Albumin-based nanoparticles as magnetic resonance contrast agents: I. Concept, first syntheses and characterisation.

To develop a platform for molecular magnetic resonance imaging, we prepared gadolinium-bearing albumin-polylactic acid nanoparticles in the size range 20-40 nm diameter. Iterative cycles of design and testing upscaled the synthesis procedures to gram amounts for physicochemical characterisation and for pharmacokinetic testing. Morphological analyses showed that the nanoparticles were spheroidal with rough surfaces. Particle sizes were measured by direct transmission electron microscopical measurements from negatively contrasted preparations, and by use of photon correlation spectroscopy; the two methods each documented nanoparticle sizes less than 100 nm and generally 10-40 nm diameter, though with significant intrabatch and interbatch variability. The particles' charge sufficed to hold them in suspension. HSA retained its tertiary structure in the particles. The nanoparticles were stable against turbulent flow conditions and against heat, though not against detergents. MRI imaging of liquid columns was possible at nanoparticle concentrations below 10 mg/ml. The particles were non-cytotoxic, non-thrombogenic and non-immunogenic in a range of assay systems developed for toxicity testing of nanoparticles. They were micellar prior to lyophilisation, but loosely structured aggregated masses after lyophilisation and subsequent resuspension. These nanoparticles provide a platform for further development, based on non-toxic materials of low immunogenicity already in clinical use, not expensive, and synthesized using methods which can be upscaled for industrial production.

Improved NlaIII digestion of PAGE-purified 102 bp ditags by addition of a single purification step in both the SAGE and microSAGE protocols.

Despite the success of microarray technologies, serial analysis of gene expression (SAGE) still remains the only technique that allows an accurate quantitative and qualitative analysis of cell transcription in a variety of physiological and pathological conditions. Nevertheless, the efficiency of SAGE is limited by the numerous gel purification steps required and these increase the possibility of contamination and reduce or inhibit the activity of the enzymes used in the protocol. In order to eliminate this problem, we have modified the original protocol by adding a single purification step before NLA:III digestion of the ditags. This allows us to increase the yield of digested ditags without reducing the amount of DNA or affecting the subsequent concatemerization.

Target-dependent plasticity of galanin and vasoactive intestinal peptide in the rat superior cervical ganglion after nerve lesion and re-innervation.

The expression of the neuropeptides galanin and vasoactive intestinal peptide (VIP) is increased in subpopulations of sympathetic neurons after axotomy of the rat superior cervical ganglion. We investigated whether postganglionic neurons innervating different targets show a prevalence for any of the two peptides in response to carotid nerve lesion. Before the respective postganglionic axons were crushed close to the ganglion, postganglionic neurons projecting either to the iris (through the internal carotid nerve) or to the submandibular gland (through both carotid branches) were identified by the retrograde tracer Fast Blue. Galanin and VIP immunoreactivities were demonstrated two and 30 days after crush and after successful regeneration of the lesioned neurons (60 days). In control ganglia, both peptides were detected in a few gland- but not in iris-projecting neurons. However, two days after crush of the respective carotid nerves, 14% of neurons within the iris and 46% within the gland population were immunoreactive for galanin. The percentage of neurons immunoreactive for VIP was significantly lower in both populations: only 3.5% of neurons projecting to the iris and 23% of the gland-projecting neuron population exhibited this peptide. After 30 days, the percentage of galanin- and VIP-positive neurons projecting to the submandibular gland was reduced (24% and 5.7%, respectively), whereas the proportion of galanin-immunoreactive neurons further increased within the iris population (55%), indicating that some neurons express galanin at later stages after the lesion. At 60 days after the crush, the percentage of galanin- or VIP-immunoreactive neurons had decreased to control levels within those neuron populations that re-innervated the iris or submandibular gland, although the total number of neurons exhibiting galanin or VIP was still increased within the ganglion, suggesting that re-establishment of target contact may play a role in down-regulation of both peptides.

