Endocytosis of CF in marginal cells of stria vascularis regulated by ROCK and MLCK signaling cascade, but not G-proteins.

Objective The endocytosis of cationized feritin (CF) via a clathrin-mediated pathway is regulated by a signaling network. Marginal cells showed the active endocytosis of CF via a clathrin-mediated pathway. The internalization of receptors through this clathrin-mediated pathway is an important regulatory event in signal transduction. Numerous kinases are involved in endocytosis, and each endocytic route is subjected to high-order regulation by cellular signaling mechanisms. In this study, we investigated whether ROCK and MLCK signaling cascades and G-proteins regulate the endocytosis of CF in marginal cells of the stria vascularis. Methods CF was infused into the cochlear duct with pertussis toxin (PTX),Clostridium botulinum C3 toxin (BTX), guanosine(g-thio)-triphosphate (GTP-γS), ML-7, Y-27632. Endocytic activity was measured at 30 min after the start of infusion under an electron microscope. Results In marginal cells, CF was internalized via a clathrin-mediated pathway that depends on F-actin and microtubules (MT). Its processes were controlled by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK), but not affected by G-protein-coupled receptor (GPCR) or the RhoA signaling cascade. Conclusion Our previous study showed that the main endocytotic pathway of microperoxidase (MPO) did not depend on the Rho/ROCK molecular switch or actin/myosin motor system, but was mainly regulated by the RhoA signaling cascade. The present study results indicate that these signaling cascades regulating CF internalization completely differ from the cascades for MPO internalization.

Experimental studies on the recovery processes from severe facial palsy and the development of its sequelae.

OBJECTIVE: To experimentally elucidate the pathogenesis of inappropriate co-contraction of facially innervated muscles after severe facial palsy. METHODS: Twenty-two guinea pigs with severe facial palsy induced by the interruption of the petrosal artery were used to follow up behavioral facial movement, including the degree of facial palsy and abnormal hyperkinetic facial movement of synkinesis and mass contracture. At the end of the follow-up, the evoked facial compound muscle action potential (evoked FCMP) and antidromically evoked facial nerve response (AFNR) were examined in a few typical cases with complete recovery and with incomplete recovery accompanied by synkinesis. After the follow-up, all animals were sacrificed for morphological studies, which consisted of a light-microscopic study (by Luxol fast blue and hematoxylin and eosin staining or toluidine blue staining) and/or an electron-microscopic study. RESULTS: The initial sign of recovery was mass contracture or spasm. This condition continued for 2 weeks or more. As voluntary facial movement recovered, the mass contracture became unnoticeable. It could not be distinguished when the so-called synkinesis developed. Synkinesis usually developed during the recovery process from severe to moderate palsy, and synkinesis persisted or progressed once it appeared. Histologically, unmyelinated fibers were intermingled with myelinated fibers in an early stage of recovery with mass contracture. In the late stage with the development of synkinesis, however, such an intermingling of unmyelinated and myelinated axons was not observed. In this stage, axons became well myelinated, but they were irregular in shape in cases with synkinesis. Especially, axons irregularly ran at the level of the G1 (at the region of the second genu) segment, and bifurcated axons were sporadically found. The axon count had a tendency to increase toward the periphery. AFNR was not detected, although evoked FCMP could be clearly detected in cases with synkinesis. CONCLUSION: Misguidance of regenerated axons is an important cause of facial synkinesis in the ischemia-induced facial palsy model. Ephaptic transmission between unmyelinated and myelinated axons is also likely to be responsible for mass contracture manifested in the early stage of the recovery process.

Behavioral and neuropathological changes in animal models of chronic painful scar.

