Journal of Cancer Research and Therapeutics

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 16  |  Issue : 7  |  Page : 1617--1624

Experimental study on embolization of rabbit renal artery with gelatin sponge microspheres


Xu Zhao1, Zhe Wang2, Guangsheng Zhao3, Yuewei Zhang4, Min Ji1,  
1 Departement of Interventional Radiology, The Affiliated Sixth Peopleæs Hospital of Dalian Medical University, Dalian 116031, P. R. China
2 Departement of Medical Oncology, Affiliated Zhongshan Hospital of Dalian University; The Key Laboratory of Biomarker High Throughput Screening and Target Translation of Breast and Gastrointestinal Tumor, Dalian University, Dalian 116001, P. R. China
3 Departement of Interventional Radiology, Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, P. R. China
4 Hepatobiliary and Pancreatic Center, Beijing Tsinghua Changgeng Hospital, No.168 Litang Road Changping District, Beijing 102218, P. R. China

Correspondence Address:
Min Ji
Departement of Interventional Radiology, The Affiliated Sixth Peopleæs Hospital of Dalian Medical University, No.269 Huibai Street, Dalian 116031
P. R. China
Yuewei Zhang
Hepatobiliary and Pancreatic Center, Beijing Tsinghua Changgeng Hospital, No.168 Litang Road Changping District, Beijing 102218
P. R. China

Abstract

Objective: The objective of this study was to evaluate the degradation characteristics and embolic effect of gelatin microspheres (GMSs) produced domestically in China through an experimental study comparing the embolization of rabbit renal arteries using GMSs and tris-acryl microspheres. Materials and Methods: Sixteen healthy adult New Zealand white rabbits were randomly divided into two groups. Group A was embolized with GMSs produced in China with a diameter of 150–200 μm (n = 8), and Group B was embolized with tris-acryl microspheres with a diameter of 100–200 μm (n = 8). The renal arteries were embolized through femoral artery puncture and catheterization. Renal artery angiography rechecks and hematoxylin and eosin staining of tissue sections were performed at 1 day, 4 days, 7 days, and 14 days after embolization, respectively, to observe vascular recanalization, degradation of microspheres, and embolic effect. Results: Group A: Digital subtraction angiography showed complete recanalization at 14 days. The changes in embolic necrotic areas at different time points after embolization were similar in the two groups. At 4 days after embolization, changes in glomerular structure were observed in the kidney on the embolic side. At 7 days after embolization, atrophy, degeneration, and necrosis of the glomeruli, as well as degeneration and inflammatory cell infiltration of the renal tubules, were observed in the kidney on the embolic side. At 14 days after embolization, extensive atrophy and hyalinization of the glomeruli were observed, and local renal tissue showed patchy fibrosis with calcification of internal tissue. Hyperplasia of fibrillar connective tissue was observed in the renal interstitium. Conclusion: The GMSs produced domestically in China can be completely degraded after embolizing blood vessels for 14 days. The GMSs are similar to tris-acryl microspheres in arterial embolization effect and are safe and effective.



How to cite this article:
Zhao X, Wang Z, Zhao G, Zhang Y, Ji M. Experimental study on embolization of rabbit renal artery with gelatin sponge microspheres.J Can Res Ther 2020;16:1617-1624


How to cite this URL:
Zhao X, Wang Z, Zhao G, Zhang Y, Ji M. Experimental study on embolization of rabbit renal artery with gelatin sponge microspheres. J Can Res Ther [serial online] 2020 [cited 2021 Apr 13 ];16:1617-1624
Available from: https://www.cancerjournal.net/text.asp?2020/16/7/1617/308753


Full Text



 Introduction



Arterial embolization is one of the most commonly used methods in interventional radiotherapy and plays an important role in the treatment of tumor and various hemorrhagic diseases.[1],[2],[3] The clinical and basic research of microparticle embolic agents is a hot topic at home and abroad.[4] There are many types of embolic microparticles or microspheres, which can be divided into two categories according to whether the embolic materials can be absorbed in the body, namely absorbable embolic agents (temporary) and nonabsorbable embolic agents (permanent). At present, permanent embolic agents are the main types of particulate embolic agents commonly used in clinical practice; in the past, our research team published some clinical research on the gelatin sponge particles as an embolic agent in the treatment of liver tumor.[5],[6] Until to now, the safety and efficacy of gelatin sponge microspheres on blood vessels in animal models has not been reported.

