Neuroprotective effect of treatment with galantamine and choline alphoscerate on brain microanatomy in spontaneously hypertensive rats

Journal of the Neurological Sciences 283 (2009) 187–194

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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s

Neuroprotective effect of treatment with galantamine and choline alphoscerate on brain microanatomy in spontaneously hypertensive rats Seyed Khosrow Tayebati ⁎, Maria Antonietta Di Tullio, Daniele Tomassoni, Francesco Amenta Section of Human Anatomy, Department of Experimental Medicine and Public Health, University of Camerino, Camerino, Italy

a r t i c l e

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Available online 21 March 2009 Keywords: Spontaneously hypertensive rats Neuroprotection Cholinesterase inhibitors Cholinergic precursors Vascular dementia

a b s t r a c t The present study was designed to assess if treatment with acetylcholinesterase inhibitor galantamine and the cholinergic precursor choline alphoscerate (alpha-glyceryl-phosphoryl-choline) alone or in association has any protective effect on brain microanatomy in spontaneously hypertensive rats (SHR) used as an animal model of vascular dementia (VaD). Thirty-two-week-old SHR and age-matched normotensive Wistar Kyoto (WKY) rats were left untreated or treated for 4 weeks with an oral dose of 3 mg/kg/day of galantamine, of 100 mg/kg/day of choline alphoscerate or their association. The number of neurons and of glial fibrillary acidic protein (GFAP) immunoreactive astrocytes, phosphorylated neurofilament, and microtubule associated protein-2 (MAP-2) and aquaporin-4 (AQP-4) was assessed by quantitative microanatomical and immunohistochemical techniques. In SHR, the number of neurons of frontal cortex, of the CA1 subfield of hippocampus and of dentate gyrus was decreased compared to WKY rats. Astrogliosis, breakdown of phosphorylated neurofilament, unchanged MAP-2 and altered AQP-4 expression were found as well. Both galantamine and choline alphoscerate countered nerve cell loss. Choline alphoscerate but not galantamine decreased astrogliosis and restored expression of AQ-4. Galantamine countered to a greater extent than choline alphoscerate phosphorylated neurofilament breakdown. The two drugs in association displayed a more remarkable effect. This study confirms a neuroprotective effect of galantamine in SHR and indicates a neuroprotective role of choline alphoscerate in the same model. A wider neuroprotective effect of the cholinergic inhibitor/precursor association was observed. These findings suggest to assess the activity of this cholinergic association in clinical trials. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Cerebrovascular diseases, as well as secondary ischemic brain injury induced by cardiovascular disorders, are common causes of adult-onset cognitive impairment. Moreover, the presence of cerebrovascular damage in Alzheimer's disease (AD) contributes to aggravate cognitive loss [1]. In the Western world, vascular dementia (VaD) is the second most common form of adult-onset dementia after Alzheimer's disease (AD). It has an overall prevalence of 1.2–4.2% in people aged 65 years or older, and accounts for 10–50% of dementia cases, depending on the diagnostic criteria and study population [2]. In terms of symptomatology, VaD is characterized by progressive cognition decline, functional ability impairment and behavioral problems. VaD results mainly from ischemic injury or oligaemia to brain areas involved in cognition, memory, and behaviour [3]. Basal forebrain cholinergic system plays an important role in cognitive function, and mainly in the domains of attention, memory

⁎ Corresponding author. Dipartimento di Medicina Sperimentale e Sanità Pubblica, Via Madonna delle Carceri, 9, 62032, Camerino (MC), Italy. Tel.: +39 0737403305; fax: +39 0737630618. E-mail address: [email protected] (S.K. Tayebati). 0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2009.02.349

