Journal of Virological Methods 136 (2006) 137–146

Assessment of calcium-binding proteins (Parvalbumin and Calbindin D-28K)and perineuronal nets in normal and scrapie-affected adult sheep brains

Enric Vidal a, Rosa Bolea b, Raul Tortosa c, Carme Costa c, Anna Domenech c, Eva Monleon b,Antonia Vargas b, Juan Jose Badiola b, Martı Pumarola a,c,d,∗

a PRIOCAT Laboratory, CReSA, Veterinary Faculty, Universitat Autonoma de Barcelona, 08193 Bellaterra (Cerdanyola del Valles), Barcelona, Spainb National Reference Centre for Transmissible Spongiform Encephalophaties, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain

c Department of Animal Medicine and Surgery, Institute of Neuroscience, Veterinary Faculty, Universitat Autonoma de Barcelona,08193 Bellaterra (Cerdanyola del Valles), Barcelona, Spain

d Animal Tissue Bank of Catalunya (BTAC), Veterinary Faculty, Universitat Autonoma de Barcelona,08193 Bellaterra (Cerdanyola del Valles), Barcelona, Spain

Received 23 November 2005; received in revised form 12 April 2006; accepted 2 May 2006Available online 7 July 2006

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bstract

Scrapie is a prion disease in small ruminants that manifests itself with neurological clinical signs amongst which are ataxia and tremors.hese signs can be explained partially by an imbalance in central inhibitory innervation. The study of the brain’s inhibitory neuronal GABAergicopulations and of their extracellular matrix has been used to define, in part, the pathogenesis of human prion diseases and scrapie models inodents.

The brain’s distribution of neuronal GABAergic subpopulations has been monitored carefully using, as markers, antibodies against the cal-ium binding proteins parvalbumin and calbindin D-28K. The distribution of this perineuronal net marker was evaluated by means of affinityistochemistry with W. floribunda agglutinin.

These techniques were performed on the brains of nine scrapie-positive sheep and on four infection-free sheep. These animals had undergonereviously a clinical follow-up as well as a lesion profile and an immunohistochemical profile of the scrapie-associated prion protein deposition inhe brain.

The study of calcium-binding proteins revealed an alteration of the parvalbumin positive GABAergic neuronal subpopulation. In scrapie-positiveases, a reduction in stained neuronal perykaria was observed, along with a marked reduction of neurite labelling. This finding was noticeable inegions such as the neocortex, particularly the motor frontal cortex, and was concomitant with a moderate PrPsc deposition and a mild degree ofesion. No changes were observed in the extracellular matrix study.

The results of the present study provide a partial explanation for the mechanisms of scrapie clinical signs due to a disturbance of the parvalbumin-ositive inhibitory neuronal population.

2006 Elsevier B.V. All rights reserved.

eywords: Scrapie; GABA; Calbindin; Parvalbumin; Extracellular matrix

. Introduction

Scrapie belongs to the group of transmissible spongiformncephalopathies (also known as TSEs or prion diseases) ofmall ruminants (sheep and goats). Clinical signs include ataxia,ead tremors, pruritus, altered mental status and other signs suchs teeth grinding, paresis, low head carriage, positive nibble

∗ Corresponding author. Tel.: +34 935811433; fax: +34 935813142.E-mail address: marti.pumarola@uab.es (M. Pumarola).

response and poor body condition (Healy et al., 2003; Houstonand Gravenor, 2003). Amongst these signs, ataxia and headtremors, which seem to have the highest incidence in naturalscrapie (Healy et al., 2003), may be explained partially by acentral inhibitory imbalance (Elble, 1996).