Gelatin sponge particles have the advantages of low price, degradability, and repeatable operation and are thus widely used in clinical practice.[7],[8] However, traditional gelatin sponge particles are cut manually, resulting in uneven size and irregular shape. Commercially available gelatin sponge particles are medium-term embolic agents. Various diameters are provided according to the size of blood vessels. The diameters of these particles are more consistent with the size of blood vessels. They have good scalability and compressibility, which enhances the target of embolism and makes them feasible for regional embolism.[9] Our study confirmed that transarterial chemoembolization using gelatin sponge particles is safe and effective in the treatment of tumors.[5],[6] However, gelatin sponge particles are irregular in shape, have a large expansion coefficient, and cannot reach accurate and uniform size; accordingly, their degradation time is inconsistent. In order to produce an embolic agent with uniform degradation time, Peking University School of Pharmaceutical Sciences prepared gelatin microspheres (GMSs) based on gelatin sponge particles, which are spherical, uniform in size, homogeneous, and degradable. Tris-acryl GMSs (trade name: Embosphere Microspheres) are a permanent spherical embolic agent with high coincidence rate of blood vessels after embolization, and the embolic effect is widely recognized.[10] In this study, the characteristics of domestically produced GMSs are preliminarily assessed by comparing the GMSs and tris-acryl microspheres in terms of degradation characteristics and embolization effect.

 Materials and Methods



Materials

The study protocol was approved by the animal experimentation committee, and the experiments were performed according to the Animal Care Guidelines of Dalian University.

GMSs produced domestically in China were prepared by Peking University School of Pharmaceutical Sciences, with a diameter of 150–200 μm and specification of 100 mg/bottle.

Tris-acryl gelatin microspheres, Embosphere icrospheres (TAGM), were purchased from BioSphere Medical (Rockland, MA, USA), with a diameter of 100–200 μ m and specification of 100 mg/bottle.

Methods

Sixteen healthy adult New Zealand white rabbits (provided by Dalian University Experimental Animal Center) weighing 2.5 ± 0.5 kg, without specification on sex, were randomly divided into Group A and Group B. The eight animals in Group A were embolized with GMSs produced in China in the right renal artery. The eight animals in Group B were embolized with Embosphere Microspheres in the right renal artery.

Anesthesia was induced by injection of 1% pentobarbital sodium 30 mg/kg into the ear vein. The experimental rabbit was then positioned on a self-made rabbit table so that its abdomen was fixed upward, and the table was placed on the digital subtraction angiography (DSA) operating table. The skin in the right inguinal region was prepared for operation, routinely disinfected, and spread with a sterile drape. An incision was made on the skin along the direction of the femoral vein, and the tissues were bluntly separated layer by layer to expose the femoral artery sheath. The femoral artery and vein were separated with gentle operation, a 15 cm surgical suture was passed horizontally through the proximal end and distal end of the femoral artery, and then, the femoral artery was punctured. The distal suture was gently lifted by the left hand, and the right hand held a 5-gauge intramuscular injection needle, which carried a 0.018 mm microguidewire for single-wall puncture. After successful puncture, the guidewire was introduced into the abdominal aorta. The guidewire was inserted into the catheter sheath, and the sutures at the proximal end of the femoral artery were cross-fixed with the purposes of hemostasis and catheter sheath fixation. The self-shaping 3F microcatheter was placed at the level of the 12th thoracic vertebra through the catheter sheath along the guidewire. Abdominal aortography was performed to confirm the status of both renal arteries. Then, the guidewire was selectively inserted into the right renal artery. The head end of the guidewire was used to locate the renal artery opening at L1 and L2 levels of the abdominal aorta. Insertion or direction change of the head end of guidewire indicated that it was entering the renal artery. The catheter was then inserted following the head end, and the guidewire was withdrawn for angiography to show the details of the renal artery branches on this side. The renal artery opening was completely embolized, and the embolic agent mixed with contrast agent was injected until the blood flow of the target vessels stagnated or refluxed. Angiography was performed again with the ultra-microcatheter placed in the renal artery trunk after an interval of 3–5 min to determine the embolization of the target vessels. After the operation, the catheter was withdrawn, the right femoral artery was ligated, the incision was sutured layer by layer, local disinfection was performed, and the surgical wound was wrapped and fixed with gauze.

