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        Experimental Study on the Safety of Abdominal Cavity Circulatory Thermal Perfusion*

        Date:2014年2月26日 16:58

        Experimental Study on the Safety of Abdominal Cavity Circulatory Thermal Perfusion*
        Wenchao Liu,  Chunbao Li, Wang Yu, Wei Li
        Correspondence to: Wenchao Liu; Email: xjcancer@fmmu.edu.cn
        Department of Oncology, Xijing Hospital, the Fourth Military Medical University, Xi’an 710032, China
        This work was supported by the National Natural Science Foundation of China (No. 30973437)
        Abstract Objective: This work investigated the effect of cavity circulatory thermal perfusion (CCTP) on the vital signs and important organs of experimental animal dogs at different temperatures. The study likewise aimed to determine the optimal temperature for chemotherapy. Methods: Dogs were used as the experimental animal models for CCTP. CCTP was performed with cisplatin using a hyperthermic intraperitoneal treatment system at 41 ℃, 42℃, and 43℃ (one course of clinical treatment). The hepatic and renal functions were detected in preserved blood samples before CCTP and at 24h after each course of the treatment. The respective morphological and pathological changes of the major abdominal organs were likewise studied at 24h as well as 2 weeks after the third course of CCTP. Results: The three courses of CCTP with a perfusion rate of 140mL/min at 41 or 42℃ did not have any observable negative effects on the hepatic and renal functions of experimental animals. On the other hand, the three courses of CCTP with a perfusion rate of 140mL/min at 43℃ had a significant negative effect on the hepatic and renal functions, with histopathological injuries in the liver, kidney, spleen, and intestines. Conclusion: CCTP with a perfusion rate of 140ml/min combined with cisplatin chemotherapy (three times/60min) was safe and feasible at 42℃, but could damage visceral organs of experimental animals at 43℃.
        Key words :Cavity circulatory thermal perfusion; Safety; Thermal injury; Thermo-chemotherapy damage
        doi:10.3969/j.issn.1000—8179.2012.22 .006
             The treatment of malignant pleural effusion mainly relies on dieresis, repeated drainage of ascites, the application of chemotherapy drugs and other methods. However, the therapeutic effect is limited. With the development of thermotherapy, the role of hyperthermia in cancer therapy is becoming more and more important. CCTP showed a good effect in the treatment of malignant effusion. The treatment performed under a warm condition with mild invasive was combined with cytotoxic drugs. Therefore its safety was more and more concerned. In this study, the safety of CCTP was assessed both in the whole body and the local of the animal models. Also the suitable temperature for CCTP was explored so as to provide the experimental evidence for the clinical application of CCTP in the treatment of malignant pleural effusion.
        1 Material and Methods
        1.1 Material
             Experimental equipment: Extracorporeal circulation perfusion machine GDPR-2100S (produced by Xi'an good doctor medical technology Co., Ltd.); RM6240 multi-channel physiological (Chengdu Instrument Factory); AI 560 digital thermometer (Xiamen Yudian Automation Co., Ltd.)
