[Clinicopathologic features of retinoblastoma and expression of VEGF and Ki-67 after comprehensive treatment].

OBJECTIVE: To investigate the clinic pathologic features of retinoblastoma (RB) after comprehensive treatment, and study the expression of vascular endothelial growth factor (VEGF) in retinoblastoma treated with chemotherapy prior to enucleation. METHODS: Retrospective analysis was performed on retinoblastoma specimens obtained consecutively between 2006 and 2008 by enucleation, and patients' clinical information and clinic pathologic features were also collected. Immunohistochemical staining and real-time PCR were performed for the expression of VEGF. Immunohistochemical staining was also performed for Ki-67. RESULT: Among the 9 chemotherapy-treated cases, six belonged to group D and three to group E of IIRC. The reasons for enucleation included extensive vitreous seeds, RB recurrence, extensive subretinal fluid/seeds, vitreous hemorrhage and total tractional detachment of the retina. During the comprehensive treatment, the main tumors regressed in all eyes. The main tumors showed a mean decrease of 43.7% in the largest basal diameter and a mean decrease of 57.9% in thickness. The average interval between the end of chemotherapy and enucleation was 5.7 months. The reason for enucleation was the recurrence of main tumor, recurrence of new tumors, recurrent vitreous seed or subretinal seed. Three eyes showed a type 1 regression pattern, one eye showed a type 2 pattern, and the other five eyes showed type 3 clinical regression patterns. The expression of VEGF was lower in eyes that underwent planned enucleation than eyes that suffered from RB recurrence. CONCLUSIONS: The main reason for enucleation was extensive subretinal fluid/seeds after the comprehensive treatment. The type 3 clinical regression patterns were most common. In retinoblastoma, higher expression of VEGF may play an important role in the recurrence of retinoblastoma after comprehensive treatment.

2-Aminoethoxydiphenyl borate administration into the nostril alleviates murine allergic rhinitis.

OBJECTIVE: Orai1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) channels and plays a key role in the store-operated Ca(2+) entry. However, little is known about the function of this pathway in allergic rhinitis (AR). In this study, we examined whether the intervention of Orai1 pathway was capable of controlling IgE-mediated allergic reactions by using AR mice models. MATERIALS AND METHODS: We used Western blotting and real-time reverse transcription polymerase chain reaction to evaluate Orai1 expression in nasal mucosa and nasal-associated lymphoid tissue (NALT) of normal, control, and 2-aminoethoxydiphenyl borate (2-APB)-treated mice. In addition, we analyzed concentrations of nasal lavage fluid leukotriene C4 (LTC4), eosinophil cation protein (ECP), ovalbumin-specific IgE, and interleukin-4 (IL-4) through enzyme-linked immunosorbent assay and measured messenger RNA (mRNA) levels of LTC4 synthase and ECP in nasal mucosa, and germline Cɛ transcription and IL-4 mRNA in NALT by using real-time reverse transcription polymerase chain reaction among groups. RESULTS: 2-Aminoethoxydiphenyl borate administration into the nostril reduced numbers of sneezing and nasal rubbing as well as counts of invasive eosinophils in treated mice compared with those in control ones. Furthermore, the administration suppressed Orai1 expression in nasal mucosa and NALT of treated mice compared with that of control ones. Similarly, 2-APB treatment restrained nasal lavage fluid LTC4, ECP, ovalbumin-specific IgE, and IL-4 and their corresponding mRNAs in the previously mentioned tissues of treated mice in comparison with those of control ones. CONCLUSION: Our results indicate that 2-APB treatment effectively alleviates murine AR through pleiotropic activities.

Up-regulation of Orai1 in murine allergic rhinitis.

