Animal Experiment of Security Evaluation of Hyperthermic Intraperitoneal Treatment System for Coelom Continued Circulatory Hyper

GUO Shan1,ZHANG He-long1
1Department of Oncology，Tangdu Hospital，Fourth Military Medical University，Xi’an 710038，China

【Abstract】
Objective: To investigate the impact on experimental animals vital signs, important organs pathology after Coelom Continued Circulatory Hyperthermia Perfusion (CCCHP) by  hyperthermic intraperitoneal treatment system on different temperature and treatment conditions, to search optimal temperature for therapy and the way to control temperature effectually and so on.  
Methods: The canine as experiment animal models of CCCHP, 41℃ (normal saline group and DDP group), 42℃ (normal saline group) and 43℃ (normal saline group) as intraperitoneal temperature, and CCCHP three times/1.0 hours (one course of clinical treatment) was performed by hyperthermic intraperitoneal treatment system respectively. Canine’s vital signs, thermodetector reading and data on panel displayed were recorded. The animals hepatic and kidney function changes were inspected before CCCHP and after CCCHP 24 hours by preserved blood specime. The general morphological changes and pathological changes of abdominal organs after CCCHP completed 24 hours and 2weeks later were inspected also. Results: CCCHP by 41℃ three times has no obvious affect on vital signs and hepatic, renal functions of experiment animals. The liver, kidney, spleen and intestine have only slightly pathological changes which can quickly return to normal. CCCHP by 41℃ three times combined DDP chemotherapy has also no obvious effect on normol physiologic function. CCCHP by 42℃ and 43℃ three times has significant effect on hepatic and renal functions of experiment animals, and hepatic,kidney,spleen and intestine were all injuried in different degree post CCCHP.
Conclusion: 41℃ intraperitoneal temperature CCCHP three times/1.0 hours is safe and feasible, so it can be used as the treatment combined DDP chemotherapy of CCCHP. 42℃ CCCHP on the same condition can cause minor injury to abdominal organs of experimental animals, so it is not suggested to be the conventional therapy temperature. 43℃ CCCHP on the same condition can cause serious damage to visceral organs of experimental animals, so it is inappropriate to be the treatment temperature of CCCHP.
【Key words】  Coelom Continued Circulatory Hyperthermia Perfusion(CCCHP); security; thermal injury; Canin

【Abstract】  Objective：

To explore the effect on experimental animal vital signs and abdominal organs in different temperature of Coelom Continued Circulatory Hyperthermia Perfusion (CCCHP), and find out the suitable treatment temperature and effective way of temperature control .

Methods:  To establish the CCCHP animal model with canines, treat each group with CCCHP 3 times (one course of clinical treatment ) at  41℃( normal saline group and the DDP group ), 42℃ (normal saline group ),43℃(normal saline group ) as intraperitoneal temperature respectively, at a time interval of 2 D and treated for 1hour each time. Record the canine vital signs, thermometer readings and the data on the panel displayed. Drew peripheral blood for test during each time of CCCHP before and 24h later. Two canines were killed in each group after 3 times of CCCHP in 24h and after 2 weeks , laparotomized and observed morphologic changes of visceral organs , and made liver, kidney and other organ sections for pathology biopsy. Results: 3 times of CCCHP at the abdominal temperature of 41 ℃ had no significant effect on canine vital signs and liver and kidney function, minor tissue damage were found in liver, kidney, spleen and intestine ; 3 times of  CCCHP  at 41℃ intraperitoneal temperature combined with cisplatin did no  apparent effect on canine normal physiological function either; performed  3 times of CCCHP at 42℃, 43℃ both  had different degree of impacts on canine vital signs and liver and kidney functions, liver, kidney, spleen and intestinal tissues were injuried at different degree.

Conclusions:  3 times of CCCHP at 41℃  intraperitoneal temperature, each time for 1.0h is safe and feasible, can be used as relatively safe treatment temperature of CCCHP combined with chemotherapy ;  Under the same condition at 42℃;  mild injury to canine abdominal major organs occured , and was not recommended as a conventional treatment temperature;  Under the same condition at 43℃; severe damage were found in canine normal physiological functions , and not suitable as   CCCHP treatment temperature.