Selective neuronal subpopulation vulnerability has beendescribed in certain human prion diseases (Belichenko et al.,1999; Ferrer et al., 1993; Guentchev et al., 1997, 1999), andscrapie experimental models in rodents (Bouzamondo et al.,2000; Guentchev et al., 1998; Lu et al., 1995) affecting GABAer-gic inhibitory neural subsets. �-Aminobutyric acid (GABA) is a

166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2006.05.008

138 E. Vidal et al. / Journal of Virological Methods 136 (2006) 137–146

major inhibitory neurotransmitter in the central nervous system(CNS) (Roberts, 1986). Parvalbumin and Calbindin D-28K arecalcium-binding proteins (CBPs) expressed mainly in inhibitoryGABAergic interneurons, and antibodies against both proteinshave been used extensively to detect these subsets of GABAer-gic neuronal populations by immunohistochemistry (Andressenet al., 1993; Celio, 1990; Polgar and Antal, 1995; Webster et al.,1990).

In certain cases, loss of a given subpopulation of neuronshas been associated to disturbances of the perineuronal netsembedding such neurons (Belichenko et al., 1999; Guentchevet al., 1999). This might be due to an activation of microglia thatreleases proteases possibly responsible for the destruction of per-ineuronal nets (Belichenko et al., 1999), as occurs with microgliaactivation in HIV-induced neuropathy. However, more recentreports on experimental TSE models suggest that involvementof the GABAergic pathways exists, but does not show selectivevulnerability, being affected instead in a similar manner to otherpathways (Bouzamondo-Bernstein et al., 2004).

Agglutinin from W. floribunda (WFA) is a lectin that bindsto N-acteil galactosamide glucidic residues forming certain glu-cosaminoglycans in the brain’s extracellular matrix proteogly-cans. It is thus a marker for perineuronal nets. It has beendemonstrated in rodents that WFA has an area-specific distri-bution within the brain (Bruckner et al., 1994) and this has beenrelated to GABAergic neural populations (Hartig et al., 1992) aswb2

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involvement of CBPs and perineuronal nets in ovine scrapie,field-case pathogenesis, a matter that has been studied widelyin several human and animal TSE models with controversialresults.

2. Materials and methods

2.1. Animals and general procedures

Two groups of animals were included in the present study:adult control animals (n = 4) and animals with scrapie (n = 9).Details on the breed, age, sex and genotype for codons 136,154 and 171 are provided in Table 1. All scrapie cases wereanimals diagnosed within the framework of the National TSESurveillance programme at the Spanish National ReferenceCentre for TSE diagnostics. Scrapie status was determined invivo by means of neurological examination and/or third eyelidbiopsy. The animals originated from five different outbreaks ofscrapie, detected either through active or passive surveillanceprogrammes. Further flock screening allowed identification andclinical examination of other concomitant scrapie cases withinthe scrapie-positive flocks. Only animals examined clinicallywere included in this study.

After necropsy, the brains were immersed in 10% phosphate-buffered formalin for 10 days. After fixation, 4 mm-thicktissue sections were blocked and immersed in 98% formicadetewtp

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ell as to glutamatergic populations, particularly those inhibitedy the gabaergic, thus rich in GABA receptors (Wegner et al.,003).

The aim of the present study firstly was to examine the patho-ogical stage of nine animals with scrapie at an advanced clinicaltage of the disease, by the lesion profile and the, PrPsc distribu-ion profile. The second objective was to describe the distributionf CBPs, as markers of the GABAergic population, and that ofhe WFA-positive perineuronal nets in normal adult sheep brains.he next step was to compare these to scrapie sheep brains andetermine if the changes could be related to the pathologicalndings detected in the first part of the study. Finally, it is con-idered whether this might somehow explain the clinical signsecorded before culling. This is a descriptive study outlining the

able 1etails of the animals included in the study

umber Case Age (years) Genotype Sex B

-73/02 SC 1 6 ARQ/ARQ F R-86/02 SC 2 3 ARQ/ARQ F R-138/03 SC 3 5 ARQ/ARQ F R-139/03 SC 4 5 ARQ/ARQ F R-248/03 SC 5 7 ARQ/ARQ F R-107/03 SC 6 Adult ARQ/ARQ F R-85/02 SC 7 4 ARQ/ARQ M R-120/03 SC 8 5 ARQ/ARQ F R-122/03 SC 9 Adult ARQ/ARQ F R-442/04 CT 1 4 ARQ/ARQ F R-441/04 CT 2 3 ARQ/ARQ F R-439/04 CT 3 5 ARQ/ARQ F R-438/04 CT 4 4 ARQ/ARQ F R

