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Soy-derived phytoestrogens as preventive and acute neuroprotectors in experimental ischemic stroke: influence of rat strain.


The ability of a soy-based high-phytoestrogen diet (nutritional intervention) or genistein (pharmacological intervention), to limit ischemic brain damage in Wistar, Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats, has been assessed. As to the nutritional intervention, two groups from each strain received either a phytoestrogen-free (PE-0) or a high-phytoestrogen (PE-600) diet from weaning to adulthood. As to the pharmacological intervention, all animals were fed the standard soy-free AIN-93G diet and subsequently separated into two groups from each strain to receive either pure genistein (aglycone form, 1 mg/kg/day intraperitoneal) or vehicle at 30 min reperfusion. After an episode of 90 min ischemia (intraluminal thread procedure) followed by 3 days reperfusion, cerebral infarct volume was measured. Arterial blood pressure (ABP) was significantly higher at the basal stage (just before ischemia) in SHR (140 [+ or -] 7 mmHg, n = 17, p< 0.05) than in Wistar (113 [+ or -]4 mmHg, n = 23) and WKY (111 [+ or -]6 mmHg, n = 14) rats. No significant differences were shown among the three stages (basal, ischemia, reperfusion) within each rat strain for both PE-0 and PE-600 diets. Wistar, but not WKY or SHR, rats fed the PE-600 diet showed significantly lower infarct volumes than their counterparts fed the PE-0 diet (30 [+ or -] 3% vs. 17 [+ or -] 3%, p< 0.01). Genistein-treated Wistar, but not WKY or SHR, rats showed significantly lower infarct volumes than their vehicle-treated controls (27 [+ or -] 2% vs. 15 [+ or -] 2%, p < 0.01). Our results demonstrate that: (1) the neuroprotective action of either chronic or acute exposure to soy isoflavones is strain-dependent, since it was shown in Wistar but not WKY or SHR rats; and (2) the soy-based diet does not prevent development of hypertension in SHR rats.

[C] 2011 Elsevier GmbH. All rights reserved.










Since a consensus on the neuroprotective effects of estrogens against ischemic stroke does not exist (Strom et al., 2009), related alternatives are being investigated. Phytoestrogens are naturally occurring plant-derived compounds structurally related to the gonadal steroid, 17[beta]-estradiol, which are present in the human diet. The interest is being focused mainly on soy-derived foods as well as on the most abundant phytoestrogens they contain: genistein and daidzein. These compounds belong to the isoflavone family of phytoestrogens, have a low estrogenic potency compared to 17[beta]-estradiol, and bind estrogen receptor alpha (ER[alpha]) and beta (ER[beta]) with different affinities. ER[alpha] and ER[beta] exist in brain, both as "classical" intranuclear receptors mediating genomic effects of sex hormones, and as membrane-associated ER mediating short-time responses. Therefore, the basis exists for phytoestrogens influencing brain function under both physiological and pathological conditions (Torand-Allerand, 2004).

As to the cerebral circulation, we have previously demonstrated that phytoestrogens relax cerebral arteries (Torregrosa et al., 2003), thus being able to improve cerebral blood flow (Salom et al., 2007). This could account, at least in part, for the reported neuroprotective action of high-soy diets in animal models of ischemic stroke (Schreihofer et al., 2005; Burguete et al., 2006; Lovekamp-Swan et al., 2007). In spite of these positive results, to the best of our knowledge the potential neuroprotective role of isoflavones in ischemic stroke associated with hypertension has not been assessed yet, even when hypertension is the first modifiable and the second most important risk factor, after age, for hemorrhagic or ischemic stroke, and correlates with both incidence and worse outcome of stroke (Veglio et al., 2009). On the other hand, the impact of individual isoflavones when administered at the acute phase of experimental ischemic stroke has not been investigated. The present study was outlined to assess both the preventive (nutritional intervention) and the acute (pharmacological intervention) neuroprotective effect of soy-derived isoflavones on three rat strains subjected to an episode of transient focal cerebral ischemia: Wistar, hypertensive (SHR), and their normotensive counterpart Wistar-Kyoto (WKY).