 【Key words 】Coelom Continued Circulatory Hperthermia Perfusion；

               security; thermal injury; canine

Malignant ascites is an abnormal aggregation of peritoneal cavity effusion when malignant tumor development to the abdominal cavity, common in breast, ovarian, gastric, bronchial, pancreas, colon and other parts of the tumor, seriously influence the patient's survival and life quality . Previous treatment mainly depended on diuretic and repeated massive paracentesis, and the routine tumor therapy such as operation, chemotherapy, and the therapeutic efficacy  were limited. In recent decades,  Coelom Continued Circulatory Hyperthermia Perfusion(CCCHP ) , as a new therapeutic method in the treatment of abdominal malignant peritoneal metastasis had achieved satisfactory curative effect, and was widely used in all over the world ^[1-3]. But at present ,the understanding of the CCCHP and the implementation method of the existing device are not uniform, the experimental data and reports in heat injury of large animals were deficient, safety and effective treatment condition remains need  further studied in clinical practice. In this research, we intended to find out the optimum temperature and other optimal conditions  by the canine model establishment of CCCHP, to explore the injury effect  of CCCHP on canines in local and systemic at different temperature and different treatment conditions, and provide gexperimental basis for the treatment of malignant ascites of the clinical application of CCCHP.

1 Material and Method

1.1  Materials

 Apparatus: Coelom Continued Circulatory Hperthermia Perfusion ( Xi'an Good Doctor Medical Science and Technology Limited Company ), Type - AI  portable high precision digital thermometer ( Xiamen Yudian automation Science and Technology Limited Company ), RM6240 multichannel physiological recorder ( Chengdu Instrument Factory ).

Experimental animals: 16 adult mongrel canines weight 25-30kg, male ( provided by experimental animal center of Tangdu Hospital of The Fourth Military Medical University ).

1.2 Methods

1.2.1 Group  Animals were randomly divided into 4 groups under different experimental conditions : 41℃ normal saline group, 42 ℃ normal saline group, 43℃ normal saline group and 41℃ cisplatin ( DDP ) group, 4 canines in each group , all CCCHP 3 times (one course of   clinical treatment ), each time 1 hour and interval was 2D.

1.2.2CCCHP  In accordance with the requirements of CCCHP, the abdominal cavity puncture needle with 50mm inner diameter was implanted in the peritoneal cavity, and the other end , as the into body passage, was connected with the inlet water pipe which was connected to the temperature sensor ,choose the single mode and unidirectionally  perfused around 3L saline into the abdominal cavity through the pressure pump , and then on the other side, implanted out of body puncture needle in the same way and connected to out of body passage , circulation model can be choosed to perform CCCHP when circulating path was established . Be sure the circulation was smooth, then punctured near by the entering and out of body puncture needles, and left, right upper abdominal respectively, insert Pt100 high precision thermometer.  Regulate the into body temperature and velocity during the circulation in order to reach the preset temperature of intraperitoneal, and maintained 1h. Detect heart rate ( HR ), breathing ( R ), mean arterial pressure ( MAP ) and central venous pressure ( CVP ).

1.2.3 Observation items   Preserved peripheral blood in each CCCHP before and after each CCCHP24h for inspection . Extracted  of peripheral blood for preparation. Recorded the 4 thermometer readings, into and out of body temperature, setting temperature and velocity displayed on the panel of the hyperthermia perfusion system per 10 min during CCCHP. Observed general conditions of animals during perfusion. Sacrificed 2 animals in each group after 3 times of CCCHP in 24h and 2 weeks,  Laparotomy observation of morphological changes of visceral organs, cutted intestine, liver, kidney  and other abdominal viscera tissues, fixed after routine paraffin embedding, sectioned, HE stained, and light microscopic observed on the histological structure.

1.2.4 Statistical analysis  Data were showed by mean± standard error,  and processed with the software of SPSS12.0 data packet , performed pairwise comparison for multiple samples with variance analysis ,   P<0.05 was marked for significant difference. Mapped curves after  temperature data handling.

2Results

2.1Changes of vital signs in experimental animals

1 hour after 41℃ CCCHP, canines body temperature increased by an average of about 0.2℃ , heart rate reached 125±5/min, and breathe rate reached16±5 /min , mean arterial pressure128mmHg. 1 hour after42℃ CCCHP, canines body temperature increased by an average of about 0.5℃ , heart rate reached 138±5/min, and breathe rate reached 23±5 /min , mean arterial pressure133mmHg. 1 hour after 43℃ CCCHP1 canines body temperature increased by an average of about 1.5℃, heart rate reached 145±5/min, and breathe rate reached 30±5 /min , mean arterial pressure decreased about 10mmHg. 1hour after 41℃ cisplatin group CCCHP, canines body temperature increased by an average of about 0.3℃ , heart rate reached 130±6/min, and breathe rate reached 20± 5 /min.

