Impaired microcirculatory perfusion in a rat model of cardiopulmonary bypass: the role of hemodilution.

Although hemodilution is attributed as the main cause of microcirculatory impairment during cardiopulmonary bypass (CPB), this relationship has never been investigated. We investigated the distinct effects of hemodilution with or without CPB on microvascular perfusion and subsequent renal tissue injury in a rat model. Male Wistar rats (375-425 g) were anesthetized, prepared for cremaster muscle intravital microscopy, and subjected to CPB (n = 9), hemodilution alone (n = 9), or a sham procedure (n = 6). Microcirculatory recordings were performed at multiple time points and analyzed for perfusion characteristics. Kidney and lung tissue were investigated for mRNA expression for genes regulating inflammation and endothelial adhesion molecule expression. Renal injury was assessed with immunohistochemistry. Hematocrit levels dropped to 0.24 ± 0.03 l/l and 0.22 ± 0.02 l/l after onset of hemodilution with or without CPB. Microcirculatory perfusion remained unaltered in sham rats. Hemodilution alone induced a 13% decrease in perfused capillaries, after which recovery was observed. Onset of CPB reduced the perfused capillaries by 40% (9.2 ± 0.9 to 5.5 ± 1.5 perfused capillaries per microscope field; P < 0.001), and this reduction persisted throughout the experiment. Endothelial and inflammatory activation and renal histological injury were increased after CPB compared with hemodilution or sham procedure. Hemodilution leads to minor and transient disturbances in microcirculatory perfusion, which cannot fully explain impaired microcirculation following cardiopulmonary bypass. CPB led to increased renal injury and endothelial adhesion molecule expression in the kidney and lung compared with hemodilution. Our findings suggest that microcirculatory impairment during CPB may play a role in the development of kidney injury.

Dopamine induces lipid accumulation, NADPH oxidase-related oxidative stress, and a proinflammatory status of the plasma membrane in H9c2 cells.

Excess catecholamine levels are suggested to be cardiotoxic and to underlie stress-induced heart failure. The cardiotoxic effects of norepinephrine and epinephrine are well recognized. However, although cardiac and circulating dopamine levels are also increased in stress cardiomyopathy patients, knowledge regarding putative toxic effects of excess dopamine levels on cardiomyocytes is scarce. We now studied the effects of elevated dopamine levels in H9c2 cardiomyoblasts. H9c2 cells were cultured and treated with dopamine (200 µM) for 6, 24, and 48 h. Subsequently, the effects on lipid accumulation, cell viability, flippase activity, reactive oxygen species (ROS) production, subcellular NADPH oxidase (NOX) protein expression, and ATP/ADP and GTP/GDP levels were analyzed. Dopamine did not result in cytotoxic effects after 6 h. However, after 24 and 48 h dopamine treatment induced a significant increase in lipid accumulation, nitrotyrosine levels, indicative of ROS production, and cell death. In addition, dopamine significantly reduced flippase activity and ATP/GTP levels, coinciding with phosphatidylserine exposure on the outer plasma membrane. Furthermore, dopamine induced a transient increase in cytoplasmic and (peri)nucleus NOX1 and NOX4 expression after 24 h that subsided after 48 h. Moreover, while dopamine induced a similar transient increase in cytoplasmic NOX2 and p47phox expression, in the (peri)nucleus this increased expression persisted for 48 h where it colocalized with ROS. Exposure of H9c2 cells to elevated dopamine levels induced lipid accumulation, oxidative stress, and a proinflammatory status of the plasma membrane. This can, in part, explain the inflammatory response in patients with stress-induced heart failure.

Atrial fibrillation coincides with the advanced glycation end product N(ε)-(carboxymethyl)lysine in the atrium.