Basic fibroblast growth factor isoforms promote axonal elongation and branching of adult sensory neurons in vitro.

Synthesis of the multifunctional cytokine basic fibroblast growth factor (FGF-2) is up-regulated after sciatic nerve lesion. In this study, the effects of low and high molecular weight FGF-2 isoforms on axonal elongation and branching of dissociated rat sensory neurons derived from adult lumbar dorsal root ganglia were investigated. These neurons express FGF receptor (FGFR) type I in the cytoplasmic/membrane compartment and in nuclear speckles. FGF-2 isoforms increase the number of axonal branches in cultures obtained from control rats, but do not promote axonal elongation. In response to a preconditioning lesion, i.e. transection of the sciatic nerve 1 week before culture, the axonal length of ipsilateral lumbar sensory neurons increases two-fold when compared with non-lesioned control rats, and this response is significantly enhanced by FGF-2 isoforms but not by nerve growth factor (NGF). Neurons dissociated from ganglia located contralaterally to the lesion exhibit a smaller increase in axon elongation (30%). The stimulating effects of FGF-2 isoforms on axon growth are fully blocked, and the enhanced regeneration of prelesioned neurons is reduced by the FGFR inhibitor SU5402 suggesting an involvement of endogenous FGF signaling in response to a lesion. The present data support a direct neurotrophic role of the 18 kD and 23 kD FGF-2 isoforms on adult axonal regeneration which may be of therapeutic value in the treatment of peripheral nerve lesions. Furthermore, evidence is provided for an enhanced regenerative capacity not only of preaxotomized neurons but also of homonymous non-axotomized neurons.

Plasticity of neuropeptide Y in the rat superior cervical ganglion in response to nerve lesion.

Axotomy of the rat superior cervical ganglion results in a two-fold increase of neuropeptide tyrosine as determined by radioimmunoassay. On the other hand, treatment of sympathetic neuron cultures with leukemia inhibitory factor, a cytokine that is known to be involved in the up-regulation of galanin after axotomy in vivo, decreases neuropeptide tyrosine messenger RNA. These, apparently contradictory findings, prompted us to investigate the regulation of neuropeptide tyrosine in the axotomized superior cervical ganglion in vivo. For comparison, the regulation of galanin was examined under the same conditions. Compared to control ganglia, the number of neuropeptide tyrosine-positive cell bodies decreased while the density of immunoreactive neuronal processes increased one week after transection of the major postganglionic nerves. The nerve fibres were identified as axons by the absence of MAP2, a somatodendritic marker protein. They extended into both carotid nerves and ramified at the lesion site. In situ hybridization revealed that, although the number of neuropeptide tyrosine messenger RNA-positive neurons was not different from controls, the average grain density/neuron decreased by 40%. When axotomized ganglia were decentralized simultaneously, a three-fold elevation of neuropeptide tyrosine immunoreactivity was detectable by radioimmunoassay and an additional increase in numerical density of neuropeptide tyrosine-immunoreactive nerve fibres was observed. Levels of neuropeptide tyrosine messenger RNA were significantly reduced within postganglionic neurons. This synergistic effect of combined axotomy and decentralization on peptide content was also detected for the neuropeptide galanin that, in contrast to neuropeptide tyrosine, is induced by axotomy or decentralization on protein and messenger RNA level. Therefore, while neuropeptide tyrosine messenger RNA is reduced in axotomized ganglia (most likely in response to leukemia inhibitory factor), the peptide accumulates in axonal processes resulting in increased peptide levels as determined by radioimmunoassay.

Expression of basic fibroblast growth factor isoforms in postmitotic sympathetic neurons: synthesis, intracellular localization and involvement in karyokinesis.

