October 2004
Volume 45, Issue 10
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Retinal Cell Biology  |   October 2004
Molecular and Cellular Reactions of Retinal Ganglion Cells and Retinal Glial Cells under Centrifugal Force Loading
Author Affiliations
  • Kenji Kashiwagi
    From the Department of Ophthalmology, University of Yamanashi Faculty of Medicine, Yamanashi, Japan; the
  • Yoko Iizuka
    From the Department of Ophthalmology, University of Yamanashi Faculty of Medicine, Yamanashi, Japan; the
  • Yuko Tanaka
    From the Department of Ophthalmology, University of Yamanashi Faculty of Medicine, Yamanashi, Japan; the
  • Makoto Araie
    School of Medicine, Tokyo University, Tokyo, Japan; and
  • Yasuyuki Suzuki
    Teikyo University Ichihara Hospital, Tokyo, Japan.
  • Shigeo Tsukahara
    From the Department of Ophthalmology, University of Yamanashi Faculty of Medicine, Yamanashi, Japan; the
Investigative Ophthalmology & Visual Science October 2004, Vol.45, 3778-3786. doi:https://doi.org/10.1167/iovs.04-0277
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      Kenji Kashiwagi, Yoko Iizuka, Yuko Tanaka, Makoto Araie, Yasuyuki Suzuki, Shigeo Tsukahara; Molecular and Cellular Reactions of Retinal Ganglion Cells and Retinal Glial Cells under Centrifugal Force Loading. Invest. Ophthalmol. Vis. Sci. 2004;45(10):3778-3786. https://doi.org/10.1167/iovs.04-0277.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To investigate changes in retinal ganglion cell (RGC) survival and morphology, retinal glial cell effects on RGC survival, and changes in mRNA expression during centrifugal force loading using a newly developed device.

methods. Changes in RGC survival and morphology were examined when isolated RGCs from 2-day-old rats were loaded with centrifugal force equivalent to 16, 28, or 33 mm Hg. The effects of cocultured retinal glial cells on RGC survival were studied in the presence of centrifugal force equivalent to 16 and 28 mm Hg for 48 hours. The microarray method and real-time polymerase chain reaction confirmed changes in mRNA expression when RGCs and retinal glial cells were loaded with centrifugal force equivalent to 28 mm Hg for 24 hours.

results. The survival of isolated RGCs and the number of neurites were significantly decreased by centrifugal force loading. Conversely, there was no significant change in the survival of isolated retinal glial cells. The survival of cocultured RGCs was significantly better than that of isolated RGCs. In contrast to the numerous changes in the mRNA expression of retinal glial cells subjected to centrifugal force loading, there was no significant change in the mRNA expression of RGCs.

conclusions. The developed device may have potential for use as an in vitro model of RGC damage. The response to centrifugal force loading varies according to cell type, and the marked changes in the mRNA expression of retinal glial cells may be involved in the improvement of RGC survival.