bbreviations: SC (scrapie), CT (control), F (female), M (male), NA (not analy

cid for 1 h to reduce prion infectivity. Tissues were thenehydrated through increasing concentrations of alcohol andmbedded in paraffin wax. Four micrometer sections werehen obtained and routinely stained with hematoxilyn andosin to evaluate morphological change. Further sectionsere then cut and mounted on triethoxysilyl propylamin-

reated glass slides for immunohistochemical and histochemicalrocedures.

The brain areas dissected for study were the following: piri-orm cortex (Pfc), hippocampus (H), occipital cortex (Oc), tem-oral cortex (Tc), parietal cortex (Pc), frontal cortex (Fc), stria-um (S), thalamus (T), mesencephalon (M), pons (P), medullablongata (Mobl), spinal cord (Sc), cerebellar medulla (Cm),erebellar hemispheres (Cc) and cerebellar vermis (Cv).

Outbreak Third eyelid biopsy Postmortem delay

ragonesa 1 Positive 30 minragonesa 2 NA 24 hragonesa 4 Positive 10 minragonesa 5 Positive 10 minragonesa 1 Negative 10 minilbilitana 1 Inconclusive 30 minragonesa 1 Positive 1 hragonesa 3 NA 10 minragonesa 3 NA 10 minragonesa – – 10 minragonesa – – 10 minragonesa – – 10 minragonesa – – 10 min

E. Vidal et al. / Journal of Virological Methods 136 (2006) 137–146 139

2.2. Immunohistochemistry

PrPsc immunohistochemistry was carried out as describedpreviously (Monleon et al., 2004). Briefly, the following pre-treatments were applied: immersion in formic acid for 15 min,proteinase K treatment for 15 min at 37 ◦C (Roche, Switzerland;4 �g/ml) and hydrated autoclaving. Monoclonal antibody L42(R-Biopharm, Germany; 1 in 500 for 30 min) as the primaryantibody, EnVisionTM (DAKO, Denmark) as the visualizationsystem and 3.3′ diaminobenzidine (DAB) as chromogen wereused.

The protocols for the remaining primary antibodies usedheat-induced epitope retrieval pretreatment with citrate buffer(pH 6.0); antibody binding was visualized with the avidinpreoxidase–biotin complex (Pierce) for the MAb Calbindin D-28K (CALB, 1 in 800, Sigma) and MAb Parvalbumin (PV, 1 in600, Sigma).

2.3. Semiquantitation and data analysis

Semiquantitation of the different parameters studied(immunolabelling – IL – and histopathological lesions) was car-ried out. Scores from 0 (absence of lesion or IL) to 5 (maximumintensity of lesion or IL) were assigned to each brain area studiedand profiles were drawn. A general estimation of the PrPsc levelwP

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with CaCl2, MgCl2·6H2O and MnCl2·4H2O, 1 mM. Finally, thebinding was carried out with the avidin–peroxidase biotin com-plex (Pierce) and developed with 3,3′-diaminobenzidine.

2.5. Double labelling

Double labelling with MAb PV and WFA was carried outfollowing a two-step protocol. The sections were processedfor MAb PV signalling as described previously, the reactionbeing visualized with DAB. After washing, the sections wereincubated with the biotinilated lectin WFA and the reactionwas developed with 0.1% benzidine dihydrocloride and 0.025%sodium nitroferricyanide in 0.001 M phosphate buffer (pH 6)and 0.005% hydrogen peroxide. The second reaction yieldeda granular bluish precipitate different from the diffuse brownstaining obtained with DAB.

