In vitro studies
Separation and isolation of stem cells
Healthy wisdom teeth in Ali Asghar and Behesht Hospitals were transferred to the cell culture laboratory of the University by soaked facial tissues. In the laboratory, the teeth were first disinfected in 70% ethanol and then washed with sterile distilled water. Then, by cutting in between the root and crown, the pulp was removed by a nerve puller 15, underneath the hood within a sterile PBS solution, followed by dividing into smaller pieces with a surgical blade. These components were placed in a 3 mg/ml collagenase solution at 60 ° C for 60 minutes and tried to isolate the cells with an insulin syringe.
By adding about 3 ml of α-MEM solution with 20% fetal Bovine Serum, the mixture was centrifuged and the resulting precipitate was placed in a sterile PBS solution, followed by re-centrifugation. By separating the precipitate, it was pipetted and then placed in an α-MEM containing 20% fetal Bovine Serum and Pen/strep 1%. Furthermore, they were placed in a filter flask and then stored in a humidified incubator at 37 ° C with 5% CO2. On average, 1: 3 passages were performed every 5 days with EDTA- trypsin solution
The Nature Of The Cells
To verify the identity of the cells in the third passage, flow cytometry was performed for measuring the positive markers of mesenchymal cells including CD90, CD105, CD73, and CD44 as well as negative markers: hematopoietic markers (CD33 and CD31).
Stem Cell Labeling
A total of 106 cells were floated in a serum-free medium and 5µg /ml of DiI was then added to their medium and slowly pipetted. After that, the medium containing the cells and the DiI was incubated for 15 minutes. To prevent additional cytoplasmic color, 5 minutes were placed in the refrigerator. The suspension was centrifuged for 5 minutes at 1500 rpm, and the liquid was removed from the precipitate, followed by suspension by PBS. In the final stages, the procedure was followed by washing twice using PBS.
In Vivo Studies
Experimental animals
In this study, 60 NMRY mice with a weight of 30–40 g were used. They were randomly selected from the animal house of Iran University of Medical Sciences. All animals were kept in standard condition (12 hours of light and 12 hours of darkness, 24 ± 1 ° C) and had enough water and food before and after ischemia. Ethic committee of Iran University of Medical Sciences approved the experiments and all experiments were performed in accordance with relevant guidelines and regulations following the recommendations in the ARRIVE guidelines.
The Ischemia Reperfusion Injury Model And Animal Grouping
The mice were intra-peritoneal anesthetized with a combination of ketamine (70 mg /kg) and xylazine (3–5 mg/kg). After fixing the hands and feet of the animal, a 2.5-cm vertical incision in the neck midline was made. Common carotid artery was carefully removed from the vagus nerve and the internal jugular vein and the arteries on both sides were blocked by using special micro-bulldog clamps for 20 minutes. Afterward, the clamps were opened in order to re-circulate the bloodstream (reperfusion).
The sham group was surgically treated but did not induce ischemia
Transplantation Of Dps Cells Into Hippocampus
After adjusting the Hamilton syringe on the Bregma point, the coordinates of points ( -2.3 mm, ML: ± 1.3 mm, DV: +1.5 mm) were adjusted using the Paxinous and Watson Atlas for the CA1 site of the Hippocampus. A total of 1 × 105 DPS cells in a 4 µl volume of PBS were delivered at each of the target coordinates. The Injection speed was 0.25 µlit/min.
Mice successfully modeled were randomly assigned (Sarveazad, Babahajian et al. 2017) in equal numbers to five groups (n = 10 per group) as follows:
-
Ischemia-reperfusion group (I/R), in which animals underwent surgery and common carotid arteries were temporarily closed to induce ischemia.
-
Vehicle group: After induction of ischemia, they received the cell carrier material
-
EPO group, in which, animals received intraperitoneal erythropoietin (1,000 IU/kg) 24 hours after induction of ischemia, repeated one day later.
-
Stem cell group, in which, dental pulp stem cells were injected into the hippocampus of animals, 4 days after induction of ischemia.
