Paul Christian Baier

The Tumorigenicity of Mouse Embryonic Stem Cells and In Vitro Differentiated Neuronal Cells Is Controlled by the Recipients' Immune Response

Embryonic stem (ES) cells have the potential to differentiate into all cell types and are considered as a valuable source of cells for transplantation therapies. A critical issue, however, is the risk of teratoma formation after transplantation. The effect of the immune response on the tumorigenicity of transplanted cells is poorly understood. We have systematically compared the tumorigenicity of mouse ES cells and in vitro differentiated neuronal cells in various recipients. Subcutaneous injection of 1×106 ES or differentiated cells into syngeneic or allogeneic immunodeficient mice resulted in teratomas in about 95% of the recipients. Both cell types did not give rise to tumors in immunocompetent allogeneic mice or xenogeneic rats. However, in 61% of cyclosporine A-treated rats teratomas developed after injection of differentiated cells. Undifferentiated ES cells did not give rise to tumors in these rats. ES cells turned out to be highly susceptible to killing by rat natural killer (NK) cells due to the expression of ligands of the activating NK receptor NKG2D on ES cells. These ligands were down-regulated on differentiated cells. The activity of NK cells which is not suppressed by cyclosporine A might contribute to the prevention of teratomas after injection of ES cells but not after inoculation of differentiated cells. These findings clearly point to the importance of the immune response in this process. Interestingly, the differentiated cells must contain a tumorigenic cell population that is not present among ES cells and which might be resistant to NK cell-mediated killing.

Association of daytime sleepiness with nigrostriatal dopaminergic degeneration in early Parkinson's disease

IntroductionMany patients with Parkinsons disease (PD) report daytime sleepiness. Its etiology, however, is still not fully understood. The aim of this study was to examine if the amount of nigrostriatal dopaminergic degeneration is associated with subjective daytime sleepiness in patients with PD.Patients and methodsWe investigated 21 patients with PD clinically and by means of [123I] FP-CIT-SPECT (DaTSCANR). Each patient filled in the Epworth sleepiness scale (ESS), the Parkinsons Disease Sleep Scale (PDSS), and the self-rating depression scale according to Zung (SDS) to assess sleepiness, sleep quality, and depressive symptoms.ResultsThe mean specific dopamine transporter binding in the 21 PD patients (60.8 ± 10.4 years, nine females, median Hoehn and Yahr stage 2.0) was decreased. Nine patients were in Hoehn and Yahr stage 1 (58.7 ± 6.6 years, four females; ESS score 7.4 ± 4.5; PDSS score 105.1 ± 30.9), the other 12 patients were in Hoehn and Yahr stage 2 (62.4 ± 12.6 years, five females; ESS score 6.7 ± 4.7, PDSS score 97.1 ± 25.6). Age, gender, ESS, and PDSS scores were not significantly different in both groups. However, ESS scores showed an inverse correlation with mean DAT binding in the striatum (r = -0.627, p = 0.03), the caudate nucleus (r = -0.708, p = 0.01), and the putamen (r = -0.599, p = 0.04) in patients with Hoehn and Yahr stage 2. There was no correlation of the ESS score with age, disease duration, UPDRS motor score, PDSS score, or depression score.ConclusionSubjective daytime sleepiness seems to be associated with dopaminergic nigrostriatal degeneration in early PD.

A highly sensitive automated complex running wheel test to detect latent motor deficits in the mouse MPTP model of Parkinson's disease

The subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP) paradigm is one of the most widely used in vivo models of Parkinsons disease (PD). However, particularly in the mouse model it has remained difficult to reliably detect behavioural correlates for PD. In the present study we apply a novel murine motor test, the motor skill sequence (MOSS) based on computerized recording of voluntary running wheel activity, and found latent motor skill deficits in the subchronic MPTP mouse model. Mice are first subjected to a 2-week training phase. The animals then receive either MPTP injections according to the standard subchronic MPTP paradigm (30 mg/kg) or vehicle injections for 5 consecutive days. Running performance transiently fell during the injection phase but returned to baseline within few days. The animals were then exposed to complex wheels with irregularly spaced crossbars demanding high central motor coordination abilities. Though both groups showed clear improvement of running performance in the learning phase on the complex wheel, MPTP animals displayed clear central motor deficits on the complex wheels, as indicated by a reduced maximum speed and running distance, despite unchanged running motivation. Our results demonstrate latent motor deficits in MPTP-treated mice, which can be unmasked by MOSS. MOSS is thus capable of detecting and quantifying central motor deficits in this widely used model of PD with high sensitivity. The automated full time data collection of several different running parameters makes it also a suitable test for efficient in vivo screening of potential therapeutic compounds in this model for PD.

