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Research Report

Effect of treatment with choline alphoscerate on hippocampusmicroanatomy and glial reaction in spontaneouslyhypertensive rats

Daniele Tomassonia, Roberto Avolab, Fiorenzo Migninia,Lucilla Parnettic, Francesco Amentaa,⁎aSezione di Anatomia Umana, Dipartimento di Medicina Sperimentale e Sanità Pubblica,Università di Camerino, Camerino, ItalybSezione di Biochimica, Dipartimento di Scienze Chimiche, Università di Catania, Catania, ItalycDipartimento di Specialità Medico Chirurgiche e Sanità Pubblica, Clinica Neurologica, Università di Perugia, Perugia, Italy

A R T I C L E I N F O

⁎ Corresponding author.Dipartimento di MediItaly. Fax: +39 0737 630618.

E-mail address: francesco.amenta@unicam

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.08.068

A B S T R A C T

Article history:Accepted 18 August 2006Available online 20 September 2006

The influence of long term treatment with choline alphoscerate on microanatomy ofhippocampus and glial reaction was assessed in spontaneously hypertensive rats (SHR)used as an animal model of cerebrovascular disease. Choline alphoscerate is a cholinergicprecursor, which has shown to be effective in countering cognitive symptoms in forms ofdementia disorders of degenerative, vascular or combined origin. Male spontaneouslyhypertensive rats (SHR) aged 6 months and age-matched normotensive Wistar–Kyoto(WKY) rats were treated for 8 weeks with an oral daily dose of 100 mg/kg of cholinealphoscerate, 285 mg/kg of phosphatidylcholine (lecithin) or vehicle. On the hippocampusof different animal groups, nerve cell number and GFAP-immunoreactive astrocytes wereassessed by neuroanatomical, immunochemical and immunohistochemical techniquesassociatedwith quantitative analysis. Treatmentwith choline alphoscerate countered nervecell loss and glial reaction primarily in the CA1 subfields and in the dentate gyrus of thehippocampus of SHR. Phosphatidylcholine did not affect hypertension-dependent changesin hippocampalmicroanatomy. Both compounds did not affect blood pressure values in SHR.These data suggest that choline alphosceratemay play a role in the countering hippocampalchanges induced by cerebrovascular involvement. The observation that treatment withcholine alphoscerate attenuates the extent of glial reaction in the hippocampus of SHRsuggests also that the compound may afford neuroprotection in this animal model ofvascular brain damage.

© 2006 Elsevier B.V. All rights reserved.

Keywords:HypertensionNeurodegenerationSpontaneously hypertensive ratVascular dementiaCholinergic precursor

cina Sperimentale e Sanità Pubblica, Università di Camerino, Via Scalzino 3, 62032 Camerino,

.it (F. Amenta).

er B.V. All rights reserved.

Table 1 – Quantitative image analysis of the number ofneurons in the hippocampus of the different animalgroups investigated

WKY(n=6)

SHR(n=6)

SHR α-GPC-treated(n=6)

SHR PDCtreated(n=6)

CA1 subfield(103/mm3)

221.9±6.7 158.6±13.3a 200.9±12.6a,b 160.7±10.9a,c

CA3 subfield(103/mm3)

101.3±5.7 97.3±6.7 96.1±6.5 99.2±5.3

Dentate gyrus(104/mm3)

110.2±4.2 100.4±4.4a 105.3±4.3a,b 100.5±3.6a,c

Data are the mean±SEM. α-GPC: α-glycerylphosphorylcholine andPDC: phosphatidylcholine.a: p<0.05 vs. WKY rats; b: p<0.05 vs. SHR rats; c: p<0.05 vs. SHRα-GPC-treated rats.

