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In the present study, we characterize the antinociceptive effects produced by the chemokine CCL4 in mice. The intraplantar administration of very low doses of CCL4 (0.1-3 pg) produced bilateral antinociception assessed by the unilateral hot-plate test (UHP) without evoking chemotactic responses at the injection site. Moreover, the subcutaneous administration of CCL4 (3-100 pg/kg) also yielded bilateral antinociception in the UHP and the paw pressure test and reduced the number of spinal neurons that express Fos protein in response to noxious stimulation. The implication of peripheral CCR5 but not CCR1 in CCL4-evoked antinociception was deduced from the inhibition produced by systemic but not intrathecal, administration of the CCR5 antagonist DAPTA, and the inefficacy of the CCR1 antagonist J113863. Besides, the inhibition observed after subcutaneous but not intrathecal administration of naloxone demonstrated the involvement of peripheral opioids and the efficacy of naltrindole but not cyprodime or nor-binaltorphimine supported the participation of δ-opioid receptors. In accordance, plasma levels of met-enkephalin, but not β-endorphin, were augmented in response to CCL4. Likewise, CCL4-evoked antinociception was blocked by the administration of an anti-met-enk antibody. Leukocyte depletion experiments performed with cyclophosphamide, anti-Ly6G, or anti-CD3 antibodies indicated that the antinociceptive effect evoked by CCL4 depends on circulating T lymphocytes. Double immunofluorescence experiments showed a four times more frequent expression of met-enk in CD4

We show here that the intraplantar administration of CCL5 in mice produces hyperalgesia at low doses but activates compensatory antinociceptive mechanisms at doses slightly higher. Thus, the injection of 3–10 ng of CCL5 evoked thermal hyperalgesia through the activation of CCR1 and CCR5 receptors, as demonstrated by the inhibitory effect exerted by the selective antagonists J113863 (0.01–0.1 μg) and DAPTA (0.3–3 μg), respectively. The prevention of this hyperalgesia by diclofenac (1–10 μg), the inhibitors of COX-1 SC-560 (0.1–1 μg) or COX-2 celecoxib (1–5 μg), the TRPV1 antagonist capsazepine (0.03–0.3 μg) or the TRPA1 antagonist HC030031 (10–50 μg) demonstrates the involvement of prostaglandin synthesis and TRP sensitization in CCL5-evoked hyperalgesia. Doses of CCL5 higher than 17 μg did not evoke hyperalgesia. However, this effect was restored by the administration of naloxone-methiodide (5 μg), nor-binaltorphimine (10 mg/kg) or an anti-dynorphin A antibody (0.62–2.5 ng). The administration of 30 ng of CCL5 also induced hyperalgesia in mice with reduced number of circulating white blood cells in response to cyclophosphamide or with selective neutrophil depletion induced by an anti-Ly6G antibody. In fact, the number of neutrophils present in paws treated with 30 ng of CCL5 was greater than in paws receiving the administration of the hyperalgesic dose of 10 ng. Finally, the expression of the endogenous opioid peptide dynorphin A was demonstrated by double immunofluorescence assays in these neutrophils attracted by CCL5. These results support previous data describing the hyperalgesic properties of CCL5 and constitute the first indication that a chemokine of the CC group can activate endogenous analgesic mechanisms.

The mammalian Period1 gene is rhythmically expressed and its proteins are found within the nucleus of the cells of the suprachiasmatic nuclei (SCN), the central circadian pacemaker in mammals; however, whether the target of the PER1 proteins is also the nucleus in the retinal peripheral clock cells is yet to be determined. Using an anti-PER1 protein antibody in Western blot analyses, we found three isoforms (75, 110 and 140 kDa) in extracts of the SCN, as well as in other different parts of the brain, whereas just two isoforms (75 and 110 kDa) were detected in the retinal extracts. We have observed that PER1 immunolabelling has a cytoplasmic location in many cells of the ganglion cell layer and in a few cells in the inner nuclear layer of the mouse retina. This cellular location was seen in any of the tissue samples taken at 4 h intervals, either in the day/night cycle or in constant darkness, of both wild type and rd mice. Unlike this situation, PER1 isoforms were nuclear proteins in the SCN cells as well as in other parts of the brain of the same animals. No circadian changes were found for these clock proteins in the neural retina. These findings suggest that PER1 proteins play roles in the retina different from those established in the SCN.

