实验动物前庭感觉毛细胞的定量观察

Quantitative observation of vestibular sensory hair cells in experimental animals

目的测量常用实验动物内耳前庭感觉区的实际面积和量化分析前庭各个感觉区的毛细胞总数或密度。方法①制作CBA/CaJ小鼠、裸鼠、SD大鼠、豚鼠、南美栗鼠、新西兰白兔和非洲黑长尾猴的球囊斑铺片和椭圆囊斑铺片及壶腹嵴铺片,所有铺片样品来自每种受试动物的6个颞骨,在放大100倍的光学显微镜下拍摄2个囊斑铺片的整体照片;②应用Image J软件的图像测量程序,测量了上述7种常用实验动物球囊斑和椭圆囊斑的实际面积;③用网格将球囊斑铺片和椭圆囊斑铺片照片上的前庭感觉区划分为一个个方块区域。在放大400倍的光学显微镜下准确计数每个方格内的毛细胞数量,然后将每个方格的毛细胞计数结果相加以获得每种受试动物球囊斑和椭圆囊斑上的毛细胞总数;④应用前庭小视野定量观察技术计算出前庭各个感觉区小视野范围内的毛细胞密度。结果①从小鼠、裸鼠、大鼠、豚鼠、南美栗鼠、白兔到猴的球囊斑面积依次为(0.193±0.009)、(0.216±0.008)、(0.323±0.010)、(0.528±0.035)、(0.687±0.065)、(1.237±0.075)、(1.371±0.032)mm 2;椭圆囊斑的面积依次为(0.193±0.020)、(0.208±0.013)、(0.321±0.011)、(0.526±0.034)、(0.795±0.017)、(1.224±0.082)、(1.388±0.048)mm 2;②从小鼠、裸鼠、大鼠、豚鼠、南美栗鼠、白兔到猴的球囊斑毛细胞的总数依次为(2476.3±64.4)、(2389.8±47.8)、(3135.3±191.6)、(4882.2±208.7)、(6128.5±242.9)、(10572.2±464.4)、(10992.7±397.4)个;椭圆囊斑毛细胞的总数依次为(2491.4±54.8)、(2368.0±46.1)、(3218.8±82.9)、(4925.3±271.1)、(7794.0±386.1)、(11347.4±435.7)、(11114.5±410.6)个;③从小鼠、大鼠、豚鼠、南美栗鼠、白兔和猴的球囊斑微纹区和周边区的毛细胞密度(毛细胞数量/0.03 mm 2)依次为101.0±5.79(微纹区)/120.8±4.15(周边区),95.5±3.91(微纹区)/109.2±5.26(周边区),78.4±6.54(微纹区)/94.8±4.38(周边区),60.0±4.74(微纹区)/84.6±2.61(周边区),57.2±3.83(微纹区)/80.0±3.54(周边区),53.8±4.21(微纹区)/68.0±4.18(周边区)。从小鼠、大鼠、豚鼠、南美栗鼠、白兔和猴的椭圆囊斑微纹区和周边区的毛细胞密度(毛细胞数量/0.03 mm 2)依次为103.8±5.02(微纹区)/119.2±3.70(周边区),91.2±2.49(微纹区)/106.4±4.16(周边区),74.1±3.54(微纹区)/90.8±3.56(周边区),60.4±4.98(微纹区)/81.6±2.07(周边区),57.8±1.92(微纹区)/77.8±3.70(周边区),54.0±2.74(微纹区)/66.4±2.51(周边区)。从小鼠、大鼠、豚鼠、南美栗鼠、白兔和猴的壶腹嵴毛细胞密度(毛细胞数量/0.03 mm 2)依次为112.4±6.38,105.5±3.51,95.2±3.42,84.0±7.16,78.2±2.86,70.8±2.39。可见由于体型较小动物毛细胞的细胞体比体型较大动物毛细胞的细胞体小,因而体型较小动物的前庭毛细胞密度高于体型较大动物的前庭毛细胞密度。另外,每种实验动物球囊斑和椭圆囊斑微纹区的毛细胞密度相似,周边区的毛细胞密度也大致相同,但是同种实验动物囊斑微纹区的毛细胞密度却低于周边区的毛细胞密度。此外,壶腹嵴毛细胞的密度与球囊斑和椭圆囊斑周边区的毛细胞密度几乎相同。鉴于某些损害因素往往具有选择性破坏囊斑微纹区毛细胞的表现,因此囊斑微纹区的毛细胞密度应该与囊斑周边区的毛细胞密度区分开来进行统计,必要时甚至需要把Ⅰ型毛细胞和Ⅱ型毛细胞也区分开来分别予以病理学改变的定量评估。结论本研究采用的前庭测量方法和获得的前庭各个感觉区的测量数据和毛细胞总数及毛细胞密度,为前庭病理学研究的定量分析提供了有益的参考经验和必要的参考数据。