3. Results

3.1. Clinical signs

The most consistent signs observed in the sheep with scrapieincluded in this study were pruritus (9/9) and wool/skin distur-bances (8/9), along with a generalised decrease in the physicalcondition. Neurological signs were also present: alteration oftaaTa2

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as performed taking into account the amount and intensity ofrPsc IL. Each area was studied as a global region for the score.

The results are plotted as a function of the anatomical area.reas are ordered in the X axis in an attempt to represent the

audorostral axis of the brain. This methodology was adaptedrom a previous study carried out on BSE-infected cattle (Vidalt al., 2005).

Origin 7.0 software was used for data analysis.

.4. Affinity histochemistry

Histochemistry using Wysteria floribunda Biotinylated Aglu-inin (WFA, Vector), which binds to N-Acetilgalactosamide, augar residue attached to proteoglycans composing the brain’serineuronal nets, was performed to evaluate the status ofhe extracellular matrix. Endogenous peroxidase activity waslocked on deparaffinised sections and these were incubatedvernight with the lectins. The washing buffer was supplemented

ig. 1. Lesion profiles. Scores for (A) spongiosis (SP), (B) intraneuronal vacuocrapie. Note the parallel shape of the three curves with a consistent peak at hears represent standard deviation. For brain area abbreviation, see paragraph 3 i

he motor function was shown as tremors (6/9), ataxia (7/9)nd recumbency (7/9). In addition, the mental status was alsoltered with some animals with marked hyperexcitability (6/9).he signs described are in keeping with previous clinical char-cterisations of Scrapie in this breed (Vargas et al., 2005,006).

.2. Neuropathological changes

Classical TSE lesions, namely neuropil spongiosis and neu-onal vacuolation, were evaluated semiquantitatively (Fig. 1),s described in the Section 2. Lesions were confined mainlyo the brain-stem region and the thalamus, the pons being onef the most affected regions. Although particular nuclei in therainstem, at the obex level, were severely affected such as theagus nerve dorsal motor nucleus, the trigeminal spinal tract orhe solitary tract, as a whole, the pons and the thalamus sec-ions evaluated showed higher lesion scores than the medulla

(VAC) and (C) PrPsc deposit in each brain area (horizontal axis) in sheep withstem region. Values are the mean of the nine scrapie-positive animals, verticaltion 2.

140 E. Vidal et al. / Journal of Virological Methods 136 (2006) 137–146

oblongata. Neuronal loss was not evaluated, although some signsof neuronal death (dark neurons) and degeneration (spheroids,pericarion swelling) were occasionally observed. An increasein the cellularity due to glial activation was also noticed, it wasparticularly heavier at the brainstem and thalamus. The lesionprofile of the positive sheep is in keeping with the classicaldescriptions of scrapie neuropathology.

3.3. PrPsc immunohistochemistry

In some cases, the IL was relatively confined to the brainstem while, in others, PrPsc deposits were widespread through-

out the encephalon. A clear tropism of the PrPsc IL was seentowards the rombencephalon, mesencephalon and diencephalonand, in contrast to this, poor tropism towards the telencephalonwas noticed, although the archicortex (represented by the Hip-pocampus region in the profiles) also showed a considerableamount of PrPsc (see Fig. 1C).

None of the control animals showed PrPsc IL in any brainarea.

It is worth mentioning that in the neocortex the PrPsc IL wasdistributed mainly in the deeper cortical layers, particularly theIV and V, adopting a fine punctate pattern in the neuropile andintraneuronal fine granular deposits.

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ig. 2. Parvalbumin immunoreactivity in control (A, C, E) and scrapie cases (B, D, Fn control brains (C), the neurites are nicely depicted by PV positivity (arrowheads), wC and D) bar 25 �m; (E and F) bar 50 �m.

). Note the reduction in stained neuronal bodies and neuropil in scrapie brains.hile this labelling is practically absent in scrapie cases. (A and B) Bar 100 �m;

E. Vidal et al. / Journal of Virological Methods 136 (2006) 137–146 141

Fig. 3. Mean semiquantitative scoring of the PV IL (from 0: absence of IL to 5: maximum IL intensity) of both pericarya and neurites was significantly reduced inscrapie affected animals (SC) in certain areas when compared to control animals (CT). Bars: standard deviation within each group.

