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Grape seed extract suppresses lipid peroxidation and reduces hypoxic ischemic brain injury in neonatal rats
Brain Research Bulletin 66 (2005) 120–127
Grape seed extract suppresses lipid peroxidation and reduces hypoxic ischemic brain injury in neonatal rats Yangzheng Feng a , Yi-Ming Liu b , Jonathan D. Fratkins c , Michael H. LeBlanc a,∗ a
Department of Pediatrics, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA b Department of Chemistry, Jackson State University, 1400 Lynch Street, Jackson, MS 39217, USA c Department of Pathology, University of Mississippi Medical Center, Jackson, MS 39216, USA Received 3 February 2005; received in revised form 22 March 2005; accepted 11 April 2005 Available online 23 May 2005
Abstract Oxygen radicals play a crucial role in brain injury. Grape seed extract is a potent anti-oxidant. Does grape seed extract reduce brain injury in the rat pup? Seven-day-old rat pups had the right carotid arteries permanently ligated followed by 2.5 h of hypoxia (8% oxygen). Grape seed extract, 50 mg/kg, or vehicle was administered by i.p. 5 min prior to hypoxia and 4 h after reoxygenation and twice daily for 1 day. Brain damage was evaluated by weight deficit of the right hemisphere at 22 days following hypoxia and by histopathology. Grape seed extract reduced brain weight loss from 20.0 ± 4.4% S.E.M. in vehicle pups (n = 21) to 3.1 ± 1.6% in treated pups (n = 20, P < 0.01). Grape seed extract improved the histopathologic brain score in cortex, hippocampus and thalamus (P < 0.05 versus vehicle). Concentrations of brain 8-isoprostaglandin F2␣ and thiobarbituric acid reacting substances significantly increased due to hypoxic ischemia. Grape seed extract reduced this increase. Treatment with grape seed extract suppresses lipid peroxidation and reduces hypoxic ischemic brain injury in neonatal rat. © 2005 Elsevier Inc. All rights reserved. Keywords: Procyanidins; Polyphenols; Stroke; Neuroprotection; 8-Isoprostaglandin F2␣ ; Thiobarbituric acid reacting substances
1. Introduction Oxygen and nitrogen free radicals are thought to play a crucial role in the pathophysiology of hypoxic ischemic brain injury (for review, see [11]). Free radical production has been detected in animal brains during cerebral ischemia and the levels of free radicals increase significantly at the onset of reperfusion [8,17,28]. Agents known to scavenge or enzymatically degrade free radicals have been shown to be neuroprotective in hypoxic ischemic brain injury [47]. Grape seed extracts contain a number of polyphenols including procyanidins and proanthocyanidins and are powerful free radical scavengers. Grape seed extracts are marketed in the United States as a dietary supplement and are reasonably safe and readily available [4,5,35]. Grape seed extract have been reported to possess a broad spec∗
Corresponding author. Tel.: +1 601 984 5260; fax: +1 601 815 3666. E-mail address: [email protected] (M.H. LeBlanc).
0361-9230/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2005.04.006
trum of pharmacological, and therapeutic effects including anti-inflammatory activity and reduced apoptotic cell death [25,40]. Grape seed extracts protect heart function and reduce infarct size in experimental cardiac ischemia [7,42]. Grape seed extracts prevent apoptotic and non-apoptotic liver cell death in acetaminophen induced liver damage [36] and reduce proteinuria in puromycin induced nephrosis [26]. Both acetaminophen induced liver damage and puromycin induced nephrosis are thought to be caused by free radicals. Recently, Hwang et al. [21] reported that grape seed extracts reduced neuronal damage in the adult gerbil after 5 min of forebrain ischemia. Hypoxic ischemic brain injury is a serious cause of death and disability in human newborns. The developmental stage of the brain of the 7-day-old rat pup resembles that of newborn humans [31]. The Rice–Vannucci–Brierley hypoxic ischemic rat pup model [37] may best match the injury caused by birth asphyxia in full-term human infants (for review, see [3]). Therefore, study of the role of neuroprotective agents in
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the neonatal hypoxic ischemic rat model may provide important information pertinent to the development of treatment for perinatal hypoxic ischemic brain damage. The neonatal rat hypoxic ischemic model [37] has been well characterized and extensively used to assess synthetic neuroprotective agents [3,15,24]. The purpose of the present study was to determine whether treatment with grape seed extract would reduce brain injury in newborn rats and to determine whether grape seed extract could attenuate the formation of oxygen free radicals, as measured by 8-isoprostaglandin F2␣ and thiobarbituric acid reacting substances in the hypoxic ischemic rat pup model. This has not previously been tested.
2. Materials and methods 2.1. Animal protocol Our institutional committee on animal use approved this protocol. Rats were cared for in accordance with National Institutes of Health guidelines. One hundred and thirty six rat pups from 10 litters were used in this experiment. The neonatal rat hypoxic ischemic procedure was performed as described by Rice et al. [37]. Because there are no differences in brain damage in 7-day-old rats between males and females in the neonatal rat hypoxic ischemic brain injury model [15], we chose 7-day-old Sprague–Dawley rat pups of either sex, weighing between 12 and 16 g (Harlan Sprague–Dawley, Indianaolis, IN) for our experiments. The rat pups were anesthetized with isoflurane (4% induction, 2% maintenance). The right common carotid artery was exposed, isolated and permanently doubly ligated. After surgery, the wound was infiltrated with Marcaine, a long acting local anesthetic and closed. The rat pups were then returned to their dams for 2–3 h recovery. Hypoxic exposure was achieved by placing the rat pups in 1.5 l sealed jars immersed 5.5 cm deep in a 37 ◦ C water bath and subjected to a warmed, humidified mixture of 8% oxygen/92% nitrogen bubbled through 37 ◦ C water and delivered at 4 l/min for 2.5 h. This results in a jar temperature of 33 ◦ C immediately above the pups. After this hypoxic exposure, the pups were returned to their dams and some pups were taken for 8-isoprostaglandin F2␣ assay and thiobarbituric acid reactive substances testing and other pups were allowed to recover and grow for 3 days for histopathology or for 22 days for estimating brain injury. 2.2. Drug treatment Pups from each litter were randomly assigned and marked to a vehicle group (n = 51), or for drug treatment (25 mg/kg, n = 25; 50 mg/kg, n = 46). Grape seed extract was prepared following a procedure previously described [2,13]. Briefly, grapes, identified as Vitis vinifera, were purchased from a local supermarket. Grape seeds were collected from the grapes and milled using a blender after being air-dried for 1 week. The powder (50 g) was macerated with 500 ml of 0.1 M
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acetate buffer (pH 5.0) prepared in water/acetone (30:70, v/v) for 12 h at room temperature. The extraction was repeated two times. The three extracts were combined and concentrated in a SpeedVac concentrator to remove acetone. The concentrated aqueous solution was extracted three times with ethyl acetate (100 ml each time). The combined ethyl acetate extracts were evaporated to dryness in a lyophilizer. Grape seed extract was obtained as a brown-red powder. Grape seeds are rich in flavonoids and contain monomers, dimers, trimers, oligomers and polymers [16]. According to the analytical results obtained by using an HPLC method previously described [33] the grape seed extract prepared in this work contained 76% of (−)-epicatechin gallate, procyanidin dimers, trimers, tetramers and their gallates, 13% of (+)catechin and (−)-epicatechin, 11% of oligomers. Compared with high molecular weight polymeric proanthocyanidines, procyanidines are less astringent, bind less strongly to proteins and are more soluble and mobile in the body [33]. Grape seed extract in doses of 25 or 50 mg/kg was dissolved in 10 l of saline per gram of body weight and administered by i.p. injection at 5 min prior to hypoxia, with additional doses given 4, 18 and 26 h after reoxygenation. The control group was given 10 l of saline per gram of body weight alone. These doses were chosen from previous studies in adult animals [32]. 2.3. Measurement of rectal temperature To evaluate whether neuroprotection by grape seed extract was dependent on systemic hypothermia, rectal temperature was measured with a 36 gauge flexible thermocouple (Omega Engineering Inc., Stamford, CT). This was done in 13 pups from 1 litter (7 from the vehicle group and 5 given 50 mg/kg of grape seed extract) prior to i.p. injection (−2.5 h), after the hypoxic period (0) and 0.25, 0.5, 1, 1.5, 2, 2.5, 3 and 4 h after hypoxia. Rat pups of this age cool rapidly once they are removed from the nest and the dam [29]. Measurement of rectal temperature immediately after hypoxia was performed immediately. Other measurements of rectal temperature were made in a 25 ◦ C room 15 min after removal of the pups from the nest. This procedure reduces the variance of the measurement caused by differences in time of measurement relative to time of removal of the pup from the cage and by differences in spontaneous positioning of the pup in the cage relative to the nest and dam. The pups’ temperature at 15 min reflects their maximal thermoregulatory capacity in a uniform cold environment. Rectal temperature and brain temperatures are almost identical and are tightly correlated [48]. Since decreased body temperature both during and after the hypoxia can effect the outcome, it is essential that both the treated and control animals maintain similar temperatures [14,48]. Body weight was measured in the rat pups from the gross pathologic score experiment (vehicle, n = 21; 25 mg/kg, n = 19; 50 mg/kg, n = 20) at 0, 1, 3, 7, 10, 14 and 22 days after injury.