Such debilitating factors as hypoxia, malnutrition, and pressure result in RGC damage. Those factors do not act directly on RGCs. Rather, they are thought to act indirectly on other cells and factors such as glial cells. 1  
In the human eye, although numerous factors are thought to be intricately involved at the molecular level in the occurrence of RGC damage, most of the studies conducted thus far have focused on those factors individually and are therefore inadequate for elucidating the complex reaction mechanisms of the human eye. With the recent advances in microarray techniques, 2 3 4 5 6 however, it has become possible to examine comprehensively the changes in the expression of numerous genes. Although studies using the microarray method to assess changes in the expression of numerous genes during the occurrence of ocular disorders have recently been reported, 7 8 9 many of them use tissue such as that from the retina as samples. Thus, we do not know whether the detected changes in gene expression originate in a specific cell, nor do we know the extent of involvement of the factors being assessed in RGC damage. To conduct a more detailed analysis of the mechanism involved in the occurrence of RGC damage, it is necessary to assess changes in gene expression comprehensively, in isolated cells. 
Intraocular pressure (IOP) is an important factor contributing to glaucomatous RGC damage. Although such devices as a closed pressurized chamber have been used in the past for reproducing a state of elevated IOP in vitro, 10 11 12 13 it is not easy to make subtle adjustments in pressure or to achieve stable loading at low pressures with those devices. In addition, the partial pressure of gas in the liquid culture may differ from that of the body. Therefore, we constructed a new experimental centrifugal force loading device that mimics the state of pressure loading by applying centrifugal force to isolated cells. 
In this study, we examined the survival and morphology of isolated RGCs subjected to centrifugal force loading using the newly developed device. In addition, the effects of retinal glial cells on RGC survival were assessed when those two types of cells were subjected to centrifugal force loading after coculture. Changes in the mRNA expression of RGCs and retinal glial cells during centrifugal force loading were assessed using the microarray method to examine the role of retinal glial cells in RGC death. 
Methods
All experiments were conducted and all laboratory animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Cell Isolation and Culture System
Isolation of RGCs and Retinal Glial Cells.
RGCs and retinal glial cells were isolated from Sprague-Dawley rats, as previously described. 14 15 16 17 18 19 20 21 Briefly, 2-day-old rats were killed to obtain approximately 60 enucleated eyes for each experiment. Isolated retinas were incubated for 20 minutes in a solution containing papain (5 mg/mL), and dissociated cells were incubated for 5 minutes with rabbit anti-macrophage antibody (Inter-Cell Technologies, Hopewell, NJ). Cell suspensions were treated for 45 minutes in 100-mm Petri dishes coated with goat antibody against rabbit IgG L and H chains (Southern Biotechnology Associates, Birmingham, AL). Suspensions containing cells that did not adhere to the Petri dish were treated for 1 hour in 100-mm Petri dishes coated with anti-Thy 1.1 antibody (from hybridoma T11D7e2; American Type Culture Collection, Manassas, VA). Cells that adhered to the second Petri dish were collected after separation by 10-minute incubation with 0.125% trypsin and were incubated in 24-well plates. Retinal glial cells were isolated according to methods described in previous reports. 20 21 Briefly, enucleated eyes of 3-day-old Sprague-Dawley rats were placed in DMEM containing 2 mM glutamate and 1:1000 penicillin-streptomycin and were stored overnight in a dark room at room temperature. The eyes were then incubated at 37°C for 30 minutes in DMEM containing 0.1% trypsin and 70 IU/mL collagenase. The eyes were transferred to a Petri dish containing DMEM and 10% fetal bovine serum (FBS). Retinas were finely dissected under a microscope, and cells were gently dissociated by a Pasteur pipette. Cells from 8 to 10 retinas were seeded onto a culture dish and cultured in DMEM containing 10% FBS, 2 mM glutamine, and 1:1000 penicillin-streptomycin at 37°C in a humidified 5% CO2 atmosphere. The medium was left unchanged for 5 to 6 days and was subsequently replaced every 3 to 4 days. Third- and fourth-passage cells were used for the experiments. 
Culture Conditions.
Culture plates were coated with 0.1 mg/mL polyornithine (Sigma-Aldrich, St. Louis, MO) for at least 5 hours. Then, the plates were additionally coated overnight with 5 μg/mL EHS-laminin (Upstate Biotechnology, Lake Placid, NY). The medium developed by Politi et al. 22 for monolayer culture of mixed mouse retinal neurons, as modified for use in this experiment, consisted of DMEM with the following additions: insulin (1.6 × 10−6 M), progesterone (4.0 × 10−8 M), selenite (6.0 × 10−8 M), transferrin (12.5 × 10−8 M), putrescine (2 × 10−4 M), hydrocortisone (1.0 × 10−7 M), cytidine-5′-diphosphocholine (5.2 × 10−6 M), cytidine-5′-diphosphoethanolamine (2.9 × 10−6 M), 40 ng/mL each of brain-derived neurotrophic factor and ciliary neurotrophic factor, and 5 μM forskolin. The seeding density was 2 × 105 cells per well. Cells were incubated at 37°C in humidified 10% CO2 and 90% air. 
Centrifugal Force Loading Device and Force Loading Conditions
A schematic of the centrifugal force loading device is shown in Figure 1A . The device is composed of a rotating vessel installed within a large incubator (model CPO2-1800; Hirasawa, Tokyo, Japan), a power supply unit, a control unit, and a cooling motor for removing heat generated by the motor installed outside the device. A high-performance motor and a rotor are installed within the incubator. The rotor rotates at 1 to 30 rotations per minute (rpm), and the rotation control accuracy is 0.01 rpm (Model H-26F; Kokusan, Tokyo, Japan). The motor unit is enclosed in a sealed box to eliminate the effects of moisture in the incubator. Within the motor box, cool air is pumped in from outside the incubator to prevent a temperature increase within the incubator, due to heat generated by the motor. Loaded pressure is calculated from the rotation speed and the distance between the center of rotation and the culture dish as the radius. The equation for calculating centrifugal force F is as follows: F (mm Hg) ≒ 1.12r × (rpm/1000) × 750, where r is radial distance (in millimeters). The culture dish is fixed at an angle θ so that the bottom of the culture dish is perpendicular to the direction of the sum of the centrifugal force and gravity vectors corresponding to an assumed rotation speed (Fig. 1B)
Effect of Centrifugal Force Loading on Cell Survival
RGC Survival.
The effect of centrifugal force loading on isolated RGC survival was assessed. After seeding isolated RGCs into a 24-well culture dish, centrifugal force equivalent to 16 or 28 mm Hg was loaded for 48 hours, followed by an assessment of the effect on cell survival by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA), as described elsewhere. 14 15 16 18  
Retinal Glial Cell Survival.
To examine the effect of centrifugal force loading on retinal glial cell survival, centrifugal force equivalent to 16, 28, or 48 mm Hg was loaded on either isolated confluent retinal glial cells or retinal glial cells cocultured with RGCs for 48 hours, followed by a cell-proliferation assay (Cell Titer 96; Aqueous One Solution Cell Proliferation Assay; Promega, Madison, WI) that is used for assaying cell survival. The following is a brief description of the cell proliferation assay. Cells were counted according to the manual provided with the assay. One hundred microliters of reagent (Cell Titer 96 One Solution Reagent; Promega) was added to the medium immediately after centrifugal force loading, and the plate was cultured for 2 hours in an incubator. Next, 400 μL of the medium was removed and transferred to a 96-well microplate reader (SpectraMax340; Molecular Devices, Sunnyvale, CA), and this was followed by absorbance measurement at 490 nm. The effect of the absence of centrifugal force loading was also assessed as the control. Because retinal glial cells are proliferative, a graded series of concentrations of RGCs (ranging from 9.4 × 106 to 4.7 × 104 cells/mL) were used for quantitative analysis. 
Effect of Retinal Glial Cells on RGC Survival
