Aims: Our aim is to investigate the effects of prenatal alcohol exposure (PAE) on the development of retinal bipolar and horizontal cells. Methods: The alterations of the retinal bipolar and horizontal cells in P7, P14 and P30 mice were observed after PAE, with immunofluorescent labeling and DiI diolistic assay. Results: The retinal development of filial pups was affected by PAE in a dose-dependent and long-term manner. The number of bipolar cells of alcohol groups was significantly lower than that of the control, and the dendritic receptive field of horizontal cells was also significantly smaller than those of the control groups (P < 0.01). Conclusion: PAE was able to cause retarded development of pup retinal neural cells.
Nowadays, widespread alcohol abuse poses significant threats to society and the health of humans. It is not unusual for pregnant women to consume alcohol excessively, which often leads to serious consequences, since alcohol can freely pass through the placenta and enter into the fetal blood circulation to damage various tissues and organs. Prenatal alcohol exposure (PAE) can cause fetal miscarriage, stillbirth or developmental abnormalities, including facial and central nervous system (CNS) ones (Ikonomidou et al., 2000; Mukherjee et al., 2005; Sancho-Tello et al., 2008). The fetal and neonatal developmental abnormalities caused by PAE are called fetal alcohol syndrome (FAS). FAS mainly includes three basic symptoms: (a) retarded development of neonates; (b) facial abnormalities and (c) developmental abnormalities of CNS. The toxicological mechanisms of alcohol on the CNS have not been fully elucidated. Some more widely accepted explanations include cellular oxidative damage, N-methyl-D-aspartate (NMDA) receptor blockage, cytokine inhibition, etc (Kotch et al., 1995; Sancho-Tello et al., 2008).
Animal models were often used to understand alcohol's toxicological mechanisms (Bonthius and West, 1988; Cudd, 2005). Almost all previous studies on PAE have mainly concentrated on the toxicological effects of alcohol on the CNS (Cummings and Kavlock, 2004). In animal models, PAE could decrease the number of dendritic synapses of cerebral visual cortical neurons and could also induce apoptosis and autophagy of neurons (Jiang et al., 2007). Across a variety of species, including humans, it has been shown that embryos exposed to ethanol display ocular abnormalities as well as deficiencies in ocular physiology and behavior. However, ethanol's toxicological effects on bipolar and horizontal cells are still not well understood. In this study, we utilized PAE animal model to study the developmental alterations of bipolar cells and horizontal cells with conventional immunofluorescent labeling and DiI diolistic assay. Therefore, our study provides new leads for understanding the pathological mechanisms of retinal diseases caused by alcohol toxicity.
MATERIALS AND METHODS
Animals and groupings
C57BL/6 mice were used in this study (Ethic authorization number: HUM006). All experiments were carried out in accordance with the guidelines and approval of the Animal Welfare and Use Committees of Henan University to ensure animal welfare during experiments. Adult male and female mice were housed in standard breeding cages with a 12 h: 12 h light: dark cycle. Females were checked each morning for the presence of a vaginal plug; a positive plug was defined as E0. Embryonic (E = day of conception, E0 = vaginal plug found in mated females) or postnatal (P = days postnatal, P0 = the first 24 h after birth) offspring were produced from timed pregnancies. Prenatal EtOH exposure was performed as described by Deng and Elberger (2003) and Jiang et al. (2007). Pregnant females were housed singly and were assigned to one of two groups. (a) EtOH treatment groups: females received a daily intragastric gavage of 25% (w/v) EtOH at a dose of either 2.0 or 4.0 g/kg, beginning on E5 and continuing to parturition. To allow the stomach to empty and to facilitate the absorption of EtOH, food was removed 2 h before EtOH dosing. Animals were weighed and gavaged at the same time each day. (b) Control groups: chowfed females were allowed free access to food and water except in the morning when food was removed for ∼2 h prior to weighing. The pairfed females were intubated with same quantity of isocaloric and isovolumetric maltose–dextrin as the EtOH treatment groups. Because there were no significant differences between the pairfed and chowfed groups, the data from the two control groups were combined in order to increase the statistical power of the analysis (Deng and Elberger, 2003). Control animals typically gave birth after E19, whereas EtOH treatment delayed birth by 1–2 days to E20 or E21. In this study, three ages (P7, P14 and P30) were taken to observe and collect data from pups, with no less than seven pups at each age. No data were collected at P0, because the morphological structures of bipolar cells and horizontal cells in retinas are not formed properly at that point.
