Reduced M2 macrophages and adventitia collagen dampen the structural integrity of blood blister–like aneurysms and induce preoperative rerupture

Abstract Objective Blood blister–like aneurysms (BBAs) are extremely rare aneurysms. They are predisposed to preoperative rerupture with a high case‐fatality rate. Here, we attempt to interrogate the distinct clinicopathology and the histological basis underlying its clinical rerupture. Methods Three middle meningeal arteries, 11 BBA (5 reruptured, 6 non‐rerupture) and 19 saccular aneurysm samples were obtained for histopathological investigation. Three reruptured BBAs, 3 non‐reruptured BBAs and 6 saccular (3 ruptured, 3 unruptured) aneurysms were obtained for quantitative flow cytometry analysis. Results Compared with true saccular aneurysms, the BBA aneurysm wall lacks arterial stroma cells including CD31+ endothelial cells and α‐SMA + smooth muscle cells. Only fibroblasts and adventitial collagen were observed in the BBA aneurysm wall. Meanwhile, BBAs were enriched with infiltrated inflammatory cells, especially polarized macrophages. Based on the rerupture status, those reruptured BBAs showed drastically reduced fibroblasts and adventitia collagen. Moreover, M2‐polarized macrophages were observed dominant in BBAs and exhibit repairing cellular functions based on their interplays with arterial fibroblasts. Reduced M2 macrophages and arterial tissue repairing modulation may be responsible for the decreasing collagen synthesis and fibrosis repairment, which potentially dampens the aneurysm integrity and induces BBA aneurysm reruputre. Conclusions BBAs poses histopathological features of occult pseudoaneurysms or dissecting aneurysms. Reduced M2 macrophages and adventitia collagen may dampen the structural integrity of BBAs and induce preoperative rerupture.


| INTRODUC TI ON
Blood blister-like aneurysm (BBA) covers a rare but treacherous subtype of intracranial aneurysms that typically grow on the nonbranching sites of internal carotid arteries (ICA). 1,2 They were first introduced as BBAs (or BBAs) in 1998 by Japan physician Ishikawa based on the blister-like appearance upon autopsy. 1 Features of BBAs have been subsequently summarized in literature, including broad neck, supra-clinoidal position and prevalence on right side and in young females. [3][4][5] Despite occasional case reports documenting their distinct aneurysm structure, the pathophysiology and aetiology remain elusive. 1,6 In contrast to the rare occurrence of approximately 1% in all ruptured brain aneurysms, BBAs exhibit an unproportionally high case-fatality rate. 1,2,[5][6][7] The main contributor was thought to be the unexpected preoperative rerupture during rapid progression. [8][9][10][11] Aneurysm rerupture, regardless of aneurysm subtype, can result in lethal consequences such as conscious deterioration and accumulation of subarachnoid haemorrhage (SAH). Additionally, it is also strongly associated with poor outcomes and increased risk of perioperative complications such as aneurysm avulsion, parent artery laceration and uncontrollable intraoperative bleeding. Hence, BBAs have been reckoned as an unneglectable risk in cerebrovascular neurosurgery. 2 Previous studies by Aoki, Frosen and Lawton have meticulously demonstrated the pathology of conventional intracranial saccular aneurysm. 12 The diverse biological functions of smooth muscle cells and macrophages have been proven pivotal throughout the aneurysm development. However, owning to the limited attention given to BBAs, it remains unknown whether, or to what extent, could similar pathological pattern be applied in BBAs. The clear pathological definition of BBAs remains controversial. Recently, we have described the pathological drives for the progression phenomenon in BBA. 3,13 Intriguingly, the initial results indicate significant discrepancies in pathology and cellular interplay between BBAs and saccular aneurysms. Likewise, irrespective of the aneurysm size, whether the rerupture of BBAs is associated with certain vasculopathy was poorly understood. Therefore, our current study aimed to illustrate the pathological features of BBAs and reveal histological basis for BBA preoperative rerupture. Relevant intercellular interplays and molecular changes were also investigated.

| Patient inclusion and exclusion
From 2017 to 2020, a total of 7,498 patients with intracranial aneurysms were screened for inclusion in our neurological centre.
Detailed inclusion criteria have been described in our previous publications. 3 Briefly, patients were included as BBAs if (1)

| Clinical information
For baseline comparison, clinical information was collected from hospital imaging system, laboratory system and history information system. Patients with BBAs were categorized into reruptured group and non-reruptured group according to the in-hospital clinical records. BBAs were considered as reruptured BBAs if patients showed CT-confirmed re-haemorrhage with/without clinical deterioration during the interval between initial subarachnoid haemorrhage and surgical intervention. BBAs were included as non-rerupture BBAs if patients were confirmed with stable during pre-operational observation and no CT angiography (CTA) or DSA-confirmed aneurysm enlargement was documented.

