Dietary Supplementation with Blueberries, Spinach, Or Spirulina Reduces Ischemic Brain Damage
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Experimental Neurology 193 (2005) 75–84 www.elsevier.com/locate/yexnr Dietary supplementation with blueberries, spinach, or spirulina reduces ischemic brain damage Yun Wanga, Chen-Fu Changa, Jenny Choua, Hui-Ling Chena, Xiaolin Denga, Brandon K. Harveya, Jean Lud Cadeta, Paula C. Bickfordb,* aNational Institute on Drug Abuse, Intramural Research Program, Baltimore, MD, 21224, USA bDepartment of Neurosurgery, University South Florida College of Medicine, James A Haley Va Hospital and Center of Excellence for Aging and Brain Repair, MDC 78, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612, USA Received 7 July 2004; revised 12 October 2004; accepted 6 December 2004 Abstract Free radicals are involved in neurodegenerative disorders, such as ischemia and aging. We have previously demonstrated that treatment with diets enriched with blueberry, spinach, or spirulina have been shown to reduce neurodegenerative changes in aged animals. The purpose of this study was to determine if these diets have neuroprotective effects in focal ischemic brain. Adult male Sprague–Dawley rats were fed with equal amounts of diets (blueberry, spinach, and spirulina) or with control diet. After 4 weeks of feeding, all animals were anesthetized with chloral hydrate. The right middle cerebral artery was ligated with a 10-O suture for 60 min. The ligature was later removed to allow reperfusional injury. Animals were sacrificed and brains were removed for caspase-3 enzymatic assays and triphenyltetrazolium chloride staining at 8 and 48 h after the onset of reperfusion. A subgroup of animals was used for locomotor behavior and biochemical assays. We found that animals which received blueberry, spinach, or spirulina enriched diets had a significant reduction in the volume of infarction in the cerebral cortex and an increase in post-stroke locomotor activity. There was no difference in blood biochemistry, blood CO2, and electrolyte levels among all groups, suggesting that the protection was not indirectly mediated through the changes in physiological functions. Animals treated with blueberry, spinach, or spirulina had significantly lower caspase-3 activity in the ischemic hemisphere. In conclusion, our data suggest that chronic treatment with blueberry, spinach, or spirulina reduces ischemia/reperfusion-induced apoptosis and cerebral infarction. Published by Elsevier Inc. Keywords: Nutrition; Diet; Ischemia; Neuroprotection; Apoptosis; Antioxidant Introduction damage during stroke. Recently, several dietary supple- ments have been reported to have strong antioxidant Stroke is an acute and progressive neurodegenerative effects and reduce neurological deficits in aged animals disorder. Besides the deprivation of oxygen and nutrients (Bickford et al., 2000; Gemma et al., 2002). Rats on during the acute ischemic phase, generation of toxic blueberry-supplemented diets for 6 weeks developed less compounds, such as free radicals, reactive oxygen or neuronal loss in the hippocampus after cerebral ischemia nitrogen species, and ONOO-, during the reperfusional (Sweeney et al., 2002). These data suggest that these stage can induce increased damage to the brain. It has been dietary supplements may have protective effect against demonstrated that pharmacological agents that possess free neurodegeneration. radical scavenging or antioxidant properties reduce brain A recent epidemiological study has shown an inverse association between the incidence of cerebral infarction and * Corresponding author. Fax: +1 813 974 1364. intake of vegetables or h-carotene in male smokers E-mail address: [email protected] (P.C. Bickford). (Hirvonen et al., 2000). The associations between incidence 0014-4886/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.expneurol.2004.12.014 76 Y. Wang et al. / Experimental Neurology 193 (2005) 75–84 of cerebral infarction and intake of fruits, berries, tea, or dants, reduce toxicity of OONO-, and furthermore, reduce wine were not significant. These data suggest that some, but cholesterol levels. All these factors have been previously not all, diets may have protective effect against cerebral reported to be involved in ischemic brain injury. The utility infarction. of spirulina diet in protection against stroke is still not Several ingredients in blueberries (Vaccinium spp.) known. have been shown to have strong antioxidant activity. In this study, we study the protective effects of three These include the pigments flavonoids, anthocyanins, and diets: blueberry, spinach, and spirulina against stroke in rats. others (Cao et al., 1999). Treatment with extracts from Our data indicate that these diets have differential effects to blueberries reduced oxidative