Presence of advanced glycation end products (AGEs) in the heart induces a proinflammatory phenotype. However, the presence of AGEs within atrial tissue of atrial fibrillation (AF) patients is unknown and was analyzed here. Left atrial appendage tissue from 33 AF patients and 9 controls was analyzed for the presence of the major AGEs N(ε)-(carboxymethyl)lysine (CML), VCAM-1, neutrophilic granulocytes, lymphocytes, and macrophages in both the fat tissue and myocardium separately. The total amount of fibrosis was also analyzed. Presence of CML was significantly higher in blood vessels of the left atrial appendage in AF patients as compared to controls, independent of diabetes mellitus. In AF patients, VCAM-1 expression in blood vessels and the numbers of infiltrated neutrophilic granulocytes, lymphocytes, and macrophages significantly increased compared to controls, and were highest in the fat tissue; there was no significant difference in fibrosis compared to controls. Interestingly, total amount of CML and fibrosis in AF and control patients correlated positively. Finally, there was no difference between AF patients based on AF type or surgical indication in the presence of CML, VCAM-1 expression, inflammatory cells, and fibrosis. Our results indicate that in AF the intramyocardial blood vessels of the left atrial appendage have an increased CML presence and proinflammatory status coinciding with a local increase in the number of inflammatory cells.

Activation of extracellular signal-related kinase in abdominal aortic aneurysm.

BACKGROUND: Extracellular matrix degeneration, caused by matrix metalloproteinase-2, facilitates smooth muscle cell migration leading to medial layer decline and, ultimately, abdominal aortic aneurysm. It remains unclear what exactly causes aneurysms to rupture, which leads to death in most patients. The extracellular signal-related kinase may be linked to the latter process. We aimed to clarify the role of extracellular signal-related kinase in aortic aneurysm development and rupture in patients. DESIGN: Aortic fragments were harvested during open repair of nonruptured (n = 20) and ruptured (n = 8) aneurysms. As control, nondilated aortas (n = 6) were obtained during autopsy. We determined levels of phosphorylated and total extracellular signal-related kinase by Western blot, matrix metalloproteinase-2 by immunohistochemistry and medial layer thickness by conventional microscopy. RESULTS: Nonruptured aneurysms had 1·8 times higher activation of extracellular signal-related kinase (ratio: phosphorylated/total) than controls (P = 0·011). However, the ruptured aneurysms had only 0·9 times the activation of controls (ns). Both nonruptured and ruptured aneurysms showed significantly higher matrix metalloproteinase-2 than controls (3·8 and 4·0-times, respectively; P < 0·005). Of the medial layer thickness in controls, the median was 1·5 mm, in nonruptured 1·0 mm and in ruptured aneurysms 0·7 mm. Activation of extracellular signal-related kinase correlated positively to medial layer thickness (Rs  = 0·48; P = 0·014), but not to matrix metalloproteinase-2 (Rs  = -0·36; P = 0·10). CONCLUSIONS: In this study, nonruptured aneurysms are associated with increased extracellular signal-related kinase activation while ruptured aneurysms are not. Extracellular signal-related kinase was not related to total matrix metalloproteinase-2 expression. We therefore speculate that increased extracellular signal-related kinase, in response to medial layer decline, could be protective against aneurysm rupture.

Monocyte subset accumulation in the human heart following acute myocardial infarction and the role of the spleen as monocyte reservoir.

AIMS: Monocytes are critical mediators of healing following acute myocardial infarction (AMI), making them an interesting target to improve myocardial repair. The purpose of this study was a gain of insight into the source and recruitment of monocytes following AMI in humans. METHODS AND RESULTS: Post-mortem tissue specimens of myocardium, spleen and bone marrow were collected from 28 patients who died at different time points after AMI. Twelve patients who died from other causes served as controls. The presence and localization of monocytes (CD14(+) cells), and their CD14(+)CD16(-) and CD14(+)CD16(+) subsets, were evaluated by immunohistochemical and immunofluorescence analyses. CD14(+) cells localized at distinct regions of the infarcted myocardium in different phases of healing following AMI. In the inflammatory phase after AMI, CD14(+) cells were predominantly located in the infarct border zone, adjacent to cardiomyocytes, and consisted for 85% (78-92%) of CD14(+)CD16(-) cells. In contrast, in the subsequent post-AMI proliferative phase, massive accumulation of CD14(+) cells was observed in the infarct core, containing comparable proportions of both the CD14(+)CD16(-) [60% (31-67%)] and CD14(+)CD16(+) subsets [40% (33-69%)]. Importantly, in AMI patients, of the number of CD14(+) cells was decreased by 39% in the bone marrow and by 58% in the spleen, in comparison with control patients (P = 0.02 and <0.001, respectively). CONCLUSIONS: Overall, this study showed a unique spatiotemporal pattern of monocyte accumulation in the human myocardium following AMI that coincides with a marked depletion of monocytes from the spleen, suggesting that the human spleen contains an important reservoir function for monocytes.

