Добірка наукової літератури з теми "Spinal cord injury; substance P; neurogenic inflammation"

Classical inflammation is a well-characterized secondary response to many acute disorders of the central nervous system. However, in recent years, the role of neurogenic inflammation in the pathogenesis of neurological diseases has gained increasing attention, with a particular focus on its effects on modulation of the blood-brain barrier BBB. The neuropeptide substance P has been shown to increase blood-brain barrier permeability following acute injury to the brain and is associated with marked cerebral edema. Its release has also been shown to modulate classical inflammation. Accordingly, blocking substance P NK1 receptors may provide a novel alternative treatment to ameliorate the deleterious effects of neurogenic inflammation in the central nervous system. The purpose of this paper is to provide an overview of the role of substance P and neurogenic inflammation in acute injury to the central nervous system following traumatic brain injury, spinal cord injury, stroke, and meningitis.

Abstract Sickle cell disease (SCD) is characterized by chronic hemolysis, inflammation, vascular dysfunction, and pain. Earlier we showed that mast cell activation contributes to neuroinflammation and pain and is accompanied by increased toll-like receptor 4 (TLR4) expression on mast cells (Vincent et al., Blood 2013), and that genetic deletion of TLR4 ameliorates neurogenic inflammation and hyperalgesia in HbSS-BERK sickle mice. Several other studies have shown increased TLR4 expression in peripheral system and its involvement in sickle pathobiology. We propose that free heme, due to hemolysis, activates TLR4 in the central nervous system in addition to peripheral activation, which further exacerbates neuroinflammation and hyperalgesia. Spinal cords of HbSS-BERK sickle mice show 3-fold mRNA transcripts for TLR4 and a 2-fold increase in hemin as compared to the spinal cords of HbAA-BERK control mice. Therefore, targeting TLR4 with pharmacological inhibitors may provide a therapeutic approach to attenuate peripheral and central inflammation and hyperalgesia. In the present study we examined the potential of pharmacological inhibition of mast cell activation, neuroinflammation and hyperalgesia in HbSS-BERK sickle mice with TLR4 inhibitor, TAK242. Sickle mice were administrated intravenously with TLR4 inhibitor TAK242 (1 mg/kg body weight/day) for 5 days. Sensory testing was performed at baseline at recruitment and periodically during the 5-day treatment and for another 8 days after concluding the treatment to evaluate mechanical hyperalgesia with von Frey filaments, thermal hyperalgesia in response to heat/cold and grip force for musculoskeletal/deep tissue hyperalgesia. Following the 5-day treatment with TAK242, release of cytokines, tryptase (marker of mast cell activation) and substance P released from skin biopsies and spinal cords were analyzed by ELISA. TAK242 significantly decreased the release of tryptase (TAK242: 5.178 ± 0.7613 pg/ml vs vehicle: 8.801 ± 0.9403 pg/ml, p = 0.0181), substance P (TAK242: 11.56 ± 1.945 pg/ml vs vehicle: 25.51 ± 4.283 pg/ml, p = 0.018), and IL-6 (TAK242: 15.59 ± 0.4541 pg/ml vs vehicle: 29.74 ± 0.8249 pg/ml, p = 0.0045) from skin biopsies, suggesting that TAK242 reduced SCD-induced mast cell activation and inflammation. TAK242 also significantly decreased substance P (TAK242: 0.7198 ± 0.0587 pg/mg vs vehicle: 0.931 ± 0.0676 pg/mg, p = 0.0462) and phosphorylation of p38/MAPK (p = 0.0184) in the spinal cord, as well as dorsal cutaneous blood flow (TAK242: 6.392 ± 0.3857 PU vs vehicle: 12.32 ± 0.5575 PU, p < 0.0001), indicating that TAK242 ameliorated SCD-evoked central and peripheral activation of inflammation and nociceptive mechanisms. Furthermore, TAK242 administration gradually reduced the mechanical, deep tissue, and thermal hyperalgesia upto 5-day treatment (p < 0.01, vs vehicle HbSS). However, discontinuation of treatment led to a gradual increase in hyperalgesia observed upto day-8 post-treatment. TAK242 also significantly decreased acute pain induced by hypoxia/reoxygenation and accelerated recovery from injury of hypoxia/reoxygenation. These data reveal the significant therapeutic effect of pharmacological inhibition of TLR4 on inflammation and hyperalgesia in sickle mice. Therapies targeting TLR4 inhibition may be potentially beneficial in ameliorating sickle pathobiology and pain. Disclosures No relevant conflicts of interest to declare.

