Alcohol and Alcoholism Advance Access originally published online on April 29, 2008
Alcohol and Alcoholism 2008 43(4):408-415; doi:10.1093/alcalc/agn024
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Red Wine, but not Port Wine, Protects Rat Hippocampal Dentate Gyrus Against Ethanol-Induced Neuronal Damage—Relevance of the Sugar Content
1 Department of Anatomy, Faculty of Medicine, University of Porto
2 Department of Chemistry, Faculty of Sciences, Center of Investigation in Chemistry, University of Porto, Porto, Portugal
* Corresponding author: Department of Anatomy, Faculty of Medicine, University of Porto, Al. Prof. Hernâni Monteiro, 4200-319 Porto, Portugal. Tel: +351-22-5513616; Fax: +351-22-5513617; E-mail: jandrade{at}med.up.pt
Received 19 September 2007; first review notified 22 November 2007; in revised form 6 December 2007; accepted 5 February 2008
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Aims: Chronic ethanol consumption leads to oxidative damage in the central nervous system inducing neuronal degeneration and impairment of brain functions. Nevertheless, it has been reported that grape polyphenols might prevent the alluded ethanol effects. We have reported that prolonged red wine intake improves hippocampal formation oxidative status, a finding not replicated using Port wine. Thus, we thought of interest to compare the effects of chronic ingestion of these wines in the morphology of dentate gyrus (DG) neurons that are particularly vulnerable to alcohol effects. Methods: Six-month-old Wistar rats were fed either with red wine or Port wine (both with 20% ethanol content, v/v), and the results were compared with 20% (v/v) ethanol-treated, ethanol/glucose and pair-fed control groups. After 6 months of treatment, the layer volumes of the DG and the total number of granule and hilar neurons were estimated. The dendritic trees of granule cells were also studied in Golgi-impregnated material. Results: The number of granule cells and the DG layer volumes were similar among all groups. However, the number of hilar neurons was reduced in Port wine, ethanol-treated and ethanol/glucose animals. Furthermore, the granule cells from these groups showed a decrease in the total dendritic length. Conclusions: Although the Port wine and red wine have similar amounts of flavanols with identical ability to protect against oxidative stress, the differences observed are probably related to the very dissimilar processes of wine production, leading in Port wine to a high content of sugars, which are known to have potent pro-oxidant effects.
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Introduction |
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TOPABSTRACTIntroductionMaterial and MethodsResultsDiscussionReferences |
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Polyphenols, the most abundant antioxidants in the human diet (Halliwell, 1992
, 2006), emerge as potential pharmacological tools that can exert a pivotal role in the protection of brain against ethanol-induced oxidative damage (Halliwell, 1992, 2006; Nordmann, 1994). One of the major sources of polyphenols is red wine that allegedly possesses beneficial antioxidant effects when ingested in moderate amounts (Soleas et al., 1997; German and Walzem, 2000) despite ethanol toxicity to almost every tissue in the body. In line with this hypothesis, we found that a mixture of flavanol oligomers extracted from grape seeds, added to a 20% (v/v) ethanol solution prevented the alcohol-induced increase in neuronal lipofuscin, a morphological marker of lipid peroxidation (de Freitas et al., 2004). Red wine is rich in different types of polyphenols (Roig et al., 1999; German and Walzem, 2000; Santos-Buelga and Scalbert, 2000), including the purified flavanols previously tested (de Freitas et al., 2004). Interestingly, it was found that prolonged intake of this beverage, with ethanol content adjusted to 20% (v/v), prevented ethanol-induced lipofuscin deposition in hippocampal pyramids and ameliorated the rat hippocampal formation oxidative status (Assunção et al., 2007b). In contrast, Port wine, a special kind of red wine that contains approximately the same amount and type of flavanols found in red wine, did not display any protective action (Assunção et al., 2007a). What makes Port wine different from common red wines is the addition of wine spirit to the must before complete fermentation, i.e. when the concentration of sugars is about half of that present in the grape. This addition increases the ethanol content of Port wine to 20% (v/v) and preserves a high concentration (90 g/l) of sugars.