-
EPO + Stem cell group, in which, animals received intraperitoneal erythropoietin (1,000 IU/kg) 24 hours after induction of ischemia and was repeated one day later followed by dental pulp stem cell injection into the hippocampus 4 days after induction of ischemia.
Spatial Memory
Eight weeks after the creation of an ischemia-reperfusion model, MWM was used to examine the spatial memory. This device is designed for assessing spatial and related forms of learning and includes a black cylinder with a diameter of 130 cm and a height of 60 cm, filled to a height of 27 cm with a temperature of 22 ± 2 ° C. The rescue platform is a 15 cm diameter, 1 cm below the water and in the northwest quarter. The MWM was in a room that was surrounded by outsider’s signs such as watches, posters, shelves, lights, tables, etc.
At the top of the pond, there was a camcorder that monitored the animal with infrared light while it was swimming. At first, animals were trained for 5 consecutive days, each day, the animal was thrown into the water from four different tank areas, each with a 60-second time, allowing the animal 60 seconds to find the submerged escape platform If the platform was not found, the animal was driven onto the submerged escape platform, and after 15 seconds of rest, it was thrown from the other area to the water. Swimming time to find a hidden platform in the learning phase was an important factor in the training days. At first, the average swimming time of animals was calculated for each target group, and each training day.
After training, experiments were performed and films from the motion of the rat were displayed through a computer monitor and a special program called Ethovision (XT version 5). The assessment included measuring the speed of the rat, the distance spent in the target quadrant, and the percentage of time spent on the platform.
Nissl Staining
The number of dark and light cells was detected by histopathology and Nissl staining .Perfusion was performed for all groups of animals. After perfusion of the animal, the brain was placed in a postfix solution, preferably formalin 10%, for one week. Then, the intermediate part of the brain was subjected to tissue processing. To prepare the tissue for embedding, it was first washed with fresh water for 15 minutes and then samples were added to the processor for removing water by dehydration with a series of alcohols, 70–95% to 100%, followed by clearing and embedding with xylene and paraffin. The coronal section of samples was provided using a rotary microtome (Leica, RM2235, and Germany) with a thickness of 5 µm and placed on gelatin slides. Finally, the specimens were placed in 0.1% Cresyl fast violet acetate or Nissl Stain solution to examine the dark and light cells, and they were glued onto the slides using Entellan mounting medium. Then, the nucleus of the pyramidal neurons was counted in the form of internal-external expansion in the CA1 region, and for each section, at least four fields with a minimum of 40 microns spacing were considered. The stained slides were examined by the OLYMPUS microscope, (AX70 70) and magnification of x40. Then cell counting was done by Image J software. The pyramidal neurons in these specimens were selected at intervals of 1.7, 1.8, 0.9, and 1.2 mm according to Paxinos atlas.
Immunohistochemistry (Dup: Abstract ?)
after applying ischemia to I/R and vehicle groups, the animals were anesthetized by intraperitoneal injection of 90 units of ketamine and xylazine (1/10). After perfusion, the brain was removed and placed in a 10% formaldehyde solution for 48 hours. After performing the steps, including dehydration, clearing, and infiltration, 5 micrometers were cut from the CA1 region and placed on 0.1% gelatin slides.
In the next step, the primary antibody (abcam18723) DCX, NeuN (abcam177487) was diluted 1: 500 with PBS and placed on tissue samples. To avoid drying the surface of the tissue, a moist chamber was used. The slices were then placed at 4 ° C for 24 hours and then exposed to secondary antibody after washing.
The DAPI was used to stain the cells for 15 minutes (1 µ/ml: 1000 µ/ml PBS), which usually brings the nuclei in blue.
Statistical analysis
All information was expressed as Mean±SE. One-way, two-way ANOVA was used for statistical analysis and behavioral tests. Furthermore, One-way ANOVA was used to analyze the number of CA1 neurons. In all calculations, P < 0.05 was considered a significant difference.