Objective To measure the actual area of the vestibular sensory area in the inner ear of commonly used experimental animals and quantitatively analyze the hair cells of each sensory area of the vestibule from the perspective of histopathological observation.Methods①The macula of saccule,macula of utricle,and crista of ampulla were prepared as flat surface preparations in experimental animals of CBA/CaJ mice,nude mice,SD rats,guinea pigs,chinchillas,New Zealand white rabbits and African vervet monkeys.All samples were obtained from 6 temporal bones of each tested animal species.The photographs of each vestibular end-organs were taken under a light microscope at 100X magnification.②Using the ImageJ software,the actual areas of the macula of saccule and the macula of utricle were measured in above seven experimental animals.③A grid was used to divide the vestibular sensory areas of macula of saccule and macula of utricle on the photos into several square areas.The number of hair cells in each grid was accurately counted under the magnified image,and the hair cell counts in each grid were then summed to obtain the total number of hair cells in the macula of saccule and macula of utricle.④The hair cell density was also calculated within the small visual field of each sensory area of the vestibule end-organs.Results①The areas of macula of saccule from CBA/CaJ mice,nude mice,rats,guinea pigs,chinchillas,white rabbits to monkeys were(0.193±0.009),(0.216±0.008),(0.323±0.01),(0.528±0.035),(0.687±0.065),(1.237±0.075),(1.371±0.032)mm 2;and the areas of macula of utricle from above animal sequence were(0.193±0.02),(0.208±0.013),(0.321±0.011),(0.526±0.034),(0.795±0.017),(1.224±0.082),(1.388±0.048)mm 2;②The total number of hair cells in the macula of saccule from mouse,nude mouse,rat,guinea pig,chinchilla,white rabbit to monkey were 2476.3±64.4,2389.8±47.8,3135.3±191.6,4882.2±208.7,6128.5±242.9,10572.2±464.4,10992.7±397.4.The total number of hair cells in macula of utricle from above animal sequence were 2491.4±54.8,2368.0±46.1,3218.8±82.9,4925.3±271.1,7794.0±386.1,11347.4±435.7,11114.5±410.6;③The hair cell density(number of hair cells/0.03 mm 2)in the striola area and marginal area of saccule in mouse,rat,guinea pig,chinchilla,rabbit and monkey were 101.0±5.79(in striola region)/120.8±4.15(in marginal region),95.5±3.91(in striola region)/109.2±5.26(in marginal region),78.4±6.54(in striola region)/94.8±4.38(in marginal region),60.0±4.74(in striola region)/84.6±2.61(in marginal region),57.2±3.83(in striola region)/80.0±3.54(in marginal region),53.8±4.21(in striola region)/68.0±4.18(in marginal region),respectively.The hair cell density(number of hair cells/0.03 mm 2)in the striola area and marginal area of utricle in mouse,rat,guinea pig,chinchilla,rabbit and monkey were 103.8±5.02(in striola region)/119.2±3.70(in marginal region),91.2±2.49(in striola region)/106.4±4.16(in marginal region),74.1±3.54(in striola region)/90.8±3.56(in marginal region),60.4±4.98(in striola region)/81.6±2.07(in marginal region),57.8±1.92(in striola region)/77.8±3.70(in marginal region),54.0±2.74(in striola region)/66.4±2.51(in marginal region),respectively.The hair cell density(number of hair cells/0.03 mm 2)in the crista of ampulla of mouse,rat,guinea pig,chinchilla,rabbit and monkey were 112.4±6.38,105.5±3.51,95.2±3.42,84.0±7.16,78.2±2.86,70.8±2.39.It can be seen that the cell size of hair cells in smaller animals is smaller than that of larger animals,the density of vestibular hair cells in smaller animals is higher than that of larger animals.In addition,the density of hair cells in the striola region between macula of saccule and macula of utricle is similar,and the hair cell density in the marginal region between the macula of saccule and macula of utricle is also quite similar,but the hair cell density in the striola area is lower than hair cell density in the marginal area.Also,the density of hair cells in the ampulla crista was almost identical to that in the marginal areas in both maculae of saccule and utricle.In view of the fact that certain damage factors often have the performance of selectively destroying the hair cells in the striola area of maculae,the hair cell density in the striola area of maculae should be distinguished from the hair cell density in the surrounding marginal zone,and if necessary,even type I hair cells and type II hair cells should be distinguished to quantify the pathological changes respectively.Conclusion The vestibular measurement method used in this study and the measurement data of each sensory area of the vestibule,the total number of hair cells and the density of hair cells provide useful reference experience and necessary reference data for the quantitative analysis of vestibular pathology.