Characterization of hyperbranched core-multishell nanocarriers as an innovative drug delivery system for the application at the oral mucosa.

BACKGROUND AND OBJECTIVES: In the oral cavity, the mucosal tissues may develop a number of different pathological conditions, such as inflammatory diseases (gingivitis, periodontitis) and autoimmune disorders (eg, oral lichen planus) that require therapy. The application of topical drugs is one common therapeutic approach. However, their efficacy is limited. Dilution effects due to saliva hinder the adherence and the penetration of drug formulations. Therefore, the bioavailability of oral topical drugs is insufficient, and patients may suffer from disease over years, if not life-long. MATERIAL AND METHODS: In the present study, we characterized core-multishell (CMS) nanocarriers for their potential use as drug delivery systems at oral mucosal tissues. For this purpose, we prepared porcine masticatory as well as buccal mucosa and performed Franz cell diffusion experiments. Penetration of fluorescently labeled CMS nanocarriers into the mucosal tissue was analyzed using confocal laser scanning microscopy. Upon exposure to CMS nanocarriers, the metabolic and proliferative activity of gingival epithelial cells was determined by MTT and sulforhodamine B assays, respectively. RESULTS: Here, we could show that the carriers penetrate into both mucosal tissues, while particles penetrate deeper into the masticatory mucosa. Electron paramagnetic resonance spectroscopy revealed that the 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy-labeled glucocorticoid dexamethasone loaded on to the CMS nanocarriers was released from the carriers in both mucosal tissues but with a higher efficiency in the buccal mucosa. The release from the nanocarriers is in both cases superior compared to the release from a conventional cream, which is normally used for the treatment of inflammatory conditions in the oral cavity. The CMS nanocarriers exhibited neither cytotoxic nor proliferative effects in vitro. CONCLUSION: These findings suggested that CMS nanocarriers might be an innovative approach for topical drug delivery in the treatment of oral inflammatory diseases.

[Can Psychometric Tests Predict Success in the Selection Interview for Medical School? A Cross-Sectional Study at One German Medical School]. / Können psychometrische Tests den Erfolg im Auswahlgespräch zum Medizinstudium vorhersagen? Eine Querschnittsstudie an einer deutschen Hochschule.

Background The final exam grade is the main selection criterion for medical school application in Germany. For academic success, it seems to be a reliable predictor. Its use as the only selection criterion is, however, criticised. At some universities, personal interviews are part of the selection process. However, these are very time consuming and are of doubtful validity. The (additional) use of appropriate psychometric instruments could reduce the cost and increase the validity. This study investigates the extent to which psychometric instruments can predict the outcome of a personal selection interview. Methods This is a cross-sectional study on the correlation of the results of psychometric instruments with those of the personal selection interview as part of the application process. As the outcome, the score of the selection interview was used. The NEO - Five Factor Inventory, the Hospital Anxiety and Depression Scale (HADS) and the questionnaire to identify work-related behaviour and experience patterns (AVEM) were used as psychometric interviews. Results There was a statistically significant correlation with the results of the personal selection interview for the sum score of the depression scale from the HADS and the sum score for the dimension of life satisfaction of the AVEM. In addition, those participants who did not previously complete an application training achieved a better result in the selection interview. Conclusion The instruments used measure different aspects than the interviews and cannot replace them. It remains to be seen whether the selected parameters are able to predict academic success.

Functional MRI activation in response to panic-specific, non-panic aversive, and neutral pictures in patients with panic disorder and healthy controls.

