植入电极记录豚鼠图形视觉诱发电位
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摘要
视觉电生理检查作为一种客观、无损伤的视觉功能评定方法,尤其适用于婴幼儿、老年人、智力低下、不合作者或者伪盲者。视觉诱发电位是其中最常用的方法之一。视觉诱发电位(visual evoked potential, VEP )是大脑皮层对视觉刺激诱发的、采用脑电图技术在头皮记录的一簇电生理信号。临床上常用的有图形视觉诱发电位(pattern VEP, P-VEP)和闪光诱发电位(Flash VEP, F-VEP)。P-VEP与视网膜对图像的识别密切相关,即与人的视力密切相关,且图形比较稳定,因此被广泛用于临床,比如视神经炎、黄斑部疾病、青光眼的诊断以及司法鉴定等。F-VEP个体间变异较大,多用于P-VEP无法记录或记录不到的情况。但是动物P-VEP信号的振幅很小,极易受到心电、肌电的干扰而难于记录。因此,探索新的记录方法,提高对P-VEP记录的稳定性,减少干扰性,对推广该技术在动物实验中的应用有着重要的意义。
豚鼠属啮齿类动物,是晚成熟动物,视网膜上视锥细胞多于一般啮齿动物(cone rich) ,与人类有着较为相似的视锥、视杆细胞比例。豚鼠的晶状体和视网膜在解剖结构、生物构成及生理功能方面都与人类及灵长类动物有相似之处,因此豚鼠可以作为一个较好的视觉研究模型动物。同时,豚鼠也是比较广泛用于各个学科基础研究的实验动物,但是有关豚鼠在视觉电生理研究的报道还比较少,因此完善豚鼠视觉功能的检查和评价,对以后的研究工作准备更加充实的资料有着重要的意义。
激光具有单色性、相干性和方向性而有别于普通光源,自一问世,即被广泛应用于各个领域,包括军事领域和航空航天领域。激光光能在时间与空间上高度集中,不同能量的激光对机体的影响是不同的,高能量激光对生物体损伤反应受多方面因素影响,诸如功率、波长、照射时间和光斑等。但如何评价激光或其他因子对动物视觉功能,特别是形觉功能的影响仍是当前视觉科学中的一个难题。本文首先探索了豚鼠P-VEP的记录方法,即与豚鼠形觉相关的功能,再探索该方法在对激光损伤评定中的应用效果。
本实验主要分为两大方面:一,建立植入电极记录豚鼠图形视觉诱发电位的方法。二,基于此方法,观察680nm激光对P-VEP的影响。
材料与方法
由第四军医大学动物实验中心提供的成年杂色雄性豚鼠,外眼和检眼镜检查屈光间质清晰;麻醉药物:速眠新,军事医学科学院军事兽医研究所,批号:(2004)005013。所有麻醉药物均有腹腔注射给药(intraperitoneal injection,ip)。棉拭子测试角膜反射消失,认为达到全麻醉状态。速眠新麻醉后进行颅骨植入电极操作,在豚鼠头顶正中线分别植入3根不锈钢螺钉作为记录电极、地极和参考电极。3天后,将清醒豚鼠固定在自制的多功能头身一体化固定台上,Ag-AgCl连接电极和RETI-port视觉电生理学记录系统( Roland Consult ,德国) ,ELSA彩色显示器(Sony),刺激方式为棋盘格(checkerboard)。记录不同条件下豚鼠的P-VEP。
半导体激光器(北方电子设备研究所研制),波长680nm,脉宽10μs,频率20Hz,平均功率1.4mW。激光器距离豚鼠眼睛1.3m。激光束与豚鼠右眼角度为45度作用于豚鼠眼睛30s,并进行P-VEP测定。实验中我们改变激光的能量,分别设置为0.3、0.5、0.8、1.1和1.4mW,观察对豚鼠视力的影响,豚鼠视力我们以当空间频率减小至某一方格无可辨认的P波出现时,其上一级方格即为能诱发出VEP的最小方格,即豚鼠的视力,用cpd来表示。每次测定根据能量大小不同间隔时间10分钟至5分钟。
结果
1.植入电极记录豚鼠清醒状态下P-VEP的特点
在豚鼠清醒状态下可以记录到稳定的P-VEP波形,呈NPN形状。空间频率对P-VEP波形有明显的影响,随着空间频率的增加,N1、P1的潜伏期都有不同程度的延长,当空间频率大于0.5cpd时,潜伏期延长明显;随着空间频率的增加振幅降低。在不同对比度(97%、60%、30%)下,P1波的振幅随着对比度的增加而提高,且任意两个对比度之间P波振幅的差异均有统计学意义(P<0.05)。在97%和60%的对比度下潜伏期差异无统计学意义(P>0.05)。不同颜色图形对P-VEP波形也有一定的影响,黑白和红绿引出的潜伏期和振幅比较明显,与红蓝和蓝绿比较,差异均有统计学意义(P<0.01);当通频带为5-50Hz时,引出的波形受干扰比较小,波形比较稳定。麻醉对P-VEP记录影响较大,速眠新麻醉条件下记录不到明显的P-VEP波形,但对F-VEP记录影响不大。
2.680nm激光对豚鼠P-VEP的影响
激光刺激对P-VEP影响较大,当刺激空间频率为0.1和0.3cpd时,与对照组比较,实验组潜伏期延长(P<0.05),其余空间频率差异不大。当空间频率大于0.05cpd时,P波振幅降低明显,与对照组比较差异有统计学意义(P<0.05)。激光对视力影响比较大,随着激光能量的增加,视力明显下降。
结论
1.建立了一种植入式电极记录清醒豚鼠P-VEP方法;豚鼠P-VEP波形记录与刺激和记录参数密切相关,在高对比度(97%)、窄通带(5-50Hz)、适当空间频率(小于0.5cpd)下可以记录稳定的P-VEP波形。
2.豚鼠P-VEP波形明显受到680nm激光的影响,表现为P波潜伏期明显延长和振幅的降低。激光对豚鼠的视力影响大,随着激光能量的增加,视力下降明显。但是低剂量短时间680nm激光对豚鼠视网膜是否有损伤还有待于深入研究。
