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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