Low and high molecular weight isoforms of the mitogen and multifunctional cytokine basic fibroblast growth factor (FGF-2) are up-regulated in neurons and glial cells in response to peripheral nerve lesion. While synthesis, regulation and functions of FGF-2 in non-neuronal cells are well established, the significance of neuronal FGF-2 remains to be investigated in the peripheral nervous system. Therefore, the expression, intracellular localization and possible effects of FGF-2 isoforms were analyzed in primary sympathetic neurons derived from the rat superior cervical ganglion. FGF-2 is detected in the nucleus and in perinuclear Golgi fields of early postnatal neurons which also express mRNA and protein for the FGF receptor type 1. Biolistic transfection of plasmids encoding FGF-2 isoforms fused to fluorescent proteins demonstrates nuclear targeting of 18 kDa FGF-2 and 23 kDa FGF-2 with prominent accumulation in the nucleolus of neurons. Neither overexpression nor treatment with FGF-2 isoforms promotes survival of sympathetic neurons deprived of nerve growth factor; however, neuronal transfection of the high molecular weight FGF-2 isoform in dissociated and slice cultures results in a bi- or multinuclear phenotype. The present study provides evidence for neuronal synthesis and targeting of FGF-2 to the nucleus and Golgi apparatus supporting a dual role of FGF-2 in the nucleus and secretory pathway of sympathetic neurons.

Immunohistochemical heterogeneity of nerve cells in the human adrenal gland with special reference to substance P.

Neurons in the human adrenal medulla, stained by the NADH-diaphorase reaction, were counted and their neurochemical markers were investigated by double labeling immunofluorescence with special reference to substance P. The findings indicate a significant participation of intramedullary nerve cell bodies in human adrenal innervation with 40.4 neurons/mm3 adrenal medulla. Substance P-immunoreactive neurons, which made up approximately 20% of all neurons, exhibited heterogeneity by co-localization of immunoreactivities for dynorphin, for cholecystokinin, and for neurofilament triplet. Substance-P-immunolabeled neurons were always nonreactive for calcitonin gene-related peptide, for vasoactive intestinal polypeptide, or for tyrosine hydroxylase, the rate-limiting enzyme of catecholamine synthesis. These chemical phenotypes of intramedullary neurons reveal immunohistochemical similarities with postganglionic neurons in parasympathetic ganglia or with enteric neurons, suggesting a hitherto unrecognized functional significance of the intrinsic nervous system in the human adrenal gland.

Expression of nicotinic acetylcholine receptors in the rat superior cervical ganglion on mRNA and protein level.

The expression of nicotinic acetylcholine receptors (nAChR) in the rat superior cervical ganglion was investigated by Western blotting, immunohistochemistry and non-radioactive in situ hybridization applying probes for the alpha 4-1 and beta 2 subunit mRNA. Immunoblot analysis of homogenized ganglia using the anti-nAChRs antibody WF6 revealed a labeled protein band of apparent molecular weight of 40 kDa which is typical for the alpha subunit of nAChRs. Applying double-labeling immunofluorescence with antibodies against tyrosine hydroxylase, nAChR-like molecules were identified in most postganglionic neurons and in a subpopulation of small intensely fluorescent (SIF) cells. alpha 4-1 and beta 2 subunit mRNAs were detected in all perikarya of postganglionic sympathetic neurons but not in SIF cells. These results suggest that antibodies raised against purified Torpedo AChR bind to nAChR in sympathetic ganglia and indicate that alpha 4-1 and beta 2 subunits are constituents of nAChRs in sympathetic postganglionic neurons but not of SIF cells.

Secretoneurin-immunoreactivity in nerve terminals apposing identified preganglionic sympathetic neurons in the rat: colocalization with substance P and enkephalin.