Abstract Pain in sickle cell anemia (SCA) is accompanied by inflammation, vascular dysfunction and ischemia/reperfusion (IR) injury. We found that activated cutaneous mast cells in HbSS-BERK sickle mice release cytokines and neuropeptides and stimulate Evans blue leakage from the vasculature resulting in neurogenic inflammation and hyperalgesia (Vincent et al., 2013, Blood). Toll-like receptor 4 (TLR4) signaling stimulates mast cell activation and plays a causative role in inflammatory and neuropathic pain. Skin mast cells and spinal cords from HbSS-BERK sickle mice showed a several-fold increase in gene expression of TLR4 transcripts as compared to control mice expressing normal human hemoglobin (p<0.001 & 0.01, respectively). We hypothesized that TLR4 mediates mast cell activation-induced neurogenic inflammation and hyperalgesia in SCA. In TLR4 knockout (KO) mice, response to lipopolysaccharide and thermal and mechanical stimuli is attenuated; and spinal glial activation and release of inflammatory cytokines are reduced with accompanying resistance to IR injury. We backcrossed HbSS-BERK mice expressing human sickle hemoglobin and HbAA-BERK control mice expressing normal human hemoglobin with TLR4-KO mice to obtain HbSS-BERK with TLR4-KO (TLR4-KO-SS), and littermate TLR4-KO, HbSS-BERK and HbAA-BERK to examine the contribution of TLR4 to chronic pain and IR-induced acute pain in SCA. Pain behaviors included grip force measurement for deep tissue pain, mechanical sensitivity to von Frey filaments for cutaneous hyperalgesia and sensitivity to heat and cold for thermal hyperalgesia, as described by us for sickle mice (Kohli et al., Blood 2010). The measure of mechanical threshold and suprathreshold to a 1.0 g von Frey fiber showed a significant reduction in mechanical sensitivity in TLR4-KO-SS as compared to HbSS-BERK (p<0.001 for both measures). Similarly, deep pain and thermal sensitivity were significantly reduced in TLR4-KO-SS as compared to HbSS-BERK (p<0.01 for each measure). All pain profiles in TLR4-KO-SS were similar to HbAA-BERK and TLR4-KO, suggestive of a contribution of TLR4 in chronic pain in sickle mice. We next examined pain evoked by hypoxia/reoxygenation (HR) simulating acute pain following vasoocclusive crisis (VOC). HR evoked a significant increase in mechanical and heat sensitivity and in deep tissue pain in HbSS-BERK mice, which was sustained for 7 days, last period of observation (p<0.05 Vs baseline at normoxia). In contrast, TLR4-KO-SS did not show a significant increase in any of the pain measures following HR, suggesting that TLR4 mediates HR-induced injury in SCA. To analyze neurogenic inflammation we quantified the leakage of Evans blue dye in response to PBS, substance P (SP) and capsaicin in the skin, 7 days after the incitement of HR. PBS-treated skin showed significantly increased leakage of Evans blue following HR in HbSS-BERK, as compared to HbSS-BERK under normoxia (p<0.01). In contrast, Evans blue leakage following HR in TLR4-KO-SS was significantly reduced as compared to HbSS-BERK under HR as well as under normoxia (p<0.01 and 0.05, respectively). In TLR4-KO-SS, SP- and capsaicin-induced Evans blue leakage was approximately 50% that of HbSS-BERK, following HR, demonstrating that TLR4 contributes to neurogenic inflammation in sickle mice. TLR4 is also expressed on endothelial cells of the vasculature, which may contribute to HR-evoked vascular dysfunction directly and also via neurogenic inflammation caused by mast cells and peripheral nerve fibers. We observed that mast cell degranulation was reduced by ∼50% in TLR4-KO-SS as compared to HbSS-BERK (p<0.001) and the number of mast cells were reduced by ∼90% in TLR4-KO-SS as compared to HbSS-BERK (p<0.001) under HR. It is likely that TLR4 also mediates the recruitment, and/or proliferation of mast cells in addition to activating the existent mast cells. Together, these data suggest that TLR4 contributes to mast cell degranulation, neurogenic inflammation and hyperalgesia in sickle mice. Therefore, targeting TLR4 with novel pharmacological antagonists/agents may reduce inflammation and pain and prevent IR injury in SCA. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Inflammation, neurogenic inflammation and pain remain challenging to treat in sickle cell disease (SCD). Alternative therapies including acupuncture have been used for centuries to reduce pain and ameliorate underlying pathobiology of many disorders. We examined the mechanisms underlying acupuncture therapy in sickle mice. To prevent the influence of anesthetics and constraint on the pathobiology we developed electroacupuncture (EA) treatment for awake/conscious freely moving mice to simulate treatment conditions in patients, and then examined the peripheral and central mechanisms of neuroinflammation and nociception. Methods: HbSS-BERK sickle and HbAA-BERK control mice were treated with four EA treatments (every 3rd day, frequency: 4 or 10 Hz, pulse width: 100 microsecond, duration: 30 min) at acupoint GB30. Untreated and sham-EA treated (acupuncture without electrical stimulation) were used as controls. Hyperalgesia was evaluated daily by determining mechanical threshold, deep tissue hyperalgesia and thermal hyperalgesia using von Frey filaments, grip force, and cold plate, respectively. Blood and tissues were collected for analysis after four sessions of treatment. Skin biopsies were incubated overnight and culture medium was analyzed for mast cell activation marker tryptase, and neuromodulatory marker substance P. Results: Varied analgesic response to EA treatment was observed in sickle mice. About 86% treated mice equally showed positive (>50% pain relief) or moderate (20-30% pain relief) response and 14% were non-responsive (<20% pain relief) to EA. In positive responders, EA significantly reduced white blood cells (p<.001 Vs moderate- and non-responders), serum amyloid protein (p<.01 Vs untreated), IL-1beta (p<.05 Vs untreated, p<.01 Vs non-responders), and substance P (p<.05 Vs untreated and p<.001 Vs non-responders and p<.05 Vs moderate-responders). Concurrently, spinal cord analysis of EA treated positive-responders showed reduced substance P (p<.05 Vs untreated and non-responders), IL-1 beta (p<.01 Vs untreated), TNF alpha (p<.05 and p<.01 Vs moderate- and non-responders, respectively). Consistent with this central and peripheral anti-inflammatory response, culture medium from skin biopsies of positive responders demonstrated reduced substance P (p<.01 Vs moderate- and non-responders) and tryptase (p<.01 Vs untreated, moderate- and non-responders), and significantly less toluidine blue stained degranulating mast cells in the skin (p<.05 Vs untreated and non-responders) suggestive of attenuation of mast cell and peripheral nervous system activation. Functionally, capsaicin and substance P-induced neurogenic inflammation were significantly attenuated in positive-responders vs non-responders (p<.05) or untreated (p<.05). Peripheral and central attenuation of inflammatory and neurogenic response to EA was accompanied by inhibition of nociceptive signaling in the spinal cord. Spinal phosphorylation of p38 MAPK decreased in EA treated mice (p<.05 Vs sham-EA and untreated control; and positive-responders Vs non-responders). Conclusions: EA treatment on conscious free-moving mice simulates clinical conditions in patients and excludes the potential influence due to restraint or anesthetics. EA leads to peripheral and central neuromodulation and anti-inflammatory response by attenuating mast cell activation, substance P, and cytokine release in the periphery and by abrogating spinal nociceptive signaling of p38MAPK and inflammation. Together, these molecular and cellular effects lead to EA-induced attenuation of neurogenic inflammation and hyperalgesia in sickle mice. Importantly, these data explain the cause of variable effectiveness of EA in SCD. Disclosures No relevant conflicts of interest to declare.

