Jörg Faulhaber

Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold

K‐Cl co‐transporters are encoded by four homologous genes and may have roles in transepithelial transport and in the regulation of cell volume and cytoplasmic chloride. KCC3, an isoform mutated in the human Anderman syndrome, is expressed in brain, epithelia and other tissues. To investigate the physiological functions of KCC3, we disrupted its gene in mice. This severely impaired cell volume regulation as assessed in renal tubules and neurons, and moderately raised intraneuronal Cl− concentration. Kcc3−/− mice showed severe motor abnormalities correlating with a progressive neurodegeneration in the peripheral and CNS. Although no spontaneous seizures were observed, Kcc3−/− mice displayed reduced seizure threshold and spike‐wave complexes on electrocorticograms. These resembled EEG abnormalities in patients with Anderman syndrome. Kcc3−/− mice also displayed arterial hypertension and a slowly progressive deafness. KCC3 was expressed in many, but not all cells of the inner ear K+ recycling pathway. These cells slowly degenerated, as did sensory hair cells. The present mouse model has revealed important cellular and systemic functions of KCC3 and is highly relevant for Anderman syndrome.

The Apolipoprotein E Knockout Mouse: A Model Documenting Accelerated Atherogenesis In Uremia

Rodents do not develop spontaneous atherosclerosis. Currently, there is no good animal model to study the effect of uremia on atherosclerosis. This study evaluated whether apolipoprotein E knockout (Apoe-/-) mice are useful to study the effect of renal dysfunction on cardiovascular risk. Apoe-/- mice have decreased serum apolipoprotein E and exhibit lipid abnormalities and atherosclerosis even on a low-cholesterol diet. Ten-wk-old Apoe-/- mice were subtotally nephrectomised (SNX Apoe-/-; n = 8), uninephrectomised (UNX Apoe-/-; n = 5), or sham-operated (sham Apoe-/-; n = 5) and compared with their genetic controls (SNX C57/BL6; UNX C57/BL6; sham C57/BL6). After 12 wk, BP was measured intraarterially, blood samples were taken, and the experiment was terminated by perfusion fixation. The heart weight was determined, and quantitative morphologic analysis of intramyocardial arteries and aortic changes was performed. At the end of the experiment, heart weight and relative left ventricular weight were comparable in all groups. Intraarterial BP was somewhat higher in Apoe-/- mice compared with controls. Baseline serum cholesterol and triglyceride levels were higher in Apoe-/- mice than in C57/BL6. Atherosclerotic plaques were not present in sham or UNX C57/BL6, but minor plaque formation was noted in some SNX control animals. In contrast, beginning plaques were seen even in untouched Apoe-/- mice, and strikingly increased plaque formation was noted in UNX and SNX Apoe-/- mice. Maximal plaque diameter (cross-section) was 37 +/- 74 micro m in SNX C57/BL6, 191 +/- 90 micro m in sham Apoe-/-, 323 +/- 66 micro m in UNX Apoe-/-, and 457 +/- 17 micro m in SNX Apoe-/-. The plaque morphology corresponded with that of early plaques characterized by foam cells and virtual absence of lymphocytes or smooth muscle cell infiltration. In conclusion, even mild renal dysfunction, i.e., after uninephrectomy, causes a dramatic increase in plaque size and aggressive morphology (foam cell rich soft plaques) in the animal model of the Apoe-/- mouse.

Regional alterations of repolarizing K+ currents among the left ventricular free wall of rats with ascending aortic stenosis

1 The effect of cardiac hypertrophy on electrocardiogram (ECG), action potential duration (APD) and repolarizing K+ currents was investigated in epicardial, midmyocardial and endocardial myocytes isolated from the rat left ventricular free wall. 2 Cardiac hypertrophy was induced by stenosis of the ascending aorta (AS), which led to an increased pressure load (+85 ± 10 mm) of the left ventricle; sham‐operated animals served as controls. 3 In ECG recordings from AS rats, the QTc interval was prolonged and the main vectors of the QRS complex and the T‐wave pointed in opposite directions, indicating an abnormal sequence of repolarization. 4 APD and K+ currents were recorded using the whole‐cell patch‐clamp technique. In the AS group, APD90 (90 % repolarization) was significantly prolonged in epicardial and midmyocardial, but not endocardial myocytes. 5 Corresponding to the increase in APD, the magnitude of the transient outward K+ current (Ito1) was significantly smaller (‐30 %) in epicardial and midmyocardial, but not endocardial myocytes. 6 Inactivation and steady‐state inactivation of Ito1 were not affected by hypertrophy. Recovery from inactivation was slightly prolonged in endocardial myocytes from AS rats. 7 No differences in delayed rectifier currents (IK) or inwardly rectifying K+ currents (IK1) were detected between myocytes of the three regions of sham‐operated or AS animals. However, both currents were reduced by AS. 8 The present data show that cardiac hypertrophy caused by pressure overload leads to an increase in APD and a decrease in Ito1 primarily in epicardial and midmyocardial myocytes, which implies a major role of alterations in Ito1 for the reduced gradient in APD. The effects of AS on IK1 and IK may slightly counteract the decrease in APD gradient. The observed changes in APD and underlying ionic currents could well explain the alterations in repolarization observed in the ECG induced by cardiac hypertrophy.

