Satoshi Kuchiiwa

Up-regulation of matrix metalloproteinase-3 in the dorsal root ganglion of rats with paclitaxel-induced neuropathy.

Paclitaxel‐induced painful peripheral neuropathy is a major dose‐limiting factor. Recently, it has been reported that macrophages accumulated in the dorsal root ganglion of paclitaxel‐treated rats, and their activation is suggested to contribute to generation and development of the neuropathy. However, the mechanism for macrophage activation is still unknown. In this study, to explore candidate genes involved in the mechanism for macrophage activation in the dorsal root ganglion of paclitaxel‐treated rats, we developed model rats for paclitaxel‐induced neuropathic pain and performed a microarray assay to analyze the changes of gene expressions in the dorsal root ganglion. Among the genes with changed expression levels, we focused on matrix metalloproteinase‐3 (MMP‐3, stromelysin‐1) and CD163, a macrophage marker. By reverse transcription–polymerase chain reaction, the expression levels of MMP‐3 and CD163 were markedly up‐regulated in paclitaxel‐treated dorsal root ganglion. As a result of immunohistochemical study, large ganglion neurons, but neither Schwann cells nor macrophages, predominantly expressed MMP‐3. This MMP‐3 up‐regulation occurred prior to macrophage accumulation in the dorsal root ganglion. In addition, recombinant MMP‐3 led to the activation of RAW264 macrophages in vitro. Taken together, the up‐regulation of MMP‐3 and following macrophage activation caused in the dorsal root ganglion might be a significant event to trigger a series of reactions developing paclitaxel‐induced peripheral neuropathic pain. (Cancer Sci 2008; 99: 1618–1625)

Morphological study of orexin neurons in the hypothalamus of the Long-Evans rat, with special reference to co-expression of orexin and NADPH-diaphorase or nitric oxide synthase activities.

Orexins, novel neuropeptides, are exclusively localized in the hypothalamus and implicated in the regulation of a variety of activities, including food intake and energy balance. Nitric oxide (NO), an unconventional neurotransmitter, is widely present in numerous brain regions including the hypothalamus, and has similar physiological roles to those of the orexins. The present study was undertaken to examine the distribution of orexin neurons and the presence of neuronal nitric oxide synthase (nNOS) in the orexin neurons to clarify whether NO interacts with the orexins in the neuronal regulation activities in the Long-Evans rat. We used two double-labeling methods: nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry in combination with orexin immunohistochemistry, and double-labeling fluorescent immunohistochemistry for orexin and nNOS. The majority of the orexin immunoreactive neurons were localized mainly in the areas of the dorsomedial hypothalamic nucleus (DMN), the dorsal part of the perifornical nucleus (PEF) and lateral hypothalamic area. The orexin immunoreactive cell bodies were medium in size, and triangular, round, elliptic, and fusiform in shape. The sizes and shapes of orexin neurons in the different parts were similar. Cell bodies coexpressing the orexin and nNOS or NADPH-d were present in the areas of the DMN and the PEF, and the nerve fibers containing orexin and nNOS were distributed in the DMN and PEF, arcuate nucleus (ARN) and ventromedial hypothalamic nucleus (VMH). These results provide morphological evidence that there exists a population of nNOS- or NADPH-d-/orexin-coexpressing neurons in the orexinergic cell group in the hypothalamus, and taken together with previous findings, suggest that NO may play a role in the mechanisms by which orexin neurons regulate food intake and energy balance.

Origins of parasympathetic postganglionic vasodilator fibers supplying the lips and gingivae; an WGA-HRP study in the cat

Application of WGA-HRP to the mandibular lip and buccal gingiva of the cat resulted in retrograde labeling in the ipsilateral otic ganglion (OG), whereas labeled neurons appeared in the pterygopalatine ganglion (PPG) as well as in the OG when the tracer was injected into the maxillary lip and buccal gingiva. The results suggest that both the facial and the glossopharyngeal preganglionic vasodilator fibers supplying the mandibular lip and buccal gingiva mediated in the OG, and those innervating the maxillary lip and buccal gingiva are mediated in the PPG and the OG.