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2.4. Gross brain damage grading Sixty-seven pups from 5 litters were used for this experiment. Twenty-five of the vehicle treated rat pups, 19 pups treated with 25 mg/kg and 23 pups treated with 50 mg/kg pups, if they survived, were anesthetized with pentobarbital and decapitated 22 days after hypoxic exposure. Brains were scored normal, mild, moderate or severe by the method of [31] by a blinded observer. Normal or “1” is no reduction in the size of the right hemisphere, mild or “2” is visible reduction in right hemisphere size, moderate or “3” is large reduction in hemisphere size from a visible infarct in the right parietal area, and severe or “4” is near total destruction of the hemisphere. After removing the cerebellum and brainstem, the brain was divided into two hemispheres and weighed. Results are presented as the percent loss of hemispheric weight of the right side relative to the left [(left − right)/left × 100]. This hypoxic ischemic model results in brain damage only on the ipsilateral side [31,37]. The loss of hemispheric weight can be used as a measure of brain damage in this model, since enough time has elapsed to allow resorption of the dead tissue and resolution of brain edema [10,15,18,24]. Since brain tissue weighs approximately 1 g/ml, the correspondence between brain weight loss and brain volume loss is obvious. Delayed neuronal injury sometimes requires more than a week to develop [46]. Three weeks following the injury, infarcts have become porencephalic cysts, sometimes occupying most of the hemisphere. Porencephalic cysts are ruptured if necessary prior to weighing to let fluid escape. There is a high degree of correspondence between the weight deficit of the injured hemisphere and histologically evaluated loss of brain tissue [1,15,18,45]. 2.5. Microscopic brain damage grading A second set of experiments using the above procedure was done to verify that the gross changes were a reflection of the expected histopathologic changes. Microscopic examination of the tissues was carried out in 13 rat pups from 1 litter, 6 pups treated with 50 mg/kg of grape seed extract and 6 vehicle treated pups. Rat pups were anesthetized with pentobarbital 3 days after injury. Their brains were perfusion fixed by cardiac puncture. They were flushed with saline then fixed with 10% buffered formalin. After removal the brains were stored in 10% buffered formalin. Sections were then embedded with paraffin. Five micron coronal sections were cut in the parietal region aiming for the equivalent of Bregma −4.3 to −4.5 mm [23], and then stained with hemotoxylin and eosin. Cerebral cortex, hippocampus and thalamus was scored by an observer blind to the treatment group of the animal from 0 to 5 by the method of Cataltepe et al. [12], where “0” is normal, “1” is 1–5% of neurons damaged, “2” is 6–25% of neurons damaged, “3” is 26–50% of neurons damaged, “4” is 51–75% of neurons damaged, “5” is >75% of neurons damaged. Damaged neurons for scores of 1–3 usually were shrunken cells with pyknotic nuclei and eosinophillic cytoplasm replacing
the healthy neurons in patchy areas of the brain. Damaged neurons for scores of 4 and 5 usually showed loss of tissue with partially replacement by inflammatory cells and connective tissue. 2.6. Measurement of 8-isoprostaglandin F2α A third set of experiments was performed to determine the effect of grape seed extract on 8-isoprostaglandin F2␣ . Eight-isoprostaglandin F2␣ was measured in 15 rat pups from 1 litter including 6 grape seed extract treated, 6 vehicle treated and 3 shams not exposed to hypoxia or ischemia. Using the above neonatal hypoxic ischemic procedure, the rat pups were treated with 50 mg/kg of grape seed extract by i.p. injection at 5 min prior to hypoxia, with a second dose given 4 h after reoxygenation. Since 8-isoprostaglandin F2␣ was measured at 24 h after injury, we could not administer the 26 h dose of drug or vehicle, and we chose to leave out the 18 h dose as well. In the experimental brain injury model, the peak brain concentration of 8-isoprostaglandin F2␣ occurs at 24 h following the brain injury [20]. Therefore, pups were anesthetized with 50 mg/kg pentobarbital at 24 h after hypoxia. The cortex in both lesioned and unlesioned hemispheres was separately dissected on ice and frozen at −80 ◦ C. Eight-isoprostaglanmdin F2␣ was only measured on the cortex since the available tissue in the hippocampus and thalamus was inadequate for this assay. Eightisoprostaglandin F2␣ was assessed as described by Hoffman et al. [20]. Each sample was passed through a C-18 Sep-Pak to purify the sample and then evaporated with N2 . Samples were reconstituted with enzyme immunoassay buffer and added to a 96-well plate for enzyme immunoassay analysis using an 8-isoprostaglandin F2␣ enzyme immunoassay kit from Cayman Chemical (Ann Arbor, MI). The plate was then incubated for 18 h at room temperature and then developed under darkness with Ellman’s reagent on a plate shaker. The concentrations of 8-isoprostaglandin F2␣ were determined colorimetrically with a microplate reader at 405 nm and calculated using a 4-parameter logistic standard curve. The amount of 8-isoprostaglandin F2␣ in the tissue was then calculated as pg of 8-isoprostaglandin F2␣ per gram of brain [20]. 2.7. Determination of thiobarbituric acid reactive substances A fourth set of experiments was performed to determine the effect of grape seed extract on thiobarbituric acid reactive substance. Thiobarbituric acid reactive substances were measured in 25 rat pups from 2 litters randomized to grape seed extract treated (25 or 50 mg/kg), vehicle treated and sham groups (vehicle, n = 7; other groups, n = 6). Using the above neonatal hypoxic ischemic procedure, the rat pups were pretreated with 25 or 50 mg/kg of grape seed extract by i.p. injection at 5 min prior to hypoxia, with a second dose given 4 h after reoxygenation. At 16 h after reoxygenation, the pups
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were decapitated, brains were removed, and right side of the cerebral cortex was frozen at −80 ◦ C. There was inadequate tissue in the hippocampus and thalamus to preform this test. Since we had shown in our previous study [15] that there was no change in thiobarbituric acid reactive substances in the contralateral hemisphere, only the right hemisphere was assayed. Thiobarbituric acid reactive substances were assessed as previously described [15]. Though the thiobarbituric acid assay is not specific for malondialdehyde and several other aldehydic products of cellular molecules can react with thiobarbituric acid, it is the most commonly used methods to determine lipid peroxidation. 2.8. Statistical analysis Categorical variables were analyzed with the χ2 -test. Ordinal variables are expressed as median (25 percentile, 75 percentile) and analyzed by Kruskal–Wallis or Mann–Whitney. Continuous variables are expressed as mean ± S.E.M. and analyzed by analysis of variance or Student’s t-test. Repeated measures analysis of variance was used for rectal temperature and body weight using the Bonferroni correction for multiple comparisons. Differences were considered significant at P < 0.05.
3. Results Rectal temperatures obtained prior to treatment and at the end of the hypoxic period, and 0.25, 0.5, 1, 1.5, 2, 2.5, 3 and 4 h after hypoxia were not significantly different between the group treated with 50 mg/kg of grape seed extract and the vehicle-treated pups at any times (Fig. 1). Body weight was measured in the rat pups from the gross pathologic score experiment (vehicle, n = 21; 25 mg/kg, n = 19; 50 mg/kg, n = 20) at 0, 1, 3, 7, 10, 14 and 22 days after injury. Body weight was not significantly different in the three groups (Fig. 2).
Fig. 1. Effect of grape seed extract (GSE) on rectal temperature. The error bars are S.E.M. Rectal temperatures obtained prior to treatment and at 0.25, 0.5, 1, 1.5, 2 and 4 h after treatment were not significantly different between 50 mg/kg of grape seed extract (n = 5) and vehicle-treated pups (n = 7) at any times.
Fig. 2. Body weight in grams (mean ± S.E.M.) for the vehicle group and pups treated with 25 or 50 mg/kg of grape seed extract (GSE). There were no statistically significant differences between the three groups.
3.1. Neuroprotective effects of treatment with grape seed extract Four of the 25 pups in the vehicle group died prior to the 22 day, two died during the hypoxic period and two between 1 and 4 days after reoxygenation. Three of the 23 pups in the 50 mg/kg grape seed extract group died. One during the hypoxic period and two between 1 and 4 days after reoxygenation. No pups in the 25 mg/kg grape seed extract group died. Of the pups that were kept alive for at least 3 days (i.e. combining the gross and microscopic pathology groups and the 25 and 50 mg/kg grape seed extract groups), 4 of 31 (13%) in the vehicle group died and 3 of 48 (6%) of the grape seed extract treated animals died. Gross neuropathologic damage was scored 22 days after injury. The proportion of pup brains scored as damaged is shown in Fig. 3. Grape seed extract decreased the percentage of brains scored as damaged (scores of 2–4) from
Fig. 3. The effect of different doses (25–50 mg/kg) of grape seed extract administered by i.p. 5 min prior to hypoxia and 4, 18 and 26 h after reoxygenation on the percentage of brains showing gross brain damage. The percentage of pups showing gross brain damage (score > 1) was determined by a blind observer 22 days after hypoxia-ischemia. Data are presented as mean ± S.D. of the proportion. Treatment with 50 mg/kg of grape seed extract improved the brain scores compared to vehicle (** P < 0.01). Treatment with 25 mg/kg of grape seed extract also trended to reduce brain damage score, but this reduction was not statistically significant (P > 0.05).
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Fig. 4. The dose–response for percentage reduction in right cerebral hemisphere weight measured using the left hemisphere weight as standard. Grape seed extract (25–50 mg/kg) was administered by i.p. at 5 min prior to hypoxia, at 4, 18 and 26 h after reoxygenation. Brain injury was evaluated 22 days later. Data are presented as mean ± S.E.M. Treatment with 25, or 50 mg/kg of grape seed extract significant decreased the percentage reduction in right hemisphere weight compared to vehicle (** P < 0.01 vs. vehicle).
Fig. 5. The microscopic brain score for grape seed extract (GSE), and vehicle treated rat pups for the cerebral cortex, hippocampus, and thalamus. Fifty milligram per kilogram of GSE was given i.p. at 5 min prior to hypoxia and at 4, 18 and 26 h after hypoxia. Brains were removed at 3 days after injury. Data is graphed as mean ± S.E.M. For all three tissues, the GSE treated animal had statistically less damage. * P < 0.05 vs. GSE, ** P < 0.01 vs. GSE. Statisical analysis was by Mann–Whitney U-test.
3.3. Brain 8-isoprostaglandin F2α 57.1% in the vehicle group (n = 21) to 5.0% with 50 mg/kg (n = 20, P < 0.01, vehicle versus 50 mg/kg). The dose of 25 mg/kg of grape seed extract (n = 19) produced a similar effect (26.3%), but this was not statistically significant (vehicle versus 25 mg/kg). Identical statistical results are obtained by treating the score as an ordinal rather than a categorical variable and analyzing with Kruskal–Wallis rather than χ2 . Left hemisphere weight was not affected by the hypoxiaischemia procedure [31] and was not significantly different from vehicle at any dose of grape seed extract. The percent reduction in right hemispheric weight is shown in Fig. 4. Grape seed extract significantly reduced the decrease in right hemisphere weight from 20.0 ± 4.4% S.E.M. (n = 21) in the vehicle group to 4.6 ± 3.1% (n = 19) in the group receiving the 25 mg/kg of grape seed extract (P < 0.01, vehicle versus 25 mg/kg) and to 3.1 ± 1.6% (n = 20) in the group receiving the 50 mg/kg of grape seed extract (P < 0.01, vehicle versus 50 mg/kg).
