Roland Blumer

Selective downregulation of VEGF-A(165), VEGF-R(1), and decreased capillary density in patients with dilative but not ischemic cardiomyopathy.

Cardiomyopathy (CM) comprises a heterogeneous group of diseases, including ischemic (ICM) and dilative (DCM) forms. The pathogenesis of primary DCM is not clearly understood. Recent studies in mice show that vascular endothelial growth factor (VEGF) is involved in ICM. Whether VEGF plays a role in human CM is unknown. We examined the mRNA and protein expression of VEGF and its receptors in hearts of patients with end-stage DCM and ICM and in healthy individuals using real-time polymerase chain reaction and Western blotting. Number of capillaries, area of myocytes, and collagen were calculated in cardiac biopsies using transmission electron microscopy. In DCM, except for VEGF-C, mRNA transcript levels of VEGF-A165, VEGF-A189, and VEGF-B and the protein level of VEGF-A and VEGF-R1 were downregulated compared with controls (P <0.05). However, in ICM, mRNA transcript levels of VEGF isoforms and protein levels of VEGF-C were upregulated. The vascular density was decreased in DCM but increased in ICM compared with controls (P <0.05). Muscular hypertrophy was not different for ICM and DCM, although DCM had more collagen (P <0.05). Blunted VEGF-A and VEGF-R1 protein expression and downregulated mRNA of the predominant isoform of VEGF-A, VEGF-A165, to our knowledge shown here for the first time, provide evidence that the VEGF-A defect in DCM is located upstream. Whether downregulation of certain VEGF isoforms in DCM is a cause or consequence of this disorder remains unclear, although upregulated VEGF levels in ICM are most likely the result of ischemia.

Impaired VE-Cadherin/β-Catenin Expression Mediates Endothelial Cell Degeneration in Dilated Cardiomyopathy

Background—The cross-talk between vascular endothelial growth factor (VEGF)-A, angiopoietin (Ang), and VE-cadherin coregulates endothelial cell (EC) survival. Cardiac expression of VEGF-A but not its receptor KDR is blunted in dilated cardiomyopathy (DCM). Whether VE-cadherin/Ang function is affected in DCM is unknown. Methods and Results—The myocardial expression of VE-cadherin/&bgr;-catenin, Ang-1, Ang-2, and their receptor Tie-2 was examined in DCM, ischemic cardiomyopathy (ICM), and in control subjects through the use of real-time RT-PCR, Western blotting, and immunocytochemistry. EC degeneration was quantified by TEM. RNA interference against VE-cadherin and VEGF deprivation and stimulation were applied to cultured DCM myocardium and human microvascular ECs to examine the interplay between VEGF, VE-cadherin/&bgr;-catenin, and Ang-2. Analysis of tissue sections with similar rates of EC degeneration in both patient groups showed that VE-cadherin/&bgr;-catenin expression was downregulated in DCM only (P <0.05). Although Ang-1 was not changed, Ang-2 expression was downregulated and Tie-2 protein expression was upregulated both in DCM and ICM (P <0.05). The ratio of degenerated to normal ECs was significantly higher in DCM versus ICM (P <0.05). Targeted VE-cadherin gene silencing in cultured human ECs resulted in similar degenerative effects observed in myocardial ECs of DCM patients. In vitro experiments indicated that VE-cadherin/&bgr;-catenin expression is independent of VEGF. Conclusions—These results indicate for the first time that the EC survival is impaired in myocardium of patients with DCM involving VE-cadherin/&bgr;-catenin, probably independent of VEGF. Targeting VE-cadherin might be of benefit to counteract the selective EC pathology in DCM.

Development of ‘No-Reflow’ Phenomenon in Ischemia/Reperfusion Injury: Failure of Active Vasomotility and Not Simply Passive Vasoconstriction

Background/Aim: Local blood flow failure (no-reflow phenomenon) during ischemia/reperfusion (I/R) injury may be mediated by interstitial edema formation (passive vasoconstriction) and/or microvascular spasm (active vasoconstriction). The development of the no-reflow phenomenon in the rabbit hind limb I/R model and the influence of treatment with L-arginine and/or antioxidative vitamins were investigated. Methods: Untreated rabbits were compared with those treated with L-arginine (4 mg/kg/min) or antioxidative vitamins (0.4 ml/kg) alone or in combination during hind limb I/R (2.5/2 h). Interstitial edema formation and microvessel diameter alterations were measured morphometrically. Capillary blood perfusion was measured continuously with laser Doppler flowmetry. Results: I/R injury was expressed by interstitial edema formation (interstitial space increase by 80%), microvascular constriction (microvessel cross-sectional area decrease by 30%), and development of no-reflow phenomenon (blood flow reduction by 60%). Treatment with antioxidative vitamins alone or L-arginine alone reduced interstitial edema by 22 and 31%, consequently, while combined L-arginine/antioxidative vitamin treatment showed a more pronounced edema reduction by 40%. Treatment with only antioxidative vitamins failed to influence the development of no-reflow, although interstitial edema formation was reduced. L-Arginine treatment alone or in combination with antioxidative vitamins prevented microvascular constriction and preserved blood flow after reperfusion without development of no-reflow despite still apparent interstitial edema. Conclusions: Affections of active vasomotility and not merely passive changes of external pressure (i.e., interstitial edema formation) should be considered important in the development of microvascular constriction during ‘no-reflow’ phenomenon.

