Vesna Radojevic

Basic Fibroblast Growth Factor Modulates Density of Blood Vessels and Preserves Tight Junctions in Organotypic Cortical Cultures of Mice: A New In Vitro Model of the Blood–Brain Barrier

This study was performed to examine the maintenance of blood vessels in vitro in cortical organotypic slice cultures of mice with special emphasis on basic fibroblast growth factor (FGF-2), which is known to promote angiogenesis and to preserve the integrity of the blood–brain barrier. Slices of neonatal day 3 or 4 mouse brain were maintained for 3, 7, or 10 d in vitro (DIV) under standard culture conditions or in the presence of FGF-2. Immunohistochemistry for factor VIII-related antigen or laminin revealed a relative low number of blood vessels under standard conditions. In contrast, moderate FGF-2 concentrations increased the number of vessels: with 0.5 ng/ml FGF-2 it was 1.4-fold higher after DIV 3 or 1.5-fold after DIV 7 compared with controls; with 5 ng/ml it was almost doubled in both cases. With an excess of 50 ng/ml, FGF-2 vessels were reduced after DIV 3 or similar to controls after DIV 7. FGF receptor 1 was preferentially found on endothelial cells; its immunolabeling was reduced in the presence of the ligand. Cell death detected by an ethidium bromide analog or the apoptosis marker caspase-3 was barely detectable during the 10 d culture period. Immunolabeling of the tight junction proteins ZO-1 (zonula occludens protein 1), occludin, claudin-5, and claudin-3 revealed evidence for structural integrity of the blood–brain barrier in the presence of moderate FGF-2 concentrations. In conclusion, FGF-2 maintains blood vessels in vitro and preserves the composition of the tight junction. Hence, we propose FGF-2-treated organotypic cortical slices as a new tool for mechanistic studies of the blood–brain barrier.

Repair of the entorhino-hippocampal projection in vitro

The repair of axonal projections and the reconstruction of neuronal circuits after CNS lesions or during neurodegenerative disease are major challenges in restorative neuroscience. We have explored the potential of transplanted immature neurons to repair a specific axonal projection in an entorhino-hippocampal slice culture model system. When slices of immature entorhinal cortex (EC) from tau-GFP transgenic mice were cultured next to slices from postnatal hippocampus, an axonal projection from the E18 embryonic entorhinal cortex to the dentate gyrus of the postnatal hippocampus developed, which was similar to that observed in control cultures. Even more immature neuronal precursors in slices from E15 developing cerebral cortex differentiated and established an axonal projection to the hippocampal slice. This projection terminated specifically in the outer molecular layer of the dentate gyrus, the normal target area of the entorhino-hippocampal projection. When embryonic tissue from the presumptive brainstem area was used, there was still a subpopulation of fibers with a specific termination in the outer molecular layer, but few specific fibers were found in cocultures with embryonic midbrain. Our results show that very immature cortical neurons are potentially able to form an entorhino-hippocampal projection that terminates in a correct lamina-specific fashion in the dentate gyrus. These findings support the idea that immature neuronal precursor cells could be used for the reconstruction of specific neuronal circuits.

Somatostatin receptor types 1 and 2 in the developing Mammalian cochlea

The neuropeptide somatostatin (SST) exerts several important physiological actions in the adult central nervous system through interactions with membrane-bound receptors. Transient expression of SST and its receptors has been described in several brain areas during early ontogeny. It is therefore believed that SST may play a role in neural maturation. The present study provides the first evidence for the developmental expression of SST receptors in the mammalian cochlea, emphasizing their possible roles in cochlear maturation. In the developing mouse cochlea, cells immunoreactive to somatostatin receptor 1 (SSTR1) and somatostatin receptor 2 (SSTR2) were located in the embryonic cochlear duct on Kolliker’s organ as early as embryonic day (E) 14 (E14). At E17, the expression of both receptors was high and already located at the hair cells and supporting cells along the length of the cochlear duct, which have become arranged into the characteristic pattern for the organ of Corti (OC) at this stage. At birth, SSTR1- and SSTR2-containing cells were only localized in the OC. In general, immunoreactivity for both receptors increased in the mouse cochlea from postnatal day (P) 0 (P0) to P10; the majority of immunostained cells were inner hair cells, outer hair cells, and supporting cells. Finally, a peak in the mRNA and protein expression of both receptors is present near the time when they respond to physiological hearing (i.e., hearing of airborne sound) at P14. At P21, SSTR1 and SSTR2 levels decrease dramatically. A similar developmental pattern was observed for SSTR1 and SSTR2 mRNA, suggesting that the expression of the SSTR1 and SSTR2 genes is controlled at the transcriptional level throughout development. In addition, we observed reduced levels of phospho-Akt and total Akt in SSTR1 knockout and SSTR1/SSTR2 double-knockout mice compared with wild-type mice. We know from previous studies that Akt is involved in hair cell survival. Taken together, the dynamic nature of SSTR1 and SSTR2 expression at a time of major developmental changes in the cochlea suggests that SSTR1 and SSTR2 (and possibly other members of this family) are involved in the maturation of the mammalian cochlea.

