Programmed death-1 promotes contused skeletal muscle regeneration by regulating Treg cells and macrophages.

Immune cells are involved in skeletal muscle regeneration. The mechanism by which Treg cells are involved in the regeneration of injured skeletal muscle is still unclear. The purpose of this study was to explore the role of programmed death-1 in contused skeletal muscle regeneration, and to clarify the regulation of programmed death-1 on Treg cell generation and macrophage polarization, in order to deepen our understanding of the relationship between the immune system and injured skeletal muscle regeneration. The results show that programmed death-1 knockdown reduced the number of Treg cells and impaired contused skeletal muscle regeneration compared with those of wild-type mice. The number of pro-inflammatory macrophages in the contused skeletal muscle of programmed death-1 knockout mice increased, and the expression of pro-inflammatory factors and oxidative stress factors increased, while the number of anti-inflammatory macrophages and the expression of anti-inflammatory factors, antioxidant stress factors, and muscle regeneration-related factors decreased. These results suggest that programmed death-1 can promote contused skeletal muscle regeneration by regulating Treg cell generation and macrophage polarization.

Bone Marrow Stromal Cell-Derived Exosomes Promote Muscle Healing Following Contusion Through Macrophage Polarization.

Skeletal muscle contusion is among the most common injuries in traumatology and clinics of sports medicine. The injured muscle is vulnerable to re-injury owing to fibrosis formation. Given that the bone marrow stromal cell-derived exosomes (BMSC-Exos) displayed promising therapeutic effect for various tissues, we used BMSC-Exos to treat skeletal muscle contusion and investigated its effects on muscle healing. In this study, the in vivo model of skeletal muscle contusion was established by subjecting the tibialis anterior of young male mice to hit injury, and the in vitro inflammation model was established by lipopolysaccharide treatment on macrophages. Macrophage depletion model was built by intraperitoneal injection with clodronate-containing liposomes. Exosomes were isolated and purified from the supernatant of BMSCs using gradient centrifugation. Nanoparticle tracking analysis, transmission electron microscope, and western blot were used to identify the exosomes. HE stain, Masson stain, immunofluorescence, and biomechanical testing were carried out on the muscle tissue. In addition, enzyme-linked immunosorbent assay (ELISA) assays, real-time qPCR, flow cytometry, and PKH67 fluorescence trace were conducted in vitro. Intramuscular injection of BMSC-Exos to mice after muscle contusion alleviated inflammation level, reduced fibrosis size, promoted muscle regeneration, and improved biomechanical property. After macrophages depletion, the effects of BMSC-Exos were inhibited. In vitro, PKH-67 fluorescence was internalized into macrophages. BMSC-Exos promoted M2 macrophages polarization both in vivo and in vitro. At the same time, BMSC-Exos reduced the production of inflammatory cytokines under the inflammatory microenvironment and upregulated anti-inflammatory factors expression. In conclusion, BMSC-Exos attenuated muscle contusion injury and promoted muscle healing in mice by modifying the polarization status of macrophages and suppressing the inflammatory reaction.

Monotrauma is associated with enhanced remote inflammatory response and organ damage, while polytrauma intensifies both in porcine trauma model.

AIM: Severely injured patients experience substantial immunological stress upon traumatic insult. Next to the direct local tissue injury also other organs, which are not directly injured such as liver and lung, are frequently affected by a so-called remote organ damage (ROD) after trauma. Thus, we studied the inflammatory response of lung and liver either after isolated femur fracture as example for ROD, or after multiple trauma in a porcine polytrauma model. METHODS: Twenty-four male pigs (Sus scrofa) underwent either isolated standardized femoral fracture (monotrauma, MT, n = 12) or polytrauma (PT, n = 12). PT consisted of a femur fracture, lung contusion, liver laceration, hemorrhagic shock, subsequent resuscitation and surgical fracture fixation. Six animals served as controls (sham). After 72 h inflammatory changes were determined by analyses of the interleukin (IL)-6 gene expression and tissue infiltration of polymorphonuclear leukocyte (PMN, myeloperoxidase staining). ROD in MT, and lung as well as liver damage in PT were assessed histologically by hematoxylin-eosin staining. Expression of phosphorylated p65 NF-κB was evaluated by immunohistology. RESULTS: IL-6 increased in lungs and liver in both groups MT and PT, respectively, compared to sham. Similarly, PMN infiltration of the lungs and liver increased significantly after both MT and PT compared to sham. Histological evaluation demonstrated tissue damage notably in lungs after MT, while tissue damage after PT was found in both lung and liver after PT. p65 NF-κB tended to an increase upon MT, and was significantly enhanced after PT in both tissues. CONCLUSION: Our data indicate that remote organ damage after MT notably in lungs was associated with an enhanced inflammatory response. Severe polytrauma substantially intensifies this response and organ damage in the underlying model.

