Distribution of glial fibrillary acidic protein (GFAP) in the cochlear nucleus of adult and aged rats

BRAIN RESEARCH Brain Research 686 (1995) 223-232

ELSEVIER

Research report

Distribution of glial fibrillary acidic protein (GFAP) in the cochlear nucleus of adult and aged rats I. Jalenques a E. Albuisson b, G. Despres a R. Romand a,, Laboratoire de neurobiologie et Physiologie du ddveloppement, Universit~ Blaise Pascal-Clermont ill, 63177 Aubi~re Cedex, France b Laboratoire de Biostatistiques, Facult~ de M~decine, Universit~ d'AuL, ergne, 28 place Henri Dunant, 63000 Clermont-Ferrand, France Accepted 28 March 1995

Abstract

The age-related change in glial fibrillary acidic protein (GFAP) immmunoreactivity was analyzed in young (3 months) and old (24 months) adult rat cochlear nuclei (CN). Quantitative analyses show a significant increase with age, in the number of GFAP positive astrocytes and processes in the old adult when compared with the young adult rat. There was also a differential distribution of GFAP immunoreactivity in the young adult CN where it predominates in the granular cell region, whereas in old rats, the GFAP immunoreactivity distribution was homogeneous in all parts of the nucleus. There was no change in the total number of neurons between these two stages in any part of the nucleus except for the antero-ventral CN, where a decrease in neuronal number was observed in the aged rats. The increase in GFAP immunoreactivity was related to an increase of both GFAP positive astrocyte number and processes. The increase of GFAP positive astrocytes may be due either to an alteration of auditory nerve fibers, changing the trophic interactions with post-synaptic cells, or to intrinsic alterations of CN neurons and local circuits reflecting aging of the CN. Keywords: Astrocyte; Intermediate filament; Auditory brainstem nucleus; Aging; Immunohistochemistry

1. Introduction

Presbycusis is the main result of progressive aging associated with peripheral auditory impairments and central modifications with various consequences on the auditory capability and cognition of aged subjects [13,38,41,50,52,56]. Two potential problems confront investigators studying the effects of aging on the central auditory system: firstly, progressive peripheral hearing loss that occurs with some consequences on the central auditory system, and secondly, aging effects that can selectively impair neural functioning [56,59]. In this report we focus on the effects of aging on the central auditory system, i.e., the cochlear nucleus (CN) where several observations have been made at the neuronal cell level in mice [57-59]. However, to understand the aging processes in the brain, one should also consider the non-neuronal cells. Astrocytes were originally thought to play only supportive roles in the brain [51]. Recent evidence suggests that they serve important functions in the

* Corresponding author. Fax: (33) 73 40 78 02. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 4 6 3 - 7

brain [18,25,26,28,45,48], therefore the study of these cells is critical when assessing age-related changes in the brain. Some data reported an age-related change in the number of astrocytes [20,46,54], while others found an increase in size and fibrous character with age, without a concomitant increase in number [17,27]. Astrogliosis refers to the change that astrocytes undergo response to central nervous system trauma or aging. Regulation of astrogliosis may be specifically linked to the expression of glial fibrillary acidic protein (GFAP) [14]. GFAP is an intermediate filament protein in astrocytes [15], as demonstrated by immunohistochemical studies; GFAP is selectively located in astrocytes [3,4,40,53]. In this report we used an antibody to GFAP in order to investigate the change of reactive astrocytes during aging in the CN. Sprague-Dawley rats were chosen because this strain presents only mild peripheral hearing loss with age [12] with reduced degeneration of sensory cells [33]. In order to decrease the effect of sensorineural hearing loss on the aging process on neuronal and non-neuronal cells of the CN, we used 24-month-old rats because the mean loss of spiral ganglion cell amounts to only 14% at 23 months [321.

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We report here that an increase of GFAP immunoreactivity was observed in aged rats, related to more numerous GFAP positive astrocytes and processes. There was also a differential distribution of GFAP immunoreactivity in the young adult CN where it predominates in the granular cell region, whereas in old rats, the GFAP immunoreactivity distribution was homogeneous in all parts of the nucleus. There was no change in the total number of neurons between these two stages in any part of the nucleus except for the antero-ventral CN, where a decrease in neuronal number was observed in the aged rats.