and emotion, and is also involved in the control of cerebral blood flow [4]. Cholinergic structures and specific brain areas such as the hippocampus are particularly sensitive to ischemic damage. This may account for a role of impaired cholinergic neurotransmission in the pathophysiology of VaD [5]. Acetylcholinesterase (AChE)/cholinesterase (ChE) inhibitors that together with memantine are the only drugs licensed for symptomatic relief of AD [6] have been proposed as a symptomatic therapy of VaD patients. Neuroprotective effects of AChE/ChE inhibitors were reported [7–12]. Preclinical studies suggest that AChE/ChE inhibitors attenuate neuronal damage and death from cytotoxic insults, and therefore might affect AD course. This effect is probably not directly mediated to their inhibition of acetylcholine catabolism [13]. Cholinergic precursor loading strategy has represented the first approach to treat cholinergic dysfunction and cognitive decline in adultonset dementia disorders, but this therapeutic option was leaved due to the lack of efficacy of several precursors investigated in clinical trials. Choline alphoscerate (L-alpha-glyceryl-phosphoryl-choline, is a cholinergic precursor studied both in preclinical paradigms and in clinical trials [14–16]. This compound increases acetylcholine release in rat hippocampus and improves memory and attention, as well as affective and somatic symptoms (fatigue, vertigo) in VaD patients [17].

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Table 1 Number of neurons, neurofilament 200 kDa immunoreactivity, number and size of GFAP-immunoreactive astrocytes in the frontal cortex of the different animal groups investigated.

Different layers Zone II Number of neurons (102/mm3) Zone III Number of neurons (102/mm3) Zone IV Number of neurons (102/mm3) Overall frontal cortex grey matter Neurofilament I.R. Astrocytes number (103/mm3) Mean immuno-reaction area μm2

WKY (N = 6)

SHR (N = 6)

SHR + GAL (N = 6)

SHR + GPC (N = 6)

SHR + ASS (N = 6)

200 ± 6

157 ± 5a

209 ± 9b

189 ± 7b

191 ± 8b

49 ± 2

32 ± 1a

48 ± 2b

47 ± 2b

52 ± 3b,c,d

96 ± 4

63 ± 3a

84 ± 4b

85 ± 3b,c

100 ± 5b,c,d

35.2 ± 1.8 17.1 ± 0.9 74.1 ± 3.5

25.9 ± 1.2a 25.4 ± 1.2a 92.2 ± 4.5a

37.4 ± 1.8b 23.6 ± 1.1a 91.8 ± 4.2a

28.7 ± 1.4a,b 19.4 ± 0.8b,c 68.7 ± 3.4b,c

33.2 ± 1.5b,c,d 18.3 ± 0.7b,c 69.3 ± 3.3b,c

Neurofilament I.R. values are expressed in arbitrary units calculated microdensitometrically as detailed in the Materials and methods section. GAL: galantamine; GPC: choline alphoscerate; ASS: galantamine plus choline alphoscerate. Data are the mean ± S.E.M. a p b 0.05 vs. WKY. b p b 0.05 vs. SHR. c p b 0.05 vs. SHR + GAL. d p b 0.05 vs. SHR + GPC.

Studies of our group have reported that combination choline alphoscerate with the AChE/ChE inhibitor rivastigmine may represent an association enhancing cholinergic neurotransmission more effectively than single compounds [16]. This association was proposed as a

possible strategy for replacement therapy in pathologies characterized by altered cholinergic neurotransmission [16]. The present study was designed to assess if treatment with the AChE inhibitor galantamine and the cholinergic precursor choline alphoscerate

Fig. 1. Sections of rat frontal cortex (zone II) stained with cresyl violet. A: control normotensive WKY rat; B: control untreated SHR; C; SHR treated with galantamine; D: SHR treated with choline alphoscerate; E: SHR treated with galantamine plus choline alphoscerate. Note in SHR the reduced number of nerve cell profiles. Neuronal loss was countered by different pharmacological treatment. Calibration bar: 50 μm.

