Johannes Schiefer

Evaluation of R6/2 HD transgenic mice for therapeutic studies in Huntington's disease: behavioral testing and impact of diabetes mellitus.

R6/2 transgenic mice express exon 1 of the human Huntingtons disease (HD) gene with an increased CAG repeat length. They develop a progressive neurological phenotype, die within 12-14 weeks of age and were also found to develop diabetes mellitus. Since R6/2 mice are broadly used to screen for potential therapies in HD, the aim of this study was (a) to search for behavioral tests that are best applicable to monitor the behavioral abnormalities in therapy studies and (b) to investigate the extent to which diabetes influences the disease phenotype. We found that the rotarod test for motor coordination and the open field test for spontaneous explorative behavior were useful to monitor the progressive behavioral deterioration of R6/2 mice. An accelerating rotarod paradigm was superior over testing with a rotarod at various fixed speeds since it leads to similar results with less repetitive daily trials so that exhaustion cannot contribute substantially to their decline in performance. With the Morris water maze, however, it was only possible to monitor cognitive decline in visuo-spatial learning in the first weeks of disease since, at later stages, mice were not able to learn the task adequately. A latent diabetes mellitus was found in all transgenic mice demonstrated by a pathological glucose tolerance. Only 26% of the mice, however, were found to develop a manifest diabetes with increasing blood glucose levels on normal diet over the disease period. R6/2 mice with manifest and latent diabetes showed no significant differences in survival, weight loss, motor coordination, or spontaneous explorative behavior. These results suggest that diabetes mellitus is not a major contributing factor to the disease phenotype.

The metabotropic glutamate receptor 5 antagonist MPEP and the mGluR2 agonist LY379268 modify disease progression in a transgenic mouse model of Huntington's disease

Chronic glutamate mediated excitotoxicity has been suggested to contribute to the pathogenesis of Huntingtons disease (HD). Both, inhibition of glutamate release through stimulation of presynaptic metabotropic glutamate receptor (mGluR) 2 and blockade of postsynaptic mGluR5 have been demonstrated to be neuroprotective against excitotoxicity. R6/2 HD transgenic mice which express an expanded CAG triplet repeat serve as a well-characterized mouse model for HD with progressing neurological abnormalities and limited survival. We treated R6/2 HD transgenic mice with either the mGluR2 agonist LY379268 (1.2 mg/kg) or with the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) (100 mg/kg) orally from a presymptomatic stage until death to investigate their potential disease modifying effects. We found that survival time in both the MPEP treated mice and the LY379268 treated mice was significantly increased in comparison to placebo treated transgenic controls (14.87+/-0.14 and 14.22+/-0.11 weeks versus 12.87+/-0.11 weeks, respectively). Additionally, the progressive decline in motor coordination of HD transgenic mice as tested with the rotarod test was significantly attenuated in MPEP- but not in LY379268-treated mice. Early pathological hyperactivity, which can be found in placebo treated HD transgenic mice, was significantly attenuated by both MPEP and LY379268 treatment. Immunohistologial examination of HD characteristic neuronal intranuclear inclusion (NII), however, demonstrated no effect on NII formation by either of the treatments applied. These data suggest that inhibition of glutamate neurotransmission via specific interaction with mGluRs might be interesting for both inhibition of disease progression as well as early symptomatic treatment in HD.

Riluzole prolongs survival time and alters nuclear inclusion formation in a transgenic mouse model of Huntington's disease

Glutamate excitotoxicity has been suggested to contribute to the pathogenesis of Huntingtons disease (HD). Riluzole is a substance with glutamate antagonistic properties that is used for neuroprotective treatment in amyotrophic lateral sclerosis and which is currently tested in clinical trials for treatment of HD. R6/2 transgenic mice, which express exon 1 of the human HD gene with an expanded CAG triplet repeat, serve as a well‐characterized mouse model for HD with progressing neurological abnormalities and limited survival. We treated R6/2 HD transgenic mice with riluzole orally beginning at a presymptomatic stage until death to investigate its potential neuroprotective effects in this mouse model and found that survival time in the riluzole group was significantly increased in comparison to placebo‐treated transgenic controls. Additionally, the progressive weight loss was delayed and significantly reduced by riluzole treatment; behavioral testing of motor coordination and spontaneous locomotor activity, however, showed no statistically significant differences. We also examined the formation of the HD characteristic neuronal intranuclear inclusions (NII) immunohistologically. At a late disease stage, striatal NII from riluzole‐treated transgenic mice showed profound changes in ubiquitination, i.e., NII were less ubiquitinated and surrounded by ubiquitinated micro‐aggregates. Staining with antibodies directed against the mutated huntingtin revealed no significant difference in this component of NII. Taken together, these data suggest that riluzole is a promising candidate for neuroprotective treatment in human HD.

