CGRP sensory neurons promote tissue healing via neutrophils and macrophages.

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4-12. However, how neuro-immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro-immune interactions has potential to treat non-healing tissues in which dysregulated neuro-immune interactions impair tissue healing.

A superior extracellular matrix binding motif to enhance the regenerative activity and safety of therapeutic proteins.

Among therapeutic proteins, cytokines and growth factors have great potential for regenerative medicine applications. However, these molecules have encountered limited clinical success due to low effectiveness and major safety concerns, highlighting the need to develop better approaches that increase efficacy and safety. Promising approaches leverage how the extracellular matrix (ECM) controls the activity of these molecules during tissue healing. Using a protein motif screening strategy, we discovered that amphiregulin possesses an exceptionally strong binding motif for ECM components. We used this motif to confer the pro-regenerative therapeutics platelet-derived growth factor-BB (PDGF-BB) and interleukin-1 receptor antagonist (IL-1Ra) a very high affinity to the ECM. In mouse models, the approach considerably extended tissue retention of the engineered therapeutics and reduced leakage in the circulation. Prolonged retention and minimal systemic diffusion of engineered PDGF-BB abolished the tumour growth-promoting adverse effect that was observed with wild-type PDGF-BB. Moreover, engineered PDGF-BB was substantially more effective at promoting diabetic wound healing and regeneration after volumetric muscle loss, compared to wild-type PDGF-BB. Finally, while local or systemic delivery of wild-type IL-1Ra showed minor effects, intramyocardial delivery of engineered IL-1Ra enhanced cardiac repair after myocardial infarction by limiting cardiomyocyte death and fibrosis. This engineering strategy highlights the key importance of exploiting interactions between ECM and therapeutic proteins for developing effective and safer regenerative therapies.

Restoration of the healing microenvironment in diabetic wounds with matrix-binding IL-1 receptor antagonist.

Chronic wounds are a major clinical problem where wound closure is prevented by pathologic factors, including immune dysregulation. To design efficient immunotherapies, an understanding of the key molecular pathways by which immunity impairs wound healing is needed. Interleukin-1 (IL-1) plays a central role in regulating the immune response to tissue injury through IL-1 receptor (IL-1R1). Generating a knockout mouse model, we demonstrate that the IL-1-IL-1R1 axis delays wound closure in diabetic conditions. We used a protein engineering approach to deliver IL-1 receptor antagonist (IL-1Ra) in a localised and sustained manner through binding extracellular matrix components. We demonstrate that matrix-binding IL-1Ra improves wound healing in diabetic mice by re-establishing a pro-healing microenvironment characterised by lower levels of pro-inflammatory cells, cytokines and senescent fibroblasts, and higher levels of anti-inflammatory cytokines and growth factors. Engineered IL-1Ra has translational potential for chronic wounds and other inflammatory conditions where IL-1R1 signalling should be dampened.

Enhancing the regenerative effectiveness of growth factors by local inhibition of interleukin-1 receptor signaling.

Although growth factors (GFs) are key molecules for regenerative medicine, their use has been limited by issues associated with suboptimal delivery systems and incomplete understanding of their signaling dynamics. Here, we explored how proinflammatory signals affect GF regenerative potential. Using bone regeneration in mouse, we found that the regenerative capacity of two clinically relevant GFs (BMP-2 and PDGF-BB) is impaired by interleukin-1 receptor (IL-1R1). Mechanistically, IL-1R1 activation in bone-forming cells desensitizes them to GFs and accelerates senescence. Moreover, administration of the GFs triggers IL-1 release by macrophages. To provide localized and sustained IL-1R1 inhibition, we engineered IL-1R antagonist (IL-1Ra) to bind the extracellular matrix (ECM) very strongly and demonstrate that codelivering GFs with ECM-binding IL-1Ra induces superior regeneration. Thus, we highlight that GF regenerative activity is hindered by proinflammatory signals, and GF-based therapies should integrate immunomodulation. Particularly, ECM-binding IL-1Ra holds clinical translational potential by enhancing efficacy of GF therapies.

