Nose to brain delivery in rats: Effect of surface charge of rhodamine B labeled nanocarriers on brain subregion localization.

Nose to brain delivery and nanotechnology are the combination of innovative strategies for molecules to reach the brain and to bypass blood brain barriers. In this work we investigated the fate of two rhodamine B labeled polymeric nanoparticles (Z-ave <250nm) of opposite surface charge in different areas of the brain after intranasal administration in rats. A preliminary screening was carried out to select the suitable positive (chitosan/poly-l-lactide-co-glycolide) nanocarrier through photon correlation spectroscopy and turbiscan. Physico-chemical and technological characterizations of poly-l-lactide-co-glycolide (negative) and chitosan/poly-l-lactide-co-glycolide (positive) fluorescent labeled nanoparticles were performed. The animals were allocated to three groups receiving negative and positive polymeric nanoparticles via single intranasal administration or no treatment. The localization of both nanocarriers in different brain areas was detected using fluorescent microscopy. Our data revealed that both nanocarriers reach the brain and are able to persist in the brain up to 48h after intranasal administration. Surface charge influenced the involved pathways in their translocation from the nasal cavity to the central nervous system. The positive charge of nanoparticles slows down brain reaching and the trigeminal pathway is involved, while the olfactory pathway may be responsible for the transport of negatively charged nanoparticles, and systemic pathways are not excluded.

Hypoglossal nucleus projections to the rat masseter muscle.

We investigated in the rat whether hypoglossal innervation extended to facial muscles other than the extrinsic musculature of the mystacial pad. Results showed that hypoglossal neurons also innervate the masseter muscle. Dil injected into the XII nucleus showed hypoglossal axons in the ipsilateral main trunk of the trigeminal nerve. After Gasser's ganglion crossing, the axons entered into the infraorbital division of the trigeminal nerve and targeted the extrinsic muscles of the mystacial pad. They also spread into the masseter branch of the trigeminal nerve to target the polar portions of the masseter muscle spindles. Retrograde double labelling, performed by injecting Dil into the pad and True Blue into the ipsilateral masseter muscle, showed labelled hypoglossal neurons in the medio-dorsal portion of the XII nucleus. The majority of these neurons were small (15-20 microm diameter), showed fluorescence for Dil and projected to the mystacial pad. Other medium-size neurons (25 microm diameter) were instead labelled with True Blue and projected to the masseter muscle. Finally, in the same area, other small hypoglossal neurons showed double labelling and projected to both structures. Functional hypotheses on the role of these hypoglossal projections have been discussed.

Hypoglossal nuclei participation in rat mystacial pad control.

Recently, we showed that extra-trigeminal axons, originating from the hypoglossal nucleus, travel with the infraorbital division of the trigeminal nerve (ION), which is known to innervate the rat mystacial pad. Dil was monolaterally injected into the rat XII nucleus to analyse the peripheral distribution of hypoglossal axons to the mystacial pad, to evaluate their involvement in facial sensory-motor control. Electromyographic responses of mystacial pad motor units to electrical stimulation of the ION were recorded, along with the evoked responses to electrical stimulation of the ipsilateral XII nucleus. The results showed that hypoglossal axon terminals target the ipsilateral extrinsic musculature of the mystacial pad, but they do not have any contact with the intrinsic muscles. ION electrical stimulation increased electromyographic activity in the ipsilateral pad extrinsic muscles, even following VII nerve transection. Hypoglossal nucleus electrical stimulation induced field potentials and monosynaptic responses in the same motor units that persisted even following VII nerve transection, these disappearing after cooling the ION. We suggest that the small hypoglossal neurons projecting to the extrinsic musculature of the mystacial pad are part of a hypoglossal-trigeminal loop that participates in the sensory-motor control of the rat vibrissae system.

Expression of pannexin2 protein in healthy and ischemized brain of adult rats.

The expression pattern of the pannexin2 protein (Px2) in healthy and ischemized brains of adult rats was investigated. A polyclonal antibody for rat Px2 was generated in chicken and purified for affinity. This antibody was used to study by Western blot, Enzyme-Linked Immunosorbent Assay, and immunohistochemistry, the expression pattern of Px2 in healthy brain of adult rats and in the hippocampus of rats submitted to bilateral clamping of carotid arteries for 20 min, followed by different times of reperfusion (I/R) (8 h, 24 h, 48 h, 72 h, 14 days and 30 days). Immunohistochemical studies visualized the wide and complex expression pattern of Px2 in the healthy brain. All Px2(+) positive cells were neurons which also showed no puncta on their cellular membranes. Both pyramidal cells and interneurons, the majority of which were positive to parvalbumin, were stained in healthy hippocampus. The number of Px2 interneurons in the hippocampus showed a progressive reduction at successive time intervals after I/R, with a negative peak of about -40% after 72 h from I/R. Interneurons which were positive for both Px2 and parvalbumin, represented about the 85% of all parvalbumin cells stained in the hippocampus. This percentage rested grossly unmodified at different time intervals after I/R in spite of the progressive neuronal depletion. Concomitantly, an intense astrogliosis occurred in the hippocampus. Most of the astroglial cells expressed de novo and for a transient time (from 24 h to 14 days from I/R), Px2. Primary co-cultures of hippocampal neurons and astrocytes were submitted to transient ischemia-like injury. This set of experiments further confirmed the in vivo results by showing that Px2 is de novo and transiently expressed in astroglial cells following a transient ischemia-like injury. These results suggested the expression of Px2 in the astrocytes may be induced either from injured neurons or by biochemical pathways internal to the astrocyte itself. In conclusion, our results showed the transient expression of Px2 in astrocytes of reactive gliosis occurring in the hippocampus following I/R injury. We hypothesize that Px2 expression in astrocytes following an ischemic insult is principally involved in the formation of hemichannels for the release of signaling molecules devoted to influence the cellular metabolism and the redox status of the surrounding environment.