Examination of visual electro-physiology is an objective and noninvade evaluation method of vision function, especially for infants, aged people, mental retardation, loners and simulated blindness. Visual evoked potential is one method of the frequently used in clinical examinations. Visual evoked potential is a bunch of electrical signals from cerebral cortex which react to visual stimulus. The type commonly used in clinic consists of P-VEP and F-VEP. P-VEP correlates closely with pictorial recognition of retina, and visual acuity. And the P-VEP form is stable, so the clinical application of P-VEP is commonly in diagnosis of visual pathway diseases, such as diagnosis of optic neuritis, macula flava diseases, glaucomatous and judicial identification. F-VEP is generally used in the condition that P-VEP can not record or record incompletely because of the great variation of F-VEP. The amplitude of P-VEP is very small and it is easily interfered by electrocardio and myoelectric. So it is very important to explore a new P-VEP recording method for increase P-VEP stability and decrease interference in order to study P-VEP deeply, especially for animal experiment.
Guinea pigs belong to rodents, and they are altricial animals. They have much more cones in retina than other rodents and they have semblable cone and rod proportion with mankind. The anatomic structure, organism construction and physiological function of crystalline lens and retina of guinea pigs are similar to mankind and primate. So guinea pigs are good models for vision research. Meanwhile guinea pigs are widespreadly used in basic research of various subjects. So they play influential role in research. But there are few reports of guinea pigs’electrophysiology research. It is very important to study the examination and evaluation of vision function of guinea pigs. And what we have done is important for future research work of guinea pig or P-VEP.
Laser is different to common light source because of monochromaticity, coherence and directionality. When it comes out, it is utilized in various scopes, including military and aerospace territory. The energy of laser is highly concentrated in time and space. Different energy of laser has different effect on body. High-energy laser is detrimental to body and its bad effects on body are related to power, wave length, exposure time and light spot. But how to evaluate animal visual function especially form sense function of animals in the condition of laser or other factors remains a tough problem in the vision science currently. We explore a recording method of P-VEP of guinea pig, which is correlated closely to vision form sense function of guinea pigs and then explore the application effect in the condition of 680nm laser .
The experiment concludes two parts: 1, To establish a new method to record pattern visual evoked potential (P-VEP) of guinea pigs. 2, To observe the effect of 680nm-laser on P-VEP.
