Role of mast cell activation in inducing microglial cells to release neurotrophin

The brain‐derived neurotrophic factor (BDNF) plays a critical role in pain hypersensitivity. BDNF is the ligand of P2X4 receptors (P2X4R) in the microglia. The causative factors involving the P2X4R over expression in the microglia remains unclear. Mast cell activation has a close relation with pain hypersensitivity. However, the underlying mechanism between mast cell activation and pain hypersensitivity is unknown. The present study aimed to elucidate the mechanism by which mast cell activation promoted the expression of P2X4R in the microglia. The results of present study showed that mast cell activation markedly promoted the expression of P2X4R and BDNF in microglial cells, which significantly enhanced the release of BDNF from microglial cells upon exposure to adenosine triphosphate. Mast cell‐derived tryptase activated PAR2 that resulted in promoting the expression of P2X4R in microglial cells. Pretreatment with antibodies against tryptase or PAR2, or using tryptase‐deficient HMC‐1 cells or PAR2‐deficient microglial cells abolished the increase in P2X4R expression and BDNF release. Increase in mitogen activated protein kinase phosphorylation was observed in the processes of mast cell‐induced BDNF release and P2X4R expression. We conclude that mast cell activation has the capacity to promote the expression of P2X4R and BDNF in microglial cells. © 2009 Wiley‐Liss, Inc.     

P2X4 receptor in the dorsal horn partially contributes to brain‐derived neurotrophic factor oversecretion and toll‐like receptor‐4 receptor activation associated with bone cancer pain

Previous studies have suggested that the microglial P2X7 purinoceptor is involved in the release of tumor necrosis factor‐α (TNFα) following activation of toll‐like receptor‐4 (TLR4), which is associated with nociceptive behavior. In addition, this progress is evoked by the activation of the P2X4 purinoceptor (P2X4R). Although P2X4R is also localized within spinal microglia in the dorsal horn, little is known about its role in cancer‐induced bone pain (CIBP), which is in some ways unique. With the present rat model of CIBP, we demonstrate a critical role of the microglial P2X4R in the enhanced nociceptive transmission, which is associated with TLR4 activation and secretion of brain‐derived neurotrophic factor (BDNF) and TNFα in the dorsal horn. We assessed mechanical threshold and spontaneous pain of CIBP rats. Moreover, P2X4R small interfering RNA (siRNA) was administered intrathecally, and real‐time PCR, Western blots, immunofluorescence histochemistry, and ELISA were used to detect the expression of P2X4R, TLR4, OX‐42, phosphorylated‐p38 MAPK (p‐p38), BDNF, and TNFα. Compared with controls, intrathecal injection of P2X4R siRNA could prevent nociceptive behavior induced by ATP plus lipopolysaccharide and CIBP and reduce the expression of P2X4R, TLR4, p‐p38, BDNF, and TNFα. In addition, the increase of BDNF protein in rat microglial cells depended on P2X4 receptor signaling, which is partially associated with TLR4 activation. The ability of microglial P2X4R to activate TLR4 in spinal cord leading to behavioral hypersensitivity and oversecretion of BDNF could provide an opportunity for the prevention and treatment of CIBP. © 2014 Wiley Periodicals, Inc.     

Release reaction of brain-derived neurotrophic factor (BDNF) through PAR1 activation and its two distinct pools in human platelets

Brain-derived neurotrophic factor (BDNF) is a cytokine that plays important roles in the survival, development, and plasticity of neurons. BDNF is also expressed in peripheral tissues and cells. In this article, we report the BDNF release reaction through thrombin stimulation and its localization in human platelets.Platelets from healthy volunteers were subjected to PAR1-AP or PAR4-AP stimulation. Release of BDNF was measured by ELISA. Localization of BDNF in resting and thrombin-activated platelets was examined by immunoelectron microscopy and sucrose gradient ultracentrifugation following western blotting.BDNF was released dose-dependently with PAR1-AP concentrations with drastic release at low PAR1-AP concentrations and gently release at high PAR1-AP concentrations. Maximum BDNF release was approximately 37% at 132 μM PAR1-AP. In contrast, 3.8% BDNF was released with 1.13 mM PAR4-AP stimulation. In immunoelectron microscopy and sucrose gradient ultracentrifugation analyses, BDNF was detected not only in α-granules but also cytoplasm in of the resting platelets, and it was distributed in the swollen open canalicular system fused to α-granules at 1 min and disappeared at 5 min after stimulation by thrombin. However, BDNF in cytoplasm remained throughout platelet activation.In conclusions, we demonstrate that BDNF is released from platelets through predominately PAR1 regulation. Furthermore, we identified two pools of BDNF in the α-granules and cytoplasm of human platelets, and only BDNF in α-granules is released through platelet activation.     