BACKGROUND: Long-lasting limb pain or back pain after surgery occasionally develops into chronic pain that leads to lower activity and a poorer quality of life for many patients. To determine the histopathological and neuropathological mechanisms that cause persistent post-operative pain, we developed an original animal model with sustained painful scars and then examined pain-related behavior and the pathological alteration of peripheral tissues and spinal nerves associated with the model. METHODS: The animal model (Scar group) was prepared in rats by extensively stripping subcutaneous tissue from the plantar in the hind paw followed by subsequent examination of pain-related behavior over the next 12 weeks. Thereafter, we conducted histological staining of the scar tissues, immunohistochemical staining of c-Fos (L5 dorsal horn), and electron microscopic analysis of the L5 spinal nerve fibers/dorsal roots. RESULTS: The mechanical pain threshold decreased specifically in the ipsilateral plantar in animals with scar. This state was maintained for 12 weeks. A collagen layer developed from fibers derma to the muscular layer in the scar tissue in which many fibroblasts were observed. No statistical differences were found for the areas of the c-Fos-immunoreactive (c-Fos-IR) neurons in the ipsilateral and contralateral sides of the L5 level of the dorsal horn in both the Scar group and Pinhole (sham operation) group. However, myelin sheath fragmentation of the nerve fibers was observed in the ipsilateral dorsal root at the L5 position. CONCLUSIONS: We created a persistent painful scar model through extensive injury of the peripheral tissues. Fibrotic thickening of the cutaneous tissues, possible sensitization, and partial degradation of the spinal nerve related to the painful scar were observed. This model should enable us to better understand the mechanism of sensitization caused by painful scar and investigate new methods for treating painful scars in humans.

Enhanced expression of WD repeat-containing protein 35 (WDR35) stimulated by domoic acid in rat hippocampus: involvement of reactive oxygen species generation and p38 mitogen-activated protein kinase activation.

BACKGROUND: Domoic acid (DA) is an excitatory amino acid analogue of kainic acid (KA) that acts via activation of glutamate receptors to elicit a rapid and potent excitotoxic response, resulting in neuronal cell death. Recently, DA was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in vitro. We have reported that WDR35, a WD-repeat protein, may mediate apoptosis in several animal models. In the present study, we administered DA to rats intraperitoneally, then used liquid chromatography/ion trap tandem mass spectrometry (LC-MS/MS) to identify and quantify DA in the brains of the rats and performed histological examinations of the hippocampus. We further investigated the potential involvement of glutamate receptors, ROS, p38 MAPK, and WDR35 in DA-induced toxicity in vivo. RESULTS: Our results showed that intraperitoneally administered DA was present in the brain and induced neurodegenerative changes including apoptosis in the CA1 region of the hippocampus. DA also increased the expression of WDR35 mRNA and protein in a dose- and time-dependent manner in the hippocampus. In experiments using glutamate receptor antagonists, the AMPA/KA receptor antagonist NBQX significantly attenuated the DA-induced increase in WDR35 protein expression, but the NMDA receptor antagonist MK-801 did not. In addition, the radical scavenger edaravone significantly attenuated the DA-induced increase in WDR35 protein expression. Furthermore, NBQX and edaravone significantly attenuated the DA-induced increase in p38 MAPK phosphorylation. CONCLUSION: In summary, our results indicated that DA activated AMPA/KA receptors and induced ROS production and p38 MAPK phosphorylation, resulting in an increase in the expression of WDR35 in vivo.

Endocytosis of cationized ferritin in marginal cells of the stria vascularis is regulated by protein kinase, protein phosphatase, and MEK/ERK and PI3-K signaling pathways.

HYPOTHESIS: The endocytosis of cationized ferritin (CF) via a clathrin-mediated pathway is regulated by a signaling network. BACKGROUND: Marginal cells showed the active endocytosis of CF via a clathrin-mediated pathway. The internalization of receptors through the clathrin-mediated pathway is an important regulatory event in signal transduction. Numerous kinases are involved in endocytosis, and each endocytic route is subjected to high-order regulation by cellular signaling mechanisms. METHODS: CF was infused into the cochlear duct with phorbol 12-myristate 13 acetate, okadaic acid, staurosporin, phenylarsine oxide, PD98059, SB20580 and wortmannin. Endocytic activity was measured at 30 minutes post-infusion by transmission electron microscopy. RESULTS: The endocytosis of CF was stimulated by a protein kinase C activator (phorbol 12-myristate 13 acetate) and a protein kinase A activator (8-bromoadenosine-3', 5'-cyclic monophosphate). It was inhibited by protein phosphatase inhibitors (okadaic acid and phenylarsine oxide), mitogen-activated protein kinase/extracellular signal-related kinase inhibitors (PD98059 and SB20580), and a phosphatidylinositol 3-kinase inhibitor (wortmannin). CONCLUSION: Our previous study showed the endocytosis of microperoxidase to be strongly dependent on protein kinase C, protein phosphatase, extracellular signal-related kinase, and phosphatidylinositol 3-kinase signaling networks but not on protein kinase A and mitogen-activated protein kinase signaling networks. The present study indicated that the signaling cascade regulating CF's internalization differed from the cascade for microperoxidase's endocytosis.