The effect of retinal glial cells on the survival of isolated RGCs during centrifugal force loading was assessed. As previously reported, 16 retinal glial cells were seeded onto a semipermeable membrane, and after reaching confluence, isolated RGCs were seeded into the bottom of the culture dish. Subsequently, centrifugal force equivalent to 16 or 28 mm Hg was loaded for 48 hours, followed by an assessment of RGC survival by flow cytometry (FACSCalibur; BD Biosciences), as described before. 14 15 16 In brief, RGCs were treated for 10 minutes by substituting the same media containing 5-chloromethylfluorescein diacetate (Molecular Probes, Eugene, OR). After this treatment, RGCs were detached from well bottoms by gentle pipetting, and RGCs in the supernatant were immediately subjected to flow cytometry. The survival rate of RGCs was quantified by measuring the amount of fluorescence. From each culture well, a total of 10,000 RGCs was subjected to analysis and the duration of evaluation was assessed. The RGCs were divided into four groups according to the intensity of their fluorescence and in regard to cell size—namely, in terms of surviving small and large RGCs and nonsurviving small and large RGCs. We used a flow cytometer system to count the number of cells in each group and calculate automatically the survival ratio of small RGCs and large RGCs. 
Changes in RGC Morphology Induced by Centrifugal Force Loading
Changes in the morphology of RGCs induced by centrifugal force loading were also examined. After isolation of RGCs, the cells were cultured for 72 hours while being loaded with force equivalent to 33 mm Hg. Cells not subjected to centrifugal force loading were used as the control. After culture, the culture medium was removed, and after the cells were washed with phosphate buffer (PB, pH 7.4), they were fixed for 40 minutes with 2% formaldehyde neutral buffer solution (Nacalai Tesque, Kyoto, Japan). After a wash with PB, the cells were treated for 10 minutes with 0.1% saponin/2% bovine serum albumin (BSA), and after the reaction was blocked for 30 minutes with 2% BSA, the cells were allowed to react overnight at 4°C with rabbit monoclonal antineurofilament 200 (NF200; Sigma-Aldrich) diluted 700-fold, as the primary antibody specific for neurofilament heavy chains (NF-H). Then, the cells were allowed to react for 2 hours with Texas-red–conjugated anti-rabbit IgG antibody diluted 200-fold, as the secondary antibody (Sigma-Aldrich). The cells were then sealed and observed with a laser confocal microscope. A total of 200 RGCs were evaluated at an arbitrary location at the center of each preparation. RGCs having neurite lengths equal to or greater than three times the cell body diameter were evaluated as those having neurite extensions, and of those RGCs, the ones exhibiting clusters of neurites were counted as those having dendritic beading processes. 
Microarray Analysis
RGCs in a 12-well culture plate and retinal glial cells in a 10-cm culture plate were subjected to centrifugal force loading for 24 hours at 28 mm Hg, followed by biomicroscopic observation. After the cells were recovered and counted, total RNA was extracted using a shredder spin column (QIA; Qiagen, Valencia, CA) and a mini-spin column (RNeasy; Qiagen) according to the manufacturer’s instructions. Using the recovered total RNA as the sample (approximately 2 μg/mL), changes in mRNA expression were examined with a rat oligo DNA chip equipped with 1099 nucleotides as probes (Hitachi, Ltd., Life Science Group, Saitama, Japan). Of the signals with intensities greater than 400 units, a rate of expression change of two times or more was considered to indicate a significant increase in mRNA expression, whereas a rate of expression change of half or less was considered to indicate a significant decrease in mRNA expression. 
Real-Time PCR
Primers were constructed for each mRNA in which significant changes were observed with the microarray method followed by real-time PCR to confirm the results obtained with the microarray method. Real-time PCR was conducted three or four times on each mRNA, and the resultant average rate of expression change was used for confirmation. 
Statistical Analysis
The survival rates under various conditions were tested with ANOVA and the post hoc method. Morphologic changes in RGCs during centrifugal force loading were tested with the Mann-Whitney test. Significant difference was defined as P < 0.05. 
Results
Centrifugal Force Loading and RGC Survival
RGC survival was significantly decreased in proportion to the centrifugal force loaded for both large and small cells (Fig. 2) . At centrifugal force equivalent to 16 mm Hg, the percentage survival was decreased by 7.3% for large RGCs and by 12.4% for small RGCs relative to the control. The percentage decrease in survival in the case of loading centrifugal forces equivalent to 28 mm Hg was 27.8% for large RGCs and 22.2% for small RGCs relative to the control. 
Effect of Cocultured Retinal Glial Cells on RGC Survival
Regarding RGCs cocultured with retinal glial cells (Fig. 3) , the percentage survival of the isolated RGCs improved significantly when centrifugal forces equivalent to 16 and 28 mm Hg were loaded. The percentage survival was improved by 3.9% for large RGCs and by 9.1% for small RGCs relative to the control at centrifugal force equivalent to 16 mm Hg. Furthermore, the percentage survival was improved by 16.2% for large RGCs and by 11.3% for small RGCs relative to the control at centrifugal force equivalent to 28 mm Hg. 
Centrifugal Force Loading and Retinal Glial Cell Survival
The cell proliferation assay showed a linear correlation between RGC concentrations and their emission intensity among the investigated range (Fig. 4) . Therefore, the number of retinal glial cells was calculated to be the same as that of RGCs. For retinal glial cell both solely cultured and cocultured with RGCs, survival was slightly reduced when cells were loaded with centrifugal force equivalent to 48 mm Hg. There were no significant changes in retinal glial cell survival caused by centrifugal force loading, and there were also no marked changes as observed by phase contrast microscopy. 
Morphologic Changes in RGCs during Centrifugal Force Loading
Morphologic observation revealed two types of RGCs: those with normal straight or cone-shaped neurites (Fig. 5A) and those with dendritic beading neurites (Fig. 5B) . Although cells in which neurite extensions were observed 72 hours after culture accounted for 38.4% of the RGCs not subjected to centrifugal force loading, that number decreased significantly (to 22.2%) in RGCs subjected to centrifugal force loading. In addition, among RGCs in which neurites were observed, for the no-force–loading group, RGCs in which no dendritic beading was observed accounted for 94.5%. In contrast, for the centrifugal-force–loading group, RGCs in which no dendritic beading was observed accounted for only 64.0%, indicating a significant decrease compared with the group in which dendritic beading was observed (Fig. 6)
Changes in mRNA Expression
Eight hundred fifty of the 1099 nucleotides studied in RGCs exhibited sufficient fluorescence and thus could be examined for changes in mRNA expression. Among those nucleotides, the only mRNA that exhibited a significant increase in expression (2-fold or more) was Rattus norvegicus neuronal differentiation related mRNA, complete cds (GenBank code: AB020022; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD), and although the increase in expression of this gene was observed by real-time PCR as well, the rate of increase was only 1.37-fold. At the same time, there were no mRNAs of which expression was decreased to half or less. In contrast, there were 960 nucleotides studied in retinal glial cells that exhibited sufficient fluorescence and thus could be examined for changes in mRNA expression. The expression of the 14 mRNAs listed in Table 1 was increased by a factor of two or more. Although a well-defined increasing trend was not observed by real-time PCR in 11 of the 14 mRNAs, the remaining 3 were observed to demonstrate an increasing trend. There were 15 genes of which mRNA expression was decreased to half or less as shown in Table 1 , and changes in the expression of 12 of those genes were confirmed by real-time PCR. Among the genes for which changes in expression were confirmed, the metabolism and growth factor/cytokine-related genes were comparatively numerous. Primers for real-time PCR are listed in Table 2
Discussion
We assessed the relationship between RGC damage and the corresponding glial cells during centrifugal force loading using a new in vitro experimental loading device. In addition, a comprehensive study was conducted of the changes in mRNA expression of RGCs and retinal glial cells during centrifugal force loading. As a result, RGC survival was found to decrease in proportion to the centrifugal force loaded, and retinal glial cells significantly inhibited the decline. In addition, the frequency of occurrence of dendritic beading in the neurites of RGCs as a result of centrifugal force loading was significantly greater than that with no loading, clearly demonstrating that loading causes not only RGC death but also morphologic changes in the surviving RGCs. In contrast to the numerous changes in the mRNA expression of retinal glial cells subjected to centrifugal force loading, there was no significant change in the mRNA expression of RGCs. 