In order to better understand how these beverages differentially affect neuronal morphology, we decided to study and compare the effects of chronic consumption of red wine and Port wine upon the main neuronal populations of the dentate gyrus (DG). This hippocampal region is particularly vulnerable to injuries caused by ethanol consumption ultimately leading to neuronal death (Andrade et al., 1992; Paula-Barbosa et al., 1993; Lukoyanov et al., 1999). Thus, using unbiased stereological methods, we verified whether the different treatments affected the volumes of DG layers, the total number of granule cells and, in Golgi-impregnated material, the organization of its dendritic arborizations. Because the neurons of the hilar region of the DG are likewise susceptible to the ethanol intake (Andrade et al., 1992; Paula-Barbosa et al., 1993; Lukoyanov et al., 1999), their total number was also estimated.
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Material and Methods |
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Animals and treatments
Studies were carried out in adult male Wistar rats (Charles River, Barcelona, Spain) that were individually housed and maintained under standard laboratory conditions (20–22°C and 12-h light/dark cycle). Prior to the beginning of the experiment, standard laboratory chow (Letica, Barcelona, Spain) and tap water were provided ad libitum. At 6 months of age, 30 animals weighing 645 ± 38 g were randomly divided into five experimental groups of 6 animals each and treated as follows:
- Ethanol (EtOH) group. Animals were treated with an aqueous ethanol solution as the only available liquid source, starting with a 5% (v/v) ethanol solution and increased progressively by 1% per day to a final concentration of 20% (v/v) 2 weeks later. These rats had free access to standard laboratory chow.
- Pair-fed control (PFC) group. Rats were given the same amount of rat chow as the EtOH group. The calories provided by ethanol were replaced by an isocaloric amount of sucrose, considering that 1 g of sucrose corresponds to 4 kcal and 1 g of ethanol to 7 kcal, and that the density of ethanol is 0.79 g/ml.
- Port wine (PW) group. Animals were given Port wine (containing 20% (v/v) ethanol, approximately 90 g/l of sugars and 174 mg/l of low-molecular-weight flavanols) as the only available drinking fluid. The introduction of Port wine was gradual, starting with a 5% ethanol (v/v) solution as with EtOH-treated rats. Animals had also free access to standard laboratory chow.
- Ethanol/glucose (EtOH/Glu) group. This group represented a nutritional control of the PW group and therefore animals were given the same amount of rat chow. As a liquid source these rats had free access to a 20% (v/v) ethanol solution in which glucose was added to replace the isocaloric value of sugars present in the Port wine. Ethanol introduction was similar to the EtOH group.
- Red wine (RW) group. Rats had free access to red wine as the sole drinking fluid in which ethanol concentration was also increased to 20% (v/v) to equalize the ethanol content in beverages ingested by the EtOH, PW and EtOH/Glu groups. Animals started drinking red wine as alluded for the other alcohol-containing groups. The content of flavanols measured in red wine was approximately 192 mg/l and animals had unrestricted access to standard laboratory chow.
In order to calculate the amounts of food and fluid intake, every other day measurements were performed. Blood ethanol concentrations were measured in all animals from the EtOH, PW, EtOH/Glu, and RW groups. Blood samples (approximately 200 µl) were collected from the dorsal tail vein of each rat, weekly in the first month of treatment and monthly thereafter, 2–4 h after the beginning of the dark period, i.e. at 22:00 hours and 00:00 hours. This schedule was chosen because the consumption of liquids in rodents is maximal during this period (Jelic et al., 1998; Assunção et al., 2007b). Estimations were made in the serum, using an enzymatic assay kit (Sigma, St Louis, USA). The handling and care of the animals were conducted according to the Portuguese Act 129/92.
The composition of flavanols (catechins and oligomeric procyanidins) in Port wine (Taylor, Fladgate and Yeatman-Vinhos, SA, Portugal) and red wine (Quinta do Vale Meão, Douro, Portugal) was assessed by high-performance liquid chromatography (HPLC) as previously described (de Freitas and Glories, 1999; Mateus et al., 2002) and is shown in Table 1.