The effect of intrathecal administration of glial activation inhibitors on dorsal horn BDNF overexpression and hind paw mechanical allodynia in spinal nerve ligated rats

Recent studies have suggested that activated glia in the spinal cord may play a vital role at different times during spinal nerve ligation (SNL)-induced neuropathic pain; therefore, glial activation inhibitors have been used as effective painkillers. Brain-derived neurotrophic factor (BDNF) is also known to be a powerful pain modulator, but it remains unclear how it contributes to the glial activation inhibitor-based treatment. This study revealed the following results: (1) intrathecal administration of minocycline (a microglial activation inhibitor) could prevent mechanical allodynia during the initiation of SNL-induced neuropathic pain, and its action was associated with the elimination of BDNF overexpression in the dorsal horn; (2) the spinal injection of fluorocitrate (an astrocytic activation inhibitor) but not minocycline could reverse mechanical allodynia during the maintenance phase of SNL-induced pain, and its action was also related to a decrease in BDNF overexpression in the dorsal horn; and (3) treatment with TrkB/Fc (a BDNF-sequestering protein) had a similar effect during both the early development and maintenance periods. These results led to the following conclusions: (1) elevated BDNF expression in the dorsal horn was required to develop and maintain neuropathic pain; (2) minocycline could only prevent mechanical allodynia in the early stages, possibly by inhibiting BDNF release from microglia; and (3) fluorocitrate could reverse existing mechanical allodynia, and its action was associated with the inhibition of BDNF upregulation induced by astrocytic activation.     

Minocycline-induced reduction of brain-derived neurotrophic factor expression in relation to cancer-induced bone pain in rats

Previous studies have suggested that the release of brain-derived neurotrophic factor (BDNF) from microglia in spinal cord is necessary for maintaining pain hypersensitivity after nerve injury. However, little is known about its role in cancer-induced bone pain (CIBP), which is in some ways unique. This study demonstrates a critical role of minocycline (a potent inhibitor of microglial activation)-modulated BDNF in the induction and maintenance of behavioral hypersensitivity in a rat model of CIBP. We assessed mechanical threshold and spontaneous pain of CIBP rats. Moreover, minocycline was administered intrathecally from day 4 to day 6 (early stage) or from day 10 to day 12 (later stage), after carcinoma cell inoculation. Real-time PCR, Western blots, and double immunofluorescence were used to detect the expression of OX-42 (marker of activated microglia), phosphorylated p38-MAPK (p-p38), and BDNF. We found that intrathecal minocycline could prevent CIBP at an early stage of tumor growth (from day 4 to day 6). However, at the late stage (from day 10 to day 12), intrathecal minocycline had no effect. Moreover, the expression of OX-42 and BDNF under CIBP, peaking on day 6, were all reduced after minocycline injection from day 4 to day 6. The ability of minocycline-induced reduction of BDNF in the induction of behavioral hypersensitivity could provide an opportunity for alleviating CIBP.     

Astrocyte-derived GDNF is a potent inhibitor of microglial activation

Neuroinflammation is recognized as a major factor in Parkinson's disease (PD) pathogenesis and increasing evidence propose that microglia is the main source of inflammation contributing to the dopaminergic degeneration observed in PD. Several studies suggest that astrocytes could act as physiological regulators preventing excessive microglia responses. However, little is known regarding how astrocytes modulate microglial activation. In the present study, using Zymosan A-stimulated midbrain microglia cultures, we showed that astrocytes secrete factors capable of modulating microglial activation, namely its phagocytic activity and the production of reactive oxygen species since both parameters were highly diminished in cells incubated with astrocytes conditioned media (ACM). Glial cell line-derived neurotrophic factor (GDNF), cerebral dopamine neurotrophic factor (CDNF) and brain-derived neurotrophic factor (BDNF), known to have a neuroprotective role in the nigrostriatal system, are among the candidates to be astrocyte-secreted molecules involved in the modulation of microglial activation. The effect of ACM on Zymosan A-induced microglial activation was abolished when the GDNF present in the ACM was abrogated using a specific antibody, but not when ACM was neutralized with anti-CDNF, anti-BDNF or with a heat-inactivated GDNF antibody. In addition, media conditioned by astrocytes silenced for GDNF were not able to prevent microglial activation, whereas supplementation of non-conditioned media with GDNF prevented the activation of microglia evoked by Zymosan A. Taken together, these results indicate that astrocyte-derived GDNF plays a major contribution to the control of midbrain microglial activation, suggesting that GDNF can protect from neurodegeneration through the inhibition of neuroinflammation.     

