Procedures were approved by the UAB IACUC. Global body-wide KL-deficient mice (KO; 129S1/SvImJ) and global body-wide KL-overexpressing mice (OE; C57BL/6J) lines were obtained from M. Kuro-o (University of Texas Southwestern, Dallas, TX), POMC-GFP(Overstreet et al. 2004 (link)) (C57BL/6J) from L. Overstreet-Wadiche (University of Alabama at Birmingham, Birmingham AL) and Nestin-GFP (Mignone et al. 2004 (link)) (C57BL/6J) from G. Enikolopov (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). WT (average weight: 3 week,11.6g; 7 week, 20.4g) and KO (average weight: 3 week,10g; 7 week, 8.1g) mice were generated by breeding heterozygotes (KL +/−, HET). KO mice die naturally at ~8 weeks of age and thus brains are harvested at or before 7 weeks as detailed. To ensure equivalent KL overexpression, mice carrying the KL overexpression cassette are bred to WT mouse from the same line. Mice were weaned on postnatal day 21. All mice were group housed and had free access to food and water at 26.6°C with humidity maintained above 40%. KO mice were supplemented with Bacon Softies or Nutra-gel (BioServ, Frenchtown, NJ). As mouse neurogenesis is not gender specific (Lagace et al. 2007 (link)), male and female mice were utilized. BrdU was intraperitoneal injected (50mg/kg) either 1x or 4x (2 hours apart) and brains collected after 30 minutes, 24 hours, 1, 2 or 3 weeks.
Bacon softies
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The Bacon Softies is a lab equipment product manufactured by Bio-Serv. It is used for handling and manipulating small samples or materials in a laboratory setting.
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12 protocols using bacon softies
1
Murine Neural Stem Cell Dynamics
2
Unilateral 6-OHDA Lesions in Rats
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Mitochondrial Antioxidant MitoQ Intervention
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MitoQ (#317102; Medkoo Bisociences) was dissolved in equal amount of ethanol: distilled sterile water to obtain a final concentration of 1 mM. Then, 100 µL of the above solution containing 68 µg of MitoQ was intraperitoneally injected into the animals on alternate days for 4 weeks at the same time each day. Control animals were injected with equal volumes of vehicle at the same time/day for the same duration. Animals were monitored for any changes in health or behavior during the experiment duration. All animals were provided with vet-approved supplements; peanut butter or Bacon Softies (Bio-Serv, Flemington, NJ, USA) for the duration of the treatment to prevent stress-induced weight loss due to increased handling. All analyses of corneal thickness following the MitoQ injections were blinded.
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Stereotactic Canula Implantation for Stress Studies
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Klotho Gene Modulation in Mice
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Kuro-o Mouse Models for KL Deficiency
7
Repeated Platinum Drug Exposure in Mice
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Mice underwent three rounds of platinum-containing drug administration separated by 10-day recovery periods (Roy et al. 2013 (link); Breglio et al. 2017 (link); Fernandez et al. 2019 (link); Fernandez et al. 2020 ) (Fig. 1b ). A cycle is defined as four consecutive days of once-daily platinum drug injections followed by 10 days of recovery. This cycle was repeated three times for a total of 42 days.
On each day of the protocol, mice received fluid and nutritional support as previously described in detail by Fernandez et al. (2019 (link)). Fluid support consisted of 1 mL of 0.9 % saline injected subcutaneously once per day and 1 mL of Normasol (Hospira, Inc., San Clemente, CA, USA) injected subcutaneously once per day, each separated by 6–8 h. Nutritional support consisted of 0.25 mL STAT high-calorie liquid supplement (PRN Pharmacal, Pensacola, FL, USA) delivered into the mouth twice per day. In addition, food pellets (Zeigler Bros, Garnders, PA, USA), bacon softies (Bio-Serv, Flemington, NJ, USA), and Diet Gel Recovery cups (Clear H20, Portland, ME, USA) were placed on each cage floor.
