Buprenovet

Chronic application of the orthosteric-like mGlu7 antagonist XAP044 (Tocris Bioscience, Bristol, UK; Figure 1B) via s.c.-implanted Alzet^® micro-osmotic pumps (pumping rate: 0.11 µL/h, Alzet^®, Model 1004, Cupertino, CA, USA) connected to an i.c.v. infusion cannula (Brain Infusion Kit 3, Alzet^®, Cupertino, CA, USA) was initiated one week before starting the CSC paradigm (day 6) in order to establish a stable baseline receptor occupancy and was continued until the end of chronic stressor exposure (day 20). XAP044 was formulated as a solution in vehicle (VEH, 5% DMSO in Ringer’s solution, Merck; Darmstadt, Germany) to ensure a continuous substance release of 1 µM, 10 µM or 100 µM of XAP044. As reported by Peterlik et al. 2017 [23 (link)], the micro-osmotic pump was implanted s.c. in the abdominal region through a 1 cm long incision at the lower neck of the mouse under isoflurane anesthesia (Baxter, GmbH, Unterschleißheim, Germany) but additionally connected via a catheter to an i.c.v. infusion cannula. Animals were treated with painkiller (Buprenovet, s.c.; 0.05 mg/kg; Bayer Health Care AG, Leverkusen, Germany) and 100 µL of antibiotics (s.c., Baytril^®, 2.5% Bayer Health Care AG, Leverkusen, Germany). Wound treatment was conducted using betaisodona (Mundipharma GmbH, Limburg, Germany).

The mice were anesthetized by inhalation of Isoflurane (Isoflurane, Abbott, Maidenhead, UK; dosages detailed in the MCAO procedure description). The spleen was removed via a dorsolateral approach, the fur around the 13th rib was shaved, the area was disinfected with 70% ethanol, and an incision was made below the 13th rib to access the spleen. It was separated from its blood vessels using electrocautery, and the wound was closed with metal clips. The animals were monitored for one day in a heated cage with analgesia using 0.1 mL of buprenorphine (Buprenovet, Bayer, Leverkusen, Germany). Full recovery from the operation was deemed after a week (22 (link),23 (link)).

Male OF1 mice (Charles River Laboratories, Saint-Germain-Nuelles, France) were anesthetized with 2% isoflurane in 100% oxygen, and the inner faces of both hindlimbs were carefully shaved. After local disinfection, an about 1-cm skin incision was made on the left hindlimb from the inguinal region to the bifurcation region of the femoral artery into the saphenous and popliteal artery. The femoral artery and vein were dissected from the nerve. The femoral artery/vein block was ligated proximally by two 8–0 ties placed just distally from the superficial epigastric artery, and distally by two 8–0 ties placed just proximally from the bifurcation of the femoral artery into the saphenous and the popliteal artery. After cutting the femoral artery/vein block between the two proximal and between the two distal ties, the femoral artery/vein block was removed. When necessary, major branches such as the lateral circumflex femoral artery were ligated to avoid bleeding.
Thereafter, subcutaneous tissue and skin were closed with non-resorbable sutures or clamped with titanium micro clips (WDT, Garbsen, Germany). Postoperative care included pain management by injection of buprenorphine (Buprenovet, Bayer; 0.1 mg/kg) once directly after surgery or flunixin meglumine (2.5 mg/kg twice daily) during 3 days and daily local wound care with an antiseptic healing cream (Dermaflon, Pfizer).

After the surgery, body weight was checked daily for as long as it took the animals to regain their preoperative bodyweight. The wellbeing of the animals (swelling, reddening, impairment of wound healing, loss of passive motion in the left hind leg) was checked daily by a scoresheet.
All animals received buprenorphine (0.3 mg/mL bw; Buprenovet^®; Bayer AG, Leverkusen, Germany) as analgesic medication perioperatively and the following 4 days.
Subsequent to the second surgery, both experimental groups received once-daily subcutaneous administration of Teriparatid 0.4 µg/kg for 42 days.
Eight weeks after the second surgery, the animals were sacrificed.

Buprenorphine (0.3 mg/mL bw; Buprenovet^®; Bayer AG, Leverkusen, Germany) was used as an analgesic medication perioperatively, as well as for the following four days every twelve hours. Body weight and body temperature, along with clinical conditions of the animals, were evaluated regularly. All animals were monitored for eight weeks after the second surgery and sacrificed.