670nm light treatment following retinal injury modulates Müller cell gliosis: Evidence from in vivo and in vitro stress models.

Photobiomodulation (PBM) with 670 nm light has been shown to accelerate wound healing in soft tissue injuries, and also to protect neuronal tissues. However, little data exist on its effects on the non-neuronal components of the retina, such as Müller cells (MCs), which are the principal macroglia of the retina that play a role in maintaining retinal homeostasis. The aim of this study was to explore the effects of 670 nm light on activated MCs using in vivo and in vitro stress models. Adult Sprague-Dawley rats were exposed to photo-oxidative damage (PD) for 24 h and treated with 670 nm light at 0, 3 and 14 days after PD. Tissue was collected at 30 days post-PD for analysis. Using the in vitro scratch model with a human MC line (MIO-M1), area coverage and cellular stress were analysed following treatment with 670 nm light. We showed that early treatment with 670 nm light after PD reduced MC activation, lowering the retinal expression of GFAP and FGF-2. 670 nm light treatment mitigated the production of MC-related pro-inflammatory cytokines (including IL-1ß), and reduced microglia/macrophage (MG/MΦ) recruitment into the outer retina following PD. This subsequently decreased photoreceptor loss, slowing the progression of retinal degeneration. In vitro, we showed that 670 nm light directly modulated MC activation, reducing rates of area coverage by suppressing cellular proliferation and spreading. This study indicates that 670 nm light treatment post-injury may have therapeutic benefit when administered shortly after retinal damage, and could be useful for retinal degenerations where MC gliosis is a feature of disease progression.

Hypoxia-induced intestinal barrier changes in balloon-assisted enteroscopy.

KEY POINTS: Balloon-assisted enteroscopy (BAE) is an emerging standard procedure by utilizing distensible balloons to facilitate deep endoscopy in the small and large intestine. Sporadic cases of bacteraemia were found after BAE. Balloon distension by BAE caused gut tissue hypoxia. The impact of balloon distension-induced hypoxia on intestinal barriers remains unclear. Murine models of BAE by colonic balloon distension showed that short- and long-term hypoxia evoked opposite effects on epithelial tight junctions (TJs). Short-term hypoxia fortified TJ integrity, whereas long-term hypoxia caused damage to barrier function. Our data showed for the first time the molecular mechanisms and signalling pathways of epithelial barrier fortification and TJ reorganization by short-term hypoxia for the maintenance of gut homeostasis. The findings suggest avoiding prolonged balloon distension during BAE to reduce the risk of hypoxia-induced gut barrier dysfunction. ABSTRACT: Balloon-assisted enteroscopy (BAE) is an emerging standard procedure that uses distensible balloons to facilitate deep endoscopy. Intestines are known to harbour an abundant microflora. Whether balloon distension causes perturbation of blood flow and gut barrier dysfunction, and elicits risk of bacterial translocation remains unknown. Our aims were to (1) conduct a prospective study to gather microbiological and molecular evidence of bacterial translocation by BAE in patients, (2) establish a murine model of colonic balloon distension to investigate tissue hypoxia and intestinal barrier, and (3) assess the effect of short- and long-term hypoxia on epithelial permeability using cell lines. Thirteen patients were enrolled for BAE procedures, and blood samples were obtained before and after BAE for paired comparison. Four of the 13 patients (30.8%) had positive bacterial DNA in blood after BAE. Post-BAE endotoxaemia was higher than the pre-BAE level. Nevertheless, no clinical symptom of sepsis or fever was reported. To mimic clinical BAE, mice were subjected to colonic balloon distension. Local tissue hypoxia was observed during balloon inflation, and reoxygenation after deflation. A trend of increased gut permeability was seen after long-term distension, whereas a significant reduction of permeability was observed by short-term distension in the proximal colon. Human colonic epithelial Caco-2 cells exposed to hypoxia for 5-20 min exhibited increased tight junctional assembly, while those exposed to longer hypoxia displayed barrier disruption. In conclusion, sporadic cases of bacteraemia were found after BAE, without septic symptoms. Short-term hypoxia by balloon distension yielded a protective effect whereas long-term hypoxia caused damage to the gut barrier.