Endogenous rhythms of mammals are controlled by the clock located in the suprachiasmatic nucleus (SCN). The molecular mechanism of a clock involves transcription/translation-based feedback loops in which the expression of the so called “clock genes” is suppressed periodically by their protein products. Previous studies reported influence of the eye itself on the circadian oscillation of the SCN, apart from the well-known photic readjustment of the central clock. With this in mind, we decided to analyze the mPer1 clock gene expression in the retinally degenerate (rd) mouse SCN by means of immunohistochemical techniques. Our objective was to detect possible alterations of the daily endogenous oscillation of PER1 protein in the SCN of these rd mice, as well as to make clear whether or not this protein was involved in the resetting of the central clock in a manner similar to wild-type animals. We found that the endogenous levels of PER1 protein were reduced in the SCN of rd mice throughout the 24-h cycle, which suggests that loss of classic photoreceptors influences somehow the main mechanism of the SCN clock. Light stimulation induced a parallel increase of Per1 expression at the subjective night, but not at the subjective day, in both rd and wild-type mice. Therefore, SCN readjustment by light in the rd mice occurs with a pattern similar to wild-type controls, despite the reduced PER1 protein levels detected. The effect of retinal degeneration on the circadian system and the possible interactions between the retinal and the SCN clocks are discussed.

Pneumolysin (PLY) is an important virulence factor of Streptococcus pneumoniae . We examined the ability of three murine monoclonal antibodies (MAbs) to PLY (PLY-4, PLY-5, and PLY-7) to affect the course of pneumococcal pneumonia in mice. The intravenous administration of antibodies PLY-4 and PLY-7 protected the mice from the lethal effect of the purified toxin. Mice treated with PLY-4 before intranasal inoculation of S. pneumoniae type 2 survived longer (median survival time, 100 h) than did untreated animals (median survival time, 60 h) ( P < 0.0001). The median survival time for mice treated with a combination of PLY-4 and PLY-7 was 130 h, significantly longer than that for mice given isotype-matched indifferent MAbs ( P = 0.0288) or nontreated mice ( P = 0.0002). The median survival time for mice treated with a combination of three MAbs was significantly longer (>480 h) than that for mice treated with PLY-5 (48 h; P < 0.0001), PLY-7 (78 h; P = 0.0007), or PLY-4 (100 h; P = 0.0443) alone. Similarly, the survival rate for mice treated with three MAbs (10 of 20 mice) was significantly higher than the survival rate obtained with PLY-5 (1 of 20; P = 0.0033), PLY-4 (2 of 20; P = 0.0138), or PLY-7 (3 of 20; P = 0.0407) alone. These results suggest that anti-PLY MAbs act with a synergistic effect. Furthermore, MAb administration was associated with a significant decrease in bacterial lung colonization and lower frequencies of bacteremia and tissue injury with respect to the results for the control groups.

The vertebrate retina is known to mediate both visual and non-image-forming photic responses. With the use of statistical analyses of sections immunohistochemically labelled with a polyclonal antiserum against the activated form of protein CREB (p-CREB), a transcription factor which participates in some neural responses to stimuli, we have observed that the piriform cortex of both wild-type and retinally degenerate (rd) mice respond to light stimulation independently of the circadian time in which the stimulus was given. Responses in visually blind (rd/rd) mice corroborate the hypothesis that there must be neural connections between the retina and cortical brain areas other than those involved in image processing, and strongly support the idea that since these mice lack rods and cones, the melanopsin retinal ganglion cells could mediate this non-visual light input.

The melanopsin-expressing retinal ganglion cells are specialized in measuring irradiance for several functions, including daily photoentrainment and regulation of pupil size. In the present study, these cells were analyzed in mice during their perinatal period, from embryonic day (E) 15 to postnatal day (P) 1. Melanopsin expression was detected at E15 in cells that did not co-express the transcription factor Brn3a. Under light/dark (LD) cycles, the number of melanopsin-expressing cells did not change between E16 and E19, while a very significant increase was observed during the short interval around birth, between E19 (the day before birth) and P0 (the day of birth). As these samples were collected after lights on, to determine whether such increase in melanopsin expression was driven by light, we also analyzed samples collected 0-4 hours after birth (during the night period), which revealed that the cell number increase was already present and, therefore, was not induced by the early post-birth light exposure. To clarify the role of ambient light conditions during this period, P1 retinas from pups under constant light or darkness conditions were also analyzed and compared to those of mice under LD cycles. No variation in the number of immunostained cells was detected among the groups studied, indicating that ambient conditions did not provoke the increase in melanopsin expression detected. Rather, it might have been induced by either a maternal or a developmental signal and is likely related to the first connections between the retina and the suprachiasmatic nucleus reported by other authors.