DSA was performed on 2 rabbits randomly selected from each group at 1 day, 4 days, 7 days, and 14 days after embolization to observe whether the blood vessels were recanalized. Euthanasia was performed at 1 day, 4 days, 7 days, and 14 days after angiography, respectively. The kidneys were removed. The specimens were cut into small pieces of tissue according to pathological section requirements in infarcted area, tissues around infarcted area, and normal tissues of kidneys at each time period after embolization. After dehydration and paraffin embedding, paraffin blocks were made. Hematoxylin and eosin staining was performed after sectioning, and the sections were observed under a light microscope. Evaluation was carried out according to the histology of microsphere degradation [Table 1], the morphological changes of GMSs and tris-acryl microspheres were observed, and renal parenchyma changes in the embolic region were assessed.{Table 1}

Statistical processing

SPSS 17.0 software package (IBM, New York, USA) was used for analysis. Measurement data were expressed as mean ± standard deviation. t-test was used for pairwise comparison, and the difference was statistically significant when P < 0.05.

 Results



Digital subtraction angiography and embolization results

All experimental rabbits were successfully embolized, and angiography was performed immediately at 5 min after embolization to show renal artery trunk truncation [Figure 1]a and [Figure 1]b. Angiography reexamination was carried out at 1, 4, 7, and 14 days after embolization, respectively. Group A: No recanalization was found at 1 and 4 days after embolization [Figure 2]a and [Figure 2]b; some vessels were recanalized at 7 days [Figure 3], and all embolized vessels were completely recanalized at 14 days [Figure 4]. Group B: No vascular recanalization was found at 1, 4, and 7 days after operation [Figure 5]a and [Figure 5]b and [Figure 6]; some vessels were recanalized after 14 days [Figure 7].{Figure 1}{Figure 2}{Figure 3}{Figure 4}{Figure 5}{Figure 6}{Figure 7}

Histopathological results

Light microscope observation

Both the groups of microspheres were mainly concentrated in the interlobular arteries, interlobar arteries, and arcuate arteries and some in the renal hilum segmental arteries. GMSs in Group A were mainly spherical under the microscope, and some could change with the shape of blood vessels, such as an oval shape, which showed pale pink after HE staining. At 1 day after embolization, observation showed that the coincidence rate of target vessel embolization was high. Most of the embolized vessels were occupied by only one microsphere, which remained spherical in the arterial lumen and had a small gap with the lumen, achieving dense embolism. No thrombus covered or occupied the surface of the microspheres, and the degradation level was 0 [Figure 8]. At 4 days after embolization, observation showed that the target vessels were still embolized, the microspheres were slightly deformed, no thrombus had formed, and the degradation level was Grade I [Figure 9]. At 7 days after embolization, the microspheres were significantly deformed, thrombosis was seen around them, and there was a gap between the microspheres and the vessel wall. The degradation level was Grade II [Figure 10]. At 14 days after embolization, no microspheres were found in the target vessels, thrombus filling was found, and the degradation level was Grade III [Figure 11]. The HE staining of the Group B tris-acryl microspheres also showed pale pink, mainly showing that the spherical shape did not change with the shape of blood vessels. At 1 day after embolization, the target vessels were embolized accurately, most of the embolized microspheres had a high coincidence rate with the vessels, a small number of microspheres aggregated, there was a gap between the microspheres and the lumen of the target vessels, compact embolism was not achieved, and no thrombus was formed [Figure 12]. At 4 days after embolization, the microspheres in the target vessels still showed spherical shape without change, and a small amount of thrombus filling was found among the microspheres [Figure 13]. At 7 days after embolization, the microspheres were not deformed, and the surrounding thrombi were aggravated [Figure 14]. At 14 days after embolization, most of the microspheres were still not deformed, and a small number were irregular and pointed, with a degradation level of 0 [Figure 15].{Figure 8}{Figure 9}{Figure 10}{Figure 11}{Figure 12}{Figure 13}{Figure 14}{Figure 15}

The changes in embolic necrotic areas at different time points after embolization were similar in the two groups. At 4 days after embolization, changes in glomerular structure were observed in the kidney on the embolic side [Figure 16]a and [Figure 16]b. At 7 days after embolization, significant atrophy, degeneration, and necrosis of the glomeruli, as well as degeneration and inflammatory cell infiltration of the renal tubules, were observed in the kidney on the embolic side [Figure 17]a and [Figure 17]b. At 14 days after embolization, extensive atrophy and hyalinization of the glomeruli were observed, and local renal tissue showed patchy fibrosis with calcification of internal tissue. Hyperplasia of fibrillar connective tissue was observed in the renal interstitium [Figure 18]a and [Figure 18]b.{Figure 16}{Figure 17}{Figure 18}

