
==== Front
Acta Cir Bras
Acta Cir Bras
acta
Acta Cirúrgica Brasileira
0102-8650
1678-2674
Sociedade Brasileira para o Desenvolvimento da Pesquisa em Cirurgia

00241
10.1590/acb397424
Original Article
Impact of daily administration of blackberry extract on gerbil model of transient cerebral ischemia
https://orcid.org/0009-0001-2083-7372
Oda Asahi Substantive scientific and intellectual contributions to the study Acquisition and analysis of data Manuscript writing 1*
https://orcid.org/0000-0003-2152-5394
Sugai Kazuhisa Substantive scientific and intellectual contributions to the study Acquisition and analysis of data Critical revision 1*
https://orcid.org/0000-0002-2343-0324
Fujisawa Masahiko Substantive scientific and intellectual contributions to the study Critical revision 1*
https://orcid.org/0000-0002-0138-6692
Hakamata Yoji Conception and design Acquisition and analysis of data Technical procedure Manuscript writing Final approval of the version to be published 12*
https://orcid.org/0009-0003-0639-8232
Kobayashi Shou Conception and design Technical procedure 3*
https://orcid.org/0000-0001-8617-3778
Kobayashi Eiji Conception and design Technical procedure Manuscript writing Final approval of the version to be published 13**
1 Nippon Veterinary and Life Science University – School of Veterinary Nursing and Technology – Department of Basic Science – Tokyo – Japan.
2 Nippon Veterinary and Life Science University – School of Veterinary Nursing and Technology – Research Center for Animal Life Science – Tokyo – Japan.
3 Kobayashi Regenerative Research Institute, LLC – Administrative Department – Wakayama – Japan.
Section editor: Edna Montero https://orcid.org/0000-0003-1437-1219

* Corresponding author: eijikoba@jikei.ac.jp
Conflict of interest: Nothing to declare.

09 9 2024
2024
39 e39742403 2 2024
08 6 2024
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT

Purpose:

Blackberries are rich in polyphenols and are a human health food continuously consumed to improve health and reduce diseases caused by aging. Herein, we evaluated the effects of daily blackberry administration before and after transient cerebral ischemia in gerbils.

Methods:

Blackberry extract (BBE) was orally administered twice a day for two weeks to protect against ischemic events during continuous administration. On the seventh day after administration, the bilateral common carotid arteries were transiently occluded for 5 min. To verify its therapeutic effect, BBE was administered after ischemia using a similar protocol without pre-administration. In both experiments, the number of viable neurons in the CA1 region of the hippocampus was assessed seven days after ischemic treatment.

Results:

The number of neurons in the group treated with BBE before ischemia was higher than that in the group treated with distilled water (p = 0.0601), and similar to that in the control group. In the BBE administration experiments after ischemia, the number of neurons was significantly reduced compared to that in the control group (p < 0.0001).

Conclusions:

Continuous BBE intake is expected to prevent or ameliorate ischemic events such as transient cerebral ischemia.

Key words

Plant Extracts Blackberries
Ischemia
Brain
Neurons
Gerbillinae
==== Body
pmcIntroduction

Berries have attracted attention as health food for humans because they contain many polyphenols, vitamins, minerals, fiber and health benefits1 , 2. Recently, clinical trials using raspberries have been conducted in the field of beauty, showing that facial skin color, elasticity, radiance, smoothness, scaliness, and wrinkles in 50-middle-aged women significantly improved3. Large-scale clinical trials are required to demonstrate the effects of health foods on humans and the preventive administration of beauty products on adverse events. However, in highly individualized human studies, it is costly and time-consuming to determine the range of efficacy4 - 6.

Numerous animal studies have demonstrated that polyphenols, which are abundant in blackberry (BB) fruit and seeds, have antioxidant7 , 8, antitumor9 , 10, anti-aging3, anti-inflammatory11 , 12, anti-lipid13, and anti-stress effects14. In many of these reports, to evaluate the effects, experimental impairment models have been established, and administration has been initiated at the same time or after impairment15. However, it has recently been reported that pretreatment with BB leaf extract for two weeks improves hepatic ischemia-reperfusion injury in rats16. We previously reported that repeated consumption of BB improves hyperlipidemia and that ingestion before the onset of adverse events is beneficial17.