Some experiments have been conducted in which pressure is applied to cultured cells to serve as an in vitro experimental model of glaucomatous neural damage. 10 12 13 23 24 25 However, because it is not easy to apply extremely low, constant pressure to cultured cells for a long time and gas partial pressure in the medium may differ from normal pressure, this model is not satisfactory for evaluating RGC damage in vitro. The device used in this study was developed for the purpose of reproducing a state that closely mimics the pressurized state by applying centrifugal force to RGCs. The use of this device makes it possible to apply mild to moderate arbitrary and constant force. In addition, by adding other loads such as hypoxic load or administering additives to the medium simultaneous to loading, it is possible to construct an intricate experimental system that closely mimics in vivo conditions. We confirmed that this device is a useful as an in vitro experimental system for studying RGC damage, as it revealed that RGC survival is decreased in proportion to the amount of centrifugal force loading and that retinal glial cells exhibit protective effects on RGCs subjected to centrifugal force loading. 
Several reports have been published regarding the effect of glial cells on RGC survival. 23 26 27 28 29 We have studied the effects of retinal glial cells on RGC survival in the absence of loading using the same coculture system, and have found that nonconfluent glial cells demonstrate a detrimental effect on RGC survival, whereas confluent glial cells had no effect on the survival. 16 It has been reported that retinal glial cells may be intimately involved in RGC survival. Kawasaki et al. 27 reported that cultured retinal glial cells protect RGCs from the neurotoxic effect of massive glutamate. Rudzinski et al. 30 reported that ocular hypertension upregulates trk receptor in rat Müller cells but not in RGCs. The finding in this study that retinal glial cells have a protective effect on RGC survival during centrifugal force loading may indicate that, whereas retinal glial cells in the mature state maintain the constancy of RGCs and have protective effects against external damage to RGCs, retinal glial cells in the growth phase have detrimental effects. Such factors as cytokines released from glial cells due to primary stimulation may have a secondary effect on neurons. In this study as well, changes in the mRNA expression of numerous genes related to cytokines and metabolism were actually observed in retinal glial cells. Miyahara et al. 8 investigated changes in mRNA expression in monkey retinas from experimental glaucoma eyes and a microarray technique revealed that very few mRNAs showed changes in expression except ceruloplasmin mRNA in Müller cells. Tezel et al. 31 reported that retinal glial cells in glaucomatous human retina are activated, whereas Yang et al. 10 and others in that laboratory 11 12 have reported hydrostatic pressure-induced changes in mRNA expression in optic astrocytes that are not consistent with the present results. Differences in cell type, loading conditions, and microarray type could be the reasons for the difference in results. These findings indicate that, regarding the protection of RGCs, the target of treatment may not be the RGCs themselves, but glial cells. 
The disappearance or reduction of neurite extensions during disturbance conditions in RGCs has been reported. 32 33 34 Pavlidis et al. 35 reported dendritic beading in RGCs from a human donor with glaucoma. Because the involvement of the blockage of axonal transport in the initial stages of RGC loss has been assumed and the obstruction of axonal transport by IOP elevation in experimental monkey models has been reported, 36 37 38 they hypothesized that the increased beading of glaucomatous axons is an effect of advanced glaucoma on axonal transport, resulting in RGC death. In this study, the significant dendritic beading observed in some neural disorders was seen as well in RGCs subjected to centrifugal force loading, consistent with previous reports. Moreover, dendritic beading has been observed in cerebral neurons and is considered to be involved in neural damage. 39 40 41 However, many aspects of the mechanism remain unclear, and whether they are characteristic of centrifugal force or pressure loading remains uncertain. Although the irregular, swollen beads along the axons may represent aggregates of transportation vesicles, their details are unclear. We have demonstrated in an experimental model of glaucoma in monkey eyes that the dephosphorylation of NF-H, which is the main skeletal protein of the axons responsible for axon strength, occurs in RGCs before the deciduation of those cells and have discussed the decrease in axon strength caused by the dephosphorylation of NF-H. 42 However, the increase in dendritic beading in the centrifugal-force–loaded RGCs observed in this study suggests the possibility of the occurrence of an abnormality in the axon skeleton during impairment, which in turn leads to RGC death. 
The changes in mRNA expression during centrifugal force loading differed considerably between RGCs and retinal glial cells, and the number of genes that exhibited significant changes was extremely low in the former. The microarray used in this study contained many of the 34 representative apoptosis-related genes. Although those genes were examined to elucidate the mechanism underlying isolated and cultured RGC death caused by centrifugal force loading, the mRNA expression of nearly all those genes was either increased by less than twofold or decreased to less than half. Although the mRNA expression of caspase-7 and tumor necrosis factor–induced protein 6 was increased by more than twofold by centrifugal force loading, the expression was low in both cases, and no significant changes were observed. In contrast, the expression of many mRNAs changed considerably in retinal glial cells. On analyzing those changes by cluster, numerous changes were observed in the gene group related to metabolism and growth factor/cytokines. Because the survival of RGCs was improved when they were cocultured with retinal glial cells, it was thought that this group of genes exhibiting changes in mRNA expression is involved in the improvement of RGC survival. Although 1099 genes were examined in this study by the microarray method, a study was also conducted of the changes in the mRNA expression of RGCs and retinal glial cells when loaded for 48 hours with centrifugal force equivalent to 33 mm Hg with this new force loading device, using the microarray manufactured by Agilent Inc. (Palo Alto, CA) on which roughly 14,000 genes are plotted. It was also confirmed in a preliminary experiment that the number of mRNAs exhibiting changes in expression in RGCs was extremely small compared with that in glial cells (unpublished data, 2004). 
We have studied the changes in NO synthase (NOS) expression during hypoxic loading, and in contrast to the significant change in NOS expression in glial cells, no significant change was observed in RGCs. 17 Taking all evidence together, we surmise that RGCs exhibit a relatively small change in mRNA expression in response to external stimulation, and that retinal glial cells and other surrounding cells are intimately involved in their survival. 
A total of 1099 mRNAs were assessed by the microarray method, which is not a particularly large number. In addition, because it is extremely difficult to collect a sufficient amount of RNA from cultured RGCs for microarray studies, the microarray study was conducted only once. Therefore, in the future, it would be beneficial to detect expression-changing genes with higher reliability by increasing the number of studies using microarrays and to conduct further studies on genes that were not detected with the microarray used in this study. 
It is also important to elucidate the mechanism by which RGCs are damaged using this newly developed centrifugal force loading device, to search for substances that protect neurons, and to proceed with further analyses of genes related to RGC death. We believe that the present results and this device would be helpful in understanding the mechanism of glaucomatous optic neuropathy and in developing new therapeutic methods. 
 
