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Citation: Joerger, A.-K.; Albrecht, C.;
Rothhammer, V.; Neuhaus, K.;
Wagner, A.; Meyer, B.; Wostrack, M.
The Role of Gut and Oral Microbiota
in the Formation and Rupture of
Intracranial Aneurysms: A Literature
Review. Int. J. Mol. Sci. 2024,25, 48.
https://doi.org/10.3390/
ijms25010048
Academic Editor: Hidenori Suzuki
Received: 12 November 2023
Revised: 14 December 2023
Accepted: 18 December 2023
Published: 19 December 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Review
The Role of Gut and Oral Microbiota in the Formation and
Rupture of Intracranial Aneurysms: A Literature Review
Ann-Kathrin Joerger 1, Carolin Albrecht 1, Veit Rothhammer 2, Klaus Neuhaus 3, Arthur Wagner 1,
Bernhard Meyer 1and Maria Wostrack 1,*
1Department of Neurosurgery, Klinikum Rechts der Isar, Technical University, 81675 Munich, Germany;
annkathrin.joerger@tum.de (A.-K.J.); bernhard.meyer@tum.de (B.M.)
2Department of Neurology, University Hospital Erlangen, Friedrich-Alexander University Erlangen
Nuremberg, 91054 Erlangen, Germany; veit.rothhammer@fau.de
3Core Facility Microbiom, ZIEL Institute for Food & Health, Technical University of Munich,
85354 Freising, Germany; neuhaus@tum.de
*Correspondence: maria.wostrack@tum.de; Tel.: +49-89-4140-2151; Fax: +49-89-4140-4889
Abstract: In recent years, there has been a growing interest in the role of the microbiome in cardiovas-
cular and cerebrovascular diseases. Emerging research highlights the potential role of the microbiome
in intracranial aneurysm (IA) formation and rupture, particularly in relation to inflammation. In
this review, we aim to explore the existing literature regarding the influence of the gut and oral
microbiome on IA formation and rupture. In the first section, we provide background information,
elucidating the connection between inflammation and aneurysm formation and presenting potential
mechanisms of gut–brain interaction. Additionally, we explain the methods for microbiome analysis.
The second section reviews existing studies that investigate the relationship between the gut and oral
microbiome and IAs. We conclude with a prospective overview, highlighting the extent to which the
microbiome is already therapeutically utilized in other fields. Furthermore, we address the challenges
associated with the context of IAs that still need to be overcome.
Keywords: oral microbiome; gut microbiome; bacteria; periodontitis; intracranial aneurysm
1. Introduction
Subarachnoid hemorrhage (SAH) resulting from the rupture of an intracranial aneurysm
(IA) is a devastating type of stroke affecting around 6/100,000 patients worldwide annually [
1
],
leading to high mortality and morbidity rates [
2
,
3
]. It harbors a case fatality rate of 50% [
4
].
Due to the young age of onset compared to ischemic stroke and intracerebral hemorrhage,
SAH is a major contributor to the stroke-related loss of productive life years despite
advancements in risk assessment, imaging techniques, and surgical and intensive care
treatment [
2
,
3
]. Most SAH survivors suffer from persistent, disabling neurological deficits;
even those who experience some degree of neurological recovery often face ongoing
psychological and cognitive impairments. As a result, 46% of SAH survivors remain
severely disabled in their activities of daily life and are unable to return to work, resulting
in a considerable socioeconomic burden [
3
,
5
]. Approximately 3% of the population harbors
an incidental IA, but only a minority will experience a rupture leading to aneurysmal
SAH [
6
]. While various risk factors for IA rupture have been identified, including smoking,
prior SAH, hypertension, hypercholesterolemia, age, gender, aneurysm location, aneurysm
size, heart disease, and aspirin use [
7
–
12
], their respective individual impact is far from
being fully investigated [13–15].
In recent years, there has been a growing interest in the role of the microbiome in
cardiovascular and cerebrovascular diseases [
16
–
22
]. The “microbiome” encompasses
all microorganisms residing in or on various parts of the human body, which includes
bacteria, fungi, and viruses (and all of their genes). The gut microbiome is particularly
Int. J. Mol. Sci. 2024,25, 48. https://doi.org/10.3390/ijms25010048 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2024,25, 48 2 of 14
susceptible to modulation by dietary habits, lifestyle, and environmental factors [23]. The
role of diet as a risk factor for cardiovascular events has been shown before [
24
]. More
recently, attention has turned to the intestinal microbiome’s role in this process, with certain
dietary components, such as carnitine from red meat and phosphatidylcholine from egg
yolk, being metabolized by gut bacteria into trimethylamine, eventually converted in the
liver into trimethylamine n-oxide (TMAO), which has been established as a risk factor for
atherosclerosis [
25
,
26
]. For aortic aneurysms there is growing evidence about a potential
role of the gut microbiome in formation and rupture [
21
,
27
–
29
]. For example, it was shown
that patients with Campylobacter gracilis or Fusobacterium in their gut microbiome had a
significantly higher incidence of aortic aneurysm-related events [27].