Melanopsin, an opsin protein expressed in mammalian retinal ganglion cells (RGCs), makes them responsive to light. Such photosensitive RGCs form the retinohypothalamic tract (RHT) that provides signals to the suprachiasmatic nucleus (SCN), the master regulator of circadian rhythms. The SCN is adjusted daily to the environmental day/night cycle by signal inputs incoming from the RHT. In the present work we have studied, using immunohistochemistry techniques, the types and number of cells which expressed melanopsin during the postnatal development of pigmented C3H/He mice maintained in a standard daily cycle (12-h light/12-h dark). Our results clearly show for the first time that the retina maintains a rather constant number of melanopsin-expressing RGCs from the first postnatal day and, thus, demonstrate that no loss of these photosensitive cells occurs during postnatal development. This supports the general idea that the non-image-forming system, in which these cells are involved, is functional at the very early postnatal stage.

The effect of cone- and rod-cell loss on the activation of transcription factor CREB (by phosphorilation at Ser133) was examined in the pacemaker of mammals, the suprachiasmatic nucleus (SCN). For this purpose, brain sections of rd/rd and wild-type C3H mice were immunolabelled with a polyclonal antibody that recognises p-CREB, i.e., the activated form of the protein. Both rd/rd and wild-type mice maintained in constant darkness showed a circadian variation of p-CREB nuclear staining: the number of immunopositive nuclear pixels at the subjective night was higher than the one observed at the subjective day. However, some differences were detected between both groups: (1) p-CREB immunolabelling in the SCN of rd/rd mice was significantly reduced throughout the 24-h cycle; (2) the time in which the activation of CREB begins to increase at the subjective night in these mice is delayed with regard to wild-type mice. When a light stimulus was given at the subjective night p-CREB inmunostaining significantly increased in the SCN of both rd/rd and wild-type mice when compared to basal levels, while no significant effect was found when the stimulus was given at the subjective day. Taken together, our results suggest that despite lower levels of p-CREB, indicating that something is altered in the SCN of rd/rd mice, the main mechanisms of the clock (e.g., circadian oscillation, readjustment by light) are still fully functional in these mice. The present study supports the idea that the CREB/CRE pathway is a component of the circadian clock molecular mechanism.

The ontogeny of photic responsiveness in the retina and the suprachiasmatic nucleus (SCN) of C57BL/6J mouse was studied using the enhanced expression of the immediate early gene c-fos as a marker of neuronal activation. c-fos expression was assessed by immunocytochemical localisation of its protein product. Light induction of Fos-like protein in the retina and SCN occurred first at postnatal day four (PD 4). At this stage of development, some cells in the inner part of the neuroblastic layer and in the ganglion cell layer showed positive immunoreaction; the number of Fos-like positive cells increased with age until it reached adult levels by PD 15. Induction of Fos-like expression at PD 4 in the SCN mainly occurred in the ventrolateral region, the region that receives the greatest density of retinal innervation. These results indicate that retinal input can activate cells in the SCN even before eyelids open, and the SCN can be stimulated by photic inputs as early as day 4 after birth.

Rd mutant mice are visually blind but they maintain the ability of synchronising their circadian rhythms to the external light-dark cycles. We used immunocytochemical procedures to detect light-induced Fos expression in the rd mice retina. We found that Fos is expressed in the rd retina in an unattenuated pattern through the entire life of the animal. Furthermore, we have found that cells expressing Fos are distributed throughout the whole retina, while opsin expression takes place only in the dorsal half of the retina in the 1-year old rd mice. Finally, we found that light induces Fos expression in the rd retina at the same levels during the subjective day as during the subjective night, whereas in the suprachiasmatic nucleus (SCN), Fos is stimulated by light only during the subjective night. Our results support the hypothesis that new, undiscovered photoreceptors are implicated in light perception for the circadian system.

An anti-Fos protein antiserum was used to elucidate the diurnal expression of Fos protein in the normal and degenerate rd/rd mice retina. We have found that Fos expression is stimulated in cells of both inner nuclear layer (INL) and ganglion cell layer (GCL) at the onset of light period and reaches its maximum after 2 h. After which, the number of stained nuclei decreases along the light/dark cycle until almost no reaction is observed at the end of dark period. This expression pattern was similar in both normal and rd/rd mice although degenerate retinas showed a much lower number of stained nuclei. Aged rd animals also show Fos expression in GCL and INL in response to light stimuli suggesting that severely degenerate retinas are still able to transduce light stimulus.