The concentrations of 8-isoprostaglandin F2␣ in left, contralateral hemisphere were 140.5 ± 9.9 pg/g (n = 3), 126.6 ± 24.8 pg/g (n = 6), and 134.5 ± 20.1 pg/g (n = 6) in the sham, 50 mg/kg of grape seed extract and vehicle treated groups, respectively. There were no significant differences among the sham, vehicle, and grape seed extract-treated groups in the left contralateral hemisphere. The concentrations of 8-isoprostaglandin F2␣ in right, ipsilateral hemisphere were significantly higher in vehicle group (369.9 ± 51.2 pg/g, n = 6) than in sham group (140.5 ± 9.9 pg/g, n = 3, P < 0.05) or in the 50 mg/kg of grape seed extract treated group (253.5 ± 26.9 pg/g, n = 6, P < 0.05 versus vehicle). No pup died prior to the planned time. Treatment with grape seed extract significantly reduced a hypoxiainduced increase in brain 8-isoprostaglandin F2␣ (Fig. 6).
3.2. Histopathology Histopathologic score was measure in six grape seed extract treated pups and six vehicle treated pups in all three tissues. No pups died prior to the planned time. The histopathologic score for the cortex was 0[0, 1.25], median[25 percentile, 75 percentile], in the 50 mg/kg of grape seed extract treated group and 5[5, 5] in the corresponding vehicle group, P < 0.05. The histopathologic score for the hippocampus was 0[0, 1.25] in the grape seed extract treated group and 5[5, 5] in the corresponding vehicle group, P < 0.05. The histopathologic score for the thalamus was 0[0, 0.75] in the grape seed extract treated group and 3.5[3, 5] in the vehicle group, P < 0.01. Treatment with grape seed extract significantly improved the outcome in the cortex, hippocampus and thalamus (Fig. 5).
Fig. 6. The effects of grape seed extract (GSE) on brain 8-isoprostaglandin F2␣ . The concentrations of 8-isoprostaglandin F2␣ in right hemisphere were significantly higher in the vehicle group than in the sham group. Treatment with 50 mg/kg of grape seed extract given 5 min prior to hypoxia and 4 h after reoxygenation significantly reduced a hypoxia-induced increase in brain 8isoprostaglandin F2␣ compared with vehicle group (* P < 0.05 vs. grape seed extract; # P < 0.05 vs. shams).
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Fig. 7. Effect of grape seed extract on brain thiobarbituric acid reactive substances (TBARS). Treatment with the grape seed extract (50 mg/kg) was given 5 min prior to hypoxia and 4 h after reoxygenation. Brain thiobarbituric acid reactive substances were assessed 16 h after reoxygenation. Data are presented as mean ± S.E.M. Treatment with 25 or 50 mg/kg of grape seed extract significantly reduced a hypoxia-induced increase in brain thiobarbituric acid reactive substances compared with vehicle group (## P < 0.01, # P < 0.05 vs. sham, ** P < 0.01 vs. vehicle).
3.4. Thiobarbituric acid reactive substance One pup in the vehicle group died during the hypoxia. The concentrations of thiobarbituric acid reactive substances in the right, ipsilateral hemisphere were significantly higher in the vehicle group (721.6 ± 44.3 pmol/g, n = 6) than in the shams (580 ± 44 pmol/g, n = 6, P < 0.01, vehicle versus sham) or in the 25 mg/kg grape seed extract treated group (579.8 ± 44.5 pmol/g, n = 6, P < 0.05, vehicle versus 25 mg/kg) or in the 50 mg/kg grape seed extract treated group (511.4 ± 28.9 pmol/g, n = 6, P < 0.01, vehicle versus 50 mg/kg). There was no increase in thiobarbituric acid reactive substances in the contralateral hemisphere in our previous studies [15], so we did not measure thiobarbituric acid reactive substances in the contralateral hemisphere. Treatment with grape seed extract significantly reduced a hypoxiainduced increase in brain thiobarbituric acid reactive substances (Fig. 7).
4. Discussion The present study demonstrates that treatment with grape seed extract significantly reduces the severity of brain injury in the focal ischemia model of the neonatal rat pups. Grape seed extract also significantly reduces a hypoxia-induced increase in brain 8-isoprostaglandin F2␣ and thiobarbituric acid reactive substances concentrations compared with the vehicle group. The histopathologic result was consistent with results of gross brain damage grading and loss of right hemisphere weight. Grape seed extract is a potent effective neuroprotectant at doses of 25 or 50 mg/kg in the rat pup. This dose is similar to the doses shown to be effective in isolated adult rat heart ischemic injury [32] and in adult gerbil transient forebrain ischemia [21].
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Grape seed extract has many possible mechanisms for neuroprotection. Grape seed extract is an effective free radical scavenger that reduces lipid peroxidation [7,41]. Grape seed extract exhibited excellent dose-dependent protective ability against 12-O-tetradecanoylphorbol-13-acetate induced hepatic and brain lipid peroxidation, and the associated DNA fragmentation in mice [5,6]. Grape seed extract provides superior anti-oxidant efficacy as compared to Vitamins C, and E at equal doses by weight [4,5]. Grape seed extract inhibits DNA oxidative damage in the gerbil forebrain ischemia model [21]. Grape seed extract can block cell death signaling mediated through the pro-apoptotic transcription factors and genes such as JNK-1 and c-JUN [40]. Chronic treatment with grape seed extract will increase expression of bcl-xl and prevent ladder like DNA fragmentation [36]. Grape seed extract also has anti-inflammatory actions in association with its oxygen free radical scavenging, and anti-lipid peroxidation activity. Grape seed extract reduces production of proinflammatory cytokines and swelling in croton oil induced ear swelling in mice and carrageenan induced paw swelling in rats [25], and decreases chemiluminescence by peritoneal macrophage in response to oxidative injury in mice [5]. Hypoxia-ischemia causes brain damage, activating a cascade of biochemical events that include loss of cellular ATP, which causes cell depolarization and glutamate release, which causes intracellular Ca2+ overload, which causes increased oxygen radical generation by mitochondria. Oxygen radicals by fostering mitochondrial permeability transition will release apoptosis inducing factor and cytochrome c from mitochondria. Cytochrome c with ATP and apoptotic protease activating factor-1 (Apaf-1) will activate procaspase-9 and initiate the caspase cascade [27]. Oxygen free radical reactions lead to the oxidation of lipids, proteins and polysaccharides and to DNA damage [11]. Lipid peroxidation has been and remains one of the most widely used indicators of oxidant/free radical formation. Thiobarbituric acid reactive substances primarily reflect production of lipid peroxides, which are broken down during the assay to yield malondialdehyde [19]. In the present study, hypoxic ischemic rat pups showed elevations in thiobarbituric acid reactive substances concentrations in the brain. Treatment with grape seed extract reduced the increase in thiobarbituric acid reactive substances. Similar results showing grape seed extract reduced formation of thiobarbituric acid reactive substances have been reported by other observers [25,39]. F2 -isoprostanes are non-enzymatic products of the oxygen radical-induced lipid peroxidation of arachidonic acid. They are relatively stable once formed. F2 -isoprostanes are formed initially as esterified fatty acids attached to phospholipids and then are released to their free form by the action of phospholipases, thus delaying the elevation of levels in the tissues [38]. Measurement of F2 -isoprostanes as indicators of lipid peroxidation is sensitive and specific [38,43]. Eight-isoprostaglandin F2␣ is an F2 -isoprostane that is often used to measure lipid peroxidation in humans. Bayir et al. [9] found that hypoxemia after traumatic brain injury
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was associated with increased F2 -isoprostane levels. Our present data showed that hypoxia-ischemia-increased brain 8-isoprostaglandin F2␣ . Treatment with grape seed extract eliminated the increase in 8-isoprostaglandin F2␣ in the brain at 24 h after reoxygenation. This result was consistent with the thiobarbituric acid reactive substances assay result and suggest that grape seed extract’s inhibition of oxidant/free radicals is an important mechanism for its neuroprotective effect. This is not the first demonstration that hypoxia and ischemia cause oxidant/free radicals in the brain of the newborn rat [8] or that scavengers of free radicals can ameliorate the brain injury in this model [44]. This is the first demonstration that grape seed extract can reduce brain lipid peroxidation and reduce injury in a newborn animal model. In conclusion our findings indicate that grape seed extract has neuroprotective properties in the neonatal rat hypoxiaischemic brain injury model. The results also indicate that the suppression of free radicals after hypoxic ischemia by grape seed extract is one potential mechanism of this neuroprotection. We suggest that grape seed extract is a novel, reasonably safe [32,4,5] and cost effective potential therapeutic agent for the treatment of brain injury. Further trials attempting to isolate the neuroprotection to one or a smaller number of compounds would be useful [36]. Human trials for this indication should await demonstration of neurofunctional as well as neuroanatomic salvage, replication of the experiment in more advanced gyrencephalic animals and demonstration that neuroprotection can be obtained when treatment is delayed at least several hours after injury. Human trials of grape seed extract for other indications have already begun [22,30,34].
