Treatment of Ruptured and Non-Ruptured Aneurysms Using a Semi-Solid Iodinated Embolic Agent

Abstract

Saccular aneurysms (SAs) are focal outpouchings from the lateral wall of an artery. Depending on their morphology and location, minimally invasive treatment options include coil embolization, flow diverter stents, stent-assisted coiling and liquid embolics. Many drawbacks are associated with these treatment options including recanalization, delayed healing, rebleeding, malpositioning of the embolic or stent, stent stenosis and even rupture of the SA. To overcome these drawbacks, we developed a nanoclay-based shear-thinning hydrogel (STH) for the endovascular treatment of SAs. Extensive in vitro testing was performed to optimize STH performance, visualization, injectability and endothelialization in cell culture. Femoral artery saccular aneurysm (FASA) models in rats and in pigs were created to test stability, efficacy, immune response, endothelialization and biocompatibility of STH in both ruptured and unruptured SA. Fluoroscopy and computed tomography imaging consistently confirmed SA occlusion without recanalization, migration, or non-target embolization; STH was also shown to outperform coil embolization of porcine aneurysms. In pigs with catastrophic bleeding due to SA rupture, STH was able to achieve instant hemostasis rescuing the pigs in long-term survival experiments. STH is a promising semi-solid iodinated embolic agent that can change the standard of medical practice and potentially save lives.

Graphical Abstract

We present a novel semi-solid gel embolization agent that can achieve instant occlusion of saccular aneurysms. This approach was developed, optimized and tested in vitro, in 3D models, in vivo in rat and in porcine arterial aneurysms. Most importantly, in ruptured porcine aneurysms causing catastrophic bleeding, the gel embolic material was able to achieve instant hemostasis that was durable in survival experiments and outperformed coil embolization.

1. Introduction

Arterial aneurysm rupture has a very high fatality rate. Aneurysms can be fusiform or saccular in shape and they can occur anywhere in the body. Saccular aneurysms (SAs) carry a greater risk of morbidity and mortality because they are prone to rupture.^[1] The true prevalence of SA is unknown; however, reports suggest an incidence of 6 million by cross-sectional imaging^[2] and 2% by autopsy series in the US.^[3] These aneurysms can be idiopathic, iatrogenic, traumatic, or atherosclerotic in etiology.^[4] Regardless of the cause, SAs warrant close imaging surveillance and timely treatment to prevent a fatal rupture.^[5] Endovascular coiling of SAs has become the standard of treatment as they are associated with higher success rates and lower incidence of peri- and post-operative complications when compared to open surgical approaches.^[6] However, acute failure of metallic coil embolization can occur. Aneurysm rupture followed by coagulopathy in a setting of massive hemorrhage, or bleeding in patients on anticoagulation therapy, can lead to failure of the embolization procedure, significant patient morbidity, and sometimes death. Another drawback to coiling is recanalization of the aneurysm sac, which can occur with inadequate packing of the sac with coils.^[7] Consequently, frequent follow-up imaging is often required to determine the need for retreatment.^[8] Non-invasive imaging with computed tomography (CT) or magnetic resonance imaging (MRI) provide limited information about the embolized aneurysm due to artifacts from the metallic coils; instead, these patients typically receive an intra-arterial catheter directed angiography to evaluate the treated aneurysms.^[9]

Depending on the size, shape and location of the aneurysm, other minimally invasive treatment options include flow diverter stents, stent-assisted coiling and liquid embolics. Flow diverter stents are deployed within the parent artery of the aneurysm without the need to place a catheter or a device inside the aneurysm sac. They work by promoting graduated thrombosis of the aneurysm sac over many months; these stents represent an alternative approach to coiling that avoids entering a potentially fragile aneurysm sac. Flow diverter stents, or other stents in general, require anticoagulation therapy such as dual anti-platelet therapy to prevent intra-stent thrombosis and stroke. However, the use of flow diverters and stents are limited in cases of ruptured aneurysms when anticoagulation is contraindicated.^[10] Moreover, delivery of these stents to the SA can sometimes be prohibitive due to atherosclerotic disease, small vessel size and tortuosity of the target arteries. Another potential drawback of using flow diverters or stents is in-stent stenosis, which may develop over-time and can require treatment using angioplasty.^[11]

Onyx liquid embolic (Medtronic, MN, USA), which is approved for the preoperative embolization of intracranial arteriovenous malformations, recently received ‘Humanitarian Use Device’ approval for aneurysm embolization.^[12] However, in April 2012, the FDA issued a safety alert regarding the dangerous possibility of cementing the catheter to the Onyx released into the aneurysm sac; in 54 reported cases, the catheter could not be removed leading to open surgical rescue and in some cases it resulted in mortality.^[13] Furthermore, Onyx has a 21% rate of incomplete aneurysm occlusion and a 8.2 % procedure- or device-related permanent neurological morbidity,^[14] and carries a 12–36 % chance of recanalization post-embolization.^[15] Major procedural complications also include hemorrhagic and ischemic stroke, distal embolic events, vasospasm, and systemic complications.^[12] These drawbacks have prevented Onyx from becoming an effective replacement for coils in the treatment of aneurysms.^{[14, 16]}

New embolic materials for the management of SAs including aneurysm liners, liquid polymers and hydrogels have also been tested. However, these new approaches have not made it to clinical practice due to a variety of complications such as intraoperative rupture, migration of the embolic agent leading to non-target embolization, incomplete occlusion, lack of biodegradation, the need for specialized delivery devices and instruments, delayed healing, and recanalization.^[17]

Here, we describe a novel formulation of a shear-thinning hydrogel (STH) optimized for enhanced visibility during catheter-based delivery for rapid and durable embolization of SAs regardless of their anatomical location. This material was extensively tested in vitro including mechanical, cell culture and 3D printed models, in vivo in rodent and in porcine aneurysm models and, more importantly, its efficacy was demonstrated in survival experiments of both ruptured and non-ruptured aneurysms mimicking the clinical scenario. Biocompatibility and biodegradation of the biomaterial over time was shown by extensive histology and microCT analysis of aneurysm sections. A biomaterial-based approach for the treatment of saccular aneurysms with or without rupture allowing rapid delivery and achieving instant and durable occlusion of the bleeding aneurysm may save lives.

2. STH-I fabrication and testing in vitro

2.1. Development of STH-I formulation for real-time visibility

Endovascular embolization of SAs require real-time visualization of the embolic material as it exits the catheter tip to prevent over-injection, which can lead to potentially fatal non-target embolization. Most common embolic material used to treat SAs are made from metallic materials; while these metallic materials demonstrate excellent visualization by fluoroscopy during the embolization procedure, they limit future assessment of the aneurysm sac for recanalization as they produce CT and MRI artifacts obscuring the aneurysm.^[9] Therefore, we developed a formulation of the STH that is visible under fluoroscopy, CT, and MRI with material properties that allow safe embolization of aneurysms. Since SA embolization procedures are performed using microcatheters, it was critical for STH to be visible under X-ray in small volumes in sub-mm catheters. STH was synthesized using gelatin and nanoclay, as previously described.^[18] Unlike previously tested biomaterials, here we mixed the biomaterial with high concentrations of iohexol (STH-I), which is a clinically used intravascular contrast agent (Figure 1a). The addition of iohexol, which is a nonionic water-soluble compound comprised of 46.4 % iodine by molecular weight,^[19] led to substantial changes to the silicate oxide spectral band on Fourier-transform infrared (FTIR) spectroscopy (Figure 1b), the appearance of the biomaterial on scanning electron microscopy (SEM) (Figure 1c) and the mechanical properties (Figure S1, Supporting Information). Consequently, STH-I was further developed, and the formulation was optimized to balance maximal visibility, to avoid non-target embolization when injected through microcatheters, with mechanical properties that favored successful embolization of SAs.

Figure 1. STH-I fabrication and characterization in vitro.

a) Image showing the injectability of STH-I through a 5F catheter. b) Attenuated total reflection Fourier-transform-infrared (ATR-FTIR) spectroscopy demonstrating significant spectral changes between STH and STH-I mediated by iohexol; Si-O absorption band, representing nanoclay, at ~1000 cm⁻¹ wavenumber is similar between the two biomaterials. c) Representative scanning electron microscopy images (SEM) demonstrating marked differences in the microstructures of STH, and STH-I. Scale bar = 100 micron. d) High level fluoroscopic (HLF) images (top) and corresponding graph (bottom) summarizing pixel intensity measured in STH-I-loaded syringes containing varying concentrations of iohexol contrast agent (n = 3). e) CT axial images (top) and corresponding graphic summary of signal intensity in Hounsfield Units (HU) of 7 syringes loaded with STH containing 0–50 wt % iohexol demonstrating enhanced intensity compared to pig aorta (top right, yellow dashed outline) (n = 3). f) Transverse views (top) and graphic summary (bottom) of standard T1- and T2-MRI weighted images of STH-I loaded syringes with varying concentrations of iohexol. There is enhanced visibility on T1-weighted image (T1WI) with peak signal intensity at 30% iohexol compared to the human aorta (yellow outline) and T2-weighted images (T2WI). g, h) Representative injection force curves and graph showing injection and break loose forces generated by STH-I1, STH-I2 and STH-I3 in a 1 cc syringe and injected through a 110 cm 2.8 F microcatheter at 1 mL min⁻¹ of injection rate (n = 5). i) Representative amplitude sweeps of STH-I1, STH-I2, and STH-I3 showing G’ and G” curves as a function of the oscillatory shear strain measured at 10 rad s⁻¹ at 37 °C. j) Graph summarizing storage modulus of STH-I1, STH-I2, and STH-I3 (n = 3). k) Flow curve demonstrating shear thinning property of STH-I2. l) Plot of shear stress versus shear rate of STH-I2, illustrating its shear thinning property. m) Frequency sweep of STH-I2 performed at 0.1 % shear strain, demonstrating STH-I2 stability. n) Thixotropy tests showing STH-I2 recoverability under repeated oscillation cycles at 10 rad s⁻¹ consisted of 5 minutes low strain (0.1 %) and 5 min high strain (100 %). o) Injection force curves generated by repeated cycles of short compression force application interrupted by pause periods through a 110 cm 2.8F catheter at a flow rate of 1mL min ⁻¹. p) Resistance pressure curve measuring the maximum pressure required to displace blood (control), coils, STH-I2, or coils + STH-I2 demonstrating STH-I2, or STH-I2 + coils ability to withstand supraphysiologic of 16 kPa (dotted red line) (n = 3). Data are mean ± SEM calculated by one-way ANOVA with Dunnett’s multiple comparison tests. * p < 0.05 and **** p < 0.0001.