Contributing factors in the development of acute lung injury in a murine double hit model.

OBJECTIVES: Blunt chest (thoracic) trauma (TxT) is known to contribute to the development of secondary pulmonary complications. Of these, acute lung injury (ALI) is common especially in multiply injured patients and might not only be due to the direct trauma itself, but seems to be caused by ongoing and multifactorial inflammatory changes. Nevertheless, the exact mechanisms and contributing factors of the development of ALI following blunt chest trauma are still elusive. METHODS: 60 CL57BL/6N mice sustained either blunt chest trauma combined with laparotomy without further interventions or a double hit (DH) including TxT and cecal ligation puncture (CLP) after 24 h to induce ALI. Animals were killed either 6 or 24 h after the second procedure. Pulmonary expression of inflammatory mediators cxcl1, cxcl5, IL-1ß and IL-6, neutrophil infiltration and lung tissue damage using the Lung Injury Score (LIS) were determined. RESULTS: Next to a moderate increase in other inflammatory mediators, a significant increase in CXCL1, neutrophil infiltration and lung injury was observed early after TxT, which returned to baseline levels after 24 h. DH induced significantly increased gene expression of cxcl1, cxcl5, IL-1ß and IL-6 after 6 h, which was followed by the postponed significant increase in the protein expression after 24 h compared to controls. Neutrophil infiltration was significantly enhanced 24 h after DH compared to all other groups, and exerted a slight decline after 24 h. LIS has shown a significant increase after both 6 and 24 h compared to both control groups as well the late TxT group. CONCLUSION: Early observed lung injury with moderate inflammatory changes after blunt chest trauma recovered quickly, and therefore, may be caused by mechanical lung injury. In contrast, lung injury in the ALI group did not undergo recovery and is closely associated with significant changes of inflammatory mediators. This model may be used for further examinations of contributing factors and therapeutic strategies to prevent ALI.

Effect of high frequency electromagnetic wave stimulation on muscle injury in a rat model.

INTRODUCTION: The aim of this study was to investigate biological changes in tissues with muscle contusion after the application of high frequency (HF) electromagnetic wave. METHODS: An acrylic pipe was placed on the right hind limb and a metallic ball was dropped inside the pipe, which resulted in a muscle contusion. After acquiring the optimal condition for muscle contusion, 20 Sprague-Dawley rats were allocated to the HF treatment (N = 10) and sham groups (N = 10), which then underwent muscle contusion injury at their right thigh. The thickness and circumference of the right thigh and the left thigh (negative control groups) were measured (day 0). HF electromagnetic wave stimulation for three days was performed on the contusion area in the HF group after one day. Thickness was measured at the thickest area of both hind limbs and the circumference was measured every day for three days. The sham group received no treatment, and the circumference and thickness were measured using the same method. After three days, Hematoxylin and eosin and immunohistochemical (IHC) staining for IL-1ß were performed and TUNEL assay was conducted for apoptosis in the skin and muscle layers. RESULTS: The thigh muscle thickness at day 1 was significantly different between groups (P = 0.018) and this difference was observed between both sham and control groups (corrected P = 0.007), and between sham and HF groups (corrected P = 0.043). Thigh circumference was significantly different at day 3 (P = 0.047) and this difference was found between sham and control groups (corrected P = 0.018), and between sham and HF groups (corrected P = 0.032). In the HF group, the inflammatory response was reduced to almost the same level as the control group. Evaluation of IL-1ß level, the inflammatory cytokine, through IHC showed marked localization of IL-1ß in muscle fibers of the sham group. However, significantly less IL-1ß was observed in the muscle of the HF treatment group. There was neither injury nor apoptosis after HF stimulation. CONCLUSIONS: Application of the HF showed therapeutic effect on muscle contusion by reducing muscle swelling. This effect might be caused by the anti-inflammatory action of the HF, which evoked energy into the injured muscle.

