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.

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.

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.

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.

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.

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.

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.

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.

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.

Assessment of anti-estrogenic activity of tamoxifen in transgenic mice expressing an enhanced green fluorescent protein gene regulated by estrogen response element.

Tamoxifen is an anti-estrogenic agent for the treatment of breast cancer, while exhibiting estrogenic activity in such tissues as the uterus. This study aimed to test whether these opposite properties of tamoxifen in the uterus can be evaluated separately in vivo. We employed two transgenic murine models named, respectively, the ERE-EGFP Ar+/+ mouse and ERE-EGFP Ar-/- mouse. Both types of mice possess an enhanced green fluorescent protein (EGFP) gene regulated by four copies of estrogen response elements (EREs), while the latter lacks a functional aromatase gene, which encodes an enzyme catalyzing conversion of androgens to estrogens. Tamoxifen clearly exhibited estrogenic activity in the uteri of ERE-EGFP Ar-/- mice, as it caused uterine wet weight gain and E2-target gene induction, as 17beta-estradiol (E2) did. However, tamoxifen did not enhance the EGFP expression in ERE-EGFP Ar-/- mice, although E2 induced it significantly. In ERE-EGFP Ar+/+ mice, tamoxifen suppressed the EGFP expression in a time- and dose-dependent manner. Thus, the present study demonstrated that estrogenic and anti-estrogenic activities of tamoxifen can be evaluated by using ERE-EGFP Ar-/- and ERE-EGFP Ar+/+ mice, respectively. Furthermore, these animal models are useful to select and evaluate estrogenic and anti-estrogenic activities of chemical compounds.

Endocytosis of microperoxidase in the marginal cells of stria vascularis.

OBJECTIVE: Endocytosis has been thought to control entry into the cell and play a crucial role in the development, immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. We investigated the basic properties of endocytosis in the marginal cells of stria vascularis (SV) to discuss whether marginal cells have a potential to maintain the endolymph homeostasis. METHODS: We perfused microperoxidase (MPO), an endocytosis tracer, into the cochlear duct. After 5-60 min of endolymphatic perfusion, the tissues were fixed and the distribution of MPO within the marginal cell was observed by transmission electron microscopy. RESULTS: Endocytosis started already at 5 min after MPO perfusion. Small MPO-loaded endosomes were observed up to 30 min after MPO perfusion. The small tubulovesicular endosomes and the plasma membrane invagination were not decorated by an electron dense bristle structure. After endocytosis, MPO labeled preendosomes were quickly transported to the large vacuolar endosomes that connected with tubular endosomes. At 60 min after MPO perfusion, MPO-loaded large vesicles that have small vesicles in the lumen were observed. CONCLUSION: The time-course of MPO-loaded endosomes was similar to that of CF-loaded endosomes in the marginal cells of SV. The strial marginal cells have vigorous endocytotic activity both in clathrin-independent and clathrin-dependent endocytosis. This high activity of endocytosis in SV seems to be needed to maintain the homeostasis of endolymph via membranous channels and/or receptors regulations.

A comparison of dehydration effects of V2-antagonist (OPC-31260) on the inner ear between systemic and round window applications.

V2-antagonist (OPC-31260 (OPC)) application to the scala tympani reduced endolymphatic hydrops. In the present study, we investigated whether systemic administration or local infusion via the round window (RW application) of OPC would be more suitable for clinical use. In Experiment 1, the increase ratios of the cross-sectional area of the scala media of experimentally induced endolymphatic hydrops were quantitatively assessed among four groups of non-OPC application, RW application of xanthan gum, systemic application of OPC and RW application of OPC. In Experiment 2, the effects of systemic and RW applications of OPC on plasma vasopressin (p-VP) concentrations and plasma osmolality (p-OSM) were investigated. In Experiment 3, endocochlear DC potential (EP) was measured in guinea pigs with the RW application of OPC. Electron microscopic observations of the stria vascularis and the hair cells were also made. Both systemic and RW applications of OPC significantly reduced endolymphatic hydrops. However, systemic application resulted in the distension of the Reissner's membrane in the non-operated ear, which seemed to be caused by elevated p-VP levels resulting from the systemic application of OPC. In contrast, RW application of OPC produced no apparent toxic effects in the inner ear, as indicated electrophysiological or morphological changes. Thus, drug delivery via the round window is more useful for the clinical application of OPC for medical decompression.

