Temperature-controlled laparoscopic laser thermal therapy (TCLTT) system
The constructed thermal endoscope consisted of a rigid endoscope (the shaft having a maximum diameter of 14 mm and length of 288 mm) (serial No. 11499, Shinko Koki, Japan), an ultra-compact infrared thermography sensor (HTPA32x32d L2.1, Heimann Sensor, Germany), and a channel for introducing an optical fiber for laser irradiation 9 (Fig. 5a).
The two-dimensional temperature distribution was visualized by the thermography sensor with a frame rate of 8.3 fps and a spatial resolution of 32 × 32 pixels (A temperature range of 20-80°C corresponds linearly to a pixel value of 0-255.). Bright field images were obtained by a CMOS camera (EO-1312C, Edmund optics, USA) connected to the rigid endoscope.
The laparoscopic laser thermal therapy system consisted of the thermal endoscope, a diode laser (BWF2 B&W Tek, wavelength of 808 nm) and a microcontroller (Arduino) controlled by a PC (Fig. 5b). The temperature information acquired by the infrared thermography sensor is transmitted to the microcontroller. Based on the temperature information, the appropriate laser irradiation output is calculated to keep the temperature of the irradiation target constant.
The laparoscopic laser thermal therapy system was used with a laparoscopic insufflation device (PNEUMO SURE, Stryker) equipped with a light source device (L10000, Stryker) (Fig. 5c).
Operation of the TCLTT system
The temperature distribution of the area observed by the thermography sensor and the bright field image of the area observed by the CMOS camera were each monitored. On the temperature monitor, the pixel with the highest temperature was displayed as a red dot or green dot: red dot when the laser is on and green dot when the laser is off. In addition, 9 x 9 pixels around the red/green pixel were automatically extracted and four vertices of the square formed by the 9 x 9 pixels were displayed as blue dots (Video 1). Simultaneously, the average of the temperatures of the 81 pixels (9 x 9 pixels) was automatically calculated, and we defined the average temperature as "temperature of the irradiated target".
A surgeon confirmed the location of the tumor on the bright field monitor and advanced the optical fiber via the channel until the fiber tip appeared on the bright field monitor. Next, the tumor was irradiated through the optical fiber in a non-contact manner. Based on the "temperature of the irradiated target", the target tumor was heated with maintenance of temperature by being automatic calculation of the appropriate power of the laser irradiation.
During the laser irradiation, the position of the laparoscopic endoscope was manually corrected to ensure that the laser irradiation site did not dislocate significantly from the tumor. When the irradiated area was far from the tumor, the laser output was stopped.
Methods for evaluating anti-tumor effects
Temperature-controlled laser thermal therapy was performed on an orthotopic hepatocellular carcinoma model rat (preparation method to be described later). The experimental animals were randomly divided into two groups: a treatment group (n=6) and a control group (n=7). After induction of general anesthesia, a 15 mm trocar (VersaOneTM15mm, COVIDIEN) was inserted into the abdominal cavity through a 1.5-cm skin incision. The thermal laparoscopic camera was inserted via the trocar and insufflation with CO2 gas (insufflation pressure of 3 mmHg was performed). For the treatment group, laser irradiation was performed at a temperature setting of 70°C for 300 seconds. In previous experiments, we confirmed that this heating setting (70°C for 300 seconds) has a therapeutic effect on the entire tumor area (supplementary information).
The rats were sacrificed one week after the thermal therapy. Liver lobes were extracted and fixed in 10% formaldehyde solution and then subjected to the process of hematoxylin and eosin (H&E) staining. The size of the tumor was measured with a digital caliper at the time of tumor extraction, and the estimated volume was calculated as follows: (length) x (width) x (height) x 1/ 6π.
Statistical methods
Statistical analysis was performed using the Mann-Whitney U test. The statistical package used was JMP® 14 (SAS Institute Inc., Cary, NC, USA). P < 0.05 was considered to be statistically significant.
Method for preparing orthotopic hepatocellular carcinoma model rats
Cell line
Rat hepatocellular carcinoma strain N1-S1 cells (CRL-1604, ATCC) were used. The culture medium was Dulbecco's Modified Eagle medium supplemented with 10% FBS, penicillin (100 U/ mL) (Thermo Fisher), streptomycin (100 µg/ mL) (Thermo Fisher), and amphotericin B (0.25 µg/ mL) (Sigma-Aldrich). The cells were incubated in an incubator at 37°C in 5% CO2 and 95% air.
Animals
Female Sprague-Dawley rats (Japan SLC, Hamamatsu, Japan) at 8 weeks of age were used in this study. Rats were housed at 3~4 per cage under controlled temperature (23–25 °C) and relative humidity (50%) with 12 h of light (7:00–19:00). All animal procedures were performed in accordance with the guidelines approved by the National Defense Medical College Animal Care and Use Committee (Permit number: 19009).
Establishment of a rat tumor model
SD rats were injected intraperitoneally with a mixture of anesthetics: medetomidine (0.3 mg/kg) (Nippon Zenyaku Kogyo Co., Ltd., Japan), midazolam (4.0 mg/kg) (Sandz Corp., Japan), and butorphanol (5.0 mg/kg) (Meiji Seika Pharma Co., Ltd., Japan). After a small laparotomy, the left lobe of the liver was led out of the body, and 20 µL of a PBS-based cell suspension (3.5 x 10E4 cells/µL) was injected by puncture with a 30 G needle under the liver capsule. One week after transplantation of the cell suspension, rats were used as liver tumor model rats.