Presence and structure of innervated myotendinous cylinders in sheep extraocular muscle

Innervated myotendinous cylinders (IMCs) were for the first time described in a sheep extraocular muscle (EOMs). They were found at the distal myotendinous junction of a medial rectus and were investigated by light and transmission electron microscopy. The IMCs are enveloped by a multi-layered capsule of fibrocytes and each contains the terminal portion of one multiply-innervated muscle fibre and its corresponding tendon. The tendinous compartment of the IMC is entered by a single nerve fibre which, inside, spreads into several terminal branches. Numerous terminal branches were found among the collagen fibrils but few on the muscle fibre tips. Nerve terminals contain mitochondria and are full of clear vesicles. Within the nerve terminals, vesicles are often concentrated in an area where the axolemma exhibits dense patches. Innervated myotendinous cylinders of sheep EOMs exhibit the same ultrastructural features as those earlier described as palisade endings or myotendinous cylinders in cat, monkey and man.

Muscle spindles and Golgi tendon organs in bovine calf extraocular muscle studied by means of double-fluorescent labeling, electron microscopy, and three-dimensional reconstruction

In the present study muscle spindles (MSps) and Golgi tendon organs (GTOs) in bovine extraocular muscles (EOMs) were analyzed in detail. The innervation pattern of these proprioceptors was investigated with transmission electron microscope and confocal laser scanning microscope after double-fluorescent labeling. Three-dimensional (3D) reconstructions were performed of GTOs. Muscle spindles. MSps are numerous, each containing two nuclear bag fibers and up to eight nuclear chain fibers. In the equatorial region and paraequatorial region thin axons enwrapping the intrafusal muscle fibers form numerous nerve contacts on the muscle fiber surface. Double staining of such nerve terminals with synaptophysin and alpha-bungarotoxin and their fine structural features confirm their sensory nature. In the encapsulated part of the polar region neuromuscular contacts have structural features of motor nerve terminals and stain positively with alpha-bungarotoxin. Golgi tendon organs. GTOs are numerous in bovine EOMs. Each GTO contains collagen bundles but frequently also intracapsular muscle fibers. Intracapsular muscle fibers either terminate inside the GTO in collagen bundles or pass through the proprioceptor. GTOs are richly supplied with sensory nerve terminals which intermingle with the collagen bundles. Nerve terminals on intracapsular muscle fibers exhibit fine structural characteristics of motor nerve terminals and are alpha-bungarotoxin positive. The 3D images of GTOs show the detailed spatial arrangement of the GTO tissue components. These new insights in the complex and specific morphology of MSps and GTOs in bovine EOMs indicate that we deal with highly developed proprioceptors. These are supposed to provide important information for EOM innervation.

Evidence that the extraocular motor nuclei innervate monkey palisade endings

Research highlights ▶ Neuronal tracer was injected into the abducens nucleus of monkey. ▶ Tracer positive axons were observed in the lateral rectus muscles. ▶ Tracer positive axons were cholinergic and established motor terminals. ▶ Palisade endings in the lateral rectus contained tracer as well. ▶ This study shows that palisade endings originate from motor nuclei.

Atomic force microscopy imaging of the human trigeminal ganglion.

This paper describes an investigation of gangliocytes via imaging semithin sections of two human trigeminal ganglia with an atomic force microscope (AFM). Whereas semithin sections are usually employed for transmission electron microscopy, we adopted this special type of sample preparation for our AFM studies to extract topographical data from the gangliocyte itself and from the nucleus, the nucleolus, the crystal-arranged lipofuscin granules, and the cell-surrounding mantle cells; simultaneously we characterized the samples with error signal mode. This AFM-related technique revealed no information concerning friction force and elasticity due to the presence of the embedding material (epoxy), but it gave additional topographical contrast. These are the first images of the human trigeminal ganglion by AFM.

