Hans Schwarzbach

The Open Microcirculation in Human Spleens: A Three-Dimensional Approach

It has long been debated whether the red pulp of human spleens harbors an open or a closed microcirculation or both. To solve this issue, the authors differentially stained the endothelium in red pulp arterial microvessels and in venous sinuses using brightfield and fluorescence immunohistology with reagents against CD34 and CD141. Three-dimensional models of red pulp arterial microvessels and sinuses were derived from serial double-stained paraffin sections with the help of license-free open-access software. In each model, arterial microvascular ends were traced and verified by reference to the original serial sections. In total, 142 ends were analyzed in the specimens of three individuals. None of these ends was connected to a sinus, suggesting that the human splenic red pulp harbors an entirely open circulatory system. Thus, the spleen is the only human organ where blood passes through spaces not lined by endothelia or other barrier-forming cells.

Phenotypic differences between red pulp capillary and sinusoidal endothelia help localizing the open splenic circulation in humans

The distribution of capillaries, sinuses and larger vessels was investigated by immunohistology in paraffin sections of 12 adult human spleens using a panel of antibodies. Double staining for CD34 and CD141 (thrombomodulin) revealed that capillary endothelia in the cords of the splenic red pulp and at the surface of follicles were CD34+CD141−, while red pulp sinus endothelia had the phenotype CD34−CD141+. Only in the direct vicinity of splenic follicles did sinus endothelial cells exhibit both antigens. Thus, splenic sinuses do not replace conventional capillaries, but exist in addition to such vessels. The endothelium in arterioles, venules and larger arteries and veins was uniformly CD34+CD141+. Anti-CD34 and anti-CD141 both additionally reacted with different types of splenic stromal cells. Differential staining of capillaries and sinuses may permit a three-dimensional reconstruction of serial sections to unequivocally delineate the “open” and “closed” splenic circulation in humans.

Morphological Alterations in Gastrocnemius and Soleus Muscles in Male and Female Mice in a Fibromyalgia Model

Background Fibromyalgia (FM) is a chronic musculoskeletal pain disorder, characterized by chronic widespread pain and bodily tenderness and is often accompanied by affective disturbances, however often with unknown etiology. According to recent reports, physical and psychological stress trigger FM. To develop new treatments for FM, experimental animal models for FM are needed to be development and characterized. Using a mouse model for FM including intermittent cold stress (ICS), we hypothesized that ICS leads to morphological alterations in skeletal muscles in mice. Methods Male and female ICS mice were kept under alternating temperature (4°C/room temperature [22°C]); mice constantly kept at room temperature served as control. After scarification, gastrocnemius and soleus muscles were removed and snap-frozen in liquid nitrogen–cooled isopentane or fixed for electron microscopy. Results In gastrocnemius/soleus muscles of male ICS mice, we found a 21.6% and 33.2% decrease of fiber cross sectional area (FCSA), which in soleus muscle concerns the loss of type IIa and IIx FCSA. This phenomenon was not seen in muscles of female ICS mice. However, this loss in male ICS mice was associated with an increase in gastrocnemius of the density of MIF+ (8.6%)-, MuRF+ (14.7%)-, Fbxo32+ (17.8%)-cells, a 12.1% loss of capillary contacts/muscle fiber as well as a 30.7% increase of damaged mitochondria in comparison with male control mice. Moreover, significant positive correlations exist among densities (n/mm2) of MIF+, MuRF+, Fbxo32+-cells in gastrocnemius/ soleus muscles of male ICS mice; these cell densities inversely correlate with FCSA especially in gastrocnemius muscle of male ICS mice. Conclusion The ICS-induced decrease of FCSA mainly concerns gastrocnemius muscle of male mice due to an increase of inflammatory and atrogenic cells. In soleus muscle of male ICS and soleus/gastrocnemius muscles of female ICS mice morphological alterations seem to occur not at all or delayed. The sex-specificity of findings, which is not easily reconciled with the epidemiology of FM (female predominance), implicate that gastrocnemius muscle of male ICS mice should preferentially be used for future investigations with FM. Moreover, we suggest to investigate morphological and/or molecular alterations at different time-points (up to two weeks) after ICS.