Toll-Like Receptor-9 (TLR9) is Requisite for Acute Inflammatory Response and Injury Following Lung Contusion.

Lung contusion (LC) is a significant risk factor for the development of acute respiratory distress syndrome. Toll-like receptor 9 (TLR9) recognizes specific unmethylated CpG motifs, which are prevalent in microbial but not vertebrate genomic DNA, leading to innate and acquired immune responses. TLR9 signaling has recently been implicated as a critical component of the inflammatory response following lung injury. The aim of the present study was to evaluate the contribution of TLR9 signaling to the acute physiologic changes following LC. Nonlethal unilateral closed-chest LC was induced in TLR9 (-/-) and wild-type (WT) mice. The mice were sacrificed at 5, 24, 48, and 72-h time points. The extent of injury was assessed by measuring bronchoalveolar lavage, cells (cytospin), albumin (permeability injury), and cytokines (inflammation). Following LC, only the TLR9 (-/-) mice showed significant reductions in the levels of albumin; release of pro-inflammatory cytokines IL-1ß, IL-6, and Keratinocyte chemoattractant; production of macrophage chemoattractant protein 5; and recruitment of alveolar macrophages and neutrophil infiltration. Histological evaluation demonstrated significantly worse injury at all-time points for WT mice. Macrophages, isolated from TLR9 (-/-) mice, exhibited increased phagocytic activity at 24 h after LC compared with those isolated from WT mice. TLR9, therefore, appears to be functionally important in the development of progressive lung injury and inflammation following LC. Our findings provide a new framework for understanding the pathogenesis of lung injury and suggest blockade of TLR9 as a new therapeutic strategy for the treatment of LC-induced lung injury.

The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats.

Muscle wound healing process is a typical inflammation-evoked event. The monoacylglycerol lipase (MAGL) inhibitor (4-nitrophenyl)4-[bis(1,3-benzodioxol -5-yl)-hydroxymethyl]piperidine-1-carboxylate (JZL184) has been previously reported to reduce inflammation in colitis and acute lung injury in mice, which provide a new strategy for primary care of skeletal muscle injury. We investigated the effect of JZL184 on inflammation in rat muscle contusion model, and found decreased neutrophil and macrophage infiltration and pro-inflammatory cytokine expression. With extension of post-traumatic interval, myofiber regeneration was significantly hindered with increased collagen types I and ІІІ mRNAfibroblast infiltration as well as promoted fibrosis. Furthermore, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-morpholin-4-ylpyrazole-3-carboxamide (AM281, a selective cannabinoid CB1 receptor antagonist) and [6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone (AM630, a selective cannabinoid CB2 receptor antagonist) treatment alleviated the anti-inflammatory effect of JZL184. Our findings demonstrate that JZL184 is able to inhibit the inflammatory response and interfere with contused muscle healing, in which the anti-inflammatory action may be mediated through cannabinoid CB1 and CB2 receptors.

Alveolar macrophage depletion increases the severity of acute inflammation following nonlethal unilateral lung contusion in mice.

BACKGROUND: Lung contusion (LC) is a common injury resulting from blunt thoracic trauma. LC is an important risk factor for the development acute lung injury, adult respiratory distress syndrome, and ventilator-associated pneumonia, all of which increase mortality from trauma. LC produces a nonspecific immune cellular response. Neutrophil recruitment is known to increase the severity of inflammation during LC. However, the exact role of macrophages in modulating the response to LC has not been well described. METHODS: We used a cortical contusion impactor to induce unilateral LC in mice. Thoracic micro computed tomographic scans of these animals were obtained to document radiologic changes over time following LC. To understand the role of macrophages during LC, liposomal clodronate was used to deplete macrophage levels before traumatic insult. Acute inflammatory attributes after LC were assessed, by measuring pressure-volume mechanics; quantifying bronchial alveolar lavage levels of leukocytes, albumin, and cytokines; and finally examining lung specimen histopathology at 5, 24, 48, and 72 hours after injury. RESULTS: After LC, alveolar macrophage numbers were significantly reduced and exhibited slowed recovery. Simultaneously, there was a significant increase in bronchial alveolar lavage neutrophil counts. The loss of macrophages could be attributed to both cellular apoptosis and necrosis. Pretreatment with clodronate increased the severity of lung inflammation as measured by worsened pulmonary compliance, increased lung permeability, amplification of neutrophil recruitment, and increases in early proinflammatory cytokine levels. CONCLUSION: The presence of regulatory alveolar macrophages plays an important role in the pathogenesis of acute inflammation following LC.