[8]
[9]
[10]
[11] [12]
[13] [14]
[15]
[16]
[17]
[18]
[19]
[20]
References [1] P. Andine, M. Thordstein, I. Kjellmer, C. Nordborg, K. Thiringer, E. Wennberg, H. Hagberg, Evaluation of brain damage in a rat model of neonatal hypoxic ischemia, J. Neurosci. Methods 35 (1990) 253–260. [2] M. Ashraf-Khorassani, L.T. Taylor, Sequential fractionation of grape seeds into oils, polyphenols, and procyanidines via a single system employing CO2 -based fluids, J. Agric. Food Chem. 52 (2004) 2440–2444. [3] S. Ashwal, W.J. Pearce, Animal models of neonatal stroke, Curr. Opin. Pediatr. 13 (2001) 506–516. [4] D. Bagchi, A. Grarg, R.L. Krohn, M. Bagchi, M.X. Tran, S.J. Stohs, Oxygen free radical scavenging abilities of Vitamins C and E, and a grape seed proanthocyanidin extract in vitro, Res. Commun. Mol. Pathol. Pharmacol. 95 (1997) 179–190. [5] D. Bagchi, A. Grarg, R.L. Krohn, M. Bagchi, D.J. Bagchi, J. Balmoori, S.J. Stohs, Protective effects of grape seed proanthocyanidins and selected antioxidants against TPA-induced hepatic and brain lipid peroxidation and DNA fragmentation, and peritoneal macrophage activation in mice, Gen. Pharmacol. 30 (1998) 771–776. [6] M. Bagchi, M. Milnes, C. Williams, J. Balmoori, X. Ye, S. Stohs, D. Bagchi, Acute and chronic stress-induced oxidative gastrointestinal injury in rats, and the protective ability of a novel grape seed proanthocyanidins extract, Nutr. Res. 19 (1999) 1189–1199. [7] D. Bagchi, C.K. Sen, S.D. Ray, D.K. Das, M. Bagchi, H.G. Preuss, J.A. Vinson, Molecular mechanisms of cardioprotection by a novel
[21]
[22]
[23]
[24]
[25]
[26]
[27]
grape seed proanthocyanidin extract, Mutat. Res. 523–524 (2003) 87–97. R. B˚agenholm, U.A. Nilsson, I. Kjellmer, Formation of free radicals in hypoxic ischemic brain damage in the neonatal rat, assessed by an endogenous spin trap and lipid peroxidation, Brain Res. 773 (1997) 132–138. H. Bayir, D.W. Marion, A.M. Puccio, S.R. Wisniewski, K.L. Janesko, R.S. Clark, P.M. Kochanek, Marked gender effect on lipid peroxidation after severe traumatic brain injury in adult patients, J. Neurotrauma 21 (2004) 1–8. E. Bona, B.B. Johansson, H. Hagberg, Sensorimotor function and neuropathology five and six weeks after hypoxia-ischemia in sevenday-old rats, Pediatr. Res. 42 (1997) 678–683. G. Buonocore, S. Perrone, R. Bracci, Free radicals and brain damage in the newborn, Biol. Neonate 79 (2001) 180–186. O.V.R. Cataltepe, D.F. Heitjan, J. Towfighi, Effect of status epilepticus on hypoxic-ischemic brain damage in the immature rat, Pediatr. Res. 38 (1995) 251–257. J. Da Silva, J. Rigaud, V. Cheynier, Procyanidine dimers and trimers from grape seeds, Phytochemistry 30 (1991) 1259–1264. Y. Feng, M.H. LeBlanc, Drug-induced hypothermia begun 5 min after injury with a poly(ADP-ribose) polymerase inhibitor reduces hypoxic brain injury in rat pups, Crit. Care Med. 30 (2002) 2420–2424. Y. Feng, J.D. Fratkin, M.H. LeBlanc, Treatment with tamoxifen reduces hypoxic-ischemic brain injury in neonatal rats, Eur. J. Pharmacol. 484 (2004) 65–74. A.M. Fine, Oligomeric proanthocyanidine complexes: history, structure, and phytopharmaceutical applications, Altern. Med. Rev. 5 (2000) 144–151. Z.Z. Guan, W.F. Yu, A. Nordberg, Dual effects of nicotine on oxidative stress and neuroprotection in PC12 cells, Neurochem. Int. 43 (2003) 243–249. H. Hagberg, E. Gilland, N.H. Diemer, P. Andin´e, Hypoxia-ischemia in the neonatal rat brain: histopathology after post-treatment with NMDA and non-NMDA receptor antagonists, Biol. Neonate 66 (1994) 205–213. J.J. Hageman, A. Bast, N.P. Vermeulen, Monitoring of oxidative free radical damage in vivo: analytical aspects, Chem. Biol. Interact. 82 (1992) 243–293. S.W. Hoffman, R.L. Roof, D.G. Stein, A reliable and sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F2␣ : a marker for lipid peroxidation after experimental brain injury, J. Neurosci. Methods 68 (1996) 133–136. I.K. Hwang, K.Y. Yoo, D.S. Kim, Y.K. Jeong, J.D. Kim, D.W. Shin, S.S. Lim, I.D. Yoo, T.C. Kang, H.K. Kim, W.K. Moon, M.H. Won, Neuroprotective effects of grape seed extract on neuronal injury by inhibiting DNA damage in the gerbil hippocampus after transient forebrain ischemia, Life Sci. 75 (2004) 1989–2001. R. Kalin, A. Righi, A. Del Rosso, D. Bagchi, S. Generini, M.M. Cerinic, D.K. Das, Activin, a grape seed-derived proanthocyanidin extract, reduces plasma levels of oxidative stress and adhesion molecules, (ICAM-1, VCAM-1, and E-selectin) in systemic sclerosis, Free Radic. Res. 36 (2002) 819–825. L. Kruger, S. Saporta, L.W. Swanson, Photographic Atlas of the Rat Brain, Cambridge University Press, Cambridge, 1995, plates 21 to 23. M.H. LeBlanc, Y. Feng, J.D. Fratkin, N-tosyl-l-phenylalanylchloromethylketone reduces hypoxic-ischemic brain injury in rat pups, Eur. J. Pharmacol. 390 (2000) 249–256. W.G. Li, X.Y. Zhang, Y.J. Wu, X. Tian, Anti-inflammatory effect and mechanism of proanthocyanidins from grape seeds, Acta. Pharmacol. Sin. 22 (2001) 1117–1120. T.K. Mattoo, L. Kovacevic, Effect of grape seed extract on puromycin-aminonucleoside-induced nephrosis in rats, Pediatr. Nephrol. 18 (2003) 872–877. M.P. Mattson, C. Culmsee, Z.F. Yu, Apoptotic and antiapoptotic mechanisms in stroke, Cell Tissue Res. 301 (2000) 173–187.
Y. Feng et al. / Brain Research Bulletin 66 (2005) 120–127 [28] J.M. McCord, Oxygen-derived free radicals in post-ischemic tissue injury, N. Engl. J. Med. 312 (1985) 159–163. [29] J.W. McDonald, C.K. Chen, W.H. Trescher, M.V. Johnston, The severity of excitotoxic brain injury is dependent on brain temperature in the immature rat, Neurosci. Lett. 126 (1991) 83–86. [30] F. Natella, F. Belelli, V. Gentili, F. Ursini, C. Saccini, Grape seed proanthocyanidins prevent plasma postprandial oxidative stress in humans, J. Agric. Food Chem. 50 (2002) 7720–7725. [31] C. Palmer, R.C. Vannucci, J. Towfighi, Reduction of perinatal hypoxic-ischemic brain damage with allopurinol, Pediatr. Res. 27 (1990) 332–336. [32] T. Pataki, I. Bak, P. Kovacs, D. Bagchi, D.K. Das, A. Tosaki, Grape seed proanthocyanidins improved cardiac recovery during reperfusion after ischemia in isolated rat hearts, Am. J. Clin. Nutr. 75 (2002) 894–899. [33] Z. Peng, Y. Hayasaka, P.G. Iland, M. Sefton, P. Hoj, E.J. Water, Quantitative analysis of polymeric procyanidins (Tannins) from grape (Vitis vinifera) seeds by reverse phase high-performance liquid chromatography, J. Agric. Food Chem. 49 (2001) 26–31. [34] H.G. Preuss, D. Wallerstedt, N. Talpur, S.O. Tutuncuoglu, B. Echard, A. Myers, M. Bui, D. Bagchi, Effects of niacin-bound chromium and grape seed proanthocyanidin extract on the lipid profile of hypercholesterolemic subjects: a pilot study, J. Med. 31 (2000) 227–246. [35] S. Ray, D. Bagchi, P.M. Lim, M. Bagchi, S.M. Gross, S.C. Kothari, H.G. Preuss, S.J. Stohs, Acute and long term safety evaluation of a novel IH636 grape seed proanthocyanidin extract, Res. Commun. Mol. Pathol. Pharmacol. 109 (2001) 165–197. [36] S.D. Ray, M.A. Kumar, D. Bagchi, A novel proanthocyanidin IH636 grape seed extract increases in vivo bcl-XL expression and prevents acetaminophen-induced programmed and unprogrammed cell death in mouse liver, Arch. Biochem. Biophys. 369 (1999) 42–58. [37] J.E. Rice, R.C. Vannucci, J.B. Brierley, The influence of immaturity on hypoxic-ischemic brain damage in the rat, Ann. Neurol. 9 (1981) 131–141. [38] L.J. Roberts, J.D. Morrow, Measurement of F2 -isoprostanes as an index of oxidative stress in vivo, Free Radic. Biol. Med. 28 (2000) 505–513.
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[39] M. Sato, G. Maulik, P.S. Ray, D. Bagchi, D.K. Das, Cardioprotective effects of grape seed proanthocyanidin against ischemic reperfusion injury, J. Mol. Cell. Cardiol. 31 (1999) 1289–1297. [40] M. Sato, D. Bagchi, A. Tosaki, D.K. Das, Grape seed proanthocyanidin reduces cardiomyocyte apoptosis by inhibiting ischemia/reperfusion-induced activation of JNK-1 and C-Jun, Free Radic. Biol. Med. 31 (2001) 729–737. [41] M. Shafiee, M.A. Carbonnneau, N. Urban, B. Descomps, C.L. Leger, Grape and grape seed extract capacities at protecting LDL against oxidation generated by Cu+2 , AAPH or SIN-1 and at decreasing superoxide THP-1 cell production: a comparison to other extracts or compounds, Free Radic. Res. 37 (2003) 573– 584. [42] Z.H. Shao, L.B. Becker, T.L. VandenHoek, P.T. Schumacker, C.Q. Li, D. Zhao, K. Wojcik, T. Anderson, Y. Qin, L. Dey, C.S. Yuan, Grape seed proanthocyanidin extract attenuates oxidant injury in cardiomyocytes, Pharmacol. Res. 47 (2003) 463–469. [43] M.M. Tarpey, D.A. Wink, M.B. Grisham, Methods for detection of reactive metabolites of oxygen and nitrogen: in vitro and in vivo considerations, Am. J. Physiol. Regul. Integr. Comp. Physiol. 286 (2004) R431–R444. [44] M. Thordstein, R. B˚agenholm, K. Thiringer, I. Kjellmer, Scavengers of free oxygen radicals in combination with magnesium ameliorate perinatal hypoxic-ischemic brain damage in the rat, Pediatr. Res. 34 (1993) 23–26. [45] J. Towfighi, C. Housman, R.C. Vannycci, D.F. Heitjan, Effect of unilateral perinatal hypoxic-ischemic brain damage on the gross development of opposite cerebral hemisphere, Biol. Neonate 65 (1994) 108–118. [46] W.H. Trescher, S. Ishiwa, M.V. Johnston, Brief post-hypoxicischemic hypothermia markedly delays neonatal brain injury, Brain Dev. 19 (1997) 326–338. [47] J.J. Volpe, Neurology of the Newborn, fourth ed., W.B. Saunders, Philadelphia, PA, 2001. [48] J. Yager, J. Towfighi, R.C. Vannucci, Influence of mild hypothermia on hypoxic-ischemic brain damage in the immature rat, Pediatr. Res. 34 (1993) 525–529.