Injurious mechanical ventilation causes kidney apoptosis and dysfunction during sepsis but not after intra-tracheal acid instillation: an experimental study.

BACKGROUND: Intratracheal aspiration and sepsis are leading causes of acute lung injury that frequently necessitate mechanical ventilation (MV), which may aggravate lung injury thereby potentially increasing the risk of acute kidney injury (AKI). We compared the effects of ventilation strategies and underlying conditions on the development of AKI. METHODS: Spraque Dawley rats were challenged by intratracheal acid instillation or 24 h of abdominal sepsis, followed by MV with a low tidal volume (LVT) and 5 cm H2O positive end-expiratory pressure (PEEP) or a high tidal volume (HVT) and no PEEP, which is known to cause more lung injury after acid instillation than in sepsis. Rats were ventilated for 4 hrs and kidney function and plasma mediator levels were measured. Kidney injury was assessed by microscopy; apoptosis was quantified by TUNEL staining. RESULTS: During sepsis, but not after acid instillation, MV with HVT caused more renal apoptosis than MV with LVT. Increased plasma active plasminogen activator inhibitor-1 correlated to kidney apoptosis in the cortex and medulla. Increased apoptosis after HVT ventilation during sepsis was associated with a 40% decrease in creatinine clearance. CONCLUSIONS: AKI is more likely to develop after MV induced lung injury during an indirect (as in sepsis) than after a direct (as after intra-tracheal instillation) insult to the lungs, since it induces kidney apoptosis during sepsis but not after acid instillation, opposite to the lung injury it caused. Our findings thus suggest using protective ventilatory strategies in human sepsis, even in the absence of overt lung injury, to protect the kidney.

Low myocardial protein kinase G activity in heart failure with preserved ejection fraction.

BACKGROUND: Prominent features of myocardial remodeling in heart failure with preserved ejection fraction (HFPEF) are high cardiomyocyte resting tension (F(passive)) and cardiomyocyte hypertrophy. In experimental models, both reacted favorably to raised protein kinase G (PKG) activity. The present study assessed myocardial PKG activity, its downstream effects on cardiomyocyte F(passive) and cardiomyocyte diameter, and its upstream control by cyclic guanosine monophosphate (cGMP), nitrosative/oxidative stress, and brain natriuretic peptide (BNP). To discern altered control of myocardial remodeling by PKG, HFPEF was compared with aortic stenosis and HF with reduced EF (HFREF). METHODS AND RESULTS: Patients with HFPEF (n=36), AS (n=67), and HFREF (n=43) were free of coronary artery disease. More HFPEF patients were obese (P<0.05) or had diabetes mellitus (P<0.05). Left ventricular myocardial biopsies were procured transvascularly in HFPEF and HFREF and perioperatively in aortic stenosis. F(passive) was measured in cardiomyocytes before and after PKG administration. Myocardial homogenates were used for assessment of PKG activity, cGMP concentration, proBNP-108 expression, and nitrotyrosine expression, a measure of nitrosative/oxidative stress. Additional quantitative immunohistochemical analysis was performed for PKG activity and nitrotyrosine expression. Lower PKG activity in HFPEF than in aortic stenosis (P<0.01) or HFREF (P<0.001) was associated with higher cardiomyocyte F(passive) (P<0.001) and related to lower cGMP concentration (P<0.001) and higher nitrosative/oxidative stress (P<0.05). Higher F(passive) in HFPEF was corrected by in vitro PKG administration. CONCLUSIONS: Low myocardial PKG activity in HFPEF was associated with raised cardiomyocyte F(passive) and was related to increased myocardial nitrosative/oxidative stress. The latter was probably induced by the high prevalence in HFPEF of metabolic comorbidities. Correction of myocardial PKG activity could be a target for specific HFPEF treatment.