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alone or in association has any protective effect on brain microanatomy in spontaneously hypertensive rats (SHR). Galantamine was chosen as an AChE inhibitor in view of its more pronounced cerebrovascular profile compared with other compounds of the same class [11]. 2. Materials and methods 2.1. Animals and tissue treatment Male SHR aged 32 weeks (n = 24) and age-matched male normotensive Wistar-Kyoto (WKY) rats (n = 6) were used. SHR were treated or not (controls) for 4 weeks with oral galantamine (3 mg/kg/day) or choline alphoscerate (150 mg/kg/day) alone or in association. Control SHR and WKY rats received the same amounts of vehicle. Rats were handled according to internationally accepted principles for care of laboratory animals (European Community Council Directive 86/609, O.J. No. L358, Dec. 18, 1986). Blood pressure values were measured once a

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week by an indirect tail-cuff method in conscious rats. Before killing animals were anaesthetised with pentobarbital sodium (50 mg/kg, i.p.) and decapitated. The brain was removed from skull, washed, weighed and divided into the two hemispheres. The left hemisphere was fixed in a Histochoice solution, embedded in a semi-synthetic paraffin. Serial consecutive 8 μm thick sections were stained with Nissl's method (cresyl violet 1.5%) for morphometric analysis and with Masson's trichromic technique for assessing the occurrence of relevant microanatomical changes. The right hemisphere was embedded in a cryoprotectant medium and stored at −80 °C until use. Serial consecutive 12 μm thick sections were cut using a microtome cryostat and processed for immunohistochemistry as detailed below. 2.2. Immunohistochemistry Frozen consecutive parasagittal sections of the right hemisphere (12 µm thick) were processed for the immunohistochemical detection

Fig. 2. Sections of the CA1 subfield (A–C) and of the dentate gyrus (D–F) of hippocampus stained with cresyl violet of control normotensive WKY rats (A and D), control untreated SHR (B and E), and SHR treated with galantamine and choline alphoscerate in association (C and F). Note in SHR the reduced number of nerve cell profiles in the CA1 subfield and in the dentate gyrus. Neuronal loss was countered by treatment with association of galantamine plus choline alphoscerate. P: Pyramidal neurons; G: granule neurons of dentate gyrus. Calibration bar: 50 μm.

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of glial fibrillary acidic protein (GFAP), neurofilament 200 kDa (NFP), microtubule associated protein-2 (MAP-2) and aquaporin-4 (AQP-4). The 1st, 5th, 10th, 15th and 20th consecutive sections were processed for GFAP immunohistochemistry using a mouse serum against GFAP (Chemicon, Millipore, Cat. No. 3402) diluted 1:500 with 0.3% PBSTriton X 100. The 2nd, 6th, 11th, 16th and 21st consecutive sections were processed for NFP immunohistochemistry by exposing them to a mouse monoclonal antibody raised against NFP (clone RT97, Chemicon, Millipore, Cat. No. 5262) at the concentration of 2 μg/ml. The 3rd 7, 12, 17th and 24th consecutive sections were processed for the immunohistochemical detection of MAP-2 immunoreactivity by exposing them to a rabbit anti MAP-2 (Sigma, USA, M4403) primary antibody diluted 1:500 after a pre-incubation in PBS-bovine serum albumin for 1 h. The 4th, 8th, 13th, 18th and 25th consecutive sections were processed for the immunohistochemical detection of AQP-4 by exposing them to a rabbit polyclonal antibody (Calbiochem, USA Cat. No. 178614) diluted 1:1000. For immunohistochemistry sections were exposed overnight in a moist chamber at 4 °C to primary antibodies and then for 30 min at 25 °C to corresponding secondary biotinylated antibodies (mouse-anti rabbit IgGs or goat-anti mouse IgGs) diluted to 1:200. The product of immune reaction was revealed using 3,3′-diaminobenzidine as a chromogen. The 2nd and 4th sections of each series were used as control and exposed to a non-immune IgG instead of the primary antibody. 2.3. Image analysis Nissl's stained sections were viewed under a light microscope at a final magnification of ×160. Via a TV connection, images were transferred from the microscope to the screen of an IAS 2000 image analyzer and used for counting the number of nerve cells of frontal cortex and hippocampus according to the procedure detailed in an earlier paper of our group [18]. For morphometric analysis, frontal cortex was divided as follows: zone I that includes the first cortical layer; zone II that includes the second, third and fourth cortical layers; zone III that includes the fifth cortical layer and zone IV that includes the sixth cortical layer. The numerical density of astrocyte profiles and their cell body area were assessed using the same procedure described above for nerve cell profiles. Astrocytes were considered cells displaying a dark-brown GFAP immunoreactivity. The density of immunoreac-

