Mouthwashes: Implications for Practice.

This is the concluding article in the supplement on the role of mouthwashes in oral care, which summarises the current guidelines across the globe regarding their acceptable adjunctive use for managing caries, gingivitis, and periodontal disease. Based on moderate evidence for clinical effectiveness, most current guidelines suggest fluoride mouthwashes for the management of dental caries, and chlorhexidine for the management of periodontal diseases. However there still appears to be gaps in the literature underpinning these recommendations. Importantly, all evidence supports such mouthwash use "adjunctively," alongside mechanical oral hygiene measures. Other antimicrobial mouthwashes such as essential oils and cetylpyridinium chloride may also be clinically effective against plaque and gingivitis, but there is a current lack of robust evidence of natural mouthwashes to recommend their adjunctive use. The authors of the current review are of the view that mouthwashes may not be of much value in those with good periodontal health or low caries risk. The reasons for this are, the potential i) risks of allergic reactions, ii) dysbiosis of the oral microbiota, iii) emergence of antimicrobial resistance, and iv) deleterious effects on the environment. There is, however, much empirical research needed on mouthwashes, particularly in vivo research derived through clinical trials. Thus, dental practitioners need to keep abreast of the evidence base on the current, and the emerging, over-the-counter mouthwashes, and pay heed to the consensus views emanating from systematic reviews, as well as international guidelines on mouthwashes.

Blended learning in undergraduate dental education: a global pilot study.

AIMS: To explore the global trends in blended learning in undergraduate dental education during the COVID pandemic and during the recovery phase by engaging with the students and faculty and evaluate the implications for dental education in the post-COVID era. METHODS: It was a pilot cross-sectional study which employed a convenience sampling technique to recruit representatives of dental faculty and undergraduate students in 80 dental institutions globally. A previously validated questionnaire consisting of a combination of closed and open-ended items was used for data collection. Responses to these online questionnaires were processed and analysed using the R statistical computing environment. RESULTS: A total of 320 dental students and 169 faculty members from 47 different dental institutions participated in the study. Video and Live Online Tutorials were considered to be the most effective method of online learning followed by online question banks by both groups. Significant differences were noted between faculty and students regarding time spent and effectiveness of online teaching and learning, respectively, both before and after the start of COVID. The results highlight the faculty need to engage more closely with the students to address their learning needs. Finally, the participants provided several recommendations regarding the future development of teaching and learning strategies as well as assessments in the post-pandemic era. CONCLUSIONS: This is the first study which explores blended learning in dental education with participants from multiple institutions in different regions of the globe. Compared to the faculty, students considered online learning to be less interactive and preferred learning activities and all assessments to be delivered face-to-face. The results underscore the need to adapt teaching practices to suit the learning needs of the students.

Effects of chlorhexidine mouthwash on the oral microbiome.

INTRODUCTION/OBJECTIVES: Chlorhexidine (CHX) is a commonly used mouthwash with potent anti-microbial effects useful for the management of oral disease. However, we are moving away from the view of simply 'killing' bacteria, towards managing oral microbial ecosystems (oral microbiome), as an integrated system, to promote oral and systemic health. Here, we aimed to review the effects of CHX mouthwash on the balance of microbial communities in the mouth in vivo in oral health and disease. SOURCES AND STUDY SECTION: The hierarchy of evidence was applied, with systematic reviews and randomised controlled trials consulted where available and case controlled studies being described thereafter. Search terms for each subject category were entered into MEDLINE, PubMed, Google Scholar and the Cochrane database. Focussing on metagenomics studies provides unique overview of the oral microbiome as an integrated system. DATA: Evidence was limited, but several next generation sequencing case-controlled studies suggested that in an integrated system, CHX may cause a shift towards lower bacterial diversity and abundance, in particular nitrate-reducing bacteria in vivo. CHX also appeared to alter salivary pH, lactate, nitrate and nitrite concentrations in saliva. Evidence regarding the effects of CHX on the oral microbiome during oral disease is still emerging. CONCLUSIONS: CHX alters the composition the oral microbiome. However, as CHX use remains widespread in dentistry to manage oral disease, urgent research using metagenomics studies of microbial communities in vivo are still needed to determine CHX mouthwash is 'good', 'bad' or otherwise for bacteria, in the context of oral and systemic health.

Current uses of chlorhexidine for management of oral disease: a narrative review.