There is evidence that besides limbic brain structures, prefrontal and insular cortical activations and deactivations are involved in the pathophysiology of panic disorder. This study investigated activation response patterns to stimulation with individually selected panic-specific pictures in patients with panic disorder with agoraphobia (PDA) and healthy control subjects using functional magnetic resonance imaging (fMRI). Structures of interest were the prefrontal, cingulate, and insular cortex, and the amygdalo-hippocampal complex. Nineteen PDA subjects (10 females, 9 males) and 21 healthy matched controls were investigated using a Siemens 3-Tesla scanner. First, PDA subjects gave Self-Assessment Manikin (SAM) ratings on 120 pictures showing characteristic panic/agoraphobia situations, of which 20 pictures with the individually highest SAM ratings were selected. Twenty matched pictures showing aversive but not panic-specific stimuli and 80 neutral pictures from the International Affective Picture System were chosen for each subject as controls. Each picture was shown twice in each of four subsequent blocks. Anxiety and depression ratings were recorded before and after the experiment. Group comparisons revealed a significantly greater activation in PDA patients than control subjects in the insular cortices, left inferior frontal gyrus, dorsomedial prefrontal cortex, the left hippocampal formation, and left caudatum, when PA and N responses were compared. Comparisons for stimulation with unspecific aversive pictures showed activation of similar brain regions in both groups. Results indicate region-specific activations to panic-specific picture stimulation in PDA patients. They also imply dysfunctionality in the processing of interoceptive cues in PDA and the regulation of negative emotionality. Therefore, differences in the functional networks between PDA patients and control subjects should be further investigated.

Visual activity is required to maintain the phenotype of supragranular NPY neurons in rat area 17.

Visual activity governs the functional maturation of the mammalian visual cortex. We report here, that visual experience is required for stabilizing the phenotype of a subset of cortical interneurons. Neurons expressing neuropeptide Y mRNA (NPY neurons) display a transiently higher expression in the early postnatal visual areas 18a and 17 that is followed by a phenotype restriction during the second postnatal month: about 50% of the NPY neurons in supragranular and infragranular layers of area 18a, and in infragranular layers of area 17 gradually stop the NPY expression. In contrast, the expression remains unchanged in supragranular layers of area 17. Dark rearing rats from birth to up to 100 days does neither prevent the developmental onset of NPY mRNA expression, nor does it prevent the phenotype restriction from occurring. In contrast, in dark reared animals NPY neurons in supragranular layers of area 17 now also undergo a phenotype restriction. Returning animals to light after variable periods of darkness results in an upregulation of NPY mRNA expression selectively in neurons in supragranular layers of area 17. These neurons acquire a constitutive expression during the second postnatal month. This suggests that the phenotypic specification of a distinct subset of cortical interneurons is regulated by visual experience which thus influences on the maturation of the neurochemical architecture of area 17.

NT-4/5 and LIF, but not NT-3 and BDNF, promote NPY mRNA expression in cortical neurons in the absence of spontaneous bioelectrical activity.

Epigenetic factors are known to influence the differentiation of neocortical neurons. The present study analyses the role of spontaneous bioelectrical activity (SBA) and neurotrophic factors on the expression of neuropeptide Y (NPY) in rat visual cortical neurons using organotypic monocultures prepared from newborn animals and in situ hybridization to detect the NPY messenger ribonucleic acid (mRNA). Spontaneously active cortex cultures display NPY mRNA expression in about 7% of all cortical neurons from 10 days in vitro (DIV) on. Blocking the SBA by chronic application of 10 mM Mg2+ for 3-30 DIV reduces the percentage of NPY neurons to about 2%. Allowing an initial phase of SBA (1-20 DIV) followed by an SBA blockade (for 21-50 DIV) results in 2% labelled neurons, indicating a dramatic reduction of NPY mRNA expression in the absence of SBA. Surprisingly, the reverse experiment (a period of SBA blockade for 1-20 DIV followed by a period of SBA recovery for 21-40 DIV) does not cause an upregulation of NPY mRNA expression. However, allowing cultures to differentiate as spontaneously active cultures, then applying a transient period of SBA blockade which is followed by a second period of SBA, does rescue the NPY mRNA expression in 7% of the cortical neurons. We conclude that SBA is a main trigger for NPY mRNA expression and it is particularly important during an early postnatal period of differentiation. We then analysed whether neurotrophic factors known to modulate cortical neuropeptide expression are able to do so in the absence of SBA. Supplementing chronically blocked cultures with the neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and the cytokine, leukaemia inhibitory factor (LIF), reveals that BDNF and NT-3 are unable to increase the percentage of NPY neurons. In contrast, LIF and NT-4/5 increase the percentage of NPY neurons to 4 and 6-7%, respectively. Moreover, neurons treated with NT-4/5 display a very high level of NPY mRNA expression in somata and in the dendritic trees. The data suggest a complex interplay and a hierarchy of epigenetic factors in regulating the neurochemical architecture of the developing neocortex.

