A series of microbiological assays resulting in positivity in intracranial aneurysms has been described, investigating the presence of microbes in intracranial aneurysms, which possibly include those originating from the microbiota. In patients with positive endotoxin test results, the levels of inflammatory factors in aneurysms were increased, suggesting that microbes may be involved in aneurysm remodeling. In this study, CLEM was used to detect microbes in intracranial aneurysmatic tissue, although this approach has rarely been described in previous literature. The IHC and CLEM tests revealed that the bacteria were localized in the cytoplasm of aneurysm vascular SMCs, which was consistent with a recent report on the tumor microbiome [16]. Regarding the contrast between positive FISH staining and negative qPCR amplification results, one possibility is that because the levels of microbiota organisms are low, it was difficult to generate reliable homogeneous test results in the present study. Additionally, a more convincing test requires observation of the process occurring during chronic inflammatory degradation and the repair of the vessel wall [1]. Most bacteria invading the aneurysm wall are cleared by immune cells, but LPS expression can be observed months after the bacteria are ingested, and its detection is feasible since the engulfment of bacterial LPS by macrophages is a slow process [17]. Some specimens tested positive for LPS but negative for LTA. The reason may be that gram-negative bacteria invade or adhere to the aneurysm wall earlier. One of the features of LPS penetration is the recruitment of inflammatory cells by infiltrating a large number of macrophages, which initiates immunopathogenic reactions and causes local vessel tissue inflammation [18], which probably start in the tunica media and spread inward to the adventitia. Inflammatory foci may predispose patients to the development of aneurysms.
Periodontitis-associated bacteria are mostly gram-negative bacteria, while endodontitis-associated bacteria are gram-positive bacteria. Periodontitis-associated bacteria may participate in the process of the formation of periodontal disease [19]. In an in vivo experiment, inhibition of the gut microbiome by oral antibiotics in mice played a protective role against the formation and rupture of aneurysms. The gut microbiome is also dominated by gram-negative bacteria, so the possibility that gram-negative bacteria from the gastrointestinal tract may cause infection cannot be excluded [6]. The probability of thrombosis is more obvious in larger-diameter aneurysms. Hemosiderin released by necrotic cells or ruptured red blood cells often readily interferes with IHC, causing false-positive staining results. At the site of deposition or necrotic cells, LPS and LTA are also more involved in adjacent areas in positive samples, and whether this is related to the occurrence of false-positive results is unknown.
Potential influences of the microbiota
Studies related to intracranial infectious aneurysms (IIAs) have suggested that infective endocarditis, meningitis, cavernous sinus thrombophlebitis and the presence of microorganisms causing definite infectious primary disease via endovascular or extravascular spread can lead to aneurysm formation, especially in the context of immunosuppression [20]. The features of IIA patients differ from those of patients with saccular cerebral artery aneurysms (SCAAs), such as younger age of onset and history of fever, and IIA patients are predisposed to distal middle cerebral artery (MCA) aneurysms. However, the pathological process of IIAs involves mechanisms similar to those that occur in SCAAs, such as intima destruction and SMC proliferation [20, 21]. Most of the specimens with dissecting and bifurcation aneurysms were obtained by conventional surgical management in the major artery that was predisposed to the development of aneurysms exclusive of any infectious process in distal brain arteries. Whether the inflammatory process is a proinflammatory or protective reaction in the context of aneurysms has not been elucidated. The bacterial microbiome has certain effects on aneurysmatic etiology, such as aneurysmatic formation and progression. Thus, characterization of the aneurysmatic microbiome may be an essential step in unraveling the effects that aneurysmatic bacteria have on various aneurysmatic hallmarks.
Endotoxins produced by bacteria in aneurysms may mediate Toll-like receptor (TLR) signaling to activate inflammatory responses. In this study, semiquantitative IHC analysis showed that CD14 expression in the LPS-positive group was significantly increased. Some IHC results revealed LPS deposition in SMCs, and both CD14 and TLR-4 had higher expression in LPS-positive aneurysm samples than in LPS-negative samples. CD14 is the LPS-binding protein receptor that responds to endotoxin. In the TLR family, TLR-2 and TLR-4 are considered the main pattern recognition receptors (PPRs) that respond to endotoxins [22]. CD14 acts as a signal-amplifying coreceptor by moving TLRs into a kinase-rich lipid raft environment [23]. The stimulation of TLR4 by LPS induces the release of critical proinflammatory cytokines that activate potent immune responses [23]. The mechanism of the effects of endotoxin in vascular atherosclerosis has been increasingly elucidated in recent years. In particular, endotoxin acts on vascular SMCs by activating the TLR signaling pathway [22, 24–26]. Endotoxin-activated CD14 mediates inflammatory responses in vascular SMCs, promoting atherosclerosis [25]; moreover, CD14 and TLR-2 are expressed and colocalized in ruptured aneurysms [12]. These results suggest that bacteria activate CD14 through endotoxins, mediating the activation of the TLR signaling pathway to produce an inflammatory response in intracranial aneurysms and thereby affecting aneurysmal progression. This evidence indicates that pathogens influence intracranial aneurysms; the pathogens do not simply coexist with the aneurysm.