3.4. CBPs immunohistochemistry

3.4.1. Calbindin D-28K immunoreactivityIn normal ovine brains, CALB IL was observed only in

the cerebellum, strongly staining the Purkinje-cell pericariaand neurites as well as basket-cell prolongations. Neurons inthe deep cerebellar nuclei showed perineuronal staining witha synaptic-like pattern. The nuclei neuropile also showed agranular/linear staining pattern due to the positive neurites,probably Purkinje-cell axons. Finally, staining of neuronal bod-ies in the olivary nucleus in the medulla oblongata was alsoobserved.

No significant differences where noticed when comparingcontrol with scrapie-affected animals.

3.4.2. Parvalbumin immunoreactivityIn normal ovine brains, PV IL was demonstrated through-

out the brain, albeit with a different distribution and intensitydepending on the region.

Staining in the neocortex was notably constant in the differ-ent cerebral lobes, except for the temporal lobe, which showed aless intense staining. IL was observed within the non-pyramidalneuronal pericaria and neurites in the deeper layers of the cor-tex; layers IV and V were those having the highest numbersof stained neuronal bodies, but some stained bodies were seenalso in layers III and VI. A neuropil-pattern staining of neurites

was also observed (maximum intensity in layers IV and V), aswas perineuronal synaptic-like staining (Fig. 2A and C). Onlya few scattered neurones were stained in the striated body, thepiriform lobe (paleocortex) and the hipocampus (archicortex).A particular nucleus, the thalamus reticular nucleus, revealedall of its neurones as immunolabelled, in addition to showingstrong neuropil staining (Fig. 2E). In the remaining brainstem,other nuclei showed moderate-to-high numbers of stained peri-caria with relevant areas of intense staining such as the mesen-cephalons coliculi. The substantia gelatinosa in the spinal cordwas also heavily stained. In the cerebellum, strong staining of thePurkinje-cell bodies and prolongations was seen similar to, butnot as strong as, that described for the CALB IL. In the molec-ular layer, basket-cell prolongations were also immunolabelled,as were stellate cells.

The neuropil and neuronal body IL-patterns were assessedsemiquantitatively. When control animals were compared toscrapie-affected cases, differences in IL were noticed (Fig. 2).The most striking of these was observed at the neocortex, par-ticularly the frontal, occipital and parietal lobes (see Fig. 3).A reduction in the number of stained pericaria was seen, butmore significant was the reduction in the neuopile and neu-rite IL. Other areas with a similar reduction of staining wereAmmon’s horn, the hippocampus and the thalamus reticularnucleus. Comparatively, the remaining brain areas showed nosignificant differences with the control animals.

142 E. Vidal et al. / Journal of Virological Methods 136 (2006) 137–146

Fig. 4. Wysteria floribunda agglutinin (WFA) staining (A–E) in frontal cortex (A and E), Parietal cortex (B) and mesenchephalon (C and D). Double-labelling withWFA (blue) and Parvalbumin (brown) in frontal cortex (F). Note preserved perineuronal staining even in vacuolated neurons in scrapie-affected brain stem sections(D). Double labelling shows that most PV+ neurons have WFA perineuronal staining (arrowhead), although a few scattered PV+ neurons do not (thin arrow). PVnegative neurons with WFA perineuronal staining are also frequent (bold arrow). (A) Bar 200 �m, (B) bar 100 �m and (C–F): bar 50 �m.

3.5. Wysteria floribunda agglutinin affinity histochemistry

In control ovine encephalon, WFA labelling was seenthroughout the brain. The staining consisted of a strong per-ineuronal pattern, also following the neurites, which percolatedin the surrounding neuropil with varying intensities, dependingon the area.

