MRI description of cerebral atrophy in mouse lemur primates

Neurobiology of Aging 21 (2000) 81– 88

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MRI description of cerebral atrophy in mouse lemur primates Marc Dhenaina,b,c,* Jean–Luc Michotc, Nicolas Privatd, Jean–Luc Picqe, Francois Bollerc, Charles Duyckaertsd, Andreas Volka a

Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) U 350, Institut Curie, 91405 Orsay, Cedex, France b California Institute of Technology, Pasadena, CA, 91125, USA c Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) U 324, 2 ter rue d’Ale´sia 75014 Paris, France d Laboratoire de Neuropathologie R. Escourolle. Hopital de la Salpeˆtrie`re. 47 Bd de l’hopital. 75651 Paris, France e Museum National d’Histoire Naturelle, Laboratoire de Conservation des Espe`ces Animales, Parc Zoologique de Paris, Paris, France Received 22 July 1999; received in revised form 15 December 1999; accepted 12 January 2000

Abstract We assessed cerebral atrophy in mouse lemur primates (Microcebus murinus) by estimating CSF volume in their brains from 4.7 Tesla T2-weighted magnetic resonance images. Thirty animals aged from 1 to 10.3 years were imaged, 14 of them were followed for up to 2 years. Seven of these animals were examined for neuropathology. In 12 out of 17 animals older than 3.5 years, CSF volumes were increased. A subgroup of six animals had severe atrophy of the temporal lobe. Another subgroup of five animals displayed diffuse atrophy in addition to the temporal atrophy. One animal had a dilation of the external part of the temporal horn of the lateral ventricle in addition to the temporal atrophy. The three animals with diffuse atrophy that could be studied for neuropathology had diffuse cerebral amyloid deposits detected by immunocytochemistry. The other animals did not display amyloid deposits. Relations between the different types of atrophy as well as their causes will have to be assessed in future studies. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Aging; Alzheimer’s disease; Amyloid deposits; Animal model; Cerebral atrophy; Microcebus murinus; Primate

1. Introduction Cerebral atrophy is reported during normal aging in humans [12,42]. It is also reported during pathological aging such as Alzheimer’s disease (AD) [10,11,27,28]. However, atrophy increases more slowly during normal aging than in AD [17,31,42]. AD is characterized by the presence of senile plaques and neurofibrillary tangles (NFT). Senile plaques are extracellular amyloid deposits surrounded by dystrophic neurites [1]. NFT are accumulations of hyperphosphorylated tau proteins in the cell body of the neuron. Senile plaques [25] and NFT [13] are also present, although in low density and in confined areas, in normal aged subjects. In aging non-human primates, cerebral weight loss has been reported in some studies [21,35] but not in all [22]. Ventricular enlargement has been reported in some aged animals [4,44], but the topography of the atrophy has never * Corresponding author. Tel.: ⫹33-1-69-86-31-64; fax: ⫹33-1-69-0753-27. E-mail address: [email protected] (M. Dhenain).

been described. In aging mice, cerebral atrophy has only been reported in SAM-P10 models of spontaneous brain atrophy, but they are models of an unknown pathologic process rather than of aging or AD like disease [39,40]. In transgenic mice producing extracellular cerebral amyloid deposits, neuritic dystrophy, and gliosis have been reported in several studies [6,18,23,24,26] but neuronal loss has been reported in only two studies [8,34], and cerebral atrophy has never been described. Mouse lemurs (Microcebus murinus) are small (about 12 cm, 100 g) lemurian primates with a lifespan of 3.5 years in the wild. In captivity their mean and maximum lifespans are 5 and 12 years, respectively [37]. They are seasonal breeders that mate during spring and summer. Young animals are sexually mature during the breeding season following the year of their birth [36]. The mouse lemur brain has an average weight of 1.7 g [5], measures 23 mm from the tip of the olfactory bulbs to the caudal end of the medulla, and has a maximum breadth of 18 mm [29]. As in other primates [19,38,41,43], age related amyloid deposits have been observed in lemurs [4]. Degenerated neurites surrounding amyloid deposits and abnormally phosphorylated tau pro-

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teins have also been reported in aged animals [3,14]. We have used magnetic resonance imaging (MRI) to detect cerebral atrophy in mouse lemurs. Different patterns of age related cerebral atrophy have been described. One of the patterns was associated with the presence of amyloid deposits in the brain.

