Introduction

Original Contribution

AGING AND THE HUMAN VESTIBULAR NUCLEUS

Ivan Lopez,* Vicente Honrubia,* and Robert W. Baloh,*t

UCLA School of Medicine, *Division of Head and Neck Surgery, tDepartment of Neurology, Los Angeles, California Reprint address: Robert W. Baloh, M.D., Department of Neurology, UCLA School of Medicine, Los Angeles, CA 90024-1769; Tel: (310) 825-5910; Fax: (310) 206-1513

0 Abstract-Degenerative changes during aging Introduction have been identified in the inner ear and in the vestibular nerve, but not in the human vestibular Complaints of dizziness and disequilibrium are nuclear complex (VNC). Therefore, the purpose of extremely common in older people (1). Associ this study was to document quantitative morpho ated falls are a major cause of morbidity and mor metric changes within the VNC in humans as a function of age. The VN C of normal human sub tality (2,3). Age-related morphological changes jects was examined for age-related changes using occur in all of the sensory systems essential for computer-based microscopy. Neuronal counts, nu maintaining balance during locomotion. Neu clear volume, neuronal density, and nuclear roanatomical studies of the peripheral vestibular length of the 4 vestibular nuclei were determined system have documented a significant loss of in 15 normal people, age 40 to 93 years. Based on a hair cells and primary vestibular neurons as a linear model, there was approximately a 3% neu function of age (4,5). Since the vestibular nu ronal loss per decade from age forty to ninety. clear complex (VNC) is a major visual-vestibu VNC volume and neuronal density also decreased lar interaction center, age-related neuronal loss significantly with age, although to a lesser degree in the VNC could have particularly important than did the number of neurons. Neuronal loss as functional implications. a percentage of the total number of neurons was greatest in the superior vestibular nucleus and There is general agreement that neuronal cell least in the medial vestibular nucleus. Despite the density in the cerebral cortex decreases with overall loss of neurons, the number of giant neu age, although the rate of decrease varies with rons (>500 11-m2) increased in older people. This the location (6-8). An age-related loss in the to increase in giant neurons could be traced to the tal number of Purkinje cells within the cerebel accumulation of lipofuscin deposits in the cell so lum is also well documented in humans (9). mata. The overall rate of neuronal loss with aging There is an average of 2.5% Purkinje-cell loss in the VNC is comparable tn that previously ob per decade over the age span of 0 to 100 years served in hair cells, primary vestibular neurons, (9). By contrast, quantitative studies of human and cerebellar Purkinje cells, but is in contrast to brain stem nuclei have found relatively little prior reports of no age-related loss of neurons in age-related neuronal loss (7, 10-14). There have other brain stem nuclei. Copyright © 1997 Elsevier been no prior studies of age-related effects on the Science Inc. human VNC, but Sturrock (30) observed a signifi cant decrease in the total number of neurons in the lateral vestibular nucleus of the aging mouse. 0 Keywords- quantitative morphometry; aging; The purpose of this study was to document vestibular nucleus. quantitative morphometric changes within the REcEIVED 12 July 1995; AccEPTED 20 August 1996. 77 78 I. Lopez et al

