J Comp Physiol A DOI 10.1007/s00359-014-0889-z
Original Paper
The comparison of sensitivity of motion sickness between retinal degeneration fast mice and normal mice Xiao‑Cheng Wang · Zhao‑Hui Shi · Ka Bian · Lei Zhang · Jun‑Hui Xue · Guo‑Qing Yang · Xue‑Song Ge · Zuo‑Ming Zhang
Received: 11 October 2013 / Accepted: 3 February 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Recent studies report that a conflict between information from the visual system and vestibular system is one of the main reasons for induction of motion sickness (MS). We may be able to clarify the integration mechanism of visual and vestibular information using an animal model with a visual defect, the retinal degeneration fast (rdf) mouse, and the role of vestibular information in the pathogenesis of MS. The rdf mice and wild-type Kunming mice were subjected to rotary stimulation to induce MS. Conditioned taste anorexia to saccharin solution and behavior score were used to observe the differences in MS sensitivity between two types of mice. The decrease in intake of saccharin solution and the behavior score in rdf mice were greater than those in normal mice. After rotatory stimulation, the reduction of intake mass and the behavior score were greater in rdf mice compared to those of normal mice. The rdf mice were more sensitive to rotation than normal mice. We conclude that visual information plays a role in the pathogenesis of MS. Visual information and vestibular information impact each other and integrate through certain channels in the central nervous system in mice.
X.-C. Wang · L. Zhang · J.-H. Xue · G.-Q. Yang · X.-S. Ge · Z.-M. Zhang (*) Department of Clinical Aerospace Medicine, Key Laboratory of Aerospace Medicine of Ministry of Education, Fourth Military Medical University, Xi’an 710032, China e-mail: [email protected] Z.-H. Shi Department of Otolaryngology Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China K. Bian Department of Otolaryngology Head and Neck Surgery, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, China
Keywords Retinal degeneration fast · Visual system · Vestibular system · Motion sickness · Sensitivity Abbreviations CTA Conditioned taste anorexia ERG Electroretinogram MS Motion sickness P Postnatal day rdf Retinal degeneration fast
Introduction Normally, sensory information accepted by different sensory systems is compatible and complementary. Sensory conflict would induce varying degrees of physiological or psychological reactions, even incapacitation (Goh and Wiegmann 2002; Holmes et al. 2003; Xu Xianrong et al. 2004; Huber 2006). The visual system and vestibular system are two very important sensory systems in maintaining normal physical and mental activities. Numerous studies have shown that conflicts between the visual and vestibular information in the central nervous system are primarily responsible for inducing motion sickness (MS) (Braithwaite et al. 1998; Jiang 2000; Goh and Wiegmann 2002; Holmes et al. 2003). MS is a common and challenging problem that impedes aviation and aerospace activities. With the innovation of transport and industry in the last 100 years, an increasing number of people suffer from MS because of a variety of motion environments (e.g., cars, boats, planes, tilting trains, funfair rides, space, and virtual reality) (Muth 2006). Due to the unclear pathogenesis of MS, effective prevention and treatment are difficult to achieve (Shupak and Gordon 2006; Buyuklu et al. 2009; Kennedy et al. 2010). Therefore, exploring integration and
13
impact mechanisms of the visual information and vestibular information in aerospace medicine is important to illuminate the pathogenesis of MS. Previous studies have shown that the pathogenic mechanisms of MS mainly occur in the vestibular system, as well as in the integration mechanisms of the visual and vestibular information in the central nervous system (Jia and Xie 2005; Golding 2006; Zajonc and Roland 2006). These studies have focused on the effects of various vestibular organs and their afferent pathways on MS susceptibility. The results suggest that vestibular information influences MS. Nevertheless, sufficient animal models and methods are not available to determine the effects of visual information on the onset of MS, as well as vestibular functions. A type of mouse with dystopia, known as retinal degeneration fast (rdf), has been identified and raised in our laboratory. We have observed the retinal morphology of the rdf mice from the day 10 after impregnation, that is E10, to postnatal day (P) 56 with the paraffin embed and hematoxylin-eosinstaining (HE) stain. Through the HE stain, it can be seen that the development of the retinas from E10 to P10 has no instinct difference between rdf and wild mice. But at P14, the number of layers in the outer nuclear layer of the rdf mice is half of the wild mice. Consequently, since P21 the outer nuclear layer, as well as the outer plexiform, photoreceptor layer cannot be distinguished from microscope. We have also measured