2. Materials and methods 2.1. Animals Thirty mouse lemurs were studied by MRI. Four females and 11 males younger than 3.5 years made up the “young age group”. Three females and 12 males older than 3.5 years constituted the “old age group”. Fourteen of them [five young, seven old animals, and two animals younger than 3.5 years during their first MRI scan and older than 3.5 during their second scan (one male and one female)] could be followed in a longitudinal way over 2 years. Practically, the MRI scans were organized into six different sessions with nine, seven, seven, nine, ten, five animals per session, respectively. All the animals were born and bred in captivity at the Museum National d’Histoire Naturelle in Brunoy and in Paris. The animals were not sacrificed at the end of the study. Neuropathology could be studied in seven animals that died from natural death. In four other animals the brains could not be examined because the post mortem delays were too long when their death was discovered (⬎12 h). The other animals are still alive. 2.2. Magnetic resonance imaging As previously described [15,16], T2-weighted images were performed on a 4.7 Tesla Bruker Biospec 47/30 system (30 cm bore horizontal magnet, 15 cm diameter gradient system (60 mT/m), TR/TE: 2000/100 ms, slice thickness: 1 mm, in plane resolution: 0.23 mm, matrix 256*256). On all animals, the position of the slices determined on series of localizer images, were very similar and images were acquired under similar conditions during the six MRI sessions. Probe adjustments, matching and tuning, were performed outside the magnet using a sweep generator connected to the probe via a return loss bridge, and optimized again inside the magnet with respect to reflected radio-frequency power. Radio-frequency pulse amplitudes (90° and 180° pulses) were calibrated manually and were found to be very similar for different animals attesting reproducible probe adjustments. The only parameter allowed to vary deliberately was the receiver gain determined automatically at the beginning of the image acquisition, variations were however small (maximum 3 dB). Images were reconstructed on the spectrometer computer (Aspect 3000), transferred to a Macintosh computer and analyzed using the N.I.H. Image software (http://rsb.info.nih.gov/nih-image/index.html). We corrected for different receiver gains by equalizing noise

level in the different images. To verify the constancy of signal to noise ratio on images acquired during different sessions, we compared the signal intensity in temporal lobe from noise adjusted images from young animals. Our previous studies had revealed that in the young age group signal intensity in this region should be rather constant [16]. The signal intensity was measured from circular regions of interest outlined bilaterally on two slices in the temporal lobe area. There was, indeed, no significant difference between the signal intensity in the temporal lobe of young animals studied during different sessions (one way ANOVA, F(4, 16) ⫽ 0.95; p ⬎ 0.1). Given the good reproducibility of experimental conditions and adjustments, images from different animals were directly compared. We assessed cerebrospinal fluid (CSF) volumes as an estimate of cerebral atrophy [33]. We used a segmentation method based on intensity thresholding to estimate pixels containing CSF [15,20]. On T2 weighted images, a voxel containing CSF is characterized by high signal intensity (bright) due to its long T2, whereas voxels containing brain matter present low signal intensity. An intensity threshold was determined to separate voxels of areas containing CSF from those containing only brain matter. The threshold was visually and simultaneously determined on the entire set of images using the high contrast between CSF containing voxels and brain tissue as well as anatomical information [5,9]. On each slice, all the pixels having an intensity superior to the threshold were automatically counted by the N.I.H. Image software. Their surfaces were converted into volume by multiplying them by the slice thickness (1 mm). A “global hyperintensity volume” estimation (CSF measure) was obtained by adding CSF volumes determined on four coronal contiguous slices passing through the whole sylvian fissure in the temporo-parietal region and through the third and lateral ventricles. This measure was used as an index of global cerebral atrophy. Measures of regional atrophy were estimated from CSF volumes in various regions: sylvian and interhemispheric fissures, bodies of the lateral ventricles, third ventricles, external and internal parts of the temporal horns of the lateral ventricles (ETH and ITH), chiasmatic fossa, interpeduncular fossa (seen only on one slice), and the cisterna around the entorhinal and piriform cortices (Ent C). Global and regional atrophies were evaluated with a four grades semi-quantitative scale (0: no atrophy; 1: slight atrophy; 2: moderate atrophy and 3: severe atrophy). To define the thresholds between the atrophy levels, adult animals younger than 3.5 years were taken as reference. The thresholds were Mcsf ⫹ (T ⫻ SDcsf) where Mcsf and SDcsf were the mean and standard deviation for CSF estimate in a given area for young adults. T was a constant equal to 2.58 for the threshold between zero and one levels, five for the threshold between one and two levels and 10 for the threshold between two and three levels. The 2.58 value corresponded to the 1% confidence interval of a gaussian population.