tion area occupied by NFP or MAP-2 was measured by image analysis in frontal cortex and hippocampus (subfields CA1, CA3 and dentate gyrus) according to the image analysis protocol detailed elsewhere [19]. The intensity of NFP immunostaining developed in the neuropil of frontal cortex and hippocampus was assessed microdensitometrically using an image analysis system calibrated taking as “zero” the background developed in sections incubated with a nonimmune serum and “100” as the conventional value of maximum intensity of staining. 2.4. Data analysis Means of different parameters investigated were calculated from single animal data, and group means ± S.E.M. were then derived from single animal values. The significance of differences between means was analyzed by analysis of variance (ANOVA) followed by the Newman–Keuls multiple range test. 3. Results Body weight values were similar in normotensive WKY or SHR either control or treated with galantamine, choline alphoscerate or the two drugs in association (data not shown). Brain weight values were lower in SHR either control or pharmacologically-treated (data not shown). Systolic blood pressure values averaged 112 ± 5.7 mmHg in WKY rats (n = 6), 201 ± 12.4 mmHg in control SHR (n = 6, P b 0.01 vs. WKY rats), 190 ± 9.8 mmHg in SHR treated with galantamine (n = 6, P b 0.01 vs. WKY rats), 190 ± 11.4 mmHg in SHR treated with choline alphoscerate (n = 6, P b 0.01 vs. WKY rats) and 199 ± 8.7 mmHg in SHR treated with galantamine plus choline alphoscerate (n = 6, P b 0.01 vs. WKY rats). These data indicate that pharmacological treatment did not lower blood pressure values in SHR. 3.1. Cerebral cortex and hippocampus microanatomy The results of microanatomical analysis of frontal cortex are summarized in Table 1. As shown, the number of neurons of various zones of frontal cortex showed a different sensitivity to hypertension, being zones II and III the areas with a lower density of nerve cell profiles in SHR compared to WKY rats (Fig. 1A and B and Table 1). Treatment with galantamine, choline alphoscerate or with the two

Table 2 Number of neurons, neurofilament 200 KDa immunoreactivity, number and size of GFAP-immunoreactive astrocytes in the subfields of hippocampus of the different animal groups investigated.

CA1 Number of neurons (103/mm3) Neurofilament I.R. Astrocytes number (103/mm3) Mean immuno-reaction area μm2 CA3 Number of neurons (103/mm3) Neurofilament I.R. Astrocytes number (103/mm3) Mean immuno-reaction area μm2 Dentate gyrus Number of neurons (103/mm3) Neurofilament I.R. Astrocytes number (103/mm3) Mean immuno-reaction area μm2

WKY (N = 6)

SHR (N = 6)

SHR + GAL (N = 6)

SHR + GPC (N = 6)

SHR + ASS (N = 6)