Photobiomodulation with 670 nm light ameliorates Müller cell-mediated activation of microglia and macrophages in retinal degeneration.

Müller cells, the supporting cells of the retina, play a key role in responding to retinal stress by releasing chemokines, including CCL2, to recruit microglia and macrophages (MG/MΦ) into the damaged retina. Photobiomodulation (PBM) with 670 nm light has been shown to reduce inflammation in models of retinal degeneration. In this study, we aimed to investigate whether 670 nm light had an effect on Müller cell-initiated inflammation under retinal photo-oxidative damage (PD) in vivo and in vitro. Sprague-Dawley rats were pre-treated with 670 nm light (9J/cm2) once daily over 5 days prior to PD. The expression of inflammatory genes including CCL2 and IL-1ß was analysed in retinas. In vitro, primary Müller cells dissociated from neonatal rat retinas were co-cultured with 661W photoreceptor cells. Co-cultures were exposed to PD, followed by 670 nm light treatment to the Müller cells only, and Müller cell stress and inflammation were assessed. Primary MG/MΦ were incubated with supernatant from the co-cultures, and collected for analysis of inflammatory activation. To further understand the mechanism of 670 nm light, the expression of COX5a and mitochondrial membrane potential (ΔΨm) were measured in Müller cells. Following PD, 670 nm light-treated Müller cells had a reduced inflammatory activation, with lower levels of CCL2, IL-1ß and IL-6. Supernatant from 670 nm light-treated co-cultures reduced activation of primary MG/MΦ, and lowered the expression of pro-inflammatory cytokines, compared to untreated PD controls. Additionally, 670 nm light-treated Müller cells had an increased expression of COX5a and an elevated ΔΨm following PD, suggesting that retrograde signaling plays a role in the effects of 670 nm light on Müller cell gene expression. Our data indicates that 670 nm light reduces Müller cell-mediated retinal inflammation, and offers a potential cellular mechanism for 670 nm light therapy in regulating inflammation associated with retinal degenerations.

Tumor Necrosis Factor α-Dependent Neutrophil Priming Prevents Intestinal Ischemia/Reperfusion-Induced Bacterial Translocation.

BACKGROUND: Intestinal ischemia/reperfusion (I/R) causes barrier impairment and bacterial influx. Protection against I/R injury in sterile organs by hypoxic preconditioning (HPC) had been attributed to erythropoietic and angiogenic responses. Our previous study showed attenuation of intestinal I/R injury by HPC for 21 days in a neutrophil-dependent manner. AIM: To investigate the underlying mechanisms of neutrophil priming by HPC, and explore whether adoptive transfer of primed neutrophils is sufficient to ameliorate intestinal I/R injury. METHODS: Rats raised in normoxia (NM) and HPC for 3 or 7 days were subjected to sham operation or superior mesenteric artery occlusion for I/R challenge. Neutrophils isolated from rats raised in NM or HPC for 21 days were intravenously injected into naïve controls prior to I/R. RESULTS: Similar to the protective effect of HPC-21d, I/R-induced mucosal damage was attenuated by HPC-7d but not by HPC-3d. Naïve rats reconstituted with neutrophils of HPC-21d rats showed increase in intestinal phagocytic infiltration and myeloperoxidase activity, and barrier protection against I/R insult. Elevated free radical production, and higher bactericidal and phagocytic activity were observed in HPC neutrophils compared to NM controls. Moreover, increased serum levels of tumor necrosis factor α (TNFα) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were seen in HPC rats. Naïve neutrophils incubated with HPC serum or recombinant TNFα, but not CINC-1, exhibited heightened respiratory burst and bactericidal activity. Lastly, neutrophil priming effect was abolished by neutralization of TNFα in HPC serum. CONCLUSIONS: TNFα-primed neutrophils by HPC act as effectors cells for enhancing barrier integrity under gut ischemia.

