Fs 2 needle

Infection of mice with MCMV was performed as previously described (Cheeran et al. 2004 (link)). Briefly, female mice (6–8 week old) were anesthetized using a combination of Ketamine and Xylazine (20 mg and 2 mg/kg body weight, respectively) and immobilized on a small animal stereotactic instrument equipped with a Cunningham mouse adapter (Stoelting Co., Wood Dale, IL). The skin and underlying connective tissue were reflected to expose reference sutures (sagittal and coronal) on the skull. The sagittal plane was adjusted such that bregma and lambda were positioned at the same coordinates on the vertical plane. Virulent, salivary gland-passaged MCMV RM461 (1.5 × 10⁵ TCID₅₀ units in 10 μl), was injected into the right lateral ventricle at 0.9 mm lateral, 0.5 mm caudal, and 3.0 mm ventral to bregma using a Hamilton syringe (10 μl) fitted to a 27 G needle. The injection was delivered over a period of 3–5 min. The opening in the skull was sealed with bone wax and the skin was closed using 4–0 silk sutures with a FS-2 needle (Ethicon).

All behavioral experiments were conducted by a blinded observer. Since HYPO and OVX female rats have no regular estrous cycles, surgical procedures on these female rats were conducted without taking into account the estrus phase. The reproductive stage of cycling intact females was determined by vaginal lavage using methods previously described [23 (link)]. The plantar incisions in rats were performed as previously described [24 (link)]. Briefly, rats were anesthetized using 2% isoflurane. The right hind paw was prepared for incision by application of antiseptic betadine solution. The incision model in rats was created by a 1-cm longitudinal incision, through skin and fascia of the plantar aspect of the foot, beginning 0.5 cm from the proximal edge of the heel and extending toward the toes. Curved forceps were used to elevate longitudinally the underlying the plantaris muscle. A mattress suture of 5-0 nylon on a FS-2 needle (Ethicon, Somerville, NJ) was used to close the incision. Antibiotic neomycin ointment (Bacitracin Zinc Ointment USP, Melville, NY) was applied to the wound and the sutures removed 2 days later. Sham treatment consisted of rats that received anesthesia, antiseptic preparation and topical antibiotic without an incision. Thermal and mechanical hypersensitivity were measured in these animals over a period of 7 days.