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1. Introduction

The role of the basal forebrain cholinergic system in cognitiveprocess is well established, particularly in associationwith thefunctional decline accompanying normal and pathologicalaging (Bartus et al., 1996), cerebral ischemia (Muralikrishna etal., 2002) and neurodegenerative disease (Mc Entee and Crook,1990; Amenta et al., 2002; Erkinjuntti et al., 2004). Lowcerebrocortical cholinergic input is well documented inAlzheimer's disease (AD) and vascular dementia (VaD)(Roman and Kalaria, 2005). Cholinergic deficits in VaD havebeen also reported, independently of any concomitant ADpathology. A cause of this impairment may be the vulner-ability of cholinergic structures to ischemic damage. Hippo-campal CA1 neurons are particularly susceptible to ischemia,and hippocampal atrophy is common in patients with VaD inthe absence of AD (Vinters et al., 2000). Loss of cholinergicneurons has been described in 70% of AD cases and in 40% ofVaD patients in neuropathological examinations. Reducedacetylcholine (ACh) concentrations in cerebral cortex, hippo-campus, striatum and cerebral spinal fluid (CSF) of VaDpatients were reported as well (Court et al., 2002).

In experimental rodent models, such as spontaneouslyhypertensive rat (SHR) and SHR stroke-prone (SP-SHR), asignificant reduction in cholinergic markers including AChoccurs in the neocortex, hippocampus and CSF (Togashi et al.,1994; Saito et al., 1995; Kimura et al., 2000; De Bruin et al., 2003;Hernandez et al., 2003). White matter infarction is one of themain causes of cholinergic impairment in these models(Kimura et al., 2000).

The hypothesis that impaired brain cholinergic transmis-sion has a key role in the deterioration of cognitive functioningin AD and/or VaD stimulated extensive research of possibletherapeutic approaches towards improvement of cholinergicneurotransmission deficits (Amenta et al., 2001). The differentpossibilities proposed or tested for relieving impaired choli-nergic neurotransmission in dementia disorders includedintervention with ACh precursors, stimulation of ACh release,the use of muscarinic or nicotinic receptors agonists andacetylcholinesterase (AChE) inhibition (Amenta et al., 2001).

Arterial hypertension is a main risk factor for cerebrovas-cular disease (Turnbull, 2003; Semplicini et al., 2006). Brain issensitive to hypertension, and cerebrovascular tree undergoesimportant hypertension-dependent changes. Impaired brainmorphology and/or functioning were reported both in hyper-tensive patients and in animal models of hypertension(Sabbatini et al., 2000; Semplicini et al., 2001). SHR that shareseveral characteristics with human essential hypertensiondevelop cognitive impairment, cholinergic deficits and brainneurodegenerative changes (Togashi et al., 1994; Saito et al.,1995; Kimura et al., 2000; De Bruin et al., 2003) consistent withtheir use as possiblemodel of VaD (Sabbatini et al., 2001, 2002).

Choline alphoscerate (L-α-glycerylphosphorylcholine α-GPC) is a semi-synthetic derivative of phosphatidylcholine(PDC or lecithin). Pre-clinical studies have demonstrated thatit increases the release of ACh in rat hippocampus (Sigala etal., 1992), facilitates learning and memory in animal models(Govoni et al., 1990), improves brain transductionmechanisms(Schettini et al., 1990) and decreases the age-dependent

structural changes occurring in rat frontal cortex and hippo-campus (Amenta et al., 1993). Clinical studies have also shownan efficacy of this cholinergic precursor in cognitive impair-ment occurring in dementia disorders of neurodegenerativeand vascular origin (Parnetti et al., 2001; De Jesus Moreno,2003). To further contribute in characterizing the potential roleof α-GPC in protecting brain from injury of vascular origin, wehave assessed the influence of long term treatment with α-GPC on microanatomy of hippocampus of SHR and comparedit with the activity of PDC.

2. Results

Body weight values in the control and either α-GPC-treatednormotensive WKY rats and hypertensive SHR are similar(data not shown). Brain weight was slightly lower, but notsignificantly different in SHR in comparisonwith age-matchednormotensive WKY rats (2.08±0.21 g WKY rats vs. 2.05±0.22 gSHR rats). In normotensive WKY rats, systolic pressure valuesaveraged 117±5 mm Hg. In control, SHR systolic bloodpressure averaged 199±7 mm Hg (p<0.05 vs. WKY rats).Treatment with α-GPC or PDC did affect blood pressure valuesin SHR (192±5 mm Hg and 200±8 mm Hg respectively).

