Francesco S. Venuti

Recombinant human erythropoietin improves angiogenesis and wound healing in experimental burn wounds

Objective:Erythropoietin interacts with vascular endothelial growth factor (VEGF) and stimulates endothelial cell mitosis and motility; thus it may be of importance in the complex phenomenon of wound healing. The purpose of this study was to investigate the effect of recombinant human erythropoietin (rHuEPO) on experimental burn wounds. Design:Randomized experiment. Setting:Research laboratory. Subjects:C57BL/6 male mice weighing 25–30 g. Interventions:Mice were immersed in 80°C water for 10 secs to achieve a deep-dermal second degree burn. Animals were randomized to receive either rHuEPO (400 units/kg/day for 14 days in 100 &mgr;L subcutaneously) or its vehicle alone (100 &mgr;l/day distilled water for 14 days subcutaneously). On day 14 the animals were killed. Burn areas were used for histologic examination, evaluation of neoangiogenesis by immunohistochemistry, and expression (Western blot) of the specific endothelial marker CD31 as well as quantification of microvessel density, measurement of VEGF wound content (enzyme-linked immunosorbent assay), expression (Western blot) of endothelial and inducible nitric oxide synthases, and determination of wound nitric oxide (NO) products. Measurements and Main Results:rHuEPO increased burn wound reepithelialization and reduced the time to final wound closure. These effects were completely abated by a passive immunization with specific antibodies against erythropoietin. rHuEPO improved healing of burn wound through increased epithelial proliferation, maturation of the extracellular matrix, and angiogenesis. The hematopoietic factor augmented neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of the specific endothelial marker CD31. Furthermore, rHuEPO enhanced the wound content of VEGF caused a marked expression of endothelial and inducible nitric oxide synthases and increased wound content of nitric oxide products. Conclusions:Our study suggests that rHuEPO may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

Attenuated Cerulein-Induced Pancreatitis in Nuclear Factor–κB–Deficient Mice

Nuclear factor (NF)-κB plays a central role in acute pancreatitis. We studied cerulein (CER)-induced pancreatitis in NF-κB knockout (KO) mice. NF-κB KO mice and normal control littermate wild-type (WT) mice were given four hyperstimulating doses of cerulein every hour to elicit secreatagogue-induced pancreatitis. Malonildialdehyde activity, glutathione levels, myeloperoxidase activity, TNF-α, and NF-κB binding activity and its inhibitory protein IκBα were studied in the pancreas. Furthermore, we measured plasma lipase and amylase and the histological damage. KO mice had reduced malonildialdehyde levels (WT + CER = 4.083 ± 0.95 μmol/g; KO + CER = 1.513 ± 0.63 μmol/g), decreased myeloperoxidase activity (WT + CER = 19.3 ± 2.39 mU/g; KO + CER = 10.21 ± 2.05 mU/g), increased glutathione levels (WT + CER 6.22 ± 2.46 μmol/g; KO + CER = 15. 516 ± 2.92 μmol/g), and reduced serum levels of amylase (WT + CER = 2519 ± 656.9 U/L; KO + CER = 916 ± 280.4 U/L) and lipase (WT + CER = 1420 ± 170 U/L; KO + CER = 861 ± 172. 3 U/L). KO mice showed reduced pancreatic NF-κB activation, decreased TNF-α tissue content, and reduced histologic alterations. Our data suggest that KO mice have an attenuated cerulein-induced pancreatitis and help to define the possible interaction between NF-κB activation and oxidative stress in this deleterious event.

Activation of the cholinergic anti-inflammatory pathway reduces NF-kappab activation, blunts TNF-alpha production, and protects againts splanchic artery occlusion shock.