2. Materials and methods

After washing in tap water, sections were then incubated 7 min in 0.05% DAB/0.01% hydrogen peroxidase in PBS solution (pH = 7.4). Some sections were prepared as described above and Nissl-counterstained, dehydrated and mounted. 2.4. Controls

Negative controls were carried out by omitting or replacing the anti-GFAP antibody by mouse IgG (Sigma). Positive controls were realized on brain sections containing the hippocampus or the cerebellum where GFAP positive astrocytes are well characterized [19,30].

2.1. Fixation

2.5. Quantitatiue analysis

Male Sprague-Dawley rats were obtained from IFFACREDO (L'Arbresle, France). The study was carried out on young (3 months; n = 12) and old (24 months; n = 3) adults. Animals were decapitated after chloroform anesthesia; brainstems were quickly dissected in absolute ethanol and fixed for 3 days. The fixative was changed twice a day. The tissue was cleared in xylene, and routinely embedded in paraffin.

Astrocytes: 3 animals were used for each age group. The CN complex of the rat is conventionally divided into ventral CN (VCN) with two divisions: the antero-ventral CN (AVCN), the postero-ventral CN (PVCN), and the dorsal CN (DCN). For detailed parcellation and cell types we refer to the works done on cat, mouse and rat [6,22,23,39,42,44]. For the molecular layer of DCN, 4 positions in the rostro-caudal axis were analyzed: the rostral one is defined as the more rostral section in which 3 principal layers of the DCN are very distinct, i.e. granular cell layer, fusiform cell layer and polymorphic layer. The caudal one, is defined as the more caudal section containing the 3 well defined layers; the 2 other positions are equidistant from these extreme poles. In the PVCN, 4 positions in the rostro-caudal axis were studied. The rostral position analyzed was at the beginning of the multipolar neurons, while the caudal was at the end of the octopus cell region. Two intermediate sections were also taken from the previous ones. In the AVCN, 3 positions --rostral at the beginning of bushy cells, caudal in the middle of PV and intermediate between these two were elicited. For each stage, 9 sections were analyzed. Only GFAP multipolar positive astrocytes were counted in a calibrated area of 16,000 / x m 2 (200 /zm X 80 /xm) from 12-/xm sections. This area was chosen to be small enough in order to fit in different regions of the CN without overlapping other regions. The counting criterion was defined as immunostained cell bodies or processes converging toward a central point without obvious cell body. No effort was made to correct measurements for shrinkage due to tissue processing. Neurons: for each age group, the number of neurons was obtained without regard to neuron type in Nissl-stained sections. Neuron numbers were counted within the calibrated area as described above when a cell displayed a full nucleus. Three sections were analyzed for each position studied in rostro-caudal and dorso-ventral axes for each animal.

2.2. Histology

Twelve-/zm sections were cut in the transversal plane. In each series, one out of every 3 sections was collected and Nissl-stained, Thus, sections for immunohistochemistry were chosen according to the structures revealed on adjacent stained sections. 2.3. Immunostaining

Brainstem sections containing cochlear nucleus were deparaffinized in xylene, hydrated through graded ethanol series and washed for 5 min in distilled water. Sections were treated for 30 min at room temperature (RT) with 0.3% hydrogen peroxide in absolute methanol to inhibit endogenous peroxidase activity. Sections were washed for 20 min in 0.1 M phosphate buffered saline (PBS) and treated with the blocking kit of Biotin/Avidin System reagents (Vector Laboratories). The avidin solution was mixed with 10% fetal calf serum to suppress background staining. Sections were then incubated for 30 min at 37°C with anti GFAP monoclonal antibody (clone G.A.5.; Sigma Chimie, France) diluted 1 / 4 0 0 in PBS containing 5% fetal calf serum and 0.1% Triton X100. Sections were washed, 3 times in PBS, 10 min each (the same washing procedure was performed between each of the following steps) and incubated for 30 min at 37°C with biotinylated anti-mouse IgG antibody diluted 1 / 8 0 0 in PBS. Then, sections were treated with a preformed avidin-biotin-peroxidase complex for 45 min at 37°C.