Moreover, the hypothesis exists that the microbiome regulates intracranial processes
like neuroinflammation, brain injury, autoimmunity, and neurogenesis via the activation
of innate and adaptive immune cells [
30
]. Several studies have demonstrated the crucial
pathophysiological role of inflammation in the formation and rupture of IAs [
31
,
32
]. By
modulating vascular inflammation, microbiota may exert both beneficial and detrimental
effects on the development and rupture of IAs. It is worth noting that a substantial
microbiome also exists in the oral cavity which could play a role in IA formation and
rupture. So far, only a few studies have investigated the correlation between the microbiome
and IAs. In this narrative review, we aim to explore the existing literature regarding the
influence of the gut and oral microbiome on IA formation and rupture.
2. Exploring Pathways: Inflammation and Cerebral Aneurysm Formation, Gut–Brain
Interactions, and Microbiome Analysis
2.1. The Role of Inflammation in Cerebral Aneurysm Formation
Increasing evidence suggests that inflammation plays a pivotal role in the formation
of IAs [
33
]. This process includes endothelial dysfunction, followed by an inflammatory
response, the phenotype shift of smooth muscle cells (SMCs), the remodeling of the ex-
tracellular matrix, and ultimately, cell death and degradation of the vessel wall [
34
,
35
].
The initial cause of endothelial dysfunction and subsequent vascular remodeling is the
result of wall shear stress [
36
]. It was shown that areas of high wall shear stress, such as
the apex of an arterial bifurcation, are especially predisposed to aneurysm formation [
37
].
Mechanical shear stress upregulates the expression of pro-inflammatory mediators, such
as the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-
κ
B) [
38
], matrix
metalloproteinases (MMPs) [
39
], interleukin-1
β
(IL-1
β
) [
40
], Ets-1, and monocyte chemoat-
tractant protein-1 (MCP-1) [
41
] and downregulates the expression of anti-inflammatory
mediators, such as nitric oxide (NO) [
40
] in endothelial cells. Pro-inflammatory media-
tors activate the inflammatory response, in which macrophages play a pivotal role [
33
].
Macrophages not only release pro-inflammatory cytokines that attract more inflammatory
cells, but also secrete MMPs that break down the extracellular matrix of the arterial wall,
causing additional damage by promoting the activation of other proteinases. For rats, it
was shown that the presence of macrophages and their derived MMPs was closely linked
to IA growth, and that the inhibition of these MMPs haltered the progression of IAs [
42
].
Similarly, Kanematsu et al. [
32
] found that mice depleted of macrophages had a significantly
reduced risk of developing IAs. Moreover, inhibiting MCP-1, a chemokine that controls the
infiltration of macrophages, prevented the development of IAs in mice [43].
Macrophages are not the sole cells participating in the inflammatory response within
the IA wall. Frosen et al. [
44
] reported in their study, which compared 42 ruptured and
24 unruptured IAs using a histological analysis, that the infiltration of the vessel wall by
both macrophages and T cells was associated with aneurysm rupture. Moreover, mast cells
may also play a role in IA formation. In rats, an elevated presence of mast cells during
IA formation was noted [
45
]. Additionally, the advancement of IA was effectively halted
when a mast cell degranulation inhibitor was administered.
SMCs, primarily located in the media layer of vessels, are the primary cells responsible
for producing the extracellular matrix in the vascular wall [
33
]. During the early stages of
Int. J. Mol. Sci. 2024,25, 48 3 of 14
aneurysm formation, SMCs migrate from the media layer into the intima layer in response
to endothelial injury and undergo proliferation, resulting in myointimal hyperplasia. As
the process continues, SMCs undergo a phenotypic shift from a specialized phenotype
focused on contraction to a dedifferentiated phenotype, which contributes to inflammation
and the breakdown of the extracellular matrix by expressing pro-inflammatory mediators
and MMPs [
34
]. Morphologically, these dedifferentiated SMCs no longer maintain their
tightly compacted spindle-like arrangement, but instead separate from each other and take
on a spider-like appearance within the aneurysm walls, leading to remodeling [46].