OBJECTIVES: Chlorhexidine (CHX) is a commonly used antiseptic mouthwash, used by dental practitioners and the public, due to its antimicrobial effects. The aim of this article was to provide a narrative review of current antimicrobial uses of CHX relevant to dentistry in the context of oral diseases, highlighting need for further studies to support its safe and appropriate use. STUDY SELECTION, DATA AND SOURCES: Randomised controlled trials, systematic reviews and national (UK and US) guidelines were consulted where available, with search terms for each subject category entered into MEDLINE, PubMed, Google Scholar and the Cochrane database. RESULTS: Some evidence existed to support adjunctive short-term use of CHX to manage dental plaque, and reduce clinical symptoms of gingivitis, dry socket, as well as reduce aerosolisation of bacteria. However, use must be weighed alongside the less desirable effects of CHX, including extrinsic staining of teeth, antimicrobial resistance to antiseptic agents and the rare, but fatal, allergic reactions to CHX. Conversely, evidence for the effectiveness of chlorhexidine to manage or prevent periodontitis, dental caries, necrotising periodontal diseases, peri-implantitis, and infections associated with extraction and aerosolised viruses remains less certain. CONCLUSIONS: The use of CHX in dentistry and oral healthcare continues to be widespread and thus it is important that dental practitioners understand that, based on its differential mechanisms of action on different microbes, appropriate clinical and dental use of CHX should be oral disease specific. However, further scientific and clinical research is required before full recommendations can be made.

Effects of Chlorhexidine mouthwash on the oral microbiome.

Following a single blind, cross-over and non-randomized design we investigated the effect of 7-day use of chlorhexidine (CHX) mouthwash on the salivary microbiome as well as several saliva and plasma biomarkers in 36 healthy individuals. They rinsed their mouth (for 1 min) twice a day for seven days with a placebo mouthwash and then repeated this protocol with CHX mouthwash for a further seven days. Saliva and blood samples were taken at the end of each treatment to analyse the abundance and diversity of oral bacteria, and pH, lactate, glucose, nitrate and nitrite concentrations. CHX significantly increased the abundance of Firmicutes and Proteobacteria, and reduced the content of Bacteroidetes, TM7, SR1 and Fusobacteria. This shift was associated with a significant decrease in saliva pH and buffering capacity, accompanied by an increase in saliva lactate and glucose levels. Lower saliva and plasma nitrite concentrations were found after using CHX, followed by a trend of increased systolic blood pressure. Overall, this study demonstrates that mouthwash containing CHX is associated with a major shift in the salivary microbiome, leading to more acidic conditions and lower nitrite availability in healthy individuals.

Effect of constant versus variable small-group facilitators on students' basic science knowledge in an enquiry-based dental curriculum.

INTRODUCTION: The role of small-group facilitators is of pivotal importance for the success of curricula based on active learning. Disorganised tutorial processes and superficial study of the problem have been identified as main hindering factors for students' learning. The aim of this study was to evaluate the influence of consistency of facilitation on students' performance in knowledge-based basic science assessments in a hybrid, enquiry-based (EBL) undergraduate dental curriculum. MATERIALS AND METHODS: This was a retrospective study of 519 first- and second-year undergraduate dental students, enrolled at Peninsula Dental School between 2013 and 2018. Twice in each academic year, students sat a 60-item single-best-answer, multiple-choice examination. Percentage and Z-scores were compared between students whose EBL groups had the same facilitator throughout the academic year, and those whose EBL group was facilitated by different members of staff. All EBL facilitators were dentally qualified but with different levels of expertise in basic dental sciences, prior EBL facilitation, involvement in the curriculum design and university affiliation. RESULTS: No statistically significant difference was observed in the percentage or Z-scores of students whose EBL sessions were supported by consistent or variable facilitators in any of the 18 MCQ tests. Z-scores of first-year students were more variable than for second-year students. In addition, pairwise comparisons revealed no statistically significant differences in students' Z-scores between any of the permanent facilitators' groups. CONCLUSIONS: The results of our study may influence the design and delivery of enquiry-based curricula as well as human resources management by shifting the focus from maintaining facilitator consistency to ensuring comparable training and approaches across facilitators.

Atorvastatin reduces endotoxin-induced microvascular inflammation via NOSII.