Data of morphometric analysis of the CA1 and CA3subfields and of the dentate gyrus are summarized in Table1. Neuron number was decreased in SHR in comparison withnormotensive WKY rats primarily at the level of the CA1subfield and to a lesser extent in the dentate gyrus (Table 1 andFig. 1). No significant changes in the number of neurons of theCA3 subfield were observed between normotensive rats andSHR (Table 1 and Fig. 1). Among the two cholinergic precursorstested, only α-GPC counterednerve cell loss in the CA1 subfieldand in the dentate gyrus of SHR (Table 1 and Fig. 1). Neitherα-GPC nor PDC affected microanatomical parameters investi-gated in normotensive WKY rats (data not shown).

Immunoblots of hippocampus for glial fibrillary acidicprotein (GFAP) were bound to a single band of approximately50 kDa (Fig. 2). Exposure of membranes to non-immune rabbitIgGs did not cause the development of bands of immunor-eactivity (data not shown). Themigration pattern of immunor-

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eactions was similar in the different animal groups investi-gated (Fig. 2). The intensity of the band of GFAP increased inSHR rats compared toWKY rats (Fig. 2). This phenomenonwascountered by treatment with α-GPC (Fig. 2).

Section processed for GFAP immunohistochemistry devel-oped dark-brown astrocytes in the CA1 and CA3 subfields ofhippocampus. The greatest accumulation of GFAP-immunor-eactive astrocytes was found primarily in the stratumradiatum and in the stratum moleculare-lacunosum (Figs.3A–C), whereas in the dentate gyrus, the higher density ofGFAP-immunoreactive astrocytes was observed in the hilumand in the molecular layer (Figs. 3D–F). These portions ofhippocampus were therefore chosen for quantitative analysis.In SHR, the number and size of astrocytes of the CA1 subfield

Fig. 1 – Sections of the CA1 subfield (A–D), CA3 subfield (E–H) anWKY rats (A, E and I), SHR (B, F and J), SHR treated with α-GPC (CCresyl violet staining. Note in SHR the reduced number of nervethe dentate gyrus. Neuronal loss was countered by treatment wiof nerve cell profiles are noticeable in the CA3 subfield of the diforiens; r: stratum radiatum: m: molecular layer; h: hilum; g: gran

(Table 2 and Fig. 3B) and to a lesser extent of the dentate gyrus(Table 2 and Fig. 3D) were increased compared to WKY. Anincreased number of GFAP-immunoreactive astrocytes wasalso observed in the CA3 subfield of SHR (Table 2). Treatmentwith α-GPC decreased the number and size of GFAP-immu-noreactive astrocytes whereas PDGwaswithout effect (Table 2and Figs. 3C and F).

3. Discussion

SHR were developed as animal model of genetic hypertensionand were largely used for investigating causes, mechanismsand pathology of hypertension as well as the influence of

d dentate gyrus (I–L) of the hippocampus of normotensive, G and K) and SHR treated with PDC (D, H and L).cell profiles in the CA1 subfield and to a lesser extent inth α-GPC and not with PDC. No changes in the numberferent animal groups. P: pyramidal neurons; o: stratumule neurons. Calibration bar: 50 μm.

Fig. 2 – Western blot of glial fibrillary acidic protein (upperlane) and β-actin (lower lane), used as internal standard, inpreparations of hippocampus. The results of densitometricanalysis are expressed as optical density values normalizedfor β-actin expression. Each value is the mean±SEM of fivedifferent independent experiments. 1: normotensive WKYrat; 2: SHR rat; 3: SHR treated withα-GPC; 4: SHR treated withPDC. *p<0.05 vs. WKY rats; **p<0.05 vs. WKY and SHR rats.

Fig. 3 – Sections of the CA1 subfield (A–C) and dentate gyrus (D–F)demonstration of glial fibrillary acidic protein (GFAP) used as a mWKY rats; Panels B and E: untreated SHR; Panels C and F: α-GPCsize of dark-brown GFAP-immunoreactive astrocytes in comparitreatment with α-GPC. P: pyramidal neurons; o: stratum oriens;g: granule neurons. Calibration bar: 25 μm.