VNC in humans as a function of age. These myelin basic protein (MBP). The immunoreac measurements will serve as normative data for a tion product was only found in cells less than 40 study on dizziness and disequilibrium in older ~J-m2 . Furthermore, specimens were immunore people, sponsored by the National Institutes of acted with antibodies against synaptophysin, Aging. Preliminary results have been reported which stains axodendritic and axosomatic ter in abstract form (15). minals. Rarely were cells of less than 40 ~J-m2 surrounded by immunoreactive terminals in young or older subjects. IVIaterials and Methods Although the counting and measuring were done automatically, the operator viewed each Fifteen human brain specimens (7 female, 8 field on a video screen and performed a variety male; ages 40 to 93 years) were obtained within of automatic editing functions. The NIH Image 6 to 24 hours postmortem from subjects who program allows the removal of vascular ele had no chronic medical illnesses or known ments and artifacts, separation of contiguous symptoms of nervous system or ear disease cell images, and increased focal detection when prior to death. At postmortem examination, the part of a cell, although visible on the video brains showed no gross evidence of pathology; screen, was not stained with sufficient intensity the cause of death in each case was not neuro to be detected automatically for measurement. logicaL The specimens were fixed for 2 weeks Among these functions, "threshold" is used to in 10% buffered formalin, embedded in paraffin, segment an image into objects of interest and and serially sectioned at 20 micrometers. Every background on the basis of gray level. Additional tenth section was stained with a modification of a editing functions like ~'sharpen" and "subtract formaldehyde-thionin technique ( 16). background" are used to increase contrast and ac The boundaries of the vestibular nuclei were centuate details of the cells (NIH Image 1.44 outlined according to the criteria of Sadjadpour manual, 1992). This process was done on every and Brodal (Figure 1) (17). The number of neu 20th section, that is, 400 1-1m apart. To compute rons in each vestibular nucleus was determined the number of neurons in each 20-section vol by using a computer-based video microscopy ume, the estimated number of neurons in each and image analysis system (NIH, 1.44, public analyzed section was multiplied by 20. To domain). An image from each brain stem sec avoid multiple counting of cells larger in diame tion in the area of the vestibular nucleus was ter than the section thickness, a correction fac digitized using a Macintosh II computer tor was used. It was implemented by dividing equipped with a frame grabber attached to a the total number of neurons with a diameter be video camera mounted on a photomicroscope. tween 1- and 2-section thickness (that is 20 to The area of each individual vestibular nucleus 40 ~J-ill) by 2, those between 2- and 3-section was manually selected and recorded using the thickness (40 to 60 ~J-ill) by 3, and so on. The cursor. Neurons within this area were identified number of neurons in the vestibular nuclear on the video screen. The number of neurons and complex was determined by adding the number the area of each neuron were automatically de of neurons of the 4 vestibular nuclei. termined by the program. The diameter was de The volume of each vestibular nucleus was termined by calculating the diameter of a circle computed by multiplying the area of each sec of equal area to the neuron using the equation d = tion by the thickness of the section (20 1-1m) and 2 Jareal 1t . Cells with areas greater than or by 10 (periodicity of the analyzed sections). The equal to 40 ~J-m2 were counted as neurons. All volume of the vestibular nuclear complex was cells less than 40 ~J-m2 were considered glia and estimated by adding together the partial volumes excluded from the analysis (19). To confirm of each block. Neuronal density was calculated this assumption, glial cells in brain stem sec by dividing the number of neurons by the vol tions of both young and older subjects were ume in cubic millimeters. identified by using monoclonal antibodies The length of each individual vestibular nu against glial fibrilar acidic protein (GF AP) and cleus was obtained by multiplying the thickness Aging in the Vestibular Nucleus 179

Figure 1. Low-magnification photomicrographs illustrating the location of the four major vestibular nuclei {dot ted lines) in the brain stem. (A) superior vestibular nucleus; (B) medial vestibular nucleus; (C) lateral vestibular nucleus; and (D) descending vestibular nucleus. Thionin staining. Bar = 1 mm.

of the section by the number of sections that Each vestibular subnucleus also showed a demonstrated the morphometric characteristics significant (p < 0.05) decrease in number of of that nucleus. The length of the VNC was de neurons as a function of age. The highest rate of termined by identifying the beginning of the neuronal loss (6% per decade) occurred in the caudal portion of the descending vestibular nu superior vestibular nucleus, and the lowest rate cleus and the rostral end of the superior vestibu of neuronal loss (3% per decade) occurred in lar nucleus. The number of slides between these the medial vestibular nucleus (Table 1). Vol levels was multiplied by the section thickness. ume and neuronal density also decreased with age in each subnucleus, but the decrease was significant (p < 0.05) in only 2 of the 4 nuclei. Results Only the length of the descending nucleus de creased significantly (p < 0.05) with age. Regression analysis documented a highly To assess the relationship between neuronal significant (p < 0. 001) decrease with age in the size and age-related neuronal loss, we subdi number of neurons in the VNC (Figure 2A). vided neurons into small (40 to 99 1-1m2), me The slope of the regression line was similar in dium (100 to 199 f-LITI 2), large (200 to 499 f-LITI2), men and women (r = 0.86 and 0.90, respec and giant (>500 f-LITI 2) (Figure 3). Small and tively). The neuronal loss in the VNC was ap medium neurons represented the largest sub proximately 3% per decade between 40 and 90 group in each nucleus and also exhibited the years of age. There was also a significant (p < greatest age-related loss. Most of the giant neu 0.05) age-related decrease in volume and neu rons were located in the lateral vestibular nu ronal density in the VNC, but not in the length cleus (where they represented 12% of the neu (Figure 2, panels B through D). ronal population) (8). The number of giant 80 I. Lopez et at

y = -874x + 297496 r = 0.845 U) 260000 c A ...0 :::J cu 240000 c 220000 -0 ...cu .c 200000 0 E :::J z 180000

y = -0. i 40x + 76.727 = 0.747 72.5 B 70 ("') E E 67.5 cu 65 E :::J 62.5 0 0 > 60