Endocytosis of microperoxidase in marginal cells is mainly regulated by RhoA signaling cascade, but not by Rho-associated protein kinase, myosin light-chain kinase and myosin phosphatase.

Endocytosis plays an important role in cell function and the activation and propagation of signaling pathways. Signaling occurs on endocytic pathways and signaling endosomes, and endocytosis is subjected to high-order regulation by cellular signaling mechanisms. Marginal cells showed active endocytosis of microperoxidase (MPO) via the clathrin-independent pathway. We examined the signaling pathway that regulates MPO endocytosis in marginal cells using specific inhibitors and activators of signaling molecules. The results showed that pertussis toxin - which inhibits the ribosylation of G-protein-coupled receptor - did not affect MPO endocytosis, but Clostridium botulinum C3 toxin - which induces RhoA inactivation resulting in extracellular-signal-related kinase inactivation - inhibited MPO endocytosis. The main endocytotic pathway of MPO did not depend on the Rho-associated protein kinase molecular switch or actin/myosin motor system, but was mainly regulated by the RhoA signaling cascade.

Endocytosis of MPO in marginal cells is regulated by PKC, protein phosphatase, ERK and PI3-K signaling cascades, but not by PKA and MEK signaling cascades.

Endocytosis of marginal cells plays a key role in maintaining the homeostasis of endocytosis and function of the organ of Corti. How the signaling cascade is involved in the regulation of endocytosis is an important issue at present. To investigate the regulation of endocytosis in marginal cells of the stria vascularis by the signaling network, we perfused MPO, an endocytosis tracer, with PMA, OA, staurosporin, PAO, PD98059, SB20580 or wortmannin into the cochlear duct. After 30 min endolymphatic perfusion, the tissues were fixed and the distribution of MPO was examined by electron microscopy. We explored the functions of PKC, RTK, PI3-K, PTP, and PP1/2A in MPO endocytosis and defined the MPO endocytic route. MPO endocytosis was strongly dependent on PKC, ERK, PTP, PP1/2A and PI3-K signaling networks, but not on PKA and MEK signaling networks. The MPO endocytic pathways are clathrin-, GPI-AP-, and caveolae-independent.

Hormonal aspects of Ménière's disease on the basis of clinical and experimental studies.

CONCLUSIONS: Endolymph homeostasis is thought to be mediated by the vasopressin-aquaporin-2 (VP-AQP2) system in the inner ear. Endolymphatic hydrops, the morphological characteristics of Ménière's disease (MD), seems to reflect the malregulation of the VP-AQP2 system in inner ear fluid. The elevation of plasma vasopressin (p-VP) level, which is often observed in MD and its related diseases, might be one of the causative factors underlying these diseases. PURPOSE OF REVIEW: Review of the role of the VP-AQP2 system in the inner ear fluid homeostasis and in the formation and development of endolymphatic hydrops. RECENT CLINICAL AND EXPERIMENTAL FINDINGS: A clinical survey has revealed that the p-VP level is often elevated in MD and its related diseases and that the increase in the p-VP level was closely linked to vertigo attacks in MD. Experimental studies have revealed that proteins and mRNAs of aquaporin-2 and vasopressin type 2 receptor were expressed in the stria vascularis of the cochlea and the epithelium of the endolymphatic sac, and that the volume of the endolymphatic compartment was mediated by the activity of the VP-AQP2 system in the inner ear.

