, serum corticosterone (CORT) levels, and HPA axis-related gene expression. The VMR during UBD was significantly higher in the shock group, compared to the sham group, at both time points. The shock-exposed groups also had significantly decreased nest building scores and mechanical withdrawal thresholds in the hind paw at 1 and 28 days post-shock. Decreased sucrose preference was observed during the shock paradigm and both groups had high mast cell degranulation rates in the bladder. Stress-induced increases in HPA axis output were also observed, evidenced as significantly elevated serum CORT levels and pro-inflammatory gene expression in the bladder of the shock group compared to sham. In summary, widespread hypersensitivity and anhedonia-like behavior were observed both acutely and persistently following foot shock stress exposure in A/J Mice. Future studies will include investigating potential physiological or pharmacological intervention to mitigate these negative outcomes.

Cultivation and identification of mouse peritoneal and bone marrow-derived mast cells

Objective To explore the in vitro culture methods for oriented differentiation of peritoneal cells and bone marrow cells into high-purity mast cells, and to identify the function of these mast cells. Methods Peritoneal cells and bone marrow cells were isolated from the peritoneal cavity lavages and femur of C57BL/6 mice, and cultured with both interleukin-3 (IL-3) and stem cell factor for 2 and 4 weeks respectively. Light microscopy was performed to observe the morphology of these cells, toluidine blue staining to identify the degree of maturity of these mast cells, and flow cytometry to measure the expression of cell surface markers CD117 and FceRⅠα. After the stimulation with compound 48/80 at different concentrations, the degranulation rate of mast cells was counted under the microscope, and β-hexosaminidase release rate was measured by spectrophotometry. Results After 2- or 4-week culture, the mouse peritoneal and bone marrow cells all manifested as refractive suspension cells of uniform size. Toluidine blue staining showed violaceous metachromatic granules in the cytoplasm of the two kinds of cells. The proportions of CD117 or FceR Ⅰ α single-positive peritoneal and bone marrow-derived mast cells were all more than 95%, and the proportions of CD117/FceRⅠα double-positive peritoneal and bone marrow-derived mast cells were 97.68% ± 0.80% and 96.12% ± 0.76% respectively. The degranulation rates of mast cells in the 100- and 1 000-mg/L compound 48/80 groups significantly differed from those in the blank control group (all P < 0.01) . Compared with the blank control group, the β-hexosaminidase release rates significantly increased in bone marrow-derived mast cells in the 100-mg/L compound 48/80 group and peritoneal mast cells in the 10- and 100-mg/L compound 48/80 groups (P < 0.01 or 0.05) . Conclusion IL-3 and stem cell factor can co-induce the directed differentiation and proliferation of mouse bone marrow stem cells and peritoneal cells, so as to harvest high-purity mature degranulated mast cells, and lay a foundation for subsequent cell biology research. Key words: Mast cells; Myeloid progenitor cells; Induced pluripotent stem cells; Mice; Cells, cultured; Interleukin-3; Stem cell factor

Effect of acupuncture at complementary acupionts on mast cells and histamine/bradykinin released by mast cells in the regions of acupionts

To observe the changes in numbers and degranulation status of local mast cells (MCs) of the complementary acupionts, as well as the concentrations of serum histamine and bradykinin after acupuncture at rat’s complementary acupionts of Yanglingquan (GB 34) and Yinlingquan (SP 10), for exploring the mechanisms of the synergistic effect of complementary acupionts. Using random number table method, 40 Wistar rats were randomly divided into a blank control group (group K), an acupuncture at Yanglingquan (GB 34) group (group A), an acupuncture at Yinlingquan (SP 9) group (group B), an acupuncture at Yanglingquan (GB 34) and Yinlingquan (SP 9) group (group AB), and an acupuncture at points near Yanglingquan (GB 34) and Yinlingquan (SP 9) group (group CD), 8 rats in each group. Group A received acupuncture at Yanglingquan (GB 34); group B received acupuncture at Yinlingquan (SP 9); group AB received acupuncture at Yanglingquan (GB 34) and Yinlingquan (SP 9); group CD received acupuncture at the control points [points 3 mm away from Yanglingquan (GB 34) and Yinlingquan (SP 9) respectively]. Acupuncture was performed at bilateral points in each group. Rats in group K were fixed using the same method as rats in the other 4 groups without acupuncture stimulation. Needles of 0.35 mm in diameter and 40 mm in length were used for acupuncture. Needle handle was connected to the G6805-II mode electroacupuncture (EA) device after needling qi was obtained, with sparse-dense wave, frequency of 2 Hz/100 Hz and current of 1 mA to keep the needle handles slightly tremulous while the rats kept quiet. Rats were continuously stimulated by EA for 20 min each time. Experimental interventions were conducted on the 1st, 3rd, 5th and 7th days when the experiment started, for 4 times in total. Specimens from rats in each group were collected 2 h after the 7th day. The levels of histamine and bradykinin in serum were detected by enzyme-linked immunosorbent assay (ELISA). Local tissues of the points were used to prepare cryosections. The changes of MCs were observed after toluidine blue staining. Compared with group K, the numbers and degranulation rates of MCs in group A, group B, group AB and group CD were significantly increased (all P group B > group A > group CD > group K, and the differences were statistically significant (all P 0.05). The number and degranulation rate of MCs of the complementary acupionts are significantly increased after acupuncture at complementary acupionts of Yanglingquan (GB 34) and Yinlingquan (SP 9) of rats, and the serum levels of histamine and bradykinin are increased, which may be one of the mechanisms of the synergism produced by the combination of complementary acupionts.