Grape seed extract suppresses lipid peroxidation and reduces hypoxic ischemic brain injury in neonatal rats Yangzheng Feng a , Yi-Ming Liu b , Jonathan D. Fratkins c , Michael H. LeBlanc a,∗ a
Department of Pediatrics, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA b Department of Chemistry, Jackson State University, 1400 Lynch Street, Jackson, MS 39217, USA c Department of Pathology, University of Mississippi Medical Center, Jackson, MS 39216, USA Received 3 February 2005; received in revised form 22 March 2005; accepted 11 April 2005 Available online 23 May 2005
Abstract Oxygen radicals play a crucial role in brain injury. Grape seed extract is a potent anti-oxidant. Does grape seed extract reduce brain injury in the rat pup? Seven-day-old rat pups had the right carotid arteries permanently ligated followed by 2.5 h of hypoxia (8% oxygen). Grape seed extract, 50 mg/kg, or vehicle was administered by i.p. 5 min prior to hypoxia and 4 h after reoxygenation and twice daily for 1 day. Brain damage was evaluated by weight deficit of the right hemisphere at 22 days following hypoxia and by histopathology. Grape seed extract reduced brain weight loss from 20.0 ± 4.4% S.E.M. in vehicle pups (n = 21) to 3.1 ± 1.6% in treated pups (n = 20, P < 0.01). Grape seed extract improved the histopathologic brain score in cortex, hippocampus and thalamus (P < 0.05 versus vehicle). Concentrations of brain 8-isoprostaglandin F2␣ and thiobarbituric acid reacting substances significantly increased due to hypoxic ischemia. Grape seed extract reduced this increase. Treatment with grape seed extract suppresses lipid peroxidation and reduces hypoxic ischemic brain injury in neonatal rat. © 2005 Elsevier Inc. All rights reserved. Keywords: Procyanidins; Polyphenols; Stroke; Neuroprotection; 8-Isoprostaglandin F2␣ ; Thiobarbituric acid reacting substances
1. Introduction Oxygen and nitrogen free radicals are thought to play a crucial role in the pathophysiology of hypoxic ischemic brain injury (for review, see [11]). Free radical production has been detected in animal brains during cerebral ischemia and the levels of free radicals increase significantly at the onset of reperfusion [8,17,28]. Agents known to scavenge or enzymatically degrade free radicals have been shown to be neuroprotective in hypoxic ischemic brain injury [47]. Grape seed extracts contain a number of polyphenols including procyanidins and proanthocyanidins and are powerful free radical scavengers. Grape seed extracts are marketed in the United States as a dietary supplement and are reasonably safe and readily available [4,5,35]. Grape seed extract have been reported to possess a broad spec∗
Corresponding author. Tel.: +1 601 984 5260; fax: +1 601 815 3666. E-mail address: [email protected] (M.H. LeBlanc).
0361-9230/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2005.04.006
trum of pharmacological, and therapeutic effects including anti-inflammatory activity and reduced apoptotic cell death [25,40]. Grape seed extracts protect heart function and reduce infarct size in experimental cardiac ischemia [7,42]. Grape seed extracts prevent apoptotic and non-apoptotic liver cell death in acetaminophen induced liver damage [36] and reduce proteinuria in puromycin induced nephrosis [26]. Both acetaminophen induced liver damage and puromycin induced nephrosis are thought to be caused by free radicals. Recently, Hwang et al. [21] reported that grape seed extracts reduced neuronal damage in the adult gerbil after 5 min of forebrain ischemia. Hypoxic ischemic brain injury is a serious cause of death and disability in human newborns. The developmental stage of the brain of the 7-day-old rat pup resembles that of newborn humans [31]. The Rice–Vannucci–Brierley hypoxic ischemic rat pup model [37] may best match the injury caused by birth asphyxia in full-term human infants (for review, see [3]). Therefore, study of the role of neuroprotective agents in
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the neonatal hypoxic ischemic rat model may provide important information pertinent to the development of treatment for perinatal hypoxic ischemic brain damage. The neonatal rat hypoxic ischemic model [37] has been well characterized and extensively used to assess synthetic neuroprotective agents [3,15,24]. The purpose of the present study was to determine whether treatment with grape seed extract would reduce brain injury in newborn rats and to determine whether grape seed extract could attenuate the formation of oxygen free radicals, as measured by 8-isoprostaglandin F2␣ and thiobarbituric acid reacting substances in the hypoxic ischemic rat pup model. This has not previously been tested.
2. Materials and methods 2.1. Animal protocol Our institutional committee on animal use approved this protocol. Rats were cared for in accordance with National Institutes of Health guidelines. One hundred and thirty six rat pups from 10 litters were used in this experiment. The neonatal rat hypoxic ischemic procedure was performed as described by Rice et al. [37]. Because there are no differences in brain damage in 7-day-old rats between males and females in the neonatal rat hypoxic ischemic brain injury model [15], we chose 7-day-old Sprague–Dawley rat pups of either sex, weighing between 12 and 16 g (Harlan Sprague–Dawley, Indianaolis, IN) for our experiments. The rat pups were anesthetized with isoflurane (4% induction, 2% maintenance). The right common carotid artery was exposed, isolated and permanently doubly ligated. After surgery, the wound was infiltrated with Marcaine, a long acting local anesthetic and closed. The rat pups were then returned to their dams for 2–3 h recovery. Hypoxic exposure was achieved by placing the rat pups in 1.5 l sealed jars immersed 5.5 cm deep in a 37 ◦ C water bath and subjected to a warmed, humidified mixture of 8% oxygen/92% nitrogen bubbled through 37 ◦ C water and delivered at 4 l/min for 2.5 h. This results in a jar temperature of 33 ◦ C immediately above the pups. After this hypoxic exposure, the pups were returned to their dams and some pups were taken for 8-isoprostaglandin F2␣ assay and thiobarbituric acid reactive substances testing and other pups were allowed to recover and grow for 3 days for histopathology or for 22 days for estimating brain injury. 2.2. Drug treatment Pups from each litter were randomly assigned and marked to a vehicle group (n = 51), or for drug treatment (25 mg/kg, n = 25; 50 mg/kg, n = 46). Grape seed extract was prepared following a procedure previously described [2,13]. Briefly, grapes, identified as Vitis vinifera, were purchased from a local supermarket. Grape seeds were collected from the grapes and milled using a blender after being air-dried for 1 week. The powder (50 g) was macerated with 500 ml of 0.1 M
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acetate buffer (pH 5.0) prepared in water/acetone (30:70, v/v) for 12 h at room temperature. The extraction was repeated two times. The three extracts were combined and concentrated in a SpeedVac concentrator to remove acetone. The concentrated aqueous solution was extracted three times with ethyl acetate (100 ml each time). The combined ethyl acetate extracts were evaporated to dryness in a lyophilizer. Grape seed extract was obtained as a brown-red powder. Grape seeds are rich in flavonoids and contain monomers, dimers, trimers, oligomers and polymers [16]. According to the analytical results obtained by using an HPLC method previously described [33] the grape seed extract prepared in this work contained 76% of (−)-epicatechin gallate, procyanidin dimers, trimers, tetramers and their gallates, 13% of (+)catechin and (−)-epicatechin, 11% of oligomers. Compared with high molecular weight polymeric proanthocyanidines, procyanidines are less astringent, bind less strongly to proteins and are more soluble and mobile in the body [33]. Grape seed extract in doses of 25 or 50 mg/kg was dissolved in 10 l of saline per gram of body weight and administered by i.p. injection at 5 min prior to hypoxia, with additional doses given 4, 18 and 26 h after reoxygenation. The control group was given 10 l of saline per gram of body weight alone. These doses were chosen from previous studies in adult animals [32]. 2.3. Measurement of rectal temperature To evaluate whether neuroprotection by grape seed extract was dependent on systemic hypothermia, rectal temperature was measured with a 36 gauge flexible thermocouple (Omega Engineering Inc., Stamford, CT). This was done in 13 pups from 1 litter (7 from the vehicle group and 5 given 50 mg/kg of grape seed extract) prior to i.p. injection (−2.5 h), after the hypoxic period (0) and 0.25, 0.5, 1, 1.5, 2, 2.5, 3 and 4 h after hypoxia. Rat pups of this age cool rapidly once they are removed from the nest and the dam [29]. Measurement of rectal temperature immediately after hypoxia was performed immediately. Other measurements of rectal temperature were made in a 25 ◦ C room 15 min after removal of the pups from the nest. This procedure reduces the variance of the measurement caused by differences in time of measurement relative to time of removal of the pup from the cage and by differences in spontaneous positioning of the pup in the cage relative to the nest and dam. The pups’ temperature at 15 min reflects their maximal thermoregulatory capacity in a uniform cold environment. Rectal temperature and brain temperatures are almost identical and are tightly correlated [48]. Since decreased body temperature both during and after the hypoxia can effect the outcome, it is essential that both the treated and control animals maintain similar temperatures [14,48]. Body weight was measured in the rat pups from the gross pathologic score experiment (vehicle, n = 21; 25 mg/kg, n = 19; 50 mg/kg, n = 20) at 0, 1, 3, 7, 10, 14 and 22 days after injury.