In this study, we aimed to evaluate the effects of daily intake of BB as a human health food in a gerbil model of transient cerebral ischemia that may occur incidentally in older people. Cerebral ischemia is an intractable disease that causes nerve cell death and motor and higher brain dysfunction. Although various animal models have been developed to elucidate the pathogenesis of cerebral ischemic injury, it can be induced in gerbils simply by occluding the common carotid artery, owing to the incomplete construction of the basilar artery, and the degree of injury can be selectively induced from neuronal cell death to cerebral infarction by the occlusion time of the vessels18. Considering that the composition of BB depending on the production area and picking season19, we used BB from the main production areas of Japan.

Methods

Animals

Mature male (n = 12) and female (n = 12) Mongolian gerbils (Meriones unguiculatus) (body weight, 70–130 g) bred in our laboratory were used in this study. All animals were kept in our laboratory and housed in an environment with 23 ± 2 °C, humidity of 55 ± 10%, and a 14-h/10-h light/dark cycle, and were provided solid mouse/rat feed (EF solid feed; Oriental Yeast Co., Ltd., Tokyo, Japan) and water ad libitum.

This study was approved by the Institute Animal Care and Use Committee (Nippon Veterinary and Life Science University, Tokyo, Japan; Permit No. 2023K-72) and followed the Nippon Veterinary and Life Science University guidelines for the care and use of laboratory animals. All sections of this study adhered to the ARRIVE guidelines for animal research20.

Preparation of blackberry extract and measurement of polyphenol concentration

BB used in this study were obtained from Amanogawa Co. (Aomori, Japan). Frozen BB were thawed in a water bath at 37 °C, homogenized using polytron-aggregate (PT1200E, Kinematica Co., Malters, Switzerland), and liquefied by sonication for 30 min using a US-Cleaner (US-2R, SND Co., Nagano, Tokyo). Liquified BB were pressed with gauze to separate the seeds, skin, and juice. The seeds and skin were dried in a dryer (NDS-520, Wakenyaku Co., Kyoto, Japan) and ground into powder in a mortar (Asvel Co., Nara, Japan). The seed and skin powders were placed in separated BB juice, dissolved in a water bath at 55 °C, and the resulting solution was regarded as BB extract (BBE) (Fig. 1). The polyphenol content of BBE was measured using the Folin–Ciocalteu method (polyphenol content: 280 mg/100 g BBE). Distilled water (DW) was used as a reference material for BBE.

Figure 1 Extraction process of blackberry extract (BBE). (a) Blackberry. (b) Blackberries were placed in a centrifuge tube and homogenized on ice. (c) Blackberries were sonicated. (d) Sample was packed into gauze and squeezed. (e) Residues were dried and ground to powder. (f) Powder was mixed with squeezed liquid to create BBE.

Source: Elaborated by the authors.

Blackberry extract administration schedule and cerebral ischemia procedure

The animals were randomly divided into three groups: control (n = 6), DW (n = 8), and BBE (n = 10).

Pre-ischemia blackberry extract administration group

Animals were divided into DW (n = 5) and BBE (n = 4) groups. Animals were orally administered DW or BBE with a probe at the dose of 10 mL/kg bodyweight twice a day (10 a.m./5 p.m.) under light isoflurane inhalation anesthesia seven days before cerebral ischemia (Fig. 2a).

Figure 2 Blackberry extract (BBE) administration and brain ischemic operation protocol. (a) Pre-ischemia administration protocol. BBE was orally administered twice a day from seven days before ischemia. BBE administration was continued for seven days after ischemic operation. On the postoperative day 7, the brain was fixed via transcardial perfusion. (b) Post-ischemia administration protocol. BBE was orally administered twice a day for seven days after ischemic operation. On the postoperative day 7, the brain was fixed via transcardial perfusion.

Source: Elaborated by the authors.

Post-ischemia blackberry extract administration group

Animals were divided into DW (n = 3) and BB (n = 6) groups. After reperfusion following cerebral ischemia, the animals were orally administered DW or BBE with a probe at the same dosage as the pre-treatment groups twice a day for three (n = 3) or seven days (n = 3) (Fig. 2b).

