Ketolar

Animal care and experimental procedures were approved by the Ethical and Animal Studies Committee of the University of Murcia (UM; Spain; Codes: A13171103, A13170110, and A13170111) and followed the Spanish and European Union regulations for the use of animals in research (Council Directive 86/609/EEC) and the ARVO statement for the use of animals in ophthalmic and vision research. In this work, we have used pigmented C57BL/6J mice (20–24 g) provided by the animal facility of the UM and housed in temperature-controlled rooms with 12-h light/dark cycles and with food and water provided ad libitum. To perform surgery on the tracing of the RGCs from the optic nerve (see below), mice were anesthetized with a mixture of intraperitoneal ketamine (60 mg/kg body weight, Ketolar; Pfizer, Alcobendas, Madrid, Spain) and xylazine (10 mg/kg Rompun; Bayer, Kiel, Germany).
A total of 37 pigmented mice were used in this study. To study the entire population of RGCs in the pigmented mouse retina, three mice (six retinas) were used to detect the total population of RGCs, both traced from the optic nerve and detected by immunohistochemistry (see below). To study the population and distribution of αRGCs and their subtypes, 30 mice (60 retinas) were used for the study of the retina in whole mounts and four additional mice (eight retinas) were used for the study of the retina in radial sections.

Ketamine (Ketolar^®, Pfizer; 10–30 mg/kg) was dissolved in saline and injected intraperitoneally (i.p.). Doses of ketamine were chosen according to literature^{45 (link)} and are expressed as free base. The volume of injection was 4 ml/kg. All other drugs and reagents used were of analytical grade.

In total, 22 NSG mice were anesthetized with 100 mg/kg ketamine (Ketolar, Pfizer, New York, NY, USA) and 10 mg/kg xylacyn (Rompun, Bayer, Barmen, Germany). Then, 10⁶ CXCR4⁺ Luciferase⁺ AN3CA cells resuspended in 25 uL of culture medium were inoculated through the myometrium into the endometrial cavity using a 29G Hamilton syringe (Microliter Serie 800, Hamilton, Reno, NV, USA), according to the procedure described in [16 (link)].
Three days after implantation, cell engraftment and the bioluminescence emission were assessed using IVIS Spectrum 200. Mice were then separated into two groups (n = 10/group) to receive either 5 µg T22-DITOX-H6 or carbonate buffer as control, three times a week. Whole body BLI was determined twice a week (Figure S3).
All mice were euthanized when at least one of them reached endpoint criteria, such as a high primary tumor or peritoneal carcinomatosis growth or abdominal distension, which happened two days after the 14th dose. All clinically relevant organs (uterus and ovaries, peritoneal carcinomatosis, lung, kidneys, liver, spleen, abdominal lymph nodes) were collected, fixed in 4% formaldehyde and paraffin-embedded for histopathological analysis, IHC evaluation of metastatic foci and possible toxicity of T22-DITOX-H6 on normal organs.

For anesthesia a mixture of xylazine (10 mg/kg body weight; Rompun; Bayer, Kiel, Germany) and ketamine (60 mg/kg body weight; Ketolar®; Pfizer, Alcobendas, Madrid, Spain) was used intraperitoneally (i.p.). After surgery, an ointment containing tobramycin (Tobrex; Alcon S. A., Barcelona, Spain) was applied on the cornea to prevent its desiccation. Rats were given oral analgesia (Buprex, Buprenorphine 0.3 mg/mL, Schering-Plough, Madrid, Spain) at 0.5 mg/kg (prepared in strawberry-flavored gelatine) the day of the surgery and during the next 3 days.
All animals were sacrificed with an i.p. injection of an overdose of pentobarbital (Dolethal, Vetoquinol, Especialidades Veterinarias, S. A., Alcobendas, Madrid, Spain).

Two-month-old female adult pigmented C57BL/6 (n = 17; 25–30 g) and albino Swiss (n = 15; 30–35 g) mice were obtained from the University of Murcia breeding colony. They were housed in a 12 h light 12 h dark light cycle with lights on at 08:00 and off at 20:00. Animal manipulations were carried out following the Spanish and European Union regulations for the use of animals in research (Council Directive 86/609/EEC), the ARVO statement for the use of animals in ophthalmic and vision research, and were approved by the Ethical and Animal Studies Committee of the University of Murcia (Murcia, Spain). For surgical manipulations, mice were anesthetized with an intraperitoneal (i.p.) injection of ketamine (70 mg/kg Ketolar^®, Pfizer, Alcobendas, Madrid, Spain) and xylazine (10 mg/kg Rompun^®, Bayer, Kiel, Germany). To prevent corneal desiccation eyes were covered with an ocular ointment (Tobrex; Alcon S. A., Barcelona, Spain). All animals were sacrificed with an overdose of sodium pentobarbital (Dolethal^® Vetoquinol, S.A., Especialidades Veterinarias, S.A., Alcobendas, Madrid, Spain). The experimental design is summarized in Table 1.

The animals were initially anesthetized with the isoflurane inhalation anesthetic (FORANE, AbbVie Farmacéutica, S.L.U.) and subsequently with a ketamine/xylacine combination (1/3), 0.1 ml/10 g body wt i.p. (KETOLAR, Pfizer and ROMPUN, Bayer). Mice were tracheotomized and connected to the ventilator according to the previously published methodology [24 (link)]. Changes in quasistatic lung compliance (C_L, ml/cmH₂O) and maximum expiratory volume (V_max, ml) at a pressure of 25 mbar after pressure-controlled ventilation were studied for each of the experimental groups.

NSG mice were anesthetized with 100 mg/kg ketamine (Ketolar, Pfizer, New york, NY, USA) and 10 mg/kg xylazine (Rompun, Bayer, Leverkusen, Germany). The lower abdomen was shaved and swabbed with povidone. A medial laparotomy was performed to expose the right uterine horn. Its irrigation system was separated, and a ligature was performed in the proximal section of the horn using 7/0 Optilene (BBraun, Melsungen, Germany).
After randomization (n = 4/group), either 10⁶ CXCR4⁺ AN3CA or CXCR4^- AN3CA cells resuspended in 25 ul of culture medium were inoculated through the myometrium into the endometrial cavity using a 29G Hamilton syringe (Microliter Serie 800, Hamilton, Reno, NV, USA).
Overall animal health conditions were monitored three times a week. All mice were left to survival, and they were sacrificed as soon as they reached human endpoint criteria (55 ± 17 days; mean ± SD). In addition to the previously stated endpoint criteria, for this experiment, high primary tumor or peritoneal carcinomatosis growth (determined by palpation or a maximum whole body bioluminiscence of 2 × 10¹⁰ p/s/cm²/sr) as well as abdominal distension, were observed.