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Fig. 2. Compression of the left temporal area probably related to a former sub dural abscess or hematoma in an aged mouse lemur (double arrow; animal 9 in Table 1).

Fig. 1. MRI (a, arrow) and neuropathological (b; slice of 1 mm width, no staining) detection of a severe unilateral dilatation of the left temporal horn of the lateral ventricle in a young adult mouse lemur. The CSF volume assessed in that animal was 59.6 (arbitrary units).

An index of temporal atrophy was calculated by adding the atrophy grades from regions bordering the temporal area (sylvian fissures, ITH, Ent C). 2.3. Neuropathology Mouse lemurs’ brains were taken and fixed in 4% formalin after the natural death of animals. The brains were examined for neuropathology by using coronal brain sections from multiple regions including isocortex (frontal, motor and occipital), basal ganglia (pallidum, putamen, caudate nucleus), thalamus, substantia nigra, hippocampus, and amygdala. Seven microns thick paraffin sections were stained with routine methods, including hematoxylin-eosin, immunostains using a commercially available polyclonal antibody against tau (Dako®; Glostrup; Denmark). Two polyclonal antibodies were used to detect amyloid deposits: E50 (generously provided by H. Akiyama from the Psychiatric Institute of Tokyo) raised to the 17–31 fraction of human synthetic amyloid peptide and FCA 42 (generous

gift of F. Checler from the Institut de Pharmacologie Mole´culaire et Cellulaire de Valbonne, France) [2]. The G Ig fraction of the E50 antibody has been purified by affinity chromatography and its specificity has been confirmed by western blot with native and synthetic A-beta peptides. Slides were pretreated with 99% formic acid during 5 min to expose amyloid epitopes. The sections were left in 10% bovine serum for 30 min before incubation with the primary antibody at room temperature overnight. Anti-tau antibodies, E50 antibodies and FCA35– 42 antibodies were respectively diluted to 1/200, 1/2000, and 1/320. The following steps were performed using ChemMate detection kit (Dako®) according to the manufacturer’s instructions: sections were incubated in the pre-diluted biotinylated secondary antibody, 25 min at room temperature. Endogenous peroxidases were blocked by incubation in 20% methanol in distilled water containing 3% H2O2 for 20 min. Sections were then treated with pre-diluted streptavidin-peroxidase, 25 min at room temperature. Immunostaining was visualized with diaminobenzidine. Sections were counterstained with Harris hematoxylin, dehydrated, and mounted in DPX®.

3. Results 3.1. Global cerebral atrophy We assessed the global atrophy by estimating the total CSF volume in four coronal temporo-parietal slices. One young adult animal of 15 tested exhibited global cerebral atrophy. It was related to a severe unilateral dilatation of the temporal horn of the lateral ventricle (Fig. 1). The neuropathological study of its brain suggested an infarction probably related to an arachnoid cyst. Its atrophy measure had not been included in the analysis. One aged animal pre-

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Fig. 3. Global CSF volume assessment (global atrophy index) plotted against age in mouse lemurs. For the 14 animals followed up in a longitudinal way, a line is drawn between dots corresponding to different MRI scans. The increases in CSF volumes are represented by the slope of the curve (in CSF unit per month). The horizontal lines correspond to the three thresholds that were used to define atrophy. The animals in which regional atrophy has been detected are labeled by numbers (see Table 1). The hollow circles correspond to animals with amyloid deposits. The solid circles correspond to animals free of amyloid deposits.

sented a compression of the left temporal area probably related to a former sub dural pyometric or hematic neocollection (Fig. 2). In this animal, we assessed the cerebral atrophy by measuring the CSF volume in the intact hemisphere; the CSF measurements were normalized by multiplying them by two. The CSF volume in the group of aged animals was significantly higher than in the young group [Student’s t-test; t ⫽ ⫺3.96; df ⫽ 28, p ⬍ 0.0005 (first scans measurements taken into account for longitudinally

assessed animals)]. According to our grading scale, nine out of the 17 animals older than 3.5 years displayed a global atrophy: six out of 13 males and three out of four females (Fig. 3). The higher prevalence of cerebral atrophy in old animals was significant when the last scans measurements were taken into account for longitudinally assessed animals (chi-squared calculated with Yates’s correction ⫽ 7.47, df ⫽ 1, p ⬍ 0.01). Four out of the seven oldest animals were not atrophied (Fig. 3). Our preliminary longitudinal studies