In a lipopolysaccharide (LPS)-induced rat model of sepsis (endotoxaemia), we previously demonstrated that pravastatin reduced microvascular inflammation via increased endothelial nitric oxide synthase III (NOSIII). This study aimed to determine whether atorvastatin, the most commonly used statin for lowering cholesterol, exerted beneficial pleiotropic effects via a similar mechanism. The mesenteric microcirculation of anaesthetised male Wistar rats (308 ± 63 g, n = 54) was prepared for fluorescent intravital microscopy. Over 4 h, animals received intravenous (i.v.) administration of either saline, LPS (150 µg kg(-1) h(-1)) or LPS + atorvastatin (200 µg kg(-1) s.c., 18 and 3 h before LPS), with/without the non-specific NOS inhibitor L-NG-Nitroarginine Methyl Ester (L-NAME) (10 µg kg(-1) h(-1)) or NOSII-specific inhibitor 1400 W (20 µg kg(-1) min(-1)). LPS decreased mean arterial blood pressure (MAP) (4 h, control 113 ± 20 mmHg; LPS 70 ± 23 mmHg), being reversed by atorvastatin (105 ± 3 mmHg) (p < 0.05). LPS also increased macromolecular leak measured after 100 mg kg(-1) of i.v FITC-BSA (arbitrary grey level adjacent to venules), which again was attenuated by atorvastatin (control 1.9 ± 4.0; LPS 12.0 ± 2.4; LPS + atorvastatin 4.5 ± 2.2) (p < 0.05). Furthermore, immunohistochemistry identified that atorvastatin decreased LPS-induced upregulation of endothelial cell NOSII expression, but NOSIII was unchanged in all groups. Atorvastatin improved MAP and reduced microvascular inflammation during endotoxaemia, associated with a reduction of pro-inflammatory NOSII. This differs from previous studies, whereby pravastatin increased expression of NOSIII. Thus preoperative statins have beneficial anti-inflammatory effects during endotoxaemia, but careful consideration must be given to the specific statin being used.

Angiopoietin-1 regulates microvascular reactivity and protects the microcirculation during acute endothelial dysfunction: role of eNOS and VE-cadherin.

The growth factor angiopoietin-1 (Ang-1) plays an essential role in angiogenesis and vascular homeostasis. Nevertheless, the role of Ang-1 in regulating vascular tone and blood flow is largely unexplored. Endothelial nitric oxide synthase (eNOS) and the junctional protein VE-cadherin are part of the complex signalling cascade initiated by Ang-1 in endothelial cells. In this study, we aimed to investigate the mechanisms underlying acute effects of Ang-1 on microvascular reactivity, permeability and blood flow, and hypothesise that eNOS and VE-cadherin underpin Ang-1 mediated vascular effects that are independent of angiogenesis and proliferation. Myography of isolated microarterioles from male C3H/HeN mice (7-10 weeks) was employed to measure vascular reactivity in vitro. Microcirculatory function in vivo was evaluated by intravital microscopy and Doppler fluximetry in dorsal window chambers. Ang-1 and its stable variant MAT.Ang-1 induced a concentration-dependent vasodilation of arterioles in vitro, which was blocked with nitric oxide (NO) synthesis inhibitor l-NAME. In vivo, MAT.Ang-1 restored to control levels l-NAME induced peripheral vasoconstriction, decreased blood flow and microvascular hyperpermeability. Tissue protein expression of VE-cadherin was reduced by NOS inhibition and restored to control levels by MAT.Ang-1, whilst VE-cadherin phosphorylation was increased by l-NAME and subsequently reduced by MAT.Ang-1 administration. Moreover, MAT.Ang-1 alone did not modulate systemic levels of angiogenetic factors. Our novel findings report that Ang-1 induces arteriolar vasodilation via release of NO, suggesting that Ang-1 is an important regulator of microvascular tone. As MAT.Ang-1 ameliorates detrimental effects on the microcirculation induced by inhibition of NO synthesis and stabilizes the endothelial barrier function through VE-cadherin, we propose that this Ang-1 variant may serve as a novel therapeutic agent to protect the microcirculation against endothelial dysfunction.

The nociceptin/orphanin FQ receptor antagonist UFP-101 reduces microvascular inflammation to lipopolysaccharide in vivo.