| Histopathological assays
Based on the included BBA patient, we obtained 11 BBA samples.
In comparison, 19 saccular aneurysms samples and 3 middle meningeal arteries were obtained from the bio-database of West China hospital cerebrovascular neurosurgery sample bank, detailed demographic information was included in Table S1. To obtain saccular aneurysm, the aneurysm dome was carefully dissected after satisfactory placement at the aneurysm neck. In BBA cases, we first reaffirm the accurate diagnosis during gross inspection. Then, we would proceed to collect the BBAs sample when aneurysm shows collectable fibrous dome after a mini-curved clip placed at the neck.
Micro-scissors were used to dissect the remnant aneurysm dome along the blade of clips as the premise of stable clipping. 3 Cryosection slides and paraffin-embedded blocks were used for histopathological studies. H&E and Masson's trichrome were used for basic pathological staining. Collagen content was calculated by ImageJ as the positive area percentage under 20× magnification.
Anti-CD31, antiα-SMA, anti-vimentin antibody were used to detect vascular epithelial cells, smooth muscle cells and adventitia fibroblasts, respectively. Anti-CD45 antibody was used to detect myeloid-derived infiltrated inflammatory cells. Iba1 was used to identify macrophages. Caspase-3 was used to detect the inflammatory apoptosis level in aneurysm wall. Matrix metalloproteinase 9 (MMP-9) was also used to evaluate the pro-inflammatory functions in BBAs. On the other hand, vascular endothelial growth factor (VEGF) was used to detect the tissue repairing process in aneurysm wall. Inducible nitric oxidate synthase (iNOS) and Arginase 1 were used to clarify the functional polarization of in situ macrophages, which also suggest the pro-inflammatory and anti-inflammatory functions of macrophages, respectively. Antibody used in this study are included in Table S2.

| Flow cytometry assays
Tissue flow cytometry was used to analyse the overall macrophage polarization status in the BBAs. Six freshly collected BBA samples (3 from berrylike BBAs and 3 non-berrylike BBAs) and 6 conventional saccular aneurysms (3 ruptured and 3 unruptured) were prospectively collected for tissue flow cytometry experiments. Fresh aneurysm tissue was collected from the operation room and transported in 4℃ to the laboratory for flow cytometry. Aneurysm tissue was dissociated in digestion buffer (digestion buffer = 0.25% trypsin + 2.5 mg/ml collagenase + 50 U/ml DNase; products were purchased from Sigma-Aldrich) into single cell for antibody staining.
Red blood cells were lysed by ACK buffer (Sigma-Aldrich) prior to antibody staining. Flowjo 10.0 was used for flow cytometry analysis and gating. CD45, CD11b, CD86 and CD163 were used separately for the identification of macrophage phenotypes. Detailed antibody information was enclosed in Table S2.
These results suggest that the progression and rerupture of BBA may not follow the conventional vascular pathological patterns in saccular aneurysms. Instead, they may be associated with arterial destruction with fibrous collagen repairment, which is mainly mediated by fibroblasts rather than endothelial cells and smooth muscle cells.