Complement mediates a primed inflammatory response after traumatic lung injury.

BACKGROUND: Pulmonary contusion (PC) is a common, potentially lethal injury that results in the priming for exaggerated responses to subsequent immune challenge such as an infection (second hit). We hypothesize a PC-induced complement (C) activation participates in the priming effect for a second hit. METHODS: Male, 8 weeks to 9 weeks, C57BL/6 mice (wild-type, C5) underwent blunt chest trauma resulting in PC. At 3 hours/24 hours after injury, the inflammatory response was measured in tissue, serum, and bronchoalveolar lavage (BAL). The thrombin inhibitor, hirudin, was used to determine if injury-induced thrombin participated in the activation of C. Injury-primed responses were tested by challenging injured mice with bacterial endotoxin (lipopolysaccharide, LPS) as a second hit. Inflammatory responses were assessed at 4 hours after LPS challenge. Data were analyzed using one-way analysis of variance with Bonferroni multiple comparison posttest (significance, p ≤ 0.05). Protocols were approved by the Institutional Animal Care and Use Committee. RESULTS: We found significantly increased levels of C5a in the BAL of injured animals as early as 24 hours, persisting for up to 72 hours after injury. Hirudin-treated injured mice had significantly decreased levels of thrombin in the BAL that correlated with reduced C5a levels. Injured mice challenged with intratracheal (IT) LPS had increased C5a and inflammatory response. Conversely, inhibition of C5a or its receptor, C5aR, before LPS challenge correlated with decreased inflammatory responses; C5a-deficient mice showed a similar loss of primed response to LPS challenge. CONCLUSION: Complement C5a levels in the BAL are increased over several days after PC. Premorbid inhibition of thrombin markedly decreases C5a levels after PC, suggesting that thrombin-induced C activation is the major pathway of activation after PC. Similarly, inhibition of C5a after PC will decrease injury-primed responses to LPS stimulation. Our findings suggest cross-talk between the coagulation and complement systems that induce immune priming after PC.

Interleukin 10 overexpression alters survival in the setting of gram-negative pneumonia following lung contusion.

OBJECTIVE: Lung contusion injury produces a vulnerable window within the inflammatory defenses of the lung that predisposes the patient to pneumonia. Interleukin 10 (IL-10) is a known anti-inflammatory mediator produced by macrophages and capable of downregulating acute lung inflammation. We investigated the impact of increased levels of IL-10 within the lung on survival and the host response to trauma in the setting of lung contusion (LC) and gram-negative pneumonia. DESIGN: A bitransgenic, tetracycline-inducible, lung-specific human IL-10 overexpression (IL-10 OE) mouse model and single transgenic (TG-) control mice were used. Mice underwent LC injury or sham injury (sham) at time -6 h. At time 0, animals were inoculated intratracheally with 500 colony-forming units of Klebsiella pneumoniae (pneu). Bronchoalveolar lavage fluid, lung tissue specimens, or purified macrophages were collected. Lung tissue and blood bacteria levels were quantified. Cytokine levels were assayed by enzyme-linked immunosorbent assay, and gene expression levels were evaluated by real-time polymerase chain reaction. Cell-type identification and quantification were done using real-time polymerase chain reaction and flow cytometry. MAIN RESULTS: Interleukin 10 OE mice demonstrated decreased 5-day survival compared with TG- mice following LC + pneu (0 vs. 30%, P < 0.0001). Interleukin 10 OE mice had significantly higher lung bacteria counts (P = 0.02) and levels of bacteremia (P = 0.001) at 24 h. The IL-10 OE mice recruited more neutrophils into the alveoli as measured in bronchoalveolar lavage fluid compared with TG- mice. Alveolar macrophages from IL-10 OE mice displayed increased alternative activation (M2 macrophages, P = 0.046), whereas macrophages from TG- mice exhibited classic activation (M1 macrophages) and much higher intracellular bacterial killing potential (P = 0.03). Interleukin 6, keratinocyte-derived chemokine, and macrophage inflammatory protein 2 levels were significantly elevated in IL-10 OE LC + pneu animals (P < 0.05). CONCLUSIONS: Lung-specific IL-10 overexpression induces alternative activation of alveolar macrophages. This shift in macrophage phenotype decreases intracellular bacterial killing, resulting in a more pronounced bacteremia and accelerated mortality in a model of LC and pneumonia.