The basement membrane of intramyocardial capillaries is thickened in patients with acute myocardial infarction.

BACKGROUND: Atherosclerotic epicardial coronary arteries are a major cause of acute myocardial infarction (AMI). Recently, we found that intramyocardial capillaries may also play a role in AMI induction. We have now evaluated intramyocardial capillaries using ultrastructural analysis in AMI patients. METHODS: 43 AMI patients (with AMI in the left ventricle) and 27 controls. No patient included in either group had diabetes mellitus. Basement membrane (BM) thickness of intramyocardial capillaries was determined using electron microscopy. BM thickness was also studied in a rat AMI model. RESULTS: BM thickness in the left ventricle of AMI patients was significantly higher than in controls (102 +/- 9 vs. 77 +/- 4 nm; p = 0.016). This increase was not found in the right ventricle. In AMI patients, BM thickness was already increased in recent infarcts and did not increase further with infarct age. No correlation was found between BM thickness and the amount of stenosis or atherosclerotic plaque stability of epicardial coronary arteries. In addition, BM thickness did not differ between control rats and AMI rats. CONCLUSIONS: These results suggest that BM thickening constitutes significant changes in the intramyocardial capillaries in patients that develop AMI. Also these changes are likely to occur prior to the induction of AMI.

Obese Older Type 2 Diabetes Mellitus Patients with Muscle Insulin Resistance Benefit from an Enriched Protein Drink during Combined Lifestyle Intervention: The PROBE Study.

(1) Background: Recent research showed that subtypes of patients with type 2 diabetes may differ in response to lifestyle interventions based on their organ-specific insulin resistance (IR). (2) Methods: 123 Subjects with type 2 diabetes were randomized into 13-week lifestyle intervention, receiving either an enriched protein drink (protein+) or an isocaloric control drink (control). Before and after the intervention, anthropometrical and physiological data was collected. An oral glucose tolerance test was used to calculate indices representing organ insulin resistance (muscle, liver, and adipose tissue) and ß-cell functioning. In 82 study-compliant subjects (per-protocol), we retrospectively examined the intervention effect in patients with muscle IR (MIR, n = 42) and without MIR (no-MIR, n = 40). (3) Results: Only in patients from the MIR subgroup that received protein+ drink, fasting plasma glucose and insulin, whole body, liver and adipose IR, and appendicular skeletal muscle mass improved versus control. Lifestyle intervention improved body weight and fat mass in both subgroups. Furthermore, for the MIR subgroup decreased systolic blood pressure and increased VO2peak and for the no-MIR subgroup, a decreased 2-h glucose concentration was found. (4) Conclusions: Enriched protein drink during combined lifestyle intervention seems to be especially effective on increasing muscle mass and improving insulin resistance in obese older, type 2 diabetes patients with muscle IR.

Classification of condom lubricants in cyanoacrylate treated fingerprints by desorption electrospray ionization mass spectrometry.

Traces of condom lubricants in fingerprints can be valuable information in cases of sexual assault. Ideally, not only confirmation of the presence of the condom but also determination of the type of condom brand used can be retrieved. Previous studies have shown to be able to retrieve information about the condom brand and type from fingerprints containing lubricants using various analytical techniques. However, in practice fingerprints often appear latent and need to be detected first, which is often achieved by cyanoacrylate fuming. In this study, we developed a desorption electrospray ionization mass spectrometry (DESI-MS) method which, combined with principal component analysis and linear discriminant analysis (PCA-LDA), allows for high accuracy classification of condom brands and types from fingerprints containing condom lubricant traces. The developed method is compatible with cyanoacrylate (CA) fuming. We collected and analyzed a representative dataset for the Netherlands comprising 32 different condoms. Distinctive lubricant components such as polyethylene glycol (PEG), polydimethylsiloxane (PDMS), octoxynol-9 and nonoxynol-9 were readily detected using the DESI-MS method. Based on the analysis of lubricant spots, a 99.0% classification accuracy was achieved. When analyzing lubricant containing fingerprints, an overall accuracy of 90.9% was obtained. Full chemical images could be generated from fingerprints, showing the distribution of lubricant components such as PEG and PDMS throughout the fingerprint, while still allowing for classification. The developed method shows potential for the development of DESI-MS based analyses of CA treated exogenous compounds from fingerprints for use in forensic science.