Orai1 is an essential pore-forming subunit of the Ca(2+) release-activated Ca(2+) channels and plays a key role in the store-operated Ca(2+) entry. However, little is known about the function of this pathway in allergic airway diseases. In this study, we evaluated Orai1 expression in normal and allergic rhinitis (AR) mice airway and spleen. AR models were established by repetitive intraperitoneal sensitization followed by intranasal challenge with ovalbumin. Sneezing was counted, and eosinophils infiltration was analyzed through Luna stain. We performed the analysis of Orai1 protein in airway and spleen by immunohistochemical staining, Western blotting and enzyme-linked immunosorbent assay, and quantitatively examined Orai1 mRNA in the above tissues by real-time reverse transcription-polymerase chain reaction. Sneezes and eosinophil counts in the AR group were increased in comparison to those in the normal group. Orai1 protein was expressed in mucosal epithelium and submucosal glands epithelium of airway, and in immune cells of spleen. The immunostaining appeared stronger in AR mice than that in normal ones. Both the Orai1 protein and mRNA levels showed up-regulation in the AR group compared with those in the normal one. Our results indicate that Orai1 is up-regulated in the airway and spleen in allergic inflammation and may participate in the pathogenesis of allergic rhinitis.

High-salt loading exacerbates increased retinal content of aquaporins AQP1 and AQP4 in rats with diabetic retinopathy.

In the neural retina, glial cells control formation of ionic gradients by mediating transmembrane water fluxes through aquaporin (AQP) water channels. Retinal content and immunolocalization of two water channels, AQP1 and AQP4, in the diabetic rat retinas during high-salt loading were examined in this study. Diabetes was induced by an intraperitoneal injection of streptozotocin. Diabetic and control animals were observed after varying lengths of exposure to normal- and high-salt conditions. Ultrathin sections of retinal tissue, stained with uranyl acetate and lead citrate, were photographed using a transmission electron microscope (TEM). Retinal wholemounts were immunostained with AQP1 and AQP4 antibody to detect the immunolocalization changes by confocal microscopy. AQP1 and AQP4 content were evaluated by Western blot analysis. In the retinas of high-salt loading diabetic animals, obviously increased intracellular edema was observed by TEM in ganglion cells and mitochondrial swelling was observed in glial cells. Immunolocalization of AQP1 increased from the posterior to peripheral retina. Western blot results indicated that a high-salt diet may cause increased retinal content of AQP4 and may exacerbate increased retinal content of AQP1 caused by diabetic retinopathy. High-salt loading may increase neural retinal edema in rats with diabetic retinopathy, and altered glial cell mediated water transport via AQP channels in the retina may play an important role in the neural retinal edema formation and resolution.

Neuroprotective effect of transcorneal electrical stimulation on light-induced photoreceptor degeneration.

Direct electrical stimulation of neural tissues is a strategic approach to treat injured axons by accelerating their outgrowth [Al-Majed, A.A., Neumann, C.M., Brushart, T.M., Gordon, T., 2000. Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J. Neurosci. 20, 2602-2608] and promoting their regeneration [Geremia, N.M., Gordon, T., Brushart, T.M., Al-Majed, A.A., Verge, V.M.K., 2007. Electrical stimulation promotes sensory neuron regeneration and growth-associated gene expression. Exp. Neurol. 205, 347-359]. Recently, transcorneal electrical stimulation (TCES), a novel less invasive method, has been shown to rescue axotomized and damaged retinal ganglion cells [Morimoto, T., Miyoshi, T., Matsuda, S., Tano, Y., Fujikado, T., Fukuda, Y., 2005. Transcorneal electrical stimulation rescues axotomized retinal ganglion cells by activating endogenous retinal IGF-1 system. Invest. Ophthalmol. Vis. Sci. 46(6), 2147-2155]. Here, we investigated the neuroprotection of TCES on light-induced photoreceptor degeneration and the underlying mechanism. Adult male Sprague-Dawley (SD) rats received TCES before (pre-TCES) or after (post-TCES) intense light exposure. After fourteen days of light exposure, retinal histology and electroretinography were performed to evaluate the neuroprotective effect of TCES. The mRNA and protein levels of apoptotic-associated genes including Bcl-2, Bax, Caspase-3 as well as ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the retinas were determined by real-time PCR and Western blot analysis. The localization of these gene products in the retinas was examined by immunohistochemistry. Both pre- and post-TCES ameliorated the progressive photoreceptor degeneration. The degree of rescue depended on the strength of the electric charge. Post-TCES showed a relatively better and longer-term protective effect than pre-TCES. Real-time PCR and Western blot analysis revealed an upregulation of Bcl-2, CNTF, and BDNF and a downregulation of Bax in the retinas after TCES. Immunohistochemical studies showed that Bcl-2 and CNTF were selectively upregulated in Müller cells. These findings provide a new therapeutic method to prevent or delay photoreceptor degeneration through activating the intrinsic survival system.