212 ± 10 33.3 ± 1.9 39.7 ± 0.8 97.8 ± 4.2

157 ± 8a 24.3 ± 1.2a 65.2 ± 2.7a 123.7 ± 5.9a

218 ± 9b 38.9 ± 1.3b 62.2 ± 2.9 109.4 ± 3.4b

220 ± 12b 32.8 ± 1.6b,c 54.1 ± 2.1a,b,c 92.1 ± 3.9b,c

219 ± 11b 36.4 ± 1.6b 50.8 ± 1.8a,b,c 87.7 ± 4.1b,c

58 ± 3 33.4 ± 1.6 45.9 ± 1.8 88.5 ± 4.0

54 ± 2 31.5 ± 1.4 71.5 ± 2.7a 101.2 ± 4.2a

50 ± 2 39.2 ± 1.7b 69.9 ± 2.6 109.2 ± 4.7b

57 ± 3 33.6 ± 1.4 55.7 ± 2.2b,c 86.3 ± 3.9b,c

56 ± 2 34.9 ± 1.6 51.2 ± 2.0b,c,d 88.8 ± 3.3b,c

627 ± 27 36.5 ± 1.2 37.4 ± 1.4 87.6 ± 3.5

466 ± 22a 26.8 ± 1.3a 49.1 ± 1.4a 104.2 ± 3.6a

621 ± 26b 38.5 ± 1.7b 46.1 ± 1.6 101.4 ± 4.7

640 ± 27b 35.8 ± 1.6b,c 39.8 ± 1.2b,c 80.4 ± 3.4b,c

636 ± 26b 34.5 ± 1.1b,c 40.9 ± 1.5b,c 85.1 ± 4.1b,c

Neurofilament I.R. values are expressed in arbitrary units calculated microdensitometrically as detailed in the Materials and methods section. For the significance of abbreviations see legend to Table 1. Data are the mean ± S.E.M. a p b 0.05 vs. WKY. b p b 0.05 vs. SHR. c p b 0.05 vs. SHR + GAL. d p b 0.05 vs. SHR + GPC.

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drugs in association affected to a different extent the number of neurons in zones II–IV of frontal cortex (Table 1). Zones II and IV were more sensitive to treatment compared to zone III (Table 1). Zone II displayed a pronounced sensitivity to both galantamine and choline alphoscerate (Table 1 and Fig. 1C–E). Treatment with the two drugs in association was most effective than single compounds in countering frontal cortex hypertension-related changes in zone IV (Table 1). In the hippocampus the CA1 subfield (Fig. 2A–C) and the dentate gyrus (Fig. 2D–F) were the areas undergoing nerve cell loss in SHR (Table 2). The number of nerve cell profiles was the same in the CA3 subfield of WKY rats and SHR either control or treated pharmacologically (data not shown). Pharmacological treatment with galantamine, choline alphoscerate or the two drugs in association (Fig. 2C and F) countered the nerve cell loss both in the CA1 subfield and in the dentate gyrus (Table 2). 3.2. Immunohistochemistry NFP immunoreactivity was located in nerve fibre-like structures within frontal cortex (Fig. 3) and hippocampus (data not shown). Quantitative image analysis revealed in frontal cortex and to a lesser extent in the hippocampus a decrease of NFP-immunoreactive structures (Fig. 3 and Tables 1 and 2). This loss was countered by

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treatment with galantamine and the association of galantamine plus choline alphoscerate and to a lesser extent by choline alphoscerate (Fig. 3). Sections processed for MAP-2 immunohistochemistry developed dark-brown staining in the dendritic tree of neurons of frontal cortex, and hippocampus (data not shown). No changes in the density and pattern of MAP-2 immunostaining were observed in the animal groups investigated (data not shown). In SHR a significant increase in the number and size of GFAP immunoreactive astrocytes was observed (Fig. 4 and Tables 1 and 2). This phenomenon was more pronounced in the CA1 subfield of hippocampus followed in the order by CA3 subfield, frontal cortex and dentate gyrus (Tables 1 and 2). This phenomenon was more pronounced in the CA1 subfield and in the frontal cortex and to a lesser extent in the dentate gyrus and in the CA3 subfield in the order (Tables 1 and 2). Treatment with choline alphoscerate alone or plus galantamine countered both numerical and volume increase of GFAPimmunoreactive astrocytes (Fig. 4 and Tables 1 and 2), whereas galantamine alone apparently did not affect astroglial reaction (Fig. 4 and Tables 1 and 2). Processing of sections for AQP-4 immunohistochemistry caused the development of a dark-brown immunoreaction around brain microvessels, confirming the localization of this water protein in

