Assessment of embolization effect with temperature-sensitive p(N-isopropylacrylamide-co-butyl methylacrylate) nanogels in rabbit renal artery by CT perfusion and pathology

The temperature-sensitive p(N-isopropylacrylamide-co-butyl methylacrylate) (PIB) nanogel is a novel embolic agent, but methods to assess its performance are limited. The study aimed to assess the effect of embolization with PIB nanogels in rabbit renal artery by non-invasive computed tomography (CT) perfusion and pathology to evaluate the feasibility of the nanogel as a blood vessel embolization agent. Ten healthy adult Japanese rabbits underwent right renal arterial embolization with PIB nanogels. CT perfusion scans were performed pre- and post-treatment at different time points (1, 4, 8, and 12 weeks). Two rabbits were euthanized and histologically examined each time, and the remaining rabbits were sacriced at 12 weeks after the embolization. The ecacy of the nanogels was further conrmed by pathological examination. The renal volume and renal blood ow of the right kidney signicantly decreased post-treatment during the 12-week CT re-examination (both P < 0.05). No recanalization or collateral circulation was observed during this period for the PIB nanogel, which dispersed in blood vessels of all levels. The CT perfusion outcome showed changes in the kidneys similar to the pathological result. The embolic effect of PIB was good dispersion and permanency, and could be evaluated by non-invasive and quantitative CT perfusion.


Introduction
With the rapid development of interventional radiological technology, blood vessel embolism materials (including metal coils, anhydrous ethanol, Lipiodol, onyx, and Polyvinyl Alcohol (PVA)) have expanded the clinical indications, such as treatment of postpartum haemorrhage, gastrointestinal bleeding 1-3 , preoperative embolotherapy of tumours 4,5 , and embolization of vascular malformations 6 . However, the clinical application of these materials has some complications, such as spring coil or gel drop caused by obstructive nephropathy 7,8 , ethanol embolism secondary pulmonary hypertension 9 , and high concentrations of onyx with neurotoxicity 10 ; additionally, Lipiodol embolization is often incomplete or totally eradicated by tissue. Therefore, nding an effective method to solve the above problems is key.
Temperature-sensitive nanogels have gained considerable attention during the last decades because they undergo reversible and rapid volume phase transitions in response to changes in ambient temperature.
First, these nanogels have good uidity in ne catheters because they remain in the liquid solution state at a lower critical solution temperature (LCST). Second, their small size enables them to overcome some biological barriers. Furthermore, in situ formation could minimize trauma and ease the administration of nanogels as implants. In addition to good dispersibility and permanent embolism, the p(Nisopropylacrylamide-co-butyl methylacrylate) (PIB) nanogels made by our team has less in ammatory vascular response, better uidity, and better operability than do PVA-embolized particles and Lipiodol 11,12 .
Temperature-sensitive p(N-isopropylacrylamide-co-butyl methylacrylate) nanogels have been used as novel blood vessel embolic materials to solve the dilemma of peripheral artery embolization and permanent embolization [11][12][13] . With the progress in preparation technology, materials have become more compatible and more convenient.
Some researchers have attempted to use digital subtraction angiography (DSA) to assess the long-term effect of embolization [11][12][13][14] . However, a major drawback of DSA is invasion. Computed tomography (CT) perfusion imaging is a new, non-invasive technology that can provide data about renal morphological changes as well as quanti ed information of bilateral renal blood ow 15 .
In our previous study 12 , PIB was rst developed as a novel temperature-sensitive blood vessel embolic agent for addressing the dilemma of owability and embolization in transarterial chemoembolization of hepatocellular carcinoma. The purpose of this study was to observe the feasibility of PIB as a blood vessel embolization agent through CT perfusion imaging and pathological examination, thus providing an experimental basis for the clinical application of a novel embolic agent.

Experimental materials
The visible embolization materials of the PIB nanogels demonstrated in this experiment were invented at the National Engineering Research Center for Nanomedicine, College of Life Science and Technology, Huazhong University of Science and Technology. PIB nanogels are new kinds of temperature-sensitive nanogels that can remain in a liquid state at a low temperature and become a gelatinous solid at temperatures above their LCST. Because it undergoes a transition from a owable sol phase to a solidi ed gel phase at its LCST (35°C), the PIB nanogel is a liquid state at low temperature (25°C) and becomes a solid white gel at high temperature (37°C) (Fig. 1).