Fate of pre-differentiated mouse embryonic stem cells transplanted in unilaterally 6-hydroxydopamine lesioned rats: Histological characterization of the grafted cells

We transplanted mouse embryonic stem (ES) cells pre-differentiated on a PA6 feeder cell layer into the striatum of 6-hydroxydopamine hemi-lesioned adult rats and studied the fate of the grafted cells 1 and 5 weeks post-grafting. At both time points, ES cell grafts contained tyrosine hydroxylase positive (TH+) and 5-HT immunoreactive cells. Between 1 and 5 weeks, there was an enlargement of the grafts and an increase in number of TH+ cells although the differences between the two time points were not significant. The mean number of TH+ neurons per striatum was 330 +/- 73 after 1 week and 1220 +/- 400 after 5 weeks. Over the same time period, mean soma profile area of the TH+ neurons increased significantly by 25.2%. Neurites were longer after 5 weeks (by 24.9%), but the difference to 1 week post-grafting was not reliable. The percentage of TH+ somata without neurites increased from 6.7% after 1 week to 38.3% after 5 weeks (not significant). After 5 weeks, two out of fifteen graft recipients had tumors indicating that pre-differentiation of mouse embryonic stem cells using this differentiation protocol is not sufficient to prevent tumor formation.

Restless Legs Syndrome induced by impairment of sensory spinal pathways

Sirs: Restless Legs Syndrome (RLS) is characterised by dysaesthetic sensations of the legs associated with an urge to move which occur exclusively at rest and can be ameliorated by walking [5]. While the primary form of the syndrome is often familial, a secondary form has been reported to be associated with a number of pathologies such as iron deficiency and uraemia (for a review, see Chaudhuri et al. [1]). There have also been reports of an association of RLS and structural spinal cord lesions [3, 4, 6]. This report presents for the first time a patient who developed symptoms of RLS following a persisting vascular lesion of sensory spinal pathways. A 52-year-old man was admitted to the hospital with sudden onset of retrosternal pain, weakness and numbness of both legs. On neurological examination the patient showed a paresis, hypaesthesia and hypalgesia of both legs with no deep tendon reflexes in the legs. The motor and sensory deficits were more pronounced in the left leg than in the right. A type-A-dissection of the ascending aorta with a consecutive ischaemia of the anterior and posterior spinal artery was diagnosed and an emergency grafting of the ascending aorta and the proximal part of the aortic arch was performed. Following surgical treatment, the patient made a nearly complete recovery of his sensory and motor deficits within half a year. When the patient first presented at our outpatient department one year after the dissection, he complained about uncomfortable sensations in both feet and lower legs. The paraesthesias had started about four weeks after the incident when he regained power and sensations in the lower limbs. The discomfort was associated with an urge to move, occurred exclusively at rest, worsened at night and could only be relieved by walking around. It was more severe in the left leg than in the right one. On clinical examination, mild dysaesthesia on touch of the lower left limb and a bilateral impairment for the sensation of pain and temperature were noted. All other clinical findings were normal. Various investigations were performed to elucidate the cause of the complaints: Magnetic resonance imaging (MRI) of the spinal cord revealed numerous small lesions of the lower thoracic spinal cord and of the medullary conus, which were interpreted as vascular myelopathy following spinal ischaemia. The cerebral MRI showed signs of an old infarction in the area of the right posterior cerebral artery with no significant difference to images obtained five years before and some days after the aortic dissection. Somatosensory-evoked potentials (SEP) of the tibial and the commune peroneal nerves showed normal spinal potentials followed by significantly delayed cortical potentials after left sided stimulation. SEP of the median nerve, motor-evoked potentials and nerve conduction studies were all normal. Thus the electrophysiological findings were in agreement with a lesion of the left dorsal funiculus. Polysomnography (PSG) was performed for two consecutive nights without medication. Polygraphic recordings included standard electroencephalographic, electrooculographic and submental electromyographic leads for visual sleep scoring and surface electromyographic leads of both tibial anterior muscles for detection of limb movements. The recordings revealed an average total sleep time of 260 min and a sleep efficiency of 56 %. Limb movements were recorded at a rate of 153/h from the left tibial muscle and of 142/h from the right tibial muscle. Periodic limb movements (PLM) were identified as described by Coleman [2] and the PLM index was found to be 89/h. Treatment was attempted with three alternative regimes: while the administration of up to 250 mg of levodopa did not have a convincing effect, 50 mg tilidin at night alleviated the symptoms. With 20 mg of zolpidem at night, the paraesthesias were well controlled and the patient now sleeps without disturbance until he wakes up in the morning. This is the first report of a patient who developed RLS after a vascular injury of the spinal cord with paraplegia and anaesthesia of the lower limbs. The patient experienced a complete recovery of his motor deficits but some sensory impairment remained. A mild dysaesthesia on touch of the left lower limb parallels the finding of a lesion of the left dorsal funiculus demonstrated on electrophysiological examination. Furthermore, there is clinical evidence of a bilateral impairment of the lateral spinothalamic tract. PSG results are typical of RLS. One of the limitations of this report is the lack of PSG data before the spinal ischaemia. However, it seems very unlikely that a sleep efficiency of 56 % and a high PLM index of 89/h existed before the spinal ischaemia LETTER TO THE EDITORS

Mice lacking the circadian modulators SHARP1 and SHARP2 display altered sleep and mixed state endophenotypes of psychiatric disorders.

