Effect of Intrastriatal 6-OHDA Lesions on Extrastriatal Brain Structures in the Mouse.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of midbrain dopaminergic neurons, resulting in motor and non-motor symptoms. The underlying pathology of non-motor symptoms is poorly understood. Discussed are pathological changes of extrastriatal brain structures. In this study, we characterized histopathological alterations of extrastriatal brain structures in the 6-hydroxydopamine (6-OHDA) PD animal model. Lesions were induced by unilateral stereotactic injections of 6-OHDA into the striatum or medial forebrain bundle of adult male mice. Loss of tyrosine hydroxylase positive (TH+) fibers as well as glia activation was quantified following stereological principles. Loss of dopaminergic innervation was further investigated by western-blotting. As expected, 6-OHDA injection into the nigrostriatal route induced retrograde degeneration of dopaminergic neurons within the substantia nigra pars compacta (SNpc), less so within the ventral tegmental area. Furthermore, we observed a region-specific drop of TH+ projection fiber density in distinct cortical regions. This pathology was most pronounced in the cingulate- and motor cortex, whereas the piriform cortex was just modestly affected. Loss of cortical TH+ fibers was not paralleled by microglia or astrocyte activation. Our results demonstrate that the loss of dopaminergic neurons within the substantia nigra pars compacta is paralleled by a cortical dopaminergic denervation in the 6-OHDA model. This model serves as a valuable tool to investigate mechanisms operant during cortical pathology in PD patients. Further studies are needed to understand why cortical dopaminergic innervation is lost in this model, and what functional consequence is associated with the observed denervation.

Optimisation of murine organotypic slice culture preparation for a novel sagittal-frontal co-culture system.

BACKGROUND: The nigrostriatal pathway is of great importance for the execution of movements, especially in the context of Parkinson's disease. In research, analysis of this pathway often requires the application of severe animal experiments. Organotypic nigrostriatal slice cultures offer a resource-saving alternative to animal experiments for research on the nigrostriatal system. NEW METHOD: We have established a time-saving protocol for the preparation of murine sagittal nigrostriatal slice cultures by using a tissue chopper and agarose embedding instead of a vibratome. Furthermore, we developed the first murine co-culture model and the first co-culture utilising sagittal slices for modelling the nigrostriatal pathway. RESULTS: Sagittal nigrostriatal slice cultures show good overall tissue preservation and a high number of morphologically unimpaired dopaminergic neurons in the substantia nigra. Sagittal-frontal co-culture demonstrates massive outgrowth of dopaminergic fibres from the substantia nigra into co-cultured tissue. COMPARISON WITH EXISTING METHODS: The use of a tissue chopper instead of a vibratome allows notable time-saving during culture preparation, therefore allowing optimisation of the preparation time. Sagittal co-cultures offer the opportunity to study dopaminergic fibres in their physiological environment and in co-cultured tissue from a different animal in the same culture system. CONCLUSION: We here present a possibility to optimise the slice culture preparation process with the simple means of using a tissue chopper and fast agarose embedding. Furthermore, our sagittal-frontal co-culture system is suitable for the observation of dopaminergic outgrowth in both co-cultured tissues.

Expression of Tgfß1 and Inflammatory Markers in the 6-hydroxydopamine Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by loss of midbrain dopaminergic (mDA) neurons in the substantia nigra (SN). Microglia-mediated neuroinflammation has been described as a common hallmark of PD and is believed to further trigger the progression of neurodegenerative events. Injections of 6-hydroxydopamine (6-OHDA) are widely used to induce degeneration of mDA neurons in rodents as an attempt to mimic PD and to study neurodegeneration, neuroinflammation as well as potential therapeutic approaches. In the present study, we addressed microglia and astroglia reactivity in the SN and the caudatoputamen (CPu) after 6-OHDA injections into the medial forebrain bundle (MFB), and further analyzed the temporal and spatial expression patterns of pro-inflammatory and anti-inflammatory markers in this mouse model of PD. We provide evidence that activated microglia as well as neurons in the lesioned SN and CPu express Transforming growth factor ß1 (Tgfß1), which overlaps with the downregulation of pro-inflammatory markers Tnfα, and iNos, and upregulation of anti-inflammatory markers Ym1 and Arg1. Taken together, the data presented in this study suggest an important role for Tgfß1 as a lesion-associated factor that might be involved in regulating microglia activation states in the 6-OHDA mouse model of PD in order to prevent degeneration of uninjured neurons by microglia-mediated release of neurotoxic factors such as Tnfα and nitric oxide (NO).

