Mitochondrial protective effects of ellagic acid in a rat model of sporadic Alzheimer's disease induced by STZ methods
Aim. Evidence-backed execution summary for Mitochondrial protective effects of ellagic acid in a rat model of sporadic Alzheimer's disease induced by STZ methods from Mitochondrial protective effects of ellagic acid in a rat model of sporadic Alzheimer's disease induced by STZ.
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This experiment, in seven questions
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rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Materials and methods
reagent used in the protocol.
- Use
- 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 P...
Animals model
reagent used in the protocol.
- Use
- 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.
Surgery and design study
reagent used in the protocol.
- Use
- 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 c...
Cresyl violet staining
reagent used in the protocol.
- Use
- After administering anesthesia, the animals were transcardially perfused with normal saline, followed by 4% paraformaldehyde in Phosphate buffer saline (PBS). The brain was isolated after perfusion and post-fixed in the same fixation solution overnight, after passing through a gradient of sucrose solutions (10%, 20%...
Biochemical analysis of brain tissue
reagent used in the protocol.
- Use
- The level of reactive oxygen species (ROS) in the brain was quantified using the technique outlined by Gupta et al. with a few adjustments. In short, brain tissues were homogenized in a 1:10 dilution with ice-cold 40 mM Tris-HCl buffer (pH = 7.4). Mix 100 µL of tissue homogenate with 1 mL of Tris-HCl buffer and...
Biochemical analysis of brain tissue
reagent used in the protocol.
- Use
- The FRAP test detects the absorbance difference at 593 nm resulting from the creation of a blue Fe II-tripyridyltriazine complex from the colorless Fe III form, facilitated by electron-donating antioxidants. In short, the active FRAP solution was made by combining 10 parts of 300 mmol/L acetate buffer (pH 3.6)...
Brain mitochondria isolation
reagent used in the protocol.
- Use
- Mitochondrial pellets were suspended in an incubation buffer (70 mM mannitol, 220 mM sucrose, 2 mM HEPES, and 0.5 mM EGTA, pH = 7.4), nevertheless for the mitochondria used to measure ROS production, mitochondrial depolarization, and mitochondrial swelling, which were suspended in respiration buffer ( 32...
Brain mitochondria isolation
reagent used in the protocol.
- Use
- Cytochrome c oxidase activity was assessed by decreasing the absorbance of reduced cytochrome c. The cytochrome c oxidation reaction was carried out in 10 mM of KH2PO4 (pH 7.0) and was incubated (at 37 °C for 3 min) with about 10-20 µg of mitochondrial protein. Absorbance was measured at 550 nm befor...
Behavioral testing
The Shuttle box has been described previously. Briefly, it was a box containing two equal spaces lighted and darkened separated by a guillotine door (11 cm in diameter). This test has two steps, the training and retention session. In the training session, which was 24 h. before the retention session, the rat was in...
- Use
- The Shuttle box has been described previously. Briefly, it was a box containing two equal spaces lighted and darkened separated by a guillotine door (11 cm in diameter). This test has two steps, the training and retention session. In the training session, which was 24 h. before the retention session, the rat was in...
Step-down test
In animal models of neurological disorders such as Alzheimer's disease, the inhibitory avoidance step-down apparatus was used as an index of memory retention. The step-down passive avoidance test was conducted in a single-compartment cage with a plastic box (30 × 30 × 40 cm high) and a wooden platfor...
- Use
- In animal models of neurological disorders such as Alzheimer's disease, the inhibitory avoidance step-down apparatus was used as an index of memory retention. The step-down passive avoidance test was conducted in a single-compartment cage with a plastic box (30 × 30 × 40 cm high) and a wooden platfor...
Stereological assessment
The samples were inspected using an Eclipse microscope (E200, Nikon, Tokyo, Japan), featuring high-numerical-aperture (NA = 1.25) × 60 oil-immersion objectives. Images were sent to a computer and an electronic microcator featuring a digital display (MT12, Heidenhain, Traunreut, Germany) with the aid...
- Use
- The samples were inspected using an Eclipse microscope (E200, Nikon, Tokyo, Japan), featuring high-numerical-aperture (NA = 1.25) × 60 oil-immersion objectives. Images were sent to a computer and an electronic microcator featuring a digital display (MT12, Heidenhain, Traunreut, Germany) with the aid...
Statistical analysis
Graph Pad Prism 6 software (Graph Pad Software Inc., San Diego, CA, USA) was used for the statistical analysis. P < 0.05 was considered to be statistically significant. Data are presented as the Mean ± SD. Data comparison was performed by the one-way analysis of variance (Pellegrini et...
- Use
- Graph Pad Prism 6 software (Graph Pad Software Inc., San Diego, CA, USA) was used for the statistical analysis. P < 0.05 was considered to be statistically significant. Data are presented as the Mean ± SD. Data comparison was performed by the one-way analysis of variance (Pellegrini et...