Nadph diaphorase neurones are evenly distributed throughout cat neocortex irrespective of functional specialization of each region

Using NADPH diaphorase histochemistry as a marker for nitric oxide synthase we investigated the possible sites of nitric oxide synthesis in cat cerebral neocortex. Intensely stained neurones were found mainly in the deep layers of the neocortex and underlying medulla. Virtually all neurones in the cerebral medulla were NADPH diaphorase positive. The density of diaphorase neurones was estimated in the cortex/medulla border zones of each neocortical gyrus. Diaphorase neurones were evenly distributed throughout the neocortex and no significant statistical difference between gyri was observed. These findings indicate that the density of diaphorase neurones is irrespective of functional specialization of each region and are more in line with the hypothesis that NADPH diaphorase neurones are involved in the control of local cortical blood flow.

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin decreases serotonin-immunoreactive neurons in raphe nuclei of male mouse offspring

Female ddY mice were administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by gavage for 8 weeks prior to pregnancy. In the male breast-fed offspring born to the TCDD-exposed mice, serotonergic neurons in the brainstem were examined using an immunocytochemical method at 42 days of age. In all offspring, a marked decrease in the intensity of immunostaining occurred in all raphe nuclei compared with the control offspring. The number of serotonin-immunoreactive neurons in each raphe nucleus was measured by computer-assisted analysis. Approximately a quarter to half of immunoreactive neurons were detected in the TCDD-exposed offspring raphe nuclei compared with the control offspring. The present findings suggest that in utero and/or lactational TCDD exposure cause a long-lasting change in the serotonergic system in the raphe nuclei of offspring.

Simultaneous measurement of parasympathetic reflex vasodilator and arterial blood pressure responses in the cat

We measured the changes in lower lip blood flow and systemic arterial blood pressure evoked by lingual nerve or trigeminal spinal nucleus (Vsp) stimulation to gain an insight into the brainstem integration of sympathetic and parasympathetic responses to nociceptive stimulation. We used artificially ventilated, cervically vago-sympathectomized cats deeply anesthetized with alpha-chloralose and urethane. A lip blood flow increase occurred in an intensity- and frequency-dependent manner following electrical stimulation of Vsp or lingual nerve regardless of whether systemic arterial blood pressure increased or decreased. In contrast, there was no apparent optimal frequency for the changes in systemic arterial blood pressure elicited by electrical stimulation of Vsp or lingual nerve. No relationship was found between the amplitude of the lip blood flow increase and that of the systemic arterial blood pressure change. Microinjection of lidocaine or kainic acid into the Vsp evoked, respectively, reversible and irreversible inhibition of the lip blood flow increase and systemic arterial blood pressure change evoked by lingual nerve stimulation. When microinjected unilaterally directly into the ipsilateral Vsp, the GABA agonist muscimol abolished both lingual nerve-evoked effects (increase in lip blood flow and changes in systemic arterial blood pressure) without changing basal systemic arterial blood pressure, suggesting the presence in the Vsp of GABA receptors serving to modulate both the parasympathetically mediated lip blood flow increase and the sympathetically mediated systemic arterial blood pressure change. Lidocaine microinjection into the salivatory nucleus caused a significant attenuation of the lingual nerve-induced blood flow increase, but had no effect on the lingual nerve-induced systemic arterial blood pressure change. Thus, the neural pathway mediating the lingual nerve-induced lip blood flow increase seems to be simple, requiring a minimum of four neurons: trigeminal afferent-Vsp-parasympathetic pre-ganglionic neurons with cell body located in the inferior salivatory nucleus-otic postganglionic neuron. On the other hand, the pathway underlying the evoked systemic arterial blood pressure changes, presumably mediated via altered sympathetic activity, seems to be more complicated and could be affected by more numerous factors.

Presence of neuronal nitric oxide synthase in autonomic and sensory ganglion neurons innervating the lacrimal glands of the cat: an immunofluorescent and retrograde tracer double-labeling study.

