Ching-Hsiang Wu

Signal transduction pathways of nitric oxide release in primary microglial culture challenged with gram-positive bacterial constituent, lipoteichoic acid

Between one-third and one-half of all cases of sepsis are known to be caused by gram-positive microorganisms through the cell wall component, e.g. lipoteichoic acid (LTA). Gram-positive bacteria are also known to induce encephalomyelitis and meningeal inflammation, and enhance the production of nitric oxide (NO) via expression of inducible nitric oxide synthase (iNOS) in murine tissue macrophages. It remains to be explored if LTA could activate microglia considered to be resident brain macrophages. We report here that LTA derived from gram-positive bacteria (Staphylococcus aureus) significantly induces NO release and iNOS expression in primary microglia. LTA-induced NO accumulation was detected at 2 h in microglial culture and was significantly attenuated by pretreatment with anti-CD14, complement receptor type 3 (CR3) or scavenger receptor (SR) antibodies. LTA activated mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase, p38 MAPK or c-Jun N-terminal kinase in cultured microglia. LTA-elicited microglial NO production was also drastically suppressed by SB203580 (p38 MAPK inhibitor) or pyrrolidine dithiocarbamate (an inhibitor of nuclear factor kappaB), indicating that p38 MAPK and nuclear factor kappaB were involved in microglial NO release after LTA challenge. These results suggest that gram-positive bacterial product such as LTA can activate microglia to release NO via the signal transduction pathway involving multiple LTA receptors (e.g. CD14, CR3 or SR), p38 MAPK and nuclear factor kappaB. The in vivo study further confirmed that administered intracerebrally LTA induced considerable noticeable iNOS, phospho-IkappaB and phospho-p38 MAPK expression in microglia/macrophages.

Microglia/macrophages responses to kainate-induced injury in the rat retina

The present study was aimed to elucidate how retinal microglia/macrophages would respond to neuronal death after intravitreal kainate injection. An increased expression of the complement receptor type 3 (CR3) and an induction of the major histocompatibility complex (MHC) class II and ED-1 antigens were mainly observed in the inner retina after kainate injection. Prominent cell death revealed by Fluoro Jade B (FJB) staining and ultrastructural examination appeared at the inner border of the inner nuclear layer (INL) at 1 day post-injection. Interestingly, some immunoreactive cells appeared at the outer segment of photoreceptor layer (OSPRL) at different time intervals. Our quantitative analysis further showed that CR3 immunoreactivity was drastically increased peaking at 7 days but subsided thereafter. MHC class II and ED-1 immunoreactivities showed a moderate but steady increase peaking at 3 days and declined thereafter. Double labeling study further revealed that retinal microglia/macrophages expressed concurrently CR3 and ED-1 antigens (OX-42+/ED-1+) or MHC class II molecules (OX-42+/OX-6+) and remained branched in shape at early stage of kainate challenge. By electron microscopy, microglia/macrophages with CR3 immunoreactivity displayed abundant cytoplasm containing a few vesicles and phagosomes. Other cells ultrastructurally similar to Müller cells or astrocytes could also engulf exogenous substances. In conclusion, retinal microglia/macrophages responded vigorously to kainate-induced neuronal cell death that may also trigger the recruitment of macrophages from neighboring tissues and induce the phagocytotic activity of cells other than retinal microglia/macrophages.

AMPA Receptor-mTOR Activation is Required for the Antidepressant-Like Effects of Sarcosine during the Forced Swim Test in Rats: Insertion of AMPA Receptor may Play a Role.

