Part IV Study On The Model Of 5-Fu/5-Fu+L-OHP-induced Bone Marrow Suppression in Mice
Oct 09, 2024
Currently, the commonly used methods for constructing animal models of bone marrow suppression include radiation modeling [1,2], cyclophosphamide intraperitoneal injection modeling [3-5], 5-fluorouracil (5-Fluorouracil, 5-FU) intraperitoneal injection modeling [6,7], radiation combined with chemotherapy drug compound modeling [8,9], anti-epileptic drug carbamazepine gavage modeling [10], etc. Different modeling methods cause different mechanisms of bone marrow suppression, and the effects on various blood cells and their degree are different; different chemotherapy drugs, due to their different mechanisms of action, also show certain differences in their effects on bone marrow hematopoiesis. Therefore, an ideal animal model of bone marrow suppression is of great significance for studying the effects of chemotherapy drugs on bone marrow hematopoiesis.
In clinical practice, adjuvant chemotherapy for colorectal cancer mainly adopts 5-FU regimen and 5-Fu combined with oxaliplatin (oxaliplatin, L-OHP) regimen. There have been relevant studies on the use of 5-Fu alone to construct an animal model of bone marrow suppression. However, the scope of 5-Fu use is relatively wide. Literature reports include 110 mg/kg, intraperitoneal injection (ip) on d1 and d5 for modeling [7, 150 mg/kg, ip, d1 for modeling 16, 11], 200 mg/kg, ip, d1 for modeling [12-15], and 250 mg/kg, ip, d1 for modeling [16]. However, there have been no reports on the use of 5-Fu combined with L-OHP to construct an animal model of bone marrow suppression. Yu Linghong et al. [17] used 5-Fu combined with L-OHP to establish an animal model of liver injury. Referring to the administration method and dosage of the literature, the team conducted a preliminary exploration of the dosage of 5-Fu combined with L-OHP to establish an animal model of bone marrow suppression, and compared the modeling method of 5-Fu 200 mg/kg, ip, d1 and the modeling method of 5-Fu 187.5 mg/kg + L-OHP 9 mg/kg, ip, d1. The results showed that the dosage of these two models was too large. Therefore, the goal of this study is to continue to explore the optimal dosage of 5-Fu-induced bone marrow suppression animal model and the dosage of 5-Fu combined with L-OHP modeling, so as to provide the most ideal animal model for the next step of studying the efficacy and mechanism of Qitu Erzhi prescription.
HERB CISTANCHE FOR KIDNEY HEALTH
Supportive Service Of Wecistanche
Email:wallence.suen@wecistanche.com
Whatsapp/Tel:+86 15292862950
Materials and methods
1 Experimental materials
1.1 Experimental animals
100 specific pathogen-free (SPF) BALB/c male mice, weighing 20g±2g, were purchased from SBEF (Beijing) Biotechnology Co., Ltd. and housed in cages at the Experimental Animal Center of Xiyuan Hospital, China Academy of Chinese Medical Sciences (certificate of qualification: SYXK (Beijing) No. 2015-0011). All mice were adaptively fed for 1 week, and had free access to water and food during the feeding period. Feeding conditions: indoor temperature 20-26℃, relative humidity 40%~60%, light/dark time 12 hrs/12 hrs. This experiment strictly complies with the relevant regulations of the Experimental Animal Management Ethics Committee of Xiyuan Hospital, China Academy of Chinese Medical Sciences and complies with animal ethics standards.
1.2 Main experimental drugs and reagents
5-Fu (fluorouracil injection) Oxaliplatin for injection
0.01M phosphate buffer (PBS buffer) Diluent for blood cell analysis (MEK-640)
Cleaning solution for blood cell analysis (MEK-680) Hemolytic agent for blood cell analysis (MEK-680) Cleaning solution for blood cell analyzer
Harris hematoxylin stain 0.5% eosin stain
Neutral gum sealing medium
Mineral acid type tissue specimen decalcification solution
1.3 Main instruments and equipment
Automatic blood cell counter (MEK6318) Capillary blood collection tube (20 μL)
1000μL pipette tip
Tissue-TEK VIP6 biological tissue dehydrator Tissue-TEK TEC biological tissue embedding machine Leica RM2235 tissue slicer
Leica HI1210 slice picker Leica HI1220 slice baking machine
Leica ST5020 automatic stainer Leica CV5030 automatic coverslipper Olympus Bx53 microscope
Path QC pathology quality control and data management system
Others: pipette, ordinary electronic scale, surgical scissors, tweezers, 75% alcohol, medical gauze, sterile cotton balls, syringes, etc.
1.4 Preparation of chemotherapy drugs
(1) Single-drug 5-Fu regimen
Dissolve 1 5-Fu (0.25 g/10 ml) in 40 ml 0.9% NaCl solution to prepare a solution with a concentration of 5 mg/ml for standby use.
(2) 5-Fu combined with L-OHP regimen
Dissolve 1 5-Fu (0.25 g/10 ml) in 40 ml 0.9% NaCl solution to prepare a solution with a concentration of 5 mg/ml for standby use. Dissolve 1 tube of L-OHP (50 mg) in 5 ml of 5% glucose solution, and continue to dilute with 5% glucose solution to a 0.5 mg/ml solution for use.