Age-related differences in skeletal muscle microvascular response to exercise as detected by contrast-enhanced ultrasound (CEUS)

Background Aging involves reductions in exercise total limb blood flow and exercise capacity. We hypothesized that this may involve early age-related impairments of skeletal muscle microvascular responsiveness as previously reported for insulin but not for exercise stimuli in humans. Methods Using an isometric exercise model, we studied the effect of age on contrast-enhanced ultrasound (CEUS) parameters, i.e. microvascular blood volume (MBV), flow velocity (MFV) and blood flow (MBF) calculated from replenishment of Sonovue contrast-agent microbubbles after their destruction. CEUS was applied to the vastus lateralis (VLat) and intermedius (VInt) muscle in 15 middle-aged (MA, 43.6±1.5 years) and 11 young (YG, 24.1±0.6 years) healthy males before, during, and after 2 min of isometric knee extension at 15% of peak torque (PT). In addition, total leg blood flow as recorded by femoral artery Doppler-flow. Moreover, fiber-type-specific and overall capillarisation as well as fiber composition were additionally assessed in Vlat biopsies obtained from CEUS site. MA and YG had similar quadriceps muscle MRT-volume or PT and maximal oxygen uptake as well as a normal cardiovascular risk factors and intima-media-thickness. Results During isometric exercise MA compared to YG reached significantly lower levels in MFV (0.123±0.016 vs. 0.208±0.036 a.u.) and MBF (0.007±0.001 vs. 0.012±0.002 a.u.). In the VInt the (post-occlusive hyperemia) post-exercise peaks in MBV and MBF were significantly lower in MA vs. YG. Capillary density, capillary fiber contacts and femoral artery Doppler were similar between MA and YG. Conclusions In the absence of significant age-related reductions in capillarisation, total leg blood flow or muscle mass, healthy middle-aged males reveal impaired skeletal muscle microcirculatory responses to isometric exercise. Whether this limits isometric muscle performance remains to be assessed.

Krill Oil-In-Water Emulsion Protects against Lipopolysaccharide-Induced Proinflammatory Activation of Macrophages In Vitro

Background: Parenteral nutrition is often a mandatory therapeutic strategy for cases of septicemia. Likewise, therapeutic application of anti-oxidants, anti-inflammatory therapy, and endotoxin lowering, by removal or inactivation, might be beneficial to ameliorate the systemic inflammatory response during the acute phases of critical illness. Concerning anti-inflammatory properties in this setting, omega-3 fatty acids of marine origin have been frequently described. This study investigated the anti-inflammatory and LPS-inactivating properties of krill oil (KO)-in-water emulsion in human macrophages in vitro. Materials and Methods: Differentiated THP-1 macrophages were activated using specific ultrapure-LPS that binds only on the toll-like receptor 4 (TLR4) in order to determine the inhibitory properties of the KO emulsion on the LPS-binding capacity, and the subsequent release of TNF-α. Results: KO emulsion inhibited the macrophage binding of LPS to the TLR4 by 50% (at 12.5 µg/mL) and 75% (at 25 µg/mL), whereas, at 50 µg/mL, completely abolished the LPS binding. Moreover, KO (12.5 µg/mL, 25 µg/mL, or 50 µg/mL) also inhibited (30%, 40%, or 75%, respectively) the TNF-α release after activation with 0.01 µg/mL LPS in comparison with LPS treatment alone. Conclusion: KO emulsion influences the LPS-induced pro-inflammatory activation of macrophages, possibly due to inactivation of the LPS binding capacity.

