Chemokine network in the nervous system: a new target for pain relief

Physical insults including but not limited to nerve damage, inflammation, visceral pathologies and cancer generate long lasting pain commonly referred as chronic pain. Recently, members of the chemokine family and their receptors emerged as key modulators in nociceptive influx transmission in neuropathic and inflammatory chronic pain models. To this day, rodents defective in specific chemokine receptors have provided evidence of the implication of chemokine in pain sensitivity. In addition, up-regulation of chemokines and their receptors at multiple levels in the central nervous (CNS) and peripheral (PNS) systems is associated in the development of chronic pain. Indeed, we point out the fact that chemokines are synthesized and released by both neuronal and non-neuronal cells and act as neuromodulators. Even if their functional roles in the CNS remain largely unknown, chemokines participate in the glial activation and modulation of neuronal excitability as well as neurotransmitter release. This review focuses on three chemokines (i.e. CCL2, CXCL12, CX3CL1) recently identified as important mediators of the initiation and maintenance of pain hypersensitivity, thus broadening the panel of new strategies for the management of chronic pain.     

Use of a liver-specific promoter reduces immune response to the transgene in adenoviral vectors.

Previous studies using adenoviral (Ad) vectors expressing human alpha1-antitrypsin (hAAT) under the control of ubiquitous promoters (RSV, mPGK) elicited the production of antibodies to hAAT in some mouse strains (C3H/HeJ and BALB/c) but not in others (C57BL/6J). In contrast, when a helper-dependent Ad vector (AdSTK109) with all viral coding sequences deleted and expressing hAAT from human genomic DNA with the endogenous promoter was used, C3H/HeJ mice failed to develop antibodies and demonstrated long-term expression. These results suggested that promoter choice and/or properties of the vector itself might influence the host immune response to the transgene product. Direct comparison of first-generation vectors expressing the hAAT cDNA from a ubiquitous mouse PGK promoter rather than from a liver-specific mouse albumin promoter demonstrated that an antibody response to hAAT occurred with the mPGK promoter but not with the albumin promoter in C3H/HeJ mice. As expected, neither vector elicits an antibody response in C57BL/6J mice. Coinjection of the two first-generation vectors containing the mPGK and albumin promoter in C3H/HeJ mice induced an antibody response with resulting loss of detectable hAAT from the sera of the injected mice in 3-4 weeks. From these data, we conclude that under certain conditions, the choice of promoter with its associated liver-specific expression can modulate the host immune response to the transgene independent of viral backbone.     

Immunohistochemical distribution of the somatostatin receptor subtype 5 in the adult rat brain: predominant expression in the basal forebrain.

Somatostatin exerts its actions by means of a family of G protein-coupled receptors, five of which have so far been cloned. Whereas mRNAs for receptor subtypes sst(1)-sst(4) have been unequivocally localized in the brain, the data concerning the fifth subtype, sst(5), are contradictory. Moreover, whereas sst(1) and sst(2A) receptor proteins have been localized by immunohistochemistry, the distribution of sst(3)-sst(5) receptor proteins and/or subtype-specific binding remains to be determined in the central nervous system. In the present study, we investigated the distribution of immunoreactive sst(5) in adult rat brain and pituitary and demonstrated the presence of this receptor protein in the central nervous system by using an affinity-purified antibody generated against the C-terminus of the receptor. The specificity of the antibody for sst(5) was established by immunoblotting experiments on membranes prepared from cells transfected with cDNA encoding different somatotropin release inhibiting (SRIF) receptor subtypes as well as from anterior pituitary. In both systems, the antibody specifically recognized a band at approximately 50 kDa molecular mass, corresponding well to the reported size of the cloned receptor (48 kDa). Immunofluorescence in COS-7 cells transfected with individual SRIF receptor subtypes as well as in sections of rat pituitary demonstrated the antibody's applicability to the immunohistochemical detection of sst(5) receptors. In rat brain sections, sst(5) immunoreactivity was predominantly associated with neuronal perikarya and primary dendrites. Immunolabeling was most prominent in the olfactory tubercle, islands of Calleja, diagonal band of Broca, substantia innominata, and magnocellular preoptic nucleus of the basal forebrain as well as in the reticular nucleus of the thalamus. Other, less intensely labeled areas included the cerebral cortex, hippocampus, amygdala, preoptic area as well as the lateroanterior nucleus of the hypothalamus. The present findings provide the first characterization of the anatomic distribution of sst(5) receptors in the rat brain. They demonstrate a prominent expression of these receptors in the basal forebrain, suggesting that they may be involved in the mediation of somatostatin effects on the sleep-wake cycle through their association with cortically projecting subcortical systems.     