High salt loading alters the expression and localization of glial aquaporins in rat retina.

In the neural retina, glial cells control the ionic concentrations in part by mediation of transmembrane water fluxes through aquaporin (AQP) water channels. The expression and immunolocalization of two water channels, AQP1 and AQP4, in the rat retina during experimental high salt loading were investigated in this study. Six-week-old Wistar rats were allowed free access to rat chow with 8% NaCl concentration. Of these rats, 6 were killed after 2, 6, 10 and 20 weeks. Twelve-week-old and 26-week-old Wistar rats with a normal diet (0.5% NaCl concentration) were used as controls. Retinal tissues were collected. Ultrathin sections stained with uranyl acetate and lead citrate were photographed using a transmission electron microscope (TEM). Retinal whole mounts and cryosections were immunostained with AQP1 and AQP4 antibodies to detect the immunolocalization changes by confocal microscopy. The AQP1 and AQP4 contents were evaluated by western blot analysis. In control tissues, no intracellular edema and mitochondria swelling were observed by TEM. The immunoreactive AQP4 was expressed by glial cells (Müller cells and astrocytes) predominantly in the inner retina, and AQP1 was expressed in the outer retina. In the retinas of high salt loading animals, obvious intracellular edema was observed by TEM in retinal ganglion cell (RGC) and mitochondria swelling was observed in glial cells. Strong expression of AQP1 was found in glial cells located in the innermost retinal layers, mainly in astrocytes. The superficial retinal vessels were surrounded by AQP4 in control retinas, but by both AQP4 and AQP1 in retina of high salt loading animals. A similar alteration in the localization of AQP1 has been described in the rat retina after transient ischemia and diabetes. Western blot results supported the conclusion that the AQP1 expression increased during high salt diet. Our findings indicate that high salt loading may induce neural retina edema, and that altered glial cell-mediated water transport via AQP channels in the retina may be one of the reasons for intracellular edema in the neural retina.

Expression of macrophage colony-stimulating factor (M-CSF) and its receptor in streptozotocin-induced diabetic rats.

PURPOSE: To demonstrate the expression and location of macrophage colony-stimulating factor (M-CSF) and its receptor (CSF-1R) in the retinas of diabetic rats, as well as in vitreous human proliferative diabetic retinopathy (PDR). METHODS: The retinas of streptozotocin-induced diabetic rat were studied. Real-time PCR was applied to evaluate M-CSF and its receptor CSF-1R mRNA expression in the retinas. The protein levels of M-CSF and CSF-1R were evaluated by Western blot analysis. Cellular sources of M-CSF and CSF-1R were determined by double-immunofluorescence staining. M-CSF levels in vitreous samples from patients with PDR were measured by ELISA. RESULTS: M-CSF and CSF-1R mRNA were upregulated in the retinas as early as two weeks after the onset of diabetes and increased over time. A similar pattern was observed for M-CSF/CSF-1R protein expression levels. Double-immunofluorescence staining revealed that increased M-CSF immunoreactivity occurred mainly in the nerve fiber layer in diabetic retinas, co-localizing with glial fibrillary acidic protein. Increased CSF-1R immunoreactivity was observed in OX-42-labeled microglia and ganglion cells in the ganglion cell layer. The vitreous level of M-CSF was elevated in patients with PDR compared to control subjects. CONCLUSIONS: The early upregulation of MCSF/CSF-1R signaling may be an important regulatory pathway among neurons, microglia, and glia in diabetic retinopathy.

[Uptake and accumulation of gentamicin by mouse hair cells from the cochlear explants in vitro].