Chromogranin A expression in human colonic adenocarcinoma.

The presence of CgA positive cells occuring in scattered elements or in clusters within human colonic adenocarcinomas has been documented in recent data. The number of these cases has ranged from 10% to 40% depending on the method of demonstration. The aim of this study is to assess the presence of CgA positive cells on a set of 60 tumours that, by standard histological procedures, were classified as well differentiated (n.5), moderately differentiated (n.48) and poorly differentiated (n.7) adenocarcinoma. 4-5 microm thick sections were processed by means of immunoperoxidase method using the primary CgA monoclonal antibody. Our results showed CgA positive cells in two cases of poorly differentiated and three cases of moderately differentiated adenocarcinoma. These specimens (8% of cases) showed diffuse, irregular areas of CgA immunoreactive cells as components of the neoplasm. These cases could be considerer as "mixed exocrine and neuroendocrine carcinoma" that develops from the same stem cell which differentiates into two atypical cell lines. Therefore, we suggest that to carry out a proper adenocarcinoma histological examination, and to establish a consequent therapy, not only common staining techniques but also immunohistochemical methods should be used.

Branching projections of catecholaminergic ventrolateral reticular neurons to the fastigial nucleus and superior colliculus in the rat: triple labelling procedure.

In this study, we employed triple fluorescent-labelling to reveal the distribution of the catecholaminergic neurons within rostral ventrolateral reticular nucleus which supply branching collateral input to the superior colliculus (SC) and to the cerebellar fastigial nucleus (FN). The catecholaminergic identity of the neurons was revealed by immunocytochemical detection of the biosynthetic enzyme, tyrosine hydroxylase. The projections were defined by injections of two retrograde tracers: rhodamine and fluoro gold in the SC and FN, respectively.

Schwann cell-derived factors support serotoninergic neuron survival and promote neurite outgrowth.

During embryogenesis and the postnatal period, neurons and glia interact in the development and differentiation of specific populations of nerve cells. Both in the peripheral (PNS) and in the central nervous system (CNS), glial cells have been shown in various experimental conditions to constitute a favorable substrate for neural adhesion, neural polarity, shape and axonal extension, while numerous soluble molecules secreted by neurons influence the survival and differentiation of the glial cells themselves. The aim of the present work was to investigate the influence of postnatal Schwann cells (SC) on embryonic serotoninergic (5-HT) neurons of the raphe, in order to study the possible influence of the peripheral glia on the CNS neurons. Cultures of SC from sciatic nerve of postnatal rats and neurons from rat embryonic rhombencephalon were successfully established and cells were immunocytochemically characterized. The number of 5-HT neurons, and the number and length of their branches were quantified in the cultures of 5-HT neurons, in cultures added with Nerve Growth Factor (NGF) and Insulin-like Growth Factor I (IGF-I), in co-cultures with SC and in cultures added with conditioned medium obtained from SC cultures. The results indicated that SC have the capacity to promote the survival and growth of 5-HT neurons in culture, and that this activity is mediated by soluble factors. Although the precise nature and mechanism of action of the growth factor or factors produced by SC in the presence of 5-HT neurons was not identified, our results add more data on the possible activity of the peripheral glia in promoting and enhancing the survival and outgrowth of the CNS neurons.

Branching serotonergic and non-serotonergic projections from caudal brainstem to the medial preoptic area and the lumbar spinal cord, in the rat.

The distribution and the chemical identity of retrogradely single and double labeled neurons in the caudal raphe nuclei were analyzed in the rat following injection of two fluorescent tracers into the medial preoptic area and the ventral/intermediate grey of the lumbar spinal cord, and serotonin immunocytochemistry. The results suggest that (1) neurons in the caudal raphe nuclei exhibit highly collateralized axons, able to simultaneously innervate rostrally- and caudally-located targets; (2) a large proportion (40-50%) of the raphe-spinal projection does not contain serotonin, which by contrast is present in more than 70% of the neurons projecting to the medial preoptic area; (3) only a small fraction of the observed collateralized projection is serotonergic. Thus, multiple transmitter systems are likely to be involved in the diffuse ascending and descending influence arising from these nuclei.

Activation of metabotropic glutamate receptors protects cultured neurons against apoptosis induced by beta-amyloid peptide.