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Tissue preparation
After 26 weeks of treatment, at 12 months of age, the animals were deeply anesthetized with sodium pentobarbital (80 mg/kg of body weight, i.p.) and killed by transcardiac perfusion with a fixative solution containing 1% paraformaldehyde (w/v) and 1% glutaraldehyde (v/v) in a 0.12 M phosphate buffer at pH 7.2. The brains were removed from the skulls, weighed, codified to allow blind estimations, separated by a midsagittal cut into right and left halves and placed in a fresh fixative solution. After the removal of the frontal and occipital poles, the blocks of tissue containing the hippocampal formations were processed for glycolmethacrylate embedding and Golgi impregnation.
After 15 days of post-fixation in the fixative solution, blocks of tissue containing the entire hippocampal formation were dehydrated through a graded series of ethanol solutions and embedded in glycolmethacrylate, as described in detail elsewhere (West et al., 1991; Madeira et al., 1997). After sectioning in the horizontal plane at a nominal thickness of 40 µm, every tenth section was collected using a systematic random sampling procedure (Gundersen and Jensen, 1987), mounted serially and stained with a modified Giemsa solution (West et al., 1991; Madeira et al., 1997).
The remaining blocks containing the hippocampal formations were post-fixed for an additional period of 15 days in the fixative solution. Golgi impregnation was performed according to the method of Stensaas (1967), with the modifications introduced by Eckenhoff and Rakic (1984). Briefly, the blocks were immersed, for 72 h, in a solution of 10 ml glutaraldehyde (25%), 10 ml formaldehyde (39%), 5 g potassium dichromate, 5–9 drops of dimethylsulfoxide and distilled water up to 100 ml. The blocks were then transferred into 1.5% silver nitrate and stored in the dark for 3–5 days. The tissue was shelled in paraffin and horizontal sections of the hippocampal formation with a nominal thickness of 100 µm were obtained, dehydrated, cleared in terpineol and mounted on slides under Damar resin with no cover slip.
Morphometric analyses
Definition of anatomical regions
The dentate molecular and granular layers are easily circumscribed due to their simple cytoarchitecture at all levels along the septotemporal axis of the hippocampal formation (Fig. 1). The border of the dentate hilus was defined by the inner edge of the granule cell layer and the lines connecting the tips of the suprapyramidal and infrapyramidal blades of the DG to the beginning of the CA3 pyramidal layer, excluding the closely packed and large CA3 pyramidal cells that frequently extend into the hilus (West et al., 1991; Amaral and Witter, 1995; Lukoyanov et al., 2004).
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Layer volumetric measurements and neuron numbers The volumes of the dentate molecular and granule cell layers and of the dentate hilus were estimated in all sampled glycolmethacrylate-embedded sections by using the principle of Cavalieri (Gundersen and Jensen, 1987
) as previously described in detail (Madeira et al., 1997; Lukoyanov et al., 2004). The total numbers of granule cells and hilar neurons were also estimated on these sections using the optical fractionator (West et al., 1991; Madeira et al., 1997; Lukoyanov et al., 2004) and utilizing the C.A.S.T.-Grid System software (Olympus DK, Denmark) and a Heidenhain MT-12 microcator (Heidenhain, Germany). This stereological methodology results in unbiased estimates of neuron numbers, i.e. the estimates are free from assumptions about neuron size and shape (West et al., 1991). Beginning at a random start position, the fields of vision were systematically sampled along the x and y axes of the sections, using a raster pattern procedure. Neurons were counted in every frame using the optical disector (West et al., 1991; Madeira et al., 1997), at a final magnification of 2000x, at the level of the monitor. Counting of neurons was based on the presence of the nucleus (the counting unit) that came into focus within the counting frame. The first, look-up, focal plane of the disector was positioned 5 µm below the upper surface of the section, thereby eliminating the problem of lost caps and avoiding counting at tissues surfaces where cells might be lost due to damage during cutting and processing (West et al., 1991). The heights of the disector used were 15 µm for the hilus and 10 µm for the granule cell layer. Glial cells, identified according to the criteria described by Ling et al. (1973), i.e. the relatively small nuclei and lack of cytoplasmic staining, were not included in the estimations. The countings obtained with the application of the optical disectors were a sample from a known fraction of the entire DG and thus used to calculate the final estimate of granule cells and hilar neurons.