Contusion injury with chronic in vivo polyphenol supplementation: leukocyte responses.

INTRODUCTION: In vivo, daily proanthocyanidolic oligomer (PCO) supplementation before and after experimental skeletal muscle contusion injury has been shown to result in a blunted neutrophil response in tissue, quicker macrophage infiltration into muscle, and faster recovery due to a left shift in time course of inflammation. The current study investigated effects of PCO on circulatory neutrophils and macrophage subpopulations as well as in vitro neutrophil migration. METHODS: Primary cultured neutrophils obtained from control animals were incubated in media with 20% conditioned plasma. To obtain conditioned media, male Wistar rats were supplemented with PCO (20 mg·kg(-1)d(-1)) or placebo (PLA) for 2 wk before a mass-drop contusion injury. Conditioned plasma was prepared from blood collected at different time points after injury (12 h, 1 d, 3 d, and 5 d). Macrophage subpopulation distribution, inflammatory cytokine, and myeloperoxidase levels were assessed for all time points. RESULTS: On day 1 postinjury, circulating neutrophil numbers were significantly lower in PLA than PCO, suggesting that extravasation from the blood was reduced by PCO. Concurrently, neutrophil migration in vitro was blunted in the presence of conditioned plasma from PCO supplemented rats compared with PLA supplemented rats. Plasma M1 and M2c macrophage numbers differed over time and between groups. M1 macrophage numbers peaked on day 3 with PCO supplementation, followed by a rise in M2c macrophages on day 5, when M1 macrophages numbers were still high in PLA. CONCLUSIONS: We conclude that PCO supplementation limits neutrophil migration capacity in vitro despite a chemotactic gradient. Furthermore, the earlier appearance of type M2 macrophages suggests a switch to an anti-inflammatory phenotype after injury even in circulation.

Mechanical ventilation increases the inflammatory response induced by lung contusion.

BACKGROUND: Posttraumatic lung contusion is common after blunt chest trauma, and patients often need ventilatory support. Lung contusion induces an inflammatory response signified by primed polymorph neutrophil granulocytes (PMNs) in blood and tissue. Mechanical ventilation (MV) can also cause an inflammatory response. The aim of this study was to develop an animal model to investigate the effect of high-volume ventilation on the inflammatory response in blunt chest trauma. MATERIALS AND METHODS: We assigned 23 male Sprague-Dawley rats to either MV or bilateral lung contusion followed by MV. We used three extra rats as controls. Lung contusion was induced by a blast generator, a device releasing a single pressure blast wave centered on the chest. We determined tissue and systemic inflammation by absolute PMN numbers in blood and bronchoalveolar lavage fluid (BALF), myeloperoxidase, interleukin (IL)-6, IL 1ß, growth-related oncogene-KC, and IL-10 in both plasma and BALF. RESULTS: Survival after blunt chest trauma was correlated to the distance to the blast generator. Compared with controls, both MV and blast plus MV rats showed increased systemic and pulmonary inflammation, expressed by higher PMNs, myeloperoxidase levels, and cytokine levels in both blood and BALF. Blast plus MV rats showed a higher systemic and pulmonary inflammatory response than MV rats. CONCLUSIONS: The blast generator generated reproducible blunt chest trauma in rats. Mechanical ventilation after lung contusion induced a larger overall inflammatory response than MV alone, which indicates that local damage contributes not only to local inflammation, but also to systemic inflammation. This emphasizes the importance of lung protective ventilation strategies after pulmonary contusion.

Curative effect of Xuebijing injection on severe pulmonary contusion.