Table 1 Semi-quantitative assessment of regional atrophy in atrophied mouse lemurs older than 3.5 years. Type of atrophy

Temporal

Diffuse

Temporal ⫹ ETH

Animals

1 2 3 4 5 6 7 8 9 10 11 12

Age

9.9 3.5 9.5 6 5 8.1 5.2 6.1 8.7 7.6 10.3 5.4

Frontoparietal areas

Temporal areas

Amyloid deposits

LV

IF

Sy

ITH

Ent C

Temp

ETH

0 0 0 0 0 0 1 1 1 2 3 1

0 0 0 0 0 0 2 1 1 2 0 0

0 0 0 0 0 3 2 2 3 3 3 0

0 2 1 3 3 1 0 3 2 3 2 2

3 1 3 1 1 3 1 1 1 2 3 0

3 3 4 4 4 7 3 6 6 8 8 2

0 0 0 0 0 0 0 0 0 0 1 3

? ? NO NO ? ? ? ? YES YES YES NO

The column “Temp” displays an index of temporal atrophy. The column “Amyloid deposits” mentions the main neuropathological findings in the animals in which we looked for amyloid deposits.

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Fig. 5. Severe ETH and moderate ITH dilatations in the 5.4-year-old animal (number 12 in Table 1).

Fig. 4. Comparison of the brains of an old atrophied animal and of a young normal animal. (a), severe dilatation of the ITH and sylvian fissures (Sy) and moderate atrophy of the Ent C area in a 7.6 years old animal (animal 10 in Table 1). This animal also presented with a diffuse atrophy characterized by a dilation of bodies of the lateral ventricles (LV) and of interhemispheric fissure (IF). Other abbreviations: 3V, third ventricles; CF, chiasmatic fossa; CP, choroid plexus. (b), brain of a 1-year and 10-monthsold animal without cerebral atrophy.

involving seven old animals revealed that in the five animals with the more severe atrophy, the high CSF volume resulted from a rapid increase in CSF volume over a few months (more than 0.75 units of volume per month) that began when the animals were 5- to 8-years-old. The two other old animals and the young animals did not present such a rapid increase in CSF volume (Fig. 3). 3.2. Regional atrophy CSF volumes were used to evaluate the regional atrophy. In young animals, the distribution of CSF volumes was the following: third ventricles: 58.5%, chiasmatic fossa: 16.9%, interpeduncular fossa: 6.4%, bodies of the lateral ventricles: 5.6%, ITH: 4.4%, interhemispheric fissure: 3.1%, ETH: 2.7%, Ent C: 1.6%, sylvian fissures: 0.6%. In 12 animals older than 3.5 years, the CSF volumes

were increased in specific areas of the brain. Three of them had no global atrophy. In nine of them, global cerebral atrophy was detectable. Different patterns of cerebral atrophy could be identified (Table 1). Six animals (animals one to six) presented a temporal atrophy shown by a dilation of the ITH, Ent C, and sometimes of the sylvian fissures. In another group of five animals (animals seven to 11), the temporal lobe atrophy was associated with a dilation of the lateral ventricles and sometimes the interhemispheric fissure revealing a diffuse atrophy (Fig. 4). One animal (animal 12) had an ETH dilation in addition to the temporal atrophy (Fig. 5). The total CSF volume in the six animals with the temporal atrophy alone was significantly lower than that in the five animals with diffuse atrophy (CSF assessment of 14.1 ⫾ 2.8 arbitrary units versus 20.5 ⫾ 4.1, Mann Whitney’s U ⫽ 2, p ⬍ 0.02) and also lower than that in the animal with the temporal atrophy associated to the ETH dilation (t-test, t ⫽ ⫺8.54, df ⫽ 5, p ⬍ 0.001). The five animals with the most severe global atrophies were four animals from the diffuse atrophy group and the animal with temporal atrophy associated to ETH dilation (Fig. 3).