On E10 and E15, the tail tip of the pregnant EtOH treatment mothers was nicked with a scissors at 60 and 120 min after gavage, and 10 μl of blood was collected into heparinized hematocrit tubes. To separate blood cells from serum, the blood was centrifuged. Approximately 5 μl of serum was obtained and tested using an ANALOX model GL5 Analyser for blood alcohol concentration (BAC) analysis following a standardization procedure. Thus, every dam had four BAC values, two each for treatment on E10 and E15. The high test of the four values was defined as each dam's or her offspring's BAC value (Deng and Elberger, 2003). The BAC of the 4 g/kg dose group was in the range of 300–500 mg/dl (429.6 ± 63.1 mg/dl), and the BAC of the 2 g/kg group was in the range of 150–300 mg/dl (238.7 ± 55.4 mg/dl). Accordingly, these groups were designated as high and moderate alcohol treatment groups, respectively.
Immunofluorescent labeling of bipolar cells
In order to visualize the retinal bipolar cells specifically, immunofluorescent assay was performed with anti-protein kinase C (PKC)-α antibody. In short, mice were anesthetized with phenobarbital (30 mg/kg) by intraperitoneal injection and were then transcardially perfusion-fixed with 4% paraformaldehyde (pH 7.4), after which the eyeballs were removed from the eye sockets. The eyeballs were postfixed in the same fixative for another 1–2 days at 4°C. Then, the retinas were removed under dissection microscope. After washing with 0.1 M phosphate buffer (PB) for three times, retina samples would be immersed in 10, 20 and 30% sucrose solutions successively overnight, and then the retinas were sectioned with cryostat for 20 μm thickness. The sections were labeled using immunohistochemistry. After washing for three times in 0.1 M PB, the sections were incubated with rabbit anti-PKC-α monoclonal antibody (1:5000, Sigma, #4334) overnight at 4°C. Then, the secondary antibody, Alexa 488 donkey anti-rabbit immunoglobulin G (1:300, Invitrogen, A21206) was added for 3 h incubation at room temperature. After washing with 0.1 M PB for three times, the sections were mounted with 65% glycerin and observed under a fluorescent microscope (BX61, Olympus, Japan).
DiI diolistic assay in spreading retinal slices
Transcardially perfusion, fixation and retina separation were described as above. Then, the ablated retinas were immersed into 4% polyformaldehyde solution at 4°C for 3–4 h for additional fixation. After washing in 0.1 M PB three times at 5 min each, the 2–3 pieces of retinas were laid upward on a Millipore filter paper evenly for DiI delivery. DiI diolistic assay was carried out according to the modification of the methods of Gan et al. (2000). At first, gene gun bullets were prepared with DiI (Sigma) and gold particles (1.6 μm diameter). For particle delivery, retinal slices on Millipore filter papers were transferred to a Petri dish, PB was drained and DiI-coated particles were delivered using the Helios Gene Gun system (BioRad) at a pressure of 150 psi. After delivery, slices were incubated in phosphate-buffered saline (0.15 M NaCl, 0.015 M sodium phosphate pH 7.2) at 4°C overnight to allow diffusion of the dye along the neuronal processes. After washing (three times), the retinal slices were coverslipped under 65% glycerol in 0.1 M PB (Deng et al., 2006). Under a microscope, various neurons and neuroglia were labeled with DiI diolistic assay. The horizontal cells were focused on and imaged under laser confocal microscopes (FV10, Olympus, Japan).
Data measurements and statistical analysis
Image J software were used to measure the retinal bipolar and horizontal cells of various pups at the different ages (P7, P14 and P30). (a) The parameter and measurement of bipolar cells: because a layer of PKC-α positive bipolar cells were compactly arranged in the retinal inner nuclear layer, we used the density of PKC-α positive cells per unit length of inner nuclear layer to quantify the development of bipolar cells. Density of bipolar cells (DBCs) = the number of PKC-α positive cells/the length of inner nuclear layer (cells/mm); (b) the parameters and measurement of horizontal cells: we used the dendritic receptive field of horizontal cells (DRFHCs) to quantify the development and the photosensitivity of horizontal cells. The DRFHC = the area covered by the dendritic processes of horizontal cells/cells (μm2/cell). DRFHC represents the ability of horizontal cells to receive signals from photosensory cells.
The values of DBC and DRFHC were obtained in the different treatments (high EtOH, moderate EtOH and controls) and at different ages (P7, P14 and P30). The data were recorded in a Microsoft Excel spreadsheet, and inter-group comparisons were performed using the one-way analysis of variance q-test. SPSS14.0 software was used to statistically analyze the data obtained. P < 0.05 was accepted for the statistical significance tests.