Materials and methods

Experiments were conducted in compliance with the Spanish legislation on "Protection of Animals used for Experimental and other Scientific Purposes", and in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

For the studies on the preventive (nutritional) intervention, 21 Wistar, 17 WKY and 22 SHR male rats were used. All the animals were purchased just after weaning (3-weeks old, 50 g body weight), and those from each strain were separated into two groups receiving either a casein-based phytoestrogen-free diet (PE-0, reference TD.04015, Harlan Teklad, Madison, WI, USA) or a soy-based high-phytoestrogen (PE-600, 600 [micro]g phytoestrogens per g of diet, reference 8604, Harlan Teklad) diet. All animals were allowed to grow for 15-17 additional weeks, at which time SHR reached a sustained hypertensive state.

For the studies on the acute (pharmacological) intervention with genistein, 24 Wistar, 13 WKY and 12 SHR male rats were used. In order to avoid a possible interference of a phytoestrogen background, all the animals were fed with the purified diet AIN-93G (reference TD.94045, Harlan Teklad), the standard soy-free diet to feed laboratory rodents as formulated by the American Institute of Nutrition. When growth was completed, two groups were established for each strain: vehicle-treated (dimethylsulfoxide, DMSO 50% in distilled water, control group) rats, and genistein-treated rats. Both the vehicle and genistein were administered 30 min after ischemia (see below) by means of a miniature osmotic pump (model 2ML1, Alzet, Durect Corp., Cupertino, CA, USA) placed intraperitoneally and delivering 42 [micro]g/kg/h (1 mg/kg/day) pure genistein in its aglycone form throughout the 3 days until euthanization.

Right transient focal cerebral ischemia was carried out by following the original intraluminal thread procedure (Longa et al., 1989), adapted to our experimental setup (Burguete et al., 2006). This includes continuous monitorization of cerebrocortical laser-Doppler flow (cortical perfusion, CP) to get evidence that both ischemia and reperfusion maneuvers were accomplished successfully, core temperature (rectal probe, T) and arterial blood pressure (ABP), and discontinuous measurement of pH, [pO.sub.2], [pCO.sub.2] and glucose (femoral artery catheter) at the three stages during surgery (pre-ischemia, ischemia and reperfusion). Once the thread was advanced and CP fell suddenly, it was maintained for 90 min after which it was carefully removed and reperfusion was monitorized for 30 min. For the studies on acute neuroprotection, the osmotic pump was placed (i.p.) at that time. At 3 days post-ischemia, brain damage was assessed by measuring infarct volume following the 2,3,5-triphenyltetrazolium chloride (TTC) vital staining method (Bederson et al., 1986), and subsequent morphometric analysis (Swanson et al., 1990). Total, cortical and subcortical infarct volumes were obtained as absolute values ([mm.sub.3]), and expressed as a percentage of the corresponding contralateral (healthy) region. Statistical analysis was carried out by the two-way ANOVA followed by the Bonferroni's test. A p value of less than 0.05 was considered significant.



Mean CP values at the ischemic stage decreased to 30-49% (range for all groups) of the basal values. After 90 min ischemia, the thread was removed and CP increased gradually reaching a sustained plateau amounting 77-204% increase (range for all groups) above the basal values. Neither intraischemic nor postischemic CP values were significantly different among the groups both in the nutritional and the pharmacological studies. As to ABP, it was significantly higher at the basal stage (just before ischemia) for SHR (140 [+ or -] 7 mmHg, n = 17, p<0.05) than for Wistar (113 [+ or -] 4 mmHg, n = 23) and WKY (111 [+ or-] 6 mmHg, n = 14) rats. No significant differences were shown among the three stages (basal, ischemia, reperfusion) within each rat strain for both PE-0 and PE-600 diets.

Cerebral infarct volume was significantly lower (by about 50% overall) for WKY than for Wistar or SHR rats (Figs. 1 and 2). As to the nutritional intervention, Wistar rats fed the PE-600 diet showed significantly lower infarct volumes than their counterparts fed the PE-0 diet. This applied to both cortical and subcortical regions. Total infarct volume decreased from 30 [+ or -] 3% to 17 [+ or -] 3% (p < 0.01). By contrast, such a neuroprotective effect was not evidenced in WKY or SHR rats (Fig. 1). As to the pharmacological intervention, genistein-treated Wistar rats showed significantly lower infarct volumes than their vehicle-treated controls. This applied to both cortical and subcortical regions. Total infarct volume decreased from 27 [+ or -] 2% to 15 [+ or-] 2% (p<0.01). By contrast, such a neuroprotective effect was not evidenced in WKY or SHR rats (Fig. 2).