| Diverse infiltrated macrophage functions underlie different aneurysm pathophysiology
Based on the pathological findings, we speculated the infil- VEGF was also increased in the BBAs compared with that in the saccular aneurysms (p < 0.05; Figure 3c, d). In contrast, the MMP-9, a pro-inflammatory extracellular lysis enzyme mainly produced F I G U R E 1 Histopathological study of blood blister-like aneurysms (BBAs). Pathological comparison among healthy middle meningeal arteries, conventional saccular aneurysms, and BBAs in H&E and Masson's trichrome stain (a, left panel). Collagen competent was significantly reduced in BBAs when compared to true saccular aneurysms (a, right histogram) (30.61% ± 4.88% vs. 54.53% ± 5.62%, p < 0.01). Immunofluorescence stain of arterial cells marked by CD31 (vascular endothelial cells, EC), α-SMA (vascular smooth muscle cells, SMC) and vimentin (fibroblasts, FB) in healthy arteries, saccular aneurysms and BBAs (b, left panel). Results showing saccular aneurysms with reduced smooth muscle cells, but elevated endothelial cells and fibroblasts (All p < 0.05). All cell types are reduced in BBAs (0.015% EC, <0.01% SMC and 1.40% FB). Only some residual fibroblasts were observed (b, right panel). All scale bars are specified in the figures F I G U R E 2 Histopathological study of non-rerupture and reruptured BBAs. Histological study indicated that the collagen level is significantly reduced in reruptured BBAs (14.6% ± 6.64% vs. 43.95% ± 4.29%, p < 0.05) (a). No significantly differences were observed between non-rerupture BBAs and reruptured BBAs in vascular endothelial cells, smooth muscle cells (EC: 0.05% ± 0.021% vs. 0.032% ± 0.019%, p = 0.208; SMC: 0.23% ± 0.2% vs. 0.12% ± 0.11%, p = 0.382). Fibroblasts are observed more in the non-rerupture BBAs (1.517% ± 0.23% vs. 1.136% ± 0.14%, p = 0.018) (b). All scale bars are specified in the figure by macrophages, was reduced in the BBAs compared with that in the saccular aneurysms (p < 0.05; Figure 3c, d). Interestingly, these data prompted that the macrophage, rather than other cell types, centres the pathophysiology of aneurysms collagen repair in BBAs. The cellular apoptosis and proliferative level in BBAs were increased, while pro-inflammatory process in saccular aneurysms was more prominent. It strongly indicated a functional dichotomization in different aneurysm phenotypes. The aetiology and pathology of BBAs have remained controversial for decades. 1,2,14,15 Most studies focusing on BBAs were case reports that lack of proper comparison. 1,10,13,16 In-depth histological investigations of aneurysm histopathology mostly excluded BBAs owning to the contentious aneurysm heterogeneity. [17][18][19] By reporting this BBA pathology cohort, our current study provided histopathological evidence that BBAs may be a special type of pseudoaneurysm that providing essential tensile strength, nutrients and inflammatory cell reservoir. 23,24 Fibroblasts are the main cell type that produce collagen fibres to maintain structure and wound healing. 23 Under pathological conditions, arteries/aneurysms experience a dynamic and delicate balance of collagen synthesis and collagen degradation mediated by fibroblasts. 25 Once the balance breaks, arterial lesion would prone to deteriorate or rupture. [17][18][19] Our current finding implied that the reduced and destructed collagen is prominent in reruptured BBAs and directly leads to the instability of its structure.

| Macrophage polarization in BBAs is associated with the rerupture
It emphasized the role of collagen components in maintaining aneurysm structure integrity and prompted a great possibility of taking it as a prediction biomarker for future study. Future collagen-specific MRI may aid the evaluation of BBA rupture prediction. 26 Additionally, the inflammatory process in BBAs was proven to be mediated by macrophages. Of note, these observations largely challenged the previous idea proposed by Ishikawa

| CON CLUS IONS
BBAs poses histopathological features of occult pseudoaneurysms or dissecting aneurysms. Reduced M2 macrophages and adventitia collagen dampen the structural integrity of BBAs and induce preoperative rerupture. Targeting M2-polarized macrophages and its interplay with arterial fibroblasts may be a future therapeutic potential.

ACK N OWLED G EM ENTS
We gratefully acknowledge the families and patients that voluntarily participate in this current study, West China Hospital, Sichuan University, Sichuan Cancer Hospital, McGill University. We thank Dr.
Jianguo Xu, Dr. Yi Liu for providing the necessary platform in performing this research. We thank Dr. Hongyu Zhou, Dr. Jack Antel and Dr. David Sinclair for their kind advice on revising the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
DK WEN contributed to drafting/revision of the manuscript for content, including medical writing for content; played major role in the acquisition of data; contributed to study concept or design; and contributed to analysis or interpretation of data. RQ Chen contributed to drafting/revision of the manuscript for content, including medical writing for content, and played major role in the acquisition of data.
H Li played major role in the acquisition of data. J Zheng contributed to drafting/revision of the manuscript for content, including medical writing for content, and played major role in the acquisition of data. Fu Wei played major role in the acquisition of data and analysis of data. ZY Shi played major role in the acquisition of data. C You contributed to drafting/revision of the manuscript for content, including medical writing for content, played major role in the acquisition of data. M Yang played major role in the acquisition of data; study concept; or design. L Ma contributed to drafting/revision of the manuscript for content, including medical writing for content; played major role in the acquisition of data; study concept or design; and analysis or interpretation of data.

D I SCLOS U R E S
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.