("') E y = -5.741x + 3932.438 = 0.528 3800 c E 0 0 II) c: 3700 0... 3600 :l oo CD 0 c: 3500 =It 3400 > ·c;;- 3300 c 3200 ccu

y = -0.011 X + 13.799 r = 0.402 1 4 D 0 0 E 13.5-1- 0 0 E 0 13- 0 u n_O 0 .c a- C) 0 - c 12.5- 0 Cl.) ...J 12- 0

11.5 I I I I I 35 45 55 65 75 85 95

Age (years)

Figure 2. Correlation between number of neurons {A), volume (B), density (C), and length (D) of the vestibular nuclear complex (VNC) versus age in 15 normal human subjects. ing in the Vestibular Nucleus 01

Table 1. Effect of Aging on the Human Vestibular System

Approximate % Loss/decade Cell type number age 40 age 40 to 90* Reference

Vestibular nucleus neurons 260,000 3 Lopez et al. (38) superior 31,000 6 lateral 30,000 5 medial 139,000 3 descending 60,000 4 Hair cells 73,000 6 Rosenhall (4) 3 cristae 22,000 9 2 maculae 51,000 5 Vestibular ganglion neurons 18,000 5.5 Richter (5) Vestibular nerve fibers 16,000 5.5 Bergstrom (39)

*Assuming a linear rate of loss neurons significantly increased (p < 0.01) glial cells. Using similar automated techniques for rather than decreased with age. To explain this counting cerebral cortical neurons, Terry and col apparent contradiction, we examined giant neu leagues ( 19) showed that almost all glial cells had rons in each of the vestibular nuclei in young cross-sectional areas <40 ~m2 , whereas nearly all and older subjects. Abundant giant neurons in neurons were larger than 40 1-1m2. Furthermore, older people were filled with lipofuscin deposits Blinkov and Ponomarev (20) counted neurons (Figure 4, panels A and C), whereas less fre and glial cells in the VNC with an oil immer quent giant cells in younger subjects were sion lens with the aid of an ocular net microme smaller and did not contain lipofuscin (Figure 4, ter in 2 normal human subjects, ages 25 and 48. panels Band D). Presumably, there was loss of They counted an average number of 245,000 neu some giant neurons with aging (as with other rons in the VNC of these subjects. By comparison, neurons), but the total number of giant neurons we found an average of 250,000 neurons in the increased due to enlargement of smaller cells VNC of our 2 youngest subjects, age 40 and 43. because of lipofuscin deposits. Diaz and colleagues (21) defined neurons as so matic profiles with a nucleus and nucleolus and found a similar number of neurons in the VNC Discussion of young normal human subjects. Are neurons dying out or are they simply In this first study of the effect of aging on the getting smaller as a function of age? If all were VNC in humans, we found a highly significant getting smaller, then one would expect that the decrease in the number of neurons as a function greatest loss would be in the largest neurons, of age. The volume and neuronal density also with medium-sized and small-sized neurons ei decreased significantly with age, although to a ther showing no decrease in number or possibly lesser degree. Most prior studies of age-related increasing in number. We found just the re loss of neurons have estimated neuronal density verse. The:' greatest age-related loss occurred in rather than the total number of neurons because small and medi urn neurons. Also there was no it can be difficult to count all neurons in a re evidence that large numbers of neurons were gion, particularly when the borders are not well shrinking below the 40 ~m 2 detection limit in defined (6,7). Brain stem structures such as the older subjects, because neurons with areas less vestibular VNC, however, have reasonably well than 40 1-1m2, identified with immunohistochemi defined borders so that the total number of neurons cal stains, were rare in younger and older sub can be estimated ( 17, 18). An important procedural jects. The number of large and giant neurons actu point in our study was that we arbitrarily defined ally increased with age. DeLacalle and colleagues cells with areas greater than 40 ~m2 as neurons, (22) found similar age-related changes in the hu assuming that the cells smaller than 40 1-1m2 were man nucleus basalis of Meynert. The total num- 82 ! . Lopez et al

y = -726.34x + 173843 = 0.632 (/) 160000 c A ...0 ::J 140000 (I) c 120000 -...0 (I) 100000 .c E ::J c 80000