Microvascular inflammation occurs during sepsis and the endogenous opioid-like peptide nociceptin/orphanin FQ (N/OFQ) is known to regulate inflammation. This study aimed to determine the inflammatory role of N/OFQ and its receptor NOP (ORL1) within the microcirculation, along with anti-inflammatory effects of the NOP antagonist UFP-101 (University of Ferrara Peptide-101) in an animal model of sepsis (endotoxemia). Male Wistar rats (220 to 300 g) were administered lipopolysaccharide (LPS) for 24 h (-24 h, 1 mg kg(-1); -2 h, 1 mg kg(-1) i.v., tail vein). They were then either anesthetised for observation of the mesenteric microcirculation using fluorescent in vivo microscopy, or isolated arterioles (~200 µm) were studied in vitro with pressure myography. 200 nM kg(-1) fluorescently labelled N/OFQ (FITC-N/OFQ, i.a., mesenteric artery) bound to specific sites on the microvascular endothelium in vivo, indicating sparse distribution of NOP receptors. In vitro, arterioles (~200 µm) dilated to intraluminal N/OFQ (10(-5)M) (32.6 + 8.4%) and this response was exaggerated with LPS (62.0 +7.9%, p=0.031). In vivo, LPS induced macromolecular leak of FITC-BSA (0.02 g kg(-1) i.v.) (LPS: 95.3 (86.7 to 97.9)%, p=0.043) from post-capillary venules (<40 µm) and increased leukocyte rolling as endotoxemia progressed (p=0.027), both being reduced by 150 nmol kg(-1) UFP-101 (i.v., jugular vein). Firstly, the rat mesenteric microcirculation expresses NOP receptors and secondly, NOP function (ability to induce dilation) is enhanced with LPS. UFP-101 also reduced microvascular inflammation to endotoxemia in vivo. Hence inhibition of the microvascular N/OFQ-NOP pathway may have therapeutic potential during sepsis and warrants further investigation.

Pkd2 mesenteric vessels exhibit a primary defect in endothelium-dependent vasodilatation restored by rosiglitazone.

Patients with autosomal dominant polycystic kidney disease have a high prevalence of hypertension and structural vascular abnormalities, such as intracranial aneurysms. Hypertension can develop in childhood and often precedes a significant reduction in the glomerular filtration rate. The major aim of this study was to investigate whether a primary endothelial defect or a vascular smooth muscle (VSM) defect was present in murine polycystic kidney disease (Pkd)2 heterozygous mesenteric vessels before the development of renal failure or hypertension. Using pressure myography, we observed a marked defect in ACh-stimulated endothelium-dependent vasodilatation in Pkd2 arterioles. In contrast, Pkd2 vessels responded normally to sodium nitroprusside, phenylephrine, KCl, and pressure, indicating unaltered VSM-dependent responses. Pretreatment with the peroxisome proliferator-activated receptor-γ agonist rosiglitazone significantly restored ACh-dependent vasodilation in Pkd2 mice. Isolated heterozygous Pkd2 endothelial cells displayed normal ACh-stimulated Ca(2+) and nitric oxide production. However, isolated Pkd2 heterozygous VSM cells displayed basal increases in superoxide and sodium nitroprusside-stimulated peroxynitrite formation, which were both suppressed by rosiglitazone. Furthermore, we observed a defective response of Pkd2 mesenteric venules to ACh in vivo, which was more marked after ischemia-reperfusion injury. In conclusion, the results of our study suggest that the defect in vasodilatation in Pkd2 heterozygous vessels is primarily due to a reduction in nitric bioavailability secondary to increased vascular oxidative stress. The ability of rosiglitazone to correct this phenotype suggests that this defect is potentially reversible in patients with autosomal dominant polycystic kidney disease.

Angiopoietin-1 variant reduces LPS-induced microvascular dysfunction in a murine model of sepsis.

INTRODUCTION: Severe sepsis is characterised by intravascular or extravascular infection with microbial agents, systemic inflammation and microcirculatory dysfunction, leading to tissue damage, organ failure and death. The growth factor angiopoietin (Ang-1) has therapeutic potential but recombinant Ang-1 tends to aggregate and has a short half-life in vivo. This study aimed to investigate the acute effects of the more stable Ang-1 variant matrilin-1-angiopoietin-1 (MAT.Ang-1) on the function of the microcirculation in an experimental model of sepsis, and whether any protection by MAT-Ang-1 was associated with modulation of inflammatory cytokines, angiogenic factors or the endothelial nitric oxide synthase (eNOS)-Akt and vascular endothelial (VE)-cadherin pathways. METHODS: Aluminium window chambers were implanted into the dorsal skinfold of male C3H/HeN mice (7 to 10 weeks old) to expose the striated muscle microcirculation. Endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (LPS, 1 mg/kg at 0 and 19 hours). MAT.Ang-1 was administered intravenously 20 hours after the onset of sepsis. Microcirculatory function was evaluated by intravital microscopy and Doppler fluximetry. RESULTS: Endotoxemia resulted in macromolecular leak, which was ameliorated by MAT.Ang-1 post-treatment. LPS induced a dramatic reduction in tissue perfusion, which was improved by MAT.Ang-1. Proteome profiler array analysis of skeletal muscle also demonstrated increased inflammatory and reduced angiogenic factors during endotoxemia. MAT.Ang-1 post-treatment reduced the level of IL-1ß but did not significantly induce the expression of angiogenic factors. MAT.Ang-1 alone did not induce leak or increase angiogenic factors but did reduce vascular endothelial growth factor expression in controls. CONCLUSION: Administration of MAT.Ang-1 after the onset of sepsis protects the microcirculation from endotoxemia-induced vascular dysfunction through reducing inflammation but without pro-angiogenic actions, thus representing a novel, potential pharmacotherapeutic agent for the treatment of sepsis.