To identify the iohexol concentration that can provide optimal visibility on various imaging platforms, image analysis of syringes filled with STH-I containing 0, 10, 20, 30, 40, or 50 wt % iohexol was performed using high-level fluoroscopy (HLF), CT, and MRI (Figure 1d–f and Figure S2, Supporting Information). Figure1d and Figure S2a shows that STH with ≥ 30 wt % iohexol demonstrates sufficient pixel intensity to allow adequate visibility for fluoroscopy procedures. Corresponding CT imaging of the same samples revealed that maximal intensity of CT imaging occurred at STH samples containing ≥ 20 % iohexol with plateauing at approximately 3000 Hounsfield units (Figure 1e and Figure S2b, Supporting Information). The CT intensity of STH with ≥ 20% iohexol in Figure 1e is uniformly bright and easily discernible when compared to the CT intensity of the porcine aorta; the intensity also demonstrates no imaging artifacts or distortions, which will allow accurate evaluation of the aneurysm sac. When imaging STH-I at various CT parameters, no discernible difference or imaging artifacts were noted when compared to 100 % iohexol alone (Figure S2c, Supporting Information). High-resolution microCT imaging of STH-I showed that the intensity was uniform and, more importantly, it did not change up to 28 days when stored at room temperature (25 °C) and did not demonstrate any phase separation (Figure S2d and e, Supporting information). These long-term in vitro studies demonstrated the stability of STH-I imaging suggesting that the interaction between nanoclay and iohexol in the STH-I create a stable nanostructure.

Next, we performed standard T1- and T2-weighted imaging of the same syringes placed over the abdomen of a volunteer using a clinical 3-Tesla MRI scanner. These images showed that T1 weighted imaging produced a brighter signal compared to the darker images on T2-weighted images (Figure 1f). On T1-weighted images (T1WI), the MRI signal from the STH-I was significantly higher than the signal in the aorta; this suggests that the STH-I signal intensity is sufficiently high without artifact to allow visualization of the STH-I within an arterial aneurysm (Figure 1f and Figure S2f, Supporting Information). On T2-weighted images (T2WI), however, the signal from the STH-I was uniformly dark (Figure 1f and Figure S2g, Supporting Information). For the first time, the unique MRI properties of STH-I may allow accurate assessment for recanalization of aneurysms following treatment using T2 based sequences where blood is bright. Based on these imaging characteristics, STH-I containing 30 wt % iohexol was selected to be the optimum formulation for maximal visibility on multiple imaging modalities without any artifacts which is of paramount importance for in vivo testing.

2.2. Evaluate the mechanical properties of STH-Is

We synthesized three STH-I formulations containing 30 wt% iohexol with increasing total solid content (5%: STH-I1, 6%: STH-I2, and 7%: STH-I3); the ratio between nanoclay and solid content was kept constant at 75 wt% (Figure S3a, Supporting Information) while the gelatin content was increased to characterize the effect of varying these component ratios on mechanical behavior and injectability of STH-I. To determine whether these STH-Is are suitable for microcatheter delivery, the injectability of STH-I formulations through a 2.8 French (F) 110 cm clinically used angiographic catheter was examined using compression testing (Figure S3b, Supporting Information). Injection force tests revealed that STH-I1 and STH-I2 are suitable for microcatheter delivery, whereas STH-I3 exceeded the maximum force of 50 N, which is considered the median injection force generated by an average hand (Figure 1g, h).^[20] In addition to injectability, STH-I mechanical properties were analyzed using rheological studies showing that STH-I2, and STH-I3 provided superior mechanical strength based on storage modulus compared to STH-I1 (Figure 1i, j). Despite differences in mechanical properties, all three formulations containing iohexol appeared to have similar microarchitecture on scanning electron microscopy (SEM) (Figure S3c, Supporting Information). Additional rheological studies showed that STH-I2 exhibited favorable shear-thinning properties, and recoverability based on frequency sweep and thixotropy testing performed under alternating high-strain and low-strain cycles (Figure 1k–n). The reversibility of the shear-thinning property of STH-Is afford clinicians the ability to inject and pause during hand injection, to assess embolization progress without the fear of occluding or cementing the catheter to the vessel, which are concerns for current liquid embolics used today.^[13] To simulate this scenario, repeated cycles of short compression followed by a pause period were performed on STH-I2 loaded into a syringe through a 2.8 F catheter showing no change in its injectability profile (Figure 1o). Furthermore, pressure-displacement tests (Figure S3d, Supporting Information) demonstrated the ability of STH-I2 alone to withstand supraphysiologic pressures without fragmentation compared to packed coils (Figure 1p). When STH-I2 was combined with packed coils inside the same channel, the displacement pressure was substantially increased (Figure 1p). Based on these mechanical tests, STH-I2 demonstrated a favorable injectability profile and excellent mechanical behavior when tested under supraphysiologic pressures.

2.3. Assessing STH-Is stability and retrievability in vitro

Clinically, morphometric parameters of SAs including neck size, aspect ratio (AR), and dome to neck ratio (DNR) are critical; these help determine the approach to the treatment of the SA.^[21] To identify the widest size of SA neck that can be effectively embolized with STH-Is without fragmentation or wash-out from the aneurysm sac under constant pulsatile flow conditions, a human cerebral SA was 3D-printed. Using a reconstructed CT-angiography scan of the aneurysm, a 3D printed model was created to compare the efficacy of STH-I embolization (STH-I, 1–3) and stability under flow conditions using a left ventricular assist device (LVAD) pump in vitro (Figure 2a). The model contained six SAs in tandem with incremental increase in the diameter of the aneurysm neck while maintaining the identical sac shape and dimensions (Figure 2b). The diameter of the SA neck was varied to reflect the AR, and DNR values used to determine treatment options of unruptured SA in clinical practice today where ratios < 2 are typically classified as wide-necked.^[1] These wide-neck aneurysms often require adjunctive therapies for treatment including stent or balloon assisted embolization to avoid non-target embolization. In fact, computer modeling of the 3D printed SAs showed that low AR values in wide-neck aneurysms demonstrate higher perfusion inside the aneurysm sac, suggesting that the embolic material may wash-outside the aneurysm sac; thus, requiring a stent, for example, to keep the embolic material such as a coil inside the SA (Figure 2c and Figure S3e, f, Supporting Information). To perform embolization in these 3D printed models, a 5 French catheter was used to deliver each of the STH-Is under fluoroscopic guidance to simulate the SA embolization procedure. Following embolization, these SA models were subjected to microCT imaging and analyzed to quantitate the STH-I volume inside the aneurysm sac at baseline and the STH-Is that remained after extreme flow conditions of 4.9 L min⁻¹ generated by the LVAD, which is approximately ten times higher than the normal cerebral arterial flow rate (Figure 2d–h).^[22] After 1- or 6-hrs of continuous perfusion, microCT imaging was performed and the volume of STH-I remaining inside the SA was calculated and compared to baseline to determine the degree of STH-I wash-out from each of the aneurysm sac. At 1-hr, 3D analysis revealed no significant wash-out rate at AR values > 2. However, at AR values < 2, all STH-I samples demonstrated significantly greater wash-out of the SA; wash-out ranged from 20–50 % at AR of 1.6. At 6-hr, STH-I1 demonstrated significantly greater wash-out at AR values < 2.3, whereas STH-I2 and 3 demonstrated similar washout at AR values down to 1.8. These data suggested that, at extreme flow rates in an in vitro setting using 3D printed models, STH-I2 and STH-I3 produced sufficient embolization with minimal wash-out at AR values > 2 at a flow rate of 4.9 L min⁻¹ (Figure S3g–i, Supporting Information). While STH-I2 and STH-I3 demonstrated similar wash-out rates in this in vitro testing, subsequent experiments focused on STH-I2 since it required a lower injection force to deliver compared to STH-I3. Furthermore, STH-I2 also showed acceptable injection profiles when using common microcatheters (1.7 F and 2.0 F) often used to embolize cerebral aneurysms; when the injectate from these microcatheters was kept in PBS, STH-I2 remained stable at room temperature up to 6 months without fragmentation or significant dissolution (Figure S4a, Supporting Information).