Pulmonary complement depositions in autopsy of critically ill patients have no relation with ARDS.

BACKGROUND: The complement system has frequently been suggested to play a role in the pathophysiology of acute respiratory distress syndrome (ARDS). The current study explored the association between pulmonary depositions of a complement activation product and the clinical diagnosis of ARDS. METHODS: Lung tissue material from autopsied critically ill patients who died whilst on invasively mechanical ventilation was collected and stained for complement C3d. The diagnosis of ARDS was by the Berlin Definition. Lung injury scores (LIS) and driving pressures were calculated, 48 and 24 h prior to death. A pathologist who remained blinded for the clinical data scored the extent of C3d depositions, using a C3d deposition score (a minimum and maximum score of 0 and 24), and of diffuse alveolar damage (DAD). The primary analysis focused on the association between the C3d deposition score and the clinical diagnosis of ARDS. Secondary analyses focused on associations between the C3d deposition score and the presence of diffuse alveolar damage (DAD) in histopathology, and LIS and driving pressures in the last 2 days before death. RESULTS: Of 36 patients of whom autopsy material was available, 12 were diagnosed as having had ARDS. In all patients, C3d depositions were found in various parts of the lungs, and to a different extent. Notably, C3d deposition scores were similar for patients with ARDS and those without ARDS (4.5 [3.3-6.8] vs. 5.0 [4.0-6.0]; not significant). C3d deposition scores were also independent from the presence or absence of DAD, and correlations between C3d scores and LIS and driving pressures prior to death were poor. CONCLUSION: Pulmonary C3d depositions are found in the lungs of all deceased ICU patients, independent of the diagnosis of ARDS. The presence of complement C3d was not associated with the presence of DAD on histopathology and had a poor correlation with ventilation characteristics prior to death.

Myocarditis in patients with subarachnoid hemorrhage: A histopathologic study.

Cardiac abnormalities after subarachnoid hemorrhage (SAH) such as electrocardiographic changes, echocardiographic wall motion abnormalities, and elevated troponin levels are independently associated with a poor prognosis. They are caused by catecholaminergic stress coinciding with influx of inflammatory cells into the heart. These abnormalities could be a sign of a myocarditis, potentially giving insight in pathophysiology and treatment options. These inflammatory cells are insufficiently characterized, and it is unknown whether myocarditis is associated with SAH. Myocardium of 25 patients who died of SAH and 18 controls was stained with antibodies identifying macrophages (CD68), lymphocytes (CD45), and neutrophil granulocytes (myeloperoxidase). Myocytolysis was visualized using complement staining (C3d). CD31 was used to identify putative thrombi. We used Mann-Whitney U testing for analysis. In the myocardium of SAH patients, the amount of myeloperoxidase-positive (P < .005), CD45-positive (P < .0005), and CD68-positive (P < .0005) cells was significantly higher compared to controls. Thrombi in intramyocardial arteries were found in 22 SAH patients and 1 control. Myocytolysis was found in 6 SAH patients but not in controls. Myocarditis, consisting of an influx of neutrophil granulocytes, lymphocytes, and macrophages, coinciding with myocytolysis and thrombi in intramyocardial arteries, occurs in patients with SAH but not in controls. These findings might explain the cardiac abnormalities after SAH and may have implications for treatment.

Ventricular myocarditis coincides with atrial myocarditis in patients.