I. Jalenques et al. / Brain Research 686 (1995) 223-232 2.6. Statistical analysis A three-way analysis of variance ( A N O V A with two factors and one repetitive factor) was used to analyse the results of this experimental design. All sections analyzed for each age group were c o m b i n e d . The aim was to explain the quantitative data (with 3 repeated values) with reference to qualitative data such as subject, side, age, nucleus and layer. Conditions of use (normality and equality of variances) were tested using the chi-squared test and the variance-ratio test. Firstly, the analysis was performed within each age group; secondly, discriminant analysis was

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performed on the whole population as a m e a n of studying the impact of age.

2. 7. Distribution o f G F A P immunoreactiue cells Distribution and position of i m m u n o r e a c t i v c multipolar astrocytes were analyzed by an image analysis workstation (Biocom) coupled to a microscope. Briefly, this system makes m o r p h o m e t r i c and cartographic m e a s u r e m e n t s of objects selected by the users at high magnification from a regional context, after first plotting the areas of interest at low magnification. All cells resulting from exploration of a

Fig. 1. GFAP immunoreactivity in the young adult rat. A: GFAP immunoreactivity in the DCN (1) and PVCN (2) of the young adult rat. The density of astrocytes seems to be more important in the granule cell domain, particularly in the lamina of granule cells (L) extending medially from the superficial layer between the PVCN and DCN and in the molecular layer. Density of global immunostaining decreases from the molecular layer to the polymorphic layer, x40. B: In the DCN, GFAP immunoreactivity decreases from molecular (m) to polymorphic (p) layer. In the molecular layer, one can observe diffuse immunostaining, partly related to many astrocyte processes below the pia. In the molecular and fusiform cell (f) layers, there are several small astrocytes and astrocyte processes lining the blood vessel walls (arrow). x 180. C: immunoreactive cell in the molecular layer with many processes. This multipolar astrocyte presents a well-immunostained cell body below the pia. One can observe the darkly stained ependymal cell layer (arrow). x 800. D: immunoreactive cell in polymorphic layer with several immunostained processes. X 800.

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cross-section are recorded on a map in high resolution cartography. Assembling maps were made with a scanner coupled to a computer with the 'Photoshop' software. These maps only give the distribution of GFAP positive cells in specific sections of the CN, and only give a qualitative picture of cell distribution for a specific section.

3. Results The density of astrocytes seems to be greater in some regions of the CN such as the granule cell domain i.e. the superficial layer of granule cells covering the free surface of the VCN, the lamina of granule cells extending medially from the superficial layer between the VCN and DCN, and

Fig. 2. GFAP immunoreactivityin the old rat. A: GFAP immunoreactivityin the DCN (1) and PVCN (2) of the aged rat can be observed. The density of global immunostainingdecreases slightly from the superficialto the deepest regions, x 40. B: GFAP immunoreactivityin the DCN of the aged rat shows numerous astrocytes in both the molecular (m) and fusiform cell (f) layers, z 180. C: immunoreactiveastrocyte from the molecular cell layer with numerous fibrous processes, x 800. D: astrocyte from the polymorphiccell layer with immunoreactiveprocesses of various length. × 800.

L Jalenques et al. /Brain Research 686 (1995) 223-232

the subpeduncular corner of granule cells located at the dorsal edge of the V C N in young adult rat (Fig. 1A) as well as in aged rats (Fig. 2A). In the CN, two types of G F A P immunoreactive cells can be observed which can be distinguished by their morphology and locations. However, this study will be restricted to multipolar astrocytes. Firstly, intensely stained unipolar cells with an immunopositive cell body can be observed and which line the inner surface of the pia mater. These cells provide thick processes of variable length which run perpendicularly to the surface of the pia mater. Another type of unipolar cells with thick processes is found close to blood vessels, these processes run perpendicularly to the blood vessel surfaces and touch them. The second type of G F A P immunostained cells are multipolar with polygonal cell bodies bearing several pro-