Preganglionic sympathetic neurons projecting to the superior cervical ganglion are innervated by nerve fibers containing classical neurotransmitters as well as neuropeptides. In this study we examined the possible participation of a novel peptide, secretoneurin (a cleavage product of secretogranin II), in regulation of sympathetic outflow to head and neck by using a retrograde labelling-technique combined with immunohistochemistry. In addition, the coexistence of secretoneurin with substance P and leu-enkephalin, peptides known to innervate preganglionic neurons, was investigated. The majority of retrogradely labeled neurons were localized in the nucleus intermediolateralis of spinal cord segments T1-T3 (maximum at T2). Nearly all of Fast Blue positive neuronal perikarya were apposed by nerve fibers and terminals exhibiting immunoreactivity to secretoneurin. The main secretoneurin-immunoreactive form found in the upper thoracic segments corresponded to the free peptide secretoneurin as revealed by chromatography and radioimmunoassay. More than half of labeled neurons were surrounded by nerve endings containing in addition substance P or leu-enkephalin which were also, however, less frequently colocalized. Our results suggest that secretoneurin influences the activity of preganglionic sympathetic neurons projecting to the superior cervical ganglion. Regarding their frequent colocalization with substance P and leu-enkephalin, functional interactions of these peptides on preganglionic sympathetic nerve activity have to be considered.

Differential display PCR reveals induction of immediate early genes by vasoactive intestinal peptide in PC12 cells.

In order to identify genes regulated by vasoactive intestinal peptide, we performed differential display PCR as originally described by Liang and Pardee. Messenger RNA of PC12 cells treated with vasoactive intestinal peptide or nerve growth factor for one hour was reverse transcribed and amplified using different sets of oligo-dT and random primers. Radioactively labeled PCR products were displayed on polyacrylamide gels and candidate cDNAs extracted from the gel, re-amplified by PCR, cloned, and sequenced. Differential expression was verified by RT-PCR applying sets of specific primers obtained from the sequence. The specificity of the PCR product was confirmed by Southern blotting using a radioactively labeled internal primer and semi-quantitative densitometric analysis. This rapid and sensitive protocol led to the isolation of two immediate early genes, pip92 and PC4, known to be increased on mRNA level by nerve growth factor in PC 12 cells.

Localization, regulation and functions of neurotransmitters and neuromodulators in cervical sympathetic ganglia.

Cervical sympathetic ganglia represent a suitable model for studying the establishment and plasticity of neurochemical organization in the nervous system since sympathetic postganglionic neurons: (1) express several neuromediators, i.e., short acting transmitters, neuropeptide modulators and radicals, in different combinations; (2) receive synaptic input from a limited number of morphologically and neurochemically well-defined neuron populations in the central and peripheral nervous systems (anterograde influence on phenotype); (3) can be classified morphologically and neurochemically by the target they innervate (retrograde influence on phenotype); (4) regenerate readily, making it possible to study changes in neuromediator content after axonal lesion and their possible influence on peripheral nerve regeneration; (5) can be maintained in vitro in order to investigate effects of soluble factors as well as of membrane bound molecules on neuromediator expression; and (6) are easily accessible. Acetylcholine and noradrenaline, as well as neuropeptides and the recently discovered radical, nitric oxide, are discussed with respect to their localization and possible functions in the mammalian superior cervical and cervicothoracic (stellate) paravertebral ganglia. Furthermore, mechanisms regulating transmitter synthesis in sympathetic neurons in vivo and in vitro, such as soluble factors, cell contact or electrical activity, are summarized, since modulation of transmitter synthesis, release and metabolism plays a key role in the neuronal response to environmental influences.

Immunohistochemistry of small intensely fluorescent (SIF) cells and of SIF cell-associated nerve fibers in the rat superior cervical ganglion.