Figure 1.
 
Schema of the centrifugal force loading device. (A) The device is composed of (a) an incubator, (b) a power supply and a control unit, (c) a cooling motor unit, (d) a motor unit for providing centrifugal force and rotor vessels, (e) culture dish trays, and (f) a cooling duct. (B) A high-performance motor (g) installed in the incubator controls the rotation with an accuracy of 0.01 rpm. The culture dish is fixed at an angle θ so that the bottom of the culture dish is perpendicular to the direction of the sum of the centrifugal force and gravity vectors corresponding to an assumed rotation speed.
Figure 1.
 
Schema of the centrifugal force loading device. (A) The device is composed of (a) an incubator, (b) a power supply and a control unit, (c) a cooling motor unit, (d) a motor unit for providing centrifugal force and rotor vessels, (e) culture dish trays, and (f) a cooling duct. (B) A high-performance motor (g) installed in the incubator controls the rotation with an accuracy of 0.01 rpm. The culture dish is fixed at an angle θ so that the bottom of the culture dish is perpendicular to the direction of the sum of the centrifugal force and gravity vectors corresponding to an assumed rotation speed.
Figure 2.
 
Effects of centrifugal force loading on RGC survival. *P < 0.01, †P = 0.02, ANOVA and the post hoc method. n = 8; bar, SE.
Figure 2.
 
Effects of centrifugal force loading on RGC survival. *P < 0.01, †P = 0.02, ANOVA and the post hoc method. n = 8; bar, SE.
Figure 3.
 
Effect of cocultured retinal glial cells on RGC survival. *P = 0.02, †P < 0.001, ‡P = 0.04, ANOVA and the post hoc method. n = 8, bar, SE.
Figure 3.
 
Effect of cocultured retinal glial cells on RGC survival. *P = 0.02, †P < 0.001, ‡P = 0.04, ANOVA and the post hoc method. n = 8, bar, SE.
Figure 4.
 
Centrifugal force loading and retinal glial cell survival. Centrifugal force did not influence the retinal glial cell survival significantly. ANOVA and the post hoc method. n = 6, bar, SE.
Figure 4.
 
Centrifugal force loading and retinal glial cell survival. Centrifugal force did not influence the retinal glial cell survival significantly. ANOVA and the post hoc method. n = 6, bar, SE.
Figure 5.
 
Centrifugal force induced morphologic changes in RGCs. There are two types of neurites in surviving RGCs: normal straight or cone-shaped (A) and dendritic beading (B) neurites. RGCs cultured with centrifugal force loading had more dendritic beading neurites than those cultured without centrifugal force loading.
Figure 5.
 
Centrifugal force induced morphologic changes in RGCs. There are two types of neurites in surviving RGCs: normal straight or cone-shaped (A) and dendritic beading (B) neurites. RGCs cultured with centrifugal force loading had more dendritic beading neurites than those cultured without centrifugal force loading.
Figure 6.
 
Effects of centrifugal force loading on RGC neurites. P < 0.01, Mann-Whitney test. n = 5, bar, SE.
Figure 6.
 
Effects of centrifugal force loading on RGC neurites. P < 0.01, Mann-Whitney test. n = 5, bar, SE.
Table 1.
 
Gravity-Related Changes in mRNA Expression of Retinal Glial Cells
Table 1.
 
Gravity-Related Changes in mRNA Expression of Retinal Glial Cells
Gene Name GenBank Number Category Change Ratio (Microarray) Change Ratio (Real-Time PCR)
Upregulated mRNAs in Retinal Glial Cells
 Rattus norvegicus UDP glycosyltransferase 1 family, polypeptide A7 NM_130407 Metabolism 4.74 2.8
 Glutathione-S-transferase, alpha type (Ye?) X78847 Metabolism 4.49 5.46
 R. norvegicus mRNA for chemokine co-receptor CKR5 Y12009 Receptor 3.49 1.36
 Arachidonate 5-lipoxygenase activating protein X52196 Metabolism 3.09 0.93
 Complement component 3a receptor 1 U86379 Receptor 2.94 0.71
 Polypeptide GalNAc transferase T1 U35890 Metabolism 2.17 4.5
 3-hydroxyisobutyrate dehydrogenase J04628 Metabolism 2.11 0.93
 Caspase 3, apoptosis related cysteine protease (ICE-like cysteine protease) U49930 Apoptosis 2.09 2.2
 Isocitrate dehydrogenase 1, soluble NM_031510 Metabolism 2.08 6.68
 ATP-binding cassette, sub-family G (WHITE), member 1 AJ303374 Transporter 2.05 0.98
 Benzodiazepin receptor (peripheral) NM_012515 Receptor 2.03 7.67
 ATP-binding cassette, sub-family A (ABC1), member 2 NM_024396 Transporter 2.01 1.03
 Programmed cell death 4 NM_022265 Apoptosis 2 2.43
 Caspase-8 NM_022277 Apoptosis 2 3.46
Downregulated mRNAs in retinal glial cells
 TGF beta 2 protein NM_031131 Growth factor 0.27 0.16
 Aldehyde dehydrogenase family 1, subfamily A1 AF001898 Metabolism 0.29 0.62
 Rattus norvegicus macrophage inflammatory protein-2 precursor, mRNA, complete cds U45965 Signal transduction 0.29 1.74
 TGFB inducible early growth response U78875 Growth factor 0.4 0.62
 Rattus norvegicus GTP-binding protein gamma subunit (Ggamma8) mRNA, complete cds L35921 Signal transduction 0.43 0.44
 Interleukin 10 X60675 Cytokine 0.43 1.01
 O6-methylguanine-DNA methyltransferase X54862 Metabolism 0.43 0.9
 Cytochrome P450, 2c37 K03501 P450 0.43 0.3
 P-glycoprotein/multidrug resistance 1 M81855 Transporter 0.44 0.37
 DNA-damage-inducible transcript 1 NM_024127 stress 0.46 0.75
 Vascular endothelial growth factor AF062644 Growth factor 0.48 0.43
 Cyclin D2 L09752 Cell Cycle 0.49 0.51
 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 J04024 Membrane 0.5 0.62
 Cyclin D1 X75207 Cell Cycle 0.5 0.5
 Macrophage inflammatory protein 1 alpha (Small inducible cytokine A3) U22414 Cytokine 0.5 1.23
Table 2.
 