Quantitative analysis of dentate granule cell dendrites
The neurons used for the analysis of the dendritic trees were sampled from the middle third of the suprapyramidal blade of the DG and selected according to the criteria suggested by de Ruiter and Uylings (1987): (1) consistent and complete impregnation throughout the extent of dendrites, i.e. the dendrites did not trail off as a series of dots of stain; (2) cell bodies located in the middle third of the section thickness to minimize cut dendritic segments at the plane of the section and (3) relative isolation of the dendritic tree from heavy clusters of dendrites from other neurons, blood vessels and stain precipitations. The overall quality of the Golgi–Stensaas impregnation was appreciated on the basis of the visualization of delicate structures, such as dendritic spines and terminal tips. The first ten cells visualized per animal fulfilling those criteria were drawn with the aid of a camera lucida at a magnification of 640x and a two-dimensional drawing of each neuron was obtained. For the metric analysis, the dendritic segments were allocated according to their different length distributions to the subgroups described by Uylings et al. (1986). Briefly, dendritic segments were classified as terminals when they were placed between the terminal tip of the dendrites and the first bifurcation point from the terminal tip and the other dendritic segments were classified as intermediate (Uylings et al., 1986). The lengths of dendritic trees of all segments were measured with a graphic pen tablet and MOP-Videoplan computer software (Kontron Elektronik GmbH, Germany) and the total dendritic length was calculated as the sum of the lengths of all dendritic segments. The mean length of the terminal and intermediate segments and the total number of segments were also calculated.
Statistical analyses
The unpaired Student's t-test was performed to assess statistical differences in daily flavanol consumption between the Port wine and red wine groups. The remaining data were analyzed using one-way ANOVA. The Newman–Keuls post hoc test was used for pairwise comparisons when appropriate. All results are expressed as means ± standard deviation (SD). Differences were considered statistically significant when P < 0.05. The coefficient of error (CE) for individual estimates of the volumes was calculated as shown by Cruz-Orive (1999) whereas the CE of the individual estimates of neuronal number was calculated according to Gundersen et al. (1999).
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Results |
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Animals and treatments
The amounts of food and fluid intake of all groups throughout the entire experimental period are summarized in Table 2. Daily food ingestion in the PW and EtOH/Glu groups was significantly higher when compared to the PFC, EtOH and RW groups (P < 0.01). Fluid ingestion in the PW and EtOH/Glu groups of animals was similar and significantly higher than that verified for the EtOH and RW groups (P < 0.01). Although all ethanol-fed groups of rats presented lower fluid ingestions when compared to PFC animals (P < 0.001), it has been previously demonstrated that rats of the same Wistar strain submitted to chronic intake of a 20% (v/v) ethanol solution did not show signs of dehydration and presented a normal rate of survival (Madeira et al., 1993
; Assunção et al., 2007b). As also depicted in Table 2, ethanol consumption expressed per kilogram of body weight was not significantly different among all ethanol-treated groups. Similarly, comparison of daily ingestion of flavanols per kilogram of body weight between PW and RW rats did not reveal differences.
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The body weight of the animals was similar among all groups at the beginning of treatment and increased gradually throughout the experiment. During the entire experimental period, animals from the PW and EtOH/Glu groups gained significantly more weight than all other groups (P < 0.001). At the end of the experiment, no significant differences were detected when the mean body weights of PFC (739 ± 30 g), EtOH (733 ± 39 g) and RW (720 ± 36 g) rats were compared. Also, no changes were observed between PW (848 ± 43 g) and EtOH/Glu (828 ± 41 g) groups. The mean brain weight was also similar among all groups (average of 1.58 ± 0.49 g).
