All experiments were performed in accordance with the guidelines of the National Institutes of Health, and all animal studies were approved by the University of South Florida (USF) Institutional Animal Care and Use Committee. Animals were humanely cared for during all experiments, and all efforts were made to minimize animal suffering. Animals were anesthetized with isoflourane (50 mg/kg) (Sigma-Aldrich, St. Louis, MO, USA) and euthanized by transcardial perfusion with ice-cold physiological saline containing heparin (10 units/ml) (Sigma-Aldrich). In this study, use of human cord blood cells was involved. Ninety-five to 98% of mononuclear cells from HUCBCs were provided by Saneron CCEL Therapeutics Inc. (Tampa, FL, USA). Saneron used de-identified HUCBC donations from certified commercial sources.
Physiological saline
Manufactured by Merck Group
Sourced in United States, United Kingdom
Physiological saline is a sterile, isotonic solution of sodium chloride in purified water. It is used as a standard diluent and rinsing agent in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
6 ohda, by Merck Group (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Heparin, by Merck Group (1 mentions)
Isoflourane, by Merck Group (1 mentions)
Ab1893, by Abcam (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Apomorphine, by Merck Group (1 mentions)
Stereotaxic apparatus, by Narishige (1 mentions)
Palmitate, by Merck Group (1 mentions)
Clonidine hydrochloride, by Merck Group (1 mentions)
Buprenorphine hydrochloride, by Merck Group (1 mentions)
Methadone hydrochloride, by Merck Group (1 mentions)
Stereotactic apparatus, by Narishige (1 mentions)
Polyvinyl alcohol (pva), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Allyl isothiocyanate, by Nacalai Tesque (1 mentions)
Mineral oil, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
25 protocols using physiological saline
1
Humanely Derived Cord Blood Cell Protocol
2
BrdU Labeling and Immunofluorescence Staining
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Animals were administered with BrdU (10 mg/mL in physiological saline; Sigma-Aldrich) twice per day by intraperitoneal injection at a dose of 350 mg per kilogram body weight, 3 days before sacrifice. After ether anesthesia, animals were transcardially perfused with 50 ml normal saline and 50 mL 4% PFA. The brains were fixed in 4% PFA overnight at 4°C, then cryoprotected in 30% sucrose and embedded in embedding medium (Tissue-Tek; Sakura Finetek, Torrance, CA). Transverse sections were cut using a cryostat (10 µm) (Leica, Wetzlar, Germany). For immunofluorescence staining, sections were re-hydrated (three PBS washes and 2 mol/L HCl for 1 h at RT). After four PBS washes, sections were treated with blocking solution (PBST and 10% goat serum) for 1 h at RT, then incubated in sheep-anti-BrdU antibody (ab1893, Abcam, Cambridge, MA) solution (1:200 in blocking solution) at 4°C overnight. After three PBS washes, sections were incubated in Alexa Fluor 488 donkey-anti-sheep antibody (1:300, Invitrogen/Molecular probe) for 2 h at RT. Following three PBST washes, the nuclei were stained with Hoechst (10 µg/mL) at RT for 20 min. The sections were mounted with GVA mounting medium. The number of BrdU-positive cells was determined from five consecutive sections (Wojtowicz and Kee 2006 (link)).
3
Cisplatin-Induced Nephrotoxicity in Rats
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The Wistar rats (average weight 260 g) in this study were obtained from the Laboratory Animal Centre of South Medical University (Guangzhou, China). The rats were housed in an alternate light–dark cycle (every 12 h) room with a temperature of 22±2 °C and a relative humidity of 50–60%. In addition, the rats were fed with a complete formula food and allowed water ad libitum. Eight-week-old female rats were used for this study after 10 days of adaption. Twenty-eight rats were randomly divided into four groups (seven rats for each group) to implement this experiment and were injected with either physiological saline (control) or cisplatin (Sigma, St. Louis, MO, USA) dissolved in 0.01 M citrate buffer at a pH of 4.5 and a dose of 10 mg/kg body weight for 1, 3 or 7 consecutive days (day 1, 3 or 7) before killing the animal. All processes involving animal treatment in this study were in accordance with the procedures of the Ethical Committee for Animal Experimentation, Jinan University.