stress and age-related reduce ischemia-induced caspase-3 activity and cerebral declines in neuronal function in vitro and in vivo infarction. (Joseph et al., 1998). On the other hand, blueberry juice, taken acutely, did not enhance the antioxidant capacity to reduce potassium nitrosodisulfonate and Materials and methods Fe(III)-2,4,6,Tri(2-pyridyl)s-trizine in plasma (Pedersen et al., 2000). Similar to blueberry, spinach leaves also Animals contain high levels of antioxidants flavonoids, p-couma- ric acid (Bergman et al., 2001), 9-cis-h-carotene (Yan et Adult male Sprague–Dawley rats, purchased from the al., 2001), and other water-soluble natural antioxidants Charles River Laboratories, were used for this study. (NAO). These ingredients can reduce LPS-induced Animals were caged singly and separated into 4 groups inflammation and necrosis (Lomnitski et al., 2000) and according to the diets: (A) control NIH 07/31 diet, (B) doxorubicin-induced cardiac damage while increasing blueberry diet (2%), (C) spinach diet (2%), and (D) superoxide dismutase activities (Breitbart et al., 2001). spirulina diet (0.33%). All animals were fed with 5 food Taken together, these data suggest that both blueberry pellets (4–6 g per each pellet) per day for 4 weeks before and spinach have certain protective effects against stroke surgery. oxidative stress and inflammation-induced damage. Spirulina, a type of blue green algae that has been Ischemia/reperfusion consumed for thousand of years as a primary food source for the Aztecs and Mayans, contains high levels of Rats were anesthetized with chloral hydrate (0.4 g/kg, antioxidants, for example, carotenoids (Annapurna et al., i.p). The ligation of the right MCA and bilateral common 1991), especially h-carotene (Careri et al., 2001), and carotids (CCAs) was performed using methods previously phycocyanin and phycocyanobilin (Reddy et al., 2000). In described (Chen et al., 1986; Wang et al., 2003). The vivo studies also indicate that chronic treatment with bilateral CCAs were identified and isolated through a spirulina diet for 6 weeks increased retinoid levels in liver ventral midline cervical incision. The CCAs were ligated (Mitchell et al., 1990). The extracts of spirulina have with non-traumatic arterial clips. A craniotomy of about 4 antioxidant activity both in vivo and in vitro (Miranda et al., mm2 was made in the right squamous bone. The right MCA 1998). Pretreatment with spirulina reduced oxidative stress was ligated with 10-O suture for 60 min. The ligature was and toxicity induced by cyclophosphamide and mitomycin removed after 60-min ischemia to allow reperfusion. Core in mice (Premkumar et al., 2001) and prevented lipid body temperature was monitored with a thermistor probe peroxidation induced by lead (Upasani et al., 2001). and maintained at 378C with a heating pad during surgery. Phycocyanin and phycocyanobilin in the spirulina were After surgery, the animals were kept in a temperature- also found to have strong anti-cyclooxygenase-2 and controlled incubator to maintained body temperature at antioxidant activity (Bhat and Madyastha, 2001; Reddy et 378C. After recovery from the anesthesia, the animals were al., 2000) and to scavenger peroxidinitrite and reduce returned to their home cages. OONO- induced oxidative damage to DNA (Bhat and Madyastha, 2001). Besides the antioxidant effect, spirulina Caspase-3 enzymatic activity has other properties that can lower the risk of damage in various tissues. Pretreatment with spirulina maxima for 13 Caspase-3 enzyme activity was measured using the weeks elicited a dose-dependent decrease in serum choles- ApoAlert kit (Clontech, Palo, Alto, CA. USA) as terol in male and female mice and increased the weight of described previously (Wang et al., 2001a). Eight hours seminal vesicles in male mice (Salazar et al., 1998). In a after the onset of reperfusion, animals were sacrificed. similar experiment, serum HDL level was increased in Brain tissue was removed, dissected, and homogenized in diabetic mice after receiving spirulina diet for 4 weeks. lysis buffer. Caspase-3 activity was determined fluoro- Such a response was not found in non-diabetic controls metrically by the formation of 7-amino-4-trifluromethyl (Rodriguez-Hernandez et al., 2001). Taken together, these coumarin (AFC) from Asp-Glu-Val-Asp-7-amino-4-tri- data suggest that chronic or subchronic treatment with fluoromethyl coumarin (DEVD-AFC). The selective spirulina can reduce lipid peroxidation, increase antioxi- caspase-3 inhibitor DEVD-aldehyde (DEVD-CHO) was Y. Wang et al. / Experimental Neurology 193 (2005) 75–84 77 included, at a concentration of 1 AM, in some assays to To label damaged nuclei, 50 AL of the TUNEL reaction ensure that the enzymatic reaction was specific. mixture was added onto each sample in a humidified chamber followed by a 60-min