OBJECTIVE: To assess the value of Ploton silver staining and phalloidin-iFlour 488 staining in observation of the morphology of osteocyte dendrites of mice at different developmental stages. METHODS: The humerus and femurs were harvested from mice at 0 (P0), 5 (P5), 15 (P15), 21 (P21), 28 (P28), and 35 days (P35) after birth to prepare cryo-sections and paraffin sections. HE staining of P35 mouse femur sections served as a reference for observing osteocytes in the trabecular bone and cortical bone. The humeral sections at different developmental stages were stained with Ploton silver staining to observe the morphology of osteocytes and canaliculi, and the canalicular lengths in the cortical and trabecular bones of the humerus of the mice in each developmental stage were recorded. The cryo-sections of the humerus from P10 and P15 mice were stained with phalloidin iFlour-488 to observe the morphology of osteocytes and measurement of the length of osteocyte dendrites in the cortical bone. RESULTS: In the trabecular bone of the humerus of P0-P15 mice, Ploton silver staining only visualized the outline of the osteocytes, and the morphology of the canaliculi was poorly defined. In P21 or older mice, Ploton silver staining revealed the morphology of the trabecular bone osteocytes and the canaliculi, which were neatly arranged and whose lengths increased significantly with age (P21 CONCLUSIONS Mouse osteocyte dendrites elongate progressively and their arrangement gradually becomes regular with age. Ploton silver staining can clearly visualize the morphology of the osteocytes and the canaliculi in adult mice but not in mice in early stages of development. Phalloidin iFlour-488 staining for labeling the cytoskeleton can be applied for mouse osteocytes at all developmental stages and allows morphological observation of mouse osteocytes in early developmental stages.
Animals ; Bone and Bones ; Dendrites ; Mice ; Osteocytes ; Phalloidine ; Silver Staining

BACKGROUND: It is well known that the loud noise exposure can lead to noise-induced hearing loss (NIHL). Drilling during mastoid surgery may result in NIHL. The noise level produced by drilling of the mastoid bone can exceed 125 dB HL (hearing level); therefore, mastoid surgery itself is associated with a lower incidence of NIHL than expected. The aim of this study was to analyze the effects of isoflurane on NIHL and hair cell morphological changes. METHODS: BALB/c mice were divided into 2 groups; a control group (n = 20) and an isoflurane group (n = 20). The mice of both groups were exposed to 120 dB SPL (sound pressure level) broadband white noise for 3 hours per day, for 3 consecutive days. The mice in the isoflurane group were anesthetized with isoflurane while exposed to the noise. The auditory brainstem response (ABR) thresholds were determined 1 day before and after the noise-exposure and then again after 7 days. Both cochlea were removed and stained using fluorescent isothiocyanate (FITC) phalloidin. RESULTS: 1 day prior to noise-exposure, the ABR thresholds were those of a normal hearing level in both the control and isoflurane groups. In the control group, the mean hearing threshold was 78.0+/-2.6 dB HL after 1 day of noise-exposure and 81.5+/-3.4 dB HL after 1 week; in the isoflurane group, the mean hearing threshold was 49+/-11.7 dB HL after 1 day and 30.5+/-9.3 dB HL after 1 week. The hearing thresholds after noise exposure in the control were significantly higher than those in the isoflurane group (P<0.05). CONCLUSIONS: The occurrence of NIHL decreased and the hair cell damage suppressed in the mice exposed to intense noise while anesthetized by isoflurane.
Animals ; Cochlea ; Evoked Potentials, Auditory, Brain Stem ; Hair ; Hearing ; Hearing Loss, Noise-Induced* ; Incidence ; Isoflurane* ; Mastoid ; Mice* ; Noise ; Phalloidine