INTRODUCTION: Atrial fibrillation (AF) is a common complication in myocarditis. Atrial inflammation has been suggested to play an important role in the pathophysiology of AF. However, little is known about the occurrence of atrial inflammation in myocarditis patients. Here, we analyzed inflammatory cell numbers in the atria of myocarditis patients without symptomatic AF. METHODS: Cardiac tissue was obtained postmortem from lymphocytic myocarditis patients (n=6), catecholamine-induced myocarditis patients (n=5), and control patients without pathological evidence of heart disease (n=5). Tissue sections of left and right ventricle and left and right atrium were stained for myeloperoxidase (neutrophilic granulocytes), CD45 (lymphocytes), and CD68 (macrophages). These cells were subsequently quantified in atrial and ventricular myocardium and atrial adipose tissue. RESULTS: In lymphocytic myocarditis patients, a significant increase was observed for lymphocytes in the left atrial adipose tissue. In catecholamine-induced myocarditis patients, significant increases were found in the atria for all three inflammatory cell types. Infiltrating inflammatory cell numbers in the atrial myocardium correlated positively with those in the ventricles, especially in catecholamine-induced myocarditis patients. CONCLUSIONS: To a varying extent, atrial myocarditis occurs concurrently with ventricular myocarditis in patients diagnosed with myocarditis of different etiology. This provides a substrate that potentially predisposes myocarditis patients to the development of AF and subsequent complications such as sudden cardiac death and heart failure.

Analysis of morphological characteristics and expression levels of extracellular matrix proteins in skin wounds to determine wound age in living subjects in forensic medicine.

OBJECTIVE: Wound age determination in living subjects is important in routine diagnostics in forensic medicine. Macroscopical description of a wound to determine wound age however is inadequate. The aim of this study was to assess whether it would be feasible to determine wound age via analysis of morphological characteristics and extracellular matrix proteins in skin biopsies of living subjects referred to a forensic outpatient clinic. METHODS: Skin biopsies (n=101), representing the border area of the wound, were taken from skin injuries of known wound age (range: 4.5h-25 days) in living subjects. All biopsies were analyzed for 3 morphological features (ulceration, parakeratosis and hemorrhage) and 3 extracellular matrix markers (collagen III, collagen IV and α-SMA). For quantification, biopsies were subdivided in 4 different timeframes: 0.2-2 days, 2-4 days, 4-10 days and 10-25 days old wounds. Subsequently, a probability scoring system was developed. RESULTS: For hemorrhage, collagen III, collagen IV and α-SMA expression no relation with wound age was found. Ulceration was only found in wounds of 0.2-2, 2-4 and 4-10 days old, implying that the probability that a wound was more than 10 days old in case of ulceration is equal to 0%. Also parakeratosis was almost exclusively found in wounds of 0.2-2, 2-4 and 4-10 days old, except for one case with a wound age of 15 days old. The probability scoring system of all analyzed markers, as depicted above, however can be used to calculate individual wound age probabilities in biopsies of skin wounds of living subjects. CONCLUSIONS: We have developed a probability scoring system, analysing morphological characteristics and extracellular matrix proteins in superficial skin biopsies of wounds in living subjects that can be applied in forensic medicine for wound age determination.

Analysis of inflammatory cells and mediators in skin wound biopsies to determine wound age in living subjects in forensic medicine.

OBJECTIVE: In forensic medicine it is important to determine the age of skin wounds in living subjects. The aim of this study was to assess whether analysis of inflammatory cells and inflammatory mediators in skin biopsies of wounds from living subjects could improve wound age determination. METHODS: Biopsies (n=101), representing the superficial border area of a skin wound, were taken from skin injuries of known wound age (range: 4.5 hours to 25 days) of living subjects. All biopsies were analyzed for 3 inflammatory cell markers (MPO, CD45 and CD68) and 4 inflammatory mediators (MIP-1, IL-8, CML and vitronectin). For quantification, biopsies were subdivided in 4 different timeframes: 0.2-2 days, 2-4 days, 4-10 days and 10-25 days old wounds. Subsequently, a probability scoring system was developed. RESULTS: MPO, CD45, MIP-1, IL-8 (inflammatory cell markers) and N(epsilon)-(carboxymethyl)lysine (CML) positivity were maximal in wounds of 0.2-2 days old and then decreased in time. Remarkably, CD45, CD68 and CML showed a minor but non-significant increase again in 10-25 days old wounds. MPO and CD68 positivity was significantly lower in 4-25 days old wounds compared to 0.2-4 days old wounds. MPO positivity was also significantly lower in 10-25 days old wounds compared to 0.2-10 days old wounds. For CD45, MIP-1, IL-8 and CML no significant differences between the age groups were found. In case of vitronectin positivity in the extravasate or when the number of MIP-1 or IL-8-positive cells was more than 10 cells/mm(2) the probability that a wound was more than 10 days old was 0%. A probability scoring system of all analyzed markers can be used to calculate individual wound age probabilities in biopsies of skin wounds of living subjects. CONCLUSIONS: We have developed a probability scoring system of inflammatory cells and mediators that can be used to determine wound age in skin biopsies of living subjects.