Cerebral ischemia was induced under isoflurane inhalation anesthesia (induction at 5%, maintained at 2%). Animals were placed on a hot plate maintained at 37°C (KN-475-3-40, Natsume Seisakusho Co., Tokyo, Japan), and bilateral common carotid arteries were occluded as previously described21. Briefly, under anesthesia, a midline incision was made in the neck, and the bilateral common carotid arteries were exposed. Both common carotid arteries were clamped for 5 min using artery clips (AS1-40; Bear Medic Co., Ibaraki, Japan). After 5 min of occlusion, the clips were removed to resume blood flow, and the incised skin was closed using wound clips (Becton Dickinson Japan Co., Tokyo, Japan). The animals in the control group underwent the same procedure as the ischemic animals, except that the bilateral common carotid arteries were not occluded. No solution was administered to the control group.

Assessment of cerebral neurons

To assess the effect of BBE on brain ischemic neuronal cell death, all animals used in this study were perfusion-fixed. Seven days after ischemia-reperfusion, the animals were deeply anesthetized with an intraperitoneal injection of pentobarbital sodium (65 mg/mL, 1 mL/kg bodyweight). The transcardial approach was used to perfuse the brains with 100 mL of heparinized saline followed by 100 mL of 10% phosphate-buffered formalin. The brains were fixed overnight in 10% phosphate-buffered formalin. The fixed brain was coronally sectioned at the pituitary level, embedded in paraffin and cut into 5-µm sections, and stained with hematoxylin and eosin (HE). Each brain section was scanned with a NanoZoomer-SQ 40× mode (0.23 µm/pixel) scanner (Hamamatsu Photonics K.K., Shizuoka, Japan), and images were analyzed by NDP.view 2 software (Hamamatsu Photonics K.K.) to measure the number of neurons in the hippocampal CA1 area. Neurons with distinct nuclear membranes and clear nucleoli were defined as viable neuronal cells and counted.

Statistical analysis

All data are presented as mean ± standard deviation. All statistical analyses were conducted using GraphPad Prism version 10.1.1. Comparisons were performed using one-way analysis of variance and Tukey’s multiple comparison test for post-hoc analysis. Statistical significance was set at p < 0.05.

Results

Effect of pre-ischemic administration of blackberry extract

The number of viable neuronal cells in the CA1 region of the hippocampus after transient cerebral ischemia in gerbils orally administered BBE for seven days is shown in Fig. 3. Remarkably, the number of viable neurons in both groups subjected to cerebral ischemia was abundant despite cerebral ischemia. The number of viable neurons in the DW group was significantly lower than that in the control group (p = 0.0110). The number of viable neuronal cells in the BBE group tended to be higher than that in the DW group, although there was no significant difference between the two groups (p = 0.0601), which was equivalent to that in the control group (p = 0.8053).

Figure 3 Effect of pre-ischemia administration of blackberry extract (BBE). (a) Images of ischemic CA1 region of hippocampus stained with hematoxylin and eosin. The image on the right is an enlarged view of the image on the left. (b) The number of viable neuronal cells in the CA1 region. The numeric values indicate p-values between each group.

Δ: typical viable neuron; ▲: dead neuron. Source: Elaborated by the authors.

Effect of post-ischemic administration of blackberry extract

The number of viable neuronal cells in the CA1 region of the hippocampus of gerbils that repeatedly underwent BBE administration after cerebral ischemia is shown in Fig. 4. The number of viable neurons in both groups subjected to cerebral ischemia was significantly lower than that in the control group (p < 0.0001). Although the number of viable neuronal cells in the BB seven days group tended to be higher than that in the BB three days group. The number of cells in both groups did not significantly differ from that in the DW group.

Figure 4 Effects of post-ischemia administration of blackberry extract (BBE). (a) Images of ischemic CA1 region of hippocampus stained with hematoxylin and eosin. The image on the right is an enlarged view of the image on the left. (b) The number of viable neuronal cells in the CA1 region. The numeric values indicate p-value between each group.

Δ: typical viable neuron; ▲: dead neuron; *p < 0.0001 vs. control group. Source: Elaborated by the authors.