Primary culture of cortical neurons, type-1 astrocytes, and microglial cells from cynomolgus monkey (Macaca fascicularis) fetuses

We established selective primary cultures of neurons, astrocytes, and microglial cells from cryopreserved fetal cerebral cortex of cynomolgus monkeys (Macaca fascicularis). At 14 days in serum-containing medium, the cell cultures of the fetal cerebral cortex consisted primarily of neurons, astrocytes, and floating microglial cells. At 21 days, we observed a small number of myelin basic protein (MBP)-positive oligodendrocytes. The addition of cytosine arabinoside (a selective DNA synthesis inhibitor) at 2 days in culture eliminated proliferative glial cells, allowing adequate numbers of neurons to survive selectively. A chemically defined serum-free medium successfully supported neuronal survival at a level equivalent to that supported by the serum-containing medium. Brain-derived neurotrophic factor (BDNF) significantly affected the survival of primate neurons. Glutamate induced a significant degree of neuronal cell death against primate neurons and MK-801, a selective N-methyl-D-aspartate receptor (NMDAR) antagonist, blocked cell death, which suggests that primate cortical neurons have NMDAR and the glutamate-induced cell toxicity is mediated by NMDAR. In the serum-free medium, type-1 astrocytes responded to dibutyryl cyclic AMP and showed a process-bearing morphology. The growth of type-1 astrocytes in the serum-free medium was stimulated by epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and hydrocortisone, which are known growth factors in rat type-1 astrocytes. Cultured microglial cells expressed CD68, a monocyte marker. Macrophage-colony stimulating factor (M-CSF) stimulated microglial cell growth in the serum-free medium. These selective primary culture systems of primate cerebral cortical cells will be useful in issues involving species specificity in neuroscience.     

Rat retinal microglial cells under normal conditions, after optic nerve section, and after optic nerve section and intravitreal injection of trophic factors or macrophage inhibitory factor

Retinal microglial cells may have a role in both degeneration and neuroprotection of retinal ganglion cells (RGC) after optic nerve (ON) section. We have used NDPase enzymohistochemistry to label adult rat retinal microglial cells and have studied these cells under normal conditions, after left ON section, and after left ON section and eye puncture or intravitreal injection of different substances: vehicle, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT3), or macrophage inhibitory factor (MIF). Resident microglial cells are present in four layers in the adult rat retina: the nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), and outer plexiform layer (OPL). Left ON section induces microglial activation in the ipsilateral and contralateral retina as manifested by stronger staining intensity in both retinas and increased microglial cell densities in the NFL, IPL, and GCL of the ipsilateral retina. Left ON section followed by left eye puncture or intravitreal injection increases microglial cell density in both retinas and induces changes in the microglial cells of the ipsilateral retina that vary depending on the substance injected: BDNF injections delay microglial activation, possibly through retinal ganglion cell neuroprotection, whereas NT3 partially inhibits microglial activation in the NFL; MIF injections have no clear effects on microglial activation. In conclusion, retinal microglial cells become activated after an ON section and react more intensely when the eye is also punctured or injected, and this response may be altered by using neurotrophic factors, although the effects of MIF are less clear.     

Overactivation of LPS-stimulated microglial cells by co-cultured neurons or neuron-conditioned medium

Microglial activation represents a well known aspect of several neuropathological diseases. However, little is known concerning the role of neurons in starting and modulating this process. In the present report, we demonstrate that differentiated, healthy neurons constitutively release in the culture medium substance(s) that are able to induce a state of overactivation in LPS-stimulated microglial cells. The neuronal factors synergize with LPS in stimulating synthesis and release of interleukin-1beta (IL-1beta) and nitric oxide by microglial cells. Prolonged exposure (72 h) to neuron-conditioned media in the presence of LPS induced microglial apoptosis, thus suggesting that neuronal overactivation of stimulated microglia favors their subsequent apoptotic elimination as part of a safety mechanism.     