Fig. 3. Sections of rat frontal cortex processed for neurofilament 200 kDa immunohistochemistry. A: control normotensive WKY rat; B: control untreated SHR; C; SHR treated with galantamine; D: SHR treated with choline alphoscerate; E: SHR treated with galantamine and choline alphoscerate in association. Neurofilament 200 kDa immunoreactive axons in the neuropil of frontal cortex were remarkably decreased in SHR. The loss of neurofilament 200 kDa immunoreactive axons was countered by treatment with galantamine and galantamine plus choline alphoscerate. Choline alphoscerate alone was less active. Roman numerals indicate the cerebrocortical zones shown. Calibration bar: 50 μm.

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Fig. 4. Sections of the CA1 subfield of hippocampus processed for glial fibrillary acidic protein (GFAP) immunohistochemistry of to stain astrocytes. A: control normotensive WKY rat; B: control untreated SHR; C; SHR treated with galantamine; D: SHR treated with choline alphoscerate; E: SHR treated with galantamine plus choline alphoscerate. Note in SHR the numerical and size increase of GFAP-immunoreactive astrocytes. This phenomenon was countered by treatment with choline alphoscerate and with galantamine plus choline alphoscerate but not with treatment with galantamine alone. P: Pyramidal neurons. Calibration bar: 50 μm.

astrocyte foot processes near blood vessels (Fig. 5). An increased expression of AQP-4 in brain vessels of SHR countered by treatment with choline alphoscerate and to a lesser extent by galantamine alone or in association with choline alphoscerate was observed (Fig. 5). 4. Discussion Protection of neurons from damage and death is a challenge for neuroscience research and may offer new perspectives in the treatment of neurodegenerative disorders such as AD and VaD. Four AChE/ChE inhibitors (tacrine, donepezil, rivastigmine and galantamine) and memantine are the only drugs licensed for symptomatic relief of AD [6]. In vitro studies on models of neurodegenerative diseases have suggested that these drugs may protect neurons from apoptosis and death [13]. Hence, the first hypothesis that AChE/ChE inhibitors, by slowing-down acetylcholine degradation, increase deficient brain levels of the neurotransmitter and therefore may correct cholinergic imbalances in adult-onset dementia with consequent clinical benefits is overly simplistic [20]. Some data are also suggestive of a disease-modifying role of these drugs [20]. The mechanism through which the above drugs may elicit neuroprotection is complex and not fully understood. It is well known that amyloid beta-protein plays an important role in the degenerative process of the disease and increases the vulnerability of cultured cortical neurons

to glutamate neurotoxicity. Glutamate contributes to amyloid betaprotein-induced cytotoxicity probably through over activation of Nmethyl-D-aspartate-sensitive (NMDA) glutamate receptor with consequent excessive Ca2+ influx via the ion channel associated with this receptor, free-radical formation and neuronal injury [21]. An interference with these mechanisms is at the basis of the activity of memantine, a non-competitive NMDA receptor antagonist [21,22]. It has been hypothesized that AChE/ChE inhibitors share a similar mechanism of neuroprotective action, countering glutamate neurotoxicity via alpha-4-beta-2 and alpha-7 nicotinic acetylcholine receptors (nAChRs) and by inhibiting apoptosis [23]. As mentioned in the Introduction, similarly as for AD, a cholinergic involvement is hypothesized in the pathophysiology of VaD [3,5]. However, different from AD, no drugs are licensed for symptomatic relief of cognitive symptoms of VaD. A recent review has concluded that in patients affected by mild to moderate VaD benefits produced in cognition domains by AChE/ChE inhibitors and memantine are small and of uncertain clinical significance. It has been concluded that these effects are not enough to support widespread use of these drugs in VaD [6]. Previous studies of our group have shown that association between an AChE/ChE inhibitor with the cholinergic precursor choline alphoscerate increased dose-dependently acetylcholine levels more effectively than single compounds [16]. In view of this, the