Efferent projections of the anterior and posterodorsal regions of the medial nucleus of the amygdala in the mouse.

The efferent projections of the anterior and posterodorsal part of the medial nucleus (MePD) in the mouse were studied by means of anterograde axonal tracing using biotinylated dextran amine. The MePD axons ran mainly via the stria terminalis and to a lesser extent via the ventral amygdalofugal pathway. The projections to the forebrain were broadly distributed and varied from very strong to scant. The most significant connections were destined to the bed nucleus of the stria terminalis in which all parts of the medial division were innervated by MePD neurons. Moderate projections reached the limbic striatum (nucleus accumbens), olfactory tubercle and the lateral septal nucleus. The substantia innominata was also innervated by the MePD, and especially the projection to its ventral portion was substantial. The profuse innervation of the medial preoptic nucleus and medial preoptic area indicated significant involvement of the MePD in sexual behavior. Many hypothalamic nuclei were innervated but to a different extent. The very strong innervation of the ventral premammillary nucleus further indicated the involvement of the MePD in the neuronal circuitry for sexual behavior. Substantial projections also reached the anterior hypothalamus and tuber cinereum, while the connections to the lateral hypothalamus were widespread but showed moderate density. MePD strongly innervated the ventrolateral part of the ventromedial hypothalamic nucleus and moderately its remaining parts. The neurosecretory hypothalamic nuclei and the arcuate nucleus contained only a few MePD terminals. The thalamic innervation was very scant and reached the lateral habenular nucleus and the nuclei of the midline. The mesencephalic connections were moderate to sparse and projected to the mesolimbic dopaminergic groups in the ventral tegmental area, the pars lateralis and the dorsal tier of the substantia nigra pars compacta, the periaqueductal gray and the dorsal raphe nucleus. The present results principally resembled data known in other rodent species; however, the efferents of the MePD often differed in extent and/or topical distribution.

In vitro characterization of embryionic ST14A-cells.

The embryonic striatal temperature sensitive immortalized ST14A-cell line was characterized in vitro by immunocytochemistry when cultured at 33 degrees C and at nonpermissive temperature of 39 degrees C for up to 14 days. At 33 degrees C in DMEM/10% FCS, cells proliferated, were extensively expressing the neural progenitor cell markers nestin and vimentin contrary to neuronal markers. However, when cultured at 39 degrees C the proliferation was delayed and cells began to increase the expression of neuronal markers, followed by a decrease of nestin and vimentin. In serum-free medium the process of neuronal differentiation became more obvious, indicating the potential to use these cells for experimental restorative therapies.

Mesencephalic human neural progenitor cells transplanted into the adult hemiparkinsonian rat striatum lack dopaminergic differentiation but improve motor behavior.

The clinical outcome of cell replacement therapies depends upon the successful survival and differentiation of transplanted cells. Here, we transplanted human neural progenitor cells derived from the ventral mesencephalon of an 8-week-old embryo into the ipsilateral (right) striatum of unilateral 6-hydroxydopamine-lesioned adult rats. To assess the therapeutic potency of grafted cells, 2 independent behavioral tests were conducted 12 weeks after transplantation: in the rotation test, a mild behavioral improvement was detected, and in the cylinder test, transplanted animals overcame the lesion-induced right forepaw preference. To address this behavioral improvement to a dopaminergic differentiation capacity of transplanted cells in vivo, immunohistochemistry for tyrosine hydroxylase was performed, showing a total lack of immunoreactivity. However, we found a considerable number of transplanted human nuclei-positive cells preferentially differentiated into neurons. In addition, glial fibrillary acidic protein-expressing cells were also detected. Our results show that behavioral improvement does not necessarily correlate with a differentiation of transplanted precursors into dopaminergic neurons, indicating other factors to be involved in a partial functional recovery. Nevertheless, for the development of a clinically useful cell therapy, it is important to overcome obstacles, namely the poor dopaminergic differentiation of human neural progenitor cells after grafting.

