Magnesium

Osteocytes were isolated from mouse long bones utilizing a modified protocol derived from the combined methods of Gu et al. and Van Der Plas et al. (33 (link),44 (link),47 (link)). Long bones (femora, tibia, and humeri) were aseptically dissected from skeletally mature 4-month-old (young) and 22-month-old (old) C57BL/6 mice (Charles River Laboratories, Wilmington, MA, USA). The bones from young and old mice were processed separately by serial digestion as described in Table 1. The bones from each individual mouse were pooled together and treated as one sample. Collagenase solution was prepared as 300 active U/mL collagenase type-IA (Sigma-Aldrich, St. Louis, MO, USA) dissolved in α-minimal essential medium (αMEM; Mediatech, Manassas, VA, USA). EDTA tetrasodium salt dehydrate (EDTA) solution (5 mM, pH = 7.4; Sigma-Aldrich) was prepared in magnesium and calcium-free Dulbecco's phosphate-buffered solution (DPBS; Mediatech) with 1% BSA (Sigma-Aldrich). All steps of the digestion took place in 8 mL solution in a six-well Petri dish, on a rotating shaker set to 200 RPM, in a 37°C and 5% CO₂ humidified incubator. Following each sequential digestion, the digest solution with suspended cells was removed from the bone pieces and kept. The bone pieces were then rinsed with Hank's balanced salt solution (HBSS) three times, and the rinsate was added to the digestion solution. The combined cell suspension solution was spun down at 200× g for 5 min, the supernatant was removed from the cell pellet, and cells were resuspended in culture medium and counted. The tissue homogenizer used in this study (Medimachine; BD Biosciences, San Jose, CA, USA) was utilized with a stainless steel mincing screen with a pore size of 50 μm.

The Rankin Focused Assessment (RFA) was developed by a working group consisting of physicians with extensive stroke clinical trial experience (JLS, SS), the head nurse coordinator (AY), and the study monitor (SC) of the NIH Field Administration of Stroke Therapy – Magnesium (FAST-MAG) phase 3 clinical trial, with additional input from the 14 nurse-coordinators performing outcome assessments at 47 participating hospitals in the trial. This mixed group of expert and novice trial staff selectively extracted, revised, and combined elements of prior instruments and generated new elements to construct the assessment tool. Important sources for tool construction were the mRS itself, the Structured Interview developed by Wilson and colleagues,7 (link), 9 (link) the videotapes and teaching booklet developed by Lees and colleagues,8 (link) and the working group’s daily experience in implementing the mRS in an ongoing trial. The assessment tool was piloted and iteratively refined in small groups of patients. The final tool was then prospectively tested in 50 consecutive trial patients.
The Rankin Focused Assessment consists of a 4 page form, accompanied by a 5 page Instruction Sheet(Supplemental Online Materials). When performed after brief review of medical records and an NIH Stroke Scale examination, the RFA is typically completed in 3–5 minutes. The assessment specifies clear, operationalized criteria to distinguish among the 7 assignable global disability levels. To determine which criteria a patient meets, the assessment permits and encourages the rater to gather data from all available useful sources, including interviews with the patient and caregivers, medical records, rehabilitation therapist notes, and the rater’s own examination of the patient. In addition to checkmark items, the Assessment tool includes text boxes in which the rater specifies the particular, concrete functional difficulties identified that led an item being checked, facilitating review of the accuracy of a particular rating and ongoing training of novice by more expert raters. Separate versions of the RFA have been developed to assess a patient’s current poststroke functional status and their historical prestroke functional status. In this study, the RFA to determine the patient’s current, poststroke mRS score was evaluated.