As the visual receptor and transmitter, the retina converts light stimulus into visual signals through retinal photosensory cells, the cone cells and rod cells. The ganglion cells are the last stage neurons to export these visual signals outside the retina. There are various interneurons to relay these visual signals between the photosensory cells and ganglion cells. The most important interneurons among them are bipolar cells and horizontal cells, which are both located in the inner nuclear layer of the retina. Bipolar cells directly transmit visual signals from photosensory cells to ganglion cells. On the other hand, horizontal cells make synaptic contacts with both photosensory cells and bipolar cells. They could regulate the transmission of visual signals from bipolar cells to ganglion cells, which is a common form of feedback synapses in the retina. Both bipolar and horizontal cells are crucial for the transmission and moderation of visual signals in the retina, the understanding of which is the focus of our study (Anderson et al., 2011).
PAE and the development of retinal bipolar cells
Retinal bipolar cells transmit neural impulses coding visual information received by photosensory cells to ganglion cells. PKC-α positive bipolar cell are located in the inner nuclear layer, with cell bodies near the edge of outer plexiform layer. Their dendrites and axons extend toward the inner and outer plexiform layers to form synaptic contacts with photosensory cells and ganglion cells, respectively (Nawy and von Gersdorff, 2011). During normal development, PKC-α positive bipolar cells could only be found in the inner nuclear layer at P5, with a few cell bodies and axons to be labeled at P7. The cell bodies of bipolar cells gradually become larger as development proceeds, with more complete axons in large numbers. Furthermore, the number of bipolar cells increased along with age as well (Fig. 1).
Although with age increase the bipolar cells in PAE groups increased in numbers similar to the control groups, there were significant differences between the control and treatment groups of the same age after close comparison: PAE dampened the increase of bipolar cells in a dose-dependent manner (Fig. 1A–F). Figure 2 shows the cell density of retinal bipolar cells of different treatment groups at P7, P14 and P30, respectively. Statistical q-test was performed among each of groups, and the P-value denotes statistical significance between alcohol treatment groups and control with dose-dependency at all time points (P7, P14 and P30), suggesting that alcohol exposure could cause long-term retarded development of retinal bipolar cells.
PAE and the development of retinal horizontal cells
Horizontal cells are also located in the retinal inner nuclear layer, making synaptic contacts with both photosensory cells and bipolar cells. Horizontal cells moderate the transmission of visual signals from photosensory cells to bipolar cells and from bipolar cells to ganglion cells, forming a feedback route of synaptic contacts among the three (Cheng et al., 2009; Razjouyan et al., 2009). Therefore, investigations into the alterations of horizontal cells have specific significance to understand the retinal pathology after PAE. With DiI diolistic assay, retinal horizontal cells could be labeled in an ablated retinal slice. During normal development, horizontal cells appear in retina at about P3–4 as oligodendrocytes with few and short dendrites. With age development, horizontal cells send out more and more dendrites radially from the cell body, and the area covered by the extending dendrites is called the dendritic receptive field, which can be used to assess the ability of horizontal cells to receive signals from photosensory cells (Greenberg et al., 2011). The dendritic receptive field becomes larger with further retinal development (Fig. 3A, D and G).
The morphological changes of horizontal cells after PAE were investigated in the study, and significant differences between the alcohol and control groups were found. PAE could significantly retard the development of the DRFHCs in a dose-dependent and long-term manner (Fig. 3A–I). The measurements of dendritic receptive field were carried out in different treatment groups (Fig. 4). Statistical q-tests were applied, and P < 0.05 was accepted for statistical significance. At P7, P14 and P30, the dendritic receptive fields in alcohol groups were smaller when compared with the control groups in a dose-dependent and long-term manner (P < 0.05). This suggests that PAE could greatly retard the dendritic development of horizontal cells and reduce the photosensitivity of horizontal cells for long-term effect with dose-dependency.