Discussion

The BB examined in this study contain anthocyanins, other phenolic compounds, flavanols, and ellagitannins, which contribute to their high antioxidant potency and other biological activities, and are consumed worldwide as a human health food3. However, large-scale randomized controlled studies are required to verify these effects in humans. In this study, we evaluated the efficacy of repeated administration of BBE on cerebral ischemia in gerbils.

In the two-week pretreatment experiment, BBE consumption dramatically reduced neuronal cell death in the CA1 region of the hippocampus with transient cerebral ischemia. The number of neurons was equivalent to that of the control group, which was not subjected to cerebral ischemia. Repeated intake of polyphenols has been reported to reduce subsequent cerebral ischemic injury in other experimental systems22 , 23. This phenomenon is known as preconditioning and is anticipated to be a new therapeutic approach for stroke in humans through healthy food consumption24.

The prominent effect of the two-week BBE administration in this study may have induced not only an improvement in the lipid system before the event, but also this preconditioning17. In the pretreatment experiment, the number of viable neurons in the BBE group showed little variation and no decrease, whereas the DW group showed a large variation among individuals. Even in the DW group, half of the animals had abundant remnant nerve cells that appeared to be resistant to ischemia. For pretreatment with BBE and DW, oral administration was performed twice a day under isoflurane inhalation anesthesia.

This anesthetic treatment may temporarily paralyze nerves and induce a decrease in blood pressure and reflexes. In some animals in the DW group, this stress may have induced a preconditioning phenomenon that reduced the cerebral ischemia-reperfusion injury. Experimental evidence of such preconditioning phenomena has been reported in the brains25 and livers26 of rat models of ischemia. Mild stress stimulates the adrenal glands, and the glucocorticoids secreted by the adrenal glands are thought to act on the brain and induce anti-inflammatory effects27. However, although numerous studies have been conducted over a long time, the comprehensive mechanism of ischemic preconditioning is still controversial, and much remains unclear28 , 29.

In therapeutic administration, BBE treatment after cerebral ischemia did not suppress neuronal cell death. Although it has been reported that polyphenols contained in BB have antioxidant effects and rescue neuronal cell death7 , 8, it remains unclear whether the results of post-administration in this study were due to insufficient BBE components or whether the ischemic injury in this model was too high.

The BB used in this study were sourced from Aomori, which has the highest production rate in Japan. Although the freeze-drying8 and acetone methods12 are common methods for extracting BB, we focused on the inclusion of all components in consideration of their generality and simplicity as health food. Frozen BB were homogenized and sonicated, and the remaining seeds and peels were ground and dissolved. The concentration of polyphenols in the extract obtained by this method was comparable to that reported previously, suggesting no discernible differences due to the extraction methods19.

Conclusion

This study demonstrates that continuous prophylactic intake of BBE stably suppresses neuronal cell death in the CA1 region of the hippocampus after transient cerebral ischemia. These results suggest that the continuous consumption of BB as a health food may be effective in developing tolerance to cerebral ischemic injury.

Acknowledgements

We appreciate Mr. Arai from Oriental Yeast Co., for advice on blackberry production and sales in Japan.

MS

PhDs

PhDs

PhDs

Researcher

PhDs

Data availability statement Raw data were generated at Nippon Veterinary and Life Science University. Derived data supporting the findings of this study are available from the author Hakamata Y upon reasonable request.

Research performed at Nippon Veterinary and Life Science University, Tokyo, Japan.

Funding: Kobayashi Regenerative Research Institute, LLC
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References