Biochemical analysis of brain tissue
The level of reactive oxygen species (ROS) in the brain was quantified using the technique outlined by Gupta et al. with a few adjustments. In short, brain tissues were homogenized in a 1:10 dilution with ice-cold 40 mM Tris-HCl buffer (pH = 7.4). Mix 100 µL of tissue homogenate with 1 mL of Tris-HCl buffer and...
- Use
- The level of reactive oxygen species (ROS) in the brain was quantified using the technique outlined by Gupta et al. with a few adjustments. In short, brain tissues were homogenized in a 1:10 dilution with ice-cold 40 mM Tris-HCl buffer (pH = 7.4). Mix 100 µL of tissue homogenate with 1 mL of Tris-HCl buffer and...
Brain mitochondria isolation
Cytochrome c oxidase activity was assessed by decreasing the absorbance of reduced cytochrome c. The cytochrome c oxidation reaction was carried out in 10 mM of KH2PO4 (pH 7.0) and was incubated (at 37 °C for 3 min) with about 10-20 µg of mitochondrial protein. Absorbance was measured at 550 nm befor...
- Use
- Cytochrome c oxidase activity was assessed by decreasing the absorbance of reduced cytochrome c. The cytochrome c oxidation reaction was carried out in 10 mM of KH2PO4 (pH 7.0) and was incubated (at 37 °C for 3 min) with about 10-20 µg of mitochondrial protein. Absorbance was measured at 550 nm befor...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Graph Pad Prism 6 software (Graph Pad Software Inc., San Diego, CA, USA) was used for the statistical analysis. P < 0.05 was considered to be statistically significant. Data are presented as the Mean ± SD. Data comparison was performed by the one-way analysis of variance (Pellegrini et...
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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.
Measurement outputs
What raw and processed outputs should exist?
The Shuttle box has been described previously. Briefly, it was a box containing two equal spaces lighted and darkened separated by a guillotine door (11 cm in diameter). This t...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
In animal models of neurological disorders such as Alzheimer's disease, the inhibitory avoidance step-down apparatus was used as an index of memory retention. The step-dow...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Thiobarbituric acid reactive substances (TBARS) were measured in brain tissues to determine the level of lipid peroxidation. The mixture contained 0.375% thiobarbituric acid (w/...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Graph Pad Prism 6 software (Graph Pad Software Inc., San Diego, CA, USA) was used for the statistical analysis. P < 0.05 was considered to be statistically signific...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Graph Pad Prism 6 software (Graph Pad Software Inc., San Diego, CA, USA) was used for the statistical analysis.
from paperScoring or quantification
Quantify the primary readouts for this experiment: The Shuttle box has been described previously. Briefly, it was a box containing two equal spaces lighted and darkened separated by a guillotine door (11 cm in diameter). This t...; In animal models of neurological disorders such as Alzheimer's disease, the inhibitory avoidance step-down apparatus was used as an index of memory retention. The step-dow...; Thiobarbituric acid reactive substances (TBARS) were measured in brain tissues to determine the level of lipid peroxidation. The mixture contained 0.375% thiobarbituric acid (w/...; Graph Pad Prism 6 software (Graph Pad Software Inc., San Diego, CA, USA) was used for the statistical analysis. P < 0.05 was considered to be statistically signific....
from paperStatistical comparison
Graph Pad Prism 6 software (Graph Pad Software Inc., San Diego, CA, USA) was used for the statistical analysis. P < 0.05 was considered to be statistically signific...; Fig. 4 Effects of ellagic acid (EA) on oxidative stress index; Brain ROS formation level ( A ), Lipid peroxidation level ( B ), Frap antioxidant capacity ( C ). Data are express...; Fig. 5 Effects of ellagic acid (EA) on mitochondrial function. Mitochondrial viability ( A ), mitochondrial ROS formation ( B ), mitochondrial membrane potential ( C ), mitochon...
from paperReporting output
Report representative outputs alongside summary comparisons for The Shuttle box has been described previously. Briefly, it was a box containing two equal spaces lighted and darkened separated by a guillotine door (11 cm in diameter). This t..., In animal models of neurological disorders such as Alzheimer's disease, the inhibitory avoidance step-down apparatus was used as an index of memory retention. The step-dow..., Thiobarbituric acid reactive substances (TBARS) were measured in brain tissues to determine the level of lipid peroxidation. The mixture contained 0.375% thiobarbituric acid (w/..., Graph Pad Prism 6 software (Graph Pad Software Inc., San Diego, CA, USA) was used for the statistical analysis. P < 0.05 was considered to be statistically signific....
inferred from protocolStructured statistical methods
Graph Pad Prism 6 software (Graph Pad Software Inc., San Diego, CA, USA) was used for the statistical analysis. P < 0.05 was considered to be statistically signific...; Fig. 4 Effects of ellagic acid (EA) on oxidative stress index; Brain ROS formation level ( A ), Lipid peroxidation level ( B ), Frap antioxidant capacity ( C ). Data are express...; Fig. 5 Effects of ellagic acid (EA) on mitochondrial function. Mitochondrial viability ( A ), mitochondrial ROS formation ( B ), mitochondrial membrane potential ( C ), mitochon...
source structuredSource and audit
What supports the facts on this page?
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.
Machine-readable layer
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