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pharmacological treatments on the development and courseof arterial hypertension (Folkow, 1982). More recently, beha-vioral studies in SHR have reported the occurrence of impairedmemory, learning and attention processes (Gattu et al., 1997a,b). Detailed exploration of memory tasks has documentedimpairment of spatial learning and working memory (Terry etal., 2000; Wyss et al., 2000).

Arterial hypertension is considered a risk factor for thedevelopment of cognitive dysfunction, for its negativeeffects on cerebral vasculature and blood flow (Birkenhageret al., 2001). In the elderly, it is a major risk factor forvascular cognitive impairment and VaD (Prince, 1997; Rigaudet al., 2000; In't Veld et al., 2001). SHR have been alsoproposed and used as a model for assessing the influence ofhypertension on cognitive functions (Meneses et al., 1996,1997) and to test drugs with potential effects for treatinglearning impairment associated with this condition (Menesesand Hong, 1998).

An interesting neuropathological observation in SHR isthat the microanatomical changes they develop occur start-ing from the 4th month of age and are obvious since the 6th

of the hippocampus processed for the immunohistochemicalarker of astrocytes. Panels A and D: control normotensive-treated SHR. Note in SHR the increase in the number andson with WKY rats. This phenomenon is countered byr: stratum radiatum: m: molecular layer; h: hilum;

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month. Hence, an age from 4 months onward represents areasonable elapse of time for assessing the effect oftherapeutic interventions on brain damage in SHR (Sabbatiniet al., 2001, 2002). In terms of involvement of braincholinergic system, SHR have reduced number of centralACh receptors which are involved in the regulation ofcerebral circulation and cognitive function (Gattu et al.,1997a; Terry et al., 2000). This suggests that similarly asfound in learning and memory alterations occurring indementia disorders of vascular origin (Avery et al., 1997;Muir, 1997; Amenta et al., 2002; Erkinjuntti et al., 2004) centralcholinergic neurotransmission is affected in SHR.

The goal of the present work was to assess in SHR theinfluence of treatment with cholinergic precursors such as α-GPC and PDC on themorphology of a key area for learning andmemory, such as hippocampus. Two quantitative microana-tomical parameters such as nerve cell number and astroglialreaction were used for evaluating the potential neuroprotec-tive activity of the two cholinergic precursors. The findingsthat the two compounds did not affect blood pressure levels inSHR indicate that any activity on brain microanatomy is notrelated to changes in blood pressure.

Based on the present data, we are unable to explain why α-GPC countered hippocampal damage noticeable in SHR,whereas PDC was without effect. Several studies havesuggested that PDC is probably not suitable for enhancingbrain cholinergic neurotransmission (for a review, see Amentaet al., 2001). This may be reason of the ineffectiveness of PDCtreatment in spite of the use of concentrations of both PDC andα-GPC containing the same amounts of choline. Beneficialeffects of α-GPCmay depend by an influence of the compoundon brain phospholipids metabolism and/or by its documentedactivity of increasing free plasma choline levels (Gatti et al.,1992) and brain ACh bioavailability and release (Sigala et al.,1992). α-GPC is involved in brain phospholipids metabolismbeing transformed by the enzyme glycerylphosphorylcholinediesterase into a molecule of choline and another of glycerol-1-phosphate. Choline can be used for synthesizing ACh,whereas glycerol-1-phosphate after phosphorylation canenter in the pool of phospholipids (Dross and Kewitz, 1972;Zaisel, 1981). Activation of these pathways could provideboth free choline and phospholipids for synthesizing AChand can reconstitute nerve cell membrane components. On

Table 2 – Number and size of GFAP-immunoreactive astrocytegroups investigated

WKY (n=6) SHR (

CA1 subfieldNumber (103/mm3) 39.6±4.2 58.3Mean immunoreaction area μm2 90.3±6.4 121.1

CA3 subfieldNumber (103/mm3) 65.6±3.1 83.3Mean immunoreaction area μm2 85.7±7.2 97.7

Dentate gyrusNumber (103/mm3) 27.6±2.3 47.3Mean immunoreaction area μm2 82.3±5.4 100.3