Bumetanide-induced enlargement of the rat intrastrial space and effects of a vasopressin type 2 antagonist.

OBJECTIVE: Water homeostasis is essential for the inner ear to maintain the function of hearing and equilibrium. Bumetanide inhibits Na(+)-K(+)-2Cl(-) co-transporter (NKCC), which is expressed in the basolateral membrane of stria vascularis marginal cell, and it causes the enlargement of intrastrial space. Aquaporin (AQP) 2 is expressed in the perilymph side of stria vascularis basal cell, and the vasopressin type 2 antagonist OPC-31260 produces downregulation of AQP2 mRNA levels in the inner ear. The aim of this study is to investigate the influence of OPC-31260 on experimentally induced enlargement of the intrastrial space caused by bumetanide. MATERIALS AND METHODS: Wistar rats were divided into two groups, a BUM group and an OPC-BUM group. The BUM group was exposed to bumetanide, and the OPC-BUM group was exposed to bumetanide after being premedicated with OPC-31260. The specimens of the stria vascularis were observed using transmission electron microscopy and analyzed quantitatively and statistically. RESULTS: Morphological changes of intrastrial space enlargement occurred in both the BUM and OPC-BUM groups. The ratio of the areas of the intrastrial space area to the stria vascularis were calculated, and the OPC-BUM group mean showed a minimal difference from the BUM-group mean. However, there is no statistical difference. CONCLUSIONS: Premedication of rats with OPC-31260 tended to reduce bumetanide-induced enlargement of the intrastrial space. This may indicate that the effect of bumetanide on the stria vascularis is much stronger than that of OPC-31260.

Time course changes of vasopressin-induced enlargement of the rat intrastrial space and the effects of a vasopressin type 2 antagonist.

CONCLUSION: The intrastrial space was enlarged remarkably at 20 min after vasopressin (VP) injection, and this enlargement of the intrastrial space was reduced by administration of OPC-31260 before VP injection. These results suggest that VP increases the influx of water from the perlymph to the basal cells via aquaporin (AQP) 2 and causes the formation of endolymphatic hydrops. OBJECTIVES: To investigate a time course of changes of the stria vascularis after VP injection and the influence of OPC-31260 on experimentally induced enlargement of the intrastrial space caused by VP injection. MATERIALS AND METHODS: In the time course study, Wistar rats were injected with 50 microg/kg of VP subcutaneously. The stria vascularis specimens were harvested at 10, 20, 30, and 60 min after VP injection. For OPC-31260 administration, animals were administered 100 mg/kg of OPC-31260 orally 1 h before receiving 50 microg/kg of VP subcutaneously. The specimens were harvested 20 min after VP injection. These specimens were observed using transmission electron microscopy. RESULTS: In the time course study, the incidence of intrastrial space enlargement was 50%, 100%, 25%, and 0% for 10, 20, 30, and 60 min, respectively. In the study with OPC-31260 administration, the stria vascularis showed no morphological changes.

Immunohistochemical and electron microscopic characterization of brush cells of the rat cecum.

Brush cells (BCs) are relatively rare cells that are sparsely distributed throughout the mammalian digestive and respiratory systems. BCs have been identified in the rodent large intestine, but these cells have not been characterized by immunocytochemistry or electron microscopy. We previously demonstrated that rat bile duct BCs had strong immunoreactivity for six proteins that function in HCO(3)(-) secretion and thus assumed that BCs secrete NaHCO(3). It is well known that the gastrointestinal (GI) tract secretes NaHCO(3), but it is not known whether BCs of the GI tract also express proteins related to HCO(3)(-) secretion. Thus, in the present study, using double immunostaining for cytokeratin 18, a specific marker for BCs, we investigated protein expression in BCs from the rodent GI tract. We show that BCs from the GI tract express six proteins related to HCO(3)(-) secretion: cystic fibrosis transmembrane conductance regulator (CFTR), Cl(-)/HCO(3)(-) exchanger, Na(+)/HCO(3)(-) cotransporter, carbonic anhydrase II, Na(+)/H(+) exchanger (NHE) 1, and NHE3. These results suggest that BCs from the GI tract secrete NaHCO(3). In addition, we examined BCs from the rat cecum using electron microscopy (EM). Transmission EM (TEM) showed that BCs have long microvilli, a well-developed tubulovesicular system, and an abundant cytoskeleton. Scanning EM revealed that BCs were scattered on the luminal surface of the cecum and had numerous long microvilli.