Methods and materials
Random coloured mature male guinea pigs which had no opacity observed by ophthalmoscope were provided by animal experiment center of the Fourth Military Medical University. Anesthesia drugs: Sumianxin, which was made by military veterinary laboratory in military medical science academy in Changchun, code: (2004)005013. All guinea pigs were treated with anesthesia drugs and vehicled by intraperitoneal injection(ip). When cornea reflection vanished which was checked by cotton swab, the animals were considered getting in the anesthesia condition. After anesthesia, the guinea pigs ' scalp surface was exposed and three stainless screws were implanted as recording electrode, ground electrode and reference electrode. 3 days later, the awaked guinea pig was fixed in multifunctional head-body apparatus. Stainless screws and RETI-port system (Roland Consult, Germany) were connected with Ag-AgCl electrode to record P-VEP in different conditions. Semiconductor laser, made in north electronic equipment research institute, was used. And its wavelength is 680nm, width was 10μs, frequency was 20Hz, mean power was 1.4mW. The distance between laser and eyes of guinea pigs was 1.3 meters. The angle between laser beam and right eye was 45 degrees, and the time was 30s. We recorded PVEP simultaneously. We changed the laser energy to 0.3, 0.5, 0.8, 1.1 and 1.4mW in the experiment and observed the effect on visual acuity of the guinea pig. Visual acuity of the guinea pig was defined as the smallest checkerboard at which P wave could just be indentified. We used cpd as its visual acuity. The span time between each animal experiment was 5 to 10mins according to the different energy.
Results
1.The characteristic of P-VEP in awaked guinea pigs
We could record steady P-VEP, and the typical wave form was NPN. The spatial frequency had an evident influence on P-VEP. With the spatial frequency increased, implicit time of N1 and P1 was delayed, especially beyond 0.5cpd, and amplitude of P1 decreased, too. Oppositely, implicit time was shorten and amplitude was augmented with contrast increased. The difference of amplitude between either was significant (P<0.05). But difference of implicit time of P wave between 97% and 60% was not significant (P>0.05). The amplitudes of B-W and R-G were separately higher than R-B and B-G (P<0.01). P-VEP wave form was more stable in the bandpass of 5-50Hz than the other. P-VEP was not recorded in the condition of anaesthesia, and F-VEP was not influenced significantly in the condition of anaesthesia.
2. The effect on P-VEP of 680nm laser
The latency of P wave of experiment group was increased significantly (P<0.05) compared with control group when the spatial frequency was in 0.1and 0.3cpd, but nonsignificant in other spatial frequencies. The amplitude of P wave of experiment group was decreased significantly (P<0.05) compared with control group when the spatial frequency was beyond 0.5cpd. 680-nm laser has an obvious effect on visual acuity. The visual acuity decreased quickly with the laser energy increased.
Conclusions
1. Established a method of recording P-VEP of awaked guinea pigs with implanted electrode. P-VEP has an close correlation with recording parameters, and can be stably recorded in the condition of high contrast(97%) ,narrow low bandpass(5-50Hz) and proper spatial frequency(below 0.5 cpd) .