2.2 General symptoms of experimental animals

In 41 ℃, 42 ℃ normal saline group: canines could  intake of food and water 1D postoperative, mental state was well 2D postoperative; In 43 ℃ normal saline group : canines were inappetency, poor mental status, drank 1D postoperative , ate little 2D postoperative; In 41 ℃ DDP group: canines could intake of food and water 1D postoperative, but poor appetite ( may be caused by side effects of cisplatin on gastrointestinal ), mental status recovery well 2D postoperative.

2.3  Liver, renal function changes of experimental animals

In 41℃( including cisplatin perfusion group ) and 42 ℃ normal saline group,  liver and kidney function changes of canines were not obvious, only slight fluctuations were found  after each time of perfusion ( P>0.05),  and returned to normal after 2 weeks ( Table 1, table 2, table 3); CCCHP 3 times in 43 ℃ abdominal temperature,there were some damage on gliver and renal function,values of ALT, AST, CR, UA were significantly elevated ( P<0.05) in 4D, and ALT, AST had not yet returned to normal after 2 weeks  ( P<0.05) ( Table 4).

 

T1: thermometer near by the into body puncture needle

T2: thermometer near by out of body puncture needle

T3: thermometer on left upper abdomen  

T4:thermometer on right upper abdomen

Fig.1  The temperature curve of 42℃ normal saline group at different times in different parts

2.4  Temperature changes in CCCHP ( Figure 1)

 In 42 ℃ normal saline group as the represention, recorded the temperature value at different sites, different time points during perfusion, Mapped with the average value of the data in the 3 tests. Temperature reached a stable level in 20-30min after perfusion basicly, the temperature difference was little in different parts of abdominal cavity (≤ 0.5 ℃).

T1: thermometer near by the into body puncture needle T2: thermometer near by out of body puncture needle T3: thermometer on left upper abdomen  T4:thermometer on right upper abdomen

2.5 General morphology changes of abdominal organs in experimental animals

Laparotomy revealed liver, kidney, spleen, intestine and other organs were not significant damaged in the killed animals in 41℃(normal saline group and DDP group ) and 42 ℃ CCCHP 3 times after 24h ,the abdominal viscera organs were no adhesion after 2 weeks, no obvious abnormalities were observed by naked eyes; abdominal cavity organ adhesion were found in 43℃ CCCHP 3 times after 24h, greater omentum mesentery vascular congestion is obvious, there were small congestion area in liver ,  purple-brown congestion and hemorrhage were found in the bowel , no obvious abnormalities in kidney were observed by naked eyes, there were still small congestion zones in parts of bowel after 2 weeks, but visceral adhesions were remissed ( Figure 2).

2.6 Pathology changes in abdominal organs (Figure 3)

2.6.1 41℃ normal saline group: Liver: hepatic cells were mild edema after 24h of CCCHP 3 times , liver parenchyma was mild congestive; recovered after 2 weeks. Kidney: little amount of inflammatory cell infiltration, some areas had mild hyperemia; 2 weeks later returned to normal.  Spleen: splenic stromal was mild hyperemia and inflammatory cell infiltration, 2 weeks later returned to normal. Intestinal: small intestinal villi structure were normal , but colonic mucosa layer were mild to moderate inflammatory cell infiltration, and there still had the infiltration in some locals after 2 weeks , mucous layer cell edema was obvious.

2.6.2 41℃ DDP group: Liver: hepatic cells were mild edema after 24h of CCCHP 3 times, vascular wall of liver portal area got thickness, mild congestion, little amount of inflammatory cell infiltration ; there were still infiltration in local areas in liver tissue after 2weeks. Kidney : mild inflammatory cell infiltration of glomerular, mild congestion; no obvious abnormalities were found after 2 weeks. Spleen: splenic stromal was mild hyperemia and inflammatory cell infiltration, no obvious abnormalities were found after 2 weeks. Intestinal: hyaline degeneration in submucosal stromal layer , vascular dilatation and congestion, moderate inflammatory cell infiltration of the mucosa; there were only a mild inflammatory cell infiltration in the mucous layer after 2 weeks.