1 Nile SH Park SW Edible berries: bioactive components and their effect on human health Nutrition 2014 30 2 134 144 10.1016/j.nut.2013.04.007 24012283
2 Afrin S Gasparrini M Forbes-Hernandez TY Reboredo-Rodriguez P Mezzetti B Varela-Lopez A Giampieri F Battino M Promising Health Benefits of the Strawberry: A Focus on Clinical Studies J Agric Food Chem 2016 64 22 4435 4449 10.1021/acs.jafc.6b00857 27172913
3 Rattanawiwatpong P Wanitphakdeedecha R Bumrungpert A Maiprasert M Anti-aging and brightening effects of a topical treatment containing vitamin C, vitamin E, and raspberry leaf cell culture extract: A split-face, randomized controlled trial J Cosmet Dermatol 2020 19 3 671 676 10.1111/jocd.13305 31975502
4 Chen GC Lv DB Pang Z Liu QF Red and processed meat consumption and risk of stroke: a meta-analysis of prospective cohort studies Eur J Clin Nutr 2013 67 1 91 95 10.1038/ejcn.2012.180 23169473
5 Kanellos PT Kaliora AC Protogerou AD Tentolouris N Perrea DN Karathanos VT The effect of raisins on biomarkers of endothelial function and oxidant damage; an open-label and randomized controlled intervention Food Res Int 2017 102 674 680 10.1016/j.foodres.2017.09.061 29195999
6 Delgado-Lista J Alcala-Diaz JF Torres-Pena JD Quintana-Navarro GM Fuentes F Garcia-Rios A Ortiz-Morales AM Gonzalez-Requero AI Perez-Caballero AI Yubero-Serrano EM Rangel-Zuniga OA Camargo A Rodriguez-Cantalejo F Lopez-Segura F Badimon L Ordovas JM Perez-Jimenez P Lopez-Miranda J CORDIOPREV Investigators C. Long-term secondary prevention of cardiovascular disease with a Mediterranean diet and a low-fat diet (CORDIOPREV): a randomised controlled trial Lancet 2022 399 10338 1876 1885 10.1016/S0140-6736(22)00122-2 35525255
7 de Mello JE Luduvico KP Dos Santos A Teixeira FC de Souza J Cardoso J de Aguiar MSS Cunico W Vizzotto M Stefanello F Spanevello R Therapeutic potential of blackberry extract in the preventing memory deficits and neurochemical alterations in the cerebral cortex, hippocampus and cerebellum of a rat model with amnesia Metab Brain Dis 2023 38 4 1261 1272 10.1007/s11011-023-01175-w 36735154
8 Oro CE Paroul N Mignoni ML Zabot GL Backes GT Dallago RM Tres MV Microencapsulation of Brazilian Cherokee blackberry extract by freeze-drying using maltodextrin, gum Arabic, and pectin as carrier materials Food Sci Technol Int 2023 29 3 255 265 10.1177/10820132211068979 34939457
9 Hsieh KY Tsai JY Lin YH Chang FR Wang HC Wu CC Golden berry 4beta-hydroxywithanolide E prevents tumor necrosis factor alpha-induced procoagulant activity with enhanced cytotoxicity against human lung cancer cells Sci Rep 2021 11 1 4610 4610 10.1038/s41598-021-84207-8 33633307
10 Mauramo M Onali T Wahbi W Vasara J Lampinen A Mauramo E Kivimaki A Martens S Haggman H Sutinen M Salo T Bilberry (Vaccinium myrtillus L.) Powder Has Anticarcinogenic Effects on Oral Carcinoma In Vitro and In Vivo Antioxidants (Basel) 2021 10 8 1319 1319 10.3390/antiox10081319 34439567
11 Van de Velde F Esposito D Grace MH Pirovani ME Lila MA Anti-inflammatory and wound healing properties of polyphenolic extracts from strawberry and blackberry fruits Food Res Int 2019 121 453 462 10.1016/j.foodres.2018.11.059 31108769
12 Choe U Li Y Yu L Gao B Wang TTY Sun J Chen P Yu L Chemical composition of cold-pressed blackberry seed flour extract and its potential health-beneficial properties Food Sci Nutr 2020 8 2 1215 1225 10.1002/fsn3.1410 32148827
13 Lim T Lee K Kim RH Cha KH Koo SY Moon EC Hwang KT Black raspberry extract can lower serum LDL cholesterol via modulation of gut microbial composition and serum bile acid profile in rats fed trimethylamine-N-oxide with a high-fat diet Food Sci Biotechnol 2022 31 8 1041 1041 10.1007/s10068-022-01079-y 35873380