2. The 680nm-laser has an evident effect on P-VEP. The latency of P wave is delayed and amplitude of P wave is decreased. The effect on visual acuity is also significant. Visual acuity is decreased quickly with the energy increasing. But whether the low-energy 680nm-laser has damage to body or not is worth researching deeply.
豚鼠属啮齿类动物,是晚成熟动物,视网膜上视锥细胞多于一般啮齿动物(cone rich) ,与人类有着较为相似的视锥、视杆细胞比例。豚鼠的晶状体和视网膜在解剖结构、生物构成及生理功能方面都与人类及灵长类动物有相似之处,因此豚鼠可以作为一个较好的视觉研究模型动物。同时,豚鼠也是比较广泛用于各个学科基础研究的实验动物,但是有关豚鼠在视觉电生理研究的报道还比较少,因此完善豚鼠视觉功能的检查和评价,对以后的研究工作准备更加充实的资料有着重要的意义。
激光具有单色性、相干性和方向性而有别于普通光源,自一问世,即被广泛应用于各个领域,包括军事领域和航空航天领域。激光光能在时间与空间上高度集中,不同能量的激光对机体的影响是不同的,高能量激光对生物体损伤反应受多方面因素影响,诸如功率、波长、照射时间和光斑等。但如何评价激光或其他因子对动物视觉功能,特别是形觉功能的影响仍是当前视觉科学中的一个难题。本文首先探索了豚鼠P-VEP的记录方法,即与豚鼠形觉相关的功能,再探索该方法在对激光损伤评定中的应用效果。
本实验主要分为两大方面:一,建立植入电极记录豚鼠图形视觉诱发电位的方法。二,基于此方法,观察680nm激光对P-VEP的影响。
材料与方法
由第四军医大学动物实验中心提供的成年杂色雄性豚鼠,外眼和检眼镜检查屈光间质清晰;麻醉药物:速眠新,军事医学科学院军事兽医研究所,批号:(2004)005013。所有麻醉药物均有腹腔注射给药(intraperitoneal injection,ip)。棉拭子测试角膜反射消失,认为达到全麻醉状态。速眠新麻醉后进行颅骨植入电极操作,在豚鼠头顶正中线分别植入3根不锈钢螺钉作为记录电极、地极和参考电极。3天后,将清醒豚鼠固定在自制的多功能头身一体化固定台上,Ag-AgCl连接电极和RETI-port视觉电生理学记录系统( Roland Consult ,德国) ,ELSA彩色显示器(Sony),刺激方式为棋盘格(checkerboard)。记录不同条件下豚鼠的P-VEP。
半导体激光器(北方电子设备研究所研制),波长680nm,脉宽10μs,频率20Hz,平均功率1.4mW。激光器距离豚鼠眼睛1.3m。激光束与豚鼠右眼角度为45度作用于豚鼠眼睛30s,并进行P-VEP测定。实验中我们改变激光的能量,分别设置为0.3、0.5、0.8、1.1和1.4mW,观察对豚鼠视力的影响,豚鼠视力我们以当空间频率减小至某一方格无可辨认的P波出现时,其上一级方格即为能诱发出VEP的最小方格,即豚鼠的视力,用cpd来表示。每次测定根据能量大小不同间隔时间10分钟至5分钟。
结果
1.植入电极记录豚鼠清醒状态下P-VEP的特点
在豚鼠清醒状态下可以记录到稳定的P-VEP波形,呈NPN形状。空间频率对P-VEP波形有明显的影响,随着空间频率的增加,N1、P1的潜伏期都有不同程度的延长,当空间频率大于0.5cpd时,潜伏期延长明显;随着空间频率的增加振幅降低。在不同对比度(97%、60%、30%)下,P1波的振幅随着对比度的增加而提高,且任意两个对比度之间P波振幅的差异均有统计学意义(P<0.05)。在97%和60%的对比度下潜伏期差异无统计学意义(P>0.05)。不同颜色图形对P-VEP波形也有一定的影响,黑白和红绿引出的潜伏期和振幅比较明显,与红蓝和蓝绿比较,差异均有统计学意义(P<0.01);当通频带为5-50Hz时,引出的波形受干扰比较小,波形比较稳定。麻醉对P-VEP记录影响较大,速眠新麻醉条件下记录不到明显的P-VEP波形,但对F-VEP记录影响不大。
2.680nm激光对豚鼠P-VEP的影响
激光刺激对P-VEP影响较大,当刺激空间频率为0.1和0.3cpd时,与对照组比较,实验组潜伏期延长(P<0.05),其余空间频率差异不大。当空间频率大于0.05cpd时,P波振幅降低明显,与对照组比较差异有统计学意义(P<0.05)。激光对视力影响比较大,随着激光能量的增加,视力明显下降。
结论
1.建立了一种植入式电极记录清醒豚鼠P-VEP方法;豚鼠P-VEP波形记录与刺激和记录参数密切相关,在高对比度(97%)、窄通带(5-50Hz)、适当空间频率(小于0.5cpd)下可以记录稳定的P-VEP波形。
2.豚鼠P-VEP波形明显受到680nm激光的影响,表现为P波潜伏期明显延长和振幅的降低。激光对豚鼠的视力影响大,随着激光能量的增加,视力下降明显。但是低剂量短时间680nm激光对豚鼠视网膜是否有损伤还有待于深入研究。
Examination of visual electro-physiology is an objective and noninvade evaluation method of vision function, especially for infants, aged people, mental retardation, loners and simulated blindness. Visual evoked potential is one method of the frequently used in clinical examinations. Visual evoked potential is a bunch of electrical signals from cerebral cortex which react to visual stimulus. The type commonly used in clinic consists of P-VEP and F-VEP. P-VEP correlates closely with pictorial recognition of retina, and visual acuity. And the P-VEP form is stable, so the clinical application of P-VEP is commonly in diagnosis of visual pathway diseases, such as diagnosis of optic neuritis, macula flava diseases, glaucomatous and judicial identification. F-VEP is generally used in the condition that P-VEP can not record or record incompletely because of the great variation of F-VEP. The amplitude of P-VEP is very small and it is easily interfered by electrocardio and myoelectric. So it is very important to explore a new P-VEP recording method for increase P-VEP stability and decrease interference in order to study P-VEP deeply, especially for animal experiment.