In the neocortex (Fig. 4A, B, and E), staining was seen arounda high number of neurones in layers III and V, as well as arounda few neurons in layers IV and VI; however, almost no stain-ing was detected in layers I and II. Neuropil staining was alsopresent at these levels, but was particularly intense in layer V

of 3 circumvolutions in the parietal lobe (Fig. 4B), namely,the marginal, ectomarginal and suprasylvian circumvolutions.In the brain stem, a strong perineuronal labelling in the thala-mus reticular nucleus was observed (when compared to othernuclei in the diencephalon). The mesencephalon (Fig. 4C andD), pons, medulla oblongata, spinal cord and deep-cerebelarnuclei were intensely stained. Particularly, the grey-matter neu-ropil staining was very intense in these regions. Neurons inthe reticular formation showed a heavier perineuronal stainingthan that of other nuclei. The cerebelar vermis and hemispheresshowed perineuronal staining of the Golgi cells at the granularlayer.

E. Vidal et al. / Journal of Virological Methods 136 (2006) 137–146 143

When compared to scrapie brains, no significant differ-ences were noticed. Moreover, even those neurons with evidentspongiform degeneration maintained their perineuronal stainingintact (Fig. 4D).

3.6. PV and WFA double labelling

To ascertain that a selective loss of perineuronal nets inPV+ neurones was not being under-reported, double-labellingof some of the most affected regions was performed (parallelto healthy control sections). It was also of interest to investi-gate whether the similar distribution showed by the labelling ofboth PV and WFA was actually due to colocalisation of bothmarkers.

A large number of PV+ neurones showed presence of WFA-labelled perineuronal nets; the alternative situation, i.e. PV+WFA-negative, was also noticed but at a lower proportion.Instead, a considerable number of WFA+ neurons without PVIL were detected (see Fig. 4F). In the diencephalon, colocali-sation of both markers was observed in the thalamus reticularnucleus. It can therefore be concluded that WFA labels a signifi-cant proportion (though not all) of perineuronal nets in the PV+subpopulation of GABAergic neurones within the ovine brain,and also that it is not an exclusive marker for such a population,since PV negative soma are seen with clear WFA+ perineuronal-net staining.

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values for both lesion and PrPsc deposition in the cerebellarregion (Benestad et al., 2003; Buschmann et al., 2004).

PrPsc deposits were spotted in virtually all regions of the brainin the nine cases, but a clear tropism towards the brain stem wasobserved. The tropism towards the brain stem described aboveis in keeping with the neuroinvasion model described in sheepby which the PrPsc would enter the CNS trough the caudal brainstem (through vagal aferetns), and then spread further within thebrain (Ryder et al., 2001; van Keulen et al., 2002) A previousPrPsc distribution study undertaken with rapid tests, also onRasa aragonesa Sheep, showed that the brain stem bore thebulk of PrPsc deposition (Bolea et al., 2005).

Individual variability can be explained by different factors,namely host breed, prnp genotype, dose and route of infection,age at time of infection, duration of clinical signs and presum-ably by the so called “strain” causing the disease (Fraser, 1993).High individual variability has been reported previously in natu-ral scrapie infections (Begara-McGorum et al., 2002; Gonzalezet al., 2002). The animals included in this study belong to theSpanish R. Aragonesa breed (except for one, which belongs tothe Roya Bilbilitana breed (Table 1)). As regards the prnp allele,all belong to the same genotype: ARQ-homozygous, being themost common genotype for the R. Aragonesa breed (Acin etal., 2004). The presence of different strains, or combinations ofthese, cannot be excluded since the animals come from five dif-ferent scrapie outbreaks, although the outbreaks were diagnosedidctd(

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The proportions of PV+ WFA+ labelled neurons were similarn scrapie and healthy animals in the different regions evaluatedi.e. frontal, parietal and occipital cerebral lobes and thalamus).