Do Normal D-dimer Levels Reliably Exclude Cerebral Sinus Thrombosis?

Background and Purpose— Cerebral sinus thrombosis (CST) needs to be considered in the differential diagnosis of all patients with acute headache. Early diagnosis is essential because early treatment may prevent morbidity and may even be life-saving. Definite exclusion, however, needs advanced neuroradiologic diagnostics, which are not readily available in many hospitals. Because measurement of D-dimers has been demonstrated to be helpful in excluding thromboembolic disease, our aim was to investigate whether D-dimers would be also sensitive enough to exclude CST. Methods— We undertook a prospective multicenter study over a 2.5-year period including all patients who came to the emergency departments with symptoms suggestive of CST. All patients were diagnosed either by magnetic resonance venography, spiral computed tomography scan venography, or intra-arterial digital subtraction angiography. D-dimer levels were measured at admission and analyzed by the same method in all patients. Results— A total of 343 patients were included. CST was diagnosed in 35 patients, of whom 34 had D-dimers above the cutoff value (>500 &mgr;g/L). From the 308 patients not having CST, D-dimers were elevated in 27. Sensitivity of D-dimers was 97.1%, with a negative predictive value of 99.6%. Specificity was 91.2%, with a positive predictive value of 55.7%. D-dimers were positively correlated with the extent of the thrombosis and negatively correlated with the duration of symptoms (Spearman rank correlation coefficients 0.76, −0.58, respectively). Conclusions— D-dimer measurement is useful in patients with suspected CST. Normal D-dimers make the presence of CST very unlikely.

Compliance with hand hygiene on surgical, medical, and neurologic intensive care units: Direct observation versus calculated disinfectant usage

BACKGROUND Hand hygiene (HH) is considered the single most effective measure to prevent and control health care-associated infections (HAIs). Although there have been several reports on compliance rates (CRs) to HH recommendations, data for intensive care units (ICUs) in general and for shift- and indication-specific opportunities in particular are scarce. METHODS The aim of this study was to collect data on ICU-, shift-, and indication-specific opportunities, activities and CRs at a surgical ICU (SICU), a medical ICU (MICU), and a neurologic ICU (NICU) at the University Hospital Aachen based on direct observation (DO) and calculated disinfectant usage (DU). RESULTS Opportunities for HH recorded over a 24-hour period were significantly higher for the SICU (188 per patient day [PD]) and MICU (163 per PD) than for the NICU (124 per PD). Directly observed CRs were 39% (73/188) in the SICU, 72% (117/163) in the MICU, and 73% (90/124) in the NICU. However, CRs calculated as a measure of DU were considerably lower: 16% (29/188) in the SICU, 21% (34/163) in the MICU, and 25% (31/124) in the NICU. Notably, CRs calculated from DO were lowest before aseptic tasks and before patient contact. CONCLUSIONS To the best of our knowledge, this study provides the first data picturing a complete day, including shift- and indication-specific analyses, and comparing directly observed CRs with those calculated based on DU, the latter of which revealed a 2.75-fold difference. Worrisomely, CRs were very low, especially concerning indications of greatest impact in preventing HAIs, such as before aseptic task. Thus, the gathering of additional data on CRs and the reasons for noncompliance is warranted.

Myopathy as a first symptom of Huntington's disease in a Marathon runner

A semi professional marathon runner at risk for Huntingtons disease (HD) (43 CAG repeats) developed signs of a slowly progressive myopathy with exercise‐induced muscle fatigue, pain, elevated creatine kinase level, and worsening of his running performance many years before first signs of chorea were detected. Muscle biopsy displayed a mild myopathy with mitochondrial pathology including a complex IV deficiency and analysis of the patients fibroblast culture demonstrated deficits in mitochondrial function. Challenging skeletal muscle by excessive training might have disclosed myopathy in HD even years before the appearance of other neurological symptoms.

Neural Cell Adhesion Molecule L1-Transfected Embryonic Stem Cells Promote Functional Recovery after Excitotoxic Lesion of the Mouse Striatum

We have generated a murine embryonic stem cell line constitutively expressing L1 at all stages of neural differentiation to investigate the effects of L1 overexpression on stem cell proliferation, migration, differentiation, cell death, and ability to influence drug-induced rotation behavior in an animal model of Huntingtons disease. L1-transfected cells showed decreased cell proliferation in vitro, enhanced neuronal differentiation in vitro and in vivo, and decreased astrocytic differentiation in vivo without influencing cell death compared with nontransfected cells. L1 overexpression also resulted in an increased yield of GABAergic neurons and enhanced migration of embryonic stem cell-derived neural precursor cells into the lesioned striatum. Mice grafted with L1-transfected cells showed recovery in rotation behavior 1 and 4 weeks, but not 8 weeks, after transplantation compared with mice that had received nontransfected cells, thus demonstrating for the first time that a recognition molecule is capable of improving functional recovery during the initial phase in a syngeneic transplantation paradigm.