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cesses of variable length. Only minor changes in the morphology of these cells are observed depending upon their locations. However, the distribution and the morphology of cell processes seem to be influenced by local architecture of the nerve cells. This type of cell is well represented in the molecular layer of DCN and in the granular cell domain• 3.1. Aged D C N and comparison with young adult D C N

Sections present a more important G F A P immunoreactivity in the aged rat which can be related partly to an increasing number of G F A P positive cells and partly to the greater and more fibrous processes of glial cells in aged rats (Fig. 1A, 2A, 1B and 2B). There is a less marked decrease in the density of global immunostaining from the superficial to the deepest regions in aged rats compared

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Fig. 3. Schematic distribution of GFAP immunoreactive cells in young adult and aged rats. A: DCN: the number and distribution of immunoreactive astrocytes change markedly from young adult (A1) to aged (A2) rats. In the young adult rat, the density of astrocytes is slightly more conspicuous in the dorsal part of the DCN than in the ventral part. There is a decrease from the molecular layer to the polymorphic layer of the DCN. The study of the schematic distribution of GFAP positive cells in an old rat (A2) shows a quite homogeneous distribution in all layers of DCN, except in some of the deepest area of the polymorphic layer. Moreover, the number of reactive astrocytes increases markedly in the old rat (A2) compared with the young adult rat (A1). B: young adult AVCN (B1): the distribution of GFAP positive astrocytes decreases in the dorso-ventral axis. Aged AVCN (B2): There is a large number of reactive astrocytes with a more or less homogeneous distribution throughout the nucleus. The comparison of the diagrams underlines the increasing number of GFAP positive astrocytes in an old rat (B2) with a more or less homogeneous distribution throughout the AVCN compared with a young adult rat (B1).

L Jalenques et al. /Brain Research 686 (1995) 223-232

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with young adults (Fig. 1A and 2A), and the distribution of immunoreactive astrocytes is quite homogeneous throughout the three layers of the aged DCN (Fig. 2B). The molecular and fusiform cell layers are more immunoreactive in aged DCN compared with young adults (Fig. 1B and 2B). The schematic distribution of GFAP positive cells confirms an homogeneous distribution in all layers of DCN in aged rats, except in the deepest area of the polymorphic layer, and a dramatic increase compared to young adults (Fig. 3A and B). Statistical analysis shows no difference in the number of immunoreactive astrocytes between layers in aged rats (Fig. 4A). The apparent contradiction with the schematic data, i.e., less stained cells in the deepest area of the polymorphic layer (Fig. 3A), is explained by the method of counting: quantitative analysis was undertaken in the central part of the polymorphic layer, which did not take into account the deepest region of this layer. A significant difference exists between young adults and aged rats when the average numbers of immunoreactive astrocytes: young adult are considered = 2.3 Labelled Cells (LC), aged rat = 6.1 LC ( P < 0.0001), as well as by comparing layers respectively (Fig. 4A). No difference was found between the average number of neurons, for the young adult and aged rats, regardless of the type of neurons. But the statistical difference in the number of neurons between the three layers of the DCN which exists in young adults was still present in aged rats, with the following results for a standard area: molecular layer = 35.6 cells; fusiform cell layer = 53.4 cells; polymorphic layer = 38.5 cells ( P < 0.0001) (Fig. 4B).

3.2. Aged PVCN and comparison with young adult PVCN The intensity of global immunostaining shows a significant increase in the aged rat which can be related to an increasing number of GFAP positive cells and to more numerous fibrous processes of glial cells (Fig. 1A and 2A). However, in both stages, the lateral border of PVCN near the pia presents a more conspicuous immunostaining than

229

the central area, while the distribution of immunoreactive astrocytes is more or less homogeneous throughout the various area of the PVCN (Fig. 1A and 2A). The statistical analysis confirms the dramatic increase in the density of immunoreactive astrocytes between the two stages: young adults present a mean of 1.5 astrocytes for the standard area while aged rats present a mean of 5.1 astrocytes ( P < 0.0001) (Fig. 4C). In aged PVCN, statistical analysis does not show any difference in the number of immunoreactive astrocytes nor neuronal cell populations between the area along the rostro-caudal axis (Fig. 4C and D). It can be underlined that the mean density of GFAP positive astrocytes in aged PVCN ( = 5.1 LC) is slightly lower than in aged DCN (6.1 LC) (Fig. 4A and C) just as the mean density of neurons in aged PVCN is lower than in aged DCN. No statistical difference is found in the number of neurons between young adult and old rats (Fig. 4D), regardless of neuron type in the PVCN.