The Role of Pyruvate in Protecting 661W Photoreceptor-Like Cells Against Light-Induced Cell Death.

PURPOSE: Light is a requirement for the function of photoreceptors in visual processing. However, prolonged light exposure can be toxic to photoreceptors, leading to increased reactive oxygen species (ROS), lipid peroxidation, and photoreceptor cell death. We used the 661W mouse cone photoreceptor-like cell line to study the effects of pyruvate in protecting these cells from light-induced toxicity. METHODS: 661W cells were exposed to 15,000 lux continuous bright light for 5 hours and incubated in Dulbecco's modified eagle medium (DMEM) with various concentrations of pyruvate. Following light damage, cells were assessed for changes in morphology, cell toxicity, viability, and ROS production. Mitochondrial respiration and anaerobic glycolysis were also assessed using a Seahorse Xfe96 extracellular flux analyzer. RESULTS: We found that cell death caused by light damage in 661W cells was dramatically reduced in the presence of pyruvate. Cells with pyruvate-supplemented media also showed attenuation of oxidative stress and maintained normal levels of ATP. We also found that alterations in the concentrations of pyruvate had no effect on mitochondrial respiration or glycolysis in light-damaged cells. CONCLUSIONS: Taken together, the results show that pyruvate is protective against light damage but does not alter the metabolic output of the cells, indicating an alternative role for pyruvate in reducing oxidative stress. Thus, sodium pyruvate is a possible candidate for the treatment against the oxidative stress component of retinal degenerations.

Persistent gut barrier damage and commensal bacterial influx following eradication of Giardia infection in mice.

BACKGROUND: Recent studies of Giardia lamblia outbreaks have indicated that 40-80% of infected patients experience long-lasting functional gastrointestinal disorders after parasitic clearance. Our aim was to assess changes in the intestinal barrier and spatial distribution of commensal bacteria in the post-clearance phase of Giardia infection. METHODS: Mice were orogastrically inoculated with G. lamblia trophozoites (strain GS/M) or pair-fed with saline and were sacrificed on post-infective (PI) days 7 (colonization phase) and 35 (post-clearance phase). Gut epithelial barrier function was assessed by Western blotting for occludin cleavage and luminal-to-serosal macromolecular permeability. Gut-associated, superficial adherent, and mucosal endocytosed bacteria were measured by agar culturing and were examined by fluorescence in situ hybridization. Intracellular bacteria cultured from isolated mucosal cells were characterized by 16S rDNA sequencing. Neutrophil-specific esterase staining, a myeloperoxidase activity assay, and enzyme-linked immunosorbent assays for cytokine concentrations were used to verify intestinal tissue inflammation. RESULTS: Tight junctional damage was detected in the intestinal mucosa of Giardia-infected mice on PI days 7 and 35. Although intestinal bacterial overgrowth was evident only during parasite colonization (PI day 7), enhanced mucosal adherence and endocytosis of bacteria were observed on PI days 7 and 35. Multiple bacterial strains, including Bacillus, Lactobacillus, Staphylococcus, and Phenylobacterium, penetrated the gut mucosa in the post-infective phase. The mucosal influx of bacteria coincided with increases in neutrophil infiltration and myeloperoxidase activity on PI days 7 and 35. Elevated intestinal IFNγ, TNFα, and IL-1ß levels also were detected on PI day 35. CONCLUSIONS: Giardia-infected mice showed persistent tight junctional damage and bacterial penetration, accompanied by mucosal inflammation, after parasite clearance. These novel findings suggest that the host's unresolved immune reactions toward its own microbiota, due to an impaired epithelial barrier, may partly contribute to the development of post-infective gut disorders.