A new method to determine wound age in early vital skin injuries: a probability scoring system using expression levels of Fibronectin, CD62p and Factor VIII in wound hemorrhage.

PURPOSE OF THE STUDY: In forensic autopsies it is important to determine the age of early vital skin wounds as accurate as possible. In addition to inflammation, coagulation is also induced in vital wounds. Analysis of blood coagulation markers in wound hemorrhage could therefore be an important additional discriminating factor in wound age determination. The aim of this study was to develop a wound age probability scoring system, based on the immunohistochemical expression levels of Fibronectin, CD62p and Factor VIII in wound hemorrhage. METHODS: Tissue samples of (A) non injured control skin (n=383), and samples of mechanically induced skin injuries of known wound age, (B) injuries inflicted shortly before death (up to a few minutes old) (n=382), and (C) injuries inflicted 15-30 min before death (n=42) were obtained at autopsy in order to validate wound age estimation. Tissue slides were stained for Fibronectin, CD62p and Factor VIII and were subsequently scored for staining intensity (IHC score) in wound hemorrhage (1=minor, 2=moderate, 3=strong positive). Finally, probability scores of these markers were calculated. RESULTS: In at most 14% of the non-injured control samples, hemorrhage was found, with mean±standard deviation IHC scores of 0.1±0.4, 0.2±0.4 and 0.2±0.5 for Fibronectin, CD62p, and Factor VIII, respectively. Expression of these markers significantly increased to mean IHC scores 1.4±0.8 (Fibronectin), 1.2±0.6 (CD62p), and 1.6±0.8 (Factor VIII) in wounds inflicted shortly before death (few minutes old) and to 2.6±0.5 (Fibronectin), 2.5±0.6 (CD62p), and 2.8±0.4 (Factor VIII) in 15-30 min old wounds. The probabilities that a wound was non vital in case of an IH score 0 were 87%, 88% and 90% for Fibronectin, CD62p, and Factor VIII, respectively. In case of an IHC score 1 or 2, the probabilities that a wound was a few minutes old were 82/90%, 82/83% and 72/93%. Finally, in case of an IHC score 3, the probabilities that a wound was 15-30 min old were 65%, 76% and 55%. CONCLUSIONS: Based on the expression of Fibronectin, CD62p and Factor VIII in wound hemorrhage, we developed a probability scoring system that can be used in forensic autopsies to improve wound age estimation in early skin injuries.

Prolonged C1 inhibitor administration improves local healing of burn wounds and reduces myocardial inflammation in a rat burn wound model.

In a previous study, the authors found persistent presence of acute inflammation markers such as C-reactive protein and complement factors locally in burn wounds. This persistence of acute inflammation may not only delay local burn wound healing but also have a systemic effect, for instance on the heart. Here, the effects of C1 esterase inhibitor (C1inh), an inhibitor of complement activation, on burn wound progression and the heart were analyzed in rats. Dorsal full-thickness burn wounds (2 × 4 cm) were induced on female Wistar rats (n = 14). The rats were divided into two groups (n = 7): a control group (just burns) and a C1inh group. C1inh was administered daily intravenously for 14 days. The burn wound, healthy skin from the hind leg (internal control), and the heart were then fixed in formalin. Tissues were analyzed for granulation tissue formation, reepithelialization, amount and type of infiltrating inflammatory cells (granulocytes and macrophages), and inflammatory markers (complement factors C3 and C4). C1inh treatment significantly reduced the amount of granulation tissue and significantly increased reepithelialization. C1inh also significantly reduced macrophage infiltration. Burns induced infiltration of macrophages into the ventricles of the heart and remarkably also into the atria of the heart. This effect could be counteracted by C1inh. These data show that systemic treatment with C1inh acts at different levels resulting in improved healing locally in burn wounds and systemically reduced inflammation in the heart. Therefore, C1inh might be a possible therapeutic intervention for burn wound patients.