3.3. Aged A VCN and comparison with young adult A VCN GFAP immunoreactivity is high in old rats, increased in comparison with young adults (Fig. 3B). This is confirmed by statistical analysis which shows a significant statistical age effect in the number of reactive astrocytes between young adult (2.5 LC) and old rats (5.6 LC) ( P < 0.0001) (Fig. 4E). The distribution of GFAP positive astrocytes in old rats is more or less homogeneous throughout the nucleus (Fig. 3B). While neurons of aged rats from Nissl staining have a normal appearance, they are stained more darkly and have a more granular nucleus. Above all, statistical analysis shows a significant decrease in the number of neurons in aged rats: young adults = 43.9 cells; aged = 34.8 cells, ( P < 0.01) (Fig. 4F). No statistical difference is found in the number of GFAP positive astrocytes and neurons in the rostro-caudal axis (Fig. 4E and F), nor between dorso-ventral regions of AVCN as far as the increase of GFAP positive astrocytes (Fig. 3B and 4E) and the decrease of neurons (Fig. 4F) are concerned.

Fig. 4. Modification of GFAP positive astrocytes and neurons number between young and old adult rat. A: number of GFAP positive astrocytes as a function of DCN layers and age of rats, counted in a reference area of 16000 ~ m : as described previously. The same area was used for the following figures. A significant statistical difference exists between layers of DCN in young adult rats ( P < 0.0001); such a difference does not appear in old rats (no significant statistical difference: NS). A significant statistical difference exists between young adult and old rats ( P < 0.0001). B: number of neurons as a function of DCN layers and age. A significant statistical difference exists between molecular, polymorphic and fusiform cell layers in young adult rats ( P < 0.0001), and in old rats ( P < 0.0001). But no significant difference appears between young adult and aged rats. C: number of immunoreactivc astrocytes as a function of PVCN rostro-caudal axis and age. No statistical significant difference appears in PVCN rostro-caudal axis either in young adult or in old rats. A significant statistical difference exists between young adult and old stages ( P < 0.0001). D: number of neurons as a function of PVCN rostro-caudal axis and age. The distribution of neurons does not show any significant variation in the rostro-caudal axis either in young adult or in old rats. No significant statistical difference appears in the number of astrocytes between young adult and old stages. E: number of GFAP positive astrocytes as a function of AVCN rostro-caudal axis without a precise relation of anatomical subdivisions. A significant statistical difference is found on this axis in young adult rats ( P < 0.001) but not in old rats. A significant statistical difference appears between young adult and old stages ( P < 0.0001). F: number of neurons as a function of AVCN rostro-caudal axis and age. No significant statistical difference appears in either young adult or in old rats. But a significant statistical decrease in the number of neurons exists between young adult and old rats ( P < 0.01).

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4. Discussion We have shown a significant effect of age on the number of GFAP positive astrocytes and processes between young adult and old rats in all three divisions of the CN. An earlier study restricted to the octopus cell area (OCA) of the PVCN of (C57 and CBA) mice showed a parallel increase of glial cells [58]. What is more, the distribution of GFAP immunoreactivity varies according to age: in adult rats, GFAP immunoreactivity predominates in the granular cell region of the CN, whereas in old animals, the GFAP immunoreactivity is distributed homogeneously. The increase in GFAP immunoreactivity seems related both to an increase of the reactive astrocyte number and to an increase in the fibrous character of glial cells. These observations agree with structural changes in glial cells occurring in other parts of the brain during aging [18,55], as well as mRNA increases [43], and after neuronal modification or degeneration [1,8,18]. The increase of GFAP immunoreactivity in the CN with aging could be related to three phenomena of varying importance acting on neuronal cells and subsequently triggering gliosis, reflecting combined effects of aging and chronic reduction of sensorineural input: + alteration of auditory nerve fibers that may change trophic interactions with postsynaptic cells, + intrinsic alterations of CN neurons and local circuits. 4.1. Alteration of auditory nerve fibers in relation to trophic interactions