        The experimental animal and grouping: 18 of adult healthy hybrid dogs (provided by The Fourth Military Medical University), regardless of gender, with body weight of (18 ±0.5) kg were randomly divided into three groups: group A (41℃ saline+ cisplatin, 6 dogs), group B (42℃ saline+ cisplatin, 6 dogs), group C (43℃ saline+ cisplatin, 6 dogs). Dogs were in separate cages.
        1.2 Methods
        1.2.1 CCTP treatment  CCTP  treatment was performed on each dogs in every treated groups, 60min for each time in every 3 days, repeated for 3 times. Intraperitoneal injection of anesthesia was used with sodium pentobarbital (50 mg/kg). Two needles were placed and fixed in both left and right sides of abdominal cavity. Two needles were respectively connected with the inlet and outlet catheter of the extracorporeal circulation perfusion machine GDPR-2100S. High precision PT100 temperature sensor was placed by the side of the catheter as well as in the left and right upper abdominal cavity. The input temperature for the lavage solution was 41℃, 42℃ and 43℃ with the flow rate of 140ml/min.  A single-path mode was firstly preferred. The input temperature for the 2500ml physiologic lavage solution containing 100mg/kg cisplatin was 41℃, 42℃ and 43℃. Then the cycle model was adapted. The whole course with constant temperatures continued for 60min.
        1.2.2 Observation The input and output temperatures were shown on GDRP-2100S. The temperature of the puncture point of both right and left upper abdomen were read by AI-5600 digital thermometer. The indexes needed to be monitored were flow rate, heart rate, blood pressure, means of arterial pressure, ascites floats and ascites colors. The temperature of four points were recorded every 10min. Peripheral blood sample were collected before and 24h after each course of the treatment. 24h as well as 2 weeks after the third course of CCTP,  2 dogs were killed in each group, collect heart, liver and spleen and kidney, fixed the tissue with 10% methanol, paraffin embed, routine HE stained and morphology observed by microscope.
        1.3 statistics
             All the data were shown as mean ±standard deviation. Statistical analysis was performed using the SPSS 12.0. ANOVA was used to investigate the different mean in each group. A p<0.05 was considered significant.
        2 Results
        2.1The change of vital signs after CCTP
             In group A, the mean body temperature of dogs rose by 0.2℃ after 1h-treatment of CCTP in 41℃, with heart rate of (120±5) beats/min, respiratory rate of (15±5) breath/min and mean arterial pressure 125mmHg.  In group B, the mean body temperature of dogs rose by 0.5℃ after 1h-treatment of CCTP in 42℃, with heart rate of (135±5) beats/min, respiratory rate of (23±5) breath/min and mean arterial pressure 130mmHg. In group C, the mean body temperature of dogs rose by 1.5℃ after 1h-treatment of CCTP in 43℃, with heart rate of (140±5) beats/min, respiratory rate of (30±5) breath/min and mean arterial pressure 120mmHg.
        2.2 The change of hepatic and renal functions after CCTP
             In group A and group B with the temperature of 41℃ and 42℃, there were little change of hepatic and renal functions after CCTP, or mild change after perfusion (p<0.05). These change returned to be normal after 2 weeks. However, in group C with the temperature of 43℃, the damage of hepatic and renal functions were found, showing that the level of glutamic-pyruvic transaminase(ALT), glutamic-oxalacetic transaminase(AST), Creatinine (Cr), Uric acid(UA) were significantly higher in 4d after CCTP(P<0.05), and still abnormal of ALT and AST in 2 weeks after CCTP (P<0.05, Table1) .