Altered Resting-State Connectivity in Huntington's Disease

Huntingtons disease (HD) is an autosomal dominantly inherited neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms. Using resting‐state fMRI (rs‐fMRI) we investigated the functional integrity of resting‐state networks (RSN) in HD. 17 HD and 19 matched control participants were examined at a 3 Tesla MR scanner. After controlling for structural degeneration by means of voxel‐based morphometry, task‐free rs‐fMRI data were analyzed using Independent Component Analysis (ICA) and a dual‐regression approach in the context of genetic and clinical parameters. Further, we evaluated HD‐related differences in interregional connectivity between networks. RSN analysis showed a significant increase in intrinsic functional connectivity in the HD sample compared with controls, including the thalamus, striatum, prefrontal, premotor, and parietal maps. A subset of the Default Mode Network (DMN) was also affected. In the HD cohort, motor impairment correlated with higher network connectivity in mainly motor and parietal cortices. Deteriorating total functional capacity was additionally associated with higher connectivity in the striatum, thalamus, insular and frontal areas. This pattern of increased activity in intrinsic functional networks might suggest a reduced ability of intra‐network differentiation with clinical disease progression in HD. Finally, results showed reduced long‐range connectivity between parietal ICA components in HD compared to controls, indicating impaired functional coupling between interregional networks in HD. Our data demonstrates that functional connectivity is profoundly altered in HD, both within and between RSN. Rs‐fMRI analysis may provide additional valuable insights into neuronal dysfunctions beyond HD‐related structural degeneration and disruptions of functional circuits in HD. Hum Brain Mapp 35:2582–2593, 2014.

Neural correlates of impaired emotion processing in manifest Huntington’s disease

The complex phenotype of Huntingtons disease (HD) encompasses motor, psychiatric and cognitive dysfunctions, including early impairments in emotion recognition. In this first functional magnetic resonance imaging study, we investigated emotion-processing deficits in 14 manifest HD patients and matched controls. An emotion recognition task comprised short video clips displaying one of six basic facial expressions (sadness, happiness, disgust, fear, anger and neutral). Structural changes between patients and controls were assessed by means of voxel-based morphometry. Along with deficient recognition of negative emotions, patients exhibited predominantly lower neural response to stimuli of negative valences in the amygdala, hippocampus, striatum, insula, cingulate and prefrontal cortices, as well as in sensorimotor, temporal and visual areas. Most of the observed reduced activity patterns could not be explained merely by regional volume loss. Reduced activity in the thalamus during fear correlated with lower thalamic volumes. During the processing of sadness, patients exhibited enhanced amygdala and hippocampal activity along with reduced recruitment of the medial prefrontal cortex. Higher amygdala activity was related to more pronounced amygdala atrophy and disease burden. Overall, the observed emotion-related dysfunctions in the context of structural neurodegeneration suggest both disruptions of striatal-thalamo-cortical loops and potential compensation mechanism with greater disease severity in manifest HD.

Tumour-cell-endothelial interactions: free radicals are mediators of melanoma-induced endothelial cell damage

Damage to vascular endothelium may play an important role during metastasis. We used a three-dimensional model of tumour cell extravasation to test the hypothesis that certain types of tumour cells are able to induce vascular endothelial cell injury. Multicellular tumour spheroids (MCTS) of 14 human cancer cell lines and spheroids from two benign cell lines were transferred onto confluent monolayers of human endothelial cells (EC). MCTS from 4 of 7 melanoma cell lines induced damage of the endothelium which was closely associated with tumour cell attachment. Endothelial cell injury became evident morphologically by loss of cell membrane integrity and sensitivity to shear stress. Similar results were obtained with EC derived from human umbilical veins, umbilical arteries and saphenous veins. Addition of the oxygen radical scavenger catalase showed a dose- and time-dependent inhibition (up to 48 h) of EC damage in the case of the melanoma cell lines ST-ML-11, ST-ML-14 and SK-MEL-28. The scavenging enzyme superoxide dismutase proved to be protective (up to 12 h) in ST-ML-12 MCTS. In contrast, allopurinol, deferoxamine mesylate, ibuprofen, nor-dihydroguaretic acid, soybean trypsin inhibitor or aprotinin had no protective effect. None of the non-melanoma cancer cell lines or benign cells induced endothelial cell damage. Endothelial injury has been shown to enhance the process of metastasis. Our results suggest that free-radical-mediated endothelial cell damage may be one of the mechanisms contributing to the devastating metastatic potential of melanoma.