Primary afferent fibers that contribute to increased substance P receptor internalization in the spinal cord after injury. Upon noxious stimulation, substance P (SP) is released from primary afferent fibers into the spinal cord where it interacts with the SP receptor (SPR). The SPR is located throughout the dorsal horn and undergoes endocytosis after agonist binding, which provides a spatial image of SPR-containing neurons that undergo agonist interaction. Under normal conditions, SPR internalization occurs only in SPR+ cell bodies and dendrites in the superficial dorsal horn after noxious stimulation. After nerve transection and inflammation, SPR immunoreactivity increases, and both noxious as well as nonnoxious stimulation produces SPR internalization in the superficial and deep dorsal horn. We investigated the primary afferent fibers that contribute to enhanced SPR internalization in the spinal cord after nerve transection and inflammation. Internalization evoked by electrical stimulation of the sciatic nerve was examined in untreated animals, at 14 days after sciatic nerve transection or sham surgery and at 3 days after hindpaw inflammation. Electrical stimulation was delivered at intensities to excite Aβ fibers only, Aβ and Aδ fibers or A and C fibers as determined by the compound action potential recorded from the tibial nerve. Electrical stimuli were delivered at a constant rate of 10 Hz for a duration of 5 min. Transection of the sciatic nerve and inflammation produced a 33.7 and 32.5% increase in SPR and immunoreactivity in lamina I, respectively. Under normal conditions, stimulation of Aδ or C fibers evoked internalization that was confined to the superficial dorsal horn. After transection or inflammation, there was a 20–24% increase in the proportion of SPR+ lamina I neurons that exhibited internalization evoked by stimulation of Aδ fibers. The proportion of lamina I SPR+ neurons that exhibited internalization after stimulation of C-fibers was not altered by transection or inflammation because this was nearly maximal under normal conditions. Moreover, electrical stimulation sufficient to excite C fibers evoked SPR internalization in 22% of SPR+ lamina III neurons after nerve transection and in 32–36% of SPR+ neurons in lamina III and IV after inflammation. Stimulation of Aβ fibers alone never evoked internalization in the superficial or deep dorsal horn. These results indicate that activation of small-caliber afferent fibers contributes to the enhanced SPR internalization in the spinal cord after nerve transection and inflammation and suggest that recruitment of neurons that possess the SPR contributes to hyperalgesia.

✓ The frequent occurrence of acute death from pulmonary failure in experimental head injury studies on Sprague-Dawley rats prompted an investigation into the manner in which acute neurogenic pulmonary edema develops in these animals as a result of an applied fluid pressure pulse to the cerebral hemispheres. Studies were performed in adult animals using histamine H1 and H2 blocking agents, or in adult animals treated as neonates with capsaicin to destroy unmyelinated C-fibers. Recordings were made of either the pulmonary arterial or the right ventricular pressure, and the left atrial and femoral arterial pressures before, during, and after injury to provide a record of the hemodynamic response throughout the development of neurogenic pulmonary edema. Head injury triggered the almost immediate development of pressure transients with and without neurogenic pulmonary edema. All rats, regardless of treatment, reacted with nearly identical systemic arterial pressure responses; however, the pulmonary responses followed a time course that was independent of systemic arterial pressure changes. Acute neurogenic pulmonary edema was always associated with a substantial increase in pulmonary arterial and left atrial pressures; conversely, pressure increases of similar magnitude were not always associated with edema. Histamine H1 and H2 blockers significantly reduced the pulmonary pressure surges only in rats free of neurogenic pulmonary edema. All capsaicin-treated rats showed suppressed pulmonary pressure responses, normal lung water content, elevated lung surface tension, and significantly reduced levels of immunoreactive substance P in the spinal cord and vagus nerve. While the pressures cannot clarify how edema influences the observed hemodynamics, they do not support the view that edema is the direct consequence of pulmonary hypertension. It is proposed that neurogenic pulmonary edema is a functional disturbance provoked by adverse stimuli from outside the lungs and that in the rat the primary afferent fiber is essential to the production of this entity.

