Isabelle Perroteau

Blood Vessels Form a Scaffold for Neuroblast Migration in the Adult Olfactory Bulb

New cells are continuously added to the rodent olfactory bulb (OB), throughout development and in adults. These cells migrate tangentially from the subventricular zone along the rostral migratory stream to the OB, where they migrate radically from the center to periphery of the OB. Although different modalities of radial migration have been described in other brain regions, the mechanisms governing radial migration in the OB are still mostly unknown. Here, we identify a new modality of migration in which neuronal precursors migrate along blood vessels toward their destination. Our results show that half of the radially migrating cells associate with the vasculature in the granule cell layer of the OB, and in vivo time-lapse imaging demonstrates that they use blood vessels as a scaffold for their migration through an interaction with the extracellular matrix and perivascular astrocyte end feet. The present data provide evidence that a new modality of migration, vasophilic migration, is occurring in the adult brain and reveals a novel role of brain vasculature.

cAMP response element-binding protein regulates differentiation and survival of newborn neurons in the olfactory bulb

The transcription factor cAMP response element-binding protein (CREB) is involved in multiple aspects of neuronal development and plasticity. Here, we demonstrate that CREB regulates specific phases of adult neurogenesis in the subventricular zone/olfactory bulb (SVZ/OB) system. Combining immunohistochemistry with bromodeoxyuridine treatments, cell tracer injections, cell transplants, and quantitative analyses, we show that although CREB is expressed by the SVZ neuroblasts throughout the neurogenic process, its phosphorylation is transient and parallels neuronal differentiation, increasing during the late phase of tangential migration and decreasing after dendrite elongation and spine formation. In vitro, inhibition of CREB function impairs morphological differentiation of SVZ-derived neuroblasts. Transgenic mice lacking CREB, in a null CREM genetic background, show reduced survival of newborn neurons in the OB. This finding is further supported by peripheral afferent denervation experiments resulting in downregulation of CREB phosphorylation in neuroblasts, the survival of which appears heavily impaired. Together, these findings provide evidence that CREB regulates differentiation and survival of newborn neurons in the OB.

Direct radioimmunoassays for plasma corticosterone and aldosterone in frog. I. Validation of the methods and evidence for daily rhythms in a natural environment

Abstract Two radioimmunoassay techniques for direct measurement of frog plasma concentrations of corticosterone after prior ethanol extraction for deproteinization, and of aldosterone without prior extraction, are described. Specific antibodies against corticosterone 21-hemisuccinate and aldosterone 18,21-diacetate-3-carboxymethoxime derivatives conjugated to bovine serum albumin are raised in rabbits. The sensitivity threshold of the assays allows the assessment of corticosterone in 10-μl and aldosterone in 5-μl samples of plasma. Sephadex LH-20 chromatography demonstrates the validity of both methods. The intra- and interassay reproducibilities and the accuracy of each assay have been studied. The conditions making it possible to reduce aldosterone fluctuations during blood taking have been ascertained. Using these techniques corticosterone and aldosterone concentrations have been assessed in the plasma of 214 frogs caught in their natural habitat at 4-hr intervals during a 40-hr period in mid-June. The existence of synchronous and reproducible 24-hr rhythms of corticosterone and aldosterone plasma levels has been demonstrated. High concentrations of both corticosteroids are recorded during the night and low concentrations are recorded during daylight. The amplitude of corticosterone fluctuations is 3.5-fold greater than that of aldosterone fluctuations. Corticosteroid rhythms are compared to activity phases of frogs during the day at this period of the year.

Acylated and unacylated ghrelin impair skeletal muscle atrophy in mice

Cachexia is a wasting syndrome associated with cancer, AIDS, multiple sclerosis, and several other disease states. It is characterized by weight loss, fatigue, loss of appetite, and skeletal muscle atrophy and is associated with poor patient prognosis, making it an important treatment target. Ghrelin is a peptide hormone that stimulates growth hormone (GH) release and positive energy balance through binding to the receptor GHSR-1a. Only acylated ghrelin (AG), but not the unacylated form (UnAG), can bind GHSR-1a; however, UnAG and AG share several GHSR-1a-independent biological activities. Here we investigated whether UnAG and AG could protect against skeletal muscle atrophy in a GHSR-1a-independent manner. We found that both AG and UnAG inhibited dexamethasone-induced skeletal muscle atrophy and atrogene expression through PI3Kβ-, mTORC2-, and p38-mediated pathways in myotubes. Upregulation of circulating UnAG in mice impaired skeletal muscle atrophy induced by either fasting or denervation without stimulating muscle hypertrophy and GHSR-1a-mediated activation of the GH/IGF-1 axis. In Ghsr-deficient mice, both AG and UnAG induced phosphorylation of Akt in skeletal muscle and impaired fasting-induced atrophy. These results demonstrate that AG and UnAG act on a common, unidentified receptor to block skeletal muscle atrophy in a GH-independent manner.