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Fig. 5. Sections of frontal cortex processed for aquaporin-4 (AQP-4) immunohistochemistry as a marker of the blood brain barrier. A: control normotensive WKY rat; B: control untreated SHR; C; SHR treated with galantamine; D: SHR treated with choline alphoscerate; E: SHR treated with galantamine and choline alphoscerate in association. Note in SHR the increase of immunoreaction along micro vessels. Arrows indicate foot processes of astrocytes. Increased expression of AQP-4 was countered by treatment with choline alphoscerate or with the association galantamine plus choline alphoscerate and to a lesser extent by galantamine alone. Calibration bar: 12 μm.

present study was designed to assess if the two compounds alone or in association countered to some extent microanatomical changes occurring in the brain of SHR. SHR represent a model of chronic hypertension sharing several similarities with human essential hypertension. This rat strain is also characterized by brain injury reminiscent of VaD. Nerve cell loss, astrogliosis, cytoskeletal breakdown and atrophy both in the cerebral cortex and in the hippocampus are common traits of middle age SHR [18,19]. Moreover, young and aged SHR exhibit reduced nAChRs compared to age-matched normotensive WKY rats in a number of important brain regions for cognitive functions such as cerebral cortex, hippocampus and thalamus [24–29]. These findings are of some relevance considering that nAChRs play a significant role in memory and attention and are reduced in AD, dementia with Lewy bodies, autism, schizophrenia, and Parkinson's disease compared to age-matched controls [30,31]. Several lines of evidence suggest that deficits of nAChRs correlate well with the level of cognitive dysfunction occurring in adult-onset dementia disorders [30,31]. Administration of galantamine or choline alphoscerate alone countered to some extent microanatomical changes occurring in the frontal cortex and hippocampus of SHR. Since the two compounds did not affect blood pressure values, neuroprotective effects observed are not related with control of elevated blood pressure. Neither hypertension nor pharmacological treatment affected MAP-2 immu-

noreactivity. MAP-2 proteins promote microtubule formation in postmitotic cells and provide support to formed protofilaments [32]. Our findings of unaltered MAP-2 immunoreactivity suggest that microtubule depolymerization and instability are not the cause of nerve cell loss observed in the animal model we have used. Both galantamine and choline alphoscerate countered nerve cell loss in selected portions of cerebrocortical areas investigated. Galantamine was more active against cytoskeletal breakdown, whereas choline alphoscerate was effective in reversing astroglial reaction and in eliciting a protective effect on blood-brain barrier characterized using AQP-4 as a marker. This suggests that the two drugs tested have different neuroprotection targets in brain of SHR and therefore probably act via different mechanisms. Interestingly, the two compounds together induced a wider neuroprotective effect influencing the majority of microanatomical parameter affected in SHR. Galantamine is an inhibitor of AChE in addition to being an allosteric modulator of nAChRs [11]. Our results of a neuroprotective effect elicited by treatment with galantamine on brain damage in SHR are consistent with findings obtained in AD-related models demonstrating an activity of the drug on glutamate and beta-amyloid toxicity in vitro and to cholinergic stress in vivo [11]. It cannot be excluded that similarly as observed in the above models, neuroprotective activity of galantamine in our experimental model is related to up-regulation of the protective protein Bcl-2 and is mediated via alpha-7 nAChR [11].