OBJECTIVE: To investigate the curative effects of Xuebijing (XBJ) injection, a Chinese patent medicine, on severe pulmonary contusion (PC). METHODS: Sixty-three patients with PC were randomized to conventional therapy plus XBJ injection (n = 33) or conventional therapy alone (n = 30). Between groups differences in corticosteroid treatment, immune regulation therapy, hemofiltration, infusion volume, transfusion volume and antibiotic period were measured, as were intensive care unit (ICU)-free time, ventilation time, 28-day mortality rate and incidence of ventilation-associated pneumonia (VAP). Serum concentrations of procalcitonin (PCT), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-10, white blood cell (WBC) counts and percentages of human leukocyte antigen DR/ CD14+ (HLA-DR/CD14+) peripheral blood mononuclear cells were compared. Markers of ventilation were determined by blood gas analysis and ventilator parameters. RESULTS: WBC counts and serum concentrations of PCT, TNF-alpha, IL-6 and IL-10 were reduced significantly more quickly, and CD14+ percentage was increased significantly earlier, in the XBJ group than in the control group (P < 0.05 each).The level of ventilation and oxygenation index were ameliorated earlier in the XBJ than in the control group (P < 0.05). XBJ treatment significantly reduced ICU-free time, ventilation time and incidence of VAP (P < 0.05 each), but had no effect on 28-day mortality rate CONCLUSION: XBJ treatment can shorten ICU-free and ventilation times and reduce the incidence of VAP, improving outcomes in patients with severe PC. XBJ may act by regulating inflammation and immunity, alleviating systemic inflammatory response syndrome induced by trauma.

Innate immune response to pulmonary contusion: identification of cell type-specific inflammatory responses.

Lung injury from pulmonary contusion is a common traumatic injury, predominantly seen after blunt chest trauma, such as in vehicular accidents. The local and systemic inflammatory response to injury includes activation of innate immune receptors, elaboration of a variety of inflammatory mediators, and recruitment of inflammatory cells to the injured lung. Using a mouse model of pulmonary contusion, we had previously shown that innate immune Toll-like receptors 2 and 4 (TLR2 and TLR4) mediate the inflammatory response to lung injury. In this study, we used chimeric mice generated by adoptive bone marrow transfer between TLR2 or TLR4 and wild-type mice. We found that, in the lung, both bone marrow-derived and nonmyeloid cells contribute to TLR-dependent inflammatory responses after injury in a cell type-specific manner. We also show a novel TLR2-dependent injury mechanism that is associated with enhanced airway epithelial cell apoptosis and increased pulmonary FasL and Fas expression in the lungs from injured mice. Thus, in addition to cardiopulmonary physiological dysfunction, cell type-specific TLR and their differential response to injury may provide novel specific targets for management of patients with pulmonary contusion.

Postcontusion polyphenol treatment alters inflammation and muscle regeneration.

PURPOSE: Given the major role that oxidants play in cellular damage, and the recent focus on antioxidants as treatment for muscle injuries, the aim of this study was to examine the effect of short-term postinjury grape seed-derived polyphenol supplementation on muscle inflammation and repair processes after contusion injury. METHODS: Experimental injury of the right gastrocnemius muscle was achieved by drop-mass method (200 g from a height of 50 cm), after which rats were gavaged with either 0.9% saline (placebo-PLA) or 20 mg·kg⁻¹·d⁻¹ of proanthocyanidolic oligomer (PCO) from 2 h after contusion injury, for up to 14 d after injury. Blood samples and injured muscle were collected at 4 h and at days 1, 3, 5, 7, and 14 after injury. RESULTS: Compared to an uninjured control group, PCO supplementation resulted in an earlier peak in number of activated satellite cells in contusion-injured muscle tissue (4 h for PCO vs day 3 for PLA, n = 4 per time point per group) and fetal myosin heavy chain expression (day 5 for PCO, P < 0.01 with no change in PLA, n = 3 per time point per group), indicative of quicker muscle regeneration. PCO supplementation limited neutrophil infiltration and facilitated earlier macrophage infiltration into the injured area (n = 4 per group). PCO also resulted in an earlier return toward control levels of muscle proinflammatory cytokines on day 3 (P < 0.01 for interleukin 6 and P < 0 05 for tumor necrosis factor α, both n = 3 per group). CONCLUSIONS: Data show that short-term postinjury PCO supplementation was able to quicken muscle regeneration by facilitating earlier recruitment of activated satellite cells and to modulate the immune system in favor of an anti-inflammatory status.

Effects of penehyclidine hydrochloride on pulmonary contusion from blunt chest trauma in rats.