Immunological detection and quantitation of alpha transforming growth factors in human breast carcinoma cells

SummaryAlpha transforming growth factors (αTGFs) were immunologically detected in the concentrated conditioned medium (CM) prepared from four human breast cancer cell lines and from primary cultures of human mammary epithelial cells, and in the tissue extracts prepared from normal, benign, and malignant breast biopsies. Immunoreactive αTGFs were quantitated by a competitive radioimmunoassay (RIA) using affinity-purified polyclonal sheep anti-rat αTGF antibodies which react with human αTGF but not with human epidermal growth factor (EGF). The relative level of RIA-detectable αTGFs in the CM from the breast cancer cell lines MCF-7, ZR-75-1, T47-D, and MDA-MB-231, and from the CM of primary cultures of human mammary epithelial cells, ranged from 0.02 to 0.85 ng/ml. MCF-7 or ZR-75-1 cells grown in the presence of 17β-estradiol (10−8 M) for 48 h were found to release two- to three-fold more αTGFs into their CM than the same cells grown in the absence of estrogen. In detergent extracts prepared from normal breast tissue, a benign fibrocystic lesion, fibroadenomas and primary breast carcinomas, the relative αTGF concentrations were found to range from 1.5 to 6 ng/mg cell protein. No significant correlations were found between the αTGF levels and the pathological state of the tissues, the estrogen receptor status of the tumors, or the relative amounts of theras gene protein p21ras in the tissues as determined by Western immunoblot analysis. The question of biological relevancy of αTGF for human mammary tumors will require further studies on (a) synthesis and turnover of αTGF, (b) the relationship between immunoreactivity and biological activity of αTGF, and (c) differences in biological responsiveness of mammary tumor cells.

Subventricular zone-derived neuroblast migration to the olfactory bulb is modulated by matrix remodelling

In the rodent brain neural progenitor cells are born in the subventricular zone and migrate along a pathway called the rostral migratory stream (RMS) into the olfactory bulb where they differentiate into several classes of interneurones. In the adult, tangential migration in the RMS takes place in ‘chains’ of cells contained within glial tubes. In contrast, neonatal neuroblasts along the RMS lack these defined glial tubes and chains, migrating instead as individual cells. Time‐lapse confocal microscopy of neuroblasts at each of these ages shows that individual cells migrate in a saltatory manner with bursts of high speed followed by periods of slower speed. Tangential migration within a glial tube is 20% faster than migration as individual cells. Neuroblasts may also interact and modify the extracellular matrix during migration through expression of a family of proteins, the matrix metalloproteinases (MMPs). MMPs are present and active along the subventricular zone–olfactory bulb pathway. In the presence of inhibitors of MMPs, neuroblast migration rates were reduced only when cells migrate individually. Chain migration in the adult was unaffected by MMP inhibitors. Taken together, these data suggest that MMPs only influence migration as individual cells and not as chains.

Schwann cell behavior after nerve repair by means of tissue‐engineered muscle‐vein combined guides