It is generally considered that parasympathetic postganglionic nerve fibers innervating the lacrimal gland (LG) arise from the pterygopalatine ganglion (PPG), while sympathetic and sensory innervations arise from the superior cervical ganglion (SCG) and trigeminal ganglion (TG), respectively. Recently, we reported for the first time that the parasympathetic innervation of the cat LG was also provided by the otic ganglion (OG) and ciliary ganglion (CG), and that the sensory innervation was also provided by the superior vagal ganglion (SVG) and superior glossopharyngeal ganglion (SGG). To determine if nitric oxide (NO) is a neurotransmitter of the autonomic and sensory neurons innervating the LG, we injected the cholera toxin B subunit (CTB) as a retrograde tracer into the cat LG, and used double-labeling fluorescent immunohistochemistry for CTB and nitric oxide synthase (NOS). We found that NOS-/CTB-immunofluorescent double-labeled perikarya were localized in the PPG, OG, TG, SVG and SGG, but not in the CG and SCG. The highest numbers of NOS-/CTB-immunofluorescent double-labeled neurons were found in the PPG and TG. In addition, we examined the presence of nitrergic nerve fibers in the LG using NADPH-d histochemistry and found that a large amount of NADPH-d-stained nerve fibers were distributed around the glandular acini and in the walls of glandular ducts and blood vessels. This study provides the first direct evidence showing that NO may act as a neurotransmitter or modulator involved in the parasympathetic and sensory regulation of lacrimal secretion and blood circulation, but may not be implicated in the sympathetic control of LG activities, and that nitrergic nerve fibers in the LG arise mainly from parasympathetic postganglionic neurons in the PPG and sensory neurons in the TG. The present results suggest that NO plays an important role in the regulation of LG activities.

Fasting-induced reduction in locomotor activity and reduced response of orexin neurons in carnitine-deficient mice.

We found reduced locomotor activity (LA) under fasting in systemic carnitine-deficient juvenile visceral steatosis (jvs(-/-)) mice. When food was withdrawn at 8:00 a.m. (lights-off at 7:00 p.m., 12h/cycle), the nocturnal LA of jvs(-/-) mice was much less than the control (jvs(+/+) and jvs(+/-)) mice. LA recovered under carnitine or sucrose administration, but not under medium-chain triglyceride. In addition, fasted jvs(-/-) mice, without any energy supply, were activated by modafinil, a stimulator of the dopamine pathway. These results suggest that the reduced LA is not adequately explained by energy deficit. As the fasted jvs(-/-) mice showed lower body core temperature (BT), we examined the central nervous system regulating LA and BT. We found lower percentage of c-Fos positive orexin neurons in the lateral hypothalamus and reduced orexin-A concentration in the cerebrospinal fluid of fasted jvs(-/-) mice. Sleep analysis revealed that fasted jvs(-/-) mice had disruption of prolonged wakefulness, with a higher frequency of brief episodes of non-REM sleep during the dark period than fasted jvs(+/+) mice. These results strongly suggest that the reduced LA in fasted jvs(-/-) mice is related to the inhibition of orexin neuronal activity.

The retinal projections to the ventral and dorsal divisions of the medial terminal nucleus and mesencephalic reticular formation in the Japanese monkey (Macaca fuscata) : a reinvestigation with cholera toxin B subunit as an anterograde tracer

After a monocular injection of the cholera toxin B subunit (CTB) into the vitreous chamber of the eye, retinal projections to the medial terminal nucleus (MTN) of the accessory optic system (AOS) were studied in the Japanese monkey. The anterogradely transported tracer was visualized with the peroxidase antibody technique by using an anti-cholera toxin antibody. One small accumulation of the CTB-immunopositive retinofugal terminals was located in a small area just medial to the medial edge of the cerebral peduncle and anterior to the attachment of the oculomotor nerve, suggesting the existence of a ventral division of the MTN of the AOS. Caudally, one very small bundle of the retinofugal fibers extending dorsally from this accumulation was seen running along the medial edge of the cerebral peduncle and substantia nigra to the small region corresponding to the dorsal division of the MTN. A few small bundles of CTB-immunopositive retinal fibers were observed to leave the superior fasciculus of the AOS at various points. These fibers coursed medially through the cerebral peduncle and substantia nigra to reach some restricted areas of the mesencephalic reticular formation between the medial lemniscus and the substantia nigra.

Sparse distribution of NADPH diaphorase neurons in the hippocampal formation of the inbred mutant strain EL mouse

The number of NADPH diaphorase-positive cells in the CA1/CA2 and CA3 regions of Ammons horn and the subiculum of the hippocampal formation of EL mice, an inbred mutant strain of the ddY mouse susceptible to convulsive seizures, was fewer than that of ddY mice. These findings suggest that smaller numbers of nitric oxide producing cells in the hippocampal formations of EL mice is related to their susceptibility to convulsive seizures.