Increasing extracellular potassium results in subthalamic neuron activity resembling that seen in a 6-hydroxydopamine lesion.

Abnormal neuronal activity in the subthalamic nucleus (STN) plays a crucial role in the pathophysiology of Parkinson's disease (PD). Although altered extracellular potassium concentration ([K+]o) and sensitivity to [K+]o modulates neuronal activity, little is known about the potassium balance in the healthy and diseased STN. In vivo measurements of [K+]o using ion-selective electrodes demonstrated a twofold increase in the decay time constant of lesion-induced [K+]o transients in the STN of adult Wistar rats with a unilateral 6-hydroxydopamine (6-OHDA) median forebrain bundle lesion, employed as a model of PD, compared with nonlesioned rats. Various [K+]o concentrations (1.5-12.5 mM) were applied to in vitro slice preparations of three experimental groups of STN slices from nonlesioned control rats, ipsilateral hemispheres, and contralateral hemispheres of lesioned rats. The majority of STN neurons of nonlesioned rats and in slices contralateral to the lesion fired spontaneously, predominantly in a regular pattern, whereas those in slices ipsilateral to the lesion fired more irregularly or even in bursts. Experimentally increased [K+]o led to an increase in the number of spontaneously firing neurons and action potential firing rates in all groups. This was accompanied by a decrease in the amplitude of post spike afterhyperpolarization (AHP) and the amplitude and duration of the posttrain AHP. Lesion effects in ipsilateral neurons at physiological [K+]o resembled the effects of elevated [K+]o in nonlesioned rats. Our data suggest that changed potassium sensitivity due to conductivity alterations and delayed clearance may be critical for shaping STN activity in parkinsonian states.

Orthotopic transplantation of immortalized mesencephalic progenitors (CSM14.1 cells) into the substantia nigra of hemiparkinsonian rats induces neuronal differentiation and motoric improvement.

Neural progenitor cell grafting is a promising therapeutic option in the treatment of Parkinson's disease. In previous experiments we grafted temperature-sensitive immortalized CSM14.1 cells, derived from the ventral mesencephalon of E14-rats, bilaterally in the caudate putamen of adult hemiparkinsonian rats. In these studies we were not able to demonstrate either a therapeutic improvement or neuronal differentiation of transplanted cells. Here we examined whether CSM14.1 cells grafted bilaterally orthotopically in the substantia nigra of hemiparkinsonian rats have the potential to differentiate into dopaminergic neurons. Adult male rats received 6-hydroxydopamine into the right medial forebrain bundle, and successful lesions were evaluated with apomorphine-induced rotations 12 days after surgery. Two weeks after a successful lesion the animals received bilateral intranigral grafts consisting of either about 50 000 PKH26-labelled undifferentiated CSM14.1 cells (n = 16) or a sham-graft (n = 9). Rotations were evaluated 3, 6, 9 and 12 weeks post-grafting. Animals were finally perfused with 4% paraformaldehyde. Cryoprotected brain slices were prepared for immunohistochemistry using the freeze-thaw technique to preserve PKH26-labelling. Slices were immunostained against neuronal epitopes (NeuN, tyrosine hydroxylase) or glial fibrillary acidic protein. The CSM14.1-cell grafts significantly reduced the apomorphine-induced rotations 12 weeks post-grafting compared to the sham-grafts (P < 0.05). There was an extensive mediolateral migration (400-700 microm) of the PKH26-labelled cells within the host substantia nigra. Colocalization with NeuN or glial fibrillary acidic protein in transplanted cells was confirmed with confocal microscopy. No tyrosine hydroxylase-immunoreactive grafted cells were detectable. The therapeutic effect of the CSM14.1 cells could be explained either by their glial cell-derived neurotrophic factor-expression or their neural differentiation with positive effects on the basal ganglia neuronal networks.

Transplantation of immortalized mesencephalic progenitors (CSM14.1 cells) into the neonatal parkinsonian rat caudate putamen.