Gene-Specific Primer Sequences for Real-Time PCR
Table 2.
 
Gene-Specific Primer Sequences for Real-Time PCR
Gene Name GenBank Number Forward Reverse
Rattus norvegicus UDP glycosyltransferase 1 family, polypeptide A7 NM_130407 5′CCG ACA TTG CCT CTG AAG TTC3′ 5′CCA ACG TGA AGT CTG TGC GTA A3′
Glutathione-S-transferase, alpha type (Yc?) X78847 5′GGA CAA GAT TAT CTC GTT GGC A3′ 5′GGT CCA TCT CTT CCA CAT GGT A3′
R. norvegicus mRNA for chemokine co-receptor CKR5 Y12009 5′ATC CTC AAC ACC CTG TTT CG3′ 5′TGT TTG CAG AAG CGT TTG AC3′
Arachidonate 5-lipoxygenase activating protein X52196 5′CGT AGA TGC GTA CCC CAC TT3′ 5′CGC TTC CGA AGA AGA AGA TG3′
Complement component 3a receptor 1 U86379 5′TGG CTT GTT CCT GTG CAA AC3′ 5′AGC AGG AAG ACA CTG GCA AAC3′
Polypeptide GalNAc transferase T1 U35890 5′GTG TCC AAA GGC CAA GTG ATC3′ 5′CAC ACC ACT GTC CTC CTG TCA T3′
3-hydroxyisobutyrate dehydrogenase J04628 5′TCG GAA CTG CTG AAG CTA TGA A3′ 5′TAA GTG TCG CTG GAC CAG CAT3′
Caspase 3, apoptosis related cysteine protease (ICE-like cysteine protease) U49930 5′CCC GGT TAC TAT TCC TGG AGA A3′ 5′GGA TGT CAT GAA TTC CAG CTT3′
Isocitrate dehydrogenase 1, soluble NM_031510 5′CGG TAC CAT CCG GAA CAT TCT3′ 5′CGT GTC GGC CAA TGA TGA T3′
ATP-binding cassette, sub-family G (WHITE), member 1 AJ303374 5′TTC CAA GTG GTG TCC CTG ATG3′ 5′CCG GTA GAC ACA TTG TCC TTG A3′
Benzodiazepin receptor (peripheral) NM_012515 5′TTT GGT GGA CCT CAT GCT TGT3′ 5′TTG AGC ATG GTG GCA AAG G3′
ATP-binding cassette, sub-family A (ABC1), member 2 NM_024396 5′GCC AGA ACT CAA GCT TCA CC3′ 5′CCA CGA TGT GCT GTA TGG TC3′
Programmed cell death 4 NM_022265 5′GAT GAG CAC AAA TGA CGT GGA A3′ 5′AAT AAA CTG GCC CAC CAA CTG T3′
Caspase-8 NM_022277 5′ACG AAC GAT CAA GCA CAG AGA3′ 5′TCC CAC ATG TCC TGC ATT TT3′
TGF beta 2 protein NM_031131 5′ATC CCG CCC ACT TTC TAC AG3′ 5′CTC CGC TCT GGT TTT CAC AA3′
Aldehyde dehydrogenase family 1, subfamily A1 AF001898 5′TGC CGA CTT GGA CAT TGC T3′ 5′GCT CGC TCA ACA CTC TTT CTC A3′
Rattus norvegicus macrophage inflammatory protein-2 precursor, mRNA, complete cds U45965 5′AGT TTG TCT CAA CCC TGA AGC C3′ 5′GGT CAG TTA GCC TTG CCT TTG T3′
TGFB inducible early growth response U78875 5′GCA CCA GAC TGT CTC CCA TT3′ 5′ACT TCC ATT TGC CAG TTT GG3′
Rattus norvegicus GTP-binding protein gamma subunit (Ggamma8) mRNA, complete cds L35921 5′AAC AGC CAT GTC CAA CAA CA3′ 5′GAG CAG GGT GCA AAA GAG TC3′
Interleukin 10 X60675 5′CCA GGT TGC TCC TTC CAT GAT3′ 5′GCT TTC GAG ACT GGA AGT GGT T3′
O6-methylguanine-DNA methyltransferase X54862 5′CTG GCT GAA ATT GAG TAA CCG T3′ 5′CCA TGA TTA CGA ATT CCC GG3′
Cytochrome P450, 2c37 K03501 5′GGA ACT GAG GAA AAC CAA TGG3′ 5′GAG CAG ATG ACA TTG CAA GGA3′
P-glycoprotein/multidrug resistance 1 M81855 5′CCA CGA TTG CCG AAA ACA TT3′ 5′CAT TGG CTT CCT TGA CAG CTT3′
DNA-damage-inducible transcript 1 NM_024127 5′TGG CTG CGG ATG AAG ATG A3′ 5′TCG CAA CAG AAA GCA CGA A3′
Vascular endothelial growth factor AF062644 5′TTC CTG CAG CAT AGC AGA TG3′ 5′AAT GCT TTC TCC GCT CTG AA3′
Cyclin D2 L09752 5′TTA CCT GGA CCG TTT CTT GG3′ 5′GGT AGC ACA CAG AGC GAT GA3′
ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 J04024 5′ACT GGT GAT GGT GTG AAC GA3′ 5′GGG GTT TGT TCA TGA TGT CC3′
Cyclin D1 X75207 5′AGG AGA CCA TTC CCC TGA CT3′ 5′TGG AAA GAA AGT GCG TTG TG3′
Macrophage inflammatory protein 1 alpha (Small inducible cytokine A3) U22414 5′GAC GGC AAA TTC CAC GAA AA3′ 5′AGG AAA ATG ACA CCC GGC T3′
Rattus norvegicus neuronal differentiation-related mRNA, complete cds AB020022 5′AGG AGC AGC TCA TCG ACA TCA3′ 5′GCA TGA CTG CAT CCC ACT CAT3′
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Figure 1.
 