Double-labelling immunofluorescence was applied on single sections of the rat superior cervical ganglion to evaluate neurochemistry and connectivity of intraganglionic SIF cells. The synaptic vesicle membrane protein synaptophysin and secretoneurin, a newly discovered neuropeptide derived from secretogranin II, proved reliable molecular markers of this cell type, whereas serotonin and tyrosine hydroxylase immunoreactivities were observed in slightly incongruent SIF cell subpopulations. Immunolabelling for vasoactive intestinal polypeptide and neuropeptide Y occurred in few SIF cells. None of the above immunoreactivities were visibly altered by preganglionic or postganglionic denervation, while some SIF cells were immunolabelled for galanin or for the neuronal microtubule-associated protein MAP2 after postganglionic denervation. SIF cells were nonreactive for the pan-neuronal marker protein gene product (PGP) 9.5 or neurofilament 160 kD. Intense staining of NADPH-diaphorase in some SIF cells, suggesting catalytic activity of nitric oxide synthase, could not be substantiated by immunoreactivity for this enzyme. SIF cells were approached by nonidentical fiber populations immunoreactive for PGP 9.5, neurofilament, or neuropeptide Y, whereas immunoreactivities for galanin and vasoactive intestinal polypeptide were colocalized in fiber meshes around SIF cells. The findings indicate (1) neurochemical SIF cell heterogeneity, (2) SIF cell plasticity in response to ganglionic perturbation, and (3) a differentiated innervation of SIF cells in the rat superior cervical ganglion.

Plasticity of postganglionic sympathetic neurons in the rat superior cervical ganglion after axotomy.

The neuropeptides galanin (GAL) and vasoactive intestinal polypeptide (VIP) are upregulated in spinal and vagal sensory as well as in cranial motor neurons after axonal transection. In this study an increase of both peptides is demonstrated in axotomized principal ganglionic neurons (PGN) of the rat sympathetic superior cervical ganglion by use of double-labeling immunofluorescence. Compared to control ganglia that do not contain more than 1% GAL- or VIP-positive cells, about 26% of all PGN exhibit GAL immunoreactivity by day 1 after transection of the major postganglionic branches. The proportion of immunoreactive neurons reaches its maximum after 30 days (40%) and decreases to about 27% within the second month after axotomy. The percentage of VIP-positive neurons is much lower than for GAL: 2% of the PGN exhibit VIP immunoreactivity at day 1 and about 7% are observed 30 and 60 days after axotomy. In order to further characterize newly GAL- and VIP-positive PGN, their cell diameters were determined 12 days after axotomy. Compared to the mean overall neuron diameter of 24.8 microns, GAL-immunoreactive neurons are predominantly of small and intermediate size (22.2 microns), whereas VIP occurs mainly in larger neurons (26.1 microns). Besides cell bodies, many intraganglionic nerve fibers stain positive for GAL or VIP, particularly at day 6. Most likely, these fibers represent axons, as indicated by the absence of MAP2, a cytoskeletal protein found in neuronal somata and dendrites. They establish direct membrane contacts with postganglionic perikarya, as revealed by pre-embedding immuno-electron microscopy. Some cell bodies and fibers contain both peptides. Colocalization of GAL or VIP with tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis, reveals a reduced immunoreactivity for TH in intensely GAL- or VIP-positive cells, and vice versa at day 6. However, no difference in staining intensity for VIP or GAL, and TH, is observed after 30 and 60 days. Possible implications of GAL and VIP for peripheral nerve regeneration and their regulation by target-derived factors are discussed.

PACAP immunoreactivity in the rat superior cervical ganglion in comparison to VIP.

The presence and regulation of pituitary adenylate cyclase activating peptide (PACAP) was investigated in the adult rat superior cervical ganglion. Double-labelling immunofluorescence using specific antibodies against PACAP and vasoactive intestinal peptide (VIP) revealed a dense innervation of postganglionic sympathetic neurones by PACAP-immunoreactive but VIP-negative nerve endings that mainly were of preganglionic origin since they disappeared after transection of the cervical sympathetic trunk. Since PACAP regulates transmitter synthesis and release in sympathetic neurones in vitro, this finding provides a morphological correlate for modulatory effects of PACAP in vivo. Few neurones contained PACAP under normal conditions. However, the number of PACAP-positive neurones increased in response to axotomy of the postganglionic nerves. Many of the newly PACAP-positive neurones also contained VIP, suggesting that both peptides are regulated by similar mechanisms.