MMPs are observed to be produced by both macrophages [42] and SMCs [45] within
the wall of the blood vessels or aneurysms. These MMPs play a role in breaking down
the extracellular matrix of the arterial wall, leading to additional damage through the
upregulation of other proteinases and angiogenic factors [47].
Given this crucial role of inflammation in the pathophysiology of IA formation, the gut
and oral microbiome could also be involved in this process by modulating the inflammatory
response.
2.2. Potential Mechanisms of Gut–Brain Interaction
In rats, after ischemic stroke, an intestinal dysregulation with a greater permeability
of the gut-blood barrier has been shown [
48
]. Consecutively, lipopolysaccharide (LPS)
from Gram-negative bacteria of the intestine is translocated to the systemic circulation [
49
],
activating inflammatory processes. Following cerebral ischemia, the disruption of the blood-
brain barrier permits the entry of LPS into the brain parenchyma. This, in turn, triggers the
activation of Toll-like receptor 4 (TLR4) and the release of inflammatory cytokines, further
intensifying the damage to the ischemic brain [
50
]. Not only after ischemic stroke, but
also after intracerebral hemorrhage (ICH), intestinal permeability increased in mice [
51
].
Moreover, T cells and monocytes originating from intestinal Peyer’s patches accumulated
in the intracerebral hematoma. The expression of pro-inflammatory markers like IL-1
β
,
inducible nitric oxide synthase, and tumor necrosis factor
α
(TNF-
α
) was significantly
elevated in the brain tissue, while this was reversed after fecal microbiota transplantation.
For IA formation, the gut–brain interaction still remains unclear. A direct transloca-
tion of bacteria or LPS to the IAs appears unlikely [
52
]; instead, an indirect mechanism
modulating the inflammatory response in the aneurysm wall is proposed [
53
] (Figure 1).
Other potential mechanisms of gut–brain interaction include the direct stimulation of the
enteric and autonomic nervous system, neuroendocrine pathways, and the production of
biochemical (neuro-)transmitters by microbiota [49].
Int.J.Mol.Sci.2024,25,xFORPEERREVIEW3of13
formationwasnoted[45].Additionally,theadvancementofIAwaseffectivelyhalted
whenamastcelldegranulationinhibitorwasadministered.
SMCs,primarilylocatedinthemedialayerofvessels,aretheprimarycellsresponsible
forproducingtheextracellularmatrixinthevascularwall[33].Duringtheearlystagesof
aneurysmformation,SMCsmigratefromthemedialayerintotheintimalayerinresponse
toendothelialinjuryandundergoproliferation,resultinginmyointimalhyperplasia.Asthe
processcontinues,SMCsundergoaphenotypicshiftfromaspecializedphenotypefocused
oncontractiontoadedifferentiatedphenotype,whichcontributestoinflammationandthe
breakdownoftheextracellularmatrixbyexpressingpro-inflammatorymediatorsand
MMPs[34].Morphologically,thesededifferentiatedSMCsnolongermaintaintheirtightly
compactedspindle-likearrangement,butinsteadseparatefromeachotherandtakeona
spider-likeappearancewithintheaneurysmwalls,leadingtoremodeling[46].
MMPsareobservedtobeproducedbybothmacrophages[42]andSMCs[45]within
thewallofthebloodvesselsoraneurysms.TheseMMPsplayaroleinbreakingdownthe
extracellularmatrixofthearterialwall,leadingtoadditionaldamagethroughtheupreg-
ulationofotherproteinasesandangiogenicfactors[47].
GiventhiscrucialroleofinflammationinthepathophysiologyofIAformation,the
gutandoralmicrobiomecouldalsobeinvolvedinthisprocessbymodulatingtheinflam-
matoryresponse.