On each day of the protocol, mice received fluid and nutritional support as previously described in detail by Fernandez et al. (2019 (link)). Fluid support consisted of 1 mL of 0.9 % saline injected subcutaneously once per day and 1 mL of Normasol (Hospira, Inc., San Clemente, CA, USA) injected subcutaneously once per day, each separated by 6–8 h. Nutritional support consisted of 0.25 mL STAT high-calorie liquid supplement (PRN Pharmacal, Pensacola, FL, USA) delivered into the mouth twice per day. In addition, food pellets (Zeigler Bros, Garnders, PA, USA), bacon softies (Bio-Serv, Flemington, NJ, USA), and Diet Gel Recovery cups (Clear H20, Portland, ME, USA) were placed on each cage floor.
8
6-OHDA Stereotactic Parkinson's Model
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Stereotactic surgeries were conducted under isoflurane anesthesia (4% induction, 2% maintenance) following the description of Thiele et al. [43 (link)]. In brief, 6-OHDA (3 µg in 0.2 µl of 0.02% ascorbate-saline) was injected into the right MFB at the following coordinates relative to the bregma or the dural surface: A/P − 1.2 mm; L/M − 1.1 mm; D/V − 5.0 mm. Lidocaine, buprenorphine (0.1 mg/kg, s.c. once before surgery), and carprofen (5 mg/kg, s.c. once after surgery) were used for pain relief. Intensive postoperative care included warm saline injections to prevent dehydration, alleviating hypothermia with heating pads, food pellets softened by soaking in water, high-energy palatable diet (Bacon Softies, Bio-Serv, Flemington, NJ, USA; Nutriplus gel, Virbac, Carros, France), and feeding by hand. One animal out of 15 was euthanized due to a poor postoperative condition (postoperative mortality 7%).
9
Cisplatin-Induced Hearing Loss in Mice
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ABR tests were performed in 2–3 months old mice 1 week before the first cisplatin injection. Following the ABR tests, mice received 2–3 ml saline (Teknova, Fishersci) 1 day before the first cisplatin injection. cisplatin (100 mg/ml; APP pharmaceuticals) was administered i.p. at 3 mg/kg each day for 4 days using a modified cisplatin protocol described by Lisa Cunningham’s group19 (link),23 (link). Following this 4-day cisplatin injection period, mice recovered for 10 days. Mice received 2–3 ml saline daily during the 4-day cisplatin injection period and daily during the 10-day recovery period. Mice were also provided with a high calorie liquid diet (STAT, PRN Pharmacal) and bacon softies (BioServ) daily. This 14-day protocol was repeated once more for a total of two cycles of cisplatin administration. Mice had an additional 7 days of recovery following the final cisplatin injection before the postcisplatin treatment ABR tests. To promote exercise and facilitate the study of healthy animals, mice were housed in cages with mouse Igloos (hut) with attached wheel (BioServ) throughout the cisplatin administration protocol.
10
Implanting Telemetric Transmitters for Cardiovascular Monitoring
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10 days prior to the start of social defeat, witness, or control manipulation, all Sprague-Dawley rats were implanted with intraperitoneal radio-telemetric transmitters that allow for collection of ECG and blood pressure recordings from awake animals (HD-S11-F0 and -F2, Data Sciences Int., St. Paul, MN). Importantly, HD-S11 F2 and F0 transmitters signal via different frequencies to allow for simultaneous recordings of both the intruder and witness animals within the same cage thereby facilitating direct comparison of cardiovascular responses to physical vs. psychological stress. Rats were anesthetized with isoflurane (2.5%) and transmitters were implanted following steps outlined by Data Sciences Int. in the Modified Lead II configuration. Post-surgery analgesia (Flunazine 2.5mg/kg) and supportive therapy of Bacon Softies (Bio-Serv, Flemington, NJ) were administered for 48 hours post-operation.
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