High-mobility Group Protein 1/ Receptor for Advanced Glycation End Products/ Nuclear Factor-κB Signalling Pathway Contributes to the Pathogenic Process of Striatal Neuron Impairment in the Rat Model of Parkinson's Disease methods
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Methods
Sprague-Dawley (SD) rats (8 weeks, 200-250 g) were obtained from the Sun Yat-sen University Center for Experimental Animals. The ethics committee of Jinan University approved all procedures for the care and handling of the rats [NO.20220313-12]. The study adhered closely to the Guide for the Care and Use of Laboratory Animals (the US National Institutes of Health guide for the care and use of laboratory animals), was closely followed during the conduct of this experiment. 19 SD rats (5 per cage) had unrestricted access to food and water, as well as a temperature of 22 ± 0.5°C, with a relative humidity of 40%-70%, and weresubjected to a 12-h light/dark cycle. The cages were cleaned and replaced weekly. All surgical procedures utilized sodium pentobarbital (50 mg/kg, i.p.) as an anesthetic to minimize suffering.
Methods
Four groups of 50 experimental rats were randomly assigned: (1) the control group ( n = 10), without any treatment; (2) the PD model group ( n = 10), which received an injection of 6-OHDA solution into the MFB; (3) the RAGE inhibitor FPS-ZM1 treatment group ( n = 10; 6-OHDA+FPS-ZM1); and (4) the sham group ( n = 20), which underwent the same procedures as the other groups but received 0.9% saline solution instead of 6-OHDA or FPS-ZM1. As a high-affinity, effective, multimodal blocker of RAGE V domain-mediated ligand binding (Ki = 25, 148, and 230 nM, respectively, against A40, HMGB1, and S100B, binding to sRAGE), FPS-ZM1 (cat. no. 553030, Sigma) is a non-toxic, blood-brain-barrier-permeant tertiary amide substance used in diverse fields of research, including diabetes, neuroinflammation, atrial fib...
PD Model
The 6-OHDA-induced DA depletion method was used to establish a PD model. (Deumens et al. ) 6-OHDA (catalog no. H116; Sigma) was dissolved in 0.9% saline to achieve a concentration of 2 µg/µL, with ascorbic acid added to reach a concentration of 0.01% to prevent oxidative degradation of 6-OHDA. Each rat was injected with 8 µL of this solution into the right middle forebrain bundle (MFB). The injection locations were as follows: medial-lateral (ML): -0.19 mm, anterior-posterior (AP): -3.6 mm, and dorsal-ventral (DV): -8.2 mm. Five-minute intervals were used to provide two 4 µL intermittent injections and after being in place for an additional 15 min, the needle was gradually withdrawn. Apomorphine (0.25 mg/kg, APO; catalog no. 2073/50, Tocris) was administered subcuta...
Behavioural Tests
We conducted the GST following the established procedures, 4 weeks post-surgery. (Shear et al. ) First, we suspended a wire with a diameter of 2 mm and a length of 35 cm, positioned 50 cm above the ground. We then timed the duration for which the rats hung onto the wire to evaluate their grip strength. The test was carried out three times a day for a total of 5 days of testing. All animals took part in these tests ( n = 10 per group). The experimental conditions for the rats were kept concealed from the observers.
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Sprague-Dawley (SD) rats (8 weeks, 200-250 g) were obtained from the Sun Yat-sen University Center for Experimental Animals. The ethics committee of Jinan University approved all procedures for the care and handling of the rats [NO.20220313-12]. The study adhered closely to the Guide for the Care and Use of Laboratory Animals (the US National Institutes of Health guide for the care and use of laboratory animals), was closely followed during the conduct of this experiment. 19 SD rats (5 per cage) had unrestricted access to food and water, as well as a temperature of 22 ± 0.5°C, with a relative humidity of 40%-70%, and weresubjected to a 12-h light/dark cycle. The cages were cleaned and replaced weekly. All surgical procedures utilized sodium pentobarbital (50 mg/kg, i.p.) as an anesthetic to minimize suffering.
Four groups of 50 experimental rats were randomly assigned: (1) the control group ( n = 10), without any treatment; (2) the PD model group ( n = 10), which received an injection of 6-OHDA solution into the MFB; (3) the RAGE inhibitor FPS-ZM1 treatment group ( n = 10; 6-OHDA+FPS-ZM1); and (4) the sham group ( n = 20), which underwent the same procedures as the other groups but received 0.9% saline solution instead of 6-OHDA or FPS-ZM1. As a high-affinity, effective, multimodal blocker of RAGE V domain-mediated ligand binding (Ki = 25, 148, and 230 nM, respectively, against A40, HMGB1, and S100B, binding to sRAGE), FPS-ZM1 (cat. no. 553030, Sigma) is a non-toxic, blood-brain-barrier-permeant tertiary amide substance used in diverse fields of research, including diabetes, neuroinflammation, atrial fibrosis, etc. (Sun et al.; Tan et al.; FPS-ZM1 ) characterizing low toxicity and high selectivity, FPS-ZM1 have not effect on exercise performance, respiration, circulation, and metabolism; thus, based on overall consideration, we have not set up a group that only injects FPS&#...
The 6-OHDA-induced DA depletion method was used to establish a PD model. (Deumens et al. ) 6-OHDA (catalog no. H116; Sigma) was dissolved in 0.9% saline to achieve a concentration of 2 µg/µL, with ascorbic acid added to reach a concentration of 0.01% to prevent oxidative degradation of 6-OHDA. Each rat was injected with 8 µL of this solution into the right middle forebrain bundle (MFB). The injection locations were as follows: medial-lateral (ML): -0.19 mm, anterior-posterior (AP): -3.6 mm, and dorsal-ventral (DV): -8.2 mm. Five-minute intervals were used to provide two 4 µL intermittent injections and after being in place for an additional 15 min, the needle was gradually withdrawn. Apomorphine (0.25 mg/kg, APO; catalog no. 2073/50, Tocris) was administered subcutaneously to the rats over the three weeks following the development of 6-OHDA lesions. Then, we counted the number of contralateral 360° rotations within a 30-min period. For further analysis, only rats that completed more than 210 total rotations were included. (EPO ) Tyrosine hydro...
We conducted the GST following the established procedures, 4 weeks post-surgery. (Shear et al. ) First, we suspended a wire with a diameter of 2 mm and a length of 35 cm, positioned 50 cm above the ground. We then timed the duration for which the rats hung onto the wire to evaluate their grip strength. The test was carried out three times a day for a total of 5 days of testing. All animals took part in these tests ( n = 10 per group). The experimental conditions for the rats were kept concealed from the observers.
Machine-readable layer
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