BACKGROUND AND OBJECTIVES: Erythropoietin (EPO) is produced in the kidney and locally in the CNS and acts through binding to erythropoietin receptor (EPO-R). Apart from playing an essential role in erythropoiesis, recent research has shown that EPO has neurotrophic and neuroprotective functions in the CNS and found EPO and EPO-R in the inner ear. The aim of this study is to investigate distribution and expression of EPO and EPO-R in the inner ear after noise exposure. MATERIALS AND METHOD: Normal guinea pigs were exposed to noise. Ten of them were sacrificed at 1 hour of the noise exposure (group B) and another 10 animals were sacrificed at day 7 (group C). Four were normal controls that were not exposed to noise (Group A). Auditory function was evaluated by ABR for 7 days. Noise-induced morphological changes of cochlea were studied by phalloidin stain. The expression of EPO and EPO-R was examined by immunofluorescence. RESULTS: The hearing threshold shift reached a level of 40 dB SPL at 8 kHz at day 1 after noise exposure and underwent a partial recovery at day 7. Increased expression of EPO and EPO-R were observed at the level of spiral ganglion cells in the noise-exposed animals. CONCLUSION: It is suggested that noise exposure affects the distribution and expression of EPO and EPO-R in the inner ear.
Animals ; Cochlea ; Ear, Inner ; Erythropoiesis ; Erythropoietin ; Guinea ; Guinea Pigs ; Hearing ; Kidney ; Noise ; Phalloidine ; Receptors, Erythropoietin ; Spiral Ganglion

BACKGROUND AND OBJECTIVES: Mechanism of inner ear hair cell distortion after noise exposure has been well described. The present study was designed to determine the response to the auditory system of a genetically well-defined laboratory mouse in preparation for examining the effect of noise on mice with specific genetic mutations. So it is important to recognize the relationship between noise exposure duration and hair cell morphological changes. We try to reveal the hearing loss and inner ear hair cell morphological changes after applying the noise protocol. SUBJECTS AND METHOD: The mice were BALB/c hybrids and aged 8 weeks. Six mice served as non-noise-exposed controls and 8 mice were exposed for 3 hours per day to white band noise with a center frequency from 0.2 kHz to 70 kHz and a sound pressure level of 120 dB. And we divided the noise exposure group into 3 subgroups(1 day, 3 day, 5 day noise exposure group). We checked the photographs of FITC phalloidin stain and scanning electron microscopy of cochlea after noise exposure. RESULTS: The hearing level of mice decreased after noise exposure. We could see the stereocilia damage in cochlea after FITC phalloidin stain in cochlea and sterocilia loss was more severe in basal turn. In scanning electron microscopy, morphological changes of stereocilia were observed to be more severe in the cochlear basal turn than other area. Significant hair cell loss in the cochlear basal turn could be calculated using cochleocytogram. CONCLUSION: 120dB broad white band noise can damage the hair cell of cochlea in mice. These changes were especially severe in the cochlear basal turn. Noise exposure duration is the other important factor in damaging cochlear hair cells. Therefore, we can guess that harmful noise level and noise exposure duration are the main risk factors that injure the inner ear hair cell.
Animals ; Cochlea ; Ear, Inner* ; Fluorescein-5-isothiocyanate ; Hair* ; Hearing ; Hearing Loss ; Mice* ; Microscopy, Electron, Scanning ; Noise* ; Phalloidine ; Risk Factors ; Stereocilia

The microfilaments of hepatocyte are distributed throughout the vicinity of cell membranes, especially numerous around the region of bile canaliculus, and provide the maintenance of cell shape, cellular wall tension, canalicular motility, the secretion for bile, etc. To evaluate the relationship between the microfilament and alteration of cell shape, we examined the morphological changes of cultured rat hepatocytes, following treatments with phalloidin or cytochalasin D with fluorescent and electron microscopes. 1. In the fluorescent micrographs, actin microfilament was distributed near the plasma membrane and bile canaliculus. 2. Both drugs, phalloidin or cytochalasin D, produce the cytoplasmic protrusions from the surface. Their shapes were pedunculated with narrow neck or bulged with broad base, respectively. 3. In the phalloidin treated group, cytoplasmic protrusion was seperated from the internal cytoplasm by microfila-ments networks at the narrow base. In contrast, in the cytochalasin D treated group, cytoplasm was bulged with broad base and kept in direct continuity with the canalicular ectoplasm. 4. Pericanalicular ectoplasm of phalloidin treated group was widened and accumulated with microfilaments. But, bile canaliculus of cytochalasin D treated group was markedly dilated and devoid of microvilli, and the ectoplasm was almost disappeared. Considering above results, dysfunction of microfilaments leads to the structural changes and inhibition of bile secretion of hepatocytes.
Actin Cytoskeleton ; Animals ; Bile ; Bile Canaliculi ; Cell Membrane ; Cell Shape ; Cytochalasin D* ; Cytoplasm ; Hepatocytes* ; Microvilli ; Neck ; Phalloidine* ; Rats*