Evaluation of fluorescent plasma markers for in vivo microscopy of the microcirculation.

This study evaluated four fluorescent-protein conjugates to monitor microcirculatory variables using the murine cremaster muscle and determined acute and long-term responses to repeated administration of FITC-BSA [conjugated at the University of Sheffield (UoS)] within a dorsal microcirculatory chamber (DMC) in rats. For analysis of the cremaster muscle, male C3H/HeN mice were anaesthetized, the cremaster muscle was exteriorized, then TRITC-BSA, TRITC-dextran, FITC-BSA, FITC-BSA (UoS) or FITC-dextran (0.25 ml/100 g) were administered systemically. The microcirculation was viewed with epi-illumination every 10 min for 120 min. For analysis of the DMC, male Wistar rats were implanted with the chamber. Three weeks later, FITC-BSA (UoS) was administered systemically, and the microcirculation response was monitored using three different protocols. In addition, in vitro stability of fluorescent conjugates was measured over 8 h. With regard to the cremaster muscle, initially no differences in interstitial fluorescence or vessel diameter were observed between the four fluorescent conjugates. By the end of the study, interstitial fluorescence from TRITC-dextran, FITC-dextran and FITC-BSA (Sigma) was significantly (p < 0.05) increased compared to FITC-BSA (UoS). With regard to the DMC, there was no interstitial fluorescence leakage after 180 min or 5 weeks despite repeated administration, but a significant (p < 0.05) leak was detected between 4 and 24 h. FITC-BSA (UoS) was the most stable fluorescent conjugate both in vitro and in vivo and was comparable with other conjugates for evaluating skeletal muscle microcirculation using fluorescent in vivo microscopy.

ROCK induced inflammation of the microcirculation during endotoxemia mediated by nitric oxide synthase.

Sepsis may be modeled using lipopolysaccharide (LPS), which alters levels of nitric oxide (NO), synthesized via endothelial and inducible nitric oxide synthase (eNOS and iNOS). This study aimed to determine whether the Rho kinase (ROCK) inhibitor fasudil protected against LPS-induced (endotoxemia) macromolecular leak and leukocyte adhesion via NOS pathways. Male Wistar rats (283±8g, n=36) were anaesthetized with thiopental and the mesentery prepared for fluorescent intravital microscopy (IVM). Animals received either (i) LPS alone (150µg kg(-1) h(-1) i.v., n=6); (ii) fasudil (FAS, 3mg kg(-1) i.v., n=6) or (iii) fasudil (10mg kg(-1) i.v., n=6), immediately prior to LPS administration, (iv) fasudil (FAS, 3mg kg(-1) i.v., n=6) alone or (v) fasudil (FAS, 10mg kg(-1) i.v., n=6) alone, or (vi) saline alone (1ml kg(-1) h(-1) i.v, n=6) for 4h (240min). LPS increased macromolecular leak (cumulative normalized grey levels, arbitrary units) from post capillary venules (<40µm) and this was reduced by 3mg kg(-1) fasudil, however, 10mg kg(-1) was less effective (t=240min, control: 3.3±1.7; LPS: 15.1±2.0; LPS+3mg kg(-1) fasudil: 3.3±1.1 (p<0.05), LPS+10mg kg(-1) fasudil: 8.4±3.2 NS). The numbers of leukocytes adhering for >1min/100µm venule were reduced by fasudil (t=240min, control: 1.8±0.7; LPS: 7.0±1.0; LPS+3mg kg(-1) fasudil: 1.75±0.25, p<0.05; LPS+10mg kg(-1) fasudil: 1.8±0.8, p<0.05). Immunohistochemistry demonstrated that fasudil increased endothelial cell expression of eNOS during sepsis, and decreased LPS-induced up-regulation of iNOS. Inhibition of ROCK in rats increases eNOS and decreases iNOS during endotoxemia, concomitantly reducing microvascular inflammation. Thus, targeting the ROCK pathway during sepsis could have therapeutic potential for reducing inflammation via a NO dependent mechanism.

Assessing adrenal status in patients before and immediately after coronary artery bypass graft surgery.

