The Veterinary Journal 186 (2010) 166–171

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The Veterinary Journal

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Magnetic resonance imaging of cerebral involutional changes in dogs as markersof aging: An innovative tool adapted from a human visual rating scale

Marco Pugliese a,*, Josep Lluís Carrasco b, Beatriz Gomez-Anson c, Carmen Andrade a, Angels Zamora d,Manuel José Rodríguez a, Joan Mascort e, Nicole Mahy a

a Unitat de Bioquímica, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spainb Unitat de Bioestadística, Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spainc Department of Radiology, Hospital de la Santa Creu i Sant Pau, IDIBAPS, 08025 Barcelona, Spaind Medical Resonancia Magnetica S. A., Sant Joan Despi, 08970 Barcelona, Spaine Servei de Neurología, Ars Veterinaria, 08023 Barcelona, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Accepted 4 August 2009

Keywords:Brain agingCanine cerebral atrophyHippocampusMagnetic resonance imagingVisual rating scale

1090-0233/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.tvjl.2009.08.009

* Corresponding author. Tel.: +34 93 402 45 25; faxE-mail address: marcopugliese@ub.edu (M. Puglie

The dog is increasingly considered as a natural animal model for the study of normal and pathologicalhuman brain aging, because it exhibits anatomical, biochemical and cognitive changes that parallel thoseseen in humans. This study presents a novel visual semi-quantitative rating scale of canine cerebral mag-netic resonance imaging (MRI). Ninety-eight dogs of both sexes from 27 pure breeds, aged 2–15 years,were used. The results suggest that (like in humans) both hippocampal and progressive global atrophyare characteristic features that correlate with aging. When classified according to head shape, cerebralatrophy was highest in mesaticephalic and brachycephalic dogs. This is the first MRI study to characterisea simple, rapid tool for studying age-related canine brain changes that can even be applied by non-expe-rienced observers. The results confirm the possibility of transferring innovative tools developed forhuman diagnosis to the veterinary field.

� 2009 Elsevier Ltd. All rights reserved.

Introduction

Several reports have indicated that dogs with a gene sequencefor amyloid precursor protein (APP) identical to that of humansand ApoE4 homogeneous may be valuable in bridging the gapbetween transgenic models and human beings (Johnstone et al.,1991; Cummings et al., 1996). Dogs over 8 years old, an age asso-ciated with the first stage of cerebral amyloid-beta depositionand high levels of oxidative stress (Head et al., 2000; Papaioannouet al., 2001), exhibit multiple geriatric behavioural modifications.These can be classified into two groups: (1) normal aging and (2)canine with cognitive dysfunction syndrome (CDS) (Puglieseet al., 2005, 2006a,b; Rofina et al., 2003). In spite of being two dif-ferent pathologies, the latter group also presents the commonneuropathological hallmarks of human cerebral aging and Alzhei-mer’s disease (AD), such as cerebral atrophy, congophilic angiopa-thy, neuronal loss and tau phosphorylation (Colle et al., 2000;Pugliese et al., 2004, 2006b).

Both global and focal brain atrophy identified by magnetic res-onance imaging (MRI) studies in humans may be studied using dif-ferent methods, such as volumetric analysis, visual rating scales,

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: +34 93 403 58 82.se).

linear measurements and co-registration techniques (Good et al.,2001; Jack et al., 2004). Each method has advantages and disadvan-tages. For example, although volumetric studies provide quantita-tive measurements of atrophy, and may seem more accurate,visual rating assessments are much quicker and allow researchersto work with a large number of scans (Bresciani et al., 2005; Schel-tens et al., 1992). Thus, a simple, widely applicable visual ratingscale allowing scores to be dichotomised (0–2 vs. 3–4) into normalvs. AD is sufficient for diagnostic purposes (Galton et al., 2001).This scale has good intra-rater and inter-rater reliability and hasbeen validated in human brain against both linear and volumetricmeasures (Scheltens et al., 1992, 1995; Vermersch et al., 1994).