OBJECTIVES: Apoptosis may play an important role in the mechanism underlying the GJB2 gene conditional knockout (cCx26) mice cochlear cell death. The objective of this study was to explore the the damage mode of the outer hair cells (OHCs) and its real time point of apoptosis and provide information to further explore the role of apoptosis in the happening of hearing loss in cCx26 mice. METHODS: Cochleae from mice at various developmental stages (P8, P12, and P21) were dissected out and first used to be observed under the scanning electron microscope (SEM). Basilar membranes from mice at P8, P14, P18, and P21 were stained by fluorescein isothiocyanate-conjugated phalloidin and propidium iodide (PI) and examined under confocal microscope. RESULTS: The loss of OHCs of cCx26 knockout mice was first set between P12 and P21 under SEM. Whole mount phalloidin and PI staining revealed that obvious apoptotic appearance of the OHCs surface morphology was observed at P18. CONCLUSION: Typical apoptotic morphology was found in the OHCs in the organ of Corti of the cCx26 mice at P18. This may provide information to further study the role of apoptosis in the occurrence of hearing loss of cCx26 mice.
Animals ; Apoptosis ; Basilar Membrane ; Cell Death ; Cochlea ; Connexins ; Electrons ; Fluorescein ; Hair ; Hair Cells, Auditory, Outer ; Hearing Loss ; Hearing Loss, Sensorineural ; Mice ; Mice, Knockout ; Organ of Corti ; Phalloidine ; Propidium

OBJECTIVES: Gentamicin (GM) is a commonly used aminoglycoside antibiotic that generates free oxygen radicals within the inner ear, which can cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. Piper longum L. (PL) is a well-known spice and traditional medicine in Asia and Pacific islands, which has been reported to exhibit a wide spectrum of activity, including antioxidant activity. In this study, we evaluated the effect of hexane:ethanol (2:8) PL extract (subfraction of PL [SPL] extract) on GM-induced hair cell loss in basal, middle and apical regions in a neonatal cochlea cultures. METHODS: The protective effects of SPL extract were measured by phalloidin staining of cultures from postnatal day 2-3 mice with GM-induced hair cell loss. The anti-apoptosis activity of SPL extract was measured using double labeling by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and myosin-7a staining. The radical-scavenging activity of SPL extract was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. RESULTS: SPL extract at a concentration of 1 microg/mL significantly inhibited GM-induced hair cell loss at basal and middle region of cochlea, while 5 microg/mL was effective against apical region hair cell loss. The protective effect of SPL extract was concentration dependent and hair cells retained their stereocilia in explants treated with SPL extract prior to treatment with 0.3 mM GM. SPL extract decreased GM-induced apoptosis of hair cells as assessed by TUNEL staining. The outer hair and inner hair counts were not decreased in SPL extract treated groups in compare to GM treated explants. Additionally, SPL extract showed concentration dependent radical scavenging activity in a DPPH assay. CONCLUSION: An anti-apoptosis effect and potent radical scavenger activity of SPL extract protects from GM-induced hair cell loss at basal, middle and apical regions in neonatal cochlea cultures.
Animals ; Apoptosis ; Asia ; Cochlea ; DNA Nucleotidylexotransferase ; Ear, Inner ; Ethanol* ; Gentamicins ; Hair* ; In Situ Nick-End Labeling ; Medicine, Traditional ; Mice ; Neurons ; Pacific Islands ; Phalloidine ; Piper* ; Reactive Oxygen Species ; Spices ; Stereocilia

OBJECTIVETo discuss the important role of actin polymerization in calcium ionophore A23187-induced human acrosome reaction and its mechanism.

METHODSEach spermatozoon specimen was divided into five groups, treated with A23187 3 micromol/L in Group A, Phalloidin 40 micromol/L and A23187 3 micromol/L in Group B, SLO 0.5 U/ml and A23187 3 micromol/L in Group C, SLO 0.5 U/ ml, Phalloidin 40 micromol/L and A23187 3 micromol/L in Group D, and nothing added in Grpup E. Then the percentage of the human acrosome reaction was assessed with Rhodamine-PSA (10 microg/ml).

RESULTSThe difference of the human spermatozoon acrosome reaction was significant (P < 0.01) among the 5 groups with or without SLO, Phalloidin and calcium ionophore A23187 but not between Groups A and B (P > 0.01).

CONCLUSIONPhalloidin does not work on the acrosome reaction of intact human spermatozoa, but in an SLO-permeabilized human spermatozoal model, it can obviously decrease the percentage of human spermatozoon acrosome reaction, which indicates that the polymerization of actin plays an important role in the course of human spermatozoon acrosome reaction, and mostly acts on the acrosome inside.