Preserved tissue structure of efferent ductules in aromatase-deficient mice.

Estrogen receptor alpha (Esr1) is proposed to play a critical role in the regulation of testicular fluid reabsorption at efferent ductules, and disruption of the Esr1 gene (Esr1(-/-)) resulted in marked dilation of the lumens of efferent ductules. This study was aimed to clarify whether disruption of the gene for aromatase (Ar), an enzyme responsible for estrogen biosynthesis, results in morphological and transcriptional alterations at efferent ductules as observed in Esr1(-/-) mice. Histology demonstrated structural preservation of the ducts in aromatase-deficient (Ar(-/-)) mice. Electron microscopic examinations reveal that endocytic apparatus and tubule-cisternal endoplasmic reticulum are present in non-ciliated cells irrespective of the genotypes. However, electron-dense and acid phosphatase-negative granules and apical tubules, which are components thought to be related to membrane recycling of endosomes, are observed only in wild-type (WT) and Ar(-/-) mice. By contrast, the Golgi complex is highly developed in Esr1(-/-) mice when compared with WT and Ar(-/-) mice. RT-PCR analysis reveals no significant differences in the expression levels of a subset of genes involved in ion transportation. Thus, from the structural and transcriptional points of view, the efferent ductules of Ar(-/-) mice are indistinguishable from those of WT mice. Moreover, data from electron microscopic examinations indicate the possible involvement of Esr1 in the regulation of vesicle recycling processes.

Expression and immunolocalization of aquaporin-6 (Aqp6) in the rat inner ear.

CONCLUSION: Since aquaporin-6 (Aqp6) protein was located in the membrane of intracellular vesicles of the stria vascularis, endolymphatic sac, and vestibule, Aqp6 might be involved in some distinct physiological function of acid-base metabolism and water balance in endolymphatic fluid homeostasis. However, its lack of expression on the plasma membrane indicates that Aqp6 does not have a direct role in water flux via the plasma membrane. OBJECTIVE: To evaluate the expression and immunolocalization of Aqp6 in the rat inner ear. MATERIALS AND METHODS: Wistar rats were used. Aqp6 mRNA expression in the rat inner ear was investigated in the vestibulum as well as in the cochlea and endolymphatic sac using the reverse transcription-polymerase chain reaction (RT-PCR) method, and detailed immunolocalization of Aqp6 in the rat inner ear was investigated using immunohistochemical methods including immunofluorescence microscopy and immunoelectron microscopy. RESULTS: We obtained novel data showing that not just Aqp6 mRNA but also Aqp6 protein is expressed in the cochlea, endolymphatic sac, and vestibule. Immunoelectron microscopic studies revealed that the immunolabelled gold was diffusely seen in the intracellular area of the stria vascularis, endolymphatic sac, and vestibule, but never in the plasma membranes.

Effects of gadolinium injected into the middle ear on the stria vascularis.

CONCLUSION: The concentration of gadolinium (Gd) used clinically showed no remarkable effects on the stria vascularis; however, a higher concentration had adverse effects. The concentration of Gd must be borne in mind when injecting Gd into the tympanic cavity. OBJECTIVE: Endolymphatic hydrops has been visualized using high resolution MRI with the intratympanic administration of Gd in patients with Meniere's disease. We attempted to investigate the effects of Gd on the stria vascularis. MATERIALS AND METHODS: Gd hydrate diluted eightfold with saline or non-diluted Gd or saline was injected into the tympanic cavity of guinea pigs. To investigate the effects of Gd on the stria vascularis, we measured endocochlear DC potential (EP) and observed the stria vascularis using transmission electron microscopy. RESULTS: Intratympanic injections of Gd hydrate diluted eightfold with saline (1/8 Gd) and saline did not cause apparent changes in the EP. Moreover, the amplitude of the EP decreased significantly 60 min after non-diluted Gd was injected. Transmission electron micrographs of the stria vascularis revealed no significant morphological difference between the ears injected with 1/8 Gd and those injected with saline. There was significant morphological change in the ear injected with non-diluted Gd. The intercellular spaces were markedly enlarged.