Schema of the centrifugal force loading device. (A) The device is composed of (a) an incubator, (b) a power supply and a control unit, (c) a cooling motor unit, (d) a motor unit for providing centrifugal force and rotor vessels, (e) culture dish trays, and (f) a cooling duct. (B) A high-performance motor (g) installed in the incubator controls the rotation with an accuracy of 0.01 rpm. The culture dish is fixed at an angle θ so that the bottom of the culture dish is perpendicular to the direction of the sum of the centrifugal force and gravity vectors corresponding to an assumed rotation speed.
Figure 1.
 
Schema of the centrifugal force loading device. (A) The device is composed of (a) an incubator, (b) a power supply and a control unit, (c) a cooling motor unit, (d) a motor unit for providing centrifugal force and rotor vessels, (e) culture dish trays, and (f) a cooling duct. (B) A high-performance motor (g) installed in the incubator controls the rotation with an accuracy of 0.01 rpm. The culture dish is fixed at an angle θ so that the bottom of the culture dish is perpendicular to the direction of the sum of the centrifugal force and gravity vectors corresponding to an assumed rotation speed.
Figure 2.
 
Effects of centrifugal force loading on RGC survival. *P < 0.01, †P = 0.02, ANOVA and the post hoc method. n = 8; bar, SE.
Figure 2.
 
Effects of centrifugal force loading on RGC survival. *P < 0.01, †P = 0.02, ANOVA and the post hoc method. n = 8; bar, SE.
Figure 3.
 
Effect of cocultured retinal glial cells on RGC survival. *P = 0.02, †P < 0.001, ‡P = 0.04, ANOVA and the post hoc method. n = 8, bar, SE.
Figure 3.
 
Effect of cocultured retinal glial cells on RGC survival. *P = 0.02, †P < 0.001, ‡P = 0.04, ANOVA and the post hoc method. n = 8, bar, SE.
Figure 4.
 
Centrifugal force loading and retinal glial cell survival. Centrifugal force did not influence the retinal glial cell survival significantly. ANOVA and the post hoc method. n = 6, bar, SE.
Figure 4.
 
Centrifugal force loading and retinal glial cell survival. Centrifugal force did not influence the retinal glial cell survival significantly. ANOVA and the post hoc method. n = 6, bar, SE.
Figure 5.
 
Centrifugal force induced morphologic changes in RGCs. There are two types of neurites in surviving RGCs: normal straight or cone-shaped (A) and dendritic beading (B) neurites. RGCs cultured with centrifugal force loading had more dendritic beading neurites than those cultured without centrifugal force loading.
Figure 5.
 
Centrifugal force induced morphologic changes in RGCs. There are two types of neurites in surviving RGCs: normal straight or cone-shaped (A) and dendritic beading (B) neurites. RGCs cultured with centrifugal force loading had more dendritic beading neurites than those cultured without centrifugal force loading.
Figure 6.
 
Effects of centrifugal force loading on RGC neurites. P < 0.01, Mann-Whitney test. n = 5, bar, SE.
Figure 6.
 
Effects of centrifugal force loading on RGC neurites. P < 0.01, Mann-Whitney test. n = 5, bar, SE.
Table 1.
 
Gravity-Related Changes in mRNA Expression of Retinal Glial Cells
Table 1.
 
Gravity-Related Changes in mRNA Expression of Retinal Glial Cells
Gene Name GenBank Number Category Change Ratio (Microarray) Change Ratio (Real-Time PCR)
Upregulated mRNAs in Retinal Glial Cells
 Rattus norvegicus UDP glycosyltransferase 1 family, polypeptide A7 NM_130407 Metabolism 4.74 2.8
 Glutathione-S-transferase, alpha type (Ye?) X78847 Metabolism 4.49 5.46
 R. norvegicus mRNA for chemokine co-receptor CKR5 Y12009 Receptor 3.49 1.36
 Arachidonate 5-lipoxygenase activating protein X52196 Metabolism 3.09 0.93
 Complement component 3a receptor 1 U86379 Receptor 2.94 0.71
 Polypeptide GalNAc transferase T1 U35890 Metabolism 2.17 4.5
 3-hydroxyisobutyrate dehydrogenase J04628 Metabolism 2.11 0.93
 Caspase 3, apoptosis related cysteine protease (ICE-like cysteine protease) U49930 Apoptosis 2.09 2.2
 Isocitrate dehydrogenase 1, soluble NM_031510 Metabolism 2.08 6.68
 ATP-binding cassette, sub-family G (WHITE), member 1 AJ303374 Transporter 2.05 0.98
 Benzodiazepin receptor (peripheral) NM_012515 Receptor 2.03 7.67
 ATP-binding cassette, sub-family A (ABC1), member 2 NM_024396 Transporter 2.01 1.03
 Programmed cell death 4 NM_022265 Apoptosis 2 2.43
 Caspase-8 NM_022277 Apoptosis 2 3.46
Downregulated mRNAs in retinal glial cells
 TGF beta 2 protein NM_031131 Growth factor 0.27 0.16
 Aldehyde dehydrogenase family 1, subfamily A1 AF001898 Metabolism 0.29 0.62
 Rattus norvegicus macrophage inflammatory protein-2 precursor, mRNA, complete cds U45965 Signal transduction 0.29 1.74
 TGFB inducible early growth response U78875 Growth factor 0.4 0.62
 Rattus norvegicus GTP-binding protein gamma subunit (Ggamma8) mRNA, complete cds L35921 Signal transduction 0.43 0.44
 Interleukin 10 X60675 Cytokine 0.43 1.01
 O6-methylguanine-DNA methyltransferase X54862 Metabolism 0.43 0.9
 Cytochrome P450, 2c37 K03501 P450 0.43 0.3
 P-glycoprotein/multidrug resistance 1 M81855 Transporter 0.44 0.37
 DNA-damage-inducible transcript 1 NM_024127 stress 0.46 0.75
 Vascular endothelial growth factor AF062644 Growth factor 0.48 0.43
 Cyclin D2 L09752 Cell Cycle 0.49 0.51
 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 J04024 Membrane 0.5 0.62
 Cyclin D1 X75207 Cell Cycle 0.5 0.5
 Macrophage inflammatory protein 1 alpha (Small inducible cytokine A3) U22414 Cytokine 0.5 1.23
Table 2.
 