Many studies showed that PAE can impair CNS development, with reduced neonatal brain weight accompanied by neuronal apoptosis and dendritic lesions. In animal models, PAE could cause high mortality and CNS developmental retardation. Previous studies have investigated the pathological alterations of dendritic spines and synapses after prenatal or postnatal exposure rats to EtOH (Gonzalez-Burgos et al., 2006). Even binge-like alcohol exposure of the rat neonate was reported to reduce spine density significantly in the apical dendrites of prefrontal cortex pyramidal cells (Whitcher and Klintsova, 2008). Prenatal ethanol exposure could affect visual function, particularly when exposure occurs during eye development. Bilotta et al. (2002) examined the effects of embryonic exposure to ethanol on visual function in zebrafish. They had assessed the visual function physiologically, via electroretinogram (ERG) recordings. Their results showed that ethanol effects on visual function were most pronounced when exposure occurred during eye development. ERG recordings from ethanol-exposed larvae differed from normal subjects both in shape of the response waveform and in visual thresholds under both light and dark adaptation. Also, ethanol-exposed larvae displayed lower visual acuity as determined from the optomotor response. In rodents, PAE could cause the abnormal development of eyes or even loss of the bulbus oculi in newborn pups (Tufan et al., 2007; Torp-Pedersen et al., 2010) as well, especially in FAS cases in human beings (Goh et al., 2011). Dursun et al. reported that binge-like ethanol exposure in mice during the early postnatal period from postnatal day 3 to 20 altered retinal ganglion cell morphology and resulted in a significant decrease in the numbers of neurons in the ganglion cell layer, as well as an increase in dendritic tortuosity and a decrease in total dendritic field (Dursun et al., 2011). However, the injuries of bipolar cell and horizontal cell after PAE were not the focus of the previous studies. Since there is a feedback circuit among retinal horizontal cells, bipolar cells and photosensory cells, bipolar cells and horizontal cells play crucial roles in visual transduction pathway, and we would like to choose them as targets to study the alcohol's effects to retinal development.
In this study, we found that PAE could indeed affect the development of bipolar cells and horizontal cells. PAE dampened the increase and development of bipolar cells in a dose-dependent manner at P7. The same pattern of differences also applies to P14 and P30 in a dose-dependent manner as well. In this study, the retinal horizontal cells were also labeled with DiI diolistic assay as well. After PAE, retarded development of the DRFHCs was found in a dose-dependent and long-term manner continuing to as far as P30. The dampened development of either retinal bipolar cells or DRFHCs by PAE is the manifestation of developmental retardation of these cells, and the decreased photosensitive field would produce great negative effects on the function of the visual transduction and a subsequent decrease in vision. In this study, we chose P30 as the furthest point to observe PAE's long-term effects on retinal development because P30 marked the beginning of sexual maturity for mice. Our data suggest that PAE caused the retarded development of retina all the way until adulthood in an irreversible manner. However, the mechanisms of how PAE causes retinal impairment are not yet clear at the moment. Several hypotheses are accepted to explain the mechanisms of alcohol exposure, such as cellular oxidative damage, NMDA receptor blockage, cytokine inhibition, etc (Kotch et al., 1995; Heaton et al., 1999). Cellular oxidative damage is the more favored explanation for the toxicological mechanism of PAE, and the oxidative damage to both bipolar cell and horizontal cell could cause cellular apoptosis and dendritic retreat (Wang and Bieberich, 2010). Through the oxidative pathway, some protein expressions, such as cellular oncogene fos, cyclooxygenase 2, nuclear factor κB, C-X-C chemokine receptor type 4, are up-regulated (Miyamoto et al., 2006; Zou and Crews, 2010) and are subsequently involved in the oxidative reactions in organisms (Lima Trajano et al., 2011). Furthermore, the oxidative damage of alcohol probably also interfered in the protein synthesis of cells, such as the synthesis of neurotropic factors (Hannigan, 1996; McIntosh and Chick, 2004). Then the lack of these neurotropic factors could cause the developmental retardation of retinal bipolar cells and horizontal cells and affect the functional transmission of visual information downstream. Our results imply that the injuries of bipolar cell and horizontal cell could eventually affect the future eyesight of alcohol-exposed offsprings—thus strict prohibition of alcohol intake is recommended during pregnancy. In addition, in the present study, the alcohol given was relatively higher than what would occur in normal binge-like alcohol exposure. Therefore, the study of lower dose alcohol exposure, such as 80–150 mg/dl, has practical and clinic significance which requires further studies in the future.
In summary, our results indicate that PAE could dampen the density increase of retinal bipolar cells, as well as causing the retarded development of the dendritic receptive field of retinal horizontal cells. The long-term effects of this retarded retinal development are probably the cause of visual nervous system abnormalities, amblyopia and strabismus in the FAS fetus.
This study was supported by the National Natural Science Foundation of China (grant no. 30771140, 31070952) and the grant for Natural Sciences of Henan University (grant no. 2010ZRZD02).