Sarcosine, an endogenous amino acid, is a competitive inhibitor of the type I glycine transporter and an N-methyl-d-aspartate receptor (NMDAR) coagonist. Recently, we found that sarcosine, an NMDAR enhancer, can improve depression-related behaviors in rodents and humans. This result differs from previous studies, which have reported antidepressant effects of NMDAR antagonists. The mechanisms underlying the therapeutic response of sarcosine remain unknown. This study examines the role of mammalian target of rapamycin (mTOR) signaling and α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR) activation, which are involved in the antidepressant-like effects of several glutamatergic system modulators. The effects of sarcosine in a forced swim test (FST) and the expression levels of phosphorylated mTOR signaling proteins were examined in the absence or presence of mTOR and AMPAR inhibitors. In addition, the influence of sarcosine on AMPAR trafficking was determined by analyzing the phosphorylation of AMPAR subunit GluR1 at the PKA site (often considered an indicator for GluR1 membrane insertion in neurons). A single injection of sarcosine exhibited antidepressant-like effects in rats in the FST and rapidly activated the mTOR signaling pathway, which were significantly blocked by mTOR inhibitor rapamycin or the AMPAR inhibitor 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) pretreatment. Moreover, NBQX pretreatment eliminated the ability of sarcosine to stimulate the phosphorylated mTOR signaling proteins. Furthermore, GluR1 phosphorylation at its PKA site was significantly increased after an acute in vivo sarcosine treatment. The results demonstrated that sarcosine exerts antidepressant-like effects by enhancing AMPAR–mTOR signaling pathway activity and facilitating AMPAR membrane insertion. Highlights– A single injection of sarcosine rapidly exerted antidepressant-like effects with a concomitant increase in the activation of the mammalian target of rapamycin mTOR signaling pathway.– The antidepressant-like effects of sarcosine occur through the activated AMPAR–mTOR signaling pathway.– Sarcosine could enhance AMPAR membrane insertion via an AMPAR throughput.

Expression of protein gene product 9.5, tyrosine hydroxylase and serotonin in the pineal gland of rats with streptozotocin-induced diabetes

Hyperglycemia is a well-known factor in reducing nocturnal pineal melatonin production. However, the mechanism underlying diabetes-induced insufficiency of pineal melatonin has remained uncertain. This study was undertaken to examine the structure, innervation and functional activity of the pineal gland in streptozotocin (STZ)-induced diabetes in rats by immunohistochemistry, Western blotting and image analysis. The number of the pinealocytes and the volume of pineal were also estimated using stereologic quantification including the optical fractionator and Cavalieris method. It has also shown a progressive reduction of the total area of the pineal gland and the nuclear size of pinealocytes beginning at 4 weeks of induced diabetes. Surprisingly, the immunoreactive intensities and protein amounts of serotonin (5-HT) and protein gene product (PGP) 9.5 in the pineal gland were progressively increased from 4 weeks of diabetes. Meanwhile, nerve fibers immunoreactive for PGP 9.5 had disappeared. Diabetes-induced neuropathy was observed in nerve fibers containing tyrosine hydroxylase (TH). The affected nerve fibers appeared swollen and smooth in outline but they showed a distribution pattern, packing density and protein levels comparable to those of the age-matched control animals. Ultrastructural observations have revealed diabetes-induced deformity of Schwann cells and basal lamina, accumulation of synaptic vesicles and deprivation of the dense-core vesicles in the axon terminals and varicosities. The increase in immunoreactivities in 5-HT and PGP 9.5 and shrinkage of pineal gland in the diabetic rats suggest an inefficient enzyme activity of the pinealocytes. This coupled with the occurrence of anomalous TH nerve fibers, may lead to an ineffective sympathetic innervation of the pinealocytes resulting in reduced melatonin production in STZ-induced diabetes.

CC-CHEMOKINE LIGAND 18/PULMONARY ACTIVATION-REGULATED CHEMOKINE EXPRESSION IN THE CNS WITH SPECIAL REFERENCE TO TRAUMATIC BRAIN INJURIES AND NEOPLASTIC DISORDERS