Presence and morphological variability of Golgi tendon organs in the distal portion of sheep extraocular muscle

This study was undertaken to demonstrate the presence of Golgi tendon organs (GTOs) in the distal portion of sheep extraocular muscle (EOM) and to describe the morphological variability of these receptors. Extraocular muscles of a young and an old sheep were perfusion fixed and/or immersion fixed. Tissue was prepared for light microscopy and transmission electron microscopy. Immunohistochemistry was done to demonstrate the myosin pattern of the intracapsular muscle fibers of the GTOs. All GTOs in the distal portions of the sheep EOMs were located in a distinct muscle layer which was designated in a former investigation as the so‐called peripheral patch layer. Each EOM of the young sheep contained GTOs; between four and 15 GTOs were counted in the rectus EOMs. Eight GTOs were found in the superior rectus of the old sheep. Golgi tendon organs in EOMs of the young and the old sheep did not differ in their morphology. In the young sheep the mean length of the GTOs was 447 ± 132 μm (n = 60) and their mean width 101 ± 26 μm (n = 60). In the old sheep values were 576 ± 188 μm (mean length, n = 8) and 103 ± 18 μm (mean width, n = 8). The GTOs were encapsulated by perineurial cells. In 12 GTOs, only collagen bundles were inside. In the remaining GTOs (56), intracapsular muscle fibers were present. Muscle fibers entered the proximal poles of the GTOs and either terminated inside the receptors or muscle fibers left the GTOs at their distal poles. These intracapsular muscle fibers were of the multiply‐innervated type. In the GTOs variably shaped nerve terminals were found which contained a high number of mitochondria. In two GTOs, additionally, nerve terminals with aggregates of densely packed vesicles were present. Anat Rec 258:359–368, 2000.

Axons Giving Rise to the Palisade Endings of Feline Extraocular Muscles Display Motor Features

Palisade endings are nerve specializations found in the extraocular muscles (EOMs) of mammals, including primates. They have long been postulated to be proprioceptors. It was recently demonstrated that palisade endings are cholinergic and that in monkeys they originate from the EOM motor nuclei. Nevertheless, there is considerable difference of opinion concerning the nature of palisade ending function. Palisade endings in EOMs were examined in cats to test whether they display motor or sensory characteristics. We injected an anterograde tracer into the oculomotor or abducens nuclei and combined tracer visualization with immunohistochemistry and α-bungarotoxin staining. Employing immunohistochemistry, we performed molecular analyses of palisade endings and trigeminal ganglia to determine whether cat palisade endings are a cholinergic trigeminal projection. We confirmed that palisade endings are cholinergic and showed, for the first time, that they, like extraocular motoneurons, are also immunoreactive for calcitonin gene-related peptide. Following tracer injection into the EOM nuclei, we observed tracer-positive palisade endings that exhibited choline acetyl transferase immunoreactivity. The tracer-positive nerve fibers supplying palisade endings also established motor terminals along the muscle fibers, as demonstrated by α-bungarotoxin. Neither the trigeminal ganglion nor the ophthalmic branch of the trigeminal nerve contained cholinergic elements. This study confirms that palisade endings originate in the EOM motor nuclei and further indicates that they are extensions of the axons supplying the muscle fiber related to the palisade. The present work excludes the possibility that they receive cholinergic trigeminal projections. These findings call into doubt the proposed proprioceptive function of palisade endings.

Multivitamin administration before ischemia reducesischemia-reperfusion injury in rabbit skeletal muscle

This study investigated the effect of a multivitamin preparation administered before ischemia or before reperfusion, on ischemia-reperfusion (I/R) injury of skeletal muscle. An in vivo hindlimb skeletal muscle I/R model (2.5 h/2 h) was carried out on adult New Zealand white rabbits. Animals used as I/R models were treated with a multivitamin preparation (0.4 ml/kg bw i.v. bolus), containing alpha-tocopherol, ascorbic acid, retinol, vitamin B complex, 30 min before starting ischemia (group MV(isc)) or 5 min before reperfusion (group MV(rep)) and compared to animals with I/R without treatment (group IR) and sham operated animals (group SHAM). Interstitial edema (muscle interfiber area, %MIFA) and changes in microvessel size (microvessel cross sectional area, MVCSA, microm(2)) were measured. Plasma malondialdehyde concentrations (MDA-TBA, nmol/ml) served as a measure of lipid peroxidation. After 2h of reperfusion, ischemia-reperfusion developed a significant microvascular constriction and an interstitial edema (IR, vs SHAM;P<< 0.01), but administration of antioxidative vitamins before the onset of ischemia reduced microvascular constriction and edema formation (P<< 0.05 vs IR group). In a similar manner, administration of vitamins before ischemia lowered plasma MDA-TBA levels as compared to the untreated group during reperfusion (p<< 0. 05). In animals treated with vitamins before reperfusion, the biochemical and morphological results showed no differences as compared to the untreated group. Antioxidative treatment with a multivitamin preparation exerted a beneficial effect on I/R injury of skeletal muscle when the aforementioned vitamins were administered before ischemia but not before the onset of reperfusion.