the electroretinogram (ERG) of the rdf mice from the seventh to eighth week postnatal. The consequence was that the ERG of rdf mice had no waves since P14, that is the extinguished ERG. On the contrary, the immature waves could be obtained from the wild mice at P14, when the photo ERGs had appeared and the scoptic ERGs were still immature. The ERG waves of the wild mice matured at P21. Therefore, we concluded that the photopic and scotopic visual functions of adult rdf mice are completely lost. We further screened and identified the causative gene of rdf mice. We chose some candidate genes and compared their expression level in the retina of rdf mice with wild-type Kunming mice. We also cloned and sequenced the selected candidate gene and analyzed the structure of the suspected causative gene, and then checked the inheritance of this suspected causative gene in several generations of rdf mouse. reverse transcription-PCR (RT-PCR) results revealed that, compared with the wild-type Kunming mice, expression of the pde6b gene in the retina was barely detectable in the adult rdf mice and was significantly decreased in P7 days rdf mice. Genomic analysis of the pde6b gene revealed that there was a long terminal repeat sequence inserted in the first intron of the pde6b gene. RT-PCR using specific primers for retrovirus revealed that retrovirus env gene was expressed in the rdf mice retina, and sequence analysis of the amplified fragment showed 100 % homology to xmv retrovirus env sequence. The insertion point was confirmed by sequence
13
J Comp Physiol A
analysis of sequences amplified using primers specific for both ends of the retrovirus to 1,505 bp in the first intron of the pde6b gene in the rdf mice. So our research identified the causative gene of rdf mouse is pde6b gene and a retrovirus insertion in the 1,505 bp of the first intron of the pde6b gene causes a defect in the gene. We also confirmed that homologous presentation of the mutant allele in the genome induces retinal degeneration phenotype. All above were similar with rd1 mice that have been identified and bred in the Jackson Lab (Long 2005). Therefore, these mice represent a good model for studying the integration mechanisms of visual and vestibular information in the central nervous system. They are also good models for studying the role of the visual function in the pathogenesis of MS and the effect of visual function on sensitivity to MS. In this study, we aim to estimate the differences in the sensitivity of MS between the rdf mice and normal wild-type Kunming mice by observing the severity of MS and the behavior score after a rotation stimulation lasting 30 min. The effect of visual function on the sensitivity of MS will be clarified, and a basic theory for the prevention and treatment of MS will be provided.
Materials and methods Animals and group A total of 28 healthy white male F19 rdf mice weighing 38.3 ± 2.1 g were supplied by the School of Aerospace Medicine of the Fourth Military Medical University of Chinese PLA. A total of 28 wild-type male Kunming mice weighing 40.7 ± 2.9 g were obtained from the Experimental Animal Center, Fourth Military Medical University. All of the rdf mice and their parents underwent ERG analysis to confirm positive phenotypes. All mice were housed at 23 ± 2 °C with free access to food and water in 12 h light/ dark cycles. All efforts were made to minimize suffering. The 28 rdf mice and 28 wild-type Kunming mice were randomly assigned to the experimental and control subgroups, respectively (n = 14). Mice in the two experimental groups were exposed to rotary motion for 30 min at a speed of 180°/s and 3 min per cycle in an alternating clockwise/counterclockwise movement. Mice in the two control groups were placed in the same experimental environment but not exposed to rotary motion. Rotatory stimulation equipment and methods A mini-type animal centrifuge unit (Yongdao Medicine Instrument Company, Japan) was used to establish an animal model of MS. The unit is composed of a generator and an arm with two suspended plexiglas cages. The radius from the center of rotation to the point of suspension of the
J Comp Physiol A
cages is 0.6 m. The angular acceleration, angular velocity, and run time are controlled by computer. The cages not only revolve around a vertical axis but also move along the direction of the arm during the rotation. The mice can move about freely in the cages (Wang et al. 2010). Mice from both experimental groups were placed in the plexiglas cages with the rotary device, which rotated in a clockwise rotation through the use of an acceleration device. When the peak was reached, the cage uniformly moved at 180°/s for 3 min and subsequently decelerated to 0°/s. The cages then spun in a counterclockwise rotation for 3 min, followed by an alternating acceleration/deceleration movement for 30 min (Fu et al. 2003; Wang et al. 2010, 2012). Mice from the control groups were placed in plexiglas cages for 30 min without any rotary motion stimulation.