Neurotrophins and their receptors (p75 and Trk family of receptors) play an important role in the survival of different populations of neurons in the central and peripheral nervous system. Expression of p75, TrkA, TrkB and TrkC was examined in mouse retinas by means immunohistochemistry in the postnatal development of normal and rd/rd mice (C57BL/6J). The rd/rd mice suffer a degeneration that causes a massive lost of photoreceptor cells. Results showed immunoreactivity to all three Trk proteins in both normal and rd/rd mice during the first 21 postnatal days, but some variations in intensity and localization were found. p75 immunoreaction was only present in rd/rd mice at the end of the degeneration process. These results could indicate a role of neurotrophins and their receptors in both the postnatal development of mouse retina and the degeneration process of rd/rd mice.

The vertebrate eye mediates both image-forming and non–image-forming photoreception. Image-forming photoreception (vision) enables the animal to detect and track objects in the environment, whereas non–image-forming photoreception is responsible for the measurement of ambient irradiance, so that, for example, the internal circadian biological clock can be synchronized with the astronomical day, a process called photoentrainment.1,2 The hypothalamic suprachiasmatic nucleus (SCN), which is considered the central circadian pacemaker of mammals, is adjusted on a daily basis to the environmental light/dark cycle1 by the detection of light by melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs).3–6 Such ipRGCs transmit this light information to the SCN by way of the retinohypothalamic tract.7,8 These cells also project to other brain areas involved in pupil constriction, promotion of sleep, gaze control, image-forming vision, and other activities.9 Moreover, ipRGCs constitute the principal conduits for rod-cone input involved in non–image-forming responses, including circadian photoentrainment.10 In fact, the destruction of these cells altered the effects of light on circadian rhythms.10–12 Therefore, the rod and cone photoreceptors and the ipRGCs are complementary in providing signals for nonvisual photoreceptive functions. In mice, at least 70% of the RGCs generated during retinal development die through programed cell death during the postnatal period13; however, as we previously demonstrated in pigmented mice, no diminution in the number of melanopsin-expressing cells occurs during postnatal development.14 ipRGCs are responsive to light from birth.15,16 Moreover, the SCN begins to function as a circadian pacemaker during late fetal development.17 Depending on the intensity of the stimulus, light was able to induce expression of the immediate early gene c-fos in the SCN at postnatal day (P) 0 to P118 or at P4.19 Taken together, these data indicate that the melanopsin-based system is functional as early as the day of birth. Previous studies have demonstrated that melanopsin expression shows daily oscillation.20–22 Such rhythm was also demonstrated in neonatal albino rats and neonatal pigmented mice,22,23 when rods and cones are not yet fully developed. Hannibal et al.21 and Mathes et al.,24 using albino rats, also reported differential regulation of melanopsin expression in response to continuous darkness (DD) or continuous light (LL). Such changes in melanopsin expression were also detected in albino rat pups.23 This suggests that ipRGCs can adapt their responsiveness to the external illumination conditions by regulating their melanopsin content even in the absence of functional rod-cone photoreceptors. Among the ipRGCs, two main morphologic types have been previously identified: M1 cells, with their dendritic arborization in the OFF sublayer of the inner plexiform layer (IPL), and M2 cells, with their dendrites forming a plexus in the ON sublayer of the IPL. Recently, two isoforms of melanopsin, Opn4S and Opn4L, have been identified. M1 cells express both melanopsin isoforms, whereas M2 cells express only the Opn4L isoform.25 Different electrophysiological responses,26 as well as different brain projections,27 were reported for these two cell subpopulations. In a previous study,22 we detected a different daily oscillation for M1 and M2 cells that was already present in the early postnatal period. Albino animals are often used as models in numerous studies concerning the retina, despite the fact that most mutations causing albinism provoke anomalous retinal development, including lower numbers of rods, incomplete development of the central retina, and chiasmatic abnormalities.28 Therefore, it should be taken into account that results obtained in albino models are not fully comparable with those of pigmented animals. To better understand the development of the ipRGCs, the present study analyzed for the first time in albino mice these cells and their main subpopulations within the postnatal period under standard 12-hour light/12-hour dark cycles. Furthermore, the effects of exposure to constant light or constant darkness on the postnatal development of these cells were also studied.