This study tested whether combined hyperbaric oxygen (HBO) and allogenic adipose-derived mesenchymal stem cells (ADMSCs) would be superior to either one for improving the locomotor recovery in rat after acute traumatic spinal cord injury (TSCI) in rat. Adult-male Sprague–Dawley rats were equally categorized into group 1 (sham-operated control), group 2 (TSCI), group 3 (TSCI + HBO for 1.5 h/day for 14 consecutive days after TSCI), group 4 (TSCI + ADMSCs/1.2 × 106 cells by intravenous injection at 3 h and days 1/2 after TSCI), and group 5 (TSCI + HBO + ADMSCs), euthanized, and spinal cord tissue was harvested by day 49 after TSCI. The protein expressions of oxidative-stress (NOX-1/NOX-2), inflammatory-signaling (TLR-4/MyD88/IL-1β/TNF-α/substance-p), cell-stress signaling (PI3K/p-AKT/p-mTOR), and the voltage-gated sodium channel (Nav1.3/1.8/1.9) biomarkers were highest in group 2, lowest in group 1, and significantly lower in group 5 than in groups 3/4 (all P <0.0001), but they did not differ between groups 3 and 4. The spinal cord damaged area, the cellular levels of inflammatory/DNA-damaged biomarkers (CD68+/GFAP+/γ-H2AX+ cells), mitogen-activated protein kinase family biomarkers (p-P38/p-JNK/p-ERK1/2), and cellular expressions of voltage-gated sodium channel (Nav.1.3, Nav.1.8, and Nav.1.9 in NF200+ cells) as well as the pain-facilitated cellular expressions (p-P38+/peripherin+ cells, p-JNK+/peripherin+ cells, p-ERK/NF200+ cells) exhibited an identical pattern of inflammation, whereas the locomotor recovery displayed an opposite pattern of inflammation among the groups (all P < 0.0001). Combined HBO-ADMSCs therapy offered additional benefits for preserving the neurological architecture and facilitated the locomotor recovery against acute TSCI.

Pressure ulcers (PUs) or sores are a secondary complication of diabetic neuropathy and traumatic spinal cord injury (SCI). PUs tend to occur in soft tissues located around bony prominences and may heal slowly or not at all. A common mechanism underlying impaired healing of PUs may be dysfunction of the local neurovascular system including deficiency of essential neuropeptides, such as substance P (SP). Previous studies indicate that disturbance in cutaneous sensory innervation leads to a defect in all stages of wound healing, as is the case after SCI. It is hypothesized that nerve fibers enhance wound healing by promoting initial inflammation via the releasing of neuropeptides such as SP. Therefore, we investigated whether exogenous SP improves skin wound healing using in vitro and in vivo models. For in vitro studies, the effects of SP on keratinocyte proliferation and wound closure after a scratch injury were studied under normoxia (pO2 ~21%) or hypoxia (pO2 ~1%) and in presence of normal serum (10% v/v) or low serum (1% v/v) concentrations. Hypoxia and low serum both significantly slowed cell proliferation and wound closure. Under combined low serum and hypoxia, used to mimic the nutrient- and oxygen-poor environment of chronic wounds, SP (10−7 M) significantly enhanced cell proliferation and wound closure rate. For in vivo studies, two full-thickness excisional wounds were created with a 5 mm biopsy punch on the dorsum on either side of the midline of 15-week-old C57BL/6J male and female mice. Immediately, wounds were treated topically with one dose of 0.5 μg SP or PBS vehicle. The data suggest a beneficial role in wound closure and reepithelization, and thus enhanced wound healing, in male and female mice. Taken together, exogenously applied neuropeptide SP enhanced wound healing via cell proliferation and migration in vitro and in vivo. Thus, exogenous SP may be a useful strategy to explore further for treating PUs in SCI and diabetic patients.

