Histopathologic analysis of hyaluronic acid composite solution following intravascular injection: Variability and safety

Although a composite solution of non‐crosslinked hyaluronic acid is generally considered safe, few studies have investigated its safety after intravascular injection.

It is unclear whether non-crosslinked hyaluronic acid injected into a vessel will have the aforementioned effects or other adverse consequences.In addition, as multiple injections of dermatologic fillers are usually required to achieve the desired esthetic outcome, the safety of non-crosslinked hyaluronic acid needs to be confirmed.
In this study, non-crosslinked hyaluronic acid composite solution was injected into the inferior epigastric artery (IEA) of rats.
The arteries were harvested for histopathologic examination at prespecified postinjection time points to assess changes in the filler characteristics in the lumen.The flap survival rate was evaluated as an index of embolus formation.

| MATERIAL S AND ME THODS
All animal procedures were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory animals.
A total of 12 male Sprague-Dawley rats were randomly divided into four groups corresponding to different postinjection time points.The animal model was established in accordance with the previous study, including time point setting and injected volume. 6ter anesthetic induction, 0.05 mL of sodium hyaluronate composite solution for injection was injected into the IEA of rats using a 32-G needle.The rats were euthanized at 0 min, 45 min, 3 h, and 24 h after injection.In addition, the distal end of the IEA was ligated and filler was injected into the lumen, and changes in vascular structure were examined.The same volume of normal saline was injected into the IEA as a control.The injected arteries were dissected stained with hematoxylin-eosin (HE staining) for histopathologic analysis.
A total of eight rats were selected for the flap survival test.
Flaps measuring 2 × 2 cm nourished only by the IEA on both sides of the lower abdomen were dissected so that only the IEA pedicle remained.A 0.05-mL volume of hyaluronic acid composite solution was then injected into one side of the IEA while the other side was left untreated as the control.The flaps were photographed at 1, 3, 5, and 7 days.In addition to erosion, necrosis, and shedding, the percent surviving area of each flap was measured using Photoshop v13.0 (Adobe Systems).The flap survival rate is expressed as the mean ± SD.Intergroup comparisons were performed with the t-test, and the difference was considered significant at P < 0.05.Data were analyzed using SPSS v20.0 software (IBM).

| Histopathologic findings
At 0 min, the vascular lumen was filled with composite solution.The intima was intact and smooth with uninterrupted endothelial and internal elastic laminae (Figure 1).

F I G U R E 3
The lumen was mostly filled with red blood cells and only a small amount of filler remained at 3 h.HE staining.Magnification: 40×.gaps were filled with red blood cells, and the filler was separated into numerous irregular fragments but without forming any clumps.The intimal structure was essentially intact and continuous, and no obvious abnormality was noted in the smooth muscle layer.There was some monocyte infiltration around the arteries, indicating a mild inflammatory reaction (Figure 2).At 3 h, there was no filler remaining in the lumen of some arteries whereas in others, traces were detected although the lumen was mostly filled with red blood cells (Figure 3).There was no significant difference in the arterial wall structure compared with the 45 min group.
At 24 h, the lumen was unobstructed and contained no filler (Figure 4).The structure of the intima was intact and the smooth muscle layer was thin but intact, with no denaturation observed.
Only a few mononuclear cells surrounded the artery.

| Flap survival
Immediately after injecting the filler into the IEA-the only blood supply of the flap-the tissue appeared pale, purple, and atrophied.
The rats had low skin temperature within 6 h in cases where the artery was completely obstructed, but not if there was transient or incomplete/no obstruction.There was no reduction in the blood supply to the flap following injection of non-crosslinked hyaluronic acid composite solution into the IEA.
After observation for 7 days, all flaps survived, and there was no significant difference in appearance between the flap and surrounding tissue (Figure 5; Table 1).There was no significant difference in the survival rate of flaps between the injected and control groups (p = 0.62).