Organization of ascending serotonin systems in the adult rat brain. A radioautographic study after intraventricular administration of [3h]5-hydroxytryptamine

Light microscope radioautography was used to visualize ascending serotonin (5-HT) systems in adult rat brain, following prolonged latero-ventricular instillation of [³H]5-HT. In paraventricular and paracisternal regions, the cell bodies, axonal projections and terminal fields of 5-HT neurons were distinctly labelled, allowing comprehensive analysis of their organizational features. Two major systems of ascending 5-HT fibers could be defined: a transtegmental system originating mainly from nucleus raphe dorsalis (B-7 and B-6) and less prominently from nucleus raphe medianus (B-8), and a periventricular system predominantly issuing from the rostral pole of nucleus raphe dorsalis (B-7). The transtegmental system converges ventral-ward across the decussation of the superior cerebellar peduncles and sweeps rostral-ward, through the ventral tegmental decussation, to enter the ventral tegmental area. The periventricular system closely follows the dorsal longitudinal fasciculus of Schu¨tz. It branches off dorsally toward the superior and inferior colliculi and the subcommissural organ, but the bulk of its fibres arch abruptly ventralward, beneath the posterior commissure, to reach the dorsal hypothalamus. Along their way, these axons most likely contribute to the dense 5-HT innervation of the periventricular gray matter of the midbrain and caudal diencephalon. Both ascending 5-HT systems merge in the medial forebrain bundle area of caudal hypothalamus, wherefrom their fibres borrow several limbic pathways to reach distant territories of innervation: fasciculus retroflexus to medial habenula; striae medullaris and terminalis toward the habenula and the amygdala, respectively; fornix to hippocampus; diagonal band of Broca to septum; induseum griseum and cingulate bundle to hippocampus and neocortex. Other fibers spread more laterally, across the internal capsule, to innervate the globus pallidus and neostriatum. Finally, at the base of anterior forebrain, some 5-HT axons traverse the anterior olfactory nucleus and end in the glomerular layer of olfactory bulb.These and additional results concerning the heterogeneous distribution of 5-HT axonal varicosities in various parts of forebrain (e.g. thalamus, hypothalamus, septal area and amygdaloid complex) complement earlier data gathered by means of other neuroanatomical techniques and make it possible to present a useful model of the general organization of ascending 5-HT systems in the mammalian brain.     

Regional and cellular distribution of low affinity neurotensin receptor mRNA in adult and developing mouse brain

Levocabastine-sensitive neurotensin receptor (NTRL) mRNAs were localized by in situ hybridization in adult and developing mouse brain. NTRL hybridization signal was widely distributed throughout the neuraxis. The highest concentrations of NTRL mRNA were detected in the olfactory system, olfactory tubercle, cerebral and cerebellar cortices, hippocampal formation, and selective hypothalamic nuclei. Moderate to dense hybridization signal was also observed in association with a variety of auditory, visual, and somatosensory relay nuclei, suggesting that the NTRL might be involved in a widespread modulation of primary afferent pathways. Finally a high expression of NTRL was evident in brainstem structures implicated in descending antinociceptive influences (e.g., the periaqueductal gray, nucleus raphe magnus, gigantocellular reticular nucleus, pars alpha, and lateral paragigantocellular nucleus) consistent with the proposed mediation of NT-induced analgesia by the NTRL. Although most of the regions found here to express NTRL mRNA were previously reported to be devoid of mRNA encoding the high affinity NT receptor (NTRH), a few areas (e.g., the anterior olfactory nucleus, medial septum, diagonal band of Broca, reticular thalamic nucleus, suprachiasmatic hypothalamic nucleus, and pontine nucleus) were enriched in both receptor subtypes, suggesting a possible coexpression of these receptors by the same cells. Ontogenic studies revealed that in the mouse brain, NTRL mRNA was detected only from postnatal day 14 and did not reach adulthood concentrations before day 30. In cerebral cortex, the developmental increase in NTRL expression was correlated over time with the decrease in NTRH expression previously documented in the rat, suggesting a progressive takeover of the latter by the former for transduction of the effects of NT in this structure. J. Comp. Neurol. 394:344–356, 1998. © 1998 Wiley-Liss, Inc.     