Spinal cord injury (SCI) is a devastating injury that commonly results in permanent physical disability. The highest incidence of SCI occurs in younger populations, causing an enormous financial burden to both individuals and society amounting to almost $1 billion annually within Australia spent on hospitalisation, treatment and rehabilitation of spinal cord injured individuals. To date, an effective clinical treatment for SCI remains elusive, highlighting the need for research aimed at developing therapeutic interventions that improve functional outcome. Spinal cord edema is recognised as a common complication of SCI which continues to develop, spreading in a rostrocaudal direction days after injury, resulting in greater tissue damage and functional deficits. Reducing edema following SCI is therefore of utmost importance and represents an attractive target for therapeutic intervention. Substance P (SP) is a neuropeptide known to facilitate the process of neurogenic inflammation, which has previously been shown to result in blood brain barrier (BBB) disruption and subsequent edema development following both traumatic brain injury (TBI) and stroke. Furthermore, inhibition of the high-affinity SP receptor, the tachykinin NK1 receptor, resulted in reduced BBB permeability, edema and improved functional outcome in both of these conditions. Accordingly, the current thesis sought to determine whether SP played a similar role as a mediator of neurogenic inflammation following traumatic SCI. Immunohistochemical assessment of human SCI demonstrated a loss of SP from the dorsal horn region, suggesting that SP release increased following injury. NK1 receptor immunoreactivity was also initially increased post-injury before declining, indicating that receptor activation and subsequent internalisation occurred. Assessment of various open experimental injury models, including the weight drop, hemisection and clip compression models, demonstrated similar SP immunoreactivity as that observed in human tissue, although NK1 receptor immunoreactivity varied in localisation and response to injury. These results highlighted the need for experimental models to accurately replicate the primary injury mechanisms observed clinically, especially the closed environment rather than the open nature of most experimental models. The balloon compression model was subsequently employed for the remainder of the study, given its closed nature and its ability to mimic primary injury mechanisms such as an initial impact followed by persisting compression. This model also proved to replicate many other facets of human injury such as severe hemorrhage, axonal injury, neuronal loss, microglial activation, as well as increased BSCB disruption, edema, intrathecal pressure (ITP) and reduced functional outcome. Balloon compression induced SCI was also associated with reduced SP immunoreactivity, suggesting increased SP release, and increased NK1 receptor immunoreactivity. Such observations implicate a potential role for SP in mediating neurogenic inflammation following SCI. However, administration of an NK1 receptor antagonist, n-acetyl tryptophan (NAT), resulted in no improvement in any assessed outcomes, suggesting that SP mediated neurogenic inflammation might not play a major role in the development of BSCB disruption following SCI. Indeed, the physiological effects of SP on the barrier may be outweighed by the substantial mechanical disruption observed. Interestingly, changes in the immunoreactivity of the water channel protein, aquaporin 4 (AQP4), were observed following both human SCI and the balloon compression model. These alterations implicate the involvement of AQP4 in facilitating the removal of excess water from the spinal cord. As such, modulation of AQP4 may represent a novel therapeutic intervention following SCI. We conclude that SP mediated neurogenic inflammation is a minor player in the injury cascade after SCI. At times, NAT administration resulted in worsened outcomes and as such raises the question as to whether SP might be beneficial following SCI rather than detrimental. Further investigation of the role of SP following SCI, especially a later time points, is required to better elucidate its potential role. Nonetheless, substantial edema development remained a serious consequence following SCI and given the observed changes in AQP4 immunoreactivity, investigation of AQP4 modulation following SCI is warranted.
Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2012