        Table1 Changes in the liver and kidney functions during CCTP with cisplatin at three temperatures

        Index

        Group

        Pre-treatment

        Post-treatment

        2d

        4d

        6d

        14d

        ALT (U/L)

        A

        37.87±5.14

        46.27±5.60

        58.12±3.11

        42.08±4.01

        39.01±5.34

         

        B

        33.87±4.32

        38.27±5.63

        43.86±6.99

        39.56±3.65

        35.16+5.03

         

        C

        33.53±5.71

        46.54±6.11

        73.18±4.63*

        68.25±4.37*

        59.13±5.49*

        AST (U/L)

        A

        41.43±5.39

        45.54±6.51

        47.56±4.36

        43.26±4.87

        42.37±4.53

         

        B

        43.19±5.27

        49.12±8.03

        50.37±5.75

        44.89±5.96

        42.96±5.11

         

        C

        41.58±5.04

        53.44±6.31

        92.06±5.12*

        76.00±4.97*

        62.79±6.22*

        Cr (µmol/L)

        A

        66.70±6.78

        70.89±6.01

        74.86±6.11

        70.88±4.68

        67.69±4.86

         

        B

        70.32±6.12

        74.82±8.06

        79.88±5.38

        73.68±7.91

        72.18±5.35

         

        C

        68.54±8.32

        74.55±4.78

        87.98±6.79*

        74.36±4.28

        70.95±54.36

        UA(µmol/L)

        A

        283.13±9.89

        275.54±13.79

        289.41±12.08

        268.30±13.96

        250.28±13.59

         

        B

        266.73±13.89

        281.19±13.42

        293.06±14.97

        282.97±13.52

        271.10±14.28

         

        C

        265.19±14.27

        282.49±15.18

        297.83±15.23*

        282.86±17.72

        266.36±15.66

         

        * compared with pre-treatment group, P<0.05. Group A: 41℃; Group B:   42℃; Group C: 43℃
        2.3 The morphological changes of abdominal organs after CCTP
             In group A and group B, no obviously damage of liver, kidney, spleen and intestine were found in 24h after three courses of CCTP, as well as 2 weeks after treatment. However, in group C, mental adhesion was found in part of organs. Mesenteric vascular congestion significantly, and also small ecchymosis and hemorrhage can be found in liver and intestinal. No obviously change was found in kidney. After 2 weeks, still small area of ecchymosis left in intestinal, however, adhesion was in remission.
        2.4 The pathological changes of abdominal organs
             In group A, No change but mild congestion was found in liver, intestinal, kidney and spleen in 24h after 3 courses of CCTP, and return to normal after 2 weeks. The pathological changes of abdominal organs in group B and group C were shown in Table2, Figure 2.
         

         

        Figure 1 General morphological changes in abdominal organs at 24h after three cycles of CCTP at 43℃ for 60 min. There was extravasated blood in a small portion of liver and intestines. The mesenteric vascular vessel was evidently congested, and the omentum adhered to most of abdominal tissues. Two weeks after CCTP. Some bowel regions were still bruised, but abdominal adhesion was relived.

        Table 2 Pathological changes of abdominal organs after CCTP at various temperatures

        Organs

        42℃

        43℃

        Liver

        Hepatic portal area moderate congestion, liver cell edema, liver parenchymal cells showed spotty necrosis in 24hr;  hepatic portal area are still mild hyperemia after 2 weeks.

        Vascular dilatation in hepatic portal area, severe congestion, widespread hydropic degeneration, focal necrosis; after still portal congestion, cell necrosis  after 2 weeks.

        intestines

        The structure of villi were normal, but the mucosa and submucosa vascular mild hyperemia, severe inflammatory cell infiltration in the mucosa, fibrinoid degeneration and hyalinization in part of submucosal interstitial in 24h; mucosal vascular still infiltration of inflammatory cells, and goblet cell hyperplasia after 2weeks.

        Large area of inflammatory cell infiltration and necrosis in The intestinal mucosa and submucosa,  congestion significantly;  hyaline degeneration and necrosis of cell debris in  submucosa, Small intestinal villus shedding;; the infiltration of inflammatory cells in mucous layer, the submucosa  loose, and small vessel hyperplasia  after 2 weeks.

        spleen

        Infiltration of inflammatory cells in splenic stromal,at 24h; vascular severe congestion, billroth's strands broadening obviously, after 2 weeks , splenic stromal moderate hyperemia and infiltration of inflammatory cells; billroth's strands increased.

        Splenic congestion seriously, cell edema, nuclear pyknosis,  some dead cell debris were visible, small vascular hyalinosis; still extravasated blood in spleen and broadening billroth's strands  increased after 2 weeks.

        Kidney

        glomerular and interstitial mild congestion at 24h, mild infiltration of inflammatory cells, a small amount of inflammatory cell infiltration after 2 weeks, with interstitial cell edema.

        Glomerular capillary moderate hyperemia, glomerular and renal tubular epithelial cells edema; moderate congestion in interstitium, inflammatory cell infiltration, brush border of renal tubular epithelial cell were loss, after 2 weeks ,glomerular still obvious congestion, moderate infiltration of inflammatory cells, glomerular and renal tubular epithelial cells widely ballooning degeneration.