Gene-Specific Primer Sequences for Real-Time PCR
Table 2.
 
Gene-Specific Primer Sequences for Real-Time PCR
Gene Name GenBank Number Forward Reverse
Rattus norvegicus UDP glycosyltransferase 1 family, polypeptide A7 NM_130407 5′CCG ACA TTG CCT CTG AAG TTC3′ 5′CCA ACG TGA AGT CTG TGC GTA A3′
Glutathione-S-transferase, alpha type (Yc?) X78847 5′GGA CAA GAT TAT CTC GTT GGC A3′ 5′GGT CCA TCT CTT CCA CAT GGT A3′
R. norvegicus mRNA for chemokine co-receptor CKR5 Y12009 5′ATC CTC AAC ACC CTG TTT CG3′ 5′TGT TTG CAG AAG CGT TTG AC3′
Arachidonate 5-lipoxygenase activating protein X52196 5′CGT AGA TGC GTA CCC CAC TT3′ 5′CGC TTC CGA AGA AGA AGA TG3′
Complement component 3a receptor 1 U86379 5′TGG CTT GTT CCT GTG CAA AC3′ 5′AGC AGG AAG ACA CTG GCA AAC3′
Polypeptide GalNAc transferase T1 U35890 5′GTG TCC AAA GGC CAA GTG ATC3′ 5′CAC ACC ACT GTC CTC CTG TCA T3′
3-hydroxyisobutyrate dehydrogenase J04628 5′TCG GAA CTG CTG AAG CTA TGA A3′ 5′TAA GTG TCG CTG GAC CAG CAT3′
Caspase 3, apoptosis related cysteine protease (ICE-like cysteine protease) U49930 5′CCC GGT TAC TAT TCC TGG AGA A3′ 5′GGA TGT CAT GAA TTC CAG CTT3′
Isocitrate dehydrogenase 1, soluble NM_031510 5′CGG TAC CAT CCG GAA CAT TCT3′ 5′CGT GTC GGC CAA TGA TGA T3′
ATP-binding cassette, sub-family G (WHITE), member 1 AJ303374 5′TTC CAA GTG GTG TCC CTG ATG3′ 5′CCG GTA GAC ACA TTG TCC TTG A3′
Benzodiazepin receptor (peripheral) NM_012515 5′TTT GGT GGA CCT CAT GCT TGT3′ 5′TTG AGC ATG GTG GCA AAG G3′
ATP-binding cassette, sub-family A (ABC1), member 2 NM_024396 5′GCC AGA ACT CAA GCT TCA CC3′ 5′CCA CGA TGT GCT GTA TGG TC3′
Programmed cell death 4 NM_022265 5′GAT GAG CAC AAA TGA CGT GGA A3′ 5′AAT AAA CTG GCC CAC CAA CTG T3′
Caspase-8 NM_022277 5′ACG AAC GAT CAA GCA CAG AGA3′ 5′TCC CAC ATG TCC TGC ATT TT3′
TGF beta 2 protein NM_031131 5′ATC CCG CCC ACT TTC TAC AG3′ 5′CTC CGC TCT GGT TTT CAC AA3′
Aldehyde dehydrogenase family 1, subfamily A1 AF001898 5′TGC CGA CTT GGA CAT TGC T3′ 5′GCT CGC TCA ACA CTC TTT CTC A3′
Rattus norvegicus macrophage inflammatory protein-2 precursor, mRNA, complete cds U45965 5′AGT TTG TCT CAA CCC TGA AGC C3′ 5′GGT CAG TTA GCC TTG CCT TTG T3′
TGFB inducible early growth response U78875 5′GCA CCA GAC TGT CTC CCA TT3′ 5′ACT TCC ATT TGC CAG TTT GG3′
Rattus norvegicus GTP-binding protein gamma subunit (Ggamma8) mRNA, complete cds L35921 5′AAC AGC CAT GTC CAA CAA CA3′ 5′GAG CAG GGT GCA AAA GAG TC3′
Interleukin 10 X60675 5′CCA GGT TGC TCC TTC CAT GAT3′ 5′GCT TTC GAG ACT GGA AGT GGT T3′
O6-methylguanine-DNA methyltransferase X54862 5′CTG GCT GAA ATT GAG TAA CCG T3′ 5′CCA TGA TTA CGA ATT CCC GG3′
Cytochrome P450, 2c37 K03501 5′GGA ACT GAG GAA AAC CAA TGG3′ 5′GAG CAG ATG ACA TTG CAA GGA3′
P-glycoprotein/multidrug resistance 1 M81855 5′CCA CGA TTG CCG AAA ACA TT3′ 5′CAT TGG CTT CCT TGA CAG CTT3′
DNA-damage-inducible transcript 1 NM_024127 5′TGG CTG CGG ATG AAG ATG A3′ 5′TCG CAA CAG AAA GCA CGA A3′
Vascular endothelial growth factor AF062644 5′TTC CTG CAG CAT AGC AGA TG3′ 5′AAT GCT TTC TCC GCT CTG AA3′
Cyclin D2 L09752 5′TTA CCT GGA CCG TTT CTT GG3′ 5′GGT AGC ACA CAG AGC GAT GA3′
ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 J04024 5′ACT GGT GAT GGT GTG AAC GA3′ 5′GGG GTT TGT TCA TGA TGT CC3′
Cyclin D1 X75207 5′AGG AGA CCA TTC CCC TGA CT3′ 5′TGG AAA GAA AGT GCG TTG TG3′
Macrophage inflammatory protein 1 alpha (Small inducible cytokine A3) U22414 5′GAC GGC AAA TTC CAC GAA AA3′ 5′AGG AAA ATG ACA CCC GGC T3′
Rattus norvegicus neuronal differentiation-related mRNA, complete cds AB020022 5′AGG AGC AGC TCA TCG ACA TCA3′ 5′GCA TGA CTG CAT CCC ACT CAT3′
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