Involvement of nitric oxide through endocannabinoids release in microglia activation during the course of CNS regeneration in the medicinal leech.

The medicinal leech is notable for its capacity to regenerate its central nervous system (CNS) following mechanical trauma. Using an electrochemical nitric oxide (NO)-selective electrode to measure NO levels, we found that the time course of NO release in the injured leech CNS is partially under the control of endocannabinoids, namely, N-arachidonyl ethanolamide (AEA) and 2-arachidonyl glycerol (2-AG). Relative quantification of these endocannabinoids was performed by stable isotope dilution (2AGd8 and AAEd8) coupled to mass spectrometry in course of regeneration process or adenosine triphosphate (ATP) treatment. Data show that 2-AG levels rose to a maximum about 30 min after injury or ATP treatment, and returned to baseline levels 4 h after injury. In same conditions, AEA levels also rapidly (within 5 min) dropped after injury or ATP treatment to the nerve cord, but did not fully return to baseline levels within 4 h of injury. In correlation with these data, chemoattraction activities of endocannabinoids on isolated leech microglial cells have been shown in vitro and in vivo reflecting that control over NO production is accompanied by the controlled chemoattraction of microglia directed from the periphery to the lesion site for neuronal repair purposes. Taken together, our results show that in the leech, after injury concurrent with ATP production, purinergic receptor activation, NO production, microglia recruitment, and accumulation to lesion site, a fine imbalance occurs in the endocannabinoid system. These events can bring explanations about the ability of the leech CNS to regenerate after a trauma and the key role of endocannabinoids in this phenomenon.

Ex-vivo detection of neural events using THz BioMEMS.

BACKGROUND: Electromagnetic frequencies up to a few terahertz (THz) can yield real-time and noninvasive measurements on biological matter. Unfortunately, strong absorption in aqueous solutions and low spatial resolution return difficult free-space investigations. A new approach based on integrated THz circuits was used. The authors designed and fabricated a BioMEMS (Biological MicroElectro-Mechanical System) compatible with microfluidic circulation and electromagnetic propagation. It is dedicated to the ex vivo detection of nitric oxide synthase (NOS) activity, which is involved in neurodegenerative phenomena. MATERIAL/METHODS: The biological model was a leech's central nervous system. After its injury, the production of NO was observed and measured in the far-THz spectral domain. The nerve cord was put inside a BioMEMS realized in polydimethylsiloxane (PDMS) sealed on a glass wafer. Glass is a good material for supporting high-frequency integrated waveguides such as coplanar waveguides (CPWs). Measurements were performed with vectorial network analyser (VNA). RESULTS: The transmission parameter in the frequency range of 0.14-0.22 THz was measured through CPWs located just below the microchannel containing the injured leech nerve cord. The lesion caused a decreased transmission coefficient due to the NOS activity. L-NAME was injected inside the microchannel and it was verified that it inhibits this activity. CONCLUSIONS: It was demonstrated that THz spectroscopy can detect a biochemical event, such as NOS activity around an injured leech's nerve cord, in real time. Future studies will be dedicated to quantitative measurements of the reaction products using the sophisticated management of several drugs allowed with microfluidic microsystems.

Molecular MALDI imaging: an emerging technology for neuroscience studies.

Mass spectrometry (MS) has become an essential tool for the detection, identification, and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of the nervous system. Generally, the application of these powerful techniques requires the destruction of the specimen under study, but recent technological advances have made it possible to apply the matrix-assisted laser desorption/ionization (MALDI) MS technique directly to tissue sections. The major advantage of direct MALDI analysis is that it enables the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification, and separation steps, which have the potential for introducing artifacts. With automation and the ability to display complex spectral data using imaging software, it is now possible to create multiple 2D maps of selected biomolecules in register with tissue sections, a method now known as MALDI Imaging, or MSI (for Mass Spectrometry Imaging). This creates, for example, an opportunity to correlate functional states, determined a priori with live recording or imaging, with the corresponding molecular maps obtained at the time the tissue is frozen and analyzed with MSI. We review the increasing application of MALDI Imaging to the analysis of molecular distributions of proteins and peptides in nervous tissues of both vertebrates and invertebrates, focusing in particular on recent studies of neurodegenerative diseases and early efforts to implement assays of neuronal development.