Schwann cells play a critical role in peripheral nerve regeneration. When a non‐nervous conduit is used to bridge a nerve defect, the conduit is soon colonized by a number of Schwann cells that make a pathway for regrowing axons. By using electron microscopy, immunohistochemistry, and reverse transcriptase‐polymerase chain reaction analysis, we have investigated the behavior of migratory glial cells along a particular type of autologous tissue‐engineered conduit made of a vein filled with fresh skeletal muscle, using the rat sciatic nerve model. With this particular type of autograft, our data show that many Schwann cells soon take up a close relationship with grafted muscle fibers, and especially with their basal lamina, which appears to serve as a migration pathway for them. The early and massive colonization of the conduit is sustained by both Schwann cell migration and proliferation, as demonstrated by PCNA immunostaining. Later, as they meet regenerating axons, Schwann cells become closely associated with them and eventually lose their connections with grafted muscle fibers because of the formation of perineurial envelopes. Because previous studies showed that α2a‐2b NRG1 is overexpressed at early stages along the muscle‐vein combined tubes, we have also investigated mRNA expression of its two receptors, erbB2 and erbB3. Both messengers are overexpressed, although with different time courses. Overall, our results provide some morphological and biochemical bases for explaining the effectiveness of fresh muscle‐vein combined nerve guides and throw an interesting light on the possible role of α2a‐2b NRG1 through the erbB2/erbB3 heterodimer receptor for nerve regeneration inside non‐nervous conduits. J. Comp. Neurol. 489:249–259, 2005.

Chapter 11: Tissue engineering of peripheral nerves.

Tissue engineering of peripheral nerves has seen an increasing interest over the last years and, similarly to many other fields of regenerative medicine, great expectations have risen within the general public to its potential clinical application in the treatment of damaged nerves. However, in spite of the scientific advancements, applications to the patients is still very limited and it appears that to optimize the strategy for the tissue engineering of the peripheral nerves in the clinical view, researchers have to strive for a new level of innovation which will bring together (in a multitranslational approach) the main pillars of tissue engineering: namely (1) microsurgery, (2) cell and tissue transplantation, (3) material science, and (4) gene transfer. This review paper provides an overview of these four key approaches to peripheral nerve tissue engineering. While some of these issues will also be specifically addressed in other papers in this special issue on peripheral nerve regeneration of the International Review of Neurobiology, in this paper we will focus on an example of successful translational research in tissue engineering, namely nerve reconstruction by muscle-vein-combined nerve scaffolds.

Functional and morphological assessment of a standardized crush injury of the rat median nerve

The availability of effective experimental models for investigating nerve regeneration and designing new strategies for promoting this unique repair process is important. The aim of this study was to standardize a rat median nerve crush injury model using a non-serrated clamp exerting a compression force of 17.02 MPa for a duration of 30s. Results showed that functional recovery, evaluated by grasping test, was already detectable at day-12 and progressively increased until day-28 after which animal performance plateaued until the end of testing (day-42), reaching a range of 75-80% of pre-operative values. Morphological analysis on the median nerve segments, distal to the crush lesion, which were withdrawn at the end of the experiment showed that regenerated nerve fibers are significantly more numerous and densely packed; they are also smaller and have a thinner myelin sheath compared to controls. Together, these results provide a baseline characterization of the crush median nerve injury experimental model for its employment in the investigation of nerve regeneration research, especially when a reproducible regeneration process is required, such as for the study of biological mechanisms of peripheral nerve fiber regeneration or development of new therapeutic agents for promoting posttraumatic nerve repair.

Employment of the mouse median nerve model for the experimental assessment of peripheral nerve regeneration.

The experimental investigation of nerve regeneration after microsurgical repair is usually carried out in rats, rather than mice, because of the larger sized peripheral nerves. Today however, the availability of genetically modified mice makes the use of this laboratory animal very intriguing for investigating nerve regeneration at a molecular level. In this study we aimed to provide a standardization of the experimental model based on microsurgical direct repair, by 12/0 suture, of the left median nerve in adult male mice. Postoperative recovery was regularly assessed by the grasping test. At day-75 postoperative, regenerated median nerve fibers were analyzed by design-based quantitative morphology and electron microscopy. Yet, sections were immuno-labelled using two axonal antibodies commonly employed for rat nerve fibers. Results indicated that functional recovery begun at day-15 and progressively increased reaching values not significantly different from normal by day-50. Quantitative morphology showed that, at day-75, the number of regenerated nerve fibers was not significantly different in comparison to controls. In contrast, differences were detected in fiber density, mean axon and fiber diameter and myelin thickness which were all significantly lower than controls. Immunohistochemistry showed that axonal markers commonly used for rat nerves studies are effective also for mouse nerves. Similar to the rat, the mouse median nerve model is superior to sciatic nerve model for the minimal impact on animal well-being and the effectiveness of the grasping test for motor function evaluation. The main limitation is the small nerve size which requires advanced microsurgical skills for performing 12/0 epineurial suturing.