Figure 2. Assessing STH-I stability and endothelialization potential in vitro.

a) Schematic depicting STH-I stability test using patient-derived 3D printed saccular aneurysm (SA) model and left ventricular assist device (LVAD). b) Image of the 3D-printed SA model containing six tandem aneurysms with identical sac height (H), and dome (D) size, but varying neck size (N) in millimeters resulting in variable H/N aspect ratio (AR), and D/N dome-to-neck size ratio (DNR). c) Representative computer simulation study visualizing laminar flow inside the SA model demonstrating higher flow magnitude and stationary field velocity inside the sac of the SA model with lower AR ratio. d) Sagittal view of 3D reconstructed microCT scan of the SA model post embolization with STH-I1, STH-I2, or STH-I3 showing remnant material relative to the sac volume (whited dotted-outline) at baseline, and after 1-, or 6-hrs of stability test. e-h) Graphs showing percent wash-out of STH-I1, STH-I2, and STH-I3 relative to the SA sac aspect ratio (AR), dome-to-neck ratio (DNR) values at 1- and 6-hrs following stability testing in vitro (n = 3). i) Photograph and schematic depicting endothelial cell (EC) adhesion and proliferation test in explanted segments of the pig aorta (Ao) containing STH-I2 overlayered with a thin layer of porcine blood. j) Fluorescence images visualizing endothelial cells (EC) on explanted Ao that contained STH-I2 overlayered with a porcine blood (upper panels) and STH-I2 alone without blood (lower panels) at 1-, 6-, or 24-hrs following EC seeding (white arrows in high power image indicate cell mitosis). k) Graph summarizing the number of EC counted per field at 1-, 6-, or 24-hrs post-EC seeding (n = 5). Data are means ± SEM. p values were calculated using two-way ANOVA with Tukey’s multiple comparison test in e to h, and one-way ANOVA with Tukey’s multiple comparison test in k. ***p < 0.001 and **** p < 0.0001. Scale bars, 100 μm in j.

The ability to retrieve or remove unwanted embolic materials during endovascular aneurysm treatment is of paramount importance to avoid intraoperative complications.^[23] To evaluate whether STH-I2 could be retrieved, the Penumbra system, which is FDA approved for stroke aspiration thrombectomy, was used. Figure S4b–f demonstrates that STH-I2 could be successfully retrieved from the parent vessel without compromising the integrity of STH-I2 inside the sac (Movie S1, Supporting Information). Retrievability is an important key feature of STH-I2 in the event of an unintended or accidental non-target embolization; today, retrievability is not possible using the current FDA-approved embolic agents.^[24]

2.4. Evaluating endothelialization potential of STH-I in vitro

To avoid recanalization or rebleeding of a treated aneurysm, rapid endothelialization of the aneurysm neck is highly desired to exclude the aneurysm from the circulation.^[25] To test whether STH-I2 enhances endothelialization, cell culture experiments were performed. To simulate the clinical scenario, fresh porcine aorta was cut into cylinders under sterile conditions to represent the orifice of the aneurysm neck. To capture the in vivo interaction with blood, the aortic cylinder was subsequently filled with STH-I2 and then overlaid with a layer of fresh porcine blood to represent the STH-I2-blood interface. These cylinders were then incubated with a fluorescently labeled endothelial cell (EC) suspension (Figure 2i); adherence and proliferation of these cells were then measured. Results showed substantial EC attachment and proliferation to the STH-I2-blood clot surface at each time-point with near complete coverage of the blood interface within the aortic cylinder with EC cells by 24 hours (Figure 2j, k). In comparison, there was no detectable EC present in the aortic cylinders containing only STH-I2 (Figure 2j). These observations illustrate that STH-I2 at the aneurysm neck produces a uniform layer of thrombus and encourages endothelialization that will likely rapidly seal the aneurysm neck in vivo, which is an important feature of STH-I2 to prevent rebleeding and recanalization. In stark contrast, incomplete endothelialization is often reported following coil embolization, which is known to produce irregular surfaces at the aneurysm neck leading to non-uniform thrombus formation and recanalization over time.^[26]

Prior to in vivo experiments, sterility of STH-I2 was also important to demonstrate. Figure S4g showed that our sterile preparation of the STH-I2 led to negative microbial growth up to 28 days of incubation at 37 °C suggesting long-term sterility and suitability for further characterization in vivo.

3. Testing STH-I2 in a rat femoral artery saccular aneurysm (FASA) model

3.1. FASA creation and morphological studies in rat

To test the efficacy of STH-I2 in vivo, a rodent model of hindlimb saccular aneurysm was created using end-to-side anastomosis between the femoral artery and the adjacent femoral vein (Figure 3a, b). The saccular aneurysm model was used to investigate the long-term biodegradation of the biomaterial within the aneurysm sac, to assess its stability, and to evaluate the host immune response to STH-I2 and whether endothelialization occurs as predicted in our in vitro experiments. In addition, this model was chosen to help determine whether fragmentation of STH-I2 occurs from the aneurysm sac; here, any fragmentation would lead to non-target embolization of the digits of the hindlimb allowing detection using a high-resolution laser speckle contrast imaging (LASCI). Such rigorous, high-resolution testing is critical in these experiments because fragmentation as small as 10 microns in a cerebral aneurysm setting may potentially lead to a stroke.^[27] To ensure reproducible AR values of the aneurysm sac measured by angiography of the rat femoral artery, we used rats (weight: 396 ± 8.5 gr, n = 28) of similar age so that the adjacent femoral vein consistently measured similar diameters (outer diameter =1.63 ± 0.13 mm). Rats with FASA were randomly divided into the treated group (n = 14) and into the untreated control group (n = 14). Each aneurysm sac of the treated group received approximately 100 μL of STH-I2; however, in the untreated, control group, the aneurysms remained for the duration of the experiment. Following the surgical creation of the FASA, each rat received LASCI analysis demonstrating consistent flow in the femoral artery, flow inside the aneurysm sac in the control group and absence of flow in the aneurysm sac of the treated group suggesting successful STH-I2 embolization (Figure 3b).

Figure 3. Assessing the embolization efficacy of STH-I2 in a rat femoral artery saccular aneurysm (FASA) model.

a) Schematic depicting femoral artery saccular aneurysm (FASA) embolization procedure and microcatheter tip placement (MC) to the aortic bifurcation (Ao) from a common carotid artery access (CCA) for digital subtraction angiography (DSA) in rats.

b) Gross view and laser speckle contrast imaging (LASCI) of tissue perfusion at baseline, during SA creation, post STH-I2 injection into SA, and at 28 days post embolization (dashed outlines indicate FASA location). c) Representative LASCI scan showing preservation of distal perfusion (dashed outlines) in the hind limb that received FASA embolization compared to the contralateral normal hind limb (non-operated) at 28 days after the procedure. d) Graph showing stable hind limb perfusion in the rat group that received FASA embolization with STH-I2 compared to the untreated control for up to 28 days post-procedure. e) Representative DSA image of a control FASA (red arrow, left panel), or a FASA that received STH-I2 embolization (black arrow, right panel) at 28 days showing complete FASA occlusion in the rat that received STH-I2 and preservation of parent and distal collateral vessels (black arrowheads). f) Representative DSA of FASA and a schematic depicting the method used for measuring; aneurysm neck (N), dome diameter (D), dome height (H), two representative vessel cross-sections located upstream of the aneurysm (D₁ at the proximal neck site and D₂ at 1.5 D₁ upstream site), and maximum dome height (H_max). g-i) Graphs summarizing FASA aspect ratio (AR = H/N), dome-to-neck ratio (DNR = D/N), and size ratio (SR = 2 H_max/(D₁+D₂) measurements based on angiographic imaging obtained at 7-, or 2 days post embolization with STH-I2 compared to control (n = 7 in each group). Data are means ± SEM. Statistical analysis was calculated using two-way ANOVA with Tukey’s multiple comparison test. ns; not significant, * p < 0.05, ** p < 0.01 and ***p < 0.001. Scale bars, 1.0 mm.

Each rat received serial LASCI up to 28 days to examine perfusion in the paws and digits of each hindlimb. Rats in the treated and control group demonstrated normal perfusion at each time point indicating that microscopic fragmentation did not occur suggesting that the STH-I2 remained within the aneurysm sac (Figure 3c, d). Prior to necropsy at day 7, or at day 28, rats in each group received catheter directed digital subtraction angiography (DSA) to evaluate patency of the aneurysm sac, to perform hind limb run-off to the digits to evaluate blood flow, and to perform measurements of the aneurysm such as AR, DNR and size ratio (SR) values. Figure 3e demonstrates representative DSA images of the treated and untreated FASA at 28 days; these images show patency of the aneurysm in the untreated group and absence of contrast opacification of the treated aneurysm sac suggesting successful embolization. These images also demonstrate normal run-off to digits implying patency of these arteries consistent with normal hind limb perfusion on LASCI from Figure 3d; these data suggest that fragmentation of the STH-I2 did not occur (Figure S5a–d and Movie S2–S5, Supporting Information). Figure 3f shows a representative angiographic image of a rat FASA with its corresponding measurements on the schematic image; these measurements were subsequently used to compare AR, DNR, and SR at day 7 and at day 28 in treated and untreated groups. In summary, all rats in the two groups survived without any evidence for aneurysm rupture despite AR, DNR and SR values indicating a high risk.^[21] Each rat at day 7 and at day 28 demonstrated significant reduction in the AR, DNR and SR values with the aneurysm sac nearly excluded following treatment suggesting successful embolization; however, some demonstrated insignificant residual contrast opacification at the orifice (for example, Figure 3e, right, black arrow). These minimal residual contrast opacification at the aneurysm orifice did not show any evidence for recanalization of the aneurysm sac and likely would be of no clinical significance in a patient (Figure 3g–i). In the untreated group, the aneurysm sacs demonstrated persistent patency and a trend toward expansion (Figure 3g, h), suggesting that without treatment the sac would likely become progressively larger over time making them more prone to rupture; this is also consistent with the clinical experience.^{[2, 28]} Despite this trend for the aneurysm to enlarge in the untreated control group, we did not observe any rupture or a sudden fatality during the testing period.