Postnatal expression pattern of calcium-binding proteins in organotypic thalamic cultures and in the dorsal thalamus in vivo.

The present study describes the postnatal expression of calbindin, calretinin and parvalbumin and glutamic acid decarboxylase (GAD) and microtubule-associated protein 2 (MAP2) in organotypic monocultures of rat dorsal thalamus compared to the thalamus in vivo. Cultures were maintained for up to 7 weeks. Cortex-conditioned medium improved the survival of thalamic cultures. MAP2-immunoreactive material was present in somata and dendrites of small and large-sized neurons throughout the cultures. Parvalbumin immunoreactivity was present in larger multipolar or bitufted neurons along the edge of a culture. These neurons also displayed strong parvalbumin mRNA and GAD mRNA expression, and GABA immunoreactivity. They likely corresponded to cells of the nucleus reticularis thalami. Parvalbumin mRNA, but neither parvalbumin protein nor GAD mRNA, was expressed in neurons with large somata within the explant. They likely represented relay cells. GAD mRNA, but not parvalbumin mRNA, was expressed in small neurons within the explants. Small neurons also displayed calbindin- and calretinin-immunoreactivity. The small neurons likely represented local circuit neurons. The time course of expression of the calcium-binding proteins revealed that all were present at birth with the predicted molecular weights. A low, but constant parvalbumin expression was observed in vitro without the developmental increase seen in vivo, which most likely represented parvalbumin from afferent sources. In contrast, the explantation transiently downregulated the calretinin and calbindin expression, but the neurons recovered the expression after 14 and 21 days, respectively. In conclusion, thalamic monocultures older than three weeks represent a stable neuronal network containing well differentiated neurons of the nucleus reticularis thalami, relay cells and local circuit neurons.

Phenotype specification of cortical neurons during a period of molecular plasticity.

The transient expression of neuropeptide transmitters is a common feature of the developing cortex. We have now analysed the role of cortical afferents in shaping the neurochemical architecture of rat visual cortex using organotypic cultures. Deafferented cortex monocultures prepared from newborn rats reveal a constant NPY mRNA expression in 6-8% of all cortical neurons up to 90 days in vitro (DIV). In contrast, afferent thalamocortical and corticocortical axonal innervation elicits a progressive reduction in the percentage of NPY mRNA expressing neurons from initially 6-8% in 30DIV cocultures to 2-3% and 3-4% respectively in 60DIV cocultures, which is maintained for up to 90DIV. This phenotype restriction is not observed in only efferently connected corticocollicular cocultures. Further, axonal innervation does not change the percentage of GAD mRNA-expressing neurons, which remains at 13% in mono- and cocultures. When feeding thalamocortical cocultures with monoculture-conditioned medium between 3-20DIV followed by normal medium up to 60DIV, the phenotype restriction fails to occur in the cocultured cortex. We conclude that cortex-derived factors secreted into the medium by a monoculture suppress the phenotype-restricting capacity of the afferents, but only when present within the first 14DIV during the period of formation of axonal connections. To elucidate the nature of the cortex-derived factors, brain-derived neurotrophic factor was applied to the medium. When applied for the first 14DIV, it does not prevent the phenotype restriction from occurring. This suggests that epigenetic factors such as axonal innervation and cortex-derived factors other than brain-derived neurotrophic factor govern a phenotype decision in neocortical neurons during a period of molecular plasticity.