Neutrophil priming by hypoxic preconditioning protects against epithelial barrier damage and enteric bacterial translocation in intestinal ischemia/reperfusion.

Intestinal ischemia/reperfusion (I/R) induces mucosal barrier dysfunction and bacterial translocation (BT). Neutrophil-derived oxidative free radicals have been incriminated in the pathogenesis of ischemic injury in various organs, but their role in the bacteria-containing intestinal tract is debatable. Primed neutrophils are characterized by a faster and higher respiratory burst activity associated with more robust bactericidal effects on exposure to a second stimulus. Hypoxic preconditioning (HPC) attenuates ischemic injury in brain, heart, lung and kidney; no reports were found in the gut. Our aim is to investigate whether neutrophil priming by HPC protects against intestinal I/R-induced barrier damage and bacterial influx. Rats were raised in normoxia (NM) or kept in a hypobaric hypoxic chamber (380 Torr) 17 h/day for 3 weeks for HPC, followed by sham operation or intestinal I/R. Gut permeability was determined by using an ex vivo macromolecular flux assay and an in vivo magnetic resonance imaging-based method. Liver and spleen homogenates were plated for bacterial culturing. Rats raised in HPC showed diminished levels of BT, and partially improved mucosal histopathology and epithelial barrier function compared with the NM groups after intestinal I/R. Augmented cytokine-induced neutrophil chemoattractant (CINC)-1 and -3 levels and myeloperoxidase activity correlated with enhanced infiltration of neutrophils in intestines of HPC-I/R compared with NM-I/R rats. HPC alone caused blood neutrophil priming, as shown by elevated production of superoxide and hydrogen peroxide on stimulation, increased membrane translocation of cytosolic p47(phox) and p67(phox), as well as augmented bacterial-killing and phagocytotic activities. Neutrophil depletion reversed the mucosal protection by HPC, and aggravated intestinal leakiness and BT following I/R. In conclusion, neutrophil priming by HPC protects against I/R-induced BT via direct antimicrobial activity by oxidative respiratory bursts and through promotion of epithelial barrier integrity for luminal confinement of enteric bacteria.

Epithelial inducible nitric oxide synthase causes bacterial translocation by impairment of enterocytic tight junctions via intracellular signals of Rho-associated kinase and protein kinase C zeta.

OBJECTIVE: Gut barrier dysfunction and bacterial translocation occur in various disorders, including intestinal obstruction. Overexpression of inducible nitric oxide synthase is implicated in the pathogenesis of bacterial translocation, of which the molecular mechanism remains unclear. Epithelial permeability is regulated by tight junction reorganization and myosin light chain phosphorylation. Our aim was to investigate the roles of Rho-associated kinase and protein kinase C ζ in epithelial nitric oxide synthase-mediated barrier damage. DESIGN: Animal study and cell cultures. SETTING: Research laboratory. SUBJECTS: BALB/c mice. INTERVENTIONS: : Mouse distal small intestine was obstructed in vivo by a 10-cm loop ligation in which vehicle, L-Nil (a nitric oxide synthase inhibitor), or Y27632 (a Rho-associated kinase inhibitor) was luminally administered. After obstruction for 24 hrs, intestinal tissues were mounted on Ussing chambers for macromolecular flux. Liver and spleen tissues were assessed for bacterial counts. Caco-2 cells were exposed to 1 mM S-nitroso-N-acetylpenicillamine (a nitric oxide donor) for 24 hrs, and transepithelial resistance and permeability were evaluated. MEASUREMENTS AND MAIN RESULTS: Mice with intestinal obstruction displayed epithelial barrier dysfunctions, such as permeability rise and bacterial translocation, associated with tight junction disruption and myosin light chain phosphorylation. Increased inducible nitric oxide synthase and phosphorylated protein kinase C ζ were observed in villus epithelium. Enteric instillation of L-Nil and Y27632 attenuated the functional and structural barrier damage caused by intestinal obstruction. L-Nil decreased intestinal obstruction-induced myosin light chain, myosin phosphatase target subunit 1, and protein kinase C ζ phosphorylation, suggesting that inducible nitric oxide synthase is upstream of Rho-associated kinase and protein kinase C ζ signaling. The intestinal phosphorylated myosin light chain level did not increase in inducible nitric oxide synthase(-/-) mice following intestinal obstruction. In vitro studies showed that S-nitroso-N-acetylpenicillamine-induced transepithelial resistance drop and permeability rise was independent of cell apoptosis. Y27632 inhibited S-nitroso-N-acetylpenicillamine-induced myosin light chain phosphorylation and permeability rise. S-nitroso-N-acetylpenicillamine also triggered phosphorylation and membrane translocation of protein kinase C ζ. Inhibitory protein kinase C ζ pseudosubstrate blocked S-nitroso-N-acetylpenicillamine-induced tight junction reorganization, but not myosin light chain phosphorylation. CONCLUSIONS: Epithelial inducible nitric oxide synthase activates two distinct signals, protein kinase C ζ and Rho-associated kinase, to disrupt tight junctions leading to bacterial influx.