OBJECTIVE: To investigate the upstaging and accumulation of gentamicin by mouse hair cells in vitro. METHODS: Cochlear explants were prepared from the microdissected neonatal mouse cochlea. Cochlear explants were cultured with gentamicin-Texas-red conjunction (GTTR) for different time. Laser confocal microscopy was used to observe the distribution of GTTR in the cochlear sensory cells after labeling with phalloidin-alexa-488. RESULTS: Soon after culture, there was diffuse red staining all tissue cells in the explants. At later time the hair cells were more staining than other cells in the explants. There was no obviously accumulation of GTTR in the supporting cells. The peak level of fluorescent density was reached at 24 hours culture. The GTTR was seen in the infracuticular zone of the hair cells. There was still accumulation of GTTR in the hair cells of the explants after 7 days culturing. CONCLUSIONS: GTTR and cochlea explants were useful methods to investigate the pharmacokinetics and mechanisms of gentamicin accumulation over time.

Neuroprotective effects of naloxone against light-induced photoreceptor degeneration through inhibiting retinal microglial activation.

PURPOSE: To determine the role of microglial activation in light-induced photoreceptor degeneration and the neuroprotective effects of naloxone as a novel microglial inhibitor. METHODS: Sprague-Dawley rats were exposed to intense blue light for 24 hours. Daily intraperitoneal injection of naloxone or PBS as a control was given 2 days before exposure to light and was continued for 2 weeks. Apoptotic cells were detected by the TUNEL assay, and anti-OX42 antibody was used to label retinal microglia. Western blot was applied to evaluate the retinal interleukin (IL)-1beta protein levels. Retinal histologic examination and electroretinography (ERG) were also performed to evaluate the effects of naloxone on light-induced photoreceptor degeneration. RESULTS: TUNEL-positive cells were noted in the outer nuclear layer (ONL) of the retina as early as 2 hours and peaked at 24 hours after exposure to light. OX42-positive microglia occurred in the ONL and subretinal space at 6 hours, peaked at 3 days, and changed morphologically from the resting ramified to the activated amoeboid. Expression of IL-1beta protein was also significantly increased at 3 days. Compared with the control, the number of microglia in the outer retina was significantly decreased in the naloxone-treated group at 3 days, and the thickness of ONL and the amplitudes of dark-adapted a- and b-waves were also well preserved at 14 days. CONCLUSIONS: The activation and migration of microglia and the expression of neurotoxic factor (IL-1beta) coincide with photoreceptor apoptosis, suggesting that activated microglia play a major role in light-induced photoreceptor degeneration. Inhibiting microglial activation by naloxone significantly reduces this degeneration.

[A study on the expression of opsins in form-deprived myopia retina of guinea pig].

OBJECTIVE: To investigate the expression of cone opsins in form-deprived myopia (FDM) of guinea pig. METHODS: Twenty-eight pigmented guinea pigs aged one-week were randomly divided into two groups: FDM group (n = 14) and control group (n = 14). White translucent vinyl diffuse attached to one randomly selected eye was used to deprive eyes of form-vision in FDM group. RT-PCR and immunohistochemistry were performed to compare the opsin mRNA expression and spatial density of photoreceptor opsin in the ventral, central and dorsal retinal areas of two groups using antibody against short and medium-wave-length-sensitive cone opsin. Slides were viewed using Leica confocal microscope and Leica light microscope. RESULTS: The distribution of opsin across the guinea pig retinae as determined by antibody labeling of cone opsin was asymmetric, with the dorsal retina dominated by M-opsin, and the ventral retinae by S-opsin. But the ventral area also showed positive staining for the M-opsin. Opsin spatial density decreased with increasing retinal eccentricity for both M and S opsins. The spatial density of S-opsin decreased most steeply for eccentricity up to one-third distance from optic disc to retinal edge. The spatial density of M-opsin gradually decreased with eccentricity. The expression of spatial density of M-opsin was higher than that of S-opsin in all three regions. The expression of density of S-opsin in normal control eye is as follow: (805.0 +/- 203.3) mm(-2) (ventral); (100.0 +/- 57.7) mm(-2) (dorsal); (1637.2 +/- 314.1) mm(-2) (Central). FDM eye: (640.9 +/- 196.8) mm(-2) (ventral); (1016.7 +/- 144.6) mm(-2) (central); (70.9 +/- 30.8) mm(-2); where The expression of spatial density of M-opsin in normal control eye are: (946.2 +/- 388.5) mm(-2) (dorsal); (1666.7 +/- 137.8) mm(-2) (central); (175.0 +/- 100.9) mm(-2) (ventral). FDM eye: (1436.7 +/- 366.0) mm(-2) (dorsal); (2780.0 +/- 180.5) mm(-2) (central); (318.2 +/- 172.7) mm(-2) (ventral). The results of RT-PCR examination showed that the relative optical density (OD) value of M-opsin and S-opsin are: 1.06 +/- 0.07 (FDM eye), 0.51 +/- 0.10 (control eye), and 0.70 +/- 0.07 (FDM eye), 1.25 +/- 0.06 (control eye) respectively. The expression of both opsins in FDM eyes was significant differences compared with control (P < 0.05). CONCLUSION: FDM can increase the expression of M-opsin and decrease the S-opsin expression in guinea pig. cone opsins may play a role in the development of myopia.