New developments in MALDI imaging for pathology proteomic studies.

With new emerging mass spectrometry technologies, it can now be demonstrated that direct tissue analysis is feasible using matrix-assisted laser desorption/ionization (MALDI) sources. A major advantage of direct MALDI analysis is to avoid time-consuming extraction, purification or separation steps, which have the potential for producing artifacts. Direct MALDI analysis of tissue sections enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. With automation and the ability to reconstruct complex spectral data using imaging software, it is now possible to produce multiplex imaging maps of selected bio-molecules within tissue sections. Thus, direct MALDI spectral data obtained from tissue sections can be converted into imaging maps, a method now known as MALDI-imaging. MALDI-imaging combines the power of mass spectrometry, namely exquisite sensitivity and unequivocal structural information, within an intact and unaltered morphological context. Critical improvements to increase image resolution are presented in this manuscript e.g., solvent treatment, new solid ionic matrices, gold sputtering, nickel support or laser focalization. One of the most important developments is the ability to carry out either direct MALDI analysis or MALDI imaging on paraffin tissue sections, thus opening the path to an archival "gold-mine" of existing pathology samples to proteomic analysis. These developments provide new avenues for biomarker hunting and diagnostic follow-up in the clinical setting. Further developments in MALDI-imaging of specific targets provide an added dimension, as validated disease-marker-gene RNA transcripts can be analyzed along with their translation by targeting their specific protein products or metabolites. Disease/health states will thus be closely molecularly monitored at protein and nucleic acids levels, with a single technique. Taken together, MALDI imaging will become a key tool for pathology proteomic studies.

Effects of perinatal maternal food restriction on pituitary-gonadal axis and plasma leptin level in rat pup at birth and weaning and on timing of puberty.

The effects of maternal 50% food restriction (FR) during the last week of gestation and/or lactation on pituitary-gonadal axis (at birth and weaning), on circulating levels of leptin (at weaning), and on the onset of puberty have been determined in rats at birth and at weaning. Maternal FR during pregnancy has no effect at term on the litter size, on the basal level of testosterone in male pups, and on the drastic surge of circulating testosterone that occurs 2 h after birth. At weaning, similar retardation of body growth is observed in male and female pups from mothers exposed to FR. This undernutrition induces the most drastic effects when it is performed during both gestation and lactation or during lactation alone. Drastic retardation of testicle growth with reduction of cross-sectional area and intratubular lumen of the seminiferous tubules is observed in male pups from mothers exposed to undernutrition during both gestation and lactation or during lactation alone. Maternal FR during the perinatal period reduces circulating levels of FSH in male pups without affecting LH and testosterone concentrations. Maternal FR does not affect circulating levels of LH, estradiol, and progesterone in female pups. Female pups from mothers exposed to FR during both gestation and lactation show a significant increase of plasma FSH as well as a drastic retardation of ovarian growth. The follicular population was also altered. The number of antral follicles of small size (vesicular follicles) was increased, although the number of antral follicles of large size (graafian follicles) was reduced. Maternal FR occurring during both late gestation and lactation (male and female pups), during lactation alone (male and female pups), or during late gestation (female pups) induces a drastic reduction of plasma leptin and fat mass in pups at weaning. The onset of puberty is delayed in pups of both sexes from mothers exposed to FR during lactation and during both gestation and lactation. In conclusion, these data demonstrate that a perinatal growth retardation induced by maternal FR has long-term consequences on both size and histology of the genitals, on plasma gonadotropins and leptin levels, on fat stores at weaning, and on the onset of puberty.