OBJECTIVE: Patients with cortisol deficiency poorly tolerate any systemic inflammatory response syndrome (SIRS), and may die if not treated with sufficient exogenous glucocorticoids. Controversy surrounds what constitutes a 'normal' adrenal response in critical illness. This study uses conventional tests for adrenal insufficiency to investigate cortisol status in patients undergoing elective coronary artery bypass surgery, a condition frequently associated with SIRS. DESIGN: A prospective, observational study. METHODS: Thirty patients with impaired left ventricular function (ejection fraction >23% <50%) underwent basal ACTH measurement, and a short cosyntropin test (250 µg, i.v.) 1 week preoperatively, and at +4 h following induction of general anaesthesia. Preoperatively, a 30 min cortisol level post cosyntropin >550 nmol/l was taken as a normal response. RESULTS: Prior to surgery, all patients had a normal response to cosyntropin. Postoperatively, eight patients (26.7%) did not achieve stimulated cortisol levels >550 nmol/l and the mean peak cortisol postoperatively was lower (1048 vs 730 nmol/l; P<0.001). There was a significant rise in ACTH after surgery (21 vs 184 ng/l; P=0.007) and reduction in Δ-cortisol post cosyntropin (579 vs 229 nmol/l; P<0.001). There was no change in basal cortisol pre- and post-operatively (447 vs 501; P=0.4). All patients underwent routine, uneventful postoperative recovery. CONCLUSION: Up to one quarter of patients with a normal cortisol status preoperatively demonstrated a raised ACTH and deficient cortisol response postoperatively. Despite these responses, all patients had uneventful outcomes. These data reinforce the need for caution when interpreting results of endocrine testing following major surgery or in the intensive care environment, and that prognostic value of these results may be of limited use.

Constriction of rat extra-splenic veins to lipopolysaccharide involves endothelin-1.

The spleen has an important role in blood volume regulation and increased resistance of post-capillary hilar veins (in mesentery adjoining the spleen) can regulate this. This study investigated whether venular constriction to lipopolysaccharide (LPS) involved endothelin-1 (ET-1). Pressure myography was used to study isolated extra-splenic (hilar) vessels from male Wistar rats (n = 111). Arteries and veins were treated with LPS (50 microg ml(-1)) for 4 h. Extra-splenic veins constricted to LPS (p < 0.05), but there was no effect on arteries. Denudation did not abolish venular constriction to LPS, indicating an endothelial independent mechanism. However, the dual ET-1 receptor antagonist bosentan (10(-5) M) and specific ET(A) and ET(B) antagonists ABT-627 (atrasentan, 6.3 x 10(-6) M) and A-192621(1.45 x 10(-6) M) completely abolished constriction of LPS-treated veins. ET-1 alone also constricted the extra-splenic arteries and veins (p < 0.05), with a greater response observed in veins (p < 0.05). ELISA also confirmed that serum and spleen levels of ET-1 increased in response to LPS (p < 0.05). That LPS-induced constriction of extra-splenic veins is mediated by ET-1. Greater constriction of post- versus pre-capillary extra-splenic vessels to LPS would result in increased intra-splenic fluid extravasation and hypovolaemia in vivo.

Macromolecular leak from extrasplenic lymphatics during endotoxemia.

BACKGROUND: The spleen has an important physiological role in maintaining blood volume; this study aimed to determine whether during pathophysiological circumstances, namely endotoxemia, the extrasplenic pathway is dysfunctional. We hypothesize that increased 'leakiness' of lymphatics in response to lipopolysaccharide (LPS) provides a route for loss of protein-rich fluid into third spaces and prevents the spleen from maintaining blood volume homeostasis. METHODS AND RESULTS: Male Wistar rats (200-280 g, n = 24) were anesthetized with thiopental (40-90 mg x kg(-1) x hr(-1), i.v.) to study the extrasplenic (vessels in mesentery adjoining the spleen) and ileal mesenteric microcirculation using fluorescently labeled albumin (66 KDa FITC-BSA, 0.02 g.100 g(-1), i.v.) with intravital microscopy. LPS (150 microg x kg(-1) x hr(-1) i.v.) induced constriction of rat extrasplenic venules (-14 +/- 2.4% from 40.4 +/- 7.8 microm, p < 0.05) and no change in arteriolar diameter (-4.6 +/- 4.7% from 32.6 +/- 4.3 microm). As the spleen is freely permeable to protein, a greater increase in venular versus arteriolar extrasplenic resistance increases intrasplenic capillary hydrostatic pressure, leading to fluid efflux into the lymphatics, draining the spleen. In agreement we report here increased extrasplenic venular resistance with LPS and lymphatic dilation to accommodate this fluid (13.5 +/- 6% from 18.5 +/- 4.8 microm, p < 0.05). However, the extrasplenic pathway then appeared to dysfunction, with macromolecular leak from extrasplenic venules (24.6 +/- 6.4%, p < 0.05) and lymphatics (12.1 +/- 3.4%, p < 0.05), indicated by increased interstitial FITC-BSA fluorescence. This was less than from ileal mesenteric venules (324 +/- 32%, p < 0.05). There was a concurrent decrease in mean arterial pressure (T(180): -15.1 +/- 6.9% from MAP of 130.3 +/- 8.8 mmHg at T(0), p < 0.05). CONCLUSION: Lymphatics are generally considered to demonstrate unidirectional and inward uptake of large molecules. However, during endotoxemia, we have demonstrated that extrasplenic lymphatics also allow the leakage of large protein molecules out into interstitial spaces. Fluid losses from extrasplenic lymphatics could therefore contribute to hypovolemia and hypotension associated with sepsis.