The present study analyzed whether grafts of the mesencephalic progenitor cell line CSM14.1 into the neonatal rat caudate putamen (CPu) differentiate into neurons and whether this is accompanied by a functional improvement in 6-hydroxydopamine (6-OHDA)-lesioned animals. As in previous studies, a neuronal differentiation of CSM14.1 cells transplanted into the CPu of adult animals could not be observed, so we here used neonatal rats, because graft location and host age seemingly are crucial parameters for neural transplant differentiation and integration. Rats bilaterally lesioned at postnatal day 1 by intraventricular 6-OHDA-injections 2 days later received 100,000 CSM14.1 cells prelabelled with the fluorescent dye PKH26 into the right CPu. Five weeks after grafting, the cylinder test was performed, and the data compared with data from age-matched intact controls and bilaterally lesioned-only animals. Brain slices immunostained for tyrosine hydroxylase (TH) were quantified by optical densitometry. We observed a significant preference of left forelimb use exclusively in transplanted animals. In these rats, TH-containing perikarya were found in the grafted CPu, presumedly leading to the significant increase of TH-immunoreactive fibers in this region. Moreover, confocal laser microscopy revealed a differentiation of transplanted PKH26-labelled CSM14.1 cells into neuronal nuclei antigen or TH-immunoreactive cells. Thus, CSM14.1 cells differentiate into TH-containing neurons, which most probably contribute to the preferred forelimb use, indicating a functional integration of CSM14.1 cells into the host basal ganglia loops during early postnatal development. These findings that are in contrast to observations in adult rats suggest instructive cues for neuronal differentiation and integration given by the neonatal microenvironment.

Mesencephalic human neural progenitor cells transplanted into the neonatal hemiparkinsonian rat striatum differentiate into neurons and improve motor behaviour.

Neural stem cell transplantation is a promising strategy for the treatment of neurodegenerative diseases. To evaluate the differentiation potential of human neural progenitor cells (hNPCs) as a prerequisite for clinical trials, we intracerebrally transplanted in vitro expanded fetal mesencephalic hNPCs into hemiparkinsonian rats. On postnatal day one (P1), 17 animals underwent a unilateral intraventricular 6-hydroxydopamine injection into the right lateral ventricle. At P3, animals (n = 10) received about 100,000 hNPCs (1 microL) in the right striatum. Five weeks after birth, animals underwent behaviour tests prior to fixation, followed by immunohistochemistry on brain slices for human nuclei, glial fibrillary acidic protein, S100beta, neuronal nuclei antigen, neuron-specific enolase and tyrosine hydroxylase. Compared with the apomorphine-induced rotations in the lesioned-only group (7.4 +/- 0.5 min(-1)), lesioned and successfully transplanted animals (0.3 +/- 0.1 min(-1)) showed a significant therapeutic improvement. Additionally, in the cylinder test, the lesioned-only animals preferred to use the ipsilateral forepaw. Conversely, the lesioned and transplanted animals showed no significant side bias similar to untreated control animals. Transplanted human nuclei-immunoreactive cells were found to survive and migrate up to 2000 microm into the host parenchyma, many containing the pan-neuronal markers neuronal nuclei antigen and neuron-specific enolase. In the striatum, tyrosine hydroxylase-immunoreactive somata were also found, indicating a dopaminergic differentiation capacity of transplanted hNPCs in vivo. However, the relative number of tyrosine hydroxylase-immunoreactive neurons in vivo seemed to be lower than in corresponding in vitro differentiation. To minimize donor tissue necessary for transplantation, further investigations will aim to enhance dopaminergic differentiation of transplanted cells in vivo.

Quinolinic acid lesions of the caudate putamen in the rat lead to a local increase of ciliary neurotrophic factor.

