Die Angiotensin II AT2-Rezeptor vermittelte funktionelle und morphologische Regeneration peripherer Nerven am Modell der experimentellen Läsion des Nervus ischiadicus der Ratte

Die vorliegende Arbeit untersuchte den Einfluß ANG II insbesondere seiner Rezeptoren AT1- und AT2 auf die funktionelle Regeneration peripherer Nerven am Modell der experimentellen Läsion des N. ischiadicus der Ratte. Nach operativer Quetschläsion des N. ischiadicus erfolgte durch einen mit einer osmotischen Minipumpe verbundenen Katheter eine kontinuierliche lokake Applikation von Wirkstoffe direkt an der Läsionsstelle. Vom 10. und 17. Tag bis zum 20. Tag post laesionem wurden zwei funktionelle Untersuchungsmethoden (Fußspannenanalyse, Elektrostimulationstest der Fußsohle) durchgeführt. Es zeigte sich gegenüber physiologischer Kochsalz-Lösung eine konzentrationsabhängige signifikante Regenerationsverbesserung durch ANG II, wobei sich eine Konzentration von 10-11 mol/l am effektivsten erwies und höhere Konzentrationen entweder weniger neurotroph (10-9 mol/l) bzw. regenerationshemmend (10-7 mol/l) wirkten. Durch ein verlängertes Untersuchungsintervall von 32 Tagen wurde deutlich, dass es sich bei dem beobachteten neurotrophen ANG II-Effekt eher um eine Beschleunigung der funktionellen Regeneration handelt. Die beobachteten Effekte konnten durch selektive Rezeptorblockade eindeutig dem AT2-Rezeptor zugeordnet werden. Am Ende des Versuchprotokolls wurden Schnitte des N. ischiadicus mittels computergestützter morphometrischer Analyse der Axone und Myelinscheiden durch Elektronenmikroskopie und Myelinscheidenfärbung mit Paraphenylendiamin, Analyse von Qualität und Quantität der regenerierten Nervenfasern, Bestimmung des Myelinisierungsgrades sowie mittels immunhistochemischer Färbung gegen „growth associated protein“ (GAP-43, ein Maker für neuaussprossende Nervenfasern) morphologische Untersuchungen durchgeführt, die bestätigten, dass ANG II als neurotropher Faktor AT2-Rezeptor-vermittelt die Nervenregeneration beschleunigen kann

The JNK Inhibitor XG-102 Protects against TNBS-Induced Colitis

The c-Jun N-terminal kinase (JNK)-inhibiting peptide D-JNKI-1, syn. XG-102 was tested for its therapeutic potential in acute inflammatory bowel disease (IBD) in mice. Rectal instillation of the chemical irritant trinitrobenzene sulfonic acid (TNBS) provoked a dramatic acute inflammation in the colon of 7–9 weeks old mice. Coincident subcutaneous application of 100 µg/kg XG-102 significantly reduced the loss of body weight, rectal bleeding and diarrhoea. After 72 h, the end of the study, the colon was removed and immuno-histochemically analysed. XG-102 significantly reduced (i) pathological changes such as ulceration or crypt deformation, (ii) immune cell pathology such as infiltration and presence of CD3- and CD68-positive cells, (iii) the production of tumor necrosis factor (TNF)-α in colon tissue cultures from TNBS-treated mice, (iv) expression of Bim, Bax, FasL, p53, and activation of caspase 3, (v) complexation of JNK2 and Bim, and (vi) expression and activation of the JNK substrate and transcription factor c-Jun. A single application of subcutaneous XG-102 was at least as effective or even better depending on the outcome parameter as the daily oral application of sulfasalazine used for treatment of IBD

Miswiring the brain:delta(9)-tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin-2 degradation pathway

Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments. Psychoactive constituents from Cannabis spp., particularly Δ(9)-tetrahydrocannabinol (THC), bind to cannabinoid receptors in the fetal brain. However, it is unknown whether THC can trigger a cannabinoid receptor-driven molecular cascade to disrupt neuronal specification. Here, we show that repeated THC exposure disrupts endocannabinoid signaling, particularly the temporal dynamics of CB(1) cannabinoid receptor, to rewire the fetal cortical circuitry. By interrogating the THC-sensitive neuronal proteome we identify Superior Cervical Ganglion 10 (SCG10)/stathmin-2, a microtubule-binding protein in axons, as a substrate of altered neuronal connectivity. We find SCG10 mRNA and protein reduced in the hippocampus of midgestational human cannabis-exposed fetuses, defining SCG10 as the first cannabis-driven molecular effector in the developing cerebrum. CB(1) cannabinoid receptor activation recruits c-Jun N-terminal kinases to phosphorylate SCG10, promoting its rapid degradation in situ in motile axons and microtubule stabilization. Thus, THC enables ectopic formation of filopodia and alters axon morphology. These data highlight the maintenance of cytoskeletal dynamics as a molecular target for cannabis, whose imbalance can limit the computational power of neuronal circuitries in affected offspring

Apoptosis.

<p>Western blot analysis of (A) caspase-3 and cleaved caspase-3, (B) Bax and Bim, and (C) FasL and p53 from colon extracts of untreated controls (co) 12 h, 24 h and 72 h following trinitrobenzene sulfonic acid (TNBS) administration without or with XG-102 (100 µg/kg sc.). These blots are representative of 3 independent experiments.</p

Production of TNF-α.

<p>TNF-α release (pg/ml) into the supernatant of organic colon culture from normal mice, following trinitrobenzene sulfonic acid (TNBS) only, and treatment with sc. 100 µg/kg XG-102. ** = p<0.01 for all groups compared with TNBS group.</p

c-Jun and phospho-c-Jun.

<p>Western blot analysis of c-Jun and phospho-c-Jun in colon homogenates from untreated controls (co) and 12 h or 24 h following trinitrobenzene sulfonic acid (TNBS) administration without or with XG-102 (100 µg/kg sc.). These blots are representative of 3 independent experiments.</p

JNK2-Bim co-precipitation.

<p>JNK1 and JNK2 immunoprecipitates (IP) from colon tissue homogenates were analyzed by Western blotting with an anti-Bim antibody. Pounceau staining demonstrated equal loading (data not shown).</p

Representative CD3 and CD68 immunofluorescence.

<p>Representative CD3 (left) and CD68 (right) immunofluorescence of the distal colon from normal mice, trinitrobenzene sulfonic acid (TNBS) administration and treatment with sc. 100 µg/kg XG-102.</p

Hematoxylin and eosin (H&E) scores.

<p>Hematoxylin and eosin (H&E) scores from distal (A) and medial (B) colon. For the tissue damage score (hatched bars), the scores of ulcer, crypts and submucosa were summed-up for each individual animal, and the mean±SEM was calculated for each group. The mean±SEM of the infiltration score (grey bar) is separately shown. ***, ** = p<0.001 and p<0.01 for all groups compared with TNBS group.</p