 Discussion



Gelatin microspheres

A new type of GMS produced in China is a degradable spherical embolic agent prepared by Peking University School of Pharmaceutical Sciences using gelatin sponge particles. It is prepared by dispersing gelatin in liquid paraffin and cross-linking with formaldehyde. The main reaction mechanism between gelatin and formaldehyde is the formation by formaldehyde of a methylene bridge between the lysine side chain ε-amino group and the arginine side chain guanidine group in gelatin peptide chain.[11] Making gelatin molecules form cross-linking bonds. Besides the structure of gelatin itself, the cross-linking degree of gelatin is mainly controlled by reaction conditions.[12],[13],[14] The degradation time of GMSs can be controlled by changing the concentration of cross-linking agent and the cross-linking time.[15]

All experimental rabbits were intubated through the right femoral artery puncture approach to embolize the right renal artery. As for the degree of embolization, the right renal artery and renal hilum segmental artery were not visualized, and the operation was successful. GMS group: No vessel recanalization was found at 1 or 4 days after operation, partial vessel recanalization was found at 7 days, and the embolized vessels were completely recanalized at 14 days. The number of microspheres in cases containing embolization material varied between cases and from slide to slide. They were seen in intravascular spaces. They could be seen individually or in clusters within the stroma. The case was consistent with previous report.[16] The recanalization time was consistent with those reported by Ohta et al. The embolized vessels were completely recanalized at 14 days. Pathological examination showed that the microspheres were completely degraded. The degradation time can be controlled from 3 days to permanent by changing the degree of cross-linking.[17] Tri-acrylic microsphere group: No vascular recanalization was found at 1, 4, and 7 days after operation, and partial vascular recanalization was found at 14 days. Pathological examination showed that at 1 day after embolization with tris-acryl microspheres, the target vessels were still embolized, microspheres aggregated in most of the embolized vessels, the gaps between the microspheres and the lumen of the target vessels were relatively large, compact embolism was not achieved, and no thrombus had formed. At 4 days after embolization, the microspheres in the target vessels maintained spherical shape without change, with a small amount of thrombosis. At 7 days after embolization, the microspheres were not deformed, and the surrounding thrombi were aggravated. At 14 days after embolization, most of the microspheres were still undeformed, while a small number of microspheres were irregular, pointed, and slightly deformed and cracked. As for the phenomenon of partial cracking and recanalization of tris-acryl microspheres as a permanent embolic agent, the results were similar to those reported by Stampfl et al.[18] However, the cause was not clear, which might be related to the unstable anchoring of microspheres to the embolic vessels, thrombus absorption, and the delayed migration of the embolic agent to distal vessels due to ischemia and hypoxia which regulated the opening of capillary network on the embolic side, resulting in redistribution of intravascular embolism. In addition, the microspheres absorbed a contrast agent to expand during embolization and became dehydrated and smaller after embolization, which might play a certain role in recanalization after embolization. Permanent embolic agents can only completely block the target tumor arteries and should not embolize arteries in the tumor region; otherwise, they will cause serious liver function damage. GMSs have the advantage of degradability; it is feasible for blocking the tumor-supplying arteries in the region, which avoids the formation of collateral-supplying arteries and serious complications such as ectopic embolism.[19] The tumor had reached liquefaction and necrosis at 3 h after embolization, and the microspheres had degraded in about 14 days; the recanalization of blood vessels was conducive to the reperfusion of tumor and control of tumor growth.[20],[21]

Embolic effect of gelatin microspheres

The necrosis process after renal embolism in the two groups was similar, and both the groups achieved complete necrosis in the embolized part of the kidney. Histologically, the embolizing agent with the same diameter showed no significant difference in the diameter of embolized blood vessels in the two groups. Both microspheres could be located in the interlobar arteries, arcuate arteries, and some in the renal hilum segmental arteries and renal artery trunk. In a study conducted by Ohta et al. on GMSs and gelatin sponge particles in rabbit renal arteries, 24 experimental rabbits were divided into 3 groups according to the different diameters of gelatin sponge microspheres, and the kidneys of experimental rabbits were embolized with GMSs (35–100 μm, 100–200 μm, and 200–300 μm). The results showed that the size of embolized necrotic area was related to the size of microspheres, and the embolism in the 100–200 μm group reached the interlobar arteries.[22] The embolic level of blood vessels studied in this experiment was consistent with the above study.

 Conclusion



The GMSs produced domestically in China were used to embolize blood vessels, the blood vessels were recanalized after 14 days, and the microspheres were completely degraded. These GMSs and tris-acryl microspheres are similar in arterial embolization effect and are safe and effective.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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