Statistical analysis Data are expressed as the mean ± SD All statistical analyses were performed on SPSS 12.0.0 statistical software package. The intake mass of 0.15 % saccharin solution before and after stimulation within each group was analyzed with one-way analysis of variance (ANOVA). The reductions of intake mass after stimulation were expressed as the percentage of the intake mass before and after stimulation, and the reductions between the groups were compared with oneway ANOVA. The scores of MS symptoms were also compared with one-way ANOVA between the groups. P values
Original Paper
The comparison of sensitivity of motion sickness between retinal degeneration fast mice and normal mice Xiao‑Cheng Wang · Zhao‑Hui Shi · Ka Bian · Lei Zhang · Jun‑Hui Xue · Guo‑Qing Yang · Xue‑Song Ge · Zuo‑Ming Zhang
Received: 11 October 2013 / Accepted: 3 February 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Recent studies report that a conflict between information from the visual system and vestibular system is one of the main reasons for induction of motion sickness (MS). We may be able to clarify the integration mechanism of visual and vestibular information using an animal model with a visual defect, the retinal degeneration fast (rdf) mouse, and the role of vestibular information in the pathogenesis of MS. The rdf mice and wild-type Kunming mice were subjected to rotary stimulation to induce MS. Conditioned taste anorexia to saccharin solution and behavior score were used to observe the differences in MS sensitivity between two types of mice. The decrease in intake of saccharin solution and the behavior score in rdf mice were greater than those in normal mice. After rotatory stimulation, the reduction of intake mass and the behavior score were greater in rdf mice compared to those of normal mice. The rdf mice were more sensitive to rotation than normal mice. We conclude that visual information plays a role in the pathogenesis of MS. Visual information and vestibular information impact each other and integrate through certain channels in the central nervous system in mice.
X.-C. Wang · L. Zhang · J.-H. Xue · G.-Q. Yang · X.-S. Ge · Z.-M. Zhang (*) Department of Clinical Aerospace Medicine, Key Laboratory of Aerospace Medicine of Ministry of Education, Fourth Military Medical University, Xi’an 710032, China e-mail: [email protected] Z.-H. Shi Department of Otolaryngology Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China K. Bian Department of Otolaryngology Head and Neck Surgery, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, China
Keywords Retinal degeneration fast · Visual system · Vestibular system · Motion sickness · Sensitivity Abbreviations CTA Conditioned taste anorexia ERG Electroretinogram MS Motion sickness P Postnatal day rdf Retinal degeneration fast
Introduction Normally, sensory information accepted by different sensory systems is compatible and complementary. Sensory conflict would induce varying degrees of physiological or psychological reactions, even incapacitation (Goh and Wiegmann 2002; Holmes et al. 2003; Xu Xianrong et al. 2004; Huber 2006). The visual system and vestibular system are two very important sensory systems in maintaining normal physical and mental activities. Numerous studies have shown that conflicts between the visual and vestibular information in the central nervous system are primarily responsible for inducing motion sickness (MS) (Braithwaite et al. 1998; Jiang 2000; Goh and Wiegmann 2002; Holmes et al. 2003). MS is a common and challenging problem that impedes aviation and aerospace activities. With the innovation of transport and industry in the last 100 years, an increasing number of people suffer from MS because of a variety of motion environments (e.g., cars, boats, planes, tilting trains, funfair rides, space, and virtual reality) (Muth 2006). Due to the unclear pathogenesis of MS, effective prevention and treatment are difficult to achieve (Shupak and Gordon 2006; Buyuklu et al. 2009; Kennedy et al. 2010). Therefore, exploring integration and
13
impact mechanisms of the visual information and vestibular information in aerospace medicine is important to illuminate the pathogenesis of MS. Previous studies have shown that the pathogenic mechanisms of MS mainly occur in the vestibular system, as well as in the integration mechanisms of the visual and vestibular information in the central nervous system (Jia and Xie 2005; Golding 2006; Zajonc and Roland 2006). These studies have focused on the effects of various vestibular organs and their afferent pathways on MS susceptibility. The results suggest that vestibular information influences MS. Nevertheless, sufficient animal models and methods are not available to determine the effects of visual information on the onset of MS, as well as vestibular functions. A type of mouse with dystopia, known as retinal degeneration fast (rdf), has been identified and raised in our laboratory. We have observed the retinal morphology of the rdf mice from the day 10 after impregnation, that is E10, to postnatal day (P) 56 with the paraffin embed and hematoxylin-eosinstaining (HE) stain. Through the HE stain, it can be seen that the development of the retinas from E10 to P10 has no instinct difference between rdf and wild mice. But at P14, the number of layers in the outer nuclear layer of the rdf mice is half of the wild mice. Consequently, since P21 the outer nuclear layer, as well as the outer plexiform, photoreceptor layer cannot be distinguished from microscope. We have also measured the electroretinogram (ERG) of the rdf mice from the seventh to eighth week postnatal. The consequence was that the ERG of rdf mice had no waves since P14, that is the extinguished ERG. On the contrary, the immature waves could be obtained from the wild mice at P14, when the photo ERGs had appeared and the scoptic ERGs were still immature. The ERG waves of the wild mice matured at P21. Therefore, we concluded that the photopic and scotopic visual functions of adult rdf mice are completely lost. We further screened and identified the causative gene of rdf mice. We chose some candidate genes and compared their expression level in the retina of rdf mice with wild-type Kunming mice. We also cloned and sequenced the selected candidate gene and analyzed the structure of the suspected causative gene, and then checked the inheritance of this suspected causative gene in several generations of rdf mouse. reverse transcription-PCR (RT-PCR) results revealed that, compared with the wild-type Kunming mice, expression of the pde6b gene in the retina was barely detectable in the adult rdf mice and was significantly decreased in P7 days rdf mice. Genomic analysis of the pde6b gene revealed that there was a long terminal repeat sequence inserted in the first intron of the pde6b gene. RT-PCR using specific primers for retrovirus revealed that retrovirus env gene was expressed in the rdf mice retina, and sequence analysis of the amplified fragment showed 100 % homology to xmv retrovirus env sequence. The insertion point was confirmed by sequence
13
J Comp Physiol A
analysis of sequences amplified using primers specific for both ends of the retrovirus to 1,505 bp in the first intron of the pde6b gene in the rdf mice. So our research identified the causative gene of rdf mouse is pde6b gene and a retrovirus insertion in the 1,505 bp of the first intron of the pde6b gene causes a defect in the gene. We also confirmed that homologous presentation of the mutant allele in the genome induces retinal degeneration phenotype. All above were similar with rd1 mice that have been identified and bred in the Jackson Lab (Long 2005). Therefore, these mice represent a good model for studying the integration mechanisms of visual and vestibular information in the central nervous system. They are also good models for studying the role of the visual function in the pathogenesis of MS and the effect of visual function on sensitivity to MS. In this study, we aim to estimate the differences in the sensitivity of MS between the rdf mice and normal wild-type Kunming mice by observing the severity of MS and the behavior score after a rotation stimulation lasting 30 min. The effect of visual function on the sensitivity of MS will be clarified, and a basic theory for the prevention and treatment of MS will be provided.
Materials and methods Animals and group A total of 28 healthy white male F19 rdf mice weighing 38.3 ± 2.1 g were supplied by the School of Aerospace Medicine of the Fourth Military Medical University of Chinese PLA. A total of 28 wild-type male Kunming mice weighing 40.7 ± 2.9 g were obtained from the Experimental Animal Center, Fourth Military Medical University. All of the rdf mice and their parents underwent ERG analysis to confirm positive phenotypes. All mice were housed at 23 ± 2 °C with free access to food and water in 12 h light/ dark cycles. All efforts were made to minimize suffering. The 28 rdf mice and 28 wild-type Kunming mice were randomly assigned to the experimental and control subgroups, respectively (n = 14). Mice in the two experimental groups were exposed to rotary motion for 30 min at a speed of 180°/s and 3 min per cycle in an alternating clockwise/counterclockwise movement. Mice in the two control groups were placed in the same experimental environment but not exposed to rotary motion. Rotatory stimulation equipment and methods A mini-type animal centrifuge unit (Yongdao Medicine Instrument Company, Japan) was used to establish an animal model of MS. The unit is composed of a generator and an arm with two suspended plexiglas cages. The radius from the center of rotation to the point of suspension of the
J Comp Physiol A
cages is 0.6 m. The angular acceleration, angular velocity, and run time are controlled by computer. The cages not only revolve around a vertical axis but also move along the direction of the arm during the rotation. The mice can move about freely in the cages (Wang et al. 2010). Mice from both experimental groups were placed in the plexiglas cages with the rotary device, which rotated in a clockwise rotation through the use of an acceleration device. When the peak was reached, the cage uniformly moved at 180°/s for 3 min and subsequently decelerated to 0°/s. The cages then spun in a counterclockwise rotation for 3 min, followed by an alternating acceleration/deceleration movement for 30 min (Fu et al. 2003; Wang et al. 2010, 2012). Mice from the control groups were placed in plexiglas cages for 30 min without any rotary motion stimulation.
Statistical analysis Data are expressed as the mean ± SD All statistical analyses were performed on SPSS 12.0.0 statistical software package. The intake mass of 0.15 % saccharin solution before and after stimulation within each group was analyzed with one-way analysis of variance (ANOVA). The reductions of intake mass after stimulation were expressed as the percentage of the intake mass before and after stimulation, and the reductions between the groups were compared with oneway ANOVA. The scores of MS symptoms were also compared with one-way ANOVA between the groups. P values