3.3. Neuropathological study The brains of 2 aged animals with temporal atrophy alone, of 3 aged animals with a diffuse atrophy associated to a temporal atrophy and of the animal with the ETH dilation in addition to the temporal atrophy were examined for neuropathology (Table 1). We also examined the brain of the young animal with the unilateral dilatation of the temporal horn of the lateral ventricle. There was no evidence of small infarcts or white matter rarefaction in any animal. None of the animals had neurons stained by the antibody against abnormal human tau proteins from Dako®. The

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Fig. 6. Amyloid deposits in mouse lemurs. (a), amyloid deposits in temporo-parietal cortex (x2) of an animal with a severe diffuse atrophy (number 11 in Table 1). (b), cortical amyloid deposits in the same animal at higher magnification (⫻40). E50 anti amyloid beta staining counterstained with Harris hematoxylin.

three animals with diffuse atrophy had amyloid deposits. The most atrophied of them (animal 11) had many diffuse amyloid deposits in the hippocampus, thalamus, temporoparietal, and frontal cortices (Fig. 6). In the two other animals (9,10) amyloid deposits were rare and involved the temporo-parietal region. One of them (10) had also rare frontal deposits. The other animals were free of amyloid deposition.

4. Discussion MRI showed a global cerebral atrophy occurring only in aged mouse lemurs. However, several very old animals were not atrophied. Atrophy appeared thus to be an agerelated pathological alteration; it does not seem to be an inevitable effect of age. The increase of CSF volume took place within a few months in the atrophied aged animals, an observation that also favors the hypothesis of a pathological event, occurring at variable ages between five and eight.

The most frequently atrophied areas were the sylvian fissures, the bodies of the lateral ventricles, the interhemispheric fissure, ITH, ETH, and Ent C areas. In a first group of six animals the dilation of sylvian fissure, ITH, and Ent C areas were particularly severe indicating a predominant temporal atrophy. In a second group of five animals the dilation also involved the bodies of the lateral ventricles and sometimes the interhemispheric fissure, suggesting a diffuse cerebral atrophy. An atypical animal had an ETH dilation in addition to the temporal atrophy. The animals with diffuse atrophy and the animal with the ETH dilation presented the most severe global atrophies. By comparison to what happens in AD where temporal areas are the first ones to be involved in the neurodegenerative process [7], the limited temporal atrophy described in mouse lemurs could be the first step of a neurodegenerative process leading to the diffuse atrophy. However, in the four longitudinally followed animals that developed a diffuse atrophy but that were not atrophied during their first scan, temporal atrophy was not an obvious intermediate stage between the non-atrophic and diffuse atrophic stages (data not shown). In a first attempt to evaluate the origin of cerebral atrophy in mouse lemurs, we considered the relationship between presence of amyloid deposits and cerebral atrophy. Animals with diffuse atrophy were the only ones to display amyloid deposits. That amyloid deposits themselves were the direct cause of cerebral atrophy seems unlikely because the cortical amyloid burden was low at least in two of the three animals and because several studies in humans [30] and transgenic mice [6,8,18,23, 24,26] suggested that diffuse amyloid deposits are not directly toxic for the brain. Amyloid deposits could be the final visible “symptom” of another neurodegenerative process that would be responsible for the diffuse atrophy in lemurs. The direct cause of atrophy remains to be determined. Studies in humans have suggested that tau pathology is an early event in the AD neurodegenerative process [7,32]. In our study none of the animals had neurons stained by antibodies detecting AD-like abnormal sites of phosphorylation on tau proteins. However, tau immunoreactivity can be detected in cortical neurons of aged animals with specific antibodies [3,14]. In future studies, we will assess the relation between tau immunoreactivity and cerebral atrophy. In conclusion, mouse lemurs display cerebral atrophy. Animals with diffuse atrophy were the only ones in which diffuse amyloid deposits were visible. Not all of the animals undergo the neurodegenerative process. The detection and follow-up of the atrophic process in vivo by MRI makes it possible to search for the biological correlates of the ongoing neurodegeneration and could help the understanding of the aging brain.

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Acknowledgments We thank M. Perret from the Laboratoire d’Ecologie Ge´ne´rale of the Museum National d’Histoire Naturelle and the staff of the Laboratoire de Conservation des Espe`ces Animales of the Zoological Park of Paris for their collaboration in this study. We thank H. Akiyama (Psychiatric Institute of Tokyo) and F. Checler (Institut de Pharmacologie Mole´culaire et Cellulaire de Valbonne) for the gifts of the E50 and FCA 35– 42 antibodies. M.D. was supported by fellowships from the France Alzheimer Association, the Chancellery of the Universities of Paris, and the Bettencourt–Schueller Foundation.

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