Inhibition of mTOR by Rapamycin Results in Auditory Hair Cell Damage and Decreased Spiral Ganglion Neuron Outgrowth and Neurite Formation In Vitro.

Rapamycin is an antifungal agent with immunosuppressive properties. Rapamycin inhibits the mammalian target of rapamycin (mTOR) by blocking the mTOR complex 1 (mTORC1). mTOR is an atypical serine/threonine protein kinase, which controls cell growth, cell proliferation, and cell metabolism. However, less is known about the mTOR pathway in the inner ear. First, we evaluated whether or not the two mTOR complexes (mTORC1 and mTORC2, resp.) are present in the mammalian cochlea. Next, tissue explants of 5-day-old rats were treated with increasing concentrations of rapamycin to explore the effects of rapamycin on auditory hair cells and spiral ganglion neurons. Auditory hair cell survival, spiral ganglion neuron number, length of neurites, and neuronal survival were analyzed in vitro. Our data indicates that both mTOR complexes are expressed in the mammalian cochlea. We observed that inhibition of mTOR by rapamycin results in a dose dependent damage of auditory hair cells. Moreover, spiral ganglion neurite number and length of neurites were significantly decreased in all concentrations used compared to control in a dose dependent manner. Our data indicate that the mTOR may play a role in the survival of hair cells and modulates spiral ganglion neuronal outgrowth and neurite formation.

Role of Somatostatin Receptor-2 in Gentamicin-Induced Auditory Hair Cell Loss in the Mammalian Inner Ear

Hair cells and spiral ganglion neurons of the mammalian auditory system do not regenerate, and their loss leads to irreversible hearing loss. Aminoglycosides induce auditory hair cell death in vitro, and evidence suggests that phosphatidylinositol-3-kinase/Akt signaling opposes gentamicin toxicity via its downstream target, the protein kinase Akt. We previously demonstrated that somatostatin—a peptide with hormone/neurotransmitter properties—can protect hair cells from gentamicin-induced hair cell death in vitro, and that somatostatin receptors are expressed in the mammalian inner ear. However, it remains unknown how this protective effect is mediated. In the present study, we show a highly significant protective effect of octreotide (a drug that mimics and is more potent than somatostatin) on gentamicin-induced hair cell death, and increased Akt phosphorylation in octreotide-treated organ of Corti explants in vitro. Moreover, we demonstrate that somatostatin receptor-1 knockout mice overexpress somatostatin receptor-2 in the organ of Corti, and are less susceptible to gentamicin-induced hair cell loss than wild-type or somatostatin-1/somatostatin-2 double-knockout mice. Finally, we show that octreotide affects auditory hair cells, enhances spiral ganglion neurite number, and decreases spiral ganglion neurite length.

Expression and localization of somatostatin receptor types 3, 4 and 5 in the wild-type, SSTR1 and SSTR1/SSTR2 knockout mouse cochlea

Somatostatin (SST) is a peptide hormone that exerts inhibitory effects mediated through binding to specific cell surface G protein-coupled receptors, of which five distinct subtypes (SSTR1-SSTR5) have been characterized. Our study performed on mouse cochlear hair cells shows the expression and localization of the three receptors (SSTR3-SSTR5) in wild-type (WT), single-knockout (SSTR1 KO) and double-knockout SSTR1/SSTR2 (DKO) mice. Similar SSTRs expression were observed in the inner hair cells (IHC), outer hair cells (OHC) and supporting cells of cultivated P7 mouse organ of Corti (OC) explants as well as in cultivated cochlear neuroepithelial supporting cells (NEsc). We found differences in the expression of SSTR3-5 in WT, SSTR1 KO and DKO mouse cochlea, which might be explained as a compensatory effect in the cochlea after the loss of SSTR1 and/or SSTR2.