= -360.29x + 98561 = 0.829 '/) 90000 c B 0 :; 85000

y = 11 0.74x + 30485 r = 0.283 (/) c 55000 c ...0 50000- ::J (I) 0 8 c 45000- 0 40000- 0 Oo - -0 0 0 ... 35000-_ oo .c(I) 0 0 E 30000- 0 ::J 0 c 25000 I I I T I I

y = 56.45x • 1832 r = 0.658 0 7000 c 0 D ...0 6000 ::J (I) c 5000 4000 -0 3000 :a.. (I) .c 2000 E 1000 ::J c: 0 30 40 50 60 70 80 90 100 Age(years)

Figure 3. Number of neurons of different sizes in the VNC versus age. (A) 40-99 fl-m2, (B) 10Q-199 fl-m2, (C) 20Q- 499 fl-m2, (D)> 500 fl-m2. ber of neurons decreased with age, but the aver speculated that this hypertrophy of remaining neu age size of remaining neurons increased. For rons occurred as non-degenerating neurons as example, in subjects aged 16 to 29 years, 54% sumed some of the function of the disappearing of the neurons had areas greater than 400 1-1m2, population. Our data suggest that deposition of whereas in subjects aged 80 to 100 years, 62% lipofuscin could account for the hypertrophy of had areas greater than 400 J,Lm2. The authors some remaining neurons. Aging in the Vestibular Nucleus 83

ceives most of its primary afferent input from the cristae, including many large diameter fi bers from the center of the cristae (24,25). the medial vestibular nucleus showed the lowest rate of neuronal dropout with age; this nucleus receives almost no large fiber primary afferent input (26). Although we, and most other investi gators, used a linear model to describe the neu ronal loss that occurs with aging, a curvilinear model is probably more appropriate. This is particularly evident in the hair cell and primary neuron data, where it appears that age-related effects are minimal until the 5th or 6th decade (4,5). We did not study any subjects under the age of 40, but the rate of neuronal loss in the age range of 40 to 90 appears curvilinear (see Figure 2A). Larger numbers of subjects are needed, however, to adequately define the rela tionship. Most prior studies on the effects of aging on neuronal populations within the human central nervous system have focused on the cerebral cortex· (6,7). The number of cerebral cortical neurons decreases with age, but the loss is re gional, affecting some areas to a greater extent Figure 4. Photomicrographs of VNC neurons in the than others. Brody (6,27) found the greatest lateral vestibular nucleus (A and B) and the medial vestibular nucleus (C and D), of an older (A and C) and age-related loss of neurons in the superior tem a younger subject (B and D). Arrowheads in photomi poral gyrus, superior frontal gyrus, precentral crographs A and C demonstrate the presence of lipo gyrus, and the area of striata. Although this neu fuscin in the cell somata of a typical giant size Dieter's neuron and three typical medial vestibular nuclear ronal loss was observed in all layers of the cor neurons. Thionin staining. Bar in A and B = 12 J.Lm; in tex, it was particularly evident in layers 2 and 4, C and D = 10 J.Lm. the external and internal granular layers. How ever, such studies have problems in separating neuronal loss from volume changes associated The rate of neuronal loss in the vestibular with tissue shrinkage during embedding, which nuclear complex with aging is comparable to is also age-related (28). Probably the most com the rate of loss of hair cells and primary vestibu plete study of a neuronal population with aging lar neurons (Table 1). Rosenhall (4) observed a was performed on cerebellar Purkinje cells by hair-cell loss of approximately 6% per decade Hall and colleagues (9). These investigators in the vestibular end organs over the age span of counted the total number of Purkinje cells in the 40 to 90 years. He noted that the hair-cell loss cerebellum of 90 normal human subjects. Al tended to be greatest at the center of the cristae, though wide individual variations in the number the area supplied by the largest diameter vestib of Purkinje cells were found at all ages, they ular nerve fibers. Bergstrom (23) found an over found a mean reduction of 2.5% per decade all vestibular nerve fiber loss of 5.5% per de over the age range of 0 to 100 years. However, cade over the same age span, but also noted that as with the vestibular hair cell and primary neu the nerve fiber dropout was greatest for the larg ron data, the relationship between the number est diameter fibers. Of note, we found that the of Purkinje cells and age appear to be curvilin highest rate of neuronal dropout occurred in the ear rather than linear, with relatively little superior vestibular nucleus, a nucleus that re- change in the number of Purkinje cells up to the 84 I. Lopez et al