ABSTRACT The cholinergic anti-inflammatory pathway has not yet been studied in splanchnic artery occlusion (SAO) shock. We investigated whether electrical stimulation (STIM) of efferent vagus nerves suppresses the inflammatory cascade in SAO shock. Animals were subjected to clamping of the splanchnic arteries for 45 min, followed by reperfusion. This surgical procedure resulted in an irreversible state of shock (SAO shock). Sham-operated animals were used as controls. Two minutes before the start of reperfusion, rats were subjected to bilateral cervical vagotomy (VGX) or sham surgical procedures. Application of constant voltage pulses to the caudal vagus ends (STIM: 5 V, 2 ms, 6 Hz for 15 min, 5 min after the beginning of reperfusion) increased survival rate (VGX + SAO + Sham STIM = 0% at 4 h of reperfusion; VGX + SAO + STIM = 90% at 4 h of reperfusion), reverted the marked hypotension, inhibited I&kgr;B&agr; liver loss, blunted the augmented nuclear factor-&kgr;B activity, decreased hepatic tumor necrosis factor (TNF)-&agr; mRNA (VGX + SAO + Sham STIM = 1.0 ± 1.9 TNF-&agr;/glyceraldehyde-3-phosphate dehydrogenase ratio; VGX + SAO + STIM = 0.3 ± 0.2 TNF-&agr;/glyceraldehyde-3-phosphate dehydrogenase ratio), reduced plasma TNF-&agr; (VGX + SAO + Sham STIM = 118 ± 19 pg/mL; VGX + SAO + STIM = 39 ± 8 pg/mL), ameliorated leukopenia, and decreased leukocyte accumulation, as revealed by means of myeloperoxidase activity in the ileum (VGX + SAO + Sham STIM = 7.9 ± 1 U/g tissue; VGX + SAO + STIM = 3.1 ± 0.7 U/g tissue) and in the lung (VGX + SAO + Sham STIM = 8.0 ± 1.0 U/g tissue; VGX + SAO + STIM = 3.2 ± 0.6 U/g tissue). Chlorisondamine, a nicotinic receptor antagonist, abated the effects of vagal stimulation. Our results show a parasympathetic inhibition of nuclear factor-&kgr;B and TNF-&agr; in SAO shock.

Effect of recombinant adeno-associated virus vector-mediated vascular endothelial growth factor gene transfer on wound healing after burn injury.

ObjectiveThe purpose of this study was to investigate the effect of recombinant adeno-associated viral (rAAV) vector-mediated human vascular endothelial growth factor (VEGF165) transfer on experimental burn wounds. DesignRandomized experiment. SettingResearch laboratory. SubjectsC57BL/6 male mice weighing 25–30 g. InterventionsMice were immersed in 80°C water for 10 secs to achieve a partial-thickness scald burn. Animals were randomized to receive at two injection sites on the edge of the burn either 1011 copies of the rAAV-VEGF165 or the vector carrying the control and inert gene &bgr;-galactosidase (rAAV-LacZ). On day 14 the animals were killed. Burn areas were used for histologic examination, evaluation of VEGF expression (immunohistochemistry) and VEGF wound content (enzyme-linked immunosorbent assay), determination of wound nitrite, and measurement of messenger RNA (mRNA) for endothelial and inducible nitric oxide synthase (eNOS and iNOS). Measurements and Main ResultsrAAV-VEGF165 increased epithelial proliferation, angiogenesis, and maturation of the extracellular matrix. Furthermore, gene transfer enhanced VEGF expression, studied by immunohistochemistry, and the wound content of the mature protein (rAAV-LacZ, 11 ± 5 pg/wound; rAAV-VEGF165, 104 ± 7 pg/wound). Moreover, VEGF165 gene transfer increased wound content of nitrate. Finally, rAAV-VEGF165 administration enhanced the messenger RNA for eNOS (rAAV-VEGF165, 1.1 ± 0.2 relative amount of eNOS mRNA; rAAV-LacZ, 0.66 ± 0.3 relative amount of eNOS mRNA) and iNOS (rAAV-VEGF165, 0.8 ± 0.09 relative amount of iNOS mRNA; rAAV-LacZ, 0.45 ± 0.05 relative amount of iNOS mRNA). ConclusionOur study suggests that rAAV-VEGF gene transfer may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

Factors associated with hypotension and bradycardia after epidural blockade

In order to identify patient-, anesthesia-, and surgery-related factors influencing the probability of hypotension and bradycardia after epidural blockade, an observational study was conducted on 1050 nonpregnant patients. Backward stepwise logistic regression was performed on the variables hypotension (systolic blood pressure <90 mm Hg) and bradycardia (heart rate <or=to45 bpm). Hypotension and bradycardia occurred in 158 and 24 patients, respectively. The probability of hypotension increased when epidural fentanyl was administered (odds ratio [OR] = 2.18; 95% confidence interval [CI] = 1.16-4.11), with body weight and spread of epidural analgesia, and decreased when a tourniquet was used (OR = 0.01, CI = 0.01-0.02) and bupivacaine instead of carbonated lidocaine was administered (OR = 0.28, CI = 0.14-0.60). Sensitivity and specificity of the model were 89% and 88%, respectively. The probability of bradycardia was less in women (OR = 0.05, CI = 0.01-0.41) and when a tourniquet was used (OR = 0.04, CI = 0.02-0.09). Sensitivity and specificity were 50% and 97%, respectively. In conclusion, our analysis can contribute to identification of patients at high risk to develop hypotension and bradycardia after epidural blockade. If bupivacaine instead of carbonated lidocaine is used and epidural fentanyl is not administered a decrease in the incidence of hypotension may be anticipated. (Anesth Analg 1996;83:1033-40)