2.6.3 42℃ normal saline group: Liver: 24h after 3 times of CCCHP , liver periportal was  moderate congestion, hepatocytes were extensive mild edema, liver parenchymal cells were spotty necrosis, cells boundaries were unclear; hepatocellular portal was still mild hyperemia after 2 weeks . Kidney: glomerular and interstitial were mild congestion, mild inflammatory cell infiltration; individual renal tubules were still mild congestion after 2 weeks, little amount of infiltration of inflammatory cells, mesenchymal cells edema obviously. Spleen: massive inflammatory cell infiltration were found in splenic stromal, vascular  dilatated congestion and hemorrhage was moderate to severe, splenic trabecula widened obviously; splenic stromal congestived and inflammatory cells infiltrated mild-to-moderate after 2 weeks, splenic trabecula increased. Intestinal: structure of the small intestine villi were normal, but the small vascular of mucosa and submucosa were mild congestion, infiltration of inflammatory cells of the mucosa was moderate to severe , parts of fibrinoid and hyaline degeneration were found in submucosal stromal; infiltration of inflammatory cells were still found in mucosal vascular after 2 weeks, goblet cells hyperplasied of large intestine .

2.6.4 43℃ normal saline group: Liver: 24h after 3 times of CCCHP, hepatic portal vascular dilatated, congestion moderate to severe , wide spread hepatocyte hydropic degeneration, point necrosis of liver cells; hepatic portal vascular dilatation after 2 weeks, congestive severly, widespread hepatocyte necrosis. Kidney: mild congestion of glomerular capillaries , micro-thrombosis, glomerular and renal tubular epithelial cells were edema, renal interstitial congestion was moderate to severe, moderate infiltration of inflammatory cells, brush border of renal tubular epithelial fell off; glomerular still obvious congestion after 2 weeks , moderate to severe inflammatory cell infiltration, widely balloon degeneration of glomerular and renal tubular epithelial cells. Spleen: : severe congestion, edema of spleen cells , nuclear pyknosis, necrosis,  some necrotic cellular debris were visible, hyaline degeneration of small vessels; the spleen was still severe congestion after 2 weeks , splenic trabecula increased. Intestinal: large inflammatory exudation and necrosis were visible in mucosa and submucosa, congestion obviously , hyaline degeneration and massive necrosis cell fragments were seen in submucosa, there were fell off of small intestinal villi structure; little amount of inflammatory cell infiltration in mucosa after 2 weeks, submucosa loosed, small vessels hyperplasied.

 

3 Conclusions

 The Coelom Continued Circulatory Hyperthermia Perfusion we used  can basically ensure the heating stability and the uniformity of intraperitoneal temperature; with the increased of the intraperitoneal perfusion solution temperature, thermal perfusion damage effect gradually aggravate, intestine and liver were heat sensitive especially , but under the condition of 41℃ ,the injury was mild, and restore in a short time, when the temperature was higher, the injury was more serious , and showed irreversible trend. So we suggest that 41℃ intraperitoneal temperature of CCCHP 3 times , each time lasted 1 h is feasible, and can be used as the relatively safe temperature in the treatment of CCCHP combined with chemotherapy.

4 Discussion

In recent years, hyperthermia has been paid more and more attention, it has become the novel method for treating tumors after operation, radiotherapy, chemotherapy, and targeted therapy. Much practice had proved that hyperthermia can not only directly kill tumor cells, inhibite drug resistance gene expression of the tumor cells ^[4] and promotes apoptosis, it can also improve immunity of the body, and mobilized the body to kill tumors. Today, a single treatment method  in patients cannot receive the very good curative effect, the comprehensive treatment of the tumor has become the consensus of people, and then  the combination of heat chemotherapy, radiotherapy and triple therapy appeared. Research had confirmed that the  hyperthermia combined with chemotherapy can shorten the time of drug to reach the effective concentration , increased drug concentration in intracellular, and promote the combination of the chemotherapy drug with the tumor cells, reduce the resistance and inhibit the tumor repairing  after chemotherapy

improve efficacy of topical treatment, reduce the adverse reactions. Hyperthermia cooperate with radiotherapy have sensitizing effect, it can synergistic killing tumor cells at different cell cycle , increased efficacy, while reducing the tolerance and side effects.

   As one kind of  hyperthermia, CCCHP mainly used for the treatment of bad general condition of patients with malignant pleural and ascites effusion in clinical, we choose the canine ( approximate to human abdominal capacity ) as experimental animal, to seek for optimum temperature and other optimal conditions of CCCHP.  According to the requirements that animal experiment should combine with clinical application, we had experiment with one canine at 46 ℃,45 ℃ temperature respectively , as the consequence that the canine died at 46℃ of one time CCCHP in 24h, and died at 45 ℃ of one time CCCHP  after 5D . Therefore, we hypothesized that the tolerable limit temperature of canine for CCCHP 1h was 44 ℃. By the perfusion experiments of 41℃,42 ℃,43 ℃ normal saline group , we hypothesized that 41℃is relatively safe treatment temperature, so we choose the 41 ℃ group for cisplatin thermal perfusion group, to observe the joint damage effect of chemotherapy drugs and temperature.