MRI techniques are currently applied to dogs in clinical practicein order to confirm a suspected lesion or to identify its extent orlocation. They are also used for the diagnosis of central nervoussystem (CNS) diseases such as neoplasia (Liu et al., 2004), inflam-mation (Lotti et al., 1999) or brain anomalies (hydrocephalus) (Sai-to et al., 2003), but are not commonly used for the prediction,diagnosis or follow-up of the canine counterpart of senile dementiaof the Alzheimer’s type (ccSDAT). The relationship between pro-gressive canine brain atrophy and aging has been evidenced in sev-eral post-mortem studies (Reifinger, 1997; Borras et al., 1999;Gonzalez-Soriano et al., 2001) and recently confirmed in houseddogs, frequently Beagles, with a variety of MRI techniques (Kimot-suki et al., 2005; Tapp et al., 2006).

M. Pugliese et al. / The Veterinary Journal 186 (2010) 166–171 167

We decided to conduct an MRI study in companion dogs usingan adapted version of a human visual rating assessment. The goalsof this study were as follows: (1) to develop and validate a reliableand rapid universal method of canine brain atrophy measurement;(2) to determine whether progressive global cerebral atrophy is re-lated to aging in these animals, and (3) to analyse hippocampalatrophy in these conditions. Because canine skulls vary more inshape and size than any other part of the skeleton, data were alsoanalysed after classification of the animals into dolicho-, mesati-,and brachycephalic breeds to determine whether brain atrophy de-pends on head shape.

Table 2Cerebral rating scale assessment.

Global cerebral atrophy (GCA)Depth of sulci 0 1Ventricular size 0 1

HippocampusWidth of temporal horn 0 1Height of hippocampus 0 1

Total scale result 0 4

Focal cerebral atrophyFrontalParietalTemporo-occipital No Yes

Total scale result: 0 = No cerebral atrophy; 1–2 = Moderate cerebral atrophy; 3–4 = Severe cerebral atrophy.

Materials and methods

Subjects

Cerebral MRIs of 98 dogs from 27 pure breeds of different weight, sex and age,as certified by their medical records from the veterinary hospital Ars Veterinaria(Barcelona), were used in this study (Table 1). The dogs were classified accordingto their cranial type by two independent and highly experienced veterinary anato-mists following the criteria given by Evans (1993). The classification resulted in thefollowing categories: brachycephalic (n = 28; Boxer [16], Yorkshire [7], Pekinese [2],French bulldog [2], Rottweiler [1]); mesaticephalic (n = 47; German shepherd [21],Poodle [11], Golden retriever [3], Miniature Schnauzer [2], French Spaniel [2], Bea-gle [2], Dalmatian [1], Standard Schnauzer [1], Alaskan Malamute [1], Bearded Col-lie [1], Bobtail [1], West Highland Terrier [1]); and dolichocephalic animals (n = 23;English Cocker Spaniel [8], Fox Terrier [3], Irish red Setter [3], English Setter [2],Rough Collie [2], Doberman [1], Pinscher [1], Giant Schnauzer [1], Basset Hound[1], Italian Pointer [1]) (Table 1). Dogs presenting with CNS alterations, such as abrain tumour, encephalitis, head trauma, or malformations that could interfere withthe measurements, were excluded.

All animals were treated in accordance with European legislation on animalhandling and experiments (86/609/EU) and procedures were approved by the Eth-ical Committee of the University of Barcelona. Every effort was made to minimiseanimal suffering and to use no more than the number of animals needed to obtainreliable scientific data. All dog owners agreed to participate in the study.

MRI

The MRI study was conducted at Medical Resonancia Magnetica, SA (Sant JoanDespi, Barcelona) with a Toshiba MR system MRT-150A, operating in a 1.5 T mag-netic field. Imaging procedure included sedation of the dogs with diazepam0.5 mg/kg IV (Valium, Roche), and induction of anaesthesia with propofol, 4 mg/kg IV (Diprivan, Astra Zeneca). After the insertion of an endotracheal tube, dogswere positioned into the magnet bore head first in a prone position with their headand neck extended. Anaesthesia was maintained by using 2% isofluorane/oxygengas mixture. Monitoring devices were used to measure heart rate, blood pressure,and blood oxygenation levels during anaesthesia.