3.2. Assessing structural and morphometric changes in explanted rat FASA

To assess remodeling of the aneurysm sac and the tissue response to STH-I2 following FASA embolization and whether endothelialization occurred at the aneurysm neck, high resolution microCT imaging, SEM and histologic evaluations were performed. In the control untreated group, microCT images demonstrated widely patent aneurysm sac in both day 7 and at day 28 (Figure 4a). In the STH-I2 treated FASA, however, the aneurysm sac remained filled with remodeled soft tissue while preserving the patency of the femoral artery (Figure 4a). On transverse microCT views of the aneurysm sac, STH-I2 enhancement inside the aneurysm sac was visible at day 7 but only minimally visible at day 28. Transverse views at day 7 and at day 28 also showed restoration of the arterial lumen architecture at the level of the aneurysm neck in the treated group suggesting healing or regeneration of the vessel wall. Segmentation and 3D reconstruction of these microCT images showed progressive decrease in the opacification of STH-I2 within the aneurysm sac suggesting biodegradation; the amount of STH-I2 inside the aneurysm sac on microCT imaging was 7-fold less at 28 days in comparison to the measured volumes at 7 days (Figure 4b). On histology, untreated aneurysms demonstrated a persistent aneurysm sac with progressive enlargement and wall thickening at day 28 when compared to day 7 (Figure 4c, d). In the treated group, consistent with microCT imaging, there is progressive replacement of the STH-I2 with fibrotic tissue via a fibroinflammatory response to the biomaterial; this is consistent with significant MPO staining in the day 7 samples compared to markedly reduced staining at day 28 (Figure 4c, e). The transient increase in inflammatory cell infiltration inside the aneurysmal sac that resolves by 28 days following embolization supports biocompatibility and may help reduce the risk of recanalization and rebleeding after endovascular treatment.^[29] Furthermore, histology images at day 7 demonstrated organized thrombus associated with the STH-I2 at the biomaterial-blood interface within the aneurysm sac, as expected based on cell culture experiments. At day 28, this organized thrombus has progressed to dense connective tissue with myofibroblasts and scattered elastin staining permanently excluding the aneurysm sac from the circulation (Figure 4c, f).

Figure 4. MicroCT and histologic evaluation of explanted rat FASA.

a) Gross images and corresponding sagittal and transverse microCT views of untreated control FASA and STH-I2 treated FASA at 7- or 28-days. * Represents STH-I2 within the sac in the treated group. b) Graph showing changes in STH-I2 volume on microCT imaging measured at 7- and 28-days post embolization. c) Representative histology sections of FASA obtained at 7- or 28-days from the control group and the STH-I2 injected group stained with H&E, myeloperoxidase (MPO), Masons’ s trichrome, or Verhoeff’s Van Gieson elastin stain. d-h) Graphs illustrating morphometric changes in sac wall thickness, MPO positive cell count, fibrosis in the aneurysm neck (F_n), fibrosis in the aneurysm sac (F_s), and total aneurysm sac area in the FASA at 7- and 28-day post embolization with STH-I2 (n = 7). Data are mean ± SEM. p value was calculated using Mann-Whitney test. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; Scale bars, 1.0 mm. FA: femoral artery. Scale bars, 1.0 mm in a and b, and 250 μm in d.

In addition, the aneurysm sac was observed to be significantly decreased in size at day 28 and replaced by fibrotic tissue indicating successful remodeling of the STH-I2 (Figure 4c, g, h). Next, the aneurysm sections treated with STH-I2 were further immunostained for alpha-smooth muscle actin (αSMA) to show the presence of smooth muscle cells and myofibroblasts at the remodeled aneurysm neck, and CD-68 to detect macrophages and monocytes. A marked increase in the detection of smooth muscle cells/myofibroblasts was observed at the aneurysm neck that paralleled an increase in macrophage/monocyte infiltration in the aneurysm sac at day 28 compared to day 7 sections (Figure 5a–f). The STH-I2 was markedly infiltrated by macrophages (CD68 staining) that may have facilitated its transformation into a granuloma. The granuloma formation progressed without affecting the ongoing remodeling process of the parent artery. These findings suggest progressive organization of the arterial wall architecture at the aneurysm neck with biodegradation of STH-I2 in the aneurysm sac likely mediated by monocytes/macrophages, which are known to play an important role in SA remodeling.^[30]

Figure 5. Histologic evaluation of FASA biodegradation and endothelialization in rats.

a, b) Representative H&E-stained sections of FASA at 7- and 28-days following embolization showing aneurysm neck (N, red dashed line) and remodeling of the thrombus (Th, black dashed line) to fibrosis tissue at the neck (F_n, red dashed line) and biodegradation of STH-I2 to fibrotic tissue in the aneurysm sac (F_s, black dashed line). c-h) Representative FASA tissue sections immunostained for alpha-smooth muscle actin (αSMA), CD68-positive macrophages, and CD31-expressing endothelial cells in STH-I2 group. i) Graph showing morphometric analysis of endothelial cell (EC) coverage assessed in CD31-stained FASA sections at D7 and D28 following embolization. j, k) Schematic and colored scanning electron microscopy (SEM) images of control or STH-I2 treated FASA at 28 days (D28) showing the aneurysm neck (white dashed line). White square in j shows the lack of EC coverage in the neck of the control FASA compared to the well-aligned spindle-shaped EC layer covering the neck area in the STH-I2 treated FASA (white square in k). Scale bar, 500 μm in a to h, and 20 μm in j and k. Data are mean ± SEM (n = 7). p value was calculated using Mann-Whitney test. *p < 0.05.

Subsequently, CD31 immunostaining was performed to determine whether the aneurysm neck had endothelialized effectively excluding the aneurysm sac from the circulation. Immunostaining for CD31 was performed in serial FASA histologic cross-sections to assess endothelial cell (EC) coverage at the FASA neck region. Morphometric analysis of CD31-stained sections identified a remarkable EC coverage (69.7 ± 9.5 %) within 7 days and complete endothelialization by 28 days in the STH-I2 group (Figure 5g–i). Moreover, on SEM imaging, the EC coverage of the SA neck was absent in the control group (Figure 5j) compared to a complete endothelialization in the STH-I2 group (Figure 5k).

In rat saccular aneurysms, STH-I2 demonstrated consistent embolization of the aneurysm with rapid endothelialization of the neck and progressive fibrosis of the sac effectively treating and healing the saccular aneurysm confirming the in vitro results and cell culture experiments. Moreover, angiography and high-resolution laser speckle imaging of the rats demonstrated the safety of STH-I2 in embolization of saccular aneurysm.

4. Assessing STH-I2 embolization efficacy in a pig model of FASA

In vitro mechanical testing, cell culture and rat experiments suggested that STH-I2 could be a potential embolic material for the treatment of saccular aneurysms even in a state of anticoagulation. A biocompatible embolic material that does not fragment and remains inside the aneurysm sac and demonstrates rapid endothelialization of the aneurysm neck are highly desirable properties for an aneurysm embolization agent. To determine whether STH-I2 could be used to embolize aneurysms in a preclinical model, FASA was created in 50–55 kg anticoagulated pigs to examine the performance of STH-I2 and its superiority over coil embolization, which is the standard of medical treatment of aneurysms with AR > 1.6.^[31] To-date, there is no embolic agent that can successfully treat and consistently exclude a saccular aneurysm in an anticoagulated state from the circulation.

4.1. FASA creation and embolization procedures in pigs

Similar to the rat aneurysm model, FASA was surgically created in pigs using end to side anastomosis of the adjacent vein to the femoral artery (Figure 6a). Following FASA creation, pigs were randomly divided into three groups; those treated with STH-I2, coils, or those that received saline injection only (i.e., untreated control group) in a state of activated clotting time ≥ 200 seconds (Figure 6b). Following a carotid artery access, a 5 French angiographic catheter was delivered to the external iliac artery and digital subtraction angiography (DSA) was performed demonstrating the FASA (Figure 6c). Following 1-week post-surgery, the FASA in all three groups received microCT imaging (Figure 6d). The baseline AR, SR, and DNR measurements of the aneurysms that were treated was not significantly different when compared to the control aneurysms (p > 0.05) (Figure 6e–g). Following embolization, both the control group and the coil embolization group showed persistent flow into the aneurysm sac, as expected due to anticoagulation mimicking the clinical scenario (Figure 6c and Figure S6, Supporting Information). However, FASA that received STH-I2 demonstrated instant occlusion of the aneurysm sac without any evidence for flow in the aneurysm suggesting complete exclusion from the circulation (Figure 6c). At 1-week post-embolization, the untreated control group showed widely patent aneurysms; the coil embolization group showed reduced but persistent flow into the aneurysm sac. However, in the FASA that received STH-I2, the aneurysm sac was excluded in the angiography images with no evidence of recanalization or flow in the aneurysm suggesting successful treatment. Furthermore, consistent with the clinical experience, DSA showing the run-off in the hind limb of the control group and in the coil embolization group showed evidence for distal embolization from thrombi produced inside the aneurysm sac (Figure S7a, b, Supporting Information). In contrast, the STH-I2 treated group showed normal hind-limb run-off (Figure S7c, Supporting Information). Following necropsy, FASA tissues were harvested and imaged using the high-resolution microCT scanner. The control group demonstrated a widely patent FASA (Figure 6d). The FASA that received coil embolization showed extensive streak artifacts limiting evaluation of the aneurysm sac or adjacent tissues (Figure 6d); this is a common drawback of coils in clinical practice. ^[32] However, the FASA that received STH-I2 demonstrated the embolic material inside the aneurysm sac without any imaging artifact allowing, for the first time, on cross-sectional images clear assessment of the aneurysm sac following embolization (Figure 6d). Furthermore, the neck region of the FASA that received STH-I2 showed newly formed soft tissue, suggesting remodeling and exclusion of the aneurysm sac from the circulation (Figure 6d). Morphometric evaluation of FASA at 1 week using the angiography images, compared to baseline, demonstrated reduced AR, SR and DNR. The coil embolization group demonstrated lower AR, SR and DNR compared to baseline, however, the values did not reach normal levels suggesting persistent flow in the aneurysm sac, which can lead to treatment failure (Figure 6e–g). Figure S6 demonstrates foci of recanalization and residual aneurysm sacs in the coil embolization group with the corresponding photographs of the neck region indicating incomplete coverage of the aneurysm neck. STH-I2 treated group demonstrated dramatic, consistent decrease of AR, SR and DNR to normal levels indicating successful treatment (Figure 6e, g).