Lipopolysaccharide alters vasodilation to atrial natriuretic peptide via nitric oxide and endothelin-1: time-dependent effects.

Nitric oxide (NO) induces vascular relaxation via cGMP in vascular smooth muscle (VSM) and is an important mediator of vascular tone during sepsis, as endothelial NO synthase (eNOS) may be upregulated during the early stages. Atrial natriuretic peptide (ANP) also stimulates cGMP via eNOS hence, this study aimed to investigate the role of NO in time-dependent altered vascular responses to ANP during the first 4h of exposure to bacterial lipopolysaccharide (LPS). We used male rat saphenous arteries [internal relaxed diameter 63-152 microm, n=48], mounted on a wire myograph and pre-constricted with phenylephrine. At 2h in the presence of LPS, there was increased relaxation to ANP in arteries exposed to LPS [16.3+/-2.4%, P<0.05]. However the response to ANP was not altered by the NOS inhibitor Nomega-nitro-l-arginine methyl ester (L-NAME, 10(-4)M) and following denudation (vessels without endothelium). At 4h there was no longer increased relaxation to ANP in the presence of LPS. Moreover the vasodilator response to ANP was significantly reduced following L-NAME or denudation [4.4+/-1.0% and 4.3+/-1.1% respectively, P<0.05]. However, the non-specific endothelin-1 (ET-1) receptor antagonist Bosentan [10(-5)M] increased dilatation in LPS exposed arteries at 1 and 2h, reaching significance at 4h [14.0+/-3.4%, P<0.05]. In summary, an endothelial and NO dependent mechanism is responsible for increased relaxation to ANP following 2h exposure to LPS. However after 4h an endothelial and NO independent process involving ET-1 is responsible for decreased relaxation to ANP. The enhanced response to ANP may exacerbate early systemic vasodilatation during early sepsis.

Reduced vascular response to phenylephrine during exposure to lipopolysaccharide in vitro involves nitric oxide and endothelin 1.

Nitric oxide (NO) and endothelin 1 (ET-1) increase significantly during the first 4 h of Escherichia coli lipopolysaccharide (LPS) exposure. The aim of this study was to investigate the role of these mediators in the reduced response to phenylephrine treatment. We used male rat saphenous arteries (internal relaxed diameter, 63-152 microm; n = 48), mounted on a wire myograph and subsequently treated with LPS. At 1 h, LPS (dose, 50 microg mL(-1)) significantly (P < 0.05) inhibited constriction to phenylephrine (concentration, 10(-1)M to 10(-6)M) (LPS concentration required for half maximal response [EC50], 10.82 +/- 1.08microM; Control EC50, 5.07 +/- 0.34microM). However, by removing the endothelium (denuded) or adding Nomega-nitro-L-arginine methyl ester (L-NAME; concentration, 10(-4) microM), the response to phenylephrine treatment was significantly improved compared with LPS only-treated arteries (LPS + denuded EC50, 7.04 +/- 1.12microM; LPS + L-NAME EC50, 2.64 +/- 0.63microM). On the other hand, denudation did not restore constriction to phenylephrine at 2 and 4 h. However, L-NAME and the nonspecific ET-1 receptor antagonist bosentan (concentration, 10(-5)M) improved constriction to phenylephrine in LPS-treated arteries (P < 0.05) at 4 h (LPS EC50, 998.50 +/- 447.10microM; LPS + L-NAME EC50, 65.23 +/- 25.61microM; LPS + bosentan EC50, 63.65 +/- 25.33microM). We conclude that endothelium-dependent mechanisms have an early role in the reduced responsiveness of vascular smooth muscle to vasoconstrictors during simulated septic conditions. Shortly after exposure to LPS (duration, 1 h), endothelium-derived NO seemed to have a role in reduced arterial constriction to phenylephrine, but later (4 h) ET-1 and endothelium-independent increase in NO seemed to contribute further to the loss of response.