Flavocoxid, a dual inhibitor of COX-2 and 5-LOX of natural origin, attenuates the inflammatory response and protects mice from sepsis

IntroductionCecal ligation and puncture (CLP) is an inflammatory condition that leads to multisystemic organ failure. Flavocoxid, a dual inhibitor of cyclooxygenase (COX-2) and 5-lipoxygenase (5-LOX), has been shown in vitro to possess antiinflammatory activity in lipopolysaccharide (LPS)-stimulated rat macrophages by reducing nuclear factor (NF)-κB activity and COX-2, 5-LOX and inducible nitric oxide synthase (iNOS) expression. The aim of this study was to evaluate the effects of flavocoxid in a murine model of CLP-induced polymicrobial sepsis.MethodsC57BL/6J mice were subjected to CLP or sham operation. In a first set of experiments, an intraperitoneal injection of flavocoxid (20 mg/kg) or vehicle was administered 1 hour after surgery and repeated every 12 hours. Survival rate was monitored every 24 hours throughout 120 hours. Furthermore, additional groups of sham and CLP mice were killed 18 hours after surgical procedures for blood-sample collection and the lung and liver were collected for biomolecular, biochemical and histopathologic studies.ResultsCOX-2, 5-LOX, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-10, extracellular-regulated-kinase 1/2 (ERK), JunN-terminal kinase (JNK), NF-κB, and β-arrestin 2 protein expression were evaluated in lung and liver with Western blot analysis. In addition, leukotriene B4 (LTB4), prostaglandin E2 (PGE2), cytokines, and lipoxin A4 serum content were measured with an enzyme-linked immunosorbent assay (ELISA). Flavocoxid administration improved survival, reduced the expression of NF-κB, COX-2, 5-LOX, TNF-α and IL-6 and increased IL-10 production. Moreover, flavocoxid inhibited the mitogen-activated protein kinases (MAPKs) pathway, preserved β-arrestin 2 expression, reduced blood LTB4, PGE2, TNF-α and IL-6, and increased IL-10 and lipoxin A4 serum levels. The treatment with flavocoxid also protected against the histologic damage induced by CLP and reduced the myeloperoxidase (MPO) activity in the lung and liver.ConclusionsFlavocoxid protects mice from sepsis, suggesting that this dual inhibitor may represent a promising approach in such a life-threatening condition.

Polydeoxyribonucleotide improves angiogenesis and wound healing in experimental thermal injury

Objective:Polydeoxyribonucleotide contains a mixture of nucleotides and interacts with adenosine receptors, stimulating vascular endothelial growth factor expression and wound healing. The purpose of this study was to investigate the effect of polydeoxyribonucleotide on experimental burn wounds. Design:Randomized experiment. Setting:Research laboratory at a university hospital. Subjects:Thermal injury in mice. Interventions:Mice were immersed in 80°C water for 10 secs to achieve a deep-dermal second-degree burn. Animals were randomized to receive either polydeoxyribonucleotide (8 mg/kg/day intraperitoneally for 14 days) or its vehicle alone (0.9% NaCl solution at 100 μL/day intraperitoneally). On days 7 and 14 the animals were killed. Blood was collected for tumor necrosis factor-α measurement; burn areas were used for histologic and immunohistochemical examination, for the evaluation of vascular endothelial growth factor and nitric oxide synthases by Western blot, and for the determination of wound nitric oxide products. Measurements and Main Results:Polydeoxyribonucleotide increased burn wound re-epithelialization and reduced the time to final wound closure. Polydeoxyribonucleotide improved healing of burn wound through increased epithelial proliferation and maturation of the extracellular matrix as confirmed by fibronectin and laminin immunostaining. Polydeoxyribonucleotide also improved neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of platelet-endothelial cell adhesion molecule-1. Furthermore, polydeoxyribonucleotide blunted serum tumor necrosis factor-α and enhanced inducible nitric oxide synthase and vascular endothelial growth factor expression and the wound content of nitric oxide products. Conclusions:Our study suggests that polydeoxyribonucleotide may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

Gene transfer of IκBα limits infarct size in a mouse model of myocardial ischemia-reperfusion injury