4
Establishing Parkinson's Disease Rat Model
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Rats were anesthetized with 7% chloral hydrate (0.5 mL/100 g, v/w), and then received an injection of a total of 8 µg of 6-OHDA (dissolved in 4 µL of 0.9% physiological saline containing 0.02% ascorbic acid [Sigma–Aldrich, St Louis, MO, USA]) using a stereotaxic apparatus (Narishige, Tokyo, Japan) equipped with a rat adaptor. The coordinates were calculated with reference to bregma for the anterioposterior (AP) and the mediolateral (ML) coordinates using the rat brain atlas20 as follows: 1) AP −3.7 mm, ML −1.7 mm; 2) AP −4.4 mm, ML −1.2 mm. The dorsoventral position of all the injections was −7.8 mm below the dura and the tooth bar was set to −2.4 mm. Three weeks after surgery, rats were tested with a subcutaneous injection of apomorphine at 0.05 mg/kg (Sigma–Aldrich, St Louis, MO, USA). Only those rats displaying a stable apomorphine-induced rotational behavior of at least seven full turns per minute contralateral to the lesioned side were selected for the next experiment. It has been previously demonstrated that rats meeting this criterion have a greater than 90% depletion of striatal DA.21 (link)
5
Impacts of Western Diet on Vascular Function
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To determine the effects of acute exposure to a more physiologically relevant stressor, we exposed a sub-set of arteries (n = 7 (YC), 8 (OC), 4 (YVR), 8 (OVR)) to an ex-vivo, simulated Western diet via intraluminal infusion for 40 minutes prior to pre-constriction followed by assessment of EDD to ACh. This ex-vivo challenge comprised warmed physiological saline containing 8mM glucose (in addition to 5mM glucose already present in physiological saline, Sigma Aldrich Corp.) and 300 μM palmitate (Sigma Aldrich Corp.), two of the major metabolites present upon consumption of a Western-style diet high in saturated fat and sugar. These concentrations were selected to simulate those reported in the circulation of rodents following chronic consumption of Western-style diets [43 (link), 78 (link)–80 (link)]. The impairments in peak EDD and AUC induced by this ex-vivo simulated Western diet were determined as the relative reduction in peak EDD or AUC in the presence versus absence of ex-vivo simulated Western diet ([PeakEDDACh-PeakEDDWD/PeakEDDACh]x100; [AUCACh-AUCWD/AUCACh]x100).
6
Thermal Resistance and Weight Loss of SWCNT/Collagen Coating
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The resistance and weight loss of the coating were studied by the method used to test artificial ligaments from PET [94 (link),95 (link)]. Thermal cycling of samples in physiological saline (Sigma-Aldrich, St. Louis, MO, USA) and blood serum was carried out. Since blood serum had a more aggressive effect on the coating, we will discuss it further. Before the start of the study, each of the 10 samples (a fragment of the PET tape coated with SWCNT/collagen) was weighed three times and placed in a vessel with blood serum. The vessel was placed in the environmental chamber (SH-641, Espec, Hudsonville, MI, USA). The serum was cooled and heated with a sample from 5 ± 1 to 55 ± 1 °C. Serum temperature was measured using an immersion temperature sensor. One cycle lasted about 3 min. Samples were thermocycled for 5000 cycles. This corresponded to six months of usage in the organism [96 (link)]. We deliberately chose such serious loads so that it could be said with confidence that the coating is not completely resorbed in the long run. The study of the changes in the structure of the samples was carried out using optical and electron microscopy. The relative weight loss of the samples after completion of thermal cycling was also evaluated. The relative mass loss was calculated as the percentage of the mass difference before and after thermal cycling to the mass before thermal cycling.