    It was commonly believed that the temperature of producing the biggest effect on tumor cells was 40-43℃ ^[5-7], temperature below 40 ℃  did little effect, and had no significant efficacy, when the temperature was higher than 43-45℃, tumor cells mainly displayed for disintegrating, necrosis, and did not show the reversal of drug resistance effect, but the experiments had confirmed that the injury of high temperature on normal tissues could not be ignored, and more often in small intestine, and liver complications^[8-10]. Of course, both the thermal sensitivity differences were large in the cases of same kind of animal in different tissues and different animal of the same tissues ^[11], the easily injured organs of canines were liver and intestine, the pathological findings we got were consistent with the foreign literatures ^[12-13]. At the same time, thermal perfusion is usually coordinated with chemoradiation in clinical, therefore it was unnecessary to excessively pursuit for high temperature when we performed coelom hyperthermia perfusion, we should minimize the side effects as far as possible when we try our best to improve the curative effect.

     the general changes and pathological changes of visceral organs in different temperature groups (CCCHP before and after CCCHP 3 times)  in the experiments had showed: at 41℃ abdominal temperature,after 3 times CCCHP ( simple normal saline group and combined with cisplatin group ), every important abdominal organs of canines were subjected to mild acute injury, but normal physiological function had not been damaged, (the function value changes of liver and kidney after 2 weeks P>0.05), so, it could be used as conventional treatment temperature of CCCHP combined with chemotherapy ; at 42 ℃ abdominal temperature  CCCHP 3 times, liver, kidney, spleen and intestinal tissues in canines were subjected to mild injury, 2 weeks later,  some acute reversible injuries recovered, liver and kidney functions were not significantly affected ( the function value changes of liver and kidney after 2 weeks P>0.05),so, it was not recommended to do CCCHP for conventional treatment temperature; at 43 ℃ abdominal temperature 3 times CCCHP,  major abdominal viscera of canines were obviously damaged,  some acute injuries recovered after 2 weeks , but the spleen, liver and intestinal tissues were severely irreversible damaged, necrotic, and did not recover after 2 weeks, so, 43℃ was not suitable for CCCHP treatment temperature.

    Since people can not measure the human coelom  temperature accurately at present, so it is difficult to control the intra-abdominal temperature in clinical hyperthermia perfusion treatment, but we grasped the law of temperature control for the application of Coelom Continued Circulatory Hyperthermia Perfusion through the expriments : because it could approximatly guarantee the fluid heat energy in the coelom distribute evenly (see Figure 1), so , when the temperature tends to be stable, ( in general, 20-30min after the circulation smoothly functioned )   intraperitoneal temperature could be estimated probably by adding 0.5-1℃ on the basis of out of  body temperature displayed on the panel . 

     To sum up, although CCCHP hasd shown better curative effect in many abdominal visceral tumor treatment, but the heat perfusion chemotherapy performed in high temperature and trauma, at the same time, the cytotoxic chemotherapeutic drugs directly functioned in the coelom  in the high temperature environment,

increased the side effects. Therefore, safety implementation conditions of hyperthermal perfusion chemotherapy  are both physician and patient concerns. Because the type of the circulating heat perfusion machine  and the device used in clinical had no unified standards at present, there were difference in the key part of the perfusion pump, heat exchanger, temperature monitor, flow regulating valve and pipeline system ^[14], thus the efficacy would be variant in different cycling operation methods^[15]. The Coelom Continued Circulatory Hyperthermia Perfusion we used could achieve the objective of precise temperature control and continuous perfusion , it utilized saline as heat carrier,  powered with pressure pump  , heated with the inductive heating tank , sensors monitored the temperature of into and out of body , adjust the temperature and velocity through the control knob , the disposable closed pipe connected with the  entry and out of body puncture needle, thereby  basicly guarantee the thermal energy and drug spread evenly over the cavity and viscera. Experiments had showed that 41 ℃ intraperitoneal temperature CCCHP 3 times, each time 1.0h is a safe, effective CCCHP  conventional treatment condition combined with chemotherapy. We also need the further animal experiments, and optimize an optimal balance conditions between the efficacy and side effects, combinedg with the large number of clinical cases and the follow-up of survival period, make the Coelom Continued Circulatory Hyperthermia Perfusion standardized and rationalized gradually , so that more patients can receive the safe, effective therapy.

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