Study design
The experiment (n = 10) was designed to assess the dispersion of the PIB nanogel and to monitor its residue time. Rabbits were treated by right renal arterial embolization (RAE) with PIB nanogels and underwent CT perfusion pre-and post-treatment at different time points (1, 4, 8, and 12 weeks). E cacy was con rmed by a pathological examination after CT perfusion.

Animal model preparation
Ten healthy adult Japanese long-eared white rabbits were provided by the experimental animal breeding plant of Tongji Medical College, Huazhong University of Science and Technology. The rabbits were aged 3-5 months, had body weights between 2.5 and 3 kg, and were in either gender. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology and are consistent with animal care guidelines.

Vascular embolization protocol
First, the animals fasted for 12 hours before each procedure. Sodium pentobarbital (2.0 wt.%, 30 mg/kg) was injected intravenously as anaesthesia. Then, interventional procedures were performed using a DSA unit with strict sterile technique. Finally, the right femoral artery was surgically isolated, and an 18-gauge sheath needle was inserted. The PIB nanogels were injected into each rabbit with an average dose of 1.5 ml per rabbit (Fig. 2).

Perfusion CT
The parameters of the perfusion CT scanning and contrast agent injection are summarized in Table 1. A 320-detector row CT scanner (Aquilion One; Toshiba Medical Systems) was used in volumetric scan mode. A 22-gauge catheter was placed in the ear vein of the rabbit, and a nonionic contrast material (Iopamiron 350; Bayer HealthCare) was administered with a power injector (Ulrich CT Plus 150, Ulrich Medical). The scan area of the perfusion CT was set to cover at least both kidneys. During the scan, the rabbits were restrained and held still but were allowed to breathe. A rumpled towel was xed against the subcostal abdominal wall of the rabbits using an elastic abdominal binder to minimize respiratory motion. Perfusion was calculated using the maximum slope model (Body Perfusion; Toshiba Medical System), and the results are shown in ml/min per 100 ml; additionally, the colour perfusion maps of renal blood ow (BF) were obtained.

Pathology
Macroscopic and microscopic pre-and post-RAE treatment At different time points (1,4,8, and 12 weeks), the rabbits were euthanised after the CT perfusion was completed, and the kidneys were harvested for general pathological observation. Then, a coronal incision of the kidneys was performed, and a dividing ruler was placed next to the kidneys. Macroscopic observation was collected at each time point to note the size, shape, texture, colour, cortex, and medullary infarction of the right kidney after the RAE, in addition to observing the presence of any surviving kidney tissue. The post-RAE renal tissues were xed in 10% formalin for 48 hours, dehydrated in graded alcohol, and embedded in para n. Then, 4-5 μm sections were cut. The tissue samples were then stained with Masson's trichrome at different time points for evaluating ultrastructural changes and estimating the effects of the treatments.

Statistical analysis
Statistical analysis was performed using SPSS 20.0 and Microsoft Excel. The mean values and standard deviations are presented as (`x ± s). Paired-samples t tests were used to assess the differences in the renal volume and BF in the right kidney pre-and post-treatment during the 12 weeks.

Dispersion and permanency
During the arterial embolization procedure, all levels of renal arteries (large, small and peripheral) could be embolized with 2 mL of PIB nanogels by adjusting the injecting rate at 0.15 ml/s. This result implied that the PIB nanogels dispersed well in the blood vessels. A perfusion CT examination was performed to obtain the correlation quantitative index of paired renal volume and renal BF. These data showed that the right renal volume decreased and that the left side compensatorily increased. The right renal BF decreased to zero after the right RAE (Fig. 3). Signi cant differences were found in the renal volume (P = 0.002) and BF (P = 0.001) of the right kidney pre-and post-treatment during the 12 weeks. In addition, during the long-term follow-up CT examination, the right kidney showed no changes in BF reperfusion and revascularization after embolization (Fig. 4). In addition, the macro-pathological pictures (Fig. 5) of RAE at four different time points (1, 4, 8, and 12 weeks) showed that the right kidney gradually shrank after embolization, combined with the compensatory enlargement of the left kidney. At the rst week, the embolized kidney was nearly yellowish white, and the renal medulla was ischaemic. At the fourth week, the embolized kidney was nearly yellow, the volume shrank, and the texture became hard, but the renal edge was yellowish white. At the eighth and twelfth weeks, the colour of the right kidney was pale, the volume was smaller, and the texture was harder. The calci cation of the right renal tissue was observed. Distinguishing between the cortex and medulla was di cult, and no abscess was formed at last. The changes in the right renal volume between gross pathology and CT perfusion imaging were the same. All of these results indicated that PIB nanogel dispersion contributed to an effective embolization within 12 weeks. The histological results of the right kidney indicated that the nanogels were immobilized in the renal arteries and branches throughout the entire experimental period (Fig. 6). The renal cells and tissues maintained their intact structure and morphology at 1 week post-embolization, while oedema and coagulative necrosis occurred (a) and began to disintegrate at 4 weeks post-embolization (b). After embolization for 8 weeks, the pyknosis, rupture, dissolution and disappearance of renal cells indicated that PIB nanogel embolization resulted in an obvious embolic gangrene, and the gel was still in the blood vessels (c). The structure and morphology of the renal tissues were entirely destroyed, and signi cant calci cations were found at 12 weeks after embolization (d).