Table 4-1 Modeling medication regimen
According to the above dosing regimen, all mice were weighed and dosed individually according to body weight. In the dual-drug group, the time interval between 5-Fu and L-OHP administration was more than 5 hours [17].
(2) Evaluation indicators of bone marrow suppression model
The evaluation indicators of bone marrow suppression model include decreased peripheral blood cell count [18-22], or peripheral blood cell count combined with bone marrow nucleated cells [9.23-25]. This study used the second method to evaluate the effect of modeling.
2.2 Observation indicators and materials
2.2.1 General condition of mice
During the experiment, the diet, defecation, activity, condition, mental state and hair condition of each group of mice were observed every day. If mice died, they were recorded in time.
2.2.2 Changes in weight of mice in each group
Weigh and record the weight of each mouse before dosing on the day of modeling (d0), and weigh and record the weight of each mouse every other day after modeling.
2.2.3 Changes in peripheral blood cell counts
Fix the mouse with a mouse fixer, expose the tail, and soak the tail in 45-50℃ hot water. After drying the tail, cut off 1-2 mm of the tail tip. Quickly and gently stroke the tail from the base to the tip 2-3 times. Use a capillary blood collection tube to collect 20μL of blood, dilute it in the prepared blood cell diluent, and perform blood cell analysis on the machine. Collect blood at a fixed time every day; all blood collection is completed by one person. The detection indicators include: white blood cells, red blood cells, hemoglobin and platelets.
2.2.4 Changes in bone marrow nucleated cell counts
One day before sampling, use a micropipette to take 1000 μL of PBS solution and dilute it to a 2 ml EP tube, and store it in a 4℃ refrigerator for later use. On d6, 8 mice were randomly selected from each group and killed by cervical dislocation. Under a sterile clean bench, surgical scissors were used to cut the fur near the femur, remove the right femur, and wrap it with medical gauze to remove the residual soft tissue. Use a 1 ml syringe needle (No. 4 needle) to extract the PBS solution in the EP tube, drill into one end of the femur to flush the bone marrow into the EP tube; repeatedly flush and aspirate several times to flush out the bone marrow cells until the femur turns white. The prepared bone marrow cell suspension was passed through a 200-mesh nylon net. Use an automatic blood cell counter to detect bone marrow nucleated cells. On d13, the remaining mice were killed in the same way and specimens were collected.
2.2.5 Bone marrow pathology HE staining
On d6, after the mice in each group were killed by cervical dislocation, surgical scissors were used to cut the fur near the femur under a sterile clean bench, remove the left femur, make wax blocks, slices and HE staining.
(1) Wax block preparation and sectioning steps
① Sampling: Fix tissue with 4% paraformaldehyde for more than 24 hours, soak in decalcification solution for 24-48 hours, prick the tissue with a pin, cut and trim the tissue after it softens, and put it into the wax block box;
② Dehydration: The tissue is sequentially placed in 70% alcohol (2 hours), 80% alcohol (1.5 hours), 95% alcohol I (1.5 hours), 95% alcohol II (1.5 hours), 100% alcohol I (0.5 hours), and 100% alcohol II (0.5 hours), and dehydrated in a gradient manner.
③ Transparency: The tissue is sequentially placed in anhydrous ethanol and xylene solution (volume ratio 1:1), xylene I, and xylene II for 40 minutes each.
④ Wax immersion: The tissue is sequentially placed in a mixture of xylene and paraffin (1:1), paraffin I, and paraffin II for 20 minutes each. Wax dipping is carried out in a constant temperature device at 58℃ to keep the paraffin melted.
⑤ Embedding: After labeling the tissue block, pre-warm the tweezers, inject the melted paraffin into the mold, and then put the tissue block in and wait for the paraffin to solidify.
⑥ Slicing: After embedding, place the tissue wax block on the slicer, adjust the angle of the slicer, and cut the tissue block into slices with a thickness of 4-5 μm.
⑦ Sticking: Spread the paraffin slices in 42℃ water, pick up the slices one by one with a slide, and stick them on the slide.
⑧ Baking: Place the stuck slide in a constant temperature box at about 70℃ and bake for 1.5 h.
(2) HE staining steps:
① Dewax by soaking in xylene I and II for 15 min each.
② Soak in 100% alcohol I and II for 5 min.
③ Soak in 95%, 85%, and 75% gradient alcohol for 3 min, and rinse with distilled water.
④ Hematoxylin staining for 1-3 min, and rinse with distilled water.
⑤ Hydrochloric acid alcohol differentiation for 2-3 s, wash with distilled water.
⑥ Immerse in ammonia water to turn blue for 1 min, wash with distilled water.
⑦ Eosin staining for 5 min.
⑧ Immerse in 75%, 85%, 95% gradient alcohol for 2 min.
⑨ Immerse in 100% alcohol I, IⅡ for 3 min each.
⑩ Immerse in xylene I, Ⅱ for 5 min each.
⑪ Seal with neutral gum.
2.3 Statistical methods
SPSS26.0 was used to perform statistical analysis on the data. Measurement data were expressed as mean ± standard deviation (x±s). First, the data were tested for normality and homogeneity of variance: For normally distributed data, one-way ANOVA was used for comparison between multiple groups: when the variance was equal, the LSD test was used for pairwise comparison; if the variance was not equal, the Tamhane's t2 test was used for pairwise comparison; if it did not conform to the normal distribution, a non-parametric test was used. P<0.05 was considered statistically significant.