When applied prior to excitotoxic lesions, ciliary neurotrophic factor (CNTF) has been shown to be neuroprotective. However, data concerning the endogenous CNTF content of the intact rat striatum are rare and have not until now been available for the quinolinic acid (QA)-lesioned striatum. Therefore, we investigated the CNTF content in the QA-lesioned rat striatum for at least 1 month using immunohistochemistry and Western blot analysis. In lesioned striata a neuronal loss was observed by Nissl staining and by a reduction of NeuN-immunoreactive cells, whereas increased glial fibrillary acidic protein immunoreactivity showed a gliotic reaction. With CNTF immunohistochemistry we found that in the QA-lesioned striatum CNTF was increased over time, whereas it was not detectable in intact and sham-lesioned striata. CNTF-immunoreactive cells had the morphology of protoplasmatic astrocytes. Furthermore, quantitative Western blotting demonstrated that the content of CNTF protein from striatal lysates containing 1 mg of whole protein 1 month after QA lesioning (2.76 +/- 1.71 ng) was significantly increased (P < 0.05, U-test) compared with sham-lesioned hemispheres (0.68 +/- 0.25 ng) and intact controls (0.55 +/- 0.25 ng). We conclude that CNTF content is correlated with glial scar formation and suggest that our results may be of relevance to cell grafting strategies for the treatment of Huntington's disease.

Comparison of contrast, sensitivity and efficiency of signal amplified and nonamplified immunohistochemical reactions suitable for videomicroscopy-based quantification and neuroimaging.

In recent years, many different technical modifications of immunohistochemical methods have been developed. The selection of a suitable technique for quantitative purposes such as mapping studies can be quite difficult. Various features of a certain method must be considered such as the sensitivity, costs, duration and practicability with respect to serial sectioned specimens. Background and foreground difference or contrast and the influence of artifacts are major problems of quantitative immunohistochemistry. It is not known which of the different modifications of immunohistochemical signal amplifications and non-amplifications gives optimal results in respect to image analytical-based quantification. However, for image analysis, it is important to analyze sections which offer a sufficient contrast between foreground and background. The sensitivity of a system is crucial when quantitative immunohistochemistry should be applied to scarce material with longer postmortem and storage times which occur often by processing human brains. In addition, the enzyme-substrate reactions have an obvious influence on this criterion; therefore, different substrates were also tested. The contrast may be as well effected by the quality and specificity of the primary antibody, the type of tissue and naturally by preparative (fixation, postmortem delay, storage) and individual factors (age, circadian effects, diseases, sex). Because all of these factors may yield to different results by combining them with different neuronal structures, we used three different antigen expressions for a specific analysis: fibrillary, granulary and perikaryal antigen distributions in brains from Wistar rats. Principally, the sensitivity of the modifications of immunohistochemical amplifications is revealed more strongly than without enhancement steps; however, the contrast between foreground and background structures does not necessary increase by applying a certain amplification technique. The lowest contrast (15%) was detected after applying the labelled streptavidin-biotin technique. All other methods offer comparable contrasts in between 30% and 40%. The catalyzed signal amplification reaction has been found to give optimal results (40% contrast) for image analysis. However, from the technical point of view and variability of protein expression, storage and postmortem delay, it was necessary to adapt the commercial CSA Kit from Dako (K1500). The modified technique, called C2 method, offers better results with respect to sensitivity, total costs, duration and contrast (60%) and variability of contrast.

Dopaminergic differentiation of the Nurr1-expressing immortalized mesencephalic cell line CSM14.1 in vitro.

The use of neural stem cells as grafts is a potential treatment for Parkinson's disease, but the potential of stem cells to differentiate into dopaminergic neurones requires investigation. The present study examined the in vitro differentiation of the temperature-sensitive immortalized mesencephalic progenitor cell line CSM14.1 under defined conditions. Cells were derived from the mesencephalic region of a 14-day-old rat embryo, retrovirally immortalized with the Large T antigen and cultured at 33 degrees C in DMEM containing 10% fetal calf serum (FCS). For differentiation, the temperature was elevated at 39 degrees C and FCS was reduced (1%). Using histology, immunocytochemical detection of the stem cell marker Nestin and the neuronal marker MAP5 and, in addition, Western blotting to determine the presence of neurone-specific enolase and the neurone nuclei antigen we demonstrated a differentiation of these cells into neuronal cells accompanied by a decrease in Nestin production. In Western blots, we detected the orphan nuclear receptor Nurr1 in these cells. This was followed by a time-dependent up-regulation of the enzymes tyrosine hydroxylase and aldehyde dehydrogenase 2 characteristic of mature dopaminergic neurones. Our in vitro model of dopaminergic cell differentiation corroborates recent in vivo observations in the developing rodent brain.