Actin filaments and microtubules regulate endocytosis in marginal cells of the stria vascularis.

CONCLUSION: Cationized ferritin (CF) was internalized via clathrin-mediated endocytosis. This process depends on clathrin, actin filaments, and microtubules. Microperoxidase (MPO) was internalized via a clathrin- and caveolin-independent endocytic pathway, which was partially dependent on microtubules but independent of clathrin and actin filaments. OBJECTIVE: We investigated the role of actin filaments and microtubules in the transport of endocytic carrier vesicles (ECVs) from the plasma membrane to the early sorting endosomes, using CF and MPO as tracers. MATERIALS AND METHODS: Fifty-five guinea pigs were used. The animals were divided into a CF endocytosis group and an MPO endocytosis group. These groups consisted of control, nocodazole-treated, cytochalasin (Cyt D)-treated, Cyt D + nocodazole-treated, and geldanamycin-treated subgroups. RESULTS: For CF endocytosis, the following results were obtained. In the nocodazole experiment, in which microtubules were disrupted to form monomeric tubulin, the number of ECVs loaded with CF was greatly decreased. In the Cyt D experiment, in which the actin filaments were disrupted to form monomers, the number of ECVs labeled with CF was also greatly decreased. In the geldanamycin experiment, in which clathrin-mediated endocytosis was regulated and actin stress fibers were dissolved, the endocytosis of CF was severely inhibited. For MPO endocytosis, in the nocodazole experiment, the endocytosis of MPO was markedly suppressed.

An animal model of ischemic facial palsy. Behavioral facial nerve function and ultrastructural changes of the facial nerve.

We investigated facial palsy which was induced by the interruption of the petrosal artery in guinea pigs. Forty animals were observed for 2 months regarding their behavioral facial nerve function and assessed by the blink reflex. Morphological changes in the intratemporal portion were observed with transmission electron microscopy in 20 animals with an interrupted petrosal artery. Facial palsy developed in 85.0% within 3 days after the interruption. The degree of palsy varied from mild to severe. Remission of palsy required 2-3 months in severe cases, 3 weeks or less in mild/moderate cases. Histological studies revealed a striking difference in the degree of degenerative changes between severe cases and mild/moderate cases. Animals with severe palsy showed extensive axonal atrophy and myelin disruption from the early stage. Meanwhile, degenerative changes were slight in cases with mild/moderate palsy. Regenerating unmyelinated fibers appeared 1 week after the interruption, but diminished in number 4 weeks later. Thereafter, new myelin was reformed on fibers. In cases of severe nerve damage, however, this regeneration process did not always seem to work well. A decrease in number and an irregular shape of the fibers were noted in animals with incomplete recovery. This animal model may be helpful for understanding the pathophysiology of ischemic facial palsy.

Presence and regulation of epithelial sodium channels in the marginal cells of stria vascularis.