PET/MRI of inflammation in myocardial infarction.

OBJECTIVES: The aim of this study was to explore post-myocardial infarction (MI) myocardial inflammation. BACKGROUND: Innate immune cells are centrally involved in infarct healing and are emerging therapeutic targets in cardiovascular disease; however, clinical tools to assess their presence in tissue are scarce. Furthermore, it is currently not known if the nonischemic remote zone recruits monocytes. METHODS: Acute inflammation was followed in mice with coronary ligation by 18-fluorodeoxyglucose ((18)FDG) positron emission tomography/magnetic resonance imaging, fluorescence-activated cell sorting, polymerase chain reaction, and histology. RESULTS: Gd-DTPA-enhanced infarcts showed high (18)FDG uptake on day 5 after MI. Cell depletion and isolation data confirmed that this largely reflected inflammation; CD11b(+) cells had 4-fold higher (18)FDG uptake than the infarct tissue from which they were isolated (p < 0.01). Surprisingly, there was considerable monocyte recruitment in the remote myocardium (approximately 10(4)/mg of myocardium, 5.6-fold increase; p < 0.01), a finding mirrored by macrophage infiltration in the remote myocardium of patients with acute MI. Temporal kinetics of cell recruitment were slower than in the infarct, with peak numbers on day 10 after ischemia. Quantitative polymerase chain reaction showed a robust increase of recruiting adhesion molecules and chemokines in the remote myocardium (e.g., 12-fold increase of monocyte chemoattractant protein-1), although levels were always lower than in the infarct. Finally, matrix metalloproteinase activity was significantly increased in noninfarcted myocardium, suggesting that monocyte recruitment to the remote zone may contribute to post-MI dilation. CONCLUSIONS: This study shed light on the innate inflammatory response in remote myocardium after MI.

Validation of ultrastructural analysis of mitochondrial deposits in cardiomyocytes as a method of detecting early acute myocardial infarction in humans.

In the present study, ultrastructural analysis of mitochondrial deposits (black dots within mitochondria) as a method for the detection of early acute myocardial infarction (AMI) was evaluated. In 24 patients with AMI and six controls, analysis was performed in the heart of infarcted patients and noninfarcted controls. In the infarction area in lactate dehydrogenase (LDH)-diagnosed AMI, the percentage of positive mitochondria was significantly higher compared to corresponding heart tissue in control patients and compared to noninfarcted areas within these patients. Also in patients with a clinically diagnosed AMI but no LDH decoloration, a significant higher percentage of positive mitochondria was found in the left ventricle compared to controls and noninfarcted areas. In patients with AMI, an increase in mitochondria with deposits was found in the infarction area compared to controls and noninfarcted tissue within the same patient, suggesting that electron microscopical changes in mitochondria can be used for the diagnosis of AMI less than 3 h old.

Acute inflammation is persistent locally in burn wounds: a pivotal role for complement and C-reactive protein.

Severe burns can cause major complications, such as infection and deforming scar formation. Burn wounds induce an excessive inflammatory response. Serum levels of complement and the acute phase reactant C-reactive protein (CRP) are upregulated in response to burn injury and have been shown to be related to the severity of burn trauma and to the clinical outcome. However, complement and CRP have not been investigated on a tissue level locally at the site of the burn trauma. Protein levels and localization of complement activation product C3d and CRP were determined semi-quantitatively in burn eschar between 2 and 46 days after injury, using immunohistochemistry. CD68 and myeloperoxidase (MPO), markers for macrophages and neutrophilic granulocytes, respectively, were also analyzed on these biopsies. Skin biopsies of very recent surgical wounds (seconds old) served as controls. C3d and CRP are present at high levels in the burn wound. Protein levels of both mediators are significantly elevated up to at least 46 days after injury in comparison with control wounds. In line with this, neutrophils and macrophages infiltrate the burn wound in high numbers up to at least 46 days after injury. The excessive presence of the inflammatory mediators, complement and CRP, and the increased infiltration of neutrophils and macrophages in burn wounds up to 46 days after injury implicate a persistent ongoing acute inflammation locally in the burn wound up to weeks after the initial trauma.