Mitochondrial protective effects of ellagic acid in a rat model of sporadic Alzheimer's disease induced by STZ methods
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Materials and methods
Streptozotocin (STZ), 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), 3-[4,5dimethylthiazol-2-yl] -2,5- diphenyltetrazolium bromide (MTT), Rhodamine123 (Rh 123), Crystal violet stain and Ellagic acid (EA) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). ATP kit ENLITEN ® was purchased from Promega (Madison, WI, USA). All salts used for making buffer solutions were of the analytical grade and obtained from Merck (Darmstadt, Germany).
Animals model
Animals: Male Sprague Dawley rats (200-250 g) were obtained from the Animal Breeding Center of Shiraz University of Medical Sciences, Shiraz Iran. Rats were housed in polypropylene cages and fed liberally with a standard diet and drinking water. The animals were kept at a temperature of 23 ± 2 °C and relative humidity ≈ of 40% under a 12:12 light/dark cycle. Animal experiments were carried out in accordance with the guidelines approved by a local ethics committee at Shiraz University of Medical Sciences, Shiraz, Iran (IR.SUMS.REC.1395.866).
Animals model
Sham group: Animals in this group underwent the same surgery process as other groups without Alzheimer's induction; they received 5 µl buffer Saline microinjection into each lateral ventricle.
Animals model
STZ + EA 5 group: After AD induction, animals received 200 µl of EA 5 mg/kg per day for 14 days orally (gavage).
Animals model
STZ + EA 50 group: After AD induction, animals received 200 µl of EA 50 mg/kg per day for 14 days orally (gavage).
Animals model
STZ + EA 100 group: After AD induction, animals received 200 µl of EA 100 mg/kg per day for 14 days orally (gavage).
Surgery and design study
On the day of surgery, the animals were anesthetized with a mixture of Ketamine (80 mg/kg) and Xylazine (10 mg/kg), administered intraperitoneal, and secured in a stereotactic frame with a heating pad to maintain their body temperature. For STZ injection, a 5 µl Hamilton syringe was connected to the injection cannula through a short piece of polyethylene tube (inside the ventricles), 3 mg/kg/5 µl of STZ or its vehicle (saline) was injected at the speed of 0.5 µl/min and the needle was left in the place for an additional 5 min to minimize the flow back of the solution. The STZ was given on days 1 and 3 (1.5 mg/kg per day). EA or saline was administered by gavage at doses of 5, 50, or 100 mg/kg/day and its first administration was started from day 4. Fourteen days after surgery (day 14), behavioral tests (Shuttle box and Step-down) were carried out. The animals were then...
Surgery and design study
Fig. 1 Study design of ellagic acid attenuates memory impairment and mitochondrial dysfunction in STZ-treated rats.
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Evidence quotes (8)
Streptozotocin (STZ), 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), 3-[4,5dimethylthiazol-2-yl] -2,5- diphenyltetrazolium bromide (MTT), Rhodamine123 (Rh 123), Crystal violet stain and Ellagic acid (EA) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). ATP kit ENLITEN ® was purchased from Promega (Madison, WI, USA). All salts used for making buffer solutions were of the analytical grade and obtained from Merck (Darmstadt, Germany).
Animals: Male Sprague Dawley rats (200-250 g) were obtained from the Animal Breeding Center of Shiraz University of Medical Sciences, Shiraz Iran. Rats were housed in polypropylene cages and fed liberally with a standard diet and drinking water. The animals were kept at a temperature of 23 ± 2 °C and relative humidity ≈ of 40% under a 12:12 light/dark cycle. Animal experiments were carried out in accordance with the guidelines approved by a local ethics committee at Shiraz University of Medical Sciences, Shiraz, Iran (IR.SUMS.REC.1395.866).
Sham group: Animals in this group underwent the same surgery process as other groups without Alzheimer's induction; they received 5 µl buffer Saline microinjection into each lateral ventricle.
STZ + EA 5 group: After AD induction, animals received 200 µl of EA 5 mg/kg per day for 14 days orally (gavage).
STZ + EA 50 group: After AD induction, animals received 200 µl of EA 50 mg/kg per day for 14 days orally (gavage).
STZ + EA 100 group: After AD induction, animals received 200 µl of EA 100 mg/kg per day for 14 days orally (gavage).
On the day of surgery, the animals were anesthetized with a mixture of Ketamine (80 mg/kg) and Xylazine (10 mg/kg), administered intraperitoneal, and secured in a stereotactic frame with a heating pad to maintain their body temperature. For STZ injection, a 5 µl Hamilton syringe was connected to the injection cannula through a short piece of polyethylene tube (inside the ventricles), 3 mg/kg/5 µl of STZ or its vehicle (saline) was injected at the speed of 0.5 µl/min and the needle was left in the place for an additional 5 min to minimize the flow back of the solution. The STZ was given on days 1 and 3 (1.5 mg/kg per day). EA or saline was administered by gavage at doses of 5, 50, or 100 mg/kg/day and its first administration was started from day 4. Fourteen days after surgery (day 14), behavioral tests (Shuttle box and Step-down) were carried out. The animals were then euthanized with ketamine/xylazine (100/10 mg/kg, i.p), and their total brain were harvested (Fig. ). Daily body weight and food intake of the mice were monitored throughout the 2-weeks feeding period. At the end of the study, the rats were euthanized using a combination of 50 mg/kg xylazine (2%) an...
Fig. 1 Study design of ellagic acid attenuates memory impairment and mitochondrial dysfunction in STZ-treated rats.
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