Anti-apoptotic PI3K/Akt signaling by sodium/glucose transporter 1 reduces epithelial barrier damage and bacterial translocation in intestinal ischemia.

Intestinal ischemia/reperfusion (I/R) causes mucosal barrier damage and bacterial translocation (BT), leading to septic complications. Previous in vitro studies showed that activation of sodium/glucose transporter 1 (SGLT1) prevented the epithelial apoptosis and permeability rise induced by microbial products. Our aim was to investigate whether luminal glucose uptake by SGLT1 protects against ischemia-induced epithelial cell death and barrier dysfunction, and to explore the glucose-mediated cellular survival pathways in vivo. Rat jejunum was luminally instilled with either vehicle, a pancaspase inhibitor ZVAD, or glucose prior to I/R challenge (occlusion of the superior mesenteric artery for 20 min and reperfusion for 60 min). Histopathology and apoptosis in the jejunum were examined by TUNEL staining and caspase-3 cleavage. Intestinal permeability was evaluated using in vivo assays measuring luminal-to-blood passage of fluorescein-dextran and portal drainage of enterally administered gadodiamide by magnetic resonance imaging. BT was determined by culturing liver and spleen homogenates. Immunofluorescent analysis and kinase assay were used to study PI3K/Akt signaling pathways. Intestinal I/R caused enterocyte apoptosis and villous destruction. Intestinal infusion with ZVAD decreased the I/R-triggered gut permeability rise and BT, suggesting that the barrier damage was partly dependent on cell apoptosis. Enteral instillation of glucose attenuated the epithelial apoptosis, barrier damage, and mucosal inflammation caused by I/R. Phloridzin (a SGLT1 inhibitor) reduced the protective effect of glucose in a dose-dependent manner. Enteral glucose increased the mucosal Akt kinase activity as evidenced by the augmented phosphorylation of exogenous GSK3. Enhanced membrane translocation and phosphorylation of Akt in epithelial cells were associated with elevated phosphorylation of mTOR, Bad, and FoxO1/3a following glucose uptake. Inhibition of PI3K/Akt signaling by LY294002 and wortmannin partially blocked the glucose-mediated rescue of cell apoptosis and barrier damage. In conclusion, SGLT1 glucose uptake alleviated I/R-induced barrier dysfunction and BT, partly by inhibiting epithelial apoptosis via activation of PI3K/Akt signaling.

Role of myosin light chain kinase in intestinal epithelial barrier defects in a rat model of bowel obstruction.