Development and activity-dependent expression of neuronal marker proteins in organotypic cultures of rat visual cortex.

We are interested in activity-dependent mechanisms which govern the structural and functional maturation of neurons in the visual cortex. We have asked whether the expression of neuronal markers microtubule-associated proteins tau, MAP-2, synaptophysin (p38), and the growth-associated protein GAP-43 are dependent on cortical afferents or spontaneous activity. As a model system we have employed organotypic monocultures of rat visual cortex (OTCs, isolated from subcortical structures) in comparison with visual cortex in vivo (innervated by thalamic and other afferents) at different postnatal ages. We know from previous work that the OTCs, like the cortex in vivo, display a high rate of spontaneously generated action potentials. Therefore, as a third objective, we have analysed OTCs grown as monocultures under chronic blockade of spontaneous action potentials. Protein expression was detected by protein blots and/or immunohistochemistry. The proteins examined in this study are expressed in OTCs, even when grown under activity blockade. However, the pattern of expression differs from the cortex in vivo. Tau is expressed much weaker in OTCs than in cortex in vivo. The expression of the major band of about 50 kDa increases over time in vivo and in OTCs. Smaller isoforms of tau are dramatically downregulated, and larger (adult) isoforms do not appear within 35 days in vitro (DIV). Under activity blockade the expression of tau reaches a maximum by 21 DIV and decreases dramatically, so that the protein is hardly detectable by 47 DIV. MAP-2-immunoreactive proteins are localized in somata and dendrites, but also persist in axons. The expression in OTCs of p38 and GAP-43 correlates well with the expression observed in vivo. Synaptophysin (p38) occurs with a similar time course and amount in OTCs as in cortex in vivo. Synaptic boutons appear in all layers, and specialized terminal elements have been observed. Activity blockade slightly affects the p38 expression, although the late postnatal decline in p38 immunoreactivity observed on protein blots from cortex in vivo and in normal OTCs appears more accentuated in activity-blocked OTCs. The GAP-43 expression is prominent from birth onwards in vivo and in OTCs. However, in normal OTCs GAP-43 is not declining as it is in vivo, although it is downregulated in activity-blocked OTCs. As a major finding we report that neuronal markers which are normally expressed in immature neurons and axons during the period of differentiation and structural plasticity are continuously expressed in OTCs, suggesting that a monocultured cortex retains the ability for growth and structural changes longer than the cortex in vivo.

cDNA cloning and expression of rsca1, the rat counterpart of the human spinocerebellar ataxia type 1 gene.

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder caused by an expanded and unstable (CAG) > 40 repeat within a gene of unknown function. We isolated the complete coding region of the rat SCA1 gene (rsca1), the 5'-untranslated region (UTR) and 1.3 kb of the 3'-UTR. The rat sequence exhibits 90% peptide identity to the human counterpart. In comparison to human, the rat (CAG)n block is reduced to two trinucleotide motifs preceded by three different proline codons not present in man. Furthermore, we investigated the expression of rsca1 in different rat tissues. The rsca1 gene is predominantly expressed in brain throughout all developmental stages. In situ hybridizations reveal high levels of expression in various regions of the adult rat brain, including cerebellum, hippocampus and cortex.

Areal differences of NPY mRNA-expressing neurons are established in the late postnatal rat visual cortex in vivo, but not in organotypic cultures.