Figure 6. Evaluating efficacy of embolization with STH-I2 in a pig model of FASA.

a) Schematic demonstrating embolization in the pig model of FASA and a representative gross image of an embolized FASA with STH-I2 (white arrows). CCA: common carotid artery; GC: guide catheter; FA: femoral artery; MC: Microcatheter; SA: saccular aneurysm. b) Graphic summary of activated clotting time (ACT) at baseline and post heparinization. c) Representative DSA images of FASA at baseline, post-treatment, and at 1 week follow up in three groups: untreated control, coil treated, or STH-I2 treated porcine aneurysms. Images demonstrate a patent FASA in the control group (black arrows), partial patency (black arrow) and recanalization of the sac at 1 week in the coil treated FASA (red arrow) and complete exclusion of the aneurysm sac in the STH-I2 treated group (black dashed circles). d) MicroCT images of control, coil or STH-I2 treated FASA showing widely open aneurysm sac in the control group, extensive streak artifact in the densely packed coil embolization group limiting evaluation, and successfully treated STH-I2 group with no artifact in the aneurysm sac (white arrow). e-g) Respective angiographic measurements of aspect ratio (AR), size ratio (SR), and dome to neck ratio (DNR) of control (n = 3), coil (n = 3), or STH-I2 (n = 5) treated FASA at baseline and at 7-days post-treatment. Data are mean ± SEM. p value was calculated using two-way ANOVA with Tukey’s multiple comparison test and paired t-test. ns, not significant, * p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001. Scale bars, 5.0 mm.

To determine whether a failed coil embolization of an aneurysm could be rescued by STH-I2, multiple coils were delivered to FASA; as expected, subsequent DSA demonstrated persistent flow (Figure S7d–f, Supporting Information). Following injection of approximately 1 cc of STH-I2 into the FASA with coils, DSA showed complete absence of flow into the aneurysm sac indicating successful treatment. Coil embolization followed by STH-I2 injection into the aneurysm sac suggests that a biomaterial approach may rescue cases of failed coil embolization (Figure S7g, h, Supporting Information).

Next, to assess whether STH-I2 could treat a ruptured aneurysm, 18–21-gauge needle was used to puncture the dome of FASA in a state of AC to cause catastrophic bleeding, which was confirmed by angiography (Figure 7a). Within 1–2 minutes of rupture, the aneurysm sac was embolized using STH-I2 producing instant hemostasis and occlusion of the aneurysm sac (Figure 7a and Movie S6, Supporting Information). These animals were subsequently survived for 1 week; the animals recovered with no evidence for hematoma while receiving 75 mg of clopidogrel daily, which is a standard anti-platelet therapy. At 1-week post-embolization, angiography was again performed, which showed durable occlusion of the aneurysm sac with no evidence for recanalization (Figure 7a). In one instance, unexpected hemodynamic instability was recognized during surgery; immediate angiography was performed demonstrating massive bleeding likely from aneurysm wall rupture following model creation (Figure 7b). Using a 5 French guide-catheter, the aneurysm sac was immediately accessed and STH-I2 was rapidly injected leading to immediate hemostasis, normalization of blood pressure and heart rate and angiographic absence of the aneurysm sac due to exclusion suggesting successful treatment (Figure 7c). After 1 week of survival, a CT angiogram was performed; these images demonstrated lack of contrast opacification of the aneurysm suggesting occlusion of the aneurysm sac. CT reconstructed images showed the hyperdense aneurysm sac filled with STH-I2 (Figure 7d–f). Morphometric analysis of angiographic images showed no difference in AR and SR values between the ruptured and the unruptured groups (p > 0.05) (Figure 7g, h). MicroCT imaging of ruptured FASA rescued by STH-I2 embolization showed similar imaging characteristics of aneurysms without rupture in the STH-I2 treated group (Figure 7a).

Figure 7. Evaluation of occlusion efficacy and stability in acutely bleeding ruptured porcine FASA with STH-I2.

a) Gross view (top panel) and corresponding angiographic images (lower panel) of a saccular aneurysm (SA) during needle puncture to induce catastrophic bleeding (black arrowhead), bleeding SA showing contrast extravasation following rupture (black arrows), instant hemostasis following endovascular embolization with STH-I2 (blue arrows). MicroCT image of ruptured SA at 1-week post embolization with STH-I2 showing STH-I2 casting the aneurysm sac (black asterisk) and a sealed aneurysm neck (white dash line) with a newly formed hypodense soft tissue that correlated with sac exclusion on DSA (a, right bottom, black dashed circle). b, c) DSA images demonstrate acute, massive bleeding of a spontaneously ruptured FASA in an anticoagulated pig (black arrowheads indicate contrast extravasation) and subsequent instant hemostasis following successful endovascular embolization using STH-I2 (black circle). d-f) Axial and 3D reconstructed images from a whole-body computed tomography (CT) imaging at one week following embolization of a ruptured FASA as shown in b) and c) demonstrate a radiodense STH-I2 filling the aneurysm sac. d) 3D reconstructed contrast enhanced CT angiography image further demonstrates complete exclusion of the aneurysm with non-enhancing soft tissue (e; white circle) in between the healed artery and the STH-I2 filled aneurysm sac. g, h) Graphs showing angiographic measurements of aspect ratio (AR), size ratio (SR) in ruptured (n = 5) or unruptured (n = 5) FASA. i) Graph comparing embolization time during catheter-mediated endovascular delivery of STH-I2 (n = 10), or coil (n = 3). Data are mean ± SEM. p value was calculated using unpaired t-test and Mann-Whitney test. ns, no significance, *** p < 0.001.

Given the acuity of these procedures, the time to catheterization and successful embolization is an important variable that can determine clinical outcome; thus, we measured the time to embolization using STH-I2 and compared this to release of coils into the SA. On average, it took coil embolization 400 ± 105.8 sec, which is about 6.7 times longer than STH-I2 embolization (61 ± 2.2 Sec) (Figure 7i).

In clinical practice, the mortality rate for rupture of aneurysms is over 40%; 66% of those who survive a rupture, suffer from major disabilities^[1]. Moreover, treatment of ruptured aneurysms with coils, fails at a rate of approximately 33.6% after an average time of 1 year,^[33] making coils inefficient for the management of ruptured SAs. In contrast, our pig studies demonstrated instant hemorrhage control and persistent sac occlusion following transcatheter embolization of ruptured aneurysm with STH-I2. These findings suggest that STH-I2 can reduce the risk of rupture, shorten the procedure time, ensure high-quality clinical outcomes, and potentially replace coils as a first-line endovascular treatment of ruptured aneurysm.

Surveillance of vital organs and vascular network distal to FASA revealed no CT imaging abnormalities; there were no abnormal lymphadenopathy or occlusion of the lower limb blood vessels (Figure S8, Supporting Information), and there were no hematologic or biochemical laboratory abnormalities pre- and post-embolization at 1 week (Table S1, Supporting Information).^[34] To rule out systemic inflammatory response to embolization with STH-I2, cytokine/chemokine array analysis was performed on serum samples obtained before embolization and at 1 week post embolization showing unremarkable changes (p > 0.05, Table S2, Supporting Information). The blood tests, and cytokine levels, attest to STH-I2 biocompatibility and safety.

4.2. Histologic evaluation of porcine FASA following embolization with STH-I2

H&E-stained histology sections of FASA tissue harvested at one-hour post embolization showed homogeneous and cell-free STH-I2 casting the aneurysm sac without any visible fragments in the parent femoral artery (Figure 8a). Histologic evaluation of untreated FASA (control) harvested at 7 days after aneurysm creation showed an open aneurysm sac with thickened walls suggesting arterialization of the aneurysm wall (Figure S9a, Supporting Information). All of the aneurysms in the control untreated group demonstrated a thrombus; these thrombi can potentially fragment and cause downstream ischemia,^[35] which is consistent with our observation at 1 week angiography (Figure S6a–c, Supporting Information).

