1. Monkeys: After induction of anesthesia, monkeys were placed in a Kopf stere- otactic head holder, and a linear incision was made from the inion to the spinous process of C2. A burr hole was made in the midline on the occipital bone 2.5 cm below the inion. The dura was opened with a scalpel blade (no. 11) and the edges of the dura were coagulated by bipolar cautery. The infusion
catheter was then inserted to a pontine target of 1.75 mm anteroposterior,
−12.5 mm dorsoventral, and 0 mm mediolateral in a standard stereotaxic atlas [29]. These coordinates were used to determine the resting depth of the catheter tip. We then mapped the linear path of the catheter for those coordinates and inserted the catheter freehand through the cerebellum at a 45° angle between the catheter and the occipital bone to a depth of ~2.25 cm from the surface of the cerebellum (see Note 4).
In animals 1 and 2, the 3-cm-long catheter was secured to the skull by placing cyanoacrylate in the burr hole. To accommodate independent move- ment between the skull and the brain, a 2.25-cm-long catheter was passed through the skull in animals 3–5. The tubing was secured to the bone with cyanoacrylate. The body of the pump was placed in a subcutaneous (SC) pocket in the low cervical/high thoracic region and connected to the catheter by the silicon tubing. The tubing was looped into the subcutaneous pocket between the pump and the burr hole (see Note 5).
2. Dogs: The animal is sedated with acetopromazine (0.2 mg/kg, i.m.) and transferred to animal-operating rooms where an intravenous line will be placed for infusion of D5NS solution. The dogs will receive a prophylactic dose of penicillin G benzathine, 30,000 IU/kg. General anesthesia will consist of sodium thiopental (10–20 mg/kg, i.v.). Sterile conditions are maintained for all proce- dures. Dogs are orally intubated for subsequent mechanical ventilation. Inhalable halothane and isofluorane are used to maintain general anesthesia. In addition to electrocardiographic monitoring, a catheter is introduced into the femoral artery for continuous monitoring of blood pressure and heart rate. For cell inoculation surgery, dogs are fasted from solid food for 12 h prior to surgery; water is available at all times. After induction of anesthesia, the dog’s head is shaved and washed with alcohol and providone-iodine solution.
An incision is made in the left frontal region commencing at the superior orbital ridge extending 2 cm and then coursing inferiorly. Scalp bleeding is controlled with electrocautery. Underlying connective tissue is removed from the frontal bone to reveal the sagittal suture. A high-speed drill is used to cre- ate a craniectomy defect in the left frontal bone that extends ~0.5 cm. A small round curette and small mastoid rongeurs are used to round out the defect. The dura is opened using a blade (no. 15) in a linear fashion. Edges are cauterized using a bipolar cautery. Approximately 30 µL of tumor cell suspension (5 mil- lion cells) is slowly (5 min) injected, using a Hamilton 50-µL syringe, ~7 mm into the cortex. The wound is covered with a piece of Gelfoam cut to the size of the defect. The temporalis muscle is closed using a running 3–0 Vicryl suture. The scalp is closed with interrupted 3–0 Prolene sutures. Prior to reversal of general anesthesia, the animal is given one injection of meperidine (2.0 mg/kg, i.m.) to abate postoperative pain and discomfort. Approximately 5 days after the cells have been implanted and an MRI imaging confirms the tumor growth, animals are prepared as described for cell inoculation surgery.
The scalp wound is identified, reshaved if necessary, and washed with alcohol and providone-iodine solution. An area between the shoulder blades of
approximately 3 cm wide × 10 cm long is shaved and washed with alcohol and providone-iodine. The sutures of the scalp wound are reopened to reveal the initial burr hole. A 5-mm-long stainless steel cannula connected to ~20 cm of silicon tubing is brought to the field. The tip of the tube is inserted to a depth of ~5 mm into the cortex. The silicon tubing connected to the tube is now in the burr hole and is sealed into place with surgical cement. A 5-cm-long inci- sion is made to open a subcutaneous pocket in the prepared area between the shoulder blades. Bleeding is controlled by electrocautery. The distal end of the tubing is passed through a tunnel under the skin created by spreading hemostat blades under the skin caudally toward the area between the shoulder blades.
A drug-containing pump is placed in the SC pocket and connected to the distal end of the silicon tubing. The temporalis muscle is closed using a running 3–0 Vicryl suture. The scalp and shoulder are closed with interrupted 3–0 Prolene sutures. Prior to reversal of general anesthesia, the animal is given one injec- tion of meperidine (2.0 mg/kg, i.m.) to abate postoperative pain and discomfort (see Note 4). Seven to nine days after the pump implant, the animal is lightly anesthetized, and the area over the shoulder blades is painted with alcohol and providone-iodine solution. The wound is opened to expose the body of the pump and the connecting tubing. The tubing is cut and sealed with a knot.
The pump is removed. The wound is resutured.
3. Rats: Animals are anesthetized with 0.65 mL of a solution containing ketamine hydrochloride (25 mg/mL), xylazine (2.5 mg/mL), and 14.25% ethyl alcohol in saline. Surgical surfaces are shaved, and washed with 70% ethyl alcohol and Betadine. With the aid of a Zeiss operating microscope, a 2-mm burr hole is made
~2 mm lateral and 1 mm anterior to the bregma. Pump cannulas are placed to a depth of 3 mm in the burr hole (see Note 4). The hole with the rigid or flexible cannula is sealed with surgical glue. The body of the pump is implanted subcutane- ously on the back of the anesthetized rodent slightly posterior to the scapulae in a pocket created by inserting and opening a hemostat into a midscapular incision and thereby spreading the subcutaneous tissue. The pocket is large enough to allow some movement of the pump, i.e., 1 cm longer than the pump. Wounds are closed with 4.0 vicryl.
In all cases to be reported, pumps were examined at the end of the infusion study to verify that the full content of drug solution had been delivered (see Note 4).
3.2 Biodistribution Measurements
1. Tissue platinum was assayed by atomic absorption spectroscopy to estimate the distribution of infused carboplatin [11, 18].
2. Doxorubicin levels were measured in clear supernatant solution obtained by centrifugation at 14,000 × g of 10% homogenates of 1-mm coronal tissue sections in saline. Fluorescence was measured at an excitation wavelength of 490 nm. Emission was measured at 594 nm [30].