14 Fernandez-Demeneghi R Rodriguez-Landa JF Guzman-Geronimo RI Acosta-Mesa HG Meza-Alvarado E Vargas-Moreno I Herrera-Meza S Effect of blackberry juice (Rubus fruticosus L.) on anxiety-like behaviour in Wistar rats Int J Food Sci Nutr 2019 70 7 856 867 10.1080/09637486.2019.1580680 30892114
15 Vahapoglu B Erskine E Gultekin Subasi B Capanoglu E Recent Studies on Berry Bioactives and Their Health-Promoting Roles Molecules 2021 27 1 108 108 10.3390/molecules27010108 35011338
16 Fathi AM Waz S Alaaeldin E Toni NDM El-Sheikh AAK Sayed AM Abdelmohsen UR Nazmy MH Blackberry-Loaded AgNPs Attenuate Hepatic Ischemia/Reperfusion Injury via PI3K/Akt/mTOR Pathway Metabolites 2023 13 3 419 419 10.3390/metabo13030419 36984859
17 Oda A Hakamata Y Kobayashi E Pre-Administration of Blackberry Extracts in Induced Ischemia Reperfusion Events in Rodents Metabolites 2023 13 11 1114 1114 10.3390/metabo13111114 37999210
18 Ito U Hakamata Y Kawakami E Oyanagi K Degeneration of astrocytic processes and their mitochondria in cerebral cortical regions peripheral to the cortical infarction: heterogeneity of their disintegration is closely associated with disseminated selective neuronal necrosis and maturation of injury Stroke 2009 40 6 2173 2181 10.1161/STROKEAHA.108.534990 19359621
19 Kaume L Howard LR Devareddy L The blackberry fruit: a review on its composition and chemistry, metabolism and bioavailability, and health benefits J Agric Food Chem 2012 60 23 5716 5727 10.1021/jf203318p 22082199
20 Percie du Hurst V Ahluwalia A Alam S Avey MT Baker M Browne WJ Clark A Cuthill IC Dirnagl U Emerson M Garner P Holgate ST Howells DW Karp NA Lazic SE Lidster K MacCallum CJ Macleod M Pearl EJ Petersen OH Rawle F Reynolds P Rooney K Sena ES Silberberg SD Steckler T Wurbel H. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research BMJ Open Sci 2020 4 1 e100115 10.1136/bmjos-2020-100115
21 Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia Brain Res. 1982 239 1 57 69 10.1016/0006-8993(82)90833-2 7093691
22 Resveratrol J Fan C Chen N Huang J Yang Q. Resveratrol pretreatment attenuates cerebral ischemic injury by upregulating expression of transcription factor Nrf2 and HO-1 in rats Neurochem Res 2011 36 12 2352 2362 10.1007/s11064-011-0561-8 21850487
23 Sumien N Huang R Chen Z Vann PH Effects of dietary 5-methoxyindole-2-carboxylic acid on brain functional recovery after ischemic stroke Behav Brain Res 2020 378 112278 112278 10.1016/j.bbr.2019.112278
24 Tauskela JS Bourourou M Blondeau N Tackling issues in the path toward clinical translation in brain conditioning: Potential offered by nutraceuticals Brain Circ 2017 3 2 78 86 10.4103/bc.bc_8_17 30276308
25 Wang Y Reis C Applegate R Stier G Martin R Zhang JH Ischemic conditioning-induced endogenous brain protection: Applications pre-, per- or post-stroke Exp Neurol 2015 272 26 40 10.1016/j.expneurol.2015.04.009 25900056
26 Yin DP Sankary HN Chong AS Ma LL Shen J Foster P Williams JW Protective effect of ischemic preconditioning on liver preservation-reperfusion injury in rats Transplantation 1998 66 2 152 157 10.1097/00007890-199807270-00002 9701256
27 Rybnikova E Nalivaeva N Glucocorticoid-Dependent Mechanisms of Brain Tolerance to Hypoxia Int J Mol Sci 2021 22 15 7982 7982 10.3390/ijms22157982 34360746
28 Murry CE Jennings RB Reimer KA Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium Circulation 1986 74 5 1124 1136 10.1161/01.cir.74.5.1124 3769170
29 Kim EN Choi JS Kim CJ Kim SR Oh SJ Role of Ischemic Preconditioning in the Cardioprotective Mechanisms of Monomeric C-Reactive Protein-Deposited Myocardium in a Rat Model J Chest Surg 2021 54 1 9 16 10.5090/kjtcs.20.096 33767007
