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Transcatheter Arterial Infusion with Heated Saline Changes the Vascular Permeability of Rabbit Hepatic Tumors

Posted Nov 30, 2011 Updated Apr 10, 2023
By Wei Cao MD , Qiang Lu MD , Jing-hua Li MD , Chang-xi Zhou MD , Jia Zhu BA , Yi Wan PhD , Yu-Feng Liu BA
10 min read

Rationale and Objectives

The vascular permeability of tumors can be changed by transarterial infusion heat, but the mechanisms remain unknown. The aim of this study was to analyze the underlying causes of changes in tumor vascular permeability after heated perfusion via two different modes.

Materials and Methods

Thirty rabbits with VX2 hepatic tumors were randomly divided into three groups of 10 rabbits each. The hepatic artery was selectively catheterized via a femoral approach, and unheated saline (control group) or heated saline (60°C) was then injected in either a continuous (transcatheter arterial continuous perfusion [TACP]) or a pulsed (transcatheter arterial pulsed perfusion [TAPP]) manner. Changes in vascular permeability in the tumors were assessed using the following markers and methods: (1) qualitative assessment by visual estimation on digital subtraction angiography performed after the heat infusion procedure on live animals and quantitative assessment by spectrophotometry using Evans blue dye extravasation on tumor and liver tissue after animals were sacrificed and (2) kinase domain receptor or vascular endothelial growth factor (VEGF), expressed in vascular endothelial cells, assessed by immunohistochemical staining, Western blot analysis, and reverse transcription polymerase chain reaction.

Results

Tumor staining increased in the TAPP group more than in the TACP group, but not in the control group, assessed on digital subtraction angiography. Extracted dye was higher in tumors in the TAPP group than in those in the TACP group; extracted dye in both groups was higher than in the control group. Kinase domain receptor protein and messenger ribonucleic acid expression were both higher in the TAPP group than in the TACP and control groups. VEGF protein expression was lower in the TAPP and TACP groups than in the control group, but VEGF messenger ribonucleic acid expression was higher in the TACP group than in the TAPP and control groups, and VEGF messenger ribonucleic acid expression was lower in the TAPP group than in the control group.

Conclusions

The vascular permeability of rabbit VX2 tumors significantly increased after arterial pulsed heated infusion, and the protein kinase domain receptor may play a key role in this increase of tumor vascular permeability.

Therapies using hyperthermia have gained acceptance for the treatment of hepatocellular carcinoma, such as radiofrequency ablation , microwave coagulation therapy , laser-induced interstitial thermotherapy , and percutaneous hot saline injection therapy . These local thermotherapy methods have proven effective in patients with single, nodular-type hepatocellular carcinomas <5 cm, and they are designed to produce heat-induced coagulation necrosis . However, during treatment for patients with hepatocellular carcinomas >5 cm or with multifocal tumors, to reach all areas of the tumor and to induce complete necrosis, percutaneous puncture must be performed repeatedly, and the insertion site must be changed , so the rate of complications, such as arterioportal shunts and bile duct injury, may be up to 12% , and a needle-track tumor-seeding rate of 12.5% has been reported in patients after percutaneous radiofrequency ablation . In contrast, if the heat is distributed throughout the tumor by transhepatic arterial infusion, the insertion site need not be changed, and the complications of these local thermotherapy methods may be avoided. Thus, transarterial infusion with heated drugs may be a safe and promising new strategy that is able to achieve a better therapeutic effect in the treatment of both large and multifocal hepatic tumors.

Recently, regional hyperthermic hepatic perfusion has been used in the clinic in combination with chemotherapy to enhance the efficacy of cancer treatment . In vitro studies have demonstrated that the best anticancer effect is achieved at 42°C to 45°C for 30 minutes , and we have verified that the temperature of heated saline (60°C) that reaches the region of the tumor is not <42°C after injection and that perfusion of 60°C saline is safe and minimally damaging to the normal liver tissue of tumor-bearing rabbits . In vivo studies have also demonstrated that if heated drugs are infused directly into the target vessel of a tumor, the heat of the infusion can change the tumor’s vascular permeability , but the mechanism or mechanisms remain unknown. Because the vascular endothelial growth factor (VEGF) and kinase domain receptor (KDR) proteins are two important factors related to vascular permeability , they were observed and evaluated in this study. The aim of this study was to analyze the cause of the change in tumor vascular permeability after different modes of heated perfusion.