Blood ethanol concentrations from EtOH, PW, EtOH/Glu and RW rats were comparable in all blood samples collected. The mean value was 0.48 ± 0.32 g/l and ranged between 0.10 and 1.20 g/l.
Qualitative observations
The general morphological features of the DG, as seen in the Giemsa-stained glycholmethacrylate-embedded material, were similar in all groups of animals. However, hilar neurons appeared to be less packed in the EtOH, PW and EtOH/Glu groups when compared to the PFC and RW groups of animals (Fig. 1). Granule cells dendritic arborizations from PFC and RW animals presented slightly more complex dendritic arborizations than those of the remaining groups (Fig. 2).
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Quantitative results
Volumetric measurements and neuron numbers The volumetric estimates of the dentate molecular and granule cell layers and of the dentate hilus are shown in Fig. 3. The mean CE for the individual estimates of the volumes was 0.09. ANOVA revealed no significant effect of treatment when the volumes of the granular [F(4, 25) = 0.53; P = 0.72], hilar [F(4, 25) = 0.61; P = 0.66] and molecular [F(4, 25) = 0.61; P = 0.77] layers were compared.
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The estimates of the total number of granule cells and hilar neurons can be seen in Fig. 4. The mean CE for the individual estimates of the neuron numbers was 0.08. ANOVA revealed no significant effect of treatment on the total number of granule cells [F(4, 25) = 0.54; P = 0.71]. However, ANOVA indicated that treatment influenced the total number of hilar neurons [F(4, 25) = 12.38; P < 0.0001]. The EtOH group showed a decrease of around 16% in the number of neurons when compared to PFC and RW animals (P < 0.05). Also, although RW animals presented approximately the same number of hilar neurons of the PFC group, they were decreased in PW rats by approximately 33% when compared to the PFC and RW groups (P < 0.01) and by 20% when compared to the EtOH group (P < 0.05). The animals from the EtOH/Glu group presented the lowest number of hilar neurons, with an approximate reduction of 41% when compared to the PFC and RW groups of animals (P < 0.001) and 30% when compared to the EtOH group (P < 0.01).
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Dendritic arborizations of granule cells
The results of the morphometric analysis of the granule cell dendritic trees are shown in Table 3. ANOVA detected a significant effect of treatment on the total dendritic length [F(4, 25) = 4.30; P < 0.02]. The animals from the PFC group presented a higher total dendritic length when compared to the EtOH, PW and EtOH/Glu groups of animals (P < 0.02), but not when compared to RW animals. ANOVA revealed no difference among groups in the total number of segments [F(4, 25) = 1.38; P = 0.24] and total number of terminal [F(4, 25) = 1.21; P = 0.31] and intermediate [F(4, 25) = 2.08; P = 0.13] segments. Likewise, the total length of terminal [F(4, 25) = 1.94; P = 0.10] and intermediate [F(4, 25) = 0.12; P = 0.88] segments remained unaltered by the treatments. ANOVA detected a significant effect of treatment on the mean length of terminal segments of granule cells [F(4, 25) = 5.06; P < 0.001]. This parameter was reduced in PW animals when compared to the PFC, EtOH, EtOH/Glu and RW groups of animals (P < 0.03). No effect of treatment was detected on the mean length of intermediate segments [F(4, 25) = 1.94; P = 0.15].
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Discussion |
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Ethanol consumption disturbs the balance between the production of reactive oxygen species and the antioxidant mechanisms of defense present in the brain (Halliwell, 1992
; Nordmann, 1994). As a consequence, there is an increase of lipid peroxidation of cell membranes, a decrease of antioxidant enzymes activity and disturbances of calcium homeostasis (Montoliu et al., 1994, 1995) and excitotoxic events (Fadda and Rossetti, 1998; Halliwell, 2006). Thus, oxidative stress has emerged as a relevant cause explaining ethanol-induced neuronal lesions (Halliwell, 1992; Nordmann, 1994; Fadda and Rossetti, 1998). For the same reason, dietary antioxidant supplementation is now regarded as a protecting measure to counteract alcohol deleterious effects.