Pulmonary activation-regulated chemokine (PARC) now designated CC-chemokine ligand 18 (CCL18) has been shown to play a significant role in the pathogenesis of various tissue injuries and diseases in a proinflammatory or immune suppressive way to limit or support the inflammation or disease. While much is known about the roles of CCL18/PARC in non-neural tissues, its expression in the CNS has remained largely unexplored and controversial. Using reverse transcription polymerase chain reaction (RT-PCR) and double immunohistochemical staining, we analyzed the expression of CCL18/PARC in the human brain with special reference to traumatic brain injuries and tumors. The RT-PCR analysis revealed the expression of CCL18/PARC mRNA both in the traumatic brain and glioma tissues examined. Immunoexpression of CCL18/PARC protein was consistently detected in all cases of traumatic brain injuries examined by immunohistochemical staining. Double immunofluorescence labeling has extended the study that CCL18/PARC positive cells were macrophages/microglia, astrocytes or neurons. The CCL18/PARC expression was localized in macrophage-like cells in two of eight glioblastoma tissues whose cancer cells were CCL18/PARC negative. Unexpectedly, CCL18/PARC mRNA weakly and constitutively expressed by glioblastoma cell line was upregulated after endotoxin stimulation. The present results indicated a significant production of CCL18/PARC in different CNS traumatic and neoplasm tissues by specific cellular elements expressing the chemokine. An anti-inflammatory mechanism jointly exerted by these cells via CCL18/PARC may be involved in the CNS immunity after traumatic injury and tumorigenesis.

Responses of microglia in vitro to the gram-positive bacterial component, lipoteichoic acid

An increase in incidence and severity of gram‐positive infections has emerged in the past decade. In this regard, attention has been focused recently on immune responses of microglial cells in the central nervous system to gram‐positive bacteria. The underlying immunological and cellular events in microglial activation induced by specific bacterial toxin of gram‐positive bacteria, however, have not yet been clarified fully. This study reports that a simple cell wall product, lipoteichoic acid (LTA), derived from gram‐positive bacteria (Staphylococcus aureus) could trigger microglial activation in vitro. Microglia challenged with LTA showed intense ruffling of plasma membrane in the form of lamellipodia or rounded up forming cell aggregates. MTT assay and Western blot analysis with anti‐proliferating cell nuclear antigen antibody showed a significant microglial proliferation that may be induced at the later phases of LTA treatment with low doses but at the early period with a high dose. Concentrated LTA also caused apoptotic death of cultured microglia showing fragmented nuclei and increased expression of annexin V or caspase 3. In response to LTA, isolated microglia increased the expression of inducible nitric oxide synthase and major histocompatibility complex class II antigen. Microglial LTA receptors such as CD14 molecule, complement receptor type 3, and macrophage scavenger receptor were upregulated concurrently. In conclusion, staphylococcal LTA can exert an immunomodulatory effect on microglial morphology, cell cycle, and immunomolecules, including its receptors.

Variant palmaris profundus enclosed by an unusual loop of the median nerve

According to the usual description in most anatomy texts, the median nerve in the forearm passes between the 2 heads of pronator teres. It continues distally between flexor digitorum superficialis and profundus almost to the retinaculum. Muscular branches leave the nerve near the elbow and supply all superficial muscles of the anterior part of the forearm except flexor carpi ulnaris. Many variations of the median nerve in the forearm have been reported (Urban & Krosman, 1992). The palmaris profundus is also a rare anomaly of the forearm (Dyreby & Engber, 1982). It originates from the radial side of the common flexor tendon in the proximal forearm and inserts into the undersurface of the palmar aponeurosis. The origin of palmaris profundus may be close to the median nerve and its branches, and may be involved in compressive neuropathy of the anterior interosseous nerve. Its tendon crossing through the carpal canal has been implicated in the carpal tunnel syndrome (reviewed by Lahey & Aulicino, 1986). In some cases, palmaris profundus was found enclosed in a common fascial sheath with the median nerve (Stark, 1992; Sahinoglu et al. 1994). To indicate its close association with the median nerve, the palmaris profundus was also named ‘musculus comitans nervi mediani’ (Sahinoglu et al. 1994). This article reports an unusual loop of the median nerve encircling an anomalous palmaris profundus in the forearm, which, to the best of our knowledge, has not been previously described.