Selective Ablation of BDNF from Microglia Reveals Novel Roles in Self-Renewal and Hippocampal Neurogenesis

Microglia, the resident immune cells of the CNS, have emerged as key regulators of neural precursor cell activity in the adult brain. However, the microglia-derived factors that mediate these effects remain largely unknown. In the present study, we investigated a role for microglial brain-derived neurotrophic factor (BDNF), a neurotrophic factor with well known effects on neuronal survival and plasticity. Surprisingly, we found that selective genetic ablation of BDNF from microglia increased the production of newborn neurons under both physiological and inflammatory conditions (e.g., LPS-induced infection and traumatic brain injury). Genetic ablation of BDNF from microglia otherwise also interfered with self-renewal/proliferation, reducing their overall density. In conclusion, we identify microglial BDNF as an important factor regulating microglia population dynamics and states, which in turn influences neurogenesis under both homeostatic and pathologic conditions.SIGNIFICANCE STATEMENT (1) Microglial BDNF contributes to self-renewal and density of microglia in the brain. (2) Selective ablation of BDNF in microglia stimulates neural precursor proliferation. (3) Loss of microglial BDNF augments working memory following traumatic brain injury. (4) Benefits of repopulating microglia on brain injury are not mediated via microglial BDNF.     

Involvement of microglia-neuron interactions in the tumor necrosis factor-alpha release,microglial activation,and neurodegeneration induced by trimethyltin

Trimethyltin (TMT) is a neurotoxicant known to induce early microglial activation. The present study was undertaken to investigate the role played by these microglial cells in the TMT-induced neurotoxicity. The effects of TMT were investigated in monolayer cultures of isolated microglia or in neuron-enriched cultures and in neuron-microglia and astrocyte-microglia cocultures. The end points used were morphological criteria; evaluation of cell death and cell proliferation; and measurements of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) release in culture supernatant. The results showed that, in cultures of microglia, TMT (10(-6) M) caused, after a 5-day treatment, an increased release of TNF-alpha, without affecting microglial shape or cell viability. When microglia were cocultured with astrocytes, TNF-alpha release was decreased to undetectable levels. In contrast, in neuron-microglia cocultures, TNF-alpha levels were found to increase at lower concentrations of TMT (i.e., 10(-8) M). Moreover, at 10(-6) M of TMT, microglia displayed further morphological activation, as suggested by process retraction and by decrease in cell size. No morphological activation was observed in cultures of isolated microglial cells and in astrocyte-microglia cocultures. With regard to neurons, 10(-6) M of TMT induced about 30% of cell death, when applied to neuron-enriched cultures, whereas close to 100% of neuronal death was observed in neuron-microglia cocultures. In conclusion, whereas astrocytes may rather dampen the microglial activation by decreasing microglial TNF-alpha production, neuronal-microglial interactions lead to enhanced microglial activation. This microglial activation, in turn, exacerbates the neurotoxic effects of TMT. TNF-alpha may play a major role in such cell-cell communications.     

Characterization of microglial cells and their response to stimulation in an organotypic retinal culture system

An organotypic culture system of the early postnatal rat retina was developed to study microglial activation within a tissue environment. One day after tissue preparation, microglial cells of the ganglion cell/nerve fiber layer revealed features of activation. Cells acquired an ameboid morphology as revealed by Bandeiraea simplicifolia lectin staining. Proliferation-as revealed by Ki67 immunocytochemistry-resulted in higher cell densities. In the supernatant, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and monocyte chemoattractant factor-1 (MCP-1) were detected by using specific enzyme-linked immunosorbent assay systems, activated microglia being the most likely source of their release. After 6 days in vitro (div), microglial cells regained their resting morphology, and cell counts returned to control levels. Concomitantly, the release activity decreased to undetectable levels. When slices were treated at this later stage of cultivation (>6 div) with bacterial lipopolysaccharide (LPS; 100 ng/ml for 24 hours), microglial cells became activated, as revealed by a change in morphology. In parallel, the LPS treatment also resulted in high levels of TNF-alpha, IL-6, and MCP-1 in the culture medium. Both the release from the tissue and the morphological changes of the microglia were reversible. Seventy-two hours after LPS removal, only microglia with ramified morphology were found, and release activities returned to baseline. These data suggest that the organotypic culture of the retina is a useful model for studying microglial activation from its resting form.     