Acrosome Reaction ; drug effects ; Actins ; physiology ; Bacterial Proteins ; pharmacology ; Calcimycin ; pharmacology ; Cells, Cultured ; Humans ; Ionophores ; pharmacology ; Male ; Phalloidine ; pharmacology ; Spermatozoa ; drug effects ; physiology ; Streptolysins ; pharmacology

The present study aimed to explore whether the stretch of ischemic myocardium could modulate the electrophysiological characteristics via mechanoelectric feedback (MEF), as well as the effect of phalloidin on the electrophysiological changes. Thirty-two Wistar rats were randomly divided into 4 groups: control group (n=9), phalloidin group (n=7), myocardial infarction (MI) group (n=9), MI + phalloidin group (n=7). The acute myocardial infarction (AMI) was conducted by ligation of the left anterior descending (LAD) coronary artery for 30 min in isolated rat heart. The volume alternation of a water-filled latex balloon in the left ventricle produced the stretch of myocardium. After perfused on Langendorff, the isolated hearts were stretched for 5 s by an inflation of 0.1, 0.2 and 0.3 mL separately and the effect of stretch was observed for 30 s, including the left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), ±dp/dt(max), monophasic action potential duration at 90% repolarization (MAPD90), and occurrence of premature ventricular beats (PVB) and ventricular tachycardia (VT). The stretch caused an increase of MAPD(90) in both control and MI rats (P<0.05, P<0.01). Moreover, MAPD(90) in MI group increased more significantly than that in the control group at the same degree of stretch (P<0.05, P<0.01). Phalloidin (1 μmol/L) had no effect on MAPD(90) in basal state. After stretch, MAPD(90) in phalloidin group slightly increased but was not significantly different from that in the control group. However, phalloidin reduced MAPD(90) in infarcted myocardium, especially when ΔV=0.3 mL (P<0.05). The incidence rates of PVB and VT in MI group were higher than that in the control group (both P<0.01). And there was no significant difference in the incidence rates of PVB and VT between phalloidin group and control group. Phalloidin inhibited the occurrence of PVB and VT in infarcted hearts (both P<0.01). LVSP and +dp/dt(max) in MI group obviously decreased (P<0.01 vs control). With application of phalloidin, LVSP slightly, but not significantly increased in infarcted hearts, while -dp/dt(max) significantly increased (P<0.05). It is suggested that MI facilitates the generation and maintenance of malignant arrhythmias, while phalloidin obviously inhibits the occurrence of arrhythmias.
Action Potentials ; Animals ; Arrhythmias, Cardiac ; prevention & control ; Coronary Vessels ; Heart ; drug effects ; physiopathology ; Heart Ventricles ; Myocardial Infarction ; physiopathology ; Phalloidine ; pharmacology ; Rats ; Rats, Wistar

BACKGROUND AND OBJECTIVES: Organotypic culture of organ of Corti maintains the basic organization of the spiral lamina and can conserve several factors responsible for the neuronal growth of the nervous components. The explant culture technique has been widely used in organ culture system, however, the floating drop method using collagen gel was also developed as a simple and reliable method. In order to study the effect of growth factors on the regenerative and protective ability of cochlear hair cells, we first had to establish an in vitro model of the inner ear. MATERIALS AND METHODS: Organ of Corti was obtained from newborn rats and cultured with the floating drop method using collagen gel. Immunohistochemical staining was used to visualize the stereocilia and scanning electron microscopic study was also carried out. RESULTS: Explants were maintained up to 10 days without contamination. Morphologically, immunofluorescent staining with phalloidin showed well preserved outer and inner hair cells with stereocilia on the second day of culture. On the tenth day of culture, the staining result showed inner and outer hair cells, although the stereocilia were poorly stained. In scanning electron microscopic examination, an explant on the tenth day of culture showed preserved outer and inner hair cells and stereocilia, although damaged hair cells and stereocilia were also observed. CONCLUSION: The floating drop method was an appropriate method for maintaining the organ of Corti in vitro with the advantage being the easiness in its manual manipulation.
Animals ; Collagen ; Culture Techniques ; Ear, Inner ; Hair ; Humans ; Infant, Newborn* ; Intercellular Signaling Peptides and Proteins ; Neurons ; Organ Culture Techniques ; Organ of Corti* ; Phalloidine ; Rats* ; Spiral Lamina ; Stereocilia