| DISCUSS ION
Non-crosslinked hyaluronic acid composite solution has been widely used for superficial tissue rejuvenation in cosmetic procedures and is generally considered safe. 8Besides local reactions, there have been no reports of serious complications associated with non-crosslinked hyaluronic acid.However, no studies have evaluated its safety when administered via intravascular injection.The composite solution is typically injected into superficial tissue and no deeper than subcutaneous tissue 9 but in practice, it is not unusual for fillers to be injected into superficial wrinkles in the face, and there is a high risk of adverse reactions associated with injection at certain sites such as the glabella. 10Therefore, the safety of non-crosslinked hyaluronic acid delivered via intravascular injection warrants clarification.
Unlike the gel-like crosslinked hyaluronic acid, currently available products with non-crosslinked hyaluronic acid are generally aqueous; as such, they are not thought to be capable of obstructing vessels.However, because of the elasticity of the smooth muscle surrounding the artery, the lumen is a narrow and tension-filled A histopathologic analysis was performed to examine changes in the filler characteristics once inside the lumen.In a previous study on the safety of crosslinked hyaluronic acid injected into the artery we observed embolization, especially the formation of complex emboli, which is consistent with the clinical process of filler-related embolization; the intravascular changes in the filler were also significant 6 compared with the present observations.In this study, there was residual filler that partly obstructed the lumen at 45 min, but filler loosening and vessel recanalization were also observed.After 3 h, the filler was looser and the lumen had started to recanalize.In other words, high-pressure injection of a large volume of non-crosslinked hyaluronic acid can cause temporary obstruction, but vessel recanalization will occur over time.
The hydrophilic, degradable filler eventually dissolved completely in the bloodstream and there was no residual filler in the lumen at 24 h.We found no serious damage to the arterial endothelium and smooth muscle layer and there was only mild inflammation around the arteries.Thus, intravascular injection of non-crosslinked hyaluronic acid is relatively safe.However, additional flap survival tests are needed to determine whether the filler forms an embolus or leads to tissue necrosis.
Because blood is only supplied by a single artery, the flap will undergo necrosis after embolization, characterized by atrophy and dehydration.Partial obstruction of the artery can lead to varying degrees of necrosis, which is generally more severe in the distal part of the flap. 11,12If blood flow is continuously interrupted for 24 h, the tissue damage will be irreversible. 13On the other hand, if the artery is recanalized early on, at least part of the flap will survive.The results of the flap survival analysis showed that the composite solution did not form an embolus that permanently obstructed the artery, and there was no statistical difference in flap survival rates between the experimental and control groups.However, animal models differ from clinical samples in terms of arterial diameter, blood pressure, and tissue tolerance to hypoxia.Moreover, the flap survival test may only simulate full skin and soft tissue intravascular injection of filler; in the case of specialized tissues such as the eye that cannot tolerate ischemia and hypoxia, even transient vascular obstruction is a safety concern. 10Although no serious complications from the injection of non-crosslinked hyaluronic acid have been reported to date, injection of non-crosslinked hyaluronic acid in high-risk areas requires caution.
A limitation of this study is that the latest time point of histopathologic observation was 24 h; at this stage, the process of dissolution of the filler was still incomplete.Further studies are needed to confirm that non-crosslinked hyaluronic acid can fully dissolve in the vessel lumen.Additionally, the volume of filler entering and the effect on vessels can vary and may be dynamic.A dynamic analysis of how much of the filler remains in the artery over time would be more convincing.The volume of filler injected into a single site is much greater in clinical practice than in an experimental model.Therefore, it is important to assess whether a larger volume carries a higher risk of adverse effects-especially in areas close to the ocular artery such as the glabella/forehead-in order to obtain more comprehensive evidence for the safety of non-crosslinked hyaluronic acid.

| CON CLUS ION
Non-crosslinked hyaluronic acid composite solution is relatively safe for intravascular injection at a minimal volume.When the distal end of the IEA was ligated, the filler remained in the vessel for at least

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no conflicts of interest to disclose.
At 45 min, there were loose, light blue-stained homogeneous fillers in the lumen, which gradually dispersed due to blood flow.The F I G U R E 1 The vascular lumen was filled with composite solution at 0 min.HE staining.Magnification: 40×.F I G U R E 2 Gaps were filled with red blood cells and the filler had separated into numerous irregular fragments at 45 min.HE staining.Magnification: 40×.

F I G U R E 4
The lumen was unobstructed and contained no filler at 24 h.HE staining.Magnification: 40×.F I G U R E 5 Results of the flap survival test.Panels (A)-(D) are representative examples from four rats.The flap with the asterix (*) represents the experimental.space.A composite solution has viscosity, such that if the filler cannot be quickly dispersed by blood flow it can remain in the lumen, resulting in different degrees of obstruction.At the same time, mechanical pressure from injection of the filler into the lumen can cause the artery to spasm, thereby reducing blood flow.Intravascular filler injection has been shown to cause injury to endothelial cells along with exposure of subendothelial collagen and activation of platelets, coagulation factors, and intrinsic coagulation cascades. 6Arterial obstruction and the downstream hemodynamic effects can increase the risk of embolism.
3 h, causing temporary and partial obstruction of the vessel, with eventual vessel recanalization.AUTH O R CO NTR I B UTI O N S Youliang Zhang, Yin Chen, Shiwei Wang, and Shengkang Luo: Contributed to study concept and design.Youliang Zhang, Yin Chen, and Hao Yu: Contributed to experiment operation and data collection.Youliang Zhang, Yin Chen, Huanyun Niu, and Shiwei Wang: Contributed to data analysis, manuscript writing, and critical review of the literature.Youliang Zhang, Yin Chen, and Shengkang Luo: Contributed to critical review of important intellectual content and final approval of the final version of the manuscript.All authors have read and approved the final manuscript.

TA B L E 1
Flap survival rate in the experimental and control groups.