BACKGROUND AND OBJECTIVE: Toll-like receptor 4 (TLR4) is widely recognised as a pattern recognition receptor (PRR) in the triggering of innate immunity. Lung inflammation and systemic innate immune responses are dependent on TLR4 activation undergoing pulmonary contusion. Therefore, the author investigated the effects of penehyclidine hydrochloride (PHC) on the expression of TLR4 and inflammatory responses of blunt chest trauma-induced pulmonary contusion. MATERIALS AND METHODS: Male Sprague-Dawley (SD) rats were randomly assigned into three groups: normal control (NC) group, pulmonary contusion (PC) group and penehyclidine hydrochloride treatment (PHC) group. Pulmonary contusion was induced in anesthetised rats at fixed chest impact energy of 2.45J. Lung injury was assessed by the histopathology changes, arterial blood gas and myeloperoxidase (MPO) activity of lung. The serum tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were measured using enzyme-linked immunosorbent assays (ELISA). The expression of TLR4 was determined by immunohistochemistry. RESULTS: Blunt chest trauma produced leucocytosis in the interstitial capillaries, hypoxemia, and increased MPO activity. The expressions of TNF-α, IL-6 and TLR4 in the lung were significantly enhanced during pulmonary contusion. PHC treatments effectively attenuated pulmonary inflammation responses, as shown by improved pulmonary oxygenation, histopathology damage, decreased the MPO activity, the expressions of TNF-α, IL-6, and TLR4 after lung injury. CONCLUSION: It might be concluded that PHC exhibit anti-inflammatory and protective effects in traumatic lung injury via the inhibition of the TLR4 pathway.

Accelerated skeletal muscle recovery after in vivo polyphenol administration.

Acute skeletal muscle damage results in fiber disruption, oxidative stress and inflammation. We investigated cell-specific contributions to the regeneration process after contusion-induced damage (rat gastrocnemius muscle) with or without chronic grape seed-derived proanthocyanidolic oligomer (PCO) administration. In this placebo-controlled study, male Wistar rats were subjected to PCO administration for 2 weeks, after which they were subjected to a standardised contusion injury. Supplementation was continued after injury. Immune and satellite cell responses were assessed, as well as oxygen radical absorption capacity and muscle regeneration. PCO administration resulted in a rapid satellite cell response with an earlier peak in activation (Pax7⁺, CD56⁺, at 4 h post-contusion) vs. placebo groups (PLA) (P<.001: CD56⁺ on Day 5 and Pax7⁺ on Day 7). Specific immune-cell responses in PLA followed expected time courses (neutrophil elevation on Day 1; sustained macrophage elevation from Days 3 to 5). PCO dramatically decreased neutrophil elevation to nonsignificant, while macrophage responses were normal in extent, but significantly earlier (peak between Days 1 and 3) and completely resolved by Day 5. Anti-inflammatory cytokine, IL-10, increased significantly only in PCO (Day 3). Muscle fiber regeneration (MHC(f) content and central nuclei) started earlier and was complete by Day 14 in PCO, but not in PLA. Thus, responses by three crucial cell types involved in muscle recovery were affected by in vivo administration of a specific purified polyphenol in magnitude (neutrophil), time course (macrophages), or time course and activation state (satellite cell), explaining faster effective regeneration in the presence of proanthocyanidolic oligomers.

Pulmonary contusion primes systemic innate immunity responses.

INTRODUCTION: Traumatic injury may result in an exaggerated response to subsequent immune stimuli such as nosocomial infection. This "second hit" phenomenon and molecular mechanism(s) of immune priming by traumatic lung injury, specifically, pulmonary contusion, remain unknown. We used an animal model of pulmonary contusion to determine whether the injury resulted in priming of the innate immune response and to test the hypothesis that resuscitation fluids could attenuate the primed response to a second hit. METHODS: Male, 8 to 9 weeks, C57/BL6 mice with a pulmonary contusion were challenged by a second hit of intratracheal administration of the Toll-like receptor 4 agonist, lipopolysaccharide (LPS, 50 microg) 24 hours after injury (injury + LPS). Other experimental groups were injury + vehicle or LPS alone. A separate group was injured and resuscitated by 4 cc/kg of hypertonic saline (HTS) or Lactated Ringer's (LR) resuscitation before LPS challenge. Mice were killed 4 hours after LPS challenge and blood, bronchoalveolar lavage, and tissue were isolated and analyzed. Data were analyzed using one-way analysis of variance with Bonferroni multiple comparison posttest for significant differences (*p < or = 0.05). RESULTS: Injury + LPS showed immune priming observed by lung injury histology and increased bronchoalveolar lavage neutrophilia, lung myeloperoxidase and serum IL-6, CXCL1, and MIP-2 levels when compared with injury + vehicle or LPS alone. After injury, resuscitation with HTS, but not Lactated Ringer's was more effective in attenuating the primed response to a second hit. CONCLUSION: Pulmonary contusion primes innate immunity for an exaggerated response to a second hit with the Toll-like receptor 4 agonist, LPS. We observed synergistic increases in inflammatory mediator expression in the blood and a more severe lung injury in injured animals challenged with LPS. This priming effect was reduced when HTS was used to resuscitate the animal after lung contusion.