Increasing evidence suggests that clock genes may be implicated in a spectrum of psychiatric diseases, including sleep and mood related disorders as well as schizophrenia. The bHLH transcription factors SHARP1/DEC2/BHLHE41 and SHARP2/DEC1/BHLHE40 are modulators of the circadian system and SHARP1/DEC2/BHLHE40 has been shown to regulate homeostatic sleep drive in humans. In this study, we characterized Sharp1 and Sharp2 double mutant mice (S1/2-/-) using online EEG recordings in living animals, behavioral assays and global gene expression profiling. EEG recordings revealed attenuated sleep/wake amplitudes and alterations of theta oscillations. Increased sleep in the dark phase is paralleled by reduced voluntary activity and cortical gene expression signatures reveal associations with psychiatric diseases. S1/2-/- mice display alterations in novelty induced activity, anxiety and curiosity. Moreover, mutant mice exhibit impaired working memory and deficits in prepulse inhibition resembling symptoms of psychiatric diseases. Network modeling indicates a connection between neural plasticity and clock genes, particularly for SHARP1 and PER1. Our findings support the hypothesis that abnormal sleep and certain (endo)phenotypes of psychiatric diseases may be caused by common mechanisms involving components of the molecular clock including SHARP1 and SHARP2.

Circadian distribution of motor-activity in unilaterally 6-hydroxy-dopamine lesioned rats

Sleep abnormalities in idiopathic Parkinson’s disease (PD) frequently consist in a reduction of total sleep time and efficacy and subsequent excessive daytime sleepiness. As it remains unclear whether these phenomena are part of the disease itself or result from pharmacological treatment, animal models for investigating the pathophysiology of sleep alterations in PD may add knowledge to this research area. In the present study, we investigate whether changes in circadian motor activity occur in 6-OHDA-lesioning model for PD, and allow a screening for disturbed sleep–waking behaviour. Activity measurements of six male Wistar rats with 6-OHDA-lesions in the medial forebrain bundle and six controls were carried out in two consecutive 12:12xa0h light–dark (LD) cycles. A computer-based video-analysis system, recording the animals’ movement tracks was used. Distance travelled and number of transitions between movement periods and resting periods were determined. Although 6-OHDA-lesioned animals show a reduced locomotor activity compared to non-lesioned rats, the circadian distribution basically remained intact. However, some lesioning effects were more pronounced in the resting phase than in the activity phase, possibly paralleling nocturnal akinesia in PD. In order to further elucidate the described phenomena, it will be necessary to perform studies combining sleep recordings with locomotor activity measurements.

Animal studies in restless legs syndrome.

Although restless legs syndrome (RLS) is a common disorder that has been studied thoroughly in the past decades, the underlying pathophysiology is still not fully understood. However, some attractive hypotheses on the pathogenesis of the disorder have been forwarded. Animal models are an important tool to verify hypotheses and to dissect out the details of pathophysiological mechanisms. Ideally they might serve the development of future treatment strategies. This review discusses the general and specific prerequisites necessary for the establishment of animal models for RLS and summarizes the approaches that have been made.

In vivo and in vitro tissue-specific expression of green fluorescent protein using the cre-lox system in mouse embryonic stem cells

Embryonic stem cells (ES) are pluripotent and may therefore serve as a source for the generation of specific cell types required for future therapies based on cell replacement. The isolation of defined cell populations from a certain lineage or tissue is a prerequisite for the analysis of the potential of such ES‐derived cells in animal transplantation studies. Here, using the Cre/loxP system, we report the generation of murine ES cells conditionally expressing the hrGFP gene at the cell surface. Such ES cells can be differentiated in vitro into neurons displaying GFP activity in neurites. Transgenic mice derived from these ES cells permit the targeting of GFP‐expression to specific tissues and provide material from the three germ layers suitable for molecular and biochemical analysis.

Growth hormone response in low-dose apomorphine test correlates with nigrostriatal dopamine transporter binding in patients with Parkinson’s disease

Summary.Challenge with low-dose apomorphine causes a rise in growth hormone (GH) in patients with Parkinson’s disease (PD). We studied 18 patients with early PD, who showed an increase of GH in the low-dose apomorphine test, by means of [123I] FP-CIT-SPECT. The mean specific dopamine transporter binding of the 18 patients was 1.50 ± 0.56 in the striatum, 1.20 ± 0.59 in the putamen, and 1.76 ± 0.59 in the caudate nucleus. The increase of GH (1.05 ± 1.01u2009ng/ml at baseline to 9.46 ± 6.36u2009ng/ml 45u2009min after apomorphine injection; p < 0.001) was significant. There was a significant negative correlation of the increase of GH with the mean specific dopamine transporter binding in all three regions (r between −0.490 and −0.587; p between 0.04 and 0.01). Challenge with low-dose apomorphine may therefore be used as an indirect tool to measure the extent of nigrostriatal neurodegeneration in early PD.