Data are the mean±SEM. α-GPC: α-glycerylphosphorylcholine and PDC: pa: p<0.05 vs. WKY rats; b: p<0.05 vs. SHR rats c: p<0.05 vs. α-GPC-treated

the other hand, choline itself can be converted in phospho-lipids via choline kinase and phosphocholine acydil transfer-ase. Brain damage in SHR is related to a reduction in cholineuptake and a consequent decline in biosynthesis of phos-pholipids (Nardella et al., 1991). Protection of neuronalmembrane in this animals model by α-GPC via this mechan-ism may contribute to the effects seen in this study. α-GPChas been found to increase ACh levels and release in rathippocampus (Sigala et al., 1992;Amenta et al., 2006). Thismay facilitate learning and memory function in animalmodels (Govoni et al., 1990) and improve brain transductionmechanisms (Schettini et al., 1990).

Another possible mechanism of the beneficial action of α-GPC on hypertensive changes occurring in rat hippocampusmay be related to its activity on astroglial reaction. Reactiveastrocytosis occurs in response to central nervous systemdisease or injury, but reactive astrocytes are also involved inneural repair and protection (Sofroniew, 2005). Enhancedcholinergic neurotransmission may prevent glutamate neu-rotoxicity via activation of nicotinic acetylcholine receptorsand phosphatidylinositol 3-kinase cascade (Akaike, 2006).Stimulation of α-7 nicotinic receptor may interfere withinflammatory responses in the central nervous system (Shytleet al., 2004), the magnitude of which can be regulated byastrocytes (Shytle et al., 2004). It cannot be excluded thatenhanced brain cholinergic activity documented for α-GPC(Amenta et al., 2006) may contribute to interfere withmechanisms of neuroprotection mediated via nicotinicreceptors.

In summary, these data confirm that SHR are an interestingmodel for screening substances with therapeutic potential fortreating brain injury of vascular origin. Moreover, our findingsindicate that treatment with α-GPC attenuates neural damageand glial reaction in hippocampus of SHR. This suggests thatthe compound may afford neuroprotection in the animalmodel investigated. Several agents, including the cholinergicprecursor CDP-choline, have been considered as neuroprotec-tive drugs in cerebrovascular disease (Hickenbottom andGrotta, 1998). Based on the above findings, we can hypothesizethat another cholinergic precursor, α-GPC, may act as aneuroprotectant. This warrants further evaluation of thecompounds in treating cognitive dysfunction of vascularorigin in appropriate clinical trials.

s in the areas of hippocampus examined in the different

n=6) SHRα-GPC treated (n=6)

SHRPDC treated (n=6)

±3.2a 45.7±3.5a,b 60.3±5.9a,c

±5.2a 104.1±9.2a,b 115.6±7.3a,c

±4.8a 72.4±4.3a,b 81.4±5.3a,c

±3.2a 90.3±7.2a,b 98.1±8.7a,c

±3.2a 33.6±3.9a,b 45.2±4.9a,c

±6.3a 93.3±6.2a,b 102.9±8.3a,c

hosphatidylcholine.rats.

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4. Experimental procedures

4.1. Animals and tissue treatment

Animals used were male SHR of 6 months of age (n=18) andage-matched male normotensive WKY rats (n=18). Animalswere handled according to internationally accepted princi-ples for care of laboratory animals (European CommunityCouncil Directive 86/609, O.J. No. L358, Dec. 18, 1986). Ratswere received 4 weeks before experiments and kept under aconstant light–dark cycle (7:00 a.m. to 7:00 p.m. light period)at an ambient temperature of 22±1 °C, with free access towater and laboratory chow. Rats were treated for 8 weekswith 100 mg/kg/day α-GPC (n=6 SHR and n=6 WKY rats) orwith 285 mg/kg PDC (n=6 SHR and n=6 WKY rats). Theseconcentrations of the two compounds contain the sameamount of choline. Drugs were added to rat drinking water,and their concentrations were adapted following bodyweight changes. The remaining 6 SHR and WKY rats wereleft untreated and served as control groups. Blood pressurevalues were measured once a week by an indirect tail-cuffmethod in conscious rats, after pre-warming at 37 °C inthermostatic cages for 30 min. Before sacrifice, animals hadblood pressure measured, were anesthetized with pentobar-bital sodium (50 mg/kg, i.p.) and killed by decapitation.Brains were removed, washed in ice-cold 0.9% salinesolution and divided into two halves by a sagittal cutthrough interhemispheric seizure. Hippocampus from theright hemisphere was dissected out, weighed, frozen inisopentane and used for Western blot analysis as detailedbelow.