Red wine is a major source of polyphenols, namely flavanols and anthocyanins, which are capable to exert widespread beneficial antioxidant effects (Soleas et al., 1997; German and Walzem, 2000; Bianchini and Vainio, 2003; Cordova et al., 2005). In the brain, antioxidants promote chelation of heavy metals, modulation of enzymes, free-radical scavenging and regulation of intraneuronal calcium (Soleas et al., 1997; German and Walzem, 2000; Youdim et al., 2004; Passamonti et al., 2005). These complex reactions support our recent data showing that chronic red wine consumption impedes an alcohol-induced progressive increase of lipofuscin, a pigment known to be an end result of lipid peroxidation, in CA3 and CA1 hippocampal pyramidal cells (Assunção et al., 2007b). Likewise, the results now obtained in the DG reinforce the view that red wine ingestion improves the overall hippocampal oxidative status (Assunção et al., 2007b) and prevents neuronal damage associated with ethanol consumption, as suggested by the morphological data observed in rats fed for long periods with red wine that apparently do not differ from pair-fed controls.
In contrast, rats ingesting 20% ethanol, Port wine and the ethanol/glucose solution displayed loss of hilar neurons, in agreement with previous results in which a reduction of these neurons was found after 6 months of ethanol intake (Andrade et al., 1992; Paula-Barbosa et al., 1993). These hilar neurons are also susceptible to ischemia (Müller et al., 2001), epilepsy (Lukoyanov et al., 2004; Zappone and Sloviter, 2004), protein deprivation (Andrade et al., 1995) and experimental diabetes (Beauquis et al., 2006). This high vulnerability of hilar neurons to aggressions can be related to alterations of their pattern of excitatory innervation, which arises from granule cells (Wenzel et al., 1997; Sloviter et al., 2003; Zappone and Sloviter, 2004). In fact, if granule cells are repetitively excited, their hilar synaptic ends degenerate and trophically destroy most of their targets in the hilus (Sloviter et al., 2003; Zappone and Sloviter, 2004). Incidentally, changes in the excitatory balance between granule and hilar cells are known to occur after the chronic consumption of ethanol (Fadda and Rossetti, 1998), and might well explain the striking hilar cell loss observed in the EtOH, PW and EtOH/Glu groups of rats.
We also found a reduction of the total dendritic length of granule cells, which indicates that the global receptive surface of these neurons in the EtOH, PW and EtOH/Glu groups is decreased. Besides, following chronic Port wine consumption, a reduction of the mean length of terminal dendritic segments was additionally detected suggesting that the afferent input from the entorhinal cortex to granule cells is markedly affected (Amaral and Witter, 1995). In addition to entorhinal neurons, some hilar cells also primarily project to these distal dendritic segments of granule cells participating in a fine modulation of the entorhinal afferents (Wenzel et al., 1997), which are of paramount importance for the regulation of the hippocampal circuitry (Amaral and Witter, 1995).