BACKGROUND: Bowel obstruction is a common cause of abdominal emergency, since the patients are at increased risk of septicemia resulting in high mortality rate. While the compartmentalized changes in enteric microfloral population and augmentation of bacterial translocation (BT) have already been reported using experimental obstruction models, alterations in epithelial permeability of the obstructed guts has not been studied in detail. Myosin light chain kinase (MLCK) is actively involved in the contraction of epithelial perijunctional actinomyosin ring and thereby increases paracellular permeability. In the current study we attempt to investigate the role of MLCK in epithelial barrier defects using a rat model of simple mechanical obstruction. METHODS: Wistar rats received intraperitoneal injection of ML-7 (a MLCK inhibitor) or vehicle at 24, 12 and 1 hrs before and 12 hrs after intestinal obstruction (IO). The distal small intestine was obstructed with a single ligature placed 10 cm proximal to the ileocecal junction in IO rats for 24 hrs. Sham-operated rats served as controls. RESULTS: Mucosal injury, such as villous blunting and increased crypt/villus ratio, was observed in the distal small intestine of IO rats. Despite massive enterocyte shedding, intestinal villi were covered with a contiguous epithelial layer without cell apoptosis. Increased transmural macromolecular flux was noticed in the distal small intestine and the proximal colon after IO. The bacterial colony forming units in the spleen and liver of IO rats were significantly higher than those of sham controls. Addition of ML-7 ameliorated the IO-triggered epithelial MLC phosphorylation, mucosal injury and macromolecular flux, but not the level of BT. CONCLUSIONS: The results suggest that IO-induced premature enterocytic sloughing and enhanced paracellular antigenic flux were mediated by epithelial MLCK activation. In addition, enteric bacteria may undergo transcytotic routes other than paracellular paths to cross the epithelium.

Magnetic resonance imaging detects intestinal barrier dysfunction in a rat model of acute mesenteric ischemia/reperfusion injury.

OBJECTIVES: To develop an in vivo intestinal permeability assay applying magnetic resonance imaging (MRI) to monitor real-time gut barrier defects in animal models of acute mesenteric ischemia/reperfusion (I/R) insult. MATERIALS AND METHODS: Twenty Wistar rats were divided to 2 groups for I/R challenge or sham controls. I/R rats received occlusion of superior mesenteric artery for 20 minutes and reperfusion for 1 hour. Sham-operation controls received laparotomy without manipulation of artery. To assess gut permeability, a 10-cm jejunal sac was created distal to the ligament of Treitz in both groups of rats after laparotomy, and a contrast agent (gadodiamide) was injected into the lumen of the ligated intestinal sac. The signals produced by gadodamide in the liver, kidney, and plasma before and after the start of reperfusion were examined by 1.5 Tesla MRI (GE Signa Excite), and the increment of signal-to-noise ratio (SNR) in these organs that parallels the luminal-to-serosal flux rate of the probe was used as an indicator of gut permeability. At the end of procedures, jejunal tissues and mucosal scrapings were collected for histologic examination and Western blotting for epithelial tight junctional proteins. Moreover, liver and spleen homogenates were cultured on fresh blood agar plates to measure the bacterial colony-forming units per gram of tissue. RESULTS: In I/R rats, disrupted villous structure and decreased epithelial tight junctional expression were seen in the jejunum associated with massive enteric bacterial translocation to the liver and spleen. The SNR in the liver of I/R rats was higher than sham controls (2.65 +/- 0.56 vs. 0.65 +/- 0.26, P < 0.01) at 15 minutes postreperfusion. Elevation of SNR in the kidney was also found in I/R rats compared with sham controls (11.61 +/- 2.07 vs. 3.06 +/- 1.15, P < 0.05). The plasma gadodiamide concentration in I/R rats was significantly increased compared with sham controls (0.220 +/- 0.044 vs. 0.006 +/- 0.004 mM, P < 0.01) at 15 minutes postreperfusion. CONCLUSIONS: This novel MRI-based intestinal permeability assay has shown a significant increase in the signal intensity in liver, kidney, and plasma samples that correlated with mucosal barrier defects in experimental models of acute mesenteric I/R.

