Claudia Heine

Changes of the GPR17 receptor, a new target for neurorepair, in neurons and glial cells in patients with traumatic brain injury

Unveiling the mechanisms participating in the damage and repair of traumatic brain injury (TBI) is fundamental to develop new therapies. The P2Y-like GPR17 receptor has recently emerged as a sensor of damage and a key actor in lesion remodeling/repair in the rodent brain, but its role in humans is totally unknown. Here, we characterized GPR17 expression in brain specimens from seven intensive care unit TBI patients undergoing neurosurgery for contusion removal and from 28 autoptic TBI cases (and 10 control subjects of matched age and gender) of two university hospitals. In both neurosurgery and autoptic samples, GPR17 expression was strong inside the contused core and progressively declined distally according to a spatio-temporal gradient. Inside and around the core, GPR17 labeled dying neurons, reactive astrocytes, and activated microglia/macrophages. In peri-contused parenchyma, GPR17 decorated oligodendrocyte precursor cells (OPCs) some of which had proliferated, indicating re-myelination attempts. In autoptic cases, GPR17 expression positively correlated with death for intracranial complications and negatively correlated with patients’ post-traumatic survival. Data indicate lesion-specific sequential involvement of GPR17 in the (a) death of irreversibly damaged neurons, (b) activation of microglia/macrophages remodeling the lesion, and (c) activation/proliferation of multipotent parenchymal progenitors (both reactive astrocytes and OPCs) starting repair processes. Data validate GPR17 as a target for neurorepair and are particularly relevant to setting up new therapies for TBI patients.

Functional Regeneration of the ex-vivo Reconstructed Mesocorticolimbic Dopaminergic System

CNS reparative-medicine therapeutic strategies need answers on the putative recapitulation of the basic rules leading to mammalian CNS development. To achieve this aim, we focus on the regeneration of functional connections in the mesocorticolimbic dopaminergic system. We used organotypic slice cocultures of ventral tegmental area/substantia nigra (VTA/SN) and prefrontal cortex (PFC) on a multielectrode array (MEA) platform to record spikes and local field potentials. The spontaneously growing synaptically based bidirectional bursting activity was followed from 2 to 28 days in vitro (DIV). A statistical analysis of excitatory and inhibitory neurons properties of the physiological firing activity demonstrated a remarkable, exponentially increasing maturation with a time constant of about 5-7 DIV. Immunohistochemistry demonstrated that the ratio of excitatory/inhibitory neurons (3:1) was in line with the functional results obtained. Exemplary pharmacology suggested that GABAA receptors were able to exert phasic and tonic inhibition typical of an adulthood network. Moreover, dopamine D2 receptor inactivation was equally inhibitory both on the spontaneous neuronal activity recorded by MEA and on patch-clamp electrophysiology in PFC pyramidal neurons. These results demonstrate that axon growth cones reach synaptic targets up to full functionality and that organotypic cocultures of the VTA/SN-PFC perfectly model their newly born dopaminergic, glutamatergic and GABAergic neuronal circuitries.

Nimodipine enhances neurite outgrowth in dopaminergic brain slice co-cultures.

Calcium ions (Ca2+) play important roles in neuroplasticity and the regeneration of nerves. Intracellular Ca2+ concentrations are regulated by Ca2+ channels, among them L‐type voltage‐gated Ca2+ channels, which are inhibited by dihydropyridines like nimodipine. The purpose of this study was to investigate the effect of nimodipine on neurite growth during development and regeneration. As an appropriate model to study neurite growth, we chose organotypic brain slice co‐cultures of the mesocortical dopaminergic projection system, consisting of the ventral tegmental area/substantia nigra and the prefrontal cortex from neonatal rat brains. Quantification of the density of the newly built neurites in the border region (region between the two cultivated slices) of the co‐cultures revealed a growth promoting effect of nimodipine at concentrations of 0.1 μM and 1 μM that was even more pronounced than the effect of the growth factor NGF.

Phosphodiesterase 2 inhibitors promote axonal outgrowth in organotypic slice co-cultures.

The development of appropriate models assessing the potential of substances for regeneration of neuronal circuits is of great importance. Here, we present procedures to analyze effects of substances on fiber outgrowth based on organotypic slice co-cultures of the nigrostriatal dopaminergic system in combination with biocytin tracing and tyrosine hydroxylase labeling and subsequent automated image quantification. Selected phosphodiesterase inhibitors (PDE-Is) were studied to identify their potential growth-promoting capacities. Immunohistochemical methods were used to visualize developing fibers in the border region between ventral tegmental area/substantia nigra co-cultivated with the striatum as well as the cellular expression of PDE2A and PDE10. The quantification shows a significant increase of fiber density in the border region induced by PDE2-Is (BAY60-7550; ND7001), comparable with the potential of the nerve growth factor and in contrast to PDE10-I (MP-10). Analysis of tyrosine hydroxylase-positive fibers indicated a significant increase after treatment with BAY60-7550 and nerve growth factor in relation to dimethyl sulfoxide. Additionally, a dose-dependent increase of intracellular cGMP levels in response to the applied PDE2-Is in PDE2-transfected HEK293 cells was found. In summary, our findings show that PDE2-Is are able to significantly promote axonal outgrowth in organotypic slice co-cultures, which are a suitable model to assess growth-related effects in neuro(re)generation.

Organotypic slice co-culture systems to study axon regeneration in the dopaminergic system ex vivo.