Cone photoreceptor topography in the retina of sexually mature Pacific salmonid fishes

We examined the retinal cone topography in sexually mature individuals from four species of Pacific salmonid fishes by using semithin plastic sections. We identified variations in cone density and cone arrangements and noted the presence of putative ultraviolet (UV) cones. Putative UV cones were found over an area extending dorsotemporally from the center of the retina. Because most of the putative UV cones are believed to disappear in early ontogeny, their presence over a large proportion (15–20%) of the surface area of the adult retina suggests that they may be reincorporated prior to or at sexual maturity, at least in rainbow trout. Cone density varied across the retina, with highest values at the peripheral margin. Relatively high densities were observed ventrotemporally (in all specimens) and, to a lesser extent, dorsonasally (7 of 11 specimens). The higher cone density in the ventrotemporal retina may represent a retinal specialization in the part of the visual field located above and in front of the animal. Lowest cone densities were found dorsocentrally and coincided approximately with the distribution of putative UV cones, raising the possibility that these cones may not be used in visual tasks requiring the higher visual acuity normally associated with higher cone densities. We also report a novel cone arrangement that consists of rows of double cones inserted between rows composed of single–double cone pairs alternating in position. J. Comp. Neurol. 383:49–59, 1997. © 1997 Wiley-Liss, Inc.     

Impact of autoclave sterilization on the activity and structure of formulated heparin

The stability of a formulated heparin was examined during its sterilization by autoclaving. A new method to follow loss in heparin binding to the serine protease inhibitor, antithrombin III, and the serine protease, thrombin, was developed using a surface plasmon resonance competitive binding assay. This loss in binding affinity correlated well with loss in antifactor IIa (thrombin) activity as well as antifactor Xa activity as measured using conventional amidolytic assays. Autoclaving also resulted in a modest breakdown of the heparin backbone as confirmed by a slight reduction in number-averaged and weight-averaged molecular weight and an increase in polydispersity. Although no clear changes were observed by nuclear magnetic resonance spectroscopy, disaccharide composition analysis using high-performance liquid chromatography-electrospray ionization-mass spectrometry suggested that loss of selected sulfo groups had taken place. It is this sulfo group loss that probably accounts for a decrease in the binding of autoclaved heparin to antithrombin III and thrombin as well as the observed decrease in its amidolytic activity.     

Involvement of NTS2 receptors in stress-induced analgesia

Stress activates multiple neural systems that suppress pain sensation. This adaptive phenomenon referred as stress-induced analgesia (SIA) is mediated by the activation of endogenous pain inhibitory systems. Both opioid and non-opioid forms of SIA have been elicited in rodents according to stressor parameters and duration. There is accumulating evidence that the endogenous neurotensin (NT) system plays an important role in SIA. Especially, NT-deficient mice were shown to exhibit reduced SIA following water avoidance or restraint stress. Since central NT produces naloxone-insensitive analgesic effects by acting on spinal and supraspinal NTS2 receptors, we hypothesized that NT might mediate non-opioid SIA through NTS2 activation. Here, we evaluated the influence of an opioid-independent severe stress produced by a cold-water swim for 3 min at 15 °C on rodent offspring's pain perception. Our results demonstrated that mice lacking NTS2 exhibit significantly reduced SIA following cold-water swim stress. Indeed, NTS2 knockout mice submitted to both acute (plantar test) and tonic (formalin test) pain stimuli show a greater sensitivity to pain in comparison to wild-type littermates. Accordingly, pretreatment with the NT receptor antagonist SR142948A results in a hyperalgesic response to stress induced by cold-water swim. Endogenous NT regulates hypothalamic-pituitary-adrenal axis activity in stress condition by increasing corticosterone plasma levels. Accordingly, the plasma levels of corticosterone measured by radioimmunoassay are significantly reduced in non-stressed and stressed NTS2-deficient mice in comparison with wild-type mice. To further investigate the site of action of NT in mediating SIA, we microinjected NTS2 agonists in lumbar spinal cord and quantified post-stress sensitivity to pain in rats using the plantar test. Exogenously administered NTS2 analogs, JMV-431, β-lactotensin and NT69L markedly enhance the magnitude and duration of stress antinociception in both 25- and 60-day-old rats. In sum, by using genetic and pharmacological approaches, we demonstrated here that NTS2 receptors mediate non-opioid SIA. Our results also revealed that the release of endogenous NT in response to stress requires the presence of NTS2 to stimulate corticotropin-releasing factor (CRF)-induced elevation of plasma corticosterone, and that NTS2 receptors localized at the lumbar spinal cord participate to the disinhibition of descending pain control pathways. Therefore, these data highlight the significance of NTS2 as a novel target for the treatment of pain and stress-related disorders.