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The other compound investigated, choline alphoscerate, is a choline-containing phospholipids extensively studied in preclinical settings both in vitro and in vivo [14,15,33–39]. Choline alphoscerate is probably, among choline-containing phospholipids, the most effective in enhancing in vivo acetylcholine release [33] and was also investigated with positive results on cognitive domain in patients affected by cerebrovascular disorders and VaD [14–17]. In the same animal model used in this study, choline alphoscerate was shown to counter hypertension-related nerve cell loss in the hippocampus and to counter glial reaction in this key area for learning and memory [39]. The present work confirms and extends the results of previous studies [39], further supporting the hypothesis that choline alphoscerate may act as a neuroprotectant in an animal model of VaD. Whereas the molecular mechanisms of neuroprotection elicited by AChE/ChE inhibitors including galantamine were investigated using several approaches [11,13,23,31], no information is available on the mechanism of action of choline alphoscerate. The remarkable increase of acetylcholine levels and release elicited by the compound [14,33] may promote trophic and/or receptor stimulating mechanisms linked to cholinergic activation, but the understanding of effects observed in the animal model of hypocholinergic/vascular neurodegeneration offered by SHR [19,24–28,40] requires further studies. The association with the AChE inhibitor galantamine and the acetylcholine precursor choline alphoscerate elicits neuroprotective effects superior than those observed with single compounds. Hence, the association between an acetylcholine breakdown inhibitor and precursor not only increases more effectively brain acetylcholine levels [16], but also confers to treatment a stronger neuroprotective effect by countering relevant aspects of brain injury in SHR. In view of the mild to moderate benefits observed in VaD by AChE/ChE inhibitors [6], clinical investigation of the effects of a cholinergic inhibitor/precursor association similar to that assessed in the present study, could contribute to identify a better pharmacotherapeutic approach to VaD based on treatments already available. References [1] Erkinjuntti T, Román G, Gauthier S, Feldman H, Rockwood K. Emerging therapies for vascular dementia and vascular cognitive impairment. Stroke 2004;35:1010–7. [2] Knopman D, Parisi J, Boeve BF, et al. Vascular dementia in a population-based autopsy study. Arch Neurol 2003;60:569–75. [3] Román GC. Vascular dementia: distinguishing characteristics, treatment, and prevention. J Am Geriatr Soc 2003;51:S296–304. [4] Everitt BJ, Robbins TW. Central cholinergic systems and cognition. Annu Rev Psychol 1997;48:649–84. [5] Brashear HR. Galantamine in the treatment of vascular dementia. Int Psychogeriatr 2003;15:187–93. [6] Kavirajan H, Schneider LS. Efficacy and adverse effects of cholinesterase inhibitors and memantine in vascular dementia: a meta-analysis of randomised controlled trials. Lancet Neurol 2007;6:782–92. [7] Sobrado M, Roda JM, López MG, Egea J, García AG. Galantamine and memantine produce different degrees of neuroprotection in rat hippocampal slices subjected to oxygen-glucose deprivation. Neurosci Lett 2004;365:132–6. [8] Arias E, Alés E, Gabilan NH, Cano-Abad MF, Villarroya M, García AG, et al. Galantamine prevents apoptosis induced by beta-amyloid and thapsigargin: involvement of nicotinic acetylcholine receptors. Neuropharmacology 2004;46:103–14. [9] Arias E, Gallego-Sandín S, Villarroya M, García AG, López MG. Unequal neuroprotection afforded by the acetylcholinesterase inhibitors galantamine, donepezil, and rivastigmine in SH-SY5Y neuroblastoma cells: role of nicotinic receptors. J Pharmacol Exp Ther 2005 Dec;315:1346–53. [10] Kume T, Sugimoto M, Takada Y, Yamaguchi T, Yonezawa A, Katsuki H, et al. Upregulation of nicotinic acetylcholine receptors by central-type acetylcholinesterase inhibitors in rat cortical neurons. Eur J Pharmacol 2005;527:77–85. [11] Geerts H. Indicators of neuroprotection with galantamine. Brain Res Bul 2005;64: 519–24. [12] Van Dam D, De Deyn PP. Cognitive evaluation of disease-modifying efficacy of galantamine and memantine in the APP23 model. Eur Neuropsychopharmacol 2006;16:59–69. [13] Francis PT, Nordberg A, Arnold SE. A preclinical view of cholinesterase inhibitors in neuroprotection: do they provide more than symptomatic benefits in Alzheimer's disease? Trends Pharmacol Sci 2005;26:104–11.

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