The existence of bacteria in intracranial aneurysms has rarely been reported in Asian populations. Evidence of the presence of bacteria in ruptured aneurysms was obtained using qPCR to verify the existence and distribution of organisms of the oral microbiota [12], but the results did not demonstrate the risk associated with rupture [13]. In an earlier NGS and qPCR analysis, the negative results of the qPCR test were not consistent with the detection of oral bacterial microbial DNA via NGS [27]. Later, it was further verified by FISH and IHC that there was a microbiome associated with the aneurysm, while semiquantitative IHC analysis showed that the presence of bacteria aggravated the inflammatory response but was not related to aneurysmal rupture. This outcome is consistent with bacterial DNA findings from a follow-up experimental study [13]. The prevalence of bacteria was 69% in ruptured aneurysm wall samples and 71% in unruptured aneurysm wall samples [28]. In this investigation, positive LPS testing was exhibited in 70.6% of cases, and positive LTA testing was exhibited in 52.9% of cases, suggesting the presence of bacterial LPS in both unruptured and ruptured aneurysms. Bacterial DNA may play a role in the pathogenesis of cerebral aneurysms in general, not merely in ruptured aneurysms. Positive FISH results, which can only be obtained with a few fresh-frozen specimens, revealed bacterial colonization of the aneurysmatic wall, which was remarkable in the constancy of both the bacteria involved and the associated histological changes and a valuable result regarding the microbial role in the etiology of aneurysms. These bacteria in association with aneurysmatic development are highly suspicious, although the pathogenicity of these bacteria remains unclear. These bacteria should be recognized and their significance investigated, colonizing aneurysmatic tissue as non or pathogenic opportunists.
A correlation between inflammatory or bacterial markers in dissecting vs. bifurcation aneurysm failed to be assessed because the sample size was not enough to compare between rupture type and/or location of the aneurysms. Negative LPS testing was observed in 29.4% of the specimens, even if LPS protein was released from gram-negative bacteria. LPS is a monoclonal antibody developed using recombinant parts of the LPS protein as a marker of bacterial infection and was used to analyze paraffin-embedded sections of fixed tissue. The LPS protein may decrease the level of immunogenicity over time. Detection of the LPS protein in the aneurysmatic wall yields imperfect sensitivity and specificity evaluating for the bacterial inflammation associated with aneurysmatic formation. Negative PCR test results may be possible when the condition of the tissue is poor, including tissues that are denatured, not large enough, and effected by an intraoperative artifact, such as cautery or crush, because the specimen was taken by the surgeon under the assertion of complete safety during the operative procedure to obtain the aneurysmatic wall tissue sample from the patients.
The prevailing theory is that atherosclerosis due to chronic inflammation affects the progression and rupture of aneurysms in addition to causing aneurysmal formatio n [5, 28, 29]. Atherosclerosis-mediated arterial vascular degradation has been accepted as the main cause of aortic aneurysm formation [30]. In a large cohort study, there was a prevalence of atherosclerosis in the population with an intracranial aneurysm that was mainly located in the MCA [31, 32]. These data obviously contrast with the prevailing theory that atherogenesis is dominant in the pathogenesis of abdominal aortic aneurysms. However, atherosclerosis is often observed in the aneurysmal roof during clipping. In an imaging study, a correlation between elevated serum lipoprotein levels in the blood associated with the aneurysm and aneurysm wall enhancement was found. Several studies have revealed that aneurysm wall enhancement in unruptured aneurysms is associated with aneurysmal instability and growth [33–35]. Animal experiments have suggested that atherosclerosis is not the dominant factor in aneurysm formation [36, 37]
There is currently no conclusion regarding causality between pathogenic microorganisms and vascular atherosclerosis [38] although previous studies revealed that infection-mediated chronic inflammation plays a role in the mechanism of atherogenesis [5, 25], Azithromycin has been used to target patients with coronary artery disease who were seropositive for Cpn [38, 39]. A significant improvement in endothelial dysfunction and a reduction in cardiovascular event rates were associated with antimicrobial application, but overall mortality was not improved. Notably, inhibition of microbial growth can reduce inflammation and protect against endothelial dysfunction [38, 40]. In a mouse model of aneurysm [6], antibiotics were shown to reduce the inflammatory response and inhibit aneurysmal formation, and this mechanism may be responsible for the improvement in endothelial dysfunction observed in an antibacterial drug trial for atherosclerotic vascular disease, whereas the pros and cons of different gut microbiota ratios in the context of the pathophysiology of aneurysms are not clear. This characteristic of the gut microbiota may serve as a biomarker to reveal the influence of environmental factors [7].
Epidemiological investigations disclosed that the oral microbiome is associated with atherosclerotic vascular disease and cerebrovascular disease and may increase the risk of adverse events [9, 41], but various conclusions are not completely consistent. Subsequent risk factor analysis performed in a large cohort further reported that oral health conditions, such as periodontitis and gingival bleeding, may be risk factors for aneurysmal formation and growth and may increase the risk of subarachnoid hemorrhage (SAH) [14]. The mechanism by which Helicobacter pylori causes stomach ulcers has been recognized [42]. This series of validations suggests that bacteria adhere to and deposit at hemodynamically unstable sites, such as intracranial vascular bifurcations where aneurysms are prone to form, easily, as observed in clinical practice, and then act as trigger points of aneurysmal formation. Then, their products, such as LPS and LTA, stimulate the TLR pathway to mediate inflammatory responses.
The limitation of this study was that the sample size was not large enough to compare ruptured and unruptured aneurysms and the type and/or location of the aneurysms and to identify a correlation between inflammatory or bacterial markers in dissecting vs. bifurcation aneurysms. The presence of follow-up data in these patients is required to evaluate whether unruptured aneurysms that are positive for markers of inflammation or the presence of bacteria are at more risk for rupture. The enrollment time span was too long to assess RNA transcriptome expression, and causality could not be determined during the final investigation. The correlation between microbial-mediated inflammation and aneurysmal rupture needs to be revealed in terms of the pathological properties, but these bacteria seem to play a critical role in aneurysmatic formation. To date, specimens obtained in the clinic have certain limitations, but animal experiments and epidemiological investigations have suggested that there is a link between pathogenic microorganisms and intracranial aneurysms. This investigation should emphasize concerns about the microbiome affecting aneurysms.