7
Synthesis and Characterization of Mitragynine
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Methadone hydrochloride, buprenorphine hydrochloride and clonidine hydrochloride were purchased from Sigma Chemicals Co. (United States). Mitragynine was extracted, isolated and verified from fresh leaves of Mitragyna speciosa at the Centre for Drug Research, Universiti Sains Malaysia as described previously (Utar et al., 2011 (link)). Purified mitragynine was confirmed by high performance liquid chromatography (HPLC) and proton nuclear magnetic resonance (1H-NMR) (400 MHz) analysis (Jamil et al., 2013 (link)). Mitragynine obtained by this procedure was approximately 98% pure (Hassan et al., 2019 (link)). Fresh stocks of methadone, buprenorphine, mitragynine and clonidine were prepared daily according to the weight of animals in the experimental design. They were dissolved in vehicle (20% (v/v) Tween 80 which was diluted with physiological saline (0.9% NaCl); Sigma Aldrich, United Kingdom) and injected intraperitoneally (i.p.).
8
Rat 6-OHDA Lesion Model Protocol
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For creation of the 6-OHDA lesion, the rat was anesthetized with an intraperitoneal (IP) injection of pentobarbital (50 mg/kg). The animal’s head was fixed in a stereotactic apparatus (Narishige, Tokyo, Japan). All animals received injections of a total of 8 μg of 6-OHDA (dissolved in 4 μl of 0.9% physiological saline containing 0.02% ascorbic acid [Sigma-Aldrich Co., St Louis, MO, USA]). The coordinates were calculated with reference to bregma for the anterioposterior and the mediolateral coordinates using the rat brain atlas16 as follows: 1) anterioposterior – 3.7, mediolateral – 1.7; 2) anterioposterior – 4.4, mediolateral – 1.2. The dorsoventral position of all injections was −7.8 mm below the dura and the tooth bar set to −2.4 mm. Three weeks after injections, the rats that exhibited a stable apomorphine-induced rotational asymmetry of at least seven full turns per minute away from the lesioned side were selected for the next experiment. It has been previously demonstrated that rats meeting this criterion have a greater than 90% depletion of striatal dopamine.17 (link)
9
Unilateral 6-OHDA Lesion Model Protocol
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Unilateral 6-OHDA lesions were performed according to our standard procedure16 (link). Briefly, 6-OHDA (2 × 16 μg dissolved in 8 μL of 0.9% physiological saline containing 0.2% ascorbic acid; Sigma-Aldrich, St Louis, MO, USA) was stereotaxically injected into the right medial forebrain bundle (MFB) of rats. The coordinates of the right medial forebrain bundle were calculated using the rat brain atlas as follows (in mm relative to the bregma and the dural surface): 1) anterior-posterior (AP), −4.4 mm, medial-lateral (ML), −1.2 mm, dorsal-ventral (DV), −7.8 mm; 2) AP, −3.7 mm, ML, −1.7, DV, −7.8. The tooth bar was set to −2.4 mm. Animals were anesthetized with ketamine (1–2 ml/kg). For the sham-operated rats, two intrastriatal injections of physiological saline containing 0.2% ascorbic acid were given at the same coordinates. Three week post lesion, the lesioned rats were screened behaviorally using an amphetamine-induced (0.5 mg/kg, i.p.) rotation test and all animals exhibited >7 full body turns/min toward the side of the lesioned side were selected for the next experiment.
10
Targeted Compound Delivery in Rodents
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BRD6688 and BRD4884 were dissolved in DMSO (5% of the total resultant solution) and then diluted in 30% Cremophor/65% in physiological saline (H2O containing 0.9% NaCl (Sigma)), for a final dosage solution of 1 mg kg–1 and 10 mg kg–1, respectively. Vehicle solutions consisted of the forementioned solution without the compounds. Solutions were prepared immediately before injection and administered daily via intraperitoneal injection for a period of 10 days prior to behaviour.
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