5th and 6th decade and then a clear dropoff in the a clear age-related decrease in VOR gain along number of Purkinje cells beyond that age. For with a decrease in the dominant time constant of comparison with our vestibular nucleus data, we the VOR (35-37). As with hair cell and neu estimated the rate of dropoff for the age range of ronal loss, the decrease in VOR gain appears to 40 to 90 years using a linear model. Hall and col be curvilinear, with relatively little decrease in leagues' (9) data suggested a 5% loss of Purkinje gain until the 5th or 6th decade, followed by a cell per decade for that age range. Torvik and rapid dropoff in VOR gain. The age-related colleagues (29) pe1fonned a morphometric anal changes in the canal ocular reflex may be ex ysis of the cerebellm· vermis in nonalcoholic men plained by the prominent age-related loss of hair between ages 39 and 94 years and showed a sig cells at the center of the cristae, the relatively se nificant decline in Purkinje cell density with in lective loss of large diameter primary vestibular creasing age in o.ll parts of the cerebeilar ver afferents, and the neuronal loss in the superior mis, particularly in the superior vermis. Finally, vestibular nucleus, a major canal ocular reflex re Sturrock (30) found a decline in the number of lay station. Age-related changes in the vestibu Purkinje cells with age in the cerebellar nodulus lospinal reflexes are more difficult to assess be of mice. cause of the prominent overlap in function with Although there have been no prior studies of other sensory motor systems. Although deterio age-related effects in the human VNC, Sturrock ration in balance and gait are well documented reported an age-related loss of neurons in the with aging, it is difficult to distinguish changes lateral vestibular nucleus of the mouse (31). in vestibulospinal reflexes from the well-docu This neuronal loss was most pronounced in the mented changes that occur in the visual and so large neurons. Loss of the large neurons was matosensory systems (1). preceded by an accumulation of lipofuscin in Is the age-related loss of sensory cells and their soma and a loss of Nissl substance (31). neurons within the vestibular system part of the Sturrock also noted age-related neuronal loss and normal aging process, or is it the result of an un similar morphologic changes in neurons from the derlying pathologic process such as progressive mesencephalic and motor nuclei of the trigemi vascular disease? None of our subjects had evi nal nerve (32) and from the facial nerve nucleus dence of small vessel disease or of infarction (33) in the mouse. By contrast, prior studies of within the brain stem. However, we did not sys other brain stem nuclei in normal human subjects, tematically assess the status of the large vessels including the ventral cochlear (12), abducens at the base of the brain. The age-related loss in (13), trochlear (14), facial (34), and inferior oli hair cells and primary and secondary vestibular vary nuclei ( 10), have not found age-related neurons, however, is so prominent and so con neuronal loss. The reason for this apparent dif sistent from study to study that inclusion of a ference in age-related effects on different brain few subjects with undetected vascular disease stem nuclei in the human is not clear, but larger could not account for the observed findings. numbers of <;ubjects must be studied with mod Hall and associates (9) systematically assessed ern quantitative techniques before a final con all postmortem specimens for small and large clusion can be reached. vessel disease, and even after exclusion of all One can only speculate on the functional im cases with vascular disease, they found a clear plications of the age-related hair cell and neu cut age-related loss of Purkinje cells within the ronal loss in normal human subjects, summa cerebellum. rized in Table 1. Age-related effects on the vestibula-ocular reflex (VOR) have been stud Acknowledgments-This work was supported by ied most extensively because the reflex is rela NIH grants AG90693 and DC01404. The authors tively easy to stimulate and there are many wish to thank Itsuko Aono for her technical assis ways to record evoked eye movements. There is tance. Aging in the Vestibular Nucleus 85

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