The following pulse-sequences were used: Spin-Echo T1-weighted (repetitiontime ms/echo time ms/matrix = 500/15/160 � 160), Fast Spin-Echo T2-weighted(repetition time ms/echo time ms/matrix = 5000/112/160 � 160). Images were ac-quired with 1–2 averages, with a 4–5 mm section thickness, and a 20% gap in thecoronal, horizontal and sagittal planes. This acquisition technique was chosenbecause of the increased spatial (about 3 mm) and contrast resolution of the

Table 1Characteristics of dogs used in the study.

Number of animals Male Female

Age

<8 years old 41 24 17>8 years old 57 30 27

Weight

<10 kg 25 10 1510–25 kg 26 11 15>25 kg 47 33 14

Breed

Brachycephalic 28 16 12Dolichocephalic 23 13 10Mesaticephalic 47 25 22Total 98 54 44

sequences, which provide adequate differentiation between grey and white mat-ters. All dogs were confirmed negative for intracranial organic lesions by a veteri-nary neuroradiologist. Once the scan was completed, dogs recovered in individualcages under veterinary control.

Visual rating assessment

Hard copies of T1 coronal MRI images of the 98 dogs were assessed by twoblinded raters (BGA who has had extensive experience in MRI evaluation, and CAwho had no experience).

The study was performed with hard copies of T1 coronal MRIs because the coro-nal images seemed easier for a non-trained observer to rate. The scans were pre-sented randomly. The method of Galton et al. (2001) was adapted to evaluatecanine atrophy, as was the scale described in detail by Scheltens et al. (1992). Inbrief, a three-point scale of global cerebral atrophy (GCA), (normal = 0, mildatrophy = 1, severe atrophy = 2) was used to qualify various cerebral sulci (marginalsulcus, middle suprasylvian sulcus, ectosylvian sulcus) and ventricular size. A three-point scale of hippocampal atrophy (no atrophy = 0, mild atrophy = 1, severe atro-phy = 2) was also used to qualify the height of the hippocampus and the size ofthe temporal horn. Focal cerebral atrophy was assessed in the frontal, parietaland temporal-occipital regions, and its presence/absence was recorded (yes/no,Table 2).

Statistical analysis

Mixed-effect models were fitted to test the association between the score andthe explanatory variables. The association was tested by the F-Wald statistic(Brown and Prescott, 1999). The normality assumption for random effects andresiduals was tested by quantile–quantile plots. The score was standardised byage and observer in all the models by including these effects as covariates. Posthoc comparisons were carried out by adjusting P values using the Tukey’s approach.The concordance between observers was calculated by the concordance correlationcoefficient (CCC) estimated by variance components (Carrasco and Jover, 2003), andthe kappa (j) statistic was used for the dichotomised scale. Precision and accuracycomponents of CCC are also reported. For the dichotomised scale the precision wasestimated by means of the phi (U) coefficient, while the accuracy was assessed by

<10 kg 10–25 kg >25 kg9 11 2116 15 26

Brachycephalic Dolichocephalic Mesaticephalic8 7 137 13 6

13 6 28

<8 years old >8 years old18 10

9 1414 33

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comparing the marginal probabilities applying the McNemar’s test. The intra-raterconcordance was evaluated by the intra-class correlation coefficient. All thehypotheses were assessed using a type I error rate of 5%.

Results

Canine cerebral atrophy

GCA and hippocampal atrophy were rated for the MRI of eachdog. Discrimination between normal and atrophied brains, takinginto account depth of sulci, ventricular size, width of temporal hornand height of hippocampus could be well differentiated in all ani-mals (Fig. 1). After standardisation by age and observer, when ani-mals were classified into younger or older than 8 years old, scoresgiven by both observers (BGA and CA) revealed a significantly posi-tive correlation between age and cerebral atrophy. This correlationwas given by the total score value (P = 0.0038), and by the GCA(P = 0.0136) or hippocampus scores (P = 0.009) separately (Fig. 2A).