. Discussion

Neuropathological changes and immunohistochemistry pat-erns described in the present study are in accordance with previ-us descriptions of scrapie neuropathology (Foster et al., 1996;raser, 1976; Hardt et al., 2000; Miller et al., 1993; Ryder et al.,001; van Keulen et al., 1995; Wood et al., 1997). Note the paral-elism between the three curves, all of them showing a peak at therainstem region (Fig. 1). The study of TSEs has been focusedrequently on the obex region. More recently, however, a num-er of studies have appeared that take into account other regionsf the CNS (Bolea et al., 2005; Gonzalez et al., 2003; Ligios etl., 2002) and even peripheral tissues (Andreoletti et al., 2002,004; Monleon et al., 2004). The whole brain of nine scrapieases was examined. The semiquantitative approach and the pro-ling methodology described in this study was adapted from arevious study performed on BSE-affected cattle (Vidal et al.,005). We believe that this approach facilitates a straightforwardnterpretation of the results and that the method is reproduciblen different laboratories.

No attempt is made to use the profiles described in this studys a tool to discriminate between TSE strains, but rather as aethod to interpret readily results. However, if such analysisere performed on certain TSE strains described recently, such

s the NOR98-type scrapie cases, differences would be observedn both the histopathological and immunohistochemical profiless a deviation of the curve to the right, that is, showing higher

n the same area. Another shortcoming in this study is that theose, route, duration and time of the infection are factors thatannot be controlled, since these are all field cases. In terms ofhe clinical stage, all animals were at fairly late stages of theisease, some even being recumbent (7/9) at the time of cullingVargas et al., 2006).

Despite the heterogeneity described above, the histopatho-ogical and immunohistochemical changes coincide, generallypeaking, with classical scrapie descriptions. Since the objectiveas to define common pathogenetic events in natural scrapie,

egarding calcium binding proteins and extracellular matrix,nly evident changes present in all the scrapie cases includedave been taken into consideration.

Few descriptions of the ovine adult brain distribution of CBPss markers for subsets of the GABAergic neuronal populationave been published (McDonald et al., 1995; Oswald et al.,001). These investigators report a widespread distribution ofalbindin D-28K in the brain of both neonatal and adult sheep.

n our case, Calbindin D-28K IR has only been demonstratedn the cerebellum, deep cerebellar nuclei and medulla oblon-ata. A similar result was obtained in a previous study carriedut on canine brains (Siso et al., 2003); however, this was nothe case with feline brains (Alcantara and Ferrer, 1995; Vidalt al., 2004) or with other species such as rodents, non-humanrimates or humans (Ferrer et al., 1992; Van Brederode et al.,990; Webster et al., 1990). An explanation of why the areasutside the cerebellum show no CALB IL, in this case, couldie with the sample processing method, since most studies pub-ished have performed immunohistochemistry against CALB inrozen tissue, whereas in our study we work with formalin-fixed,ormic acid inactivated, paraffin-embedded tissue.

144 E. Vidal et al. / Journal of Virological Methods 136 (2006) 137–146

Again, few studies were found describing PV IL in adult ovinebrains (Oswald et al., 2001), in the present study, the normaldistribution of PV immunolabelled neurons in the whole brainfor four control animals is described.

When comparing the CBP status between control and scrapieanimals, no evident changes were detected in CALB IL, but somesignificant change in the PV labelling was observed. An apparentreduction of PV+ pericaria is reported accompanied by a signif-icant reduction of the neurite staining in certain brain regions(frontal, occipital and parietal cortical lobes, Ammon’s horn inthe hippocampus and the thalamus reticular nucleus (TRN)).

These regions were affected by a mild degree of spongio-sis (scores between 1 and 2) and PrPsc deposition was presentthroughout. The punctate PrPsc IL deposits in the neuropilobserved in these regions correspond to neurite membrane PrPscaggregates according to ultrastructural studies done in scrapieinfected mice (Jeffrey et al., 1994). It is thus tempting to thinkthat neurites of PV+ cortical neurons are susceptible to deposi-tion of PrPsc aggregates in ovine scrapie. Dendritic spine losshas been clearly identified early in the pathogenesis of murinescrapie (Brown et al., 2001; Jeffrey et al., 2000). Also the similardistribution shown by PrPsc intraneuronal IL and PV IL in thedeep neocortical layers supports the hypothesis that this neuralsubpopulation is selectively affected by PrPsc depostits: doubleIL techniques should be conducted to verify that both markersare staining the same neural population. Similar findings havebeeaiaosism

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be hypothesized that perineuronal nets disruption is not a keyfeature in our sheep scrapie, field-case pathogenesis.