PACAP deficiency aggravates atherosclerosis in ApoE deficient mice

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) plays an important role in cytoprotection, inflammation and cardiovascular regulation. Thus, we studied the involvement of PACAP in atherogenesis. Differentiated human THP-1 macrophages (MΦ) were stimulated with oxidized low-density lipoproteins (oxLDL) and the influence of PACAP38 treatment on lipid content and TNF release was determined. To test the effect of PACAP deficiency (PACAP-/-) on the development of atherosclerosis under standard chow (SC) or cholesterol-enriched diet (CED) in vivo, PACAP-/- mice were crossbred with ApoE-/- to generate PACAP-/-/ApoE-/- mice. Blood cholesterol and triglyceride levels were quantified. Lumen stenosis in the brachiocephalic trunk, cellularity and amounts of pro-inflammatory as well as autophagy-, apoptosis- and necroptosis-relevant proteins were analysed in atherosclerotic plaques by quantitative immunohistochemistry. In vitro, PACAP38 inhibited oxLDL-induced intracellular lipid storage as well as TNF release in MФ. In vivo, after SC, but not under CED, PACAP-/-/ApoE-/- mice showed an increased lumen stenosis compared to ApoE-/- mice. In atherosclerotic plaques of PACAP-/-/ApoE-/- mice, the immunoreactive areas of TNF+, IL-1β+, autophagic, apoptotic and necroptotic cells were increased. In contrast, the overall cell density was decreased compared to ApoE-/- under SC, while no differences were seen under CED. Similar plasma cholesterol levels were observed in PACAP-/-/ApoE-/- and ApoE-/- mice under the respective feeding regime. Thus, PACAP-/-/ApoE-/- mice represent a novel mouse model of accelerated atherosclerosis where CED is not required. Our data indicate that PACAP acts as an endogenous atheroprotective neuropeptide. Thus, stable PACAP agonists may have potential as anti-atherosclerotic therapeutics. The specific PACAP receptor(s) mediating atheroprotection remain(s) to be identified.

Neurotrophic, Cytoprotective, and Anti-inflammatory Effects of St. John's Wort Extract on Differentiated Mouse Hippocampal HT-22 Neurons

Introduction: Since ancient times Hypericum perforatum L. named St. Johns wort (SJW), has been used in the management of a wide range of applications, including nervous disorders. Development of mood disorders are due to alterations in glutamate metabolism, initiation of inflammatory pathways, and changes of the neuronal plasticity. Previous studies suggest that the glutamatergic system contributes to the pathophysiology of depression. Extracts of SJW have been recommended for the treatment of depression. The aim of the present in vitro study was to evaluate the action of STW3-VI, a special SJW extract in differentiated mouse hippocampal HT-22 neurons. We evaluated the stimulation of neurogenesis, the protective effect against glutamate or N-methyl-D-aspartate receptor induced-excitotoxicity and its anti-inflammatory properties in LPS-activated human macrophages. Results: After 48 h treatment, STW3-VI stimulated the neurite formation by 25% in comparison with the control and showed protective effects against glutamate- or NMDA-induced cytotoxicity by significantly increasing the viability about +25 or +50%. In conjunction with these effects, after pretreatment with STW3-VI, the intracellular reduced glutathione content was significantly 2.3-fold increased compared with the neurons incubated with glutamate alone. Additionally, pre-treatment of human macrophages with STW3-VI showed anti-inflammatory effects after 24 or 48 h concerning inhibition of LPS-induced TNF release by −47.3 and −53.8% (24 h) or −25.0 to −64.8% (48 h). Conclusions: Our data provide new evidence that STW3-VI protects hippocampal cells from NMDA- or glutamate-induced cytotoxicity. Moreover, our results indicate a morphological remodeling by increasing neurite outgrowth and activation of the anti-inflammatory defense by inhibition of the cytokine production in human macrophages after STW3-VI treatment. These protective, neurotrophic and anti-inflammatory properties may be beneficial in the treatment of depressive disorders.