Chronic ETA Receptor Blockade Attenuates Cardiac Hypertrophy Independently of Blood Pressure Effects in Renovascular Hypertensive Rats

In isolated cardiac myocytes, the direct effects of angiotensin II on cellular growth and gene expression were shown to be mediated by endothelin via the endothelin subtype A (ETA) receptor. To determine whether this pathway is also involved in the cardiovascular adaptations to a chronic activation of the renin-angiotensin system in vivo, the effects of a selective ETA receptor antagonist (LU 127043) were investigated in adult rats with renal artery stenosis. Four groups of rats (n=107) were studied over a period of 10 days after surgery: (1) sham-operated animals with saline administration, (2) rats subjected to left renal artery clipping with saline administration, (3) sham-operated rats with LU 127043 administration, and (4) rats subjected to left renal artery clipping with LU 127043 administration. LU 127043 (50 mg/kg) or saline was given by gavage twice daily starting 1 day before the operation. In clipped rats with saline administration, plasma renin activity, the ratio of left ventricular weight to body weight, and mRNAs for beta-myosin heavy chain and atrial natriuretic peptide were significantly elevated as early as 2 days after surgery. Blood pressure started to rise on the third postoperative day and attained a steady state hypertensive level by day 6. Blockade of ETA receptors had no effects on plasma renin activity or the time course of hypertension in clipped animals but completely prevented left ventricular hypertrophy and the re-expression of the beta-myosin heavy chain and atrial natriuretic peptide genes on day 2. While the expressions of the beta-myosin heavy chain and atrial natriuretic peptide genes were not different from saline-treated, clipped animals after day 4, the development of left ventricular hypertrophy remained markedly blunted (-50%) during ETA receptor blockade until day 10. These results show that a continuous blockade of ETA receptors significantly attenuates the development of left ventricular hypertrophy and, more transiently, fetal gene expression in the early phase of renovascular hypertension. Since neither blood pressure nor the increase in plasma renin activity was significantly altered by ETA receptor blockade, the inhibitory influences of the ETA receptor antagonist on left ventricular hypertrophy and gene expression were mediated most likely through a direct blockade of myocardial ETA receptors.

NADPH Oxidase Accounts for Enhanced Superoxide Production and Impaired Endothelium-Dependent Smooth Muscle Relaxation in BKβ1−/− Mice

Objective—Nitric oxide (NO)-induced vasorelaxation involves activation of large conductance Ca2+-activated K+ channels (BK). A regulatory BK&bgr;1 subunit confers Ca2+, voltage, and NO/cGMP sensitivity to the BK channel. We investigated whether endothelial function and NO/cGMP signaling is affected by a deletion of the &bgr;1-subunit. Methods and Results—Vascular superoxide in BK&bgr;1−/− was measured using the fluorescent dye hydroethidine and lucigenin-enhanced chemiluminescence. Vascular NO formation was analyzed using electron paramagnetic resonance (EPR), expression of NADPH oxidase subunits, the endothelial NO synthase (eNOS), the soluble guanylyl cyclase (sGC), as well as the activity and expression of the cyclic GMP-dependent kinase I (cGK-I) were assessed by Western blotting technique. eNOS, sGC, cGK-I expression and acetylcholine-induced NO production were unaltered in Bk&bgr;1−/− animals, whereas endothelial function was impaired and the activity of the cGK-I was reduced. Vascular O2− and expression of the NADPH oxidase subunits p67phox and Nox1 were increased. Endothelial dysfunction was normalized by the NADPH oxidase inhibitor apocynin. Potassium chloride- and iberiotoxin-induced depolarization mimicked the effect of BK&bgr;1-deletion by increasing vascular O2− in an NADPH-dependent fashion. Conclusions—The deletion of BK&bgr;1 causes endothelial dysfunction by increasing O2− formation via increasing activity and expression of the vascular NADPH oxidase.

Removal of the Outer Table of the Skull for Reconstruction of Full-Thickness Scalp Defects with a Dermal Regeneration Template

BACKGROUND The reconstruction of large defects of the scalp after wide excisional surgery of cutaneous malignancies is challenging. When the pericranium must be resected due to safety considerations, the exposed bone complicates reconstructive approaches. OBJECTIVE The objective was to develop an improved technique for reconstructive surgery of full-thickness scalp defects. METHODS Full-thickness scalp defects of up to 126 cm2 in size with exposure of denuded bone were treated by partial removal of the outer table of the skull. The diploic space exposed by this treatment showed petechial bleeding and was covered with a dermal regeneration template (Integra, Integra Lifesciences Corp., Plainsboro, NJ). After transformation of the template by vascularization and by proliferation of fibroblasts, an ultrathin skin graft was transplanted onto the neodermis. RESULTS Thirteen patients with cutaneous malignancies of the scalp were treated using this technique. The defects were transplanted within a median postoperative time period of 29 days (± 4 days) and showed good cosmetic results and stable scars. After a follow-up period of 6 months, no local recurrences were observed. CONCLUSION This reconstructive procedure allows closure and rapid healing of large scalp defects in which the pericranium had to be resected.