Expression pattern of candidate cell death effector proteins Bax, Bcl-2, Bcl-X, and c-Jun in sensory and motor neurons following sciatic nerve transection in the rat.

Numerous studies have demonstrated a prolonged expression of c-Jun transcription factor in neurons following axotomy, and it has been hypothesized that c-Jun may be causally involved in neuroregeneration in vivo. By contrast, there is growing evidence from in vitro studies that induction of c-Jun may be necessary for neuronal cell death induced by growth factor starvation. It has been demonstrated that protein levels of cell death repressor Bcl-2 and cell death promotor Bax determine the threshold for neuronal cell death and that their expression is dynamically modulated at the onset of neurodegeneration. In the present study, we investigated by double-immunolabeling methods activation of c-Jun transcription factor and expression of members of the Bcl-2 family of cell death effector proteins in axotomized neurons. Six days after transection of the sciatic nerve in young rats, when axotomized neurons start to degenerate, strong nuclear Jun immunostaining in spinal cord motoneurons was associated with intense cytoplasmic Bax labeling and signs of neuronal atrophy. Bcl-2 and Bcl-X proteins were present only at moderate to low levels. In situ end-labeling by terminal transferase revealed nuclear DNA fragmentation in scattered motoneurons of the ipsilateral ventral horn (1 or 2 labeled nuclei per section). In the L5 dorsal root ganglia (DRG) levels of Bax, Bcl-2, and Bcl-X proteins were highly variable. High levels of Bax immunoreactivity together with intense Jun immunofluorescence were frequently observed in small-diameter sensory neurons. RT-PCR analysis revealed expression of exclusively the anti-apoptotic bcl-xL mRNA isoform in rat DRG which decreased significantly following sciatic nerve transection. These findings indicate that the high susceptibility of central neurons and small-sized DRG neurons to axotomy-induced cell death might be related to their low ratio of cell death repressor Bcl-2 and Bcl-XL to cell death promotor Bax expression. It should be noted, however, that numerous strongly Jun-positive DRG neurons contained low levels of Bax or high levels of Bcl-2 and Bcl-X immunoreactivity. Thus, high levels of c-Jun protein in axotomized neurons do not necessarily suggest a destination to die, and other factors may determine the outcome of axotomy.

Regulation of galanin in rat sympathetic neurons in vitro.

The neuropeptide galanin is induced in sensory and autonomic neurons after peripheral nerve lesion. Leukemia inhibitory factor (LIF) has been suggested to be involved in the up-regulation of galanin. A direct effect of LIF on galanin content in pure sympathetic neuron cultures dissociated from newborn rat superior cervical ganglia was investigated by radioimmunoassay and immunohistochemistry. Galanin increases in sympathetic neurons during a 12 day culture period in the presence of NGF (10 ng/ml). Five days after addition of LIF (10 ng/ml) a 7-fold elevation is observed when compared to control cultures. Furthermore, galanin increases significantly in the presence of non-neuronal cells and in response to potassium-induced depolarization. The proportion of galanin-immunoreactive neurons in mixed cultures is similar to that found in adult rat superior cervical ganglia after transection of the major postganglionic branches. The results corroborate the hypothesis that LIF, presumably released from ganglionic satellite cells, induces galanin in a subpopulation of sympathetic neurons in vivo and in vitro.

Increased innervation of rat preganglionic sympathetic neurons by substance P containing nerve fibers in response to spinal cord injury.