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2.4. Gross brain damage grading Sixty-seven pups from 5 litters were used for this experiment. Twenty-five of the vehicle treated rat pups, 19 pups treated with 25 mg/kg and 23 pups treated with 50 mg/kg pups, if they survived, were anesthetized with pentobarbital and decapitated 22 days after hypoxic exposure. Brains were scored normal, mild, moderate or severe by the method of [31] by a blinded observer. Normal or “1” is no reduction in the size of the right hemisphere, mild or “2” is visible reduction in right hemisphere size, moderate or “3” is large reduction in hemisphere size from a visible infarct in the right parietal area, and severe or “4” is near total destruction of the hemisphere. After removing the cerebellum and brainstem, the brain was divided into two hemispheres and weighed. Results are presented as the percent loss of hemispheric weight of the right side relative to the left [(left − right)/left × 100]. This hypoxic ischemic model results in brain damage only on the ipsilateral side [31,37]. The loss of hemispheric weight can be used as a measure of brain damage in this model, since enough time has elapsed to allow resorption of the dead tissue and resolution of brain edema [10,15,18,24]. Since brain tissue weighs approximately 1 g/ml, the correspondence between brain weight loss and brain volume loss is obvious. Delayed neuronal injury sometimes requires more than a week to develop [46]. Three weeks following the injury, infarcts have become porencephalic cysts, sometimes occupying most of the hemisphere. Porencephalic cysts are ruptured if necessary prior to weighing to let fluid escape. There is a high degree of correspondence between the weight deficit of the injured hemisphere and histologically evaluated loss of brain tissue [1,15,18,45]. 2.5. Microscopic brain damage grading A second set of experiments using the above procedure was done to verify that the gross changes were a reflection of the expected histopathologic changes. Microscopic examination of the tissues was carried out in 13 rat pups from 1 litter, 6 pups treated with 50 mg/kg of grape seed extract and 6 vehicle treated pups. Rat pups were anesthetized with pentobarbital 3 days after injury. Their brains were perfusion fixed by cardiac puncture. They were flushed with saline then fixed with 10% buffered formalin. After removal the brains were stored in 10% buffered formalin. Sections were then embedded with paraffin. Five micron coronal sections were cut in the parietal region aiming for the equivalent of Bregma −4.3 to −4.5 mm [23], and then stained with hemotoxylin and eosin. Cerebral cortex, hippocampus and thalamus was scored by an observer blind to the treatment group of the animal from 0 to 5 by the method of Cataltepe et al. [12], where “0” is normal, “1” is 1–5% of neurons damaged, “2” is 6–25% of neurons damaged, “3” is 26–50% of neurons damaged, “4” is 51–75% of neurons damaged, “5” is >75% of neurons damaged. Damaged neurons for scores of 1–3 usually were shrunken cells with pyknotic nuclei and eosinophillic cytoplasm replacing
the healthy neurons in patchy areas of the brain. Damaged neurons for scores of 4 and 5 usually showed loss of tissue with partially replacement by inflammatory cells and connective tissue. 2.6. Measurement of 8-isoprostaglandin F2α A third set of experiments was performed to determine the effect of grape seed extract on 8-isoprostaglandin F2␣ . Eight-isoprostaglandin F2␣ was measured in 15 rat pups from 1 litter including 6 grape seed extract treated, 6 vehicle treated and 3 shams not exposed to hypoxia or ischemia. Using the above neonatal hypoxic ischemic procedure, the rat pups were treated with 50 mg/kg of grape seed extract by i.p. injection at 5 min prior to hypoxia, with a second dose given 4 h after reoxygenation. Since 8-isoprostaglandin F2␣ was measured at 24 h after injury, we could not administer the 26 h dose of drug or vehicle, and we chose to leave out the 18 h dose as well. In the experimental brain injury model, the peak brain concentration of 8-isoprostaglandin F2␣ occurs at 24 h following the brain injury [20]. Therefore, pups were anesthetized with 50 mg/kg pentobarbital at 24 h after hypoxia. The cortex in both lesioned and unlesioned hemispheres was separately dissected on ice and frozen at −80 ◦ C. Eight-isoprostaglanmdin F2␣ was only measured on the cortex since the available tissue in the hippocampus and thalamus was inadequate for this assay. Eightisoprostaglandin F2␣ was assessed as described by Hoffman et al. [20]. Each sample was passed through a C-18 Sep-Pak to purify the sample and then evaporated with N2 . Samples were reconstituted with enzyme immunoassay buffer and added to a 96-well plate for enzyme immunoassay analysis using an 8-isoprostaglandin F2␣ enzyme immunoassay kit from Cayman Chemical (Ann Arbor, MI). The plate was then incubated for 18 h at room temperature and then developed under darkness with Ellman’s reagent on a plate shaker. The concentrations of 8-isoprostaglandin F2␣ were determined colorimetrically with a microplate reader at 405 nm and calculated using a 4-parameter logistic standard curve. The amount of 8-isoprostaglandin F2␣ in the tissue was then calculated as pg of 8-isoprostaglandin F2␣ per gram of brain [20]. 2.7. Determination of thiobarbituric acid reactive substances A fourth set of experiments was performed to determine the effect of grape seed extract on thiobarbituric acid reactive substance. Thiobarbituric acid reactive substances were measured in 25 rat pups from 2 litters randomized to grape seed extract treated (25 or 50 mg/kg), vehicle treated and sham groups (vehicle, n = 7; other groups, n = 6). Using the above neonatal hypoxic ischemic procedure, the rat pups were pretreated with 25 or 50 mg/kg of grape seed extract by i.p. injection at 5 min prior to hypoxia, with a second dose given 4 h after reoxygenation. At 16 h after reoxygenation, the pups
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were decapitated, brains were removed, and right side of the cerebral cortex was frozen at −80 ◦ C. There was inadequate tissue in the hippocampus and thalamus to preform this test. Since we had shown in our previous study [15] that there was no change in thiobarbituric acid reactive substances in the contralateral hemisphere, only the right hemisphere was assayed. Thiobarbituric acid reactive substances were assessed as previously described [15]. Though the thiobarbituric acid assay is not specific for malondialdehyde and several other aldehydic products of cellular molecules can react with thiobarbituric acid, it is the most commonly used methods to determine lipid peroxidation. 2.8. Statistical analysis Categorical variables were analyzed with the χ2 -test. Ordinal variables are expressed as median (25 percentile, 75 percentile) and analyzed by Kruskal–Wallis or Mann–Whitney. Continuous variables are expressed as mean ± S.E.M. and analyzed by analysis of variance or Student’s t-test. Repeated measures analysis of variance was used for rectal temperature and body weight using the Bonferroni correction for multiple comparisons. Differences were considered significant at P < 0.05.
3. Results Rectal temperatures obtained prior to treatment and at the end of the hypoxic period, and 0.25, 0.5, 1, 1.5, 2, 2.5, 3 and 4 h after hypoxia were not significantly different between the group treated with 50 mg/kg of grape seed extract and the vehicle-treated pups at any times (Fig. 1). Body weight was measured in the rat pups from the gross pathologic score experiment (vehicle, n = 21; 25 mg/kg, n = 19; 50 mg/kg, n = 20) at 0, 1, 3, 7, 10, 14 and 22 days after injury. Body weight was not significantly different in the three groups (Fig. 2).
Fig. 1. Effect of grape seed extract (GSE) on rectal temperature. The error bars are S.E.M. Rectal temperatures obtained prior to treatment and at 0.25, 0.5, 1, 1.5, 2 and 4 h after treatment were not significantly different between 50 mg/kg of grape seed extract (n = 5) and vehicle-treated pups (n = 7) at any times.
Fig. 2. Body weight in grams (mean ± S.E.M.) for the vehicle group and pups treated with 25 or 50 mg/kg of grape seed extract (GSE). There were no statistically significant differences between the three groups.
3.1. Neuroprotective effects of treatment with grape seed extract Four of the 25 pups in the vehicle group died prior to the 22 day, two died during the hypoxic period and two between 1 and 4 days after reoxygenation. Three of the 23 pups in the 50 mg/kg grape seed extract group died. One during the hypoxic period and two between 1 and 4 days after reoxygenation. No pups in the 25 mg/kg grape seed extract group died. Of the pups that were kept alive for at least 3 days (i.e. combining the gross and microscopic pathology groups and the 25 and 50 mg/kg grape seed extract groups), 4 of 31 (13%) in the vehicle group died and 3 of 48 (6%) of the grape seed extract treated animals died. Gross neuropathologic damage was scored 22 days after injury. The proportion of pup brains scored as damaged is shown in Fig. 3. Grape seed extract decreased the percentage of brains scored as damaged (scores of 2–4) from
Fig. 3. The effect of different doses (25–50 mg/kg) of grape seed extract administered by i.p. 5 min prior to hypoxia and 4, 18 and 26 h after reoxygenation on the percentage of brains showing gross brain damage. The percentage of pups showing gross brain damage (score > 1) was determined by a blind observer 22 days after hypoxia-ischemia. Data are presented as mean ± S.D. of the proportion. Treatment with 50 mg/kg of grape seed extract improved the brain scores compared to vehicle (** P < 0.01). Treatment with 25 mg/kg of grape seed extract also trended to reduce brain damage score, but this reduction was not statistically significant (P > 0.05).
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Fig. 4. The dose–response for percentage reduction in right cerebral hemisphere weight measured using the left hemisphere weight as standard. Grape seed extract (25–50 mg/kg) was administered by i.p. at 5 min prior to hypoxia, at 4, 18 and 26 h after reoxygenation. Brain injury was evaluated 22 days later. Data are presented as mean ± S.E.M. Treatment with 25, or 50 mg/kg of grape seed extract significant decreased the percentage reduction in right hemisphere weight compared to vehicle (** P < 0.01 vs. vehicle).
Fig. 5. The microscopic brain score for grape seed extract (GSE), and vehicle treated rat pups for the cerebral cortex, hippocampus, and thalamus. Fifty milligram per kilogram of GSE was given i.p. at 5 min prior to hypoxia and at 4, 18 and 26 h after hypoxia. Brains were removed at 3 days after injury. Data is graphed as mean ± S.E.M. For all three tissues, the GSE treated animal had statistically less damage. * P < 0.05 vs. GSE, ** P < 0.01 vs. GSE. Statisical analysis was by Mann–Whitney U-test.
3.3. Brain 8-isoprostaglandin F2α 57.1% in the vehicle group (n = 21) to 5.0% with 50 mg/kg (n = 20, P < 0.01, vehicle versus 50 mg/kg). The dose of 25 mg/kg of grape seed extract (n = 19) produced a similar effect (26.3%), but this was not statistically significant (vehicle versus 25 mg/kg). Identical statistical results are obtained by treating the score as an ordinal rather than a categorical variable and analyzing with Kruskal–Wallis rather than χ2 . Left hemisphere weight was not affected by the hypoxiaischemia procedure [31] and was not significantly different from vehicle at any dose of grape seed extract. The percent reduction in right hemispheric weight is shown in Fig. 4. Grape seed extract significantly reduced the decrease in right hemisphere weight from 20.0 ± 4.4% S.E.M. (n = 21) in the vehicle group to 4.6 ± 3.1% (n = 19) in the group receiving the 25 mg/kg of grape seed extract (P < 0.01, vehicle versus 25 mg/kg) and to 3.1 ± 1.6% (n = 20) in the group receiving the 50 mg/kg of grape seed extract (P < 0.01, vehicle versus 50 mg/kg).