Studies on the auditory system have revealed a variety of morphological degenerative changes with aging [5,7,9,31,33,34,49,56-59]. These changes affect the peripheral as well as the central portions of the auditory system. In the peripheral auditory system, degeneration of hair cells is a well known phenomenon [5,24,33,49] with a consistent loss of spiral ganglion cells [34]. Changes also occur in the central auditory system: some morphological features differ with age irrespective of cell types or location in auditory nuclei; other morphological changes seem to be dependent on strain, species, cell type and location of neurons within these nuclei [7,9,59]. The reduction of afferent system activity could be responsible for the changes observed in AVCN where a decrease in neuron number was observed associated with an increase of GFAP reactivity. This observation could be related to the major direct input from the auditory nerve producing a transneuronal degenerative change as observed in octopus cell area (OCA) of mice [58] and in the polymorphic layer of DCN [59]. However, in our case, it is reasonable to postulate that the reduction of afferent system activity is not very much involved in modifications observed in aging CN. In fact, it has been shown [32] that a mean of only 14% of spiral ganglion cells are lost at 23

months in the same strain of rat. So, it could be assumed that GFAP positive astrocytic changes with aging in the CN in the rat are not as dependent upon a peripheral sensorineural loss because the density of neuronal cells is unchanged except for the AVCN. Controversial results exist regarding loss of neurons in the aging brain [16] as well as in the CN. This problem was addressed by Willot et al. [57] in AVCN with results depending on the strains of mice concerning peripheral and central presbycusis. Aging may be associated with rather different control effects depending on cell type, regional organization of the brain, genetic background and trophic factors involved. A study performed with the same strain of rat that we used [10] which presented a significant neuron loss in the medial nucleus of the trapezoid body may be related to decreased input from the controlateral CN, or an age-related devascularization [11]. 4.2. Intrinsic changes in the CN with aging

The report of Willott and Seegers-Bross [58], dealing with the OCA in C57 and CBA mice, shows that the volume of the OCA declines substantially during the second half of life. The packing density of octopus cells changes little during this time and so a loss of octopus cells is associated with the reduction in OCA volume late in life. The packing density of glial cells increases as octopus cells are lost. The modification of glial cell density is likely to correspond with our GFAP increase of immunoreactivity in most regions of the CN. According to Willott and Seegers-Bross [58] observations made in OCA are more likely to be associated with aging affecting selectively the CN and not with presbycusis. The age-related changes in GFAP immunoreactivity and GFAP mRNA affect the major part of the central nervous system in aged animals [18,29,53] as well as in the aged human brain [21,36]. 4.3. Functional significance

The functional significance of changes in GFAP expression with aging in the CN is unknown. It may be that an increase in the fibrous nature and number of GFAP positive astrocytes may reflect astrocytes undergoing reactive gliosis. Gliosis may be a reaction to intrinsic modifications in the CN such as degeneration of neighboring synapses, dendrites or entire neurons [1,17,35] as observed in the CN [58,59]. Alternatively, the astrocytic reaction could act to promote long-term neuronal survival by diffusible neurotrophic factors [45,47]. It is known that trophic interactions exist between astrocytes and neurons [2,37] that could slow down the dying process of aging neurons, and may explain the low number of dying cells in aged CN. Therefore, it would be worthwhile to probe the role of trophic factors in the aging process of the CN.

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Acknowledgements W e t h a n k Dr. A. work was supported m 6 d i c a l e ' as w e l l as and the 'Association

Hafidi for his valuable comments. The b y ' L a F o n d a t i o n p o u r la R e c h e r c h e b y ' A s s o c i a t i o n R e c h e r c h e et P a r t a g e ' of European Psychiatrists'.

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