Nuclear factor-κB (NF-κB) plays a central role in myocardial ischemia-reperfusion (MI/R) injury. The inhibitory protein IκBα prevents its activation. We investigated the effects of adeno-associated viral vector-mediated IκBα gene transfer in MI/R injury. Male C57BL/6 mice were randomized to receive a recombinant adeno-associated virus (rAAV) encoding the gene for the NF-κB inhibitory protein IκBα (rAAV- IκBα) or the β-galactosidase gene (a control and inert gene; rAAV-LacZ), both at a dose of 1011 copies. Four weeks later anesthetized animals were subjected to total occlusion (45 minutes) of the left main coronary artery followed by 5 hours of reperfusion. MI/R produced a wide infarct size (IF/area-at-risk = 56 ± 8%; IF/left ventricle = 44 ± 5%) and tissue neutrophil infiltration, studied by means of elastase activity (area-at-risk = 2.5 ± 0.4 μg/gm tissue; infarct area = 2.9 ± 0.6 μg/gm tissue). Furthermore MI/R caused peak message for intercellular adhesion molecule-1 (ICAM-1) in the area-at-risk at 3 hours of reperfusion (1.2 ± 0.4 relative amount of cardiac ICAM-1 mRNA). NF-κB activation was evident at 0.5 hours of reperfusion and reached its maximum increase at 2 hours of reperfusion. rAAV-IκBα injection reduced infarct size (IF/area-at-risk = 19 ± 3%; IF/left ventricle = 10 ± 2%; p < 0.001), blocked NF-κB activation, diminished cardiac ICAM-1 expression (0.4 ± 0.02 relative amount of cardiac ICAM-1 mRNA; p < 0.001), and blunted leukocyte accumulation (area-at-risk = 0.6 ± 0.05 μg/gm tissue; infarct area = 0.4 ± 0.02 μg/gm tissue; p < 0.001). Our data indicate that rAAV-IκBα may be useful for MI/R gene therapy.

Lipid peroxidation inhibition reduces NF-κB activation and attenuates cerulein-induced pancreatitis

Increased lipid peroxidation, enhanced nuclear factor kappa-B (NF- s B) activation and augmented tumor necrosis factor- f (TNF- f ) production have been implicated in cerulein-induced pancreatitis. We investigated whether lipid peroxidation inhibition might reduce NF- s B activation and the inflammatory response in cerulein-induced pancreatitis. Male Sprague-Dawley rats of 230-250 g body weight received administration of cerulein (80 w g/kg s.c. for each of four injections at hourly intervals). A control group received four s.c. injections of 0.9% saline at hourly intervals. Animals were randomized to receive either raxofelast, an inhibitor of lipid peroxidation (20 mg/kg i.p. administered with the first cerulein injection) or its vehicle (1 ml/kg of a 10% DMSO/NaCl solution). All these rats were sacrificed 2 h after the last injection of either cerulein or its vehicle. Raxofelast administration (20 mg/kg i.p. with the first cerulein) significantly reduced malondialdehyde (MDA) levels, an index of lipid peroxidation (CER+DMSO=3.075 - 0.54 w mol/g; CER+raxofelast= 0.693 - 0.18 w mol/g; p <0.001 ), decreased myeloperoxidase (MPO) activity ( CER+DMSO=22.2 - 3.54 mU/g; CER+raxofelast=9.07 - 2.05 mU/g; p <0.01 ), increased glutathione levels (GSH) (CER+DMSO= 5.21 - 1.79 w mol/g; CER+raxofelast=15.71 - 2.14 w mol/g; p <0.001 ), and reduced acinar cell damage evaluated by means of histology and serum levels of both amylase ( CER+DMSO=4063 - 707.9 U/l; CER+raxofelast=1198 - 214.4 U/l; p <0.001 ), and lipase (CER+DMSO=1654 - 330 U/l; CER+raxofelast= 386 - 118.2 U/l; p <0.001 ), Furthermore, raxofelast reduced pancreatic NF- s B activation and the TNF- f mRNA levels and tissue content of mature protein in the pancreas. Indeed, lipid peroxidation inhibition might be considered a potential therapeutic approach to prevent the severe damage in acute pancreatitis.

A Multifactorial analysis of the spread of epidural analgesia

The controversies about the factors determining the spread of epidural analgesia are partly due to inappropriate methodology or sample size of previous studies. We performed a multivariate regression analysis on 803 ASA class 1–2 non–atherosclerotic adults, undergoing lumbar epidural anaesthesia according to a predefined standardised procedure. The spread of epidural analgesia is more accurately studied by analysing dose/ segment (R2 = 0.671) instead of spread (R2 = 0.271) as dependent variable. The impact of local anaesthetic (2% lidocaine C02 or 0.5% bupivacaine) and addition of adrenaline is not significant. Spread significantly increases with increasing age, weight, body–mass index, dose of local anaesthetic, addition of fentanyl, higher site of injection, and decreasing body height. The impact of age and dose is higher under the age of 40 and at doses lower than 20 ml. Increasing the total dose increases the dose needed to block one spinal segment. Unknown idiosyncratic factors still determine a certain proportion of the sample variance. The addition of adrenaline to lidocaine and the use of bupivacaine improve the predictability of spread. In conclusion, we found clinically significant correlations between a group of factors and epidural spread. Alternative anaesthetic solutions lead to different degrees of predictability.