In order to learn about the factors regulating the postnatal development of neocortical peptidergic neuron populations, we have analysed neurons expressing neuropeptide Y (NPY) by immunohistochemistry and in situ hybridization in developing and adult rat visual cortical areas 17 and 18a in vivo, and in organotypic slice cultures of rat visual cortex. For quantitative analysis, the percentage of NPY mRNA-expressing neurons was determined in supragranular layers I-IV, in infragranular layers V and VI and in the white matter. In vivo, this percentage increased in visual areas 17 and 18a until postnatal day 21 in supra- and infragranular layers. Initially, in both areas the neurons were about equally distributed in supra- and infragranular layers (a ratio of 1:1). During the second postnatal month, the percentage of NPY mRNA-expressing neurons in area 18a declined by approximately 50% in both supra- and infragranular layers, so that the ratio of 1:1 remained constant. In contrast, in area 17 the percentage of neurons in supragranular layers remained fairly constant, but it declined to 50% in infragranular layers, so that by postnatal day 70 the ratio was gradually shifted to 2:1. Throughout development, area 18a contained significantly more NPY mRNA-expressing neurons than area 17. In organotypic slice cultures, a high density of NPY mRNA-expressing neurons had appeared by 10 days in vitro. A much higher percentage of neurons expressed NPY mRNA. The ratio of labelled neurons in supra- versus infragranular layers was 1:1. Both ratio and percentage remained constant from 10-85 days in vitro. The decline in vivo was not caused by an elimination of transient cell types. All cell types persisted into adulthood. Four NPY peptide-immunoreactive neuronal types were classified by axonal morphology in organotypic slice cultures and in vivo; they include (i) cells in layer VI/white matter with horizontal axons and ascending collaterals, (ii) cells in layers V/VI with descending axon and horizontal collaterals, (iii) Martinotti cells in layers V/VI with ascending axons, and (iv) cells in layers III-V with columnar axons. Two further types, bipolar cells with axons descending from dendrites and small basket cells with short horizontal axons, both found in vivo in layers II/III, could not be unequivocally identified in organotypic slice cultures. The NPY-immunoreactive neuron types had already formed a dense innervation of the cultures by 10 days in vitro, which remained stable for up to 85 days in vitro, and resembled the innervation observed in vivo. NPY peptide-immunoreactive neurons in organotypic slice cultures and in vivo were distributed in cortical layers II/III, V and VI and the white matter, but rarely in layers I and IV, which corresponded to the distribution of NPY mRNA-expressing neurons. However, with in situ hybridization more neurons were detectable, especially in layers II/III. A majority of NPY mRNA-expressing neurons co-localized NPY peptide, somatostatin and calbindin. We conclude that intrinsic cues were sufficient to drive the molecular expression of the NPY phenotype, the morphological differentiation and the stabilization of an organotypic NPY innervation in organotypic slice cultures. However, the area- and lamina-specific changes observed in vivo were not observed under monoculture conditions.

Oligodendrocytes differentiate in organotypic cultures of rat visual cortex and myelinate efferent axons.

We have investigated the presence and function of glia cells, especially of oligodendrocytes (OL) in organotypic cultures of rat visual cortex grown for 1-6 weeks in vitro. OL identified by strong Galactocerebroside-immunoreactivity (GalC-ir) displayed rather small somata and elaborately ramified processes. They were most concentrated in layers VIa and VIb and the remnant of the white matter. Silver staining revealed long descending or oblique processes in layers V and VI, which were often arranged in patches, and horizontal processes in the white matter. Proximal processes of OL cell bodies were connected to these long processes. DiI-labeling revealed very similar patches of processes, termed OL domains. They were identified as membraneous sheaths formed by processes of single OL around axons passing the OL domain. Confocal microscopy revealed single axons running through the membrane sheaths. We compared the molecular differentiation of glial cells in cultures to the in vivo situation with protein blots and immunohistochemistry for glial cell marker molecules. In homogenates of visual cortex in vivo, protein blots revealed the increase in expression by OL of myelin basic protein (MBP) during the fourth postnatal week. The astrocytic marker glial fibrillary acidic protein (GFAP), blotted as a control, increased over time in vivo, beginning at P14, indicating the differentiation of astrocytes. In homogenates of organotypic cortex cultures, the times course of expression of GFAP was very similar: it increased dramatically during the first 10 DIV, and remained fairly constant in older cultures.(ABSTRACT TRUNCATED AT 250 WORDS)