When the correlation between score and animal weight wasanalysed, significant differences were found between the 10–25 kg animal group and <10 kg animal group (P = 0.0211), mostlydue to lower hippocampus scores (P = 0.006) (Fig. 2B). When thescore analysis was related to the canine skull, dolichocephalic dogs

Fig. 1. T1-coronal MR images of dogs with three different head shapes with their ratinhorns, and hippocampal height were evaluated (0 or 1) for assessment of cerebral atrop(ventricular size 1 and cerebral sulci 1); score 3 (ventricular size 1, cerebral sulci 1, anhippocampus 1, and width of temporal horn 1). (A) T1-representative coronal MR imageold); Score 2 (Cocker, 7 years old); Score 3 (Cocker, 10 years old); Score 4 (Fox Terrier, 10(German shepherd, 3 years old); Score 1 (German shepherd, 4 years old); Score 2 (Alaska15 years old). (C) T1-representative coronal MR images of brachycephalic dogs: Score 0 (old); Score 3 (Boxer, 7 years old); Score 4 (French Bulldog, 11 years old).

presented significantly lower total scores than the brachycephalicand mesaticephalic animals (P = 0.0158) due to their GCA score(P = 0.0168; Fig. 2C). Finally, no focal atrophy was observed inany of the 98 MRIs.

Intra-rater reliability

For the total value, the two observers presented an excellentsimilar intra-rater repeatability (ICC) of 0.97. For the GCA rating,CA presented a higher repeatability (ICC � 1) than BGA(ICC = 0.91), and for the hippocampal assessment the observershad an excellent similar intra-rater repeatability (BGA: ICC =0.96; CA: ICC = 0.94).

Inter-rater agreement

The inter-rater agreement was fair (BGA vs. CA, CCC = 0.65, CI95% 0.52–0.74) for the total value. The precision component was0.67 whilst the accuracy component was 0.96. The closeness to 1of the accuracy component reflects the proximity of the meanvalues of each rater that was estimated in 0.38 units. For theGCA and hippocampal ratings, the inter-rater reliability was

gs of cerebral atrophy (0–4). Cerebral sulci, ventricular size, width of the temporalhy (specified in A). Score 0 (each parameter 0); score 1 (ventricular size 1); score 2d height of hippocampus 1); score 4 (ventricular size 1, cerebral sulci 1, height ofs of dolicocephalic dogs: Score 0 (Dobermann, 4 years old); Score 1 (Cocker, 5 yearsyears old). (B) T1-representative coronal MR images of mesaticephalic dogs: Score 0n Malamute, 9 years old); Score 3 (German shepherd, 9 years old); Score 4 (Poodle,

Boxer, 5 years old); Score 1 (Yorkshire, 7 years old); Score 2 (French Bulldog, 9 years

Fig. 2. (A) Significant correlation between cerebral atrophy and age using both GCA and hippocampal scores. (B) Significant correlation between cerebral atrophy and <10 kganimal group mostly due to the hippocampal scores. (C) Significant correlation between cerebral atrophy and brachycephalic and mesaticephalic breeds, mostly due to higherGCA scores. *P < 0.05; **P < 0.01.

M. Pugliese et al. / The Veterinary Journal 186 (2010) 166–171 169

slightly lower (BGA vs. CA, CCC = 0.51 and 0.5, respectively), withthe accuracy component around 0.96. Dichotomisation of the scaleinto normal to moderate atrophy (scores 0–2) vs. severe atrophy(scores 3–4) gave a low agreement between raters (j = 0.49), witha CI 95% of [0.32; 0.66]. The U coefficient gave a value of 0.5 whilethe difference between marginal probabilities was 0.09 resulting ina non-significant difference (P = 0.07).

Discussion

This is the first MRI study to describe the use by a non-experi-enced observer of a tool for studying age-related changes in dogs asa model for human brain aging. The study was carried out in anextensive, heterogeneous group of dogs, including 27 pure breedsof different ages and sex, to determine whether involutional brainchanges correlate with aging. To do this, we developed a newmethod of visual rating assessments of MRI in dogs using a scalesimilar to one previously applied in humans (Galton et al., 2001).Challenging previous results by other authors (Reifinger, 1997),our data clearly demonstrate a progressive cerebral atrophy withcanine aging, which also involves the hippocampal formation andis similar to the changes present in aging humans.

The significant atrophy beginning around the age of 8 yearsconfirmed this border value as important for canine brain agingand the formation of neuropathological markers (Head et al.,

2000; Pugliese et al., 2006a,b). Thus, brain atrophy due to agingwas significantly higher in the mesaticephalic and brachycephalicdogs, mostly due to the GCA score.