CONCLUSION: This study indicates that epithelial Na(+)-selective channels (ENaC) recycle Na(+) via clathrin-mediated endocytosis in the marginal cells of the stria vascularis and that clathrin-independent endocytosis appeared to be modulated by the amount of Na(+) transported. These results suggest the presence of ENaC in the luminal membrane of marginal cells and that ENaC are an efficient pathway for the uptake of Na(+) from the endolymph. OBJECTIVE: The ENaC found in many transporting epithelia play a key role in the regulation of salts and water homeostasis, cellular pH, cell volume, and cell function. Both biochemical and physiological approaches have been used to identify, characterize, and quantify this important channel, but its location in the marginal cells of the stria vascularis has not been fully clarified. The aim of this study was to determine the localization and regulation of ENaC. MATERIALS AND METHODS: Forty healthy female guinea pigs were used: 20 for the control experiment, 10 for the amiloride experiment, and 10 for the aldosterone experiment. We perfused cationized ferritin (CF) and microperoxidase (MPO) as tracers for clathrin-mediated and clathrin-independent endocytosis, respectively, into the cochlear duct. After 30 min of endolymphatic perfusion, the tissues were fixed and CF- and MPO-loaded endosomes within the marginal cell were observed by transmission electron microscopy. The numbers of CF- and MPO-loaded endosomes were compared between the three groups. RESULTS: In the amiloride group, the numbers of CF- and MPO-loaded endosomes decreased in comparison with the control. In the aldosterone group, the numbers of CF- and MPO-loaded endosomes decreased and increased, respectively. Recently, it has been reported that ENaC are endocytosed via clathrin-mediated endosomes and aldosterone decreases the rate of endocytosis of ENaC. In this study, the results of the aldosterone experiment were consistent with those of recent studies.

Ancient divergence of Paragonimus westermani in Sri Lanka.

Adult flukes of Paragonimus species were obtained from lungs of experimental animals infected with metacercariae found in field-collected freshwater crabs in Sri Lanka. Morphological studies of adult worms under a scanning electron microscope as well as a ordinary microscope were performed in the present study. All of morphological features observed clearly indicated that this species is P. westermani. On the other hand, the shapes of metacercariae were found to be mainly oval, but semioval and spherical ones also coexisted. In spite of the variety of their morphology of the metacercariae, there is no correlation between their shapes of metacercariae and the deoxyribonucleic acid (DNA) sequences. Molecular phylogenetic analyses using two DNA regions (partial mitochondrial cytochrome c oxidase subunit 1 and second internal transcribed spacer of the nuclear ribosomal gene repeat) placed the adult flukes of P. westermani from Sri Lanka basal in a tree including all specimens of P. westermani from various areas in Asia and P. siamensis from Thailand. The present study showed that P. westermani from Sri Lanka is an ancestral form.

Protective effects of edaravone against ischemia-induced facial palsy.

OBJECTIVE: The protective effect of edaravone, an inhibitor of reactive oxygen species (ROS), against the development of ischemia-induced facial palsy was investigated. METHODS: Experimental ischemic facial palsy was induced by interruption of the petrosal artery (PA) in guinea pigs. The application of edaravone was carried out by daily intraperitoneal injection for 1 week. The behavioral facial movement, fluorescence intensity of ROS, and morphological changes were investigated. RESULTS: Edaravone injection from immediately after PA interruption significantly reduced dihydrotetramethylrosamine fluorescence intensity (indicative of ROS) in the facial nerve of the interrupted ear and attenuated the development of ischemia-induced facial palsy. Edaravone injection from the 2nd day following PA interruption also reduced the incidence of facial palsy. Light and electron microscopy revealed that edaravone application tended to prevent the degenerative changes of the facial nerve caused by ischemia. CONCLUSIONS: Edaravone suppressed the production of ROS and had a remarkable protective effect against the development of mild to moderate facial palsy. Morphologically, nerve damage was also decreased by edaravone injection.

Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: pushing pixels to explore biological phenomena.

Quantitative colocalization analysis is an advanced digital imaging tool to examine antigens of interest in immunofluorescence images obtained using confocal microscopes. It employs specialized algorithms to estimate the degree of overlap of fluorescence signals and thus enables acquiring important new information not otherwise obtainable using qualitative approaches alone. As raw confocal images have high levels of background, they should be prepared to become suitable for reliable calculation of colocalization coefficients by correcting it. We provide concise theoretical basis of quantitative colocalization analysis, discuss its limitations, and describe proper use of the technique. The use of quantitative colocalization analysis is demonstrated by studying bile salt export pump and multidrug resistance associated protein 2 in the liver and major basic protein and platelet activating factor receptor antigens in conjunctiva. The review is focused on the applicability and correct interpretation of the results of colocalization coefficients calculations.