Pulmonary contusion induces alveolar type 2 epithelial cell apoptosis: role of alveolar macrophages and neutrophils.

Alveolar type 2 (AT-2) cell apoptosis is an important mechanism during lung inflammation, lung injury, and regeneration. Blunt chest trauma has been shown to activate inflammatory cells such as alveolar macrophages (AMs) or neutrophils (polymorphonuclear granulocytes [PMNs]), resulting in an inflammatory response. The present study was performed to determine the capacity of different components/cells of the alveolar compartment (AMs, PMNs, or bronchoalveolar lavage [BAL] fluids) to induce apoptosis in AT-2 cells following blunt chest trauma. To study this, male Sprague-Dawley rats were subjected to either sham procedure or blunt chest trauma induced by a single blast wave. Various time points after injury (6 h to 7 d), the lungs were analyzed by immunohistochemistry, for AT-2 cells, or with antibodies directed against caspase 3, caspase 8, Fas, Fas ligand (FasL), BAX, and BCL-2. Bronchoalveolar lavage concentrations of TNF-alpha, IL-1beta, and soluble FasL were determined by enzyme-linked immunosorbent assay. Furthermore, cultures of AT-2 cells isolated from healthy rats were incubated with supernatants of AMs, PMNs, or BAL fluids obtained from either trauma or sham-operated animals in the presence or absence of oxidative stress. Annexin V staining or TUNEL (terminal deoxynucleotidyl transferase) assay was used to detect apoptotic AT-2 cells. Histological evaluation revealed that the total number of AT-2 cells was significantly reduced at 48 h following trauma. Fas, FasL, active caspase 8, and active caspase 3 were markedly up-regulated in AT-2 cells after chest trauma. BAX and BCL-2 did not show any significant changes between sham and trauma. IL-1beta, but not TNF-alpha, levels were markedly increased at 24 h after the injury, and soluble FasL concentrations were significantly enhanced at 6, 12, 24, and 48 h after the insult. Apoptosis of AT-2 cells incubated with supernatants from cultured AMs, isolated at 48 h following chest trauma was markedly increased when compared with shams. In contrast, no apoptosis was induced in AT-2 cells incubated with supernatants of activated PMNs or BAL fluids of traumatized animals. In summary, blunt chest trauma induced apoptosis in AT-2 cells, possibly involving the extrinsic death receptor pathway. Furthermore, mediators released by AMs appeared to be involved in the induction of AT-2 cell apoptosis.

The extent of myelin pathology differs following contusion and transection spinal cord injury.

Demyelination is a prominent feature of spinal cord injury (SCI) and is followed by incomplete remyelination, which may contribute to physiological impairment. Demyelination has been documented in several species including humans, but the extent of demyelination and its functional consequence remain unknown. In this report, we document and compare the extent of tissue pathology, white matter apoptosis, demyelination, and remyelination 2 months following injury in rat contusion and transection models of SCI. Moreover, we document and compare the macrophage response 3 and 14 days post contusion and transection SCI. Contusion injury resulted in widespread tissue pathology, white matter apoptosis, demyelination, incomplete remyelination, and robust macrophage response extending several millimeters cranial and caudal to the epicenter of injury. In contrast, transection injury resulted in focal tissue pathology with white matter apoptosis, demyelination, incomplete remyelination, and robust macrophage response at the epicenter of injury, and little pathologic features at a distance from the epicenter of injury, as indicated by the lack of apoptosis and demyelination. These data indicate for the first time that myelin pathology differs substantially following contusion and transection SCI.