Guinea pigs belong to rodents, and they are altricial animals. They have much more cones in retina than other rodents and they have semblable cone and rod proportion with mankind. The anatomic structure, organism construction and physiological function of crystalline lens and retina of guinea pigs are similar to mankind and primate. So guinea pigs are good models for vision research. Meanwhile guinea pigs are widespreadly used in basic research of various subjects. So they play influential role in research. But there are few reports of guinea pigs’electrophysiology research. It is very important to study the examination and evaluation of vision function of guinea pigs. And what we have done is important for future research work of guinea pig or P-VEP.
Laser is different to common light source because of monochromaticity, coherence and directionality. When it comes out, it is utilized in various scopes, including military and aerospace territory. The energy of laser is highly concentrated in time and space. Different energy of laser has different effect on body. High-energy laser is detrimental to body and its bad effects on body are related to power, wave length, exposure time and light spot. But how to evaluate animal visual function especially form sense function of animals in the condition of laser or other factors remains a tough problem in the vision science currently. We explore a recording method of P-VEP of guinea pig, which is correlated closely to vision form sense function of guinea pigs and then explore the application effect in the condition of 680nm laser .
The experiment concludes two parts: 1, To establish a new method to record pattern visual evoked potential (P-VEP) of guinea pigs. 2, To observe the effect of 680nm-laser on P-VEP.
Methods and materials
Random coloured mature male guinea pigs which had no opacity observed by ophthalmoscope were provided by animal experiment center of the Fourth Military Medical University. Anesthesia drugs: Sumianxin, which was made by military veterinary laboratory in military medical science academy in Changchun, code: (2004)005013. All guinea pigs were treated with anesthesia drugs and vehicled by intraperitoneal injection(ip). When cornea reflection vanished which was checked by cotton swab, the animals were considered getting in the anesthesia condition. After anesthesia, the guinea pigs ' scalp surface was exposed and three stainless screws were implanted as recording electrode, ground electrode and reference electrode. 3 days later, the awaked guinea pig was fixed in multifunctional head-body apparatus. Stainless screws and RETI-port system (Roland Consult, Germany) were connected with Ag-AgCl electrode to record P-VEP in different conditions. Semiconductor laser, made in north electronic equipment research institute, was used. And its wavelength is 680nm, width was 10μs, frequency was 20Hz, mean power was 1.4mW. The distance between laser and eyes of guinea pigs was 1.3 meters. The angle between laser beam and right eye was 45 degrees, and the time was 30s. We recorded PVEP simultaneously. We changed the laser energy to 0.3, 0.5, 0.8, 1.1 and 1.4mW in the experiment and observed the effect on visual acuity of the guinea pig. Visual acuity of the guinea pig was defined as the smallest checkerboard at which P wave could just be indentified. We used cpd as its visual acuity. The span time between each animal experiment was 5 to 10mins according to the different energy.
Results
1.The characteristic of P-VEP in awaked guinea pigs
We could record steady P-VEP, and the typical wave form was NPN. The spatial frequency had an evident influence on P-VEP. With the spatial frequency increased, implicit time of N1 and P1 was delayed, especially beyond 0.5cpd, and amplitude of P1 decreased, too. Oppositely, implicit time was shorten and amplitude was augmented with contrast increased. The difference of amplitude between either was significant (P<0.05). But difference of implicit time of P wave between 97% and 60% was not significant (P>0.05). The amplitudes of B-W and R-G were separately higher than R-B and B-G (P<0.01). P-VEP wave form was more stable in the bandpass of 5-50Hz than the other. P-VEP was not recorded in the condition of anaesthesia, and F-VEP was not influenced significantly in the condition of anaesthesia.
2. The effect on P-VEP of 680nm laser
The latency of P wave of experiment group was increased significantly (P<0.05) compared with control group when the spatial frequency was in 0.1and 0.3cpd, but nonsignificant in other spatial frequencies. The amplitude of P wave of experiment group was decreased significantly (P<0.05) compared with control group when the spatial frequency was beyond 0.5cpd. 680-nm laser has an obvious effect on visual acuity. The visual acuity decreased quickly with the laser energy increased.
Conclusions
1. Established a method of recording P-VEP of awaked guinea pigs with implanted electrode. P-VEP has an close correlation with recording parameters, and can be stably recorded in the condition of high contrast(97%) ,narrow low bandpass(5-50Hz) and proper spatial frequency(below 0.5 cpd) .
2. The 680nm-laser has an evident effect on P-VEP. The latency of P wave is delayed and amplitude of P wave is decreased. The effect on visual acuity is also significant. Visual acuity is decreased quickly with the energy increasing. But whether the low-energy 680nm-laser has damage to body or not is worth researching deeply.
引文
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