The concentrations of 8-isoprostaglandin F2␣ in left, contralateral hemisphere were 140.5 ± 9.9 pg/g (n = 3), 126.6 ± 24.8 pg/g (n = 6), and 134.5 ± 20.1 pg/g (n = 6) in the sham, 50 mg/kg of grape seed extract and vehicle treated groups, respectively. There were no significant differences among the sham, vehicle, and grape seed extract-treated groups in the left contralateral hemisphere. The concentrations of 8-isoprostaglandin F2␣ in right, ipsilateral hemisphere were significantly higher in vehicle group (369.9 ± 51.2 pg/g, n = 6) than in sham group (140.5 ± 9.9 pg/g, n = 3, P < 0.05) or in the 50 mg/kg of grape seed extract treated group (253.5 ± 26.9 pg/g, n = 6, P < 0.05 versus vehicle). No pup died prior to the planned time. Treatment with grape seed extract significantly reduced a hypoxiainduced increase in brain 8-isoprostaglandin F2␣ (Fig. 6).
3.2. Histopathology Histopathologic score was measure in six grape seed extract treated pups and six vehicle treated pups in all three tissues. No pups died prior to the planned time. The histopathologic score for the cortex was 0[0, 1.25], median[25 percentile, 75 percentile], in the 50 mg/kg of grape seed extract treated group and 5[5, 5] in the corresponding vehicle group, P < 0.05. The histopathologic score for the hippocampus was 0[0, 1.25] in the grape seed extract treated group and 5[5, 5] in the corresponding vehicle group, P < 0.05. The histopathologic score for the thalamus was 0[0, 0.75] in the grape seed extract treated group and 3.5[3, 5] in the vehicle group, P < 0.01. Treatment with grape seed extract significantly improved the outcome in the cortex, hippocampus and thalamus (Fig. 5).
Fig. 6. The effects of grape seed extract (GSE) on brain 8-isoprostaglandin F2␣ . The concentrations of 8-isoprostaglandin F2␣ in right hemisphere were significantly higher in the vehicle group than in the sham group. Treatment with 50 mg/kg of grape seed extract given 5 min prior to hypoxia and 4 h after reoxygenation significantly reduced a hypoxia-induced increase in brain 8isoprostaglandin F2␣ compared with vehicle group (* P < 0.05 vs. grape seed extract; # P < 0.05 vs. shams).
Y. Feng et al. / Brain Research Bulletin 66 (2005) 120–127
Fig. 7. Effect of grape seed extract on brain thiobarbituric acid reactive substances (TBARS). Treatment with the grape seed extract (50 mg/kg) was given 5 min prior to hypoxia and 4 h after reoxygenation. Brain thiobarbituric acid reactive substances were assessed 16 h after reoxygenation. Data are presented as mean ± S.E.M. Treatment with 25 or 50 mg/kg of grape seed extract significantly reduced a hypoxia-induced increase in brain thiobarbituric acid reactive substances compared with vehicle group (## P < 0.01, # P < 0.05 vs. sham, ** P < 0.01 vs. vehicle).
3.4. Thiobarbituric acid reactive substance One pup in the vehicle group died during the hypoxia. The concentrations of thiobarbituric acid reactive substances in the right, ipsilateral hemisphere were significantly higher in the vehicle group (721.6 ± 44.3 pmol/g, n = 6) than in the shams (580 ± 44 pmol/g, n = 6, P < 0.01, vehicle versus sham) or in the 25 mg/kg grape seed extract treated group (579.8 ± 44.5 pmol/g, n = 6, P < 0.05, vehicle versus 25 mg/kg) or in the 50 mg/kg grape seed extract treated group (511.4 ± 28.9 pmol/g, n = 6, P < 0.01, vehicle versus 50 mg/kg). There was no increase in thiobarbituric acid reactive substances in the contralateral hemisphere in our previous studies [15], so we did not measure thiobarbituric acid reactive substances in the contralateral hemisphere. Treatment with grape seed extract significantly reduced a hypoxiainduced increase in brain thiobarbituric acid reactive substances (Fig. 7).
4. Discussion The present study demonstrates that treatment with grape seed extract significantly reduces the severity of brain injury in the focal ischemia model of the neonatal rat pups. Grape seed extract also significantly reduces a hypoxia-induced increase in brain 8-isoprostaglandin F2␣ and thiobarbituric acid reactive substances concentrations compared with the vehicle group. The histopathologic result was consistent with results of gross brain damage grading and loss of right hemisphere weight. Grape seed extract is a potent effective neuroprotectant at doses of 25 or 50 mg/kg in the rat pup. This dose is similar to the doses shown to be effective in isolated adult rat heart ischemic injury [32] and in adult gerbil transient forebrain ischemia [21].
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Grape seed extract has many possible mechanisms for neuroprotection. Grape seed extract is an effective free radical scavenger that reduces lipid peroxidation [7,41]. Grape seed extract exhibited excellent dose-dependent protective ability against 12-O-tetradecanoylphorbol-13-acetate induced hepatic and brain lipid peroxidation, and the associated DNA fragmentation in mice [5,6]. Grape seed extract provides superior anti-oxidant efficacy as compared to Vitamins C, and E at equal doses by weight [4,5]. Grape seed extract inhibits DNA oxidative damage in the gerbil forebrain ischemia model [21]. Grape seed extract can block cell death signaling mediated through the pro-apoptotic transcription factors and genes such as JNK-1 and c-JUN [40]. Chronic treatment with grape seed extract will increase expression of bcl-xl and prevent ladder like DNA fragmentation [36]. Grape seed extract also has anti-inflammatory actions in association with its oxygen free radical scavenging, and anti-lipid peroxidation activity. Grape seed extract reduces production of proinflammatory cytokines and swelling in croton oil induced ear swelling in mice and carrageenan induced paw swelling in rats [25], and decreases chemiluminescence by peritoneal macrophage in response to oxidative injury in mice [5]. Hypoxia-ischemia causes brain damage, activating a cascade of biochemical events that include loss of cellular ATP, which causes cell depolarization and glutamate release, which causes intracellular Ca2+ overload, which causes increased oxygen radical generation by mitochondria. Oxygen radicals by fostering mitochondrial permeability transition will release apoptosis inducing factor and cytochrome c from mitochondria. Cytochrome c with ATP and apoptotic protease activating factor-1 (Apaf-1) will activate procaspase-9 and initiate the caspase cascade [27]. Oxygen free radical reactions lead to the oxidation of lipids, proteins and polysaccharides and to DNA damage [11]. Lipid peroxidation has been and remains one of the most widely used indicators of oxidant/free radical formation. Thiobarbituric acid reactive substances primarily reflect production of lipid peroxides, which are broken down during the assay to yield malondialdehyde [19]. In the present study, hypoxic ischemic rat pups showed elevations in thiobarbituric acid reactive substances concentrations in the brain. Treatment with grape seed extract reduced the increase in thiobarbituric acid reactive substances. Similar results showing grape seed extract reduced formation of thiobarbituric acid reactive substances have been reported by other observers [25,39]. F2 -isoprostanes are non-enzymatic products of the oxygen radical-induced lipid peroxidation of arachidonic acid. They are relatively stable once formed. F2 -isoprostanes are formed initially as esterified fatty acids attached to phospholipids and then are released to their free form by the action of phospholipases, thus delaying the elevation of levels in the tissues [38]. Measurement of F2 -isoprostanes as indicators of lipid peroxidation is sensitive and specific [38,43]. Eight-isoprostaglandin F2␣ is an F2 -isoprostane that is often used to measure lipid peroxidation in humans. Bayir et al. [9] found that hypoxemia after traumatic brain injury
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was associated with increased F2 -isoprostane levels. Our present data showed that hypoxia-ischemia-increased brain 8-isoprostaglandin F2␣ . Treatment with grape seed extract eliminated the increase in 8-isoprostaglandin F2␣ in the brain at 24 h after reoxygenation. This result was consistent with the thiobarbituric acid reactive substances assay result and suggest that grape seed extract’s inhibition of oxidant/free radicals is an important mechanism for its neuroprotective effect. This is not the first demonstration that hypoxia and ischemia cause oxidant/free radicals in the brain of the newborn rat [8] or that scavengers of free radicals can ameliorate the brain injury in this model [44]. This is the first demonstration that grape seed extract can reduce brain lipid peroxidation and reduce injury in a newborn animal model. In conclusion our findings indicate that grape seed extract has neuroprotective properties in the neonatal rat hypoxiaischemic brain injury model. The results also indicate that the suppression of free radicals after hypoxic ischemia by grape seed extract is one potential mechanism of this neuroprotection. We suggest that grape seed extract is a novel, reasonably safe [32,4,5] and cost effective potential therapeutic agent for the treatment of brain injury. Further trials attempting to isolate the neuroprotection to one or a smaller number of compounds would be useful [36]. Human trials for this indication should await demonstration of neurofunctional as well as neuroanatomic salvage, replication of the experiment in more advanced gyrencephalic animals and demonstration that neuroprotection can be obtained when treatment is delayed at least several hours after injury. Human trials of grape seed extract for other indications have already begun [22,30,34].