Materials and methods

Animal Models

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Experimental Grouping

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Arterial Catheterization

Temperature Probe Placement

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Hepatic Artery Catheterization

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Digital Subtraction Angiography

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Artery Perfusion

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Evans Blue (EB) Dye Injection

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Catheter Removal

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Animal Sacrifice

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Extravasation of EB Dye

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Hematoxylin and Eosin (H&E) and Immunohistochemistry

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Reverse Transcription Polymerase Chain Reaction (RT-PCR)

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Western Blot Analysis

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Statistical Analysis

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Results

Heating Efficiency

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Changes in Tumor Vascular Permeability

Digital Subtraction Angiographic Findings

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Figure 1, A hepatic artery from the gastric artery of a rabbit (a,d) . As detected with digital subtraction angiographic imaging, the contrast agent (e,f) was more concentrated in the tumor region of the liver ( arrow ) after heated perfusion compared to before perfusion (b,c) , but the tumor staining in the control group displayed no significant changes from before (a) to after (d) perfusion. The tumor staining in the transcatheter arterial pulsed perfusion group (f) was markedly more intense and clear than that in the transcatheter arterial continuous perfusion group (e) after heated perfusion.

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EB Dye Extravasation

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Figure 2, All tumors were located in the rabbits' left hepatic lobes, and the tumors showed nodular growth (a–c) . Typical Evans blue (EB) staining ( arrows ) was evident in the tumor tissue slices of all three groups (d) . Heated perfusion in the pulsed mode significantly increased the EB levels in tumor and normal tissues compared to the other groups (e) . Data are shown as mean ± standard deviation on the basis of 10 independent experiments. ∗ P < .01 versus control group; # P < .01 versus normal tissue; ▾ P < .01 versus continuous group.

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H&E Staining

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Figure 3, Hematoxylin and eosin staining showed no obvious signs of tissue destruction in normal liver tissue ( a , 400×) or tumor tissue in the three groups ( b , 400×; c , 200×; d , 200×). Coagulated blood or plasma was seen in the cavities of tumor microarteries ( arrows ) in the transcatheter arterial continuous perfusion (TACP) group ( c , 200×). Appreciable infiltration by erythrocytes was found in the periphery of the tumors in the transcatheter arterial pulsed perfusion (TAPP) group ( d , 200×). As detected by immunohistochemistry, kinase domain receptor (KDR) protein was mainly expressed in tumor vascular endothelial cells ( e , 400×) ( brown ), and vascular endothelial growth factor (VEGF) protein expression was detected mainly in viable VX2 tumor cells ( f , 400×) ( brown ).

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KDR Protein and Messenger Ribonucleic Acid (mRNA) in Tumor Tissue

Immunohistochemical Staining

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Table 1

Comparisons of KDR and VEGF Protein Levels between Groups After Perfusion

Group KDR Protein Level ∗ VEGF Protein Level † ± + ++ 0 1+ 2+ 3+ Control (37°C) ( n = 10) 4 6 0 0 4 3 3 TACP (60°C) ( n = 10) 0 7 3 1 7 2 0 TAPP (60°C) ( n = 10) 0 2 8 0 8 2 0

KDR, kinase domain receptor; TACP, transcatheter arterial continuous perfusion; TAPP, transcatheter arterial pulsed perfusion; VEGF, vascular endothelial growth factor.

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Western Blot Analysis

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Figure 4, Relative changes in kinase domain receptor (KDR) and vascular endothelial growth factor (VEGF) protein and messenger ribonucleic acid levels in tumor tissue were detected by Western blot analysis and reverse transcription polymerase chain reaction (PCR) after treatment in each group ( n = 10). Bands from protein electrophoresis (a) and PCR products (b) of KDR and VEGF were observed in the different experimental groups. Integrated light density value (IDV) statistical analysis of the KDR and VEGF proteins was performed for the different experimental groups (c,d) . Data are shown as mean ± standard deviation on the basis of 10 independent experiments. ∗ P < .05 versus control group; ▾ P < .05 versus continuous group; # P > .05 versus continuous group.

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RT-PCR

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VEGF Protein and mRNA in Tumor Tissue

Immunohistochemical Staining

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Western Blot Analysis

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RT-PCR

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Discussion

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Conclusions

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Academic Radiology, Volume 18, Issue 12
Journals General Radiology
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