LPS abolishes extrasplenic vasoconstriction to atrial natriuretic peptide: the role of NO and endothelin 1.

Sepsis causes changes in vascular resistance and hypovolemia. Previous studies have demonstrated that the spleen regulates blood volume via atrial natiuretic peptide (ANP). We hypothesized that LPS alters extrasplenic responses to ANP via endothelial-dependent mechanisms and studied the role of NO and endothelin 1 (ET-1). Isolated extrasplenic arteries and veins (vessels in mesentery adjoining spleen) were obtained from male Wistar rats weighing 200 to 280 g (n = 102) and mounted on a pressure myograph to determine intraluminal diameter for 4 h. Isolated vessels constricted in response to the half-maximum response of ANP (veins, 30% +/- 1.7%; arteries, 34.5 +/- 1.7%; P < 0.05), and this was abolished by the NO donor S-nitroso-N-acetylpenicillamine (SNAP 75 microM). Arteries and veins incubated with LPS (50 microg mL(-1) for 4 h) were unresponsive to ANP, and constriction was not restored by the NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME 100 microM). However, venular constriction returned in the presence of the ET-1 antagonist Bosentan, increasing from -1.5 +/- 1.2 (10 min) to -10 +/- 2.5% (4 h) with LPS + Bosentan (3 x 10(-6) M) compared with -2.3 +/- 1.2 and 0% with LPS alone. In conclusion, LPS abolished endothelial-dependent extrasplenic venular constriction to ANP partially due to increased ET-1, whereas NO seemed to modulate vascular responses to ANP.

Proinflammatory and vasodilator effects of nociceptin/orphanin FQ in the rat mesenteric microcirculation are mediated by histamine.

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). N/OFQ causes hypotension and vasodilation, and we aimed to determine the role of histamine in inflammatory microvascular responses to N/OFQ. Male Wistar rats (220-300 g, n = 72) were anesthetized with thiopental (30 mg/kg bolus, 40-90 mg x kg(-1) x h(-1) iv), and the mesentery was prepared for fluorescent intravital microscopy using fluorescein isothiocyanate-conjugated BSA (FITC-BSA, 0.25 ml/100 g iv) or 1 microm fluorescently labeled microspheres. N/OFQ (0.6-60 nmol/kg iv) caused hypotension (SAP, baseline: 154 +/- 11 mmHg, 15 nmol/kg N/OFQ: 112 +/- 10 mmHg, P = 0.009), vasodilation (venules: 23.9 +/- 1.2 microm, 26.7 +/- 1.2 microm, P = 0.006), macromolecular leak (interstitial gray level FITC-BSA: 103.7 +/- 3.4, 123.5 +/- 11.8, P = 0.009), and leukocyte adhesion (2.0 +/- 0.9, 15.2 +/- 0.9/100 microm, P = 0.036). Microsphere velocity also decreased (venules: 1,230 +/- 370 microm/s, P = 0.037), but there were no significant changes in blood flow. Flow cytometry measured a concurrent increase in neutrophil expression of cd11b with N/OFQ vs. controls (Geo mean fluorescence: 4.19 +/- 0.13 vs. 2.06 +/- 0.38, P < 0.05). The NOP antagonist [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-101; 60 and 150 nmol/kg iv), H(1) and H(2)antagonists pyrilamine (mepyramine, 1 mg/kg iv) and ranitidine (1 mg/kg iv), and mast cell stabilizer cromolyn (1 mg x kg(-1) x min(-1)) also abolished vasodilation and macromolecular leak to N/OFQ in vivo (P < 0.05), but did not affect hypotension. Isolated mesenteric arteries (approximately 200 microm, n = 25) preconstricted with U-46619 were also mounted on a pressure myograph (60 mmHg), and both intraluminally and extraluminally administered N/OFQ (10(-5) M) caused dilation, inhibited by pyrilamine in the extraluminal but not the intraluminal (control: -6.9 +/- 3.8%; N/OFQ: 32.6 +/- 8.4%; pyrilamine: 31.5 +/- 6.8%, n = 18, P < 0.05) experiments. We conclude that, in vivo, mesenteric microvascular dilation and macromolecular leak occur via N/OFQ-NOP-mediated release of histamine from mast cells. Therefore, N/OFQ-NOP has an important role in microvascular inflammation, and this may be targeted during disease, particularly as we have proven that UFP-101 is an effective antagonist of microvascular responses in vivo.