Figure 8. Histologic evaluation of FASA following embolization with STH-I2 in pigs.

a) Images of H&E stained histology sections of FASA at day-0 (D0) following embolization showing STH-I2 casting the aneurysm sac (black asterisk) and complete embolization of the aneurysm including the neck region (black arrows). b, c) Images of H&E stained histology sections of ruptured or unruptured FASA at D7 post embolization showing organized thrombus in the region of the aneurysm neck (#) with granulomatous tissue formed from remodeled STH-I2 (asterisk). d) Images of CD31 immunostained FASA tissue sections obtained at D0 or D7 post embolization showing no EC coverage at D0 and extensive endothelialization at D7 (white arrowheads). e) Graph showing endothelial cell (EC) coverage in the sealed neck region in rupture or unruptured FASA groups (n = 5). f) Graph showing the percentage of EC coverage in the aneurysm neck in rats (n = 7) and pigs (n = 10) at D7 following FASA embolization with STH-I2. Data are mean ± SEM. Statistical analysis was performed using unpaired t-test, or Welch’s t-test. ns, not significant. Scale bars, 500 μm.

Tissue sections of ruptured and unruptured FASA at 7 days post-embolization with STH-I2 showed that the aneurysm sac contained organized thrombus at the neck region overlaying a remodeled STH-I2 filled region forming granuloma without evidence of recanalization or fragmentation (Figure 8b, c and Figure S9b, c, Supporting Information). Furthermore, FASA tissue sections were immunostained for CD31 to determine whether the neck area of the ruptured and unruptured FASA that was embolized with STH-I2 demonstrated endothelialization at 7 days (Figure 8d). CD31 chromogenic and immunofluorescence analysis of the neck region showed that an average of 76.63 % EC coverage occurred in both ruptured and unruptured FASA treated with STH-I2 with no significant difference between the two groups (Figure 8e). Additionally, the rate of EC coverage at 7 days following embolization in pigs was comparable to the findings in rats (Figure 8f).

Complete EC coverage in the neck area post embolization is an important process for restoration of vascular function in the parent vessel bed preventing degeneration, recanalization, and potential rupture following embolization.^[36] In pig SAs, the results consistently showed that STH-I2 led to rapid endothelialization, complete embolization and exclusion of the aneurysm sac from the circulation with no evidence for non-target embolization or recanalization by CT imaging and by angiography. More importantly, in ruptured aneurysms causing catastrophic bleeding, STH-I2 was able to achieve instant hemostasis and embolization of the SA. In both survival and non-survival porcine experiments, STH-I2 outperformed coil embolization.

There are several limitations to our study; large animals were allowed to survive up to 1 week. To have a better understanding of the durability of the biomaterial in embolizing SAs, longer survival studies such as 6 months or to a year will be required. In addition, aneurysms were created in the hindlimbs, instead of the more desirable cerebral circulation, to allow for better assessment of the outcome measures. Aneurysm creation in pig cerebral circulation would be challenging given its small size and limited surgical access to the arteries and assessment for fragmentation would require more complex brain CT or MRIs. In addition, flow diverter stents were not used to compare outcomes to STH-I2 because of their prohibitive cost. We also did not include survival experiments for testing the combination of STH-I2 and coils in the embolization of porcine aneurysms; this scenario among others will be the focus of future studies.

5.0. Conclusions

Saccular aneurysms are common and, depending on the aneurysm location and morphology, they may require close imaging surveillance or prompt treatment; when they rupture, especially in a coagulopathic patient, treatment options are often limited and can be ineffective. Following in vitro optimization, STH-I2 was shown to be visible on multiple imaging modalities without any artifacts, injectable in microcatheters as small as 570 microns at lengths of 180 cm and stable especially when tested in extreme high flow conditions in 3D printed models of aneurysms with narrow neck. STH-I2 also promoted rapid endothelialization in cell culture suggesting that it will likely seal the neck of the aneurysm in vivo preventing rebleeding and recanalization, which can be common when coils are used to treat aneurysms. In saccular aneurysms in rats, STH-I2 demonstrated consistent embolization of the aneurysm with rapid endothelialization of the neck and progressive fibrosis of the sac effectively treating the saccular aneurysm. Moreover, angiography of these rats and high-resolution laser speckle microperfusion imaging both revealed that there was no evidence for fragmentation of the STH-I2 from the aneurysm sac to cause non-target embolization in the hind limb. In saccular aneurysms in pigs, the results were consistent; STH-I2 led to rapid endothelialization, complete embolization and exclusion of the aneurysm sac from the circulation with no evidence for non-target embolization by CT imaging and by angiography. More importantly, STH-I2 in survival experiments was able to successfully treat ruptured aneurysms causing catastrophic bleeding by instant embolization and durable occlusion of the aneurysm. STH-I2 significantly outperformed coil embolization in both survival and non-survival experiments. These pre-clinical data suggest that a hydrogel-based approach to the treatment of aneurysms may save lives as they can lead to instant occlusion and work in anticoagulated states and may lead to a more rapid-adoption as they are easy to use without requiring specific catheters or additional tools for delivery. Hydrogel based approaches may also allow the treatment of aneurysms arising from distal, smaller arteries that are often unreachable for the delivery of flow diverter stents; with the advent of smaller microcatheters, these aneurysms can now be safely accessed and potentially treated using bioengineered embolic materials avoiding open surgery.

6.0. Experimental Section/Methods

STH-I preparation:

Various STH-I formulations were prepared by mixing silicate nanoplatelets (Laponite XLG, BYK USA Inc., Rochester Hills, MI, USA), gelatin (Type A, Sigma Aldrich, St. Louis, MO, USA), and iohexol (Omnipaque 350 mgI ml⁻¹, GE HealthCare, MA) according to previously developed protocol^[37]. Briefly, stock of 37 °C warm gelatin solution (18 wt %), and 4 °C cold nanoplatelets hydrogel (9 wt %) were prepared in ultrapure water. Iohexol-free STH was prepared by mixing gelatin and nanoplatelets and ultrapure water at a weight ratio of 1:6:5. For imaging characterization, iohexol was included in the formulation using gradient concentrations of 0 wt %, 10 wt %, 20 wt %, 30 wt %, 40 wt %, and 50 wt %, respectively, to render STH-I radiopaque under X-ray based imaging modalities, such as fluoroscopy and computed tomography (CT). Homogenous mixing of STH-Is was achieved by using a SpeedMixer (FlackTek Inc., Landrum, SC, USA). Subsequently, three types of STH-Is were prepared for in vitro and in vivo characterization and testing were synthesized using the following formulations: STH-I1 (1.25 wt % gelatin, 3.75 wt % nanoclay, and 30 wt % iohexol), STH-I2 (1.5 wt % gelatin, 4.5 wt % nanoclay, and 30 wt % iohexol), and STH-I3 (1.8 wt % gelatin, 5.2 wt % nanoclay and 30 wt % of iohexol). For all three STH-Is, the total solid contents (gelatin + nanoclay) were 5 wt %, 6 wt %, and 7 wt %, respectively, whereas the ratio between nanoclay and the solid content was kept at 75 %.

Fourier Transform Infrared Spectroscopy (FTIR):

Infrared spectra of STH, or STH-I was obtained using FTIR spectrometer fitted with a diamond crystal (Nicolet, iZ10, ThermoFisher Scientific, Waltham, MA). Each spectra was acquired using attenuated total internal reflectance (ATR) mode and the absorbance was recorded spanning 4000 to 650 cm⁻¹ wavelength at a resolution of 4 cm⁻¹ which was plotted using OMNIC software. Each material was measured at least three times by random sampling from the bulk to ensure consistency.

Scanning electron microscopy (SEM):

SEM was used to assess STH, STH-I, and from harvested FASA tissue. To perform SEM, aliquots of STH, STH-Is were frozen at −80 °C and lyophilized using a freeze dryer (Labconco, 0.120 mBar, and −50 °C). The lyophilized samples were then sputter-coated with 7 nm of gold/palladium (Leica, Wetzlar, Germany) and examined using an SEM (JEOL, JCM-6000plus, Jeol Inc.). To examine the long-term effect of FASA embolization with STH-I2 on the aneurysm neck, a rat femoral artery segment harboring the saccular aneurysm was longitudinally cut to expose and visualize the aneurysm neck directly from the luminal aspect. Tissues were fixed in 10% formalin for 24 hours followed by post-fixation in 1.0 % osmium tetroxide for 1 hour. The samples were then rinsed in ultrapure water, dehydrated in gradient ethanol of 70%, 80%, 95%, and 100% for 5 minutes each, and dehydrated using a critical point dryer (Leica, Wetzlar, Germany). The specimens were then sputter-coated with 7 nm of gold/palladium and imaged using the SEM system.

STH-I imaging:

Aliquots of STH-I containing 0, 10, 30, 40, or 50 wt % iohexol contrast-agent were loaded into 1cc syringes to assess their imaging characteristics using high level fluoroscopy (HLF), axial computed tomography (CT), and magnetic resonance imaging (MRI) modalities. For X-ray-based imaging a syringe containing pure Omnipaque (350mgI ml⁻¹, GE HealthCare, MA) was used as a control. X-ray imaging modality include high-level fluoroscopy (HLF) and CT imaging. HLF signal intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA) in five regions of interest (ROI) in each syringe. CT imaging was performed using a clinical scanner (SOMATOM Force, Siemens Healthineers, Germany). A no-contrast protocol of 80 and 150 kVp energy level with a 3.0 mm detector size configuration was applied to examine the intensity profile of biomaterials and calculate Hounsfield Units (HU) in five different sections in each sample and was compared to the pig aorta. MRI was performed using MAGNETOM Vida (Siemens Healthineers, Germany) to acquire the T1 weighted image (T1WI) (TR: 3.96, TE:1.42, GR: 324 × 384, thickness: 3 mm) and T2 weighted image (TR:550, TE:134, SE: 324 × 384, thickness: 4 mm, SP/5 mm). The samples placed were placed on the abdomen of an adult volunteer to acquire and compare the signal with the soft tissues of the human body. Five scan sections were selected to calculate signal intensity using ImageJ (National Institutes of Health, Bethesda, Maryland, USA) for each sample and aorta.