The main findings in the current study are: (1) Wistar, but not WKY or SHR rats, underwent significantly lower ischemic brain damage under either chronic or acute exposure to soy isoflavones; and (2) the isoflavone-enriched diet did not prevent development of hypertension in SHR rats. Overall, our results emphasize the need to carry out preclinical studies on putative neuroprotective strategies in different experimental models, before starting clinical trials.

As to the nutritional intervention, our present results from Wistar rats confirm previous results from our own group and others showing neuroprotective effects of high-isoflavone diets in stroke (Schreihofer et al., 2005; Burguete et al., 2006; Lovekamp-Swan et al., 2007). By contrast, such a neuroprotective action was not evidenced in WKY or SHR rats. As far as we know, no results have been previously reported from these two rat strains. As to the pharmacological intervention, our results show for the first time that genistein, when administered at the dose of 1 mg/kg/day under a sustained slow-release regime early after the ischemia-reperfusion episode, is able to reduce cerebral infarct volume to a similar extent than the soy-based diet in Wistar rats. Once again, WKY and SHR rats were refractory to the beneficial effects of isoflavones.


Why ischemic WKY and SHR rats did not benefit from the exposure to soy isoflavones deserves further research. A striking finding in the current study is the lower infarct volume in WKY compared to Wistar and SHR rats. It has been suggested that both structural and functional changes in cerebral arteries from SHR rats impair tissue perfusion and also limit the ability of cerebral arteries (particularly small arterioles) to dilate in response to ischemia. This could lead to an inadequate collateral circulation and higher infarctions (Amenta, 2003). However, such argument does not explain why brain lesions are almost identical in SHR and normotensive Wistar rats. Instead, one can suggest that WKY is not the most suitable nor-motensive control of SHR rats in stroke research, since it would be rather difficult to reduce such a small cerebral infarct. As to the SHR, some views claim to elevate blood pressure in the sequence of an acute ischemic stroke (Messerli, 2007). Most probably, mild hypertension as displayed by SHR rats in our current study could have exerted some neuroprotection by increasing collateral blood flow in the ischemic territory, as reported previously (Shin et al., 2008). Under such condition, exposure to isoflavones may be do not confer further neuroprotection.

Since ABP just before surgery did not differ between SHR rats fed either the PE-0 or the PE-600 diets, our results do not lend support to the use of soy-enriched diets in preventing hypertension. An antihypertensive effect of soy-based diets has been reported in experimental studies with SHR rats (Nevala et al., 2000; Martin et al., 2001). Interestingly, some of these studies also reported a lack of antihypertensive effect in certain experimental groups. The same controversy can be seen among studies in humans, in which soy either lowers (Rivas et al., 2002) or does not alter (Teede et al., 2006) ABP in hypertensive subjects.


The present work was partially supported by Instituto de Salud Carlos III through: (1) grant P106/0981 (responsible researcher Dr. German Torregrosa), (2) "Red Neurovascular (RENEVAS)" (RD06/0026/0006). included in the program "Redes Tematicas de Investigation Cooperativa en Salud (RETICS)", and (3) pre-doctoral grant to Maria Castello-Ruiz. The authors thank Maria C. Manez and Sonia Ruiz for technical assistance.


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M. Castello-Ruiz (a), (b), G. Torregrosa (a), (b), *, M.C. Burguete (b), J.B. Salom (a), (b), J.V. Gil (c), F.J. Miranda (b), T. Jover-Mengual (b), V.G. Marrachelli (b), E. Alborch (a), (b)

(a) Centro de Investigacion, Hospital Universitario La Fe, Ave. Campanar 21, 46009-Valencia, Spain

(b) Departamento de Fisiologia, Universidad de Valencia, Ave. Blasco Ibanez 15, 46010-Valencia, Spain

(c) Departamento de Medicina Preventiva y Salud Publica, Universidad de Valencia, Ave. Vicent Andres Estelles s/n, 46100-Burjassot, Valencia, Spain

* Corresponding author at: Hospital Universitario La Fe, Centra de Investigacion, Ave, Campanar, 21, 46009-Valencia, Spain. Tel.: +34 963862797; fax: +34 961973018.

E-mail address: (G. Torregrosa).

0944-7113/$ - see front matter [C] 2011 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2011.02.001
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Article Details
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Author:Castello-Ruiz, M.; Torregrosa, G.; Burguete, M.C.; Salom, J.B.; Gil, J.V.; Miranda, F.J.; Jover-Meng
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUSP
Date:Apr 15, 2011
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