The effects of different auditory activity on the expression of phosphorylated c-Jun in the auditory system.

CONCLUSION: The data revealed that calcium influx via the NMDA receptor up-regulated the expression of phosphorylated c-Jun in the primary auditory cortices following sensory stimulation and after different neural injury stimulations which guide activity-dependent changes in gene expression and neural plasticity. OBJECTIVES: Activator protein-1 (AP-1) transcription factor, which is mainly composed of c-Fos and c-Jun proteins, is believed to be a key participant in molecular processes that guide activity-dependent changes in gene expression. Our previous study had shown that the expression of NMDAR2A gene on synaptosomes membrane of auditory cortical neurons varied by electrical intracochlear stimulation (EIS) and neural injury induced by acoustic trauma. In this study, we investigated the role of the NMDA receptor (NMDAR) in regulating the expression of phosphorylated c-Jun in the primary auditory cortex (AI). The modulation factors observed for gene expression included EIS and noise traumas. MATERIALS AND METHODS: EIS was applied in rats with early postnatal auditory deprivation. The impact of the noise traumas on the ultrastructures of spiral ganglion neurons (SGNs) and their innervations to inner hair cells (IHCs) were verified by transmission electron microscopy (EM). These changes include a decrease in subcellular organelles, the swelling of mitochondria and endoplasmic reticulum, the morphological changes in cell nuclei, and damage in the afferent synapse. RESULTS: Immunohistochemistry observations showed that the expression of phosphorylated c-jun and active caspase-3 in hair cells and SGNs varied with amount of noise. Immunocytochemistry and Western blotting showed that the auditory cortex began to express phosphorylated c-jun 24 h after 2 h of EIS. However, this expression was not changed by EIS if NMDAR antagonist was applied. The level of phosphorylated c-Jun was remarkably increased in AI after noise overstimulation at 115 dB SPL for 3 h. Again, such an increase was not seen if NMDAR antagonist 3-(2 carboxypiperazin-4yl) propyl-1-phosphonic acid (CPP, 10 mg/kg, i.p.) was applied 30 min before the noise exposure.

[Distribution of gentamicin in inner ear after intratympanic gentamicin injection].

OBJECTIVE: To investigate uptake and accumulation of gentamicin by cells in the guinea pig inner ear after intratympanic injection using a fluorescent probe--gentamicin-Texas-red conjunction (GTTR). METHODS: Adult guinea pigs (n = 80) were administered a single dose of GTrR to the middle ear cavity through the intact membrane and survived for 12 h, 24 h, 48 h, 3 d, 4 d, 7 d, 14 d and 28 d. The distribution of GTTR in the cochlear and vestibular cells was observed after staining with phalloidin-alexa-488. Texas Red and DMSO were injected into the tympanum as control. RESULTS: Diffuse staining of gentamicin in the labyrinth was observed initially after local drug administration. At later time point the outer hair cells and sensory cells of vestibular organ were staining more densely than the support cells in the inner ear. The peak level of fluorescent density was reached 3 days after local injection. The GTTR was observed in the infracuticular zone. CONCLUSIONS: GTTR was a potential fluorescent probe to investigate the pharmacokinetics and mechanisms of gentamicin accumulation in local application.