These two groups were most affected, probably due to their ge-netic predisposition for developing ventricle dilatation, in spite ofthe major discrepancies in the literature regarding this classifica-tion (Brehm et al., 1985; Evans, 1993; Seiferle, 1966). In our study,classification was agreed by two independent and highly experi-enced veterinary anatomists. Even though the ventricle enlarge-ment and asymmetry frequently observed in brachycephalicbreeds (Esteve-Ratsch et al., 2001) may have caused both observersto award excessively high GCA values, they cannot explain the hip-pocampal atrophy results.

The data showed similar results in the mesaticephalic group,which presents no specific predisposition for developing ventricleenlargement. In fact, GCA appeared related to canine head shapeand aging, whereas hippocampal atrophy varies only with aging,especially in dogs weighing <10 kg. Because of the large diversityof breeds present in each group (e.g. Pekinese and Boxer breeds be-long to the brachycephalic animals), bodyweight did not correlatewith the head shape classification. Aging appears to be the com-mon factor of atrophy in all groups of animals. However, themarked hippocampal atrophy observed in the <10 kg dogs wasnot related to any other factor and could reflect the difficulty ofevaluating smaller brains.

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The major interest of our new scale comes from its ease of useand its reliability with trained observers. Progressive brain atrophyhas been described and quantified using MRI studies in humans(Chan et al., 2003). In contrast to normally aging subjects, AD pa-tients have predominantly an early atrophy of medial temporallobe structures (mainly the entorhinal cortex and hippocampus)(Du et al., 2003). This involvement of specific brain regions is usefulfor early AD detection by MRI (Silbert et al., 2003). Our study dem-onstrates that hippocampal formation plays a significant part in ca-nine cerebral atrophy and can be considered an early marker ofcerebral aging.

Qualitative ratings of human focal brain atrophy judged by anexperienced observer are considered to be of more utility thanquantitative measures. This is especially true in conditions inwhich there is hippocampal atrophy, such as normal aging (Lyeet al., 2004) or mesial temporal sclerosis (Cheon et al., 1998). Pla-nimetric and volumetric evaluations show good discrimination,but are far more demanding in terms of time and training require-ments and are too expensive for veterinary use (Tapp et al., 2006).Using rating scales, an inexperienced examiner can also correctlydetect hippocampal and global cerebral atrophy, as was shown inour study. So our visual rating scale allows the application ofMRI techniques to the evaluation of canine brain aging.

The intra-rater repeatability of both observers was high. CA pre-sented higher reliability in the evaluation of GCA, whereas BGAshowed similar reliability in the evaluation of both GCA and hippo-campus. This indicates the possibility of easily following up theprogression of cerebral atrophy by an inexperienced observer.The higher GCA mean values given by both observers suggest ahigher impairment in aging that makes it easier to evaluate. Thebetter GCA discrimination presented by the non-experienced ob-server supports this possibility. As a result, semi-quantitativeGCA ratings may be sufficient to grade changes appearing withaging, and may be more reliable than hippocampus measurementsmade by a non-trained observer.

Overall, the inter-rater agreement was fair and the accuracycomponent good; the main source of discordance was the lack ofprecision. For that reason, supervision by an experienced observeris recommended for the widespread use of this new instrument.

Conclusions

Semi-quantitative MRI assessment of canine brain using thisnew, easy and quick rating scale provided measures of cerebralatrophy of high repeatability that correlated with aging. The studyreinforced the notion that the brain aging process in dogs and hu-mans is similar, and shows the possibility of transferring innova-tive tools developed for diagnosis in human medicine to theveterinary field. Finally, as also has been shown by others (Hase-gawa et al., 2005), our data confirmed the possibility of using com-panion dogs to investigate cerebral atrophy in man.

Conflict of interest statement

None of the authors of this paper has a financial or personalrelationship with other people or organisations that could inappro-priately influence or bias the content of the paper.

Acknowledgements

The research projects CIBERNED of the Spanish Ministerio deCiencia e Innovación, SAF2005-04314 of the Spanish Ministeriode Educación y Ciencia and 2005SGR00609 of the Generalitat ofCatalunya, Spain, supported this study.

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