Protease activated receptor 2 controls myelin development,resiliency and repair

Oligodendrocytes are essential regulators of axonal energy homeostasis and electrical conduction and emerging target cells for restoration of neurological function. Here we investigate the role of protease activated receptor 2 (PAR2), a unique protease activated G protein‐coupled receptor, in myelin development and repair using the spinal cord as a model. Results demonstrate that genetic deletion of PAR2 accelerates myelin production, including higher proteolipid protein (PLP) levels in the spinal cord at birth and higher levels of myelin basic protein and thickened myelin sheaths in adulthood. Enhancements in spinal cord myelin with PAR2 loss‐of‐function were accompanied by increased numbers of Olig2‐ and CC1‐positive oligodendrocytes, as well as in levels of cyclic adenosine monophosphate (cAMP), and extracellular signal related kinase 1/2 (ERK1/2) signaling. Parallel promyelinating effects were observed after blocking PAR2 expression in purified oligodendrocyte cultures, whereas inhibiting adenylate cyclase reversed these effects. Conversely, PAR2 activation reduced PLP expression and this effect was prevented by brain derived neurotrophic factor (BDNF), a promyelinating growth factor that signals through cAMP. PAR2 knockout mice also showed improved myelin resiliency after traumatic spinal cord injury and an accelerated pattern of myelin regeneration after focal demyelination. These findings suggest that PAR2 is an important controller of myelin production and regeneration, both in the developing and adult spinal cord.     

Brain-derived neurotrophic factor contributes to spinal long-term potentiation and mechanical hypersensitivity by activation of spinal microglia in rat

It has been shown that following peripheral nerve injury brain-derived neurotrophic factor (BDNF) released by activated microglia contributes to neuropathic pain, but whether BDNF affects the function of microglia is still unknown. In the present work we found that spinal application of BDNF, which induced long-term potentiation (LTP) of C-fiber evoked field potentials, activated spinal microglia in naïve animals, while pretreatment with microglia inhibitor minocycline blocked BDNF-induced LTP. In addition, following LTP induction by BDNF, both phosphorylated Src-family kinases (p-SFKs) and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) were up-regulated only in spinal microglia but not in neurons and astrocytes, whilst spinal application of SFKs inhibitor (PP2 or SU6656) or p38 MAPK inhibitor (SB203580) blocked BDNF-induced LTP and suppressed microglial activation. As spinal LTP at C-fiber synapses is considered to underlie neuropathic pain, we subsequently examined whether BDNF may contribute to mechanical hypersensitivity by activation of spinal microglia using spared nerve injury (SNI) model. Following SNI BDNF and TrkB receptor were up-regulated mainly in dorsal horn neurons and in activated microglia, and p-SFKs and p-p38 MAPK were increased exclusively in microglia. Intrathecal injection of BDNF scavenger TrkB-Fc starting before SNI, which prevented the behavioral sign of neuropathic pain, suppressed both microglial activation and the up-regulation of p-SFKs and p-p38 MAPK produced by SNI. Thus, the increased BDNF/TrkB signaling in spinal dorsal horn may contribute to neuropathic pain by activation of microglia following peripheral nerve injury and inhibition of SFKs or p38 MAPK may selectively inhibit microglia in spinal dorsal horn.     

The role of CaMKII in BDNF-mediated neuroprotection of retinal ganglion cells (RGC-5)

The purpose of the study is to determine if expression or secretion of brain-derived neurotrophic factor (BDNF) in retinal ganglion cells (RGC-5) is mediated by NFkappaB or Ca2+/calmodulin-dependent protein kinase II (CaMKII). RGC-5 cells were exposed to 1 mM glutamate for various periods of time, in the presence or absence of prospective regulatory molecules. BDNF mRNA and protein expression were assessed with the aid of real-time PCR and immunoblots, respectively, and BDNF secretion was determined by ELISA. The NFkappaB inhibitor (TLCK and PTD-p65), or a specific CaMKII inhibitor (m-AIP), was used to study association of NFkappaB or CaMKII with BDNF expression/secretion in RGC-5 cells. Glutamate stimulated a transient increase in BDNF mRNA and protein in RGC-5 cells, and also stimulated an early release of BDNF into the culture media. Neutralizing the BDNF or blocking the TrkB receptor enhanced the glutamate-induced cytotoxicity. NFkappaB nuclear translocation was revealed in response to glutamate treatment. Application of TLCK or PTD-p65 inhibited the glutamate-induced BDNF expression and secretion. Inhibition of CaMKII by m-AIP did not affect expression but significantly enhanced the release of BDNF from glutamate challenged cells. Our data suggest that glutamate treatment may stimulate expression of BDNF in RGC-5 cells through NFkappaB activation. A novel mechanism for neuroprotection is proposed for the CaMKII inhibitor, AIP, which appears to protect RGC-5 cells from cytotoxicity by enhancing the release of BDNF from glutamate challenged cells.     