Thus, in contrast to red wine intake, our data show that Port wine consumption does not display protective effects upon the DG although this beverage contains similar amounts of flavanols. As Port wine production noticeably differs from common red wines, we cannot reject the possibility that the content in other polyphenols or different kinds of compounds is markedly dissimilar, justifying the observed lack of protection in the central nervous system (de Freitas et al., 2004; Assunção et al., 2007a,b). More importantly, the presence of high amounts of carbohydrates in Port wine cannot be discarded as underlying the lack of protective effects when compared to red wine and 20% ethanol. This is supported by the reduced length of terminal dendritic segments found in PW rats that display a more severe toxic action when compared to EtOH/Glu animals. Indeed, a direct interaction between polyphenols and sugars has been described and this complexation may hamper their intestinal absorption (Scalbert and Williamson, 2000; Williamson and Manach, 2005). However, the existence of direct pro-oxidant effects of the carbohydrates not efficiently counterbalanced by antioxidant compounds cannot be excluded (Busserolles et al., 2002; Vincent et al., 2004; Beauquis et al., 2006). Actually, it has been demonstrated that hyperglycemia of long duration, as observed in glucose-infused animals and in experimental diabetes, enhanced and accelerated neuronal damage induced by transient experimental hypoxia or focal cerebral ischemia and facilitated the induction of seizures (Smith et al., 1984; Li et al., 1998; Muranyi and Li, 2006). In fact, hyperglycemia in these hypoxic conditions increased both the formation of reactive oxygen species and mitochondrial dysfunction, and activated neuronal death pathways in accordance with an increased loss of neurons in several brain regions, including the hippocampal formation (Ding et al., 2004; Shi and Liu, 2006). Thus, it is not surprising that diabetic rodents presented a 30% reduction of the number of hilar neurons (Beauquis et al., 2006). These data fit nicely with our results, where both the EtOH/Glu and the PW groups displayed a 41% and 33% reduction of hilar neurons, respectively, when compared to PFC and RW animals. Moreover, the hilar neuronal loss in both EtOH/Glu and PW groups was significantly greater than EtOH rats further supporting the implication of the high sugar content in the difference between red wine and Port wine neuronal effects. This neuronal death was not accompanied with a reduction of the volume of the hilus, a conjunction that has also been shown in diabetic rodents (Beauquis et al., 2006), after ethanol consumption and withdrawal (Brandão et al., 1995) and in experimental epilepsy in Wistar rats (Lukoyanov et al., 2004; Xu et al., 2004). In this last experimental setting, it was suggested that the enhanced growth and sprouting of the axons of granule cells could be involved in the absence of hilar volumetric changes (Xu et al., 2004) that, if accompanied by proliferation of glial cells (Borges et al., 2006), could eventually explain the same finding in all ethanol-containing groups of the present experiment.
Whatsoever the mechanisms involved in the lack of protection displayed by Port wine in the DG, the present data are in accordance with previous reports showing that chronic Port wine consumption failed to prevent the deposition of lipofuscin in CA3 and CA1 hippocampal pyramids (Assunção et al., 2007a) contrary to what has been described following the intake of an ethanolic solution containing purified grape seed flavanols (de Freitas et al., 2004) and red wine consumption (Assunção et al., 2007b).
The regressive changes in the dendrites of granule cells and hilar neuronal loss found in the EtOH, PW and EtOH/Glu groups are very likely to interfere in the interneuronal connectivity of the DG. For example, it is well established that the morphological integrity of the hippocampal trisynaptic circuitry including the normal number of regulatory hilar neurons is essential for the maintenance of cognitive activity (Amaral and Witter, 1995; Lukoyanov et al., 2004). Accordingly, we have previously reported that EtOH animals had significant spatial memory deficits whereas RW rats displayed no behavioral deficits when compared to the PFC group (Assunção et al., 2007b). Therefore, we can hypothesize that given the still higher hilar neuronal loss found in the PW and EtOH/Glu groups of rats, cognitive functions are probably worse than those found in EtOH animals (Assunção et al., 2007b).
In conclusion, we did not find major alterations in the structure of the DG following red wine ingestion. Hence, we can suggest that the antioxidant compounds present in this alcoholic beverage protected DG neurons from the ethanol-induced oxidative stress. In contrast, Port wine consumption did not impede, but even enhanced, ethanol-induced hilar neuronal loss and the regressive dendritic changes of granule cells. As the two alcoholic beverages contain approximately the same amount of flavanols and therefore an identical potential to protect against oxidative stress, the differences observed must relate to other factors like the higher content of sugar in Port wine.
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ACKNOWLEDGEMENTS |
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This work was supported by Fundação para a Ciência e a Tecnologia (FCT)-Unit 121/94. The Port wine was a gift from Taylor, Fladgate and Yeatman-Vinhos, SA, Portugal and the red wine was kindly provided by Quinta do Vale Meão, Douro, Portugal.
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