Rheology:

The rheological properties of STH-Is were performed using a rheometer (MCR 302, Anton Paar, Graz, Austria). All rheology tests were completed at 37°C using 25 mm sand-blasted plate geometry at a 1 mm gap. A solvent trap was used to prevent STH-Is from drying. Amplitude sweeps test at 10 rad s⁻¹ was performed on all three STH-Is which was repeated three times. In addition, rheological properties of STH-I2 were further examined by flow curve and frequency sweep tests at 0.1% fixed shear rate. For shear recovery experiments at 10 rad s⁻¹, the shear rate oscillated via application of 0.1% low strain for 5 min, followed by 100% high strain for 5 min.

Injection force testing:

STH-I injectability testing was performed according to a previously established protocol. ^[18] Briefly, the force to pass STH-I loaded in a 1cc BD syringe through a 2.8 French 110 cm microcatheter (Cook medical, Indiana, USA) at a flow rate of 1 mL min⁻¹ was recorded using a mechanical tester (Instron Model 5942, Massachusetts, USA). For interrupted delivery of the STH-Is, repeated cycles of short compression force lasting 10 seconds followed by a 5 second pause over a 90 second period was performed using a syringe loaded with STH-I2 injected through a 2.8 F catheter. The injection profile, the break-loose, and injection forces were documented using Bluehill version 3 Software (Instron, Norwood, MA, US). Injection force tests was repeated five times for each formulation.

In vitro model of vascular occlusion:

To document the maximum ability of STH-I2, coils, STH-I2, and coils + STH-I2 to withstand supraphysiologic pressures, an in vitro occlusion model was used according to a previously established protocol.^[18] A syringe pump (GenieTouch, Kent Scientific Corporation, Torrington, CT) was used to generate a flow rate of 50 mL min⁻¹ to deliver anticoagulated pig blood inside the system and cause displacement of the embolic agent which was placed inside the tubing. The maximum pressure that required to displace blood alone (baseline control), 1 mL of STH-I2, pact coil fibers, 1 mL of STH-I2 combined with coil fibers was recorded using a pressure gauge sensor (Omega Engineering, Norwalk, CT). Tests were repeated three times for each condition.

In vitro model of saccular aneurysm embolization:

A CT angiographic scan of a de-identified intracranial saccular aneurysm (SA) was segmented using Materialise 3-Matic software (Materialise 3-Matic, Materialise, Belgium) to create a 3D model using CAD software (Fusion 360, Autodesk, USA). The SA model was repeated to produce six tandemly aligned aneurysm sacs with equivalent aneurysm sac dimension and varying neck sizes of 8.6, 7.7, 6.5, 5.8, 5.2-, and 4.0-mm diameter, respectively. The model was printed at 30 μm resolution and 16 μm layer height using VeroClear UV cure transparent RGD810 resin and Objet260 Connex3 3D printer (Stratasys, USA). Six aneurysms were molded inside the model which was fitted with Luer lock port at each end that connect to the main channel representing the parent vessel. In the same fashion, a single 3D aneurysm sac model was 3D printed with a 3 mm inner diameter for the retrievability test.

STH-I stability testing in vitro:

The stability of the STH-I was tested using a 3D printed SA model that was interpositioned inside a tubing that was connected to an FDA approved left ventricular assist device pump (LVAD) (Heart-mate-3, Abbot Laboratories, IL, USA). The sacs of the 3D printed model were embolized with STH-I via a 5 French cobra catheter (Cook Medical, IN, USA) then scanned using a microCT system (SkyScan 1276, Bruker, Belgium) to measure the STH-I volume at baseline. MicroCT imaging was performed using a 0.25 mm aluminum filter and the following scanning parameters: 45 kV, 200 μA, and 10 μm voxel resolution. After scanning, the 3D model was connected to the LVAD pump to generate a cardiac output of 4.9 L min⁻¹ (Q = heart rate (70 bpm) × stroke volume (70 ml)) that simulates the normal hemodynamic state. All model testing was performed using the circulation of 3 liters of phosphate-buffered saline (PBS, pH = 7.4) that was circulated through the pump. At 1- and 6-hrs after the start of stability testing, the models were removed and microCT imaging was repeated to calculate the wash-out rate in each aneurysm sac. Each formulated STH-I was tested three times.

Endothelial cell adhesion and proliferation in vitro:

Porcine aorta endothelial cells (PAEC) (CAT#96052, CellBiologics, Chicago, IL, USA) were incubated in tissue culture medium (CAT#1168, CellBiologics, Chicago, IL, USA) inside a 5 % CO₂ tissue culture incubator at 37 °C. PAEC were fluorescently stained using CellTracker™ CMFDA reagent for 45 minutes according to manufacturer instructions (C2925, CellBiologics, Chicago, IL, USA). To evaluate adherence and proliferation of endothelial cells, a freshly harvested porcine aorta preserved in Hank’s balanced salt solution, was cut into cylinders under sterile conditions to create half-inch long aortic cylinder. The aortic cylinders were subsequently placed vertically inside a Petri dishe (100 mm × 15 mm). In selected aortic cylinders, STH-I2 was used to fill half of the lumen depth then over layered with calcium-activated uncoagulated fresh pig blood collected from the same animal and incubated for 10 minutes to induce a thrombus. A designated group of aortic cylinders were filled with STH-I2 alone and used as control. The surface of each aortic cylinder was seeded with 5 × 10⁴ PAEC suspension in growth medium and incubated for 3, 6, and 24 hours. At the end of the incubation period, the media was removed, and the cylinders were briefly rinsed in PBS and then fixed with 4% buffered paraformaldehyde (Electron Microscopy Science, PA, USA) for 10 minutes, rinsed twice with PBS, then placed downward on a slide chamber containing Vectashield antifade mounting medium (Vector Lab, Burlingame, CA). The fluorescently labeled cells were visualized and imaged using fluorescent microscopy (Evos FL AUTO2, ThermoFisher Scientific, MA, USA). Stained cells were counted and averaged in five randomized fields at 20× magnification. Data were expressed as mean PAEC per field.

STH-I retrievability test in vitro:

The retrievability of STH-I2 from the 3D aneurysm model was performed using a 5 French catheter to embolize the aneurysm sac and occlude a 6 cm segment of the main channel representing the parent vessel. A 6F suction catheter was connected to an aspiration pump (CAT6, Penumbra System, CA, USA) that generated a vacuum pressure of −25 mmHg. The catheter was gradually advanced inside the main channel under fluoroscopic guidance to aspirate STH-I2 from the lumen until all the material was fully retrieved.

Animal studies:

In vivo studies were approved by the institutional animal care and use committee (IACUC) at Mayo Clinic and conducted according to federal and institutional regulations and guidelines.

Rat model of femoral artery saccular aneurysm (FASA):

Male Sprague Dawley rats weighing 395.6 ± 44.9 grams (n = 28) were randomly divided into STH-I2 treatment group (n = 14) and an untreated control group (n = 14) and subjected to femoral artery saccular aneurysm surgery using a Zeiss surgical microscope (Extaro 300 system). Anesthetized rats were placed in a supine position on a warming platform. The inguinal area was prepped then a 2 cm groin incision was made to expose the femoral vessels using a combination of sharp and blunt dissection. A segment of the exposed vessels was dissected away from the femoral nerve. Two 7–0 silk (Teleflex medical, CT, USA) slipknot sutures were placed proximally and distally to temporarily control flow in the femoral artery during the procedure. Three 7–0 silk ties were positioned on the femoral vein to create two separated segments, a longer distal segment that was used for the anastomosis to the side of the femoral artery, and a short proximal segment that was used to divide the femoral vein and free the proximal end of the longer segment to form the aneurysm sac. The longer femoral vein segment was cut transversally at a location closer to the distal end of the vein segment. Arteriotomy on the side of the femoral artery was made that extended to 1.5 times of the formal vein diameter using a microscissor. The lumens of both vessels were flushed with heparinized saline then the distal end of the vein was sutured to the open side of the femoral artery using equidistantly placed 10–0 monofilament sutures (Demetech Corp, FL, USA). Following anastomosis, the slipknots of the femoral artery were removed to resume blood flow in the femoral artery and the newly created aneurysm sac. Following FASA creation, rats in the treatment group received a 200 μL injection of STH-I2 directly through the sac dome using a 29-gauge needle to embolize the FASA whereas rats in the control group received FASA surgery only without treatment. Following embolization, the inguinal incision was closed in layers using 5–0 Vicryl sutures and Vetbond glue (3M, MN, USA). All rats received a subcutaneous injection Buprenorphine SR (ZooPharm LLC, WN, USA) 1 mg/kg once for analgesia and 150 IU/kg heparin (Mylan, PA, USA) that was administrated for three days post-surgery. Subgroups of rats in each group survived for 7, or 28 days after the procedure.

