Ultra fine nano pen needles

The University of New Mexico Institutional Animal Care and Use Committee approved all experiments involving animals. Female, CD hairless Crl rats (Charles River Laboratories, Wilmington, MA), were anesthetized with 2.0% Isoflurane and 0.2 l per min oxygen. Sterile, 4 mm × 32 G, Ultra-fine Nano pen needles (BD, Franklin Lakes, NJ) were then placed into 3D-printed microneedle array holders. Furthermore, 1–5 µl calibrated pipet capillary tubes (Drummond Scientific Co., Broomall, PA) were then attached to each pen needle in the holder. The microneedle array was then gently pressed into the abdomen and/or flank dermal tissue. A small clamp was placed over the center of the holder and the array was held in place until sufficient quantity of ISF had been collected. If a decrease in ISF flow was noted, the array was removed and re-inserted in an adjacent area of the skin. In a typical experiment, moving the array multiple times allowed for collection of 30–60 µl of ISF in a 1- to 2-h period. The ISF was recovered from the capillary tubes of the microneedle array into a microcentrifuge tube on ice, an equal volume of TRIzol LS reagent (Ambion, Carlsbad, CA) was mixed in, and the sample was flash frozen using liquid nitrogen and stored at −80 °C for transcriptomic profiling.

ISF was extracted using our previously published methods [8 (link),9 (link),10 (link)]. Briefly, animals were anesthetized using 2% isoflurane. Microneedle arrays were then applied to extract ISF. Ultra-fine Nano PEN needles (BD, Franklin Lakes, NJ, USA) were placed into 3D-printed microneedle array holders. Each needle was attached to a 1–5 µL calibrated pipet capillary tube (Drummond Scientific Co., Broomall, PA, USA). The array assembly was then pressed into the dermal tissue of the rats and held in place until enough ISF was collected. ISF was transferred from the capillary tubes onto the glucose or ketone meter strips using a small plunger that is supplied with the capillary tubing. Glucose and ketone measurements were immediately recorded using a Nova Max Plus glucose/ketone meter (Nova Biomedical, Waltham, MA, USA) with Nova Max Plus glucose and ketone test strips (Nova Biomedical, Waltham, MA, USA), and another ISF extraction was then performed. This routine was repeated for 1.8 h for multiple glucose and ketone measurements of both blood and ISF. The blood was obtained from a small tail snip, where a drop of blood from the tail was periodically placed on the glucose and ketone test strips to measure the blood concentrations correlating with the times of ISF measurement. All animals had a terminal cardiac puncture performed at the conclusion of each experiment.

The animal care and use program of the University Of New Mexico (UNM) is accredited by AAALAC International and the UNM’s animal care and use committee approved all experiments. CD hairless, Crl:CD-Prss8hr, rats (Charles River Laboratories, Wilmington, MA) were used for the studies. ISF was extracted using our previously published methods (Tran et al. 2018 (link)). Briefly, animals were anesthetized using 2% isoflurane, and MAs were applied to extract ISF. Ultra-fine Nano PEN needles (BD, Franklin Lakes, NJ) were placed into the 3D-printed MN holders to form the MA. Each needle was attached to a 1–5 µl calibrated pipet capillary tube (Drummond Scientific Co., Broomall, PA). The array assembly was then pressed into the dermal tissue of the rats and held in place for exactly 2.0 min per extraction. The volume of ISF extracted in each needle and the total ISF extracted per MA was recorded for each extraction. All animals had a terminal cardiac puncture performed at the conclusion of the experiments.