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[9]
[10]
[11] [12]
[13] [14]
[15]
[16]
[17]
[18]
[19]
[20]
References [1] P. Andine, M. Thordstein, I. Kjellmer, C. Nordborg, K. Thiringer, E. Wennberg, H. Hagberg, Evaluation of brain damage in a rat model of neonatal hypoxic ischemia, J. Neurosci. Methods 35 (1990) 253–260. [2] M. Ashraf-Khorassani, L.T. Taylor, Sequential fractionation of grape seeds into oils, polyphenols, and procyanidines via a single system employing CO2 -based fluids, J. Agric. Food Chem. 52 (2004) 2440–2444. [3] S. Ashwal, W.J. Pearce, Animal models of neonatal stroke, Curr. Opin. Pediatr. 13 (2001) 506–516. [4] D. Bagchi, A. Grarg, R.L. Krohn, M. Bagchi, M.X. Tran, S.J. Stohs, Oxygen free radical scavenging abilities of Vitamins C and E, and a grape seed proanthocyanidin extract in vitro, Res. Commun. Mol. Pathol. Pharmacol. 95 (1997) 179–190. [5] D. Bagchi, A. Grarg, R.L. Krohn, M. Bagchi, D.J. Bagchi, J. Balmoori, S.J. Stohs, Protective effects of grape seed proanthocyanidins and selected antioxidants against TPA-induced hepatic and brain lipid peroxidation and DNA fragmentation, and peritoneal macrophage activation in mice, Gen. Pharmacol. 30 (1998) 771–776. [6] M. Bagchi, M. Milnes, C. Williams, J. Balmoori, X. Ye, S. Stohs, D. Bagchi, Acute and chronic stress-induced oxidative gastrointestinal injury in rats, and the protective ability of a novel grape seed proanthocyanidins extract, Nutr. Res. 19 (1999) 1189–1199. [7] D. Bagchi, C.K. Sen, S.D. Ray, D.K. Das, M. Bagchi, H.G. Preuss, J.A. Vinson, Molecular mechanisms of cardioprotection by a novel
[21]
[22]
[23]
[24]
[25]
[26]
[27]
grape seed proanthocyanidin extract, Mutat. Res. 523–524 (2003) 87–97. R. B˚agenholm, U.A. Nilsson, I. Kjellmer, Formation of free radicals in hypoxic ischemic brain damage in the neonatal rat, assessed by an endogenous spin trap and lipid peroxidation, Brain Res. 773 (1997) 132–138. H. Bayir, D.W. Marion, A.M. Puccio, S.R. Wisniewski, K.L. Janesko, R.S. Clark, P.M. Kochanek, Marked gender effect on lipid peroxidation after severe traumatic brain injury in adult patients, J. Neurotrauma 21 (2004) 1–8. E. Bona, B.B. Johansson, H. Hagberg, Sensorimotor function and neuropathology five and six weeks after hypoxia-ischemia in sevenday-old rats, Pediatr. Res. 42 (1997) 678–683. G. Buonocore, S. Perrone, R. Bracci, Free radicals and brain damage in the newborn, Biol. Neonate 79 (2001) 180–186. O.V.R. Cataltepe, D.F. Heitjan, J. Towfighi, Effect of status epilepticus on hypoxic-ischemic brain damage in the immature rat, Pediatr. Res. 38 (1995) 251–257. J. Da Silva, J. Rigaud, V. Cheynier, Procyanidine dimers and trimers from grape seeds, Phytochemistry 30 (1991) 1259–1264. Y. Feng, M.H. LeBlanc, Drug-induced hypothermia begun 5 min after injury with a poly(ADP-ribose) polymerase inhibitor reduces hypoxic brain injury in rat pups, Crit. Care Med. 30 (2002) 2420–2424. Y. Feng, J.D. Fratkin, M.H. LeBlanc, Treatment with tamoxifen reduces hypoxic-ischemic brain injury in neonatal rats, Eur. J. Pharmacol. 484 (2004) 65–74. A.M. Fine, Oligomeric proanthocyanidine complexes: history, structure, and phytopharmaceutical applications, Altern. Med. Rev. 5 (2000) 144–151. Z.Z. Guan, W.F. Yu, A. Nordberg, Dual effects of nicotine on oxidative stress and neuroprotection in PC12 cells, Neurochem. Int. 43 (2003) 243–249. H. Hagberg, E. Gilland, N.H. Diemer, P. Andin´e, Hypoxia-ischemia in the neonatal rat brain: histopathology after post-treatment with NMDA and non-NMDA receptor antagonists, Biol. Neonate 66 (1994) 205–213. J.J. Hageman, A. Bast, N.P. Vermeulen, Monitoring of oxidative free radical damage in vivo: analytical aspects, Chem. Biol. Interact. 82 (1992) 243–293. S.W. Hoffman, R.L. Roof, D.G. Stein, A reliable and sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F2␣ : a marker for lipid peroxidation after experimental brain injury, J. Neurosci. Methods 68 (1996) 133–136. I.K. Hwang, K.Y. Yoo, D.S. Kim, Y.K. Jeong, J.D. Kim, D.W. Shin, S.S. Lim, I.D. Yoo, T.C. Kang, H.K. Kim, W.K. Moon, M.H. Won, Neuroprotective effects of grape seed extract on neuronal injury by inhibiting DNA damage in the gerbil hippocampus after transient forebrain ischemia, Life Sci. 75 (2004) 1989–2001. R. Kalin, A. Righi, A. Del Rosso, D. Bagchi, S. Generini, M.M. Cerinic, D.K. Das, Activin, a grape seed-derived proanthocyanidin extract, reduces plasma levels of oxidative stress and adhesion molecules, (ICAM-1, VCAM-1, and E-selectin) in systemic sclerosis, Free Radic. Res. 36 (2002) 819–825. L. Kruger, S. Saporta, L.W. Swanson, Photographic Atlas of the Rat Brain, Cambridge University Press, Cambridge, 1995, plates 21 to 23. M.H. LeBlanc, Y. Feng, J.D. Fratkin, N-tosyl-l-phenylalanylchloromethylketone reduces hypoxic-ischemic brain injury in rat pups, Eur. J. Pharmacol. 390 (2000) 249–256. W.G. Li, X.Y. Zhang, Y.J. Wu, X. Tian, Anti-inflammatory effect and mechanism of proanthocyanidins from grape seeds, Acta. Pharmacol. Sin. 22 (2001) 1117–1120. T.K. Mattoo, L. Kovacevic, Effect of grape seed extract on puromycin-aminonucleoside-induced nephrosis in rats, Pediatr. Nephrol. 18 (2003) 872–877. M.P. Mattson, C. Culmsee, Z.F. Yu, Apoptotic and antiapoptotic mechanisms in stroke, Cell Tissue Res. 301 (2000) 173–187.
Y. Feng et al. / Brain Research Bulletin 66 (2005) 120–127 [28] J.M. McCord, Oxygen-derived free radicals in post-ischemic tissue injury, N. Engl. J. Med. 312 (1985) 159–163. [29] J.W. McDonald, C.K. Chen, W.H. Trescher, M.V. Johnston, The severity of excitotoxic brain injury is dependent on brain temperature in the immature rat, Neurosci. Lett. 126 (1991) 83–86. [30] F. Natella, F. Belelli, V. Gentili, F. Ursini, C. Saccini, Grape seed proanthocyanidins prevent plasma postprandial oxidative stress in humans, J. Agric. Food Chem. 50 (2002) 7720–7725. [31] C. Palmer, R.C. Vannucci, J. Towfighi, Reduction of perinatal hypoxic-ischemic brain damage with allopurinol, Pediatr. Res. 27 (1990) 332–336. [32] T. Pataki, I. Bak, P. Kovacs, D. Bagchi, D.K. Das, A. Tosaki, Grape seed proanthocyanidins improved cardiac recovery during reperfusion after ischemia in isolated rat hearts, Am. J. Clin. Nutr. 75 (2002) 894–899. [33] Z. Peng, Y. Hayasaka, P.G. Iland, M. Sefton, P. Hoj, E.J. Water, Quantitative analysis of polymeric procyanidins (Tannins) from grape (Vitis vinifera) seeds by reverse phase high-performance liquid chromatography, J. Agric. Food Chem. 49 (2001) 26–31. [34] H.G. Preuss, D. Wallerstedt, N. Talpur, S.O. Tutuncuoglu, B. Echard, A. Myers, M. Bui, D. Bagchi, Effects of niacin-bound chromium and grape seed proanthocyanidin extract on the lipid profile of hypercholesterolemic subjects: a pilot study, J. Med. 31 (2000) 227–246. [35] S. Ray, D. Bagchi, P.M. Lim, M. Bagchi, S.M. Gross, S.C. Kothari, H.G. Preuss, S.J. Stohs, Acute and long term safety evaluation of a novel IH636 grape seed proanthocyanidin extract, Res. Commun. Mol. Pathol. Pharmacol. 109 (2001) 165–197. [36] S.D. Ray, M.A. Kumar, D. Bagchi, A novel proanthocyanidin IH636 grape seed extract increases in vivo bcl-XL expression and prevents acetaminophen-induced programmed and unprogrammed cell death in mouse liver, Arch. Biochem. Biophys. 369 (1999) 42–58. [37] J.E. Rice, R.C. Vannucci, J.B. Brierley, The influence of immaturity on hypoxic-ischemic brain damage in the rat, Ann. Neurol. 9 (1981) 131–141. [38] L.J. Roberts, J.D. Morrow, Measurement of F2 -isoprostanes as an index of oxidative stress in vivo, Free Radic. Biol. Med. 28 (2000) 505–513.
127
[39] M. Sato, G. Maulik, P.S. Ray, D. Bagchi, D.K. Das, Cardioprotective effects of grape seed proanthocyanidin against ischemic reperfusion injury, J. Mol. Cell. Cardiol. 31 (1999) 1289–1297. [40] M. Sato, D. Bagchi, A. Tosaki, D.K. Das, Grape seed proanthocyanidin reduces cardiomyocyte apoptosis by inhibiting ischemia/reperfusion-induced activation of JNK-1 and C-Jun, Free Radic. Biol. Med. 31 (2001) 729–737. [41] M. Shafiee, M.A. Carbonnneau, N. Urban, B. Descomps, C.L. Leger, Grape and grape seed extract capacities at protecting LDL against oxidation generated by Cu+2 , AAPH or SIN-1 and at decreasing superoxide THP-1 cell production: a comparison to other extracts or compounds, Free Radic. Res. 37 (2003) 573– 584. [42] Z.H. Shao, L.B. Becker, T.L. VandenHoek, P.T. Schumacker, C.Q. Li, D. Zhao, K. Wojcik, T. Anderson, Y. Qin, L. Dey, C.S. Yuan, Grape seed proanthocyanidin extract attenuates oxidant injury in cardiomyocytes, Pharmacol. Res. 47 (2003) 463–469. [43] M.M. Tarpey, D.A. Wink, M.B. Grisham, Methods for detection of reactive metabolites of oxygen and nitrogen: in vitro and in vivo considerations, Am. J. Physiol. Regul. Integr. Comp. Physiol. 286 (2004) R431–R444. [44] M. Thordstein, R. B˚agenholm, K. Thiringer, I. Kjellmer, Scavengers of free oxygen radicals in combination with magnesium ameliorate perinatal hypoxic-ischemic brain damage in the rat, Pediatr. Res. 34 (1993) 23–26. [45] J. Towfighi, C. Housman, R.C. Vannycci, D.F. Heitjan, Effect of unilateral perinatal hypoxic-ischemic brain damage on the gross development of opposite cerebral hemisphere, Biol. Neonate 65 (1994) 108–118. [46] W.H. Trescher, S. Ishiwa, M.V. Johnston, Brief post-hypoxicischemic hypothermia markedly delays neonatal brain injury, Brain Dev. 19 (1997) 326–338. [47] J.J. Volpe, Neurology of the Newborn, fourth ed., W.B. Saunders, Philadelphia, PA, 2001. [48] J. Yager, J. Towfighi, R.C. Vannucci, Influence of mild hypothermia on hypoxic-ischemic brain damage in the immature rat, Pediatr. Res. 34 (1993) 525–529.