Protease‐activated receptor 4 activation increases the expression of calcitonin gene‐related peptide mRNA and protein in dorsal root ganglion neurons

Accumulating evidence demonstrates that nociceptor activation evokes a rapid change in mRNA and protein levels of calcitonin gene‐related peptide (CGRP) in dorsal root ganglion (DRG) neurons. Although the colocalization of CGRP and protease‐activated receptor‐4 (PAR4), a potent modulator of pain processing and inflammation, was detected in DRG neurons, the role of PAR4 activation in the expression of CGRP has not been investigated. In the present study, the expression of CGRP and activation (phosphorylation) of extracellular signal‐regulated kinases 1 and 2 (ERK1/2) in rat DRG neurons were measured by immunofluorescence, real‐time PCR, and Western blotting after AYPGKF‐NH2 (selective PAR4‐activating peptide; PAR4‐AP) intraplantar injection or treatment of cultured DRG neurons. The expression of CGRP in cultured DRG neurons was also assessed after treatment with AYPGKF‐NH2 with preaddition of PD98059 (an inhibitor for ERK1/2 pathway). Results showed that PAR4‐AP intraplantar injection or treatment of cultured DRG neurons evoked significant increases in DRG cells displaying CGRP immunoreactivity and cytoplasmic and nuclear staining for phospho‐ERK1/2 (p‐ERK1/2). Percentages of total DRG neurons expressing both CGRP and PAR4 or p‐ERK1/2 also increased significantly at 2 hr after PAR4‐AP treatment. Real‐time PCR and Western blotting showed that PAR4‐AP treatment significantly increased expression of CGRP mRNA and protein levels in DRG neurons. The PAR4 activation‐evoked CGRP expression both at mRNA and at protein levels was significantly inhibited after p‐ERK1/2 was inhibited by PD98059. These results provide evidence that activation of PAR4 upregulates the expression of CGRP mRNA and protein levels in DRG neurons via the p‐ERK1/2 signal pathway. © 2013 Wiley Periodicals, Inc.     

Protease activated receptor 4 status of mast cells in post infectious irritable bowel syndrome

Background Growing evidence suggests that protease activated receptors (PARs) are mediators of persistent neuropathic pain, but their possible function as mediators in patients with post infectious irritable bowel syndrome (PI-IBS) remains to be further explored. This article aims to investigate the expression of PAR(2) and PAR(4) in the colonic mucosa of patients with PI-IBS, focusing on correlation with mast cell activation status. Methods A total of 17 normal controls and 23 patients with PI-IBS volunteered the study. The expression and localization of PAR(2) and PAR(4) were investigated by RT-PCR and immunohistochemistry, and the expression of PAR(2) and PAR(4) in the mast cells was examined using double-immunofluorescence staining. Key Results The immunohistochemical study revealed that epithelial and submucosal cells showed immunoreactivity for both PAR(2) and PAR(4) . Protease activated receptor 4 mRNA expression and immunoreactivity were down-regulated in PI-IBS compared with the control group. Specifically, a reduced immunoreactivity for PAR(4) was observed in mast cells of PI-IBS compared with normal controls, whereas there are no significant differences shown in PAR(2) between the PI-IBS and the control group. It is also found that the PAR(4) immunoreactivity decreases, while the activity of mast cells increases in PI-IBS rather than normal controls. Conclusions & Inferences This study outlines the down-regulation of PAR(4) in the mast cells of PI-IBS. It could be of considerable interests in understanding the mechanisms involved in the persistent colonic hypersensitivity and their potential role as therapeutic targets for PI-IBS.