Neurogenic mechanisms contribute to hypertension in mice with disruption of the K-Cl cotransporter KCC3

The neurodegenerative disorder Andermann syndrome is caused by mutations of the K-Cl cotransporter KCC3. Mice with a targeted disruption of the corresponding gene, Slc12a6, reproduce neurodegeneration of the peripheral and central nervous system (CNS) and display arterial hypertension. Kcc3 is expressed in numerous tissues, including the CNS and vascular smooth muscle cells. As the intracellular chloride concentration may influence myogenic tone and hence blood pressure, we measured the chloride concentration in vascular smooth muscle cells. It was indeed increased in superficial brain arteries and saphenous arteries of Kcc3−/− mice. Isolated saphenous arteries and their third-order branches, however, reacted indistinguishably to changes in intravascular pressure, stimulation of &agr;1-adrenoreceptors, exogenous nitric oxide, or blockade of calcium-activated chloride channels. Likewise, the responses to &agr;1-adrenergic stimulation or exogenous nitric oxide in vivo were identical in both genotypes. These results argue against a major vascular-intrinsic component of arterial hypertension in Kcc3−/− mice. In contrast, either &agr;1-adrenergic blockade or inhibition of ganglionic transmission abolished the difference in arterial blood pressure between both genotypes. This demonstrates a neurogenic component in the maintenance of this phenotype, which is further supported by an increase of urinary norepinephrine and epinephrine excretion in Kcc3−/− mice. Our data indicate that local control of myogenic tone does not require KCC3 and that hypertension in Kcc3−/− mice depends on an elevated sympathetic tone.

Endothelin Mediates Local and Systemic Disease Sequelae in Severe Experimental Pancreatitis

Endothelin-1 has been shown to reduce pancreatic blood flow and cause focal acinar cell necrosis similar to those seen in acute pancreatitis (AP), whereas therapy with endothelin receptor antagonists enhanced pancreatic capillary blood flow (PCBF) and decreased mortality rates. The current study evaluated the role of endothelin in the development of severe AP. Trypsinogen activation peptides, acinar cell necrosis, and PCBF were used as local indicators of disease severity, fluid sequestration, cardiorespiratory and renal parameters, and colonic capillary blood flow as systemic disease indicators. The following groups of animals were examined: 1) rats with mild edematous AP and 2) severe necrotizing AP treated with and without endothelin, 3) transgenic rats overexpressing endothelin with severe AP, and 4) rats with severe AP prophylactically treated with endothelin receptor antagonists. The following observations were made: endothelin superimposed on mild AP caused hemoconcentration, a decrease in PCBF, and necrosis and ascites not seen in this model without endothelin exposure. Endothelin superimposed on severe AP had no significant effects. After induction of severe AP, less PCBF and more acinar cell necrosis were observed in transgenic rats than in their normal littermates. Prophylactic endothelin receptor antagonists improved local (acinar necrosis, PCBF) and systemic parameters (ascites, urine production, colonic capillary blood flow) of disease severity in animals with severe AP. These observations underscore the role of endothelin as a mediator of disease severity in AP and suggest that endothelin receptor blockade may become a promising therapeutic tool in this disease.

One-stage reconstruction of deep facial defects with a single layer dermal regeneration template.

Background  The reconstruction of deep facial wounds in oncological surgery is challenging. Especially for elderly multimorbid patients, a rapid procedure with acceptable aesthetic and reliable functional outcome is required. Recently, a new single layer skin substitute was developed. Integra® dermal regeneration template single layer (IDRT‐SL) allows one‐stage surgery in combination with split thickness skin grafting. However, no study has yet analysed the efficiency of IDRT‐SL treatment.

Functional and Aesthetic Reconstruction of Full-Thickness Defects of the Lower Lip After Tumor Resection: Analysis of 59 Cases and Discussion of a Surgical Approach

BACKGROUND Carcinoma of the lower lip is a common cancer of the head and neck region. The accepted standard of care is surgical resection and reconstruction. The delicate location poses a challenge to surgeons because they have to aim for conflicting goals: complete oncological resection and functional and aesthetic restoration. For over a century, this challenge led to the development of more than 100 different reconstructive procedures. OBJECTIVE To develop a standardized surgical approach to simplify selection of an adequate reconstructive procedure. MATERIALS AND METHODS We performed a review of the literature and a retrospective analysis of 59 patients treated according to a standardized surgical approach over the past 5 years. RESULTS By taking into account the anatomic location, the width of the defect, and the amount of tissue loss, three flowcharts have been developed to guide the physician to a selection of suitable reconstructive procedures for each case. CONCLUSION This surgical approach facilitated the successful reconstruction of all encountered defects, and over a mean period of 31 months, no recurrences or major complications were seen. The authors have indicated no significant interest with commercial supporters.