Laser speckle contrasts analysis imaging:

Laser speckle contrast imaging (LASCI) (PeriScan PIM3, Stockholm, Sweden) was used to assess perfusion directly in the surgical field during FASA creation in anesthetized rats that were placed in a supine position. Additionally, distal perfusion was serially assessed in the paws of the hind limbs at baseline before FASA creation and at 1, 3, 7, 14, 21, and 28 days after surgery. Hind limb perfusion was performed in anesthetized rats that were placed in a prone position on a warming platform to maintain core temperature at 37°C. Following 5 minutes stabilization period, the laser was positioned over the rat and images were acquired using the following parameters: working distance = 10 cm, recording duration =1 minute, image resolution = 0.3 mm, frame rate = 25 images s⁻¹, scan area = 2 cm × 2 cm for the groin region and 5 cm × 5 cm for the hind limbs, intensity filter was set at 0.30–10. The region of interest (ROI) was outlined in each hind limb paw to obtain perfusion unit value. The ratio of perfusion values measured in the ipsilateral hind limb that had FASA to the contralateral non-operated hind limb was calculated and expressed as a percentage of baseline perfusion ratio in each animal.

Femoral artery angiography in rats:

Angiography was performed in via common carotid artery access at 7 days or 28 days following procedure. Anesthetized rats were placed in a supine position on an X-ray compatible table followed by common carotid artery cut down to deliver a 5 F catheter sheath and a 2 F microcatheter (Cook medical, Indiana, USA) that was advanced into the femoral artery and positioned proximally to the FASA location under fluoroscopic guidance (OEC 9800 Plus, GE healthcare, IL, USA). To prevent blood clotting during the procedure, 150 IU kg⁻¹ heparin diluted in normal saline was administered through the catheter. Digital subtraction angiography (DSA) was performed by injecting 0.5 mL contrast media (Omnipaque 350 mg mL⁻¹, GE health care, IL, USA) via the microcatheter to opacify the arteries and the aneurysm sac and fluoroscopic images were obtained at 15 frames s⁻¹. FASA dimensions were measured using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). Following angiography, rats were euthanized, and tissues were harvested and fixed for microCT imaging and histology.

Femoral artery saccular aneurysm embolization in pigs:

Male Yorkshire pigs (S&S Farms, Brentwood, CA) weighing 52 ± 4.0 kg were divided into non-survival group (D0, n = 4), or 7 days survival group (D7, n = 10). The survival group included 5 pigs that had FASA surgery without rupture (unruptured) followed by embolization, and 5 pigs had FASA creation then the aneurysm sac was ruptured using an 18-, or 21-gauge needle after aneurysm creation to simulate aneurysm rupture (rupture group) followed by embolization. Anesthesia was induced using intramuscular injection of 5 mg kg⁻¹ tiletamine-zolazepam (Telazol, Zoetis, NJ, USA), 2mg ml⁻¹ xylazine (Vedco inc., MO, USA), and 0.02 mg kg⁻¹ glycopyrrolate (Wyeth, NJ, USA) followed by endotracheal intubation. Animals were placed in a supine position and anesthesia was maintained using inhalation of 1–3 % isoflurane in 100 % O₂. Electrocardiogram, transcutaneous oxyhemoglobin saturation (SpO₂), end-tidal CO₂ concentration, inspired oxygen fraction, and core temperature were monitored and documented throughout the procedure. Ultrasound-guided (Butterfly iQ+, Butterfly Network Inc. Guilford, CT) common carotid access was performed to place a 5 French artery sheath (Cook medical, IN, USA) followed by introducing a 5 French Cobra catheter (Cook medical, IN, USA). The femoral artery angiography of the respective surgical side prior to incision was performed at baseline with the catheter positioned proximally to the location of the anastomosis. Using ultrasound, the femoral artery and vein were identified and traced on the skin overlying the vessels. A vertical incision was made using electrocautery in the inguinal region where the pig’s leg meets the abdomen. The subcutaneous tissues were dissected down to the femoral sheath using electrocautery. Scissors were used to enter the sheath and the femoral artery was dissected then encircled with vessel loops proximally and distally to temporarily control blood flow in the artery. The femoral vein was dissected, and all vein branches were ligated using 5–0 silk (Teleflex medical, CT, USA). The vein was transected proximally and distally with one end of the vein remaining ligated. Arteriotomy was made to the sidewall of the femoral artery and the vein graft was anastomosed to the femoral artery using 6–0 Prolene sutures in a running fashion. A bolus of 150 IU kg⁻¹ Heparin (Mylan, PA, USA) was administered intravenously to induce anticoagulation which was titrated using an iSTAT system (Abbot Laboratories, IL, USA) to measure activated clotting time. The vessel loops were relaxed to restore blood flow and verify saccular aneurysm formation. FASA embolization was achieved by positioning the microcatheter tip inside the aneurysm to deliver STH-I2 to fill the aneurysm sac and seal the neck under real-time fluoroscopy guidance (OEC Elite C-Arm, GE Healthcare Systems, Chicago, IL). Coil embolization was performed using a similar approach through a 2.8 French microcatheter (Cook Medical, IN, USA) which was manipulated into the dome of the saccular aneurysm. Under fluoroscopy guiding, micro coils (Tornado Microcoil, Cook Medical, IN, USA) were pushed to fully pack the sac. Following embolization, or control procedures, DSA was performed to assess FASA as well as local and distal vessel patency. In selected groups of pigs, aneurysm rupture was simulated using a 18 or 21-gauge standard access needle puncture (Cook Medical, IN, USA) after FASA creation. Gross observation and DSA were used to document aneurysm bleeding followed by embolization with STH-I2. Once embolization was completed, the subcutaneous tissue was closed using running absorbable 2–0 polyglactin sutures, and the skin layer was approximated using running subcuticular 4–0 polyglactin sutures (Vicryl, Ethicon, Inc., NJ, USA). Dermabond (Ethicon, Inc. NJ, USA) was applied to cover the incision. Following recovery, the animals were allowed to survive 1 week after the procedure.

Rescue of coil embolization with STH-I in pigs:

Embolization rescue with STH-I2 was performed on a non-survival animal in the same fashion as above. In brief, micro-coils were used to pack the aneurysm sac that was created in anticoagulated pig followed by the DSA to visualize recanalization. A 2.8 French microcatheter was positioned in the proximity of the residual aneurysm sac cavity. STH-I2 was delivered to fill the gap until it reached the interface of the sac with the femoral artery. DSA was repeated to confirm sac exclusion. At 1-hour post embolization, tissues were harvested for morphological examination.

Whole Body CT scan and Analysis:

Whole-body CT scanning was performed on pigs in the survival group at 7 days following embolization of FASA with STH-I2 using a dual-source scanner (SOMATOM, Siemens Healthineers, Germany) to assess embolization efficacy, assess vessel patency and rule out distal embolization and tissue ischemia. During the scan, CT angiography (CTA) was performed by administrating contrast media (Omnipaque, 350mgI mL⁻¹, GE HealthCare, MA) intravascularly to visualize vasculature roadmap. The spiral scan was performed at 80 and 150 kVp energy levels, respectively, with a 0.6 mm detector size configuration. 3D rendering of the imaging stacks was visualized using Visage 7.1 (Visage Imaging Inc., San Diego, CA, USA).

Ex vivo MicroCT scanning:

Excised rat and pig FASA tissues were fixed in 10% buffered formalin and preserved in 70% ethanol then scanned with a microCT system (Skyscan 1276, Bruker Corporation, Kontich, Belgium). Scanning parameters consisted of 45 kV and 200 μA current, 10 μm voxel resolution, 0.4° rotational steps, and 2 frames averaging. The 3D image stacks were reconstructed from the rotation image projections, using the NRecon software (Bruker Corp., Kontich, Belgium) which allowed for adjustment of reconstruction parameters: smoothing, beam-hardening correction, and ring artifact reduction. CTvox software (Bruker Corporation, Kontich, Belgium) was used for 3D rendering of the reconstructed image stacks. 3D morphometric analysis tools of the CTAn software was used to calculate the volume of STH-I2 following segmentation of the volume of interest by applying a standardized global threshold set at a fixed greyscale range of 130–220, representing an X-ray attenuation value which optimally segmented STH-I2 inside the sac in all of the reconstructed scans.

Histopathology and immunohistochemistry:

Paraffin-embedded sections were stained with H&E, Mason’s trichrome or EVG elastin stain and immunostaining for myeloperoxidase (MPO; Ab208670, Abcam), CD68 (ab125212, Abcam), CD31 (Ab182981, Abcam), and alpha-smooth muscle cells actin (αSMA; A2547, Millipore Sigma) as previously described ^{[18, 37]}. Morphometric analysis was performed by an operator blinded to the study using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).

Blood collection and analysis in pigs:

Blood samples were collected via the arterial sheath side port during the procedure and prior to euthanasia. The blood samples were either collected in EDTA-Vacuette tube (Greiner Bio-One, North American, Inc., Monroe, NC), or allowed to clot in a Vacuette tube for 30 minutes at room temperature and then centrifuged at 1,500 xg for 5 minutes to obtain serum aliquots. Complete blood count and serum levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), creatinine (CRE), blood urea nitrogen (BUN), glucose (Glu) were measured using an automated analyzer (HemaTrue, Heska, Loveland, CO) and a Veterinary Chemistry Analyzer (DRI-CHEM 4000, Heska, Loveland, CO), respectively. Serum samples were analyzed using the porcine cytokine/chemokine array 13-plex (Eve Technologies, Calgary, CA) and the analyte concentrations were expressed in picogram per mL.

Statistical analysis:

All data were expressed as mean ± SEM unless otherwise noted. Comparison between two groups were calculated by student t-test and analysis of variance (ANOVA) with post hoc tests used for comparisons between three or more datasets. Statistical significance was evaluated with Prism-9 software (GraphPad Inc., CA, USA). A P < 0.05 was considered statistically significant.