Protection of ischemic brain cells is dependent on astrocyte-derived growth factors and their receptors.

An in vitro ischemia model (oxygen, glucose, and serum deprivation) is used to investigate the possible cellular and molecular mechanisms responsible for cerebral ischemia. We have previously demonstrated that supernatants derived from ischemic microglia can protect ischemic brain cells by releasing GDNF and TGF-beta1. In the present study, we investigate whether products of ischemic astrocytes can also protect ischemic microglia, astrocytes, and neurons in a similar manner. Supernatants from ischemic astrocytes were collected after various periods of ischemia and incubated with microglia, astrocytes, or neurons individually, under in vitro ischemic conditions. The components responsible for the protective effects of astrocyte-derived supernatants were then identified by Western blot, ELISA, trypan blue dye exclusion, and immunoblocking assays. Results showed that under conditions of in vitro ischemia the number of surviving microglia, astrocytes, and neurons was significantly increased by the incorporation of the astrocyte-derived supernatants. Astrocyte supernatant-mediated protection of ischemic microglia was dependent on TGF-beta1 and NT-3, ischemic astrocytes were protected by GDNF, and ischemic neurons were protected by NT-3. In addition, protein expression of TGF-beta1 and NT-3 receptors in microglia, GDNF receptors in astrocytes, and NT-3 receptors in neurons was increased by in vitro ischemia. These results suggest that astrocyte-derived protection of ischemic brain cells is dependent not only on factors released from the ischemic astrocytes, but also on the type of receptor present on the responding cells. Therapeutic potential of TGF-beta1, GDNF, and NT-3 in the control of cerebral ischemia is further suggested.

Bax-regulated mitochondria-mediated apoptosis is responsible for the in vitro ischemia induced neuronal cell death of Sprague Dawley rat.

An in vitro ischemia model was used to determine the molecular mechanisms responsible for the ischemia-induced neuronal cell death. Additionally, the neuronal protective mechanisms of anti-apoptotic drugs against ischemia were also evaluated. In this study, the primary neuronal cultures were incubated in an anoxic chamber with 95% of N2 and 5% of CO2 for various times. The death rate, degree of the apoptotic damage, reduction of mitochondrial membrane potential, translocation of Bax, release of cytochrome C and activation of caspase-9 and -3 were determined at each time point. Results showed that a Bax-regulated mitochondria- mediated apoptosis is responsible for the in vitro ischemia-induced neuronal death. Reduction in mitochondrial membrane potential plays no role in triggering this apoptosis. Furthermore, the anti-apoptotic drugs: furosemide (a Bax blocker) and ZVAD-fmk (caspase inhibitor) but not cyclosporine A (a MPT pore blocker), significantly protected the neurons against ischemia-induced damage. This provides an additional consideration in the future selection of new anti-ischemic drugs.

Molecular mechanisms responsible for microglia-derived protection of Sprague-Dawley rat brain cells during in vitro ischemia.

Microglia-derived protection of brain cells (microglia, astrocytes, and neurons) during in vitro ischemic stress (deprivation of glucose, oxygen, and serum) was determined. Trypan blue exclusion assay, immunoblocking assay, Western blot analysis, and ELISA assay were used to determine the molecular mechanisms responsible for the microglia-derived protection. Results demonstrated that supernatants from the ischemic microglia protected all three cell-types from ischemia-induced damage by releasing the transforming growth factor-beta1 (TGF-beta1) and glial cell line-derived neurotrophic factor (GDNF). The protection of microglia was TGF-beta1 related, whereas astrocytes protection was GDNF-dependent. The protection of neurons was TGF-beta1 and GDNF independent, and the molecular nature responsible for their protection remains to be determined. These results indicate contribution from the surrounding cells and the types of receptors expressed on different brain cells probably also play an important role in determining their fate against ischemia.