In conclusion, in scrapie-affected ovine brains, a reductionhas been described in the number of pericaria and neurite stain-ing by PV in certain regions related to the presence of mildspongiform change, evident deposition of PrPsc but no apparentchange in the perineuronal nets, as stained by the WFA.

The fact that the perineuronal nets are preserved suggeststhat the reduction of parvalbumin signalling is due to a downregulation of parvalbumin expression rather than a loss of thisneural subpopulation. In murine scrapie models, perineuronalnet disruption is associated closely with gliosis (own observa-tion), in this study gliosis has been assessed morphologicallyand, is rather mild at the regions were changes in parvalbuminchanges have been described.

The neurological signs described in this study, typically asso-ciated with scrapie, could be justified in part with the presentfindings in that the alteration of GABAergic neurites might havecaused an inhibitory imbalance in areas related to motor function(frontal cortex), and other areas related to mental state (TRN,hippocampus). Thus, signs such as tremors or hyperexcitabil-ity might be explained. However, involvement of other path-ways cannot be excluded, since signs such as the ataxia mainlyreflect an involvement of cerebellar structures, which showedno changes in their GABA population in the present study.

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een described previously in the context of human prion dis-ases (Ferrer et al., 1993; Guentchev et al., 1997, 1999) andxperimental scrapie models (Guentchev et al., 1998). Indeed,recent report on intracerebrally scrapie-challenged hamsters

ndicate that presynaptic buttons in GABAergic neurons areltered (Bouzamondo-Bernstein et al., 2004). However, no lossf PV+ or GABA+ neurons was observed in this model. A veryhort incubation period (60–70 days post inoculation) and annoculation route far too different from the natural, which isupposedly oral, makes it difficult to compare results from thisodel to natural occurring cases.To our knowledge, this description is the first to describe dis-

ribution in the adult ovine brain of the WFA labelling. This lectinas permitted the evaluation of the perineuronal nets of certainrain regions; a close relationship has been reported betweenhe population labelled by WFA and those labelled by PV inhat the majority of PV+ neurones showed a WFA-labelled per-neuronal net. Although an evident morphological change haseen detected in PV+ neurons in certain areas, it has not beenossible to correlate such change with a disturbance of the per-neuronal nets associated to them. Even neuronal bodies withvident spongiform change seem to maintain the WFA labellingattern of their perineuronal nets. These results are in contrastith those from similar studies conducted in human brains withJD (Belichenko et al., 1999) and in a murine scrapie model

Ferrer et al., 1993; Guentchev et al., 1997, 1999). In those stud-es, an early destruction of the perineuronal nets is suggested toe one of the first steps leading to neurodegeneration. Since thearkers used in those studies are either the same as that used

n the present study (WFA) or Isolectin B4 (from Vicia villosa)lectin that binds to the same sugars as does WFA, it might

cknowledgements

The authors wish to acknowledge the excellent technicalupport provided by Sierra Espinar and Anna Maria Llorensf the Priocat Laboratory, CReSA and that of Silvia Ruız,uria Segovia and Santiago Becerra from the National Refer-

nce Centre for TSE diagnostic (CNREETs) at the Universityf Zaragoza. The present study was possible, thanks to thenancial support of the Departament de Salut, the Departament’Agricultura Ramaderia i Pesca of the Catalan GovernmentGeneralitat de Catalunya), the Aragon Government and the Net-ork of Excellence for Prion Diseases (Animal Tissues Bank).

t was also partially financed by project ET2002-05168-C04-01f the Spanish Ministerio de Ciencia y Tecnologia (MCyT).

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