Left hemisphere sagittal slices (L1490-L240, König andKlippel, 1963) 3 mm apart were cut fixed in Histochoicesolution (Medite, DiaPath, Martinengo Italy), dehydrated inethanol, embedded in a semi-synthetic paraffin (Histowax,Reichert-Jung, Vienna, Austria) and cut serially using arotatory microtome. Groups of three serial consecutive12 μm were used for morphometric analysis and immuno-histochemistry as detailed below. The first section of eachgroups was stained with Nissl's staining (cresyl violet 1.5%),the second was processed for GFAP immunohistochemistry;the third was used as a control of immunohistochemicalanalysis.

4.2. Morphometric analysis and nerve cell number

The first of each group of three consecutive sections of lefthemisphere was stained with a 1.5% cresyl violet solutionand used for assessing the volume of hippocampus accord-ing to the procedure detailed elsewhere (Sabbatini et al.,2000). The same sections were also used for counting thenumber of nerve cells. Neurons were considered cell profileswith nuclei pale and a granular background, surrounded by aring of visible cytoplasm and displaying one or more clearlyvisible nucleolus. For assessing the number of nerve cell,consecutive sections were examined at a final ×125 magni-fication, with a light microscope connected via a TV camerato an IAS 2000 image analyzer (Delta Sistemi, Rome, Italy).Neuron numerical density was then evaluated indepen-

dently in the CA1 and CA3 subfields and in the dentate gyrus(Sabbatini et al., 2000).

4.3. Western blotting

GFAP immunoreactivity was assessed in samples of hippo-campus of different animal groups by Western blotting usingthe protocol reported in an earlier paper (Tomassoni et al.,2004). Briefly, samples of hippocampus (0.1±0.02 g) were lysedand after two centrifugations aliquots of the supernatant wereused for protein assay against a standard of bovine serumalbumin. Equal amounts of protein (40 μg) were separated by10% sodium dodecyl sulfate polyacrylamide gel electrophor-esis and transferred to nitrocellulose membrane by electro-blotting in Towbin buffer. Transblotted membranes wereincubated with GFAP antibodies diluted 1:1000 in PBS 0.1 M,BSA (1%) and Tween-20 (0.05%). The specificity of immunereaction was assessed using antibodies pre-adsorbed with thepeptide used for generating them.

4.4. Glial fibrillary acidic protein (GFAP)immunohistochemistry

The second of each group of three consecutive sections of theleft hemisphere was used for the immunohistochemicaldetection of GFAP immunoreactivity (test section), whereasthe third one was used to assess the specificity of immunereaction (control section). Test sections were processed forGFAP immunohistochemistry by exposing them to a primaryantibody anti-GFAP diluted 1:1000. The product of immunor-eaction was revealed by indirect avidin–biotin immunohisto-chemistry. The numerical density of astrocyte profilesdisplaying a dark-brown staining and their area were thenassessed in the CA1 and CA3 subfields and in the dentategyrus by image analysis. The number of astrocytes wasreferred to the volume of corresponding brain areas. Forfurther details on image analysis of GFAP immunohistochem-istry, see Tomassoni et al. (2004). Analysis was limited toparenchymal astrocytes that were more diffused than peri-vascular astrocytes (Tomassoni et al., 2004).

4.5. Data analysis and chemicals

Values for individual animals within the groups investigatedare means of measurements of the parameters considered.Group means were then derived from individual means.Statistical analysis was performed by analysis of variance(ANOVA). The significance of differences between means wasassessed by Duncan's multiple range test, taking p<0.05 asthe minimum level of significance. Chemicals were obtainedfrom the same source recently indicated (Tomassoni et al.,2004).

Acknowledgments

The present study was supported by a grant from ItalianMinistero Istruzione, Università e Ricerca Scientifica (MIUR),Project COFIN 2001, No. 20011054379.

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