Organotypic slice co-cultures are suitable tools to study axonal regeneration and development (growth or regrowth) of different projection systems of the CNS under ex vivo conditions.In this chapter, we describe in detail the reconstruction of the mesocortical and nigrostriatal dopaminergic projection system culturing tissue slices from the ventral tegmental area/substantia nigra (VTA/SN) with the prefrontal cortex (PFC) or the striatum (STR). The protocol includes the detailed slice preparation and incubation. Moreover, different application possibilities of the ex vivo model are mentioned; as an example, the substance treatment procedure and biocytin tracing are described to reveal the effect of applied substances on fiber outgrowth.

P2Y1 receptor mediated neuronal fibre outgrowth in organotypic brain slice co-cultures

Extracellular purines have multiple functional roles in development, plastic remodelling, and regeneration of the CNS by stimulating certain P2X/Y receptor (R) subtypes. In the present study we elucidated the involvement of P2YRs in neuronal fibre outgrowth in the developing nervous system. We particularly focused on the P2Y1R subtype and the dopaminergic system, respectively. For this purpose, we used organotypic slice co-cultures consisting of the ventral tegmental area/substantia nigra (VTA/SN) and the prefrontal cortex (PFC). After detecting the presence of the P2Y1R in VTA/SN, PFC, and on outgrowing fibres in the border region (e.g. on glial processes) connecting both brain slices, we could show that pharmacological modulation of the receptor influenced neuronal fibre outgrowth. Biocytin-tracing and tyrosine hydroxylase-immunolabelling together with quantitative image analysis revealed a significant increase in fibre growth in the border region of the co-cultures after treatment with ADPβS (P2Y1,12,13R agonist). The observed stimulatory potential of ADPβS was inhibited by pre-treatment with the P2X/YR antagonist PPADS. In P2Y1R knockout (P2Y1R(-/-)) mice, the ADPβS-induced stimulatory effect was absent, while growth was significantly enhanced in the co-cultures of the respective wild-type. This observation was confirmed in entorhino-hippocampal co-cultures, an example of a different projection system, expressing the P2Y1R. Using wortmannin and PD98059 we further showed that PI3K/Akt and MAPK/ERK cascades are involved in the mechanism underlying ADPβS-induced fibre growth. In conclusion, the data of this study clearly indicate that activation of the P2Y1R stimulates fibre growth and thereby emphasises the general role of this particular receptor subtype during development and regeneration.

Optimized polyethylenimine (PEI)-based nanoparticles for siRNA delivery, analyzed in vitro and in an ex vivo tumor tissue slice culture model.

The non-viral delivery of small RNA molecules like siRNAs still poses a major bottleneck for their successful application in vivo. This is particularly true with regard to crossing physiological barriers upon systemic administration. We have previously established polyethylenimine (PEI)-based complexes for therapeutic RNA formulation. These nanoplexes mediate full RNA protection against nucleolytic degradation, delivery to target tissues as well as cellular uptake, intracellular release and therapeutic efficacy in preclinical in vivo models. We herein present data on different polyplex modifications for the defined improvement of physicochemical and biological nanoparticle properties and for targeted delivery. (i) By non-covalent modifications of PEI polyplexes with phospholipid liposomes, ternary complexes (“lipopolyplexes”) are obtained that combine the favorable features of PEI and lipid systems. Decreased cytotoxicity and highly efficient delivery of siRNA is achieved. Some lipopolyplexes also allow prolonged storage, thus providing formulations with higher stability. (ii) Novel tyrosine modifications of low molecular weight PEI offer further improvement of stability, biocompatibility, and knockdown efficacy of resulting nanoparticles. (iii) For ligand-mediated uptake, the shielding of surface charges is a critical requirement. This is achieved by PEI grafting with polyethylene glycol (PEG), prior to covalent coupling of anti-HER1 antibodies (Erbitux®) as ligand for targeted delivery and uptake. Beyond tumor cell culture, analyses are extended towards tumor slice cultures from tumor xenograft tissues which reflect more realistically the in vivo situation. The determination of siRNA-mediated knockdown of endogenous target genes, i.e., the oncogenic survival factor survivin and the oncogenic receptor tyrosine kinase HER2, reveals nanoparticle penetration and biological efficacy also under intact tissue and stroma conditions.

Segmentation of Axonal Fibres in Tissue Slices

This work focuses on the segmentation of axonal structures in digital images of organotypic slice co-cultures. An image processing chain is presented, which relies on anisotropic diffusion for preprocessing of the images and the intelligent scissors method for segmentation. This method requires manual user interaction to set the starting points. To overcome this drawback, the initial parameters for the intelligent scissors are automatically extracted from the images by a graph-based approach.

Inhibitors of the phosphodiesterase 2 increased axonal fibre growth in a dopaminergic organotypic ex vivo slice co-culture model

Background Due to the necessity for therapeutic strategies promoting neuro-(re)generation the development of appropriate models appraising the potential of substances for regeneration and repair of neuronal circuits are of great importance. Here we present a procedure for the quantification of fibre outgrowth on the basis of organotypic slice co-cultures of the dopaminergic projection system, appropriate for characterising the effects on axonal growth after treatment with well known controls and growth factors e.g. nerve growth factor (NGF) and new drug candidates. Therefore co-culture preparations were used, consisting of at least two slices of different parts of the brain, namely the ventral tegmental area/substantia nigra (VTA/SN) and the striatum (STR), whereas while incubation fibres projections regenerate and new connections were built, linking the two slices [1,2].