Discussion
In this study, we successfully performed embolization with PIB nanogels dispersion in rabbit renal arteries through CT perfusion scan and histological examinations before and after embolization.
Thermally-induced gelling systems have gained growing attention over the last decade. A smart polymer material can remain in a liquid solution state at LCST and become a gelatinous solid above LCST. With a transition temperature close to physiological temperature, smart polymer materials have been reported in blood vessel embolic materials 13,16 , drug delivery [17][18][19] , precise cancer therapy 20,21 , tissue engineering 22 , and cell sheet engineering 23,24 . Most of the studies have focused on phase behaviour theory, drug delivery and promising biomedical applications. However, clinical follow-up of the treatment effect is equally important, and there are few animal experiments in this area.
The ideal vascular embolization material has the following characteristics: good dispersibility, radiopacity, biocompatibility and embolism permanency 16,25 . Recently, gel performance has been greatly improved over the past decades, in terms of the viscosity, safety, radiopacity, phase transition temperature. In previous studies, PIB nanogels were con rmed to be nonadhesive, controllable, and not likely to cause severe in ammatory reactions of the vessels 11 . The TAE treatment is highly dependent on the dispersion and integration of the embolization material. The experimental results showed that the application of PIB nanogels dispersion on right renal embolization was effective, uniform, and persistent. This phenomenon led to a signi cant drop in the right kidney volume and BF after embolization. BF, atrophy and calci cation were observed in the right kidneys. The PIB nanogels remained in the blood vessel during the 12-week follow-up by evidence of pathologic examination. Zhao 11 studied the dispersion of the gel by different injection rates, while the long-term effect of the treatment was not observed by imaging methods and was unknown. In this study, we observed the dispersion of PIB in the right kidney and evaluated both the long-term effect of treatment by CT and pathology in a 12-week follow-up examination.
Assessing treatment e cacy is critical for any embolic material. In this experiment, we evaluated the embolization effect of the right renal artery using CT perfusion, a major advantage of which is noninvasion. In addition, this method can not only depict morphologic characteristics of the kidney but also provide additional quantitative factors that could in uence the embolization effect. During follow-up, the volume of the right kidney decreased gradually after embolization, while the volume of the left kidney increased compensatively after the operation and then decreased slightly. After embolization of the right renal artery, the right renal BF decreased signi cantly to 0, and the left renal BF increased compensatively.
The lack of vascular enhancement re ects necrosis and is regarded as successful treatment 26 , demonstrating that the gelling agent has excellent permanency. The results of pathology further verify that the judgement of CT perfusion is correct, so evaluating the embolic effect is feasible by CT perfusion.
Nevertheless, there were some limitations to the current study. Not all the catheters are of good quality, and there were other personal factors. Moreover, the sample quantity was limited, the results were authentic yet still limited, and the sample quantity needs expanding to be compensated in a further study. In addition, we have not added chemotoxic agents to PIB to evaluate the rate of drug use. Studies regarding these properties of our new gel are now ongoing.

Conclusion
The effect of embolization on PIB was good, with good dispersion and permanency. The nanogel can reach the peripheral blood vessel and remain in it for a long time; thus, this gel is a good blood vessel embolic material and can be used in vivo. CT perfusion can be used to non-invasively and quantitatively evaluate the embolization effect of temperature-sensitive nanogels.