T-cadherin in the mammalian cochlea

Cadherins are a superfamily of transmembrane glycoproteins, which mediate calcium‐dependent intercellular adhesions. T‐cadherin is an atypical member of the cadherin family in regard to its structure; it acts as a signalling receptor rather than an adhesion molecule. In this study we examine the role of T‐cadherin in the mammalian cochlea.

Directed Fiber Outgrowth from Transplanted Embryonic Cortex-Derived Neurospheres in the Adult Mouse Brain

Neural transplantation has emerged as an attractive strategy for the replacement of neurons that have been lost in the central nervous system. Multipotent neural progenitor cells are potentially useful as donor cells to repopulate the degenerated regions. One important aspect of a transplantation strategy is whether transplanted cells are capable of fiber outgrowth with the aim of rebuilding axonal connections within the host brain. To address this issue, we expanded neuronal progenitor from the cortex of embryonic day 15 ubiquitously green fluorescent protein-expressing transgenic mice as neurospheres in vitro and grafted them into the entorhinal cortex of 8-week-old mice immediately after a perforant pathway lesion. After transplantation into a host brain with a lesion of the entorhino-hippocampal projection, the neurosphere-derived cells extended long fiber projections directed towards the dentate gyrus. Our results indicate that transplantation of neurosphere-derived cells might be a promising strategy to replace lost or damaged axonal projections.

Activin signaling disruption in the cochlea does not influence hearing in adult mice.

Activin, a member of the TGF-F superfamily, was found to play an important role in the development, repair and apoptosis of different tissues and organs. Accordingly, activin signaling is involved in the development of the cochlea. Activin binds to its receptor ActRII, then dimerizes with ActRI and induces a signaling pathway resulting in gene expression. A study reported a case of fibrodysplasia ossificans progressiva with an unusual mutation in the ActRI gene leading to sensorineural hearing loss. This draws attention to the role of activin and its receptors in the developed cochlea. To date, only the expression of ActRII is known in the adult mammalian cochlea. In this study, we present for the first time the presence of activin A and ActRIB in the adult cochlea. Transgenic mice with postnatal dominant-negative ActRIB expression causing disruption of activin signaling in vivo were used for assessing cochlear morphology and hearing ability through the auditory brainstem response (ABR) threshold. Nonfunctioning ActRIB did not affect the ABR thresholds and did not alter the microscopic anatomy of the cochlea. We conclude, therefore, that activin signaling is not necessary for hearing in adult mice under physiological conditions but may be important during and after damaging events in the inner ear. i 2014 S. Karger AG, Basel

TARGETING THE SOMATOSTATIN RECEPTORS AS A THERAPEUTIC APPROACH FOR THE PRESERVATION AND PROTECTION OF THE MAMMALIAN COCHLEA FROM EXCITOTOXICITY

The neuropeptide somatostatin (SST) is an important modulator of neurotransmission in the central nervous system (CNS) and binds to G-protein-coupled receptors (SSTR1-5) on target cells. Little is known about the expression and function of the somatostatinergic system in the mammalian cochlea. We analyzed the expression of SSTR1-SSTR5 in the immature mammalian cochlea. The peak in the expression of SSTR1 and SSTR2 at mRNA and protein level is around the onset of hearing to airborne sound, at postnatal day (P)14. This suggests their involvement in the maturation of the mammalian cochlea. We demonstrated that all five receptors are expressed in the inner hair cells (IHC) and outer hear cells (OHC) as well as in defined supporting cells of the organ of Corti (OC) in the adult mouse cochlea. A similar expression of the SSTRs in the IHC and OHC was found in cultivated P6 mouse OC explants as well as in neuroepithelial cell culture. In order to learn more about the regulation of SSTRs, we used mice with either a deletion of SSTR1, SSTR2 or SSTR1/SSTR2 double knock out (DKO). In DKO mice, SSTR5 was up-regulated and SSTR3 and SSTR4 were down regulated. These findings provide evidence of a compensatory regulation in the mammalian cochlea as a consequence of a receptor subtype deletion. In addition, we observed reduced levels of phospho-Akt and total-Akt in SSTR1 KO and DKO mice as compared to wild type (WT) mice. Akt is likely to be involved in hair cell survival. Most importantly, we found improved hair cell survival in somatostatin and octreotide treated OC explants that had been exposed to gentamicin compared to those explants exposed to gentamicin alone. These findings propose that the somatostatinergic system within the cochlea may have neuroprotective properties.