Expression of the estrogen-inducible EGFP gene in aromatase-null mice reveals differential tissue responses to estrogenic compounds.

Aromatase is an enzyme responsible for the conversion of androgen to estrogen. We genetically engineered an aromatase-deficient mouse (Ar(-/-) mouse) to express an enhanced green fluorescent protein (EGFP) gene in the uterus, ovary, adrenal and pituitary glands in a 17beta-estradiol (E2)-inducible manner. In this study, we analyzed estrogenic activities of diethylstilbestrol, genistein, daidzein and E2 in the Ar(-/-) tissues by using the EGFP expression as an indicator. These analyses manifest differential responses of the tissues to the compounds and also allow to determine the relative estrogenic potency of the compounds to that of E2 in vivo. Furthermore, analyses of the EGFP expression in ERalpha-deficient mice suggested that the expression is ERalpha-dependent in the uterus and pituitary gland. In conclusion, the Ar(-/-) mouse carrying the E2-inducible EGFP gene is a valuable tool for quantitative analyses of natural and synthetic estrogenic compounds in vivo.

Experimental LPS-induced cholestasis alters subcellular distribution and affects colocalization of Mrp2 and Bsep proteins: a quantitative colocalization study.

Quantitative colocalization analysis is a powerful tool for reliable estimation of the colocalization of antigens. We employed it to determine the changes of colocalization of multidrug resistance protein 2 (Mrp2) and bile salt export pump (Bsep) in confocal immunofluorescence microscopy images of rat liver following lipopolysaccharide (LPS) administration. Samples were taken 2, 24, 48 hours, and 1 week after LPS challenge. Pearson's correlation coefficient (PCC), an overlap coefficient according to Manders' (MOC), and overlap coefficients k1 and k2 were used to explore changes of the degree of colocalization. In intact animals, confocal microscopy showed tight colocalization of Mrp2 and Bsep proteins exclusively at the bile canaliculi. High degree of colocalization was confirmed quantitatively. Injection of LPS resulted in the appearance of fuzzy-looking areas of fluorescence of both proteins around bile canaliculi 2 and 24 hours after administration and relocation of Mrp2 protein to the basolateral domain of hepatocytes at 48 hours. By 1 week, canalicular localization was restored morphologically. Quantitative colocalization analysis of canalicular regions showed a steady decrease of the degree of colocalization of Mrp2 and Bsep up to 48 hours with the slight increase of its value by 1 week. These findings demonstrate that Mrp2, in contrast to Bsep, is partially and reversibly relocated from canalicular to basolateral domain of hepatocytes after LPS challenge.

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.

Aromatase-knockout mouse carrying an estrogen-inducible enhanced green fluorescent protein gene facilitates detection of estrogen actions in vivo.

Aromatase is an enzyme that converts androgen to estrogen in the gonads and also at extragonadal sites, including the brain. In this study we developed a transgenic mouse that carries an enhanced green fluorescent protein (EGFP) gene inducible by estrogen through an estrogen response element to facilitate detection of estrogen actions in vivo. The expression of EGFP in aromatase-deficient (Ar(-/-)) female mice was significantly suppressed at the pituitary gland, ovary, uterus, and gonadal fat pad and was induced by dietary 17beta-estradiol to wild-type (Ar(+/+)) levels or higher. These results demonstrate that the expression of the EGFP gene is tissue selective and estrogen dependent in vivo. Employing this transgenic mouse, we examined whether estrogen synthesis in the extragonadal sites is necessary for reproduction in female mice. When ovaries of Ar(-/-) mice were replaced with Ar(+/+) ovaries, a significant induction of EGFP expression in the pituitary gland and uterus was observed. Histological examinations showed the presence of antral follicles in the replaced ovaries, indicating that the transplants are functional in Ar(-/-) mice. After crossing with males, three of 10 Ar(-/-)females with Ar(+/+) ovaries became pregnant and fed their pups. Collectively, these observations indicate that estrogen synthesis in the ovary is sufficient for supporting female reproduction, and that infertility of Ar(-/-) females is primarily due to a defect in estrogen synthesis in the ovary.