Trimetazidine Increases Cell Survival and Inhibits the Activation of Inflammatory Response in Sodium Taurocholate–Induced Acute Pancreatitis

Objective: To evaluate the therapeutic effects of trimetazidine (TMZ) in an experimental acute pancreatitis (AP) model induced with sodium taurocholate (STC). Summary of Background Data: At present, AP is considered a disease with no specific treatment. Preventing mitochondrial dysfunction in acinar cells may be an option for specific treatment of AP. TMZ is an anti-ischemic drug with anti-inflammatory, antioxidant, and mitochondrial modulatory effects. Methods: Rats were divided into 4 groups. AP was induced in the AP (n = 7) and AP + TMZ (n = 7) groups by an injection of 4% sodium taurocholate to the pancreatic duct. The sham (n = 6) and drug (n = 6) groups were designated as control groups. The AP + TMZ and drug groups were administered TMZ. Samples were taken at 72 hours, and histopathologic changes as well as biochemical parameters were analyzed. Results: Serum amylase, tissue myeloperoxidase activity, malondialdehyde levels, serum cytokine levels, and mast cell degranulation rates were elevated after induction of AP, whereas tissue antioxidant enzyme activities and cell viability rates [determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay] decreased. These parameters were found to be different in the AP group compared with those in all other groups (P &amp;lt; 0.05). A significant improvement of all parameters was achieved with the TMZ treatment of AP. Histologically, significant differences were found between the AP and AP + TMZ groups in terms of leukocyte infiltration, necrosis, and apoptotic cell counts. Conclusions: In this study, we demonstrated that TMZ treatment protected the mitochondrial function and prevented the activation of the inflammatory cascade in the sodium taurocholate–induced AP model.

Differential Ca2+ mobilization and mast cell degranulation by FcεRI- and GPCR-mediated signaling

Mast cells play a primary role in allergic diseases. During an allergic reaction, mast cell activation is initiated by cross-linking IgE-FcεRI complex by multivalent antigen resulting in degranulation. Additionally, G protein-coupled receptors also induce degranulation upon activation. However, the spatio-temporal relationship between Ca2+ mobilization and mast cell degranulation is not well understood. We investigated the relationship between oscillations in Ca2+ level and mast cell degranulation upon stimulation in rat RBL-2H3 cells. Nile red and Fluo-4 were used as probes for monitoring histamine and intracellular Ca2+ levels, respectively. Histamine release and Ca2+ oscillations in real-time were monitored using total internal reflection fluorescence microscopy (TIRFM). Mast cell degranulation followed immediately after FcεRI and GPCR-mediated Ca2+ increase. FcεRI-induced Ca2+ increase was higher and more sustained than that induced by GPCRs. However, no significant difference in mast cell degranulation rates was observed. Although intracellular Ca2+ release was both necessary and sufficient for mast cell degranulation, extracellular Ca2+ influx enhanced the process. Furthermore, cytosolic Ca2+ levels and mast cell degranulation were significantly decreased by downregulation of store-operated Ca2+ entry (SOCE) via Orai1 knockdown, 2-aminoethyl diphenylborinate (2-APB) or tubastatin A (TSA) treatment. Collectively, this study has demonstrated the role of Ca2+ signaling in regulating histamine degranulation.

Comparison of Electroacupuncture and Moxibustion for Relieving Visceral Hypersensitivity in Rats with Constipation-Predominant Irritable Bowel Syndrome.

Aim. To compare whether there is different effect between electroacupuncture (EA) and moxibustion (Mox) on visceral hypersensitivity (their analgesic effects) in constipation-predominant irritable bowel syndrome (C-IBS). Methods. EA at 1 mA and 3 mA and Mox at 43°C and 46°C were applied to the Shangjuxu (ST37, bilateral) acupoint in rats with C-IBS and normal rats. An abdominal withdrawal reflex (AWR) score was used to assess visceral hypersensitivity. Toluidine blue staining was used to assess mast cell (MC) activity in colon of rats. Immunochemistry was used to measure 5-HT and 5-HT4 receptor expression in the colon. Results. AWR scores in all EA (1 mA and 3 mA) and Mox (43°C and 46°C) treatment groups after colorectal distention (CRD) stimulation pressure of 20, 40, 60, and 80 mmHg were significantly lower than those of the model (MC) group (P all < 0.01). The MC counts and degranulation rates in the colon of all EA and Mox treatment groups and the MC group were significantly higher than those of the NC group (P all < 0.01). MC degranulation rates in the colon of all EA and Mox treatment groups were lower than those of the MC group (P all < 0.05). 5-HT expression in colon of all EA and Mox treatment groups was significantly lower than that of the MC group (P all < 0.01), and 5-HT4R expression in colon of both EA groups was significantly higher than that of the MC group (P both < 0.01). Conclusion. EA and Mox treatments may both ameliorate visceral hypersensitivity at different degree in rats with C-IBS, and EA treatment was better than Mox.