Prolonged exposure of cultured cortical cells or cultured cerebellar granule cells to the residue 25-35 fragment of beta-amyloid peptide (beta AP), beta AP(25-35), induced neuronal apoptosis, as revealed by morphological analysis, fluorescent chromatin staining, and immunodetection of oligonucleosomes released from the nucleus into the cytoplasm. beta AP(25-35)-induced apoptosis was insensitive to ionotropic glutamate receptor antagonists but was substantially attenuated by the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid. The neuroprotective action of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid was antagonized by (RS)-alpha-methyl-4-carboxyphenylglycine and was mimicked by (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (a selective agonist of mGluR2 and -3 subtypes) and by L-2-amino-4-phosphobutanoate and L-serine-O-phosphate (selective agonists of mGluR4, -6, and -7 subtypes). However, whereas all of these drugs behaved as neuroprotectants in cultured cortical cells, only L-2-amino-4-phosphobutanoate and L-serine-O-phosphate [and not (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine] reduced beta AP(25-35)-induced apoptosis in cultured cerebellar granule cells. The neuroprotective activity of mGluR agonists may be related to their ability to inhibit membrane Ca2+ conductance, because drugs that block voltage-sensitive Ca2+ channels, such as nimodipine or Co2+, could also attenuate beta AP(25-35)-induced apoptosis.

Basic fibroblast growth factor promotes the survival of embryonic ventral mesencephalic dopaminergic neurons--I. Effects in vitro.

We have studied the effects of basic fibroblast growth factor on rat embryonic mesencephalic neurons in vitro. Basic fibroblast growth factor promotes the survival of dopaminergic neurons in vitro, the effect increasing with dose and reaching a maximum at 10 ng/ml. In the absence of basic fibroblast growth factor the number of tyrosine hydroxylase-stained (tyrosine hydroxylase positive) neurons declines to almost zero within 14 days, whereas in the presence of basic fibroblast growth factor numbers remain almost constant from three to 28 days in vitro. This effect of basic fibroblast growth factor is abolished by preventing non-neuronal cells from appearing in the cultures, apart from a basic fibroblast growth factor-mediated increase in the numbers of tyrosine hydroxylase-positive cells during the first two days in vitro. The presence or absence of non-neuronal cells also influences dopaminergic neuronal morphology, the neurons having more, longer, and more varicose processes in the absence of astrocytes. Survival of dopaminergic neurons in vitro in the absence of basic fibroblast growth factor is very dependent on plating cell density, but in the presence of basic fibroblast growth factor this dependency vanishes. It is also possible to make survival independent of plating density by growing the cultures on inverted coverslips, which have the effect of concentrating secreted molecules in the thin layer of medium between coverslip and dish. Our conclusions from these experiments on plating density are that astrocytes probably constitutively secrete a small amount of a trophic factor which promotes survival of dopaminergic neurons, and that the rate of production of this factor is greatly increased by basic fibroblast growth factor. If basic fibroblast growth factor is withdrawn from cultures after two or seven days the dopaminergic neurons soon die. However, if basic fibroblast growth factor is withdrawn after 14 days, after the period of naturally occurring cell death of these neurons, there is no increase in dopaminergic neuronal death compared to controls in which basic fibroblast growth factor treatment is maintained. If basic fibroblast growth factor is used to improve the survival of dopaminergic neurons grafted in vivo, it should therefore be sufficient to treat the grafts for 14 days.

Neurons in raphe nuclei pontis and magnus have branching axons that project to medial preoptic area and cervical spinal cord. A fluorescent retrograde double labeling study in the rat.

In this study, we utilized a double retrograde axonal tracing technique to investigate the possible existence of collateralized axonal projections from raphe nuclei 'pontis' and 'magnus' to both medial preoptic area (MPA) and cervical spinal cord (C1-C2). Following microinjections of fluorescent tracers Fast blue (FB) and Diamidino yellow (DY) within MPA and C1-C2, substantial numbers of FB and DY single-labeled neurons, as well as FB-DY double-labeled neurons have been found within raphe nuclei 'pontis' and 'magnus'.

Branching projections from subcoeruleus area neurons to medial preoptic area and cervical spinal cord revealed by double retrograde neuronal labeling.

In this study we utilized a double retrograde axonal tracing technique to investigate the possible existence of collateralized axonal projections of subcoeruleus area neurons to both 'medial preoptic area (MPA) and cervical (C1-C3) spinal cord'. Following microinjections of fluorescent tracers (Fast blue (FB) and Diamidino yellow (DY) within MPA and C1-C3, substantial numbers of FB and DY single-labeled neurons as well as FB-DY double-labeled branched neurons have been found within subcoeruleus area.

Branching axons from subcoeruleus area project to the nucleus raphe pontis and hypothalamic zona incerta, as studied with the double fluorescent retrograde tracing technique.

In this study we utilized a double retrograde axonal tracing technique to investigate the possible existence of collateralized axonal projections of subcoeruleus area neurons to both raphe pontis nucleus and hypothalamic zona incerta. Following microinjections of fluorescent tracers (Fast blue (FB) and Diamidino yellow (DY] within raphe pontis and zone incerta, substantial numbers of double-labeled branched neurons have been found within the subcoeruleus area.