Substance P (SP) is elevated in the intermediate zone caudal to a spinal cord lesion presumably due to sprouting of intraspinal and primary afferent axons. It is unclear, however, if axon terminals are in direct contact with preganglionic neurons located within the different autonomic subnuclei. Therefore, the innervation of preganglionic sympathetic neurons by SP was quantified using confocal imaging and morphometric image analysis. The number of SP-immunoreactive varicosities apposed to nitric oxide synthase-positive neurons significantly increased bilaterally in all sympathetic areas of segment T2 one week after low cervical hemisection at C6/7. Consequently, direct excitatory effects of SP on preganglionic neurons may play an important role in the dysregulation of arterial blood pressure observed in patients with spinal cord injury at the cervical or upper thoracic level.

Vasoactive intestinal peptide but not galanin promotes survival of neonatal rat sympathetic neurons and neurite outgrowth of PC12 cells.

The synthesis of the neuropeptide galanin (GAL) is greatly enhanced after axonal lesion in different neuron populations of the peripheral and central nervous system. In sympathetic ganglia, GAL-immunoreactive nerve fiber baskets have been found surrounding postganglionic neurons after axotomy. Until now, it is unclear if GAL may be involved in neuronal survival or regeneration as suggested for vasoactive intestinal peptide (VIP) that is also upregulated after nerve lesion. We have, therefore, studied the effects of GAL on survival of sympathetic neurons dissociated from newborn rat superior cervical ganglia and on neurite outgrowth of PC12 cells. These effects were compared to those elicited by VIP. Whereas VIP promoted survival of about 10% of sympathetic neurons 2 days after nerve growth factor deprivation and induced neurite outgrowth of PC12 cells already at 6 h after addition of the peptide, GAL had no effect in either of these culture systems. While the induction of VIP may be beneficial for axotomized neurons, the functional significance of increased GAL levels remains to be established.

Ultrastructure of alpha-synuclein-positive aggregations in U373 astrocytoma and rat primary glial cells.

Abnormal alpha-synuclein-positive glial cytoplasmic inclusions are found in Parkinson's disease, multiple system atrophy and dementia with Lewy bodies. We have recently developed an in vitro model of alpha-synuclein-immunoreactive aggregations in U373 astrocytoma cells. We have additionally overexpressed wild-type and a C-terminally truncated form of alpha-synuclein in primary rat glial cells. Astrocytes and oligodendrocytes were found to form alpha-synuclein-positive aggregations in vitro perinuclearly or in the processes of the cells. The morphological studies presented here demonstrate that the aggregations we have observed in vitro are not limited by a membrane but have unclear borders. They have an amorphous dense core that is intensely alpha-synuclein-immunopositive and a predominantly filamentous halo around. Mainly filamentous structures at the border area between the halo and the core are alpha-synuclein-immunoreactive. We conclude that this in vitro model of alpha-synuclein-positive glial aggregations mimics the morphology of the abnormal glial inclusions described in neurodegenerative disorders and could be a suitable model for studying their role in the pathogenesis of these diseases.

Postnatal development of the locus coeruleus in trisomy 19 mice: morphological and morphometric study.

To study the effect of trisomy upon a brain region that is generated very early during development, the locus coeruleus (LC) has been examined morphologically and morphometrically in 23 Trisomy 19 (Ts19) mice and their chromosomally balanced control littermates aged 2-18 days postpartum. Gross morphological alterations of the Ts19 LC could neither be observed by light nor by electron microscopy. The LC was properly located. Ultrastructural features indicating increasing protein synthesis such as nucleolus-like bodies and a rise in the amount of granular endoplasmic reticulum and in the size of the nucleoli have been observed both in Ts19 and control mice. Maturation of the LC was delayed in Ts19. Morphometric studies on the volume, cell number, and cell density revealed that, apart from a 2-day delay in development, the Ts19 LC was of normal size. The present study supports the observation that the noradrenergic system is not affected in the Ts19 CNS. Taking into account that the cerebellum of Ts19 mice is markedly hypoplastic, the results indicate a differential pathogenic effect of trisomy upon different neural systems.