NOVEL DISTRIBUTION OF CLUSTER OF DIFFERENTIATION 200 ADHESION MOLECULE IN GLIAL CELLS OF THE PERIPHERAL NERVOUS SYSTEM OF RATS AND ITS MODULATION AFTER NERVE INJURY

This study examined CD200 expression in different peripheral nerves and ganglia. Intense CD200 immunoreactivity was consistently localized in unmyelinated nerve fibers as opposed to a faint immunostaining in the myelinated nerve fibers. By light microscopy, structures resembling the node of Ranvier and Schmidt-Lanterman incisures in the myelinated nerve fibers displayed CD200 immunoreactivity. Ultrastructural study revealed CD200 expression on the neurilemma of Schwann cells whose microvilli and paranodal loops at the node of Ranvier were immunoreactive. The CD200 immunoexpression was also localized in the satellite glial cells of sensory and autonomic ganglia and in the enteric glial cells. Double labeling of CD200 with specific antigens of satellite glia or Schwann cells in the primary cultures of dorsal root ganglia had shown a differential expression of CD200 in the peripheral glial cells. The existence of CD200 in glial cells in the peripheral nervous system (PNS) was corroborated by the expression of CD200 mRNA and protein in a rat Schwann cell line RSC96. Using the model of crush or transected sciatic nerve, it was found that CD200 expression was attenuated or diminished at the site of lesion. A remarkable feature, however, was an increase in incidence of CD200-labelled Schmidt-Lanterman incisures proximal to the injured site at 7 days postlesion. Because CD200 has been reported to impart immunosuppressive signal, we suggest that its localization in PNS glial cells may play a novel inhibitory role in immune homeostasis in both normal and pathological conditions.

Ginkgo biloba extract enhances noncontact erection in rats: the role of dopamine in the paraventricular nucleus and the mesolimbic system

Penile erection is essential for successful copulation in males. Dopaminergic projections from the paraventricular nucleus (PVN) to the ventral tegmental area (VTA) and from the VTA to the nucleus accumbens (NAc) are thought to exert a facilitatory effect on penile erection. Our previous study showed that treatment with an extract of Ginkgo biloba leaves (EGb 761) enhances noncontact erection (NCE) in male rats. However, the relationship between NCE and dopaminergic activity in the PVN, VTA, and NAc remains unknown. The present study examined the relationship between NCE and central dopaminergic activity following EGb 761 treatment. We report here that, in comparison with the controls, there was a significant increase in the number of NCEs in rats after treatment with 50 mg/kg of EGb 761 for 14 days. EGb 761-treated rats also showed more NCEs than the same group before EGb 761 treatment. A significant increase in the expression of catecholaminergic neurons in the PVN and the VTA was seen by means of tyrosine hydroxylase immunohistochemistry, and tissue levels of dopamine and 3,4-dihydroxyphenylacetic acid in the NAc were also markedly increased in the EGb 761-treated animals. However, the norepinephrine tissue levels in the PVN and the NAc in the EGb 761-treated group were not significantly different from those in the controls. Together, these results suggest that administration of EGb 761 increases dopaminergic activity in the PVN and the mesolimbic system to facilitate NCE in male rats.

Hyperbaric oxygen upregulates cochlear constitutive nitric oxide synthase

BackgroundHyperbaric oxygen therapy (HBOT) is a known adjuvant for treating ischemia-related inner ear diseases. Controversies still exist in the role of HBOT in cochlear diseases. Few studies to date have investigated the cellular changes that occur in inner ears after HBOT. Nitric oxide, which is synthesized by nitric oxide synthase (NOS), is an important signaling molecule in cochlear physiology and pathology. Here we investigated the effects of hyperbaric oxygen on eardrum morphology, cochlear function and expression of NOS isoforms in cochlear substructures after repetitive HBOT in guinea pigs.ResultsMinor changes in the eardrum were observed after repetitive HBOT, which did not result in a significant hearing threshold shift by tone burst auditory brainstem responses. A differential effect of HBOT on the expression of NOS isoforms was identified. Upregulation of constitutive NOS (nNOS and eNOS) was found in the substructures of the cochlea after HBOT, but inducible NOS was not found in normal or HBOT animals, as shown by immunohistochemistry. There was no obvious DNA fragmentation present in this HBOT animal model.ConclusionsThe present evidence indicates that the customary HBOT protocol may increase constitutive NOS expression but such upregulation did not cause cell death in the treated cochlea. The cochlear morphology and auditory function are consequently not changed through the protocol.