         

         

        A: Liver; B intestines; C: spleen; D: Kidney
        Figure2 Pathological changes of abdominal organs in the group that was treated at 43℃

        3 Discussion
             Peritoneal metastasis is an important metastasis route of digestive system tumor, especially in gastrointestinal tumor, and it is also the main causes of death. Thermo chemotherapy is considered to be one of an important therapy ways for digestive system tumor.
              Cavity thermal perfusion can be divided into abdominal irradiation heating in vitro and intraperitoneal circulation heating. The perfusion solution did not flow in former while it is continuous flowing in later. There was study found that, the relative safe temperature for non-cycle perfusion is 43℃ for 30 min. It is also supported by a in vitro study shown that 43℃ for 40min is a safety tipping point to organs and tissues [1]. However, the safe temperature for cycle perfusion was unclear.  Therefore, the study aimed to determine the optimal temperature for cavity circulatory thermal perfusion combined with chemotherapy.
             Dogs were selected as experimental animal to explore the optimal temperature for CCTP. According to the experimental animal and clinical application, 1 dog was perfused by pure physiology saline at the perfusion flow rate of140mL/min for 45℃. Finally, the dog is dead at 3d after CCTP. Therefore, 43℃ and 60 min were suspected to be the tolerated limiting for dog perfused by saline combined with cisplatin.  Based on this study, 42℃ is a safety temperature among 41℃, 42℃ and 43℃.
             According to the pathological changes of organs in this study, no obviously effect were found after three courses of  CCTP with (physiological saline +cisplatin) condition of 41℃, 140mL/min and 60min. It can be used as the temperature for prophylactic treatment. Under the CCTP (physiological saline +cisplatin) with condition of 42℃, 140ml/min flow rate for 60min, mile damage were found in liver, spleen, kidney and intestines of dogs. And it can recover at 2 weeks, as a reversible injury. So it can also be used for CCTP treatment. An irreversible injury of liver, spleen and intestines can be found after CCTP treatment under the condition of 43℃, 140mL/min of flow rate for 60min, and it cannot recover at 2 weeks after treatment. So 43℃ cannot be used as CCTP treatment.
             42℃~43℃ was considered to be the ideal temperature [2-4], under which tissues and cells can obtain best effect. The effect is not obvious when the temperature is below 40℃. While the tissues may be injured when the temperature is above 43~ 45℃ and no effect of reversal of drug resistance was found [5-6]. CCTP treatment with the temperature of 43℃ for 60min induced irreversible injury on cells. The study found that the damage mechanism of heating of 41~42℃ includes the change of tumor cell membrane permeability [7], degrade the extracellular matrix [8], up-regulation of cell adhesion ability [9.13]. Additionally, heat can promote D N A and platinum binding, and increase the intracellular concentration of platinum drugs, and inhibit cisplatin-induced cell damage repair mechanism [10, 14]. So synergistic effect can be found when cisplatin was combined with hyperthermia [11]. The organ injured temperature obtained from this study was lower than the temperature reported previously. It possibly because of different technology of temperature monitor; secondly, tissue fluid erosion damage caused by perfusion flow (flow rate 140mL/min).  The faster of flow rate the more damage might be made. Thirdly, chemotherapy drugs had the directly effect on tissues. All the above suggested that the tissue injured during perfusion were not only caused by high temperature (43℃), but also by fluid power and drug.
        Recently, different kinds of circulating perfusion machine with distinct temperature control method and heating methods were used. So the different model of thermal damage may happen. The GDRP-2100 we used in this study can precisely control and measure temperature and maintain a constant peritoneal temperature after heating rapidly, and guarantee the heat and drug dispersed in body and organ evenly. There were three main conclusions in this study, CCTP with the condition of 41℃, at flow rate of 140mL/min for 60min had no obviously injure effect; with 42℃, reversible injury happened; and with 43℃, serious and irreversible injury happened. In conclusion, for dogs, the safety condition of CCTP is 42℃, with the flow rate or 140mL/min, 3 times, and 60min per course.

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