Carprofen s c

Mice were anesthetized using isoflurane (Covetrus, Portland, ME) and provided analgesics with 5 mg/kg Carprofen s.c. (Zoetis, Troy Hills, NJ). A craniotomy was performed above the target region and a glass pipette was stereotaxically lowered to the desired depth. Injections were performed using a nano-inject II apparatus (Drummond Scientific), with 25 nl of solution delivered every 15 s until a total amount of 200 nl was reached. The pipette was retracted after 5 min. Viruses were injected bilaterally, at a titer of 2 × 10¹² vg/ml, with injection coordinates AP –2.0, ML ±1.5, DV –1.4 (in millimeters from Bregma). One single virus injection was performed per hemisphere, and each hemisphere counted as an independent injection site.
For virus construction, Addgene plasmid #44362 (pAAV-hSyn-DIO-hM4D(Gi)-mCherry) was cut with restriction enzymes NheI and AscI, and the hM4D(Gi)-mCherry cDNA was replaced with mouse HCN1 cDNA including an N-terminal HA tag (YPYDVPDYA) immediately following the starting methionine. Site-directed mutagenesis was performed by Applied Biological Materials Inc (Richmond, BC, Canada) and each viral DNA packaged into AAV serotype 8 and serotype 9 by the Duke University Viral Vector Core (Durham, NC). Experiments performed with either virus serotype produced analogous results.

Between day 26 and 90 after injection (depending on the respective cell line), mice were anesthetized using a bodyweight-adapted i.p. injection scheme of ketamine hydrochloride (100 mg/mL; 1.2 mL/kg; Graeub, Bern, Switzerland) and xylazine hydrochloride (20 mg/mL; 0.8 mL/kg; Bayer, Leverkusen, Germany). PTs were excised under sterile conditions and immediately fixed in 3.7% neutral buffered formalin for 48 h or stored in liquid nitrogen (fresh frozen samples). Skin defects were clipped with disposable skin staples (3M; Health Care, Borken, Germany) and mice received carprofen s.c. (5 mg/kg; Zoetis, Berlin, Germany) directly after surgery as well as for two days following surgery. Subsequently, all mice were inspected daily and the overall clinical condition, including appearance (posture, behavior) and physiological responses as well as food and water intake were assessed. Mice were sacrificed when they failed the United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR )health score system [39 ].

As a further modification of the protocol, beginning after passage 6, additional NSG mice were engrafted with C5 tumors and tumors were surgically resected when reached about 1.5 cm³. Briefly, mice anesthetized with ketamine/xylazin were depilated in the tumor area and placed on a warming mat. The skin above the tumor was incised, the tumor was mobilized and cut off the underlying connective tissue and supplying microvessels using an electrocautery device. Skin closure was performed with disposable skin staples (3 M Health Care, Borken, Germany) and mice received Carprofen s.c. (5 mg/mL, 5 mg/kg, Zoetis, Berlin, Germany) immediately after surgery as well as on the first and second post-operative day. Skin staples were removed on the seventh post-operative day. Mice were monitored after surgery for at least the primary tumor growth span before they were sacrificed and distant organs harvested for detection of metastatic cells as described above. All animal experiments described in this study were approved by the local animal experiment approval committee (Behörde für Gesundheit und Verbraucherschutz, Amt für Verbraucherschutz, Lebensmittelsicherheit und Veterinärwesen, assigned project No. 88/09, 19/15 and 55/16), supervised by the institutional animal welfare officer and performed in accordance with relevant guidelines and regulations.