2.2.PotentialMechanismsofGut–BrainInteraction
Inrats,afterischemicstroke,anintestinaldysregulationwithagreaterpermeability
ofthegut-bloodbarrierhasbeenshown[48].Consecutively,lipopolysaccharide(LPS)
fromGram-negativebacteriaoftheintestineistranslocatedtothesystemiccirculation[49],
activatinginflammatoryprocesses.Followingcerebralischemia,thedisruptionoftheblood-
brainbarrierpermitstheentryofLPSintothebrainparenchyma.This,inturn,triggersthe
activationofToll-likereceptor4(TLR4)andthereleaseofinflammatorycytokines,further
intensifyingthedamagetotheischemicbrain[50].Notonlyafterischemicstroke,butalso
afterintracerebralhemorrhage(ICH),intestinalpermeabilityincreasedinmice[51].More-
over,TcellsandmonocytesoriginatingfromintestinalPeyer’spatchesaccumulatedinthe
intracerebralhematoma.Theexpressionofpro-inflammatorymarkerslikeIL-1β,inducible
nitricoxidesynthase,andtumornecrosisfactorα(TNF-α)wassignificantlyelevatedinthe
braintissue,whilethiswasreversedafterfecalmicrobiotatransplantation.
ForIAformation,thegut–braininteractionstillremainsunclear.Adirecttransloca-
tionofbacteriaorLPStotheIAsappearsunlikely[52];instead,anindirectmechanism
modulatingtheinflammatoryresponseintheaneurysmwallisproposed[53](Figure1).
Otherpotentialmechanismsofgut–braininteractionincludethedirectstimulationofthe
entericandautonomicnervoussystem,neuroendocrinepathways,andtheproductionof
biochemical(neuro-)transmiersbymicrobiota[49].
Figure1.Gut–braininteraction.Figureshowsapotentialmechanismofgut–braininteraction.Gut
dysbiosisleadstodysregulationofthegut–bloodbarrierandLPStranslocationtosystemic
Figure 1. Gut–brain interaction. Figure shows a potential mechanism of gut–brain interaction. Gut
dysbiosis leads to dysregulation of the gut–blood barrier and LPS translocation to systemic circulation,
consecutively activating the immune system. The immune cells enter the intracranial vessels through
the systemic circulation and exert stress on the vascular endothelium here through inflammatory
mediators. TLR = Toll-like receptor, IL = interleukin, TNF = tumor necrosis factor, iNOS = inducible
nitric oxide synthase, IFN = interferon. Created with BioRender.com.
Int. J. Mol. Sci. 2024,25, 48 4 of 14
2.3. Methods of Analyzing the Microbiome
The two currently predominant approaches for microbial identification in microbiome
samples involve the next-generation sequencing (NGS) of gene amplicons from marker
genes, such as 16S rRNA, or shotgun metagenomics [54].
16S-rRNA gene amplicon sequencing: This approach is a targeted approach, i.e., with
the help of the polymerase chain reaction (PCR), a marker gene of interest is amplified.
The amplicons are then sequenced in high throughput and the sequences are used to
identify an organism. The primary target for bacterial identification is normally the 16S-
rRNA gene [
54
]. Due to its critical role in the ribosome, it is a well-conserved gene and
suitable for the taxonomic classification of bacteria [
55
]. The 16S-rRNA gene sequence can
be divided into invariable regions and nine variable regions (V1–V9). PCR is used with
specific primers, which bind in the conserved regions. However, the most-used current
sequencing machines only cover 2
×
300 bp, and therefore, only one to three (adjacent)
variable regions are amplified. In the medical context, many 16S rRNA-based genotyping
protocols focus on V1–V3, V3–V4, or the V4 regions, while, for instance, V5–V6 or V6–V8
are used more often in other fields (e.g., soil samples). Of note, despite their name, the
invariable regions are also not completely fixed and primer bias occurs, since some primers
may or may not bind certain taxa [
56
]. Novel long-read sequencers may cover the entire
length of the 16S-rRNA gene, which can increase species-level resolution [
57
]. In any case,
after sequencing, the data are used to identify and categorize microbial profiles for alpha
and beta diversity and further advanced analyses (see [
58
] on Type 2 diabetes as example).
Finally, a note of caution. Detecting bacteria, which may cause aneurysms but are only
present in low numbers using an amplicon-based approach, is challenging [
59
,
60
]. For
instance, the placenta microbiome has turned out to be purely due to contamination [
61
].
Future research must therefore use proper controls and care to avoid false conclusions.
Metagenome sequencing: This approach is an untargeted approach; i.e., where possi-
ble, the complete DNA of a given sample is isolated, fragmented, and sequenced (shotgun
sequencing; [
54
]). Due to its untargeted nature, it could, in principle, detect all organisms
present; however, this is limited by sequencing depth. For instance, biopsies might contain
too-low numbers of bacteria and their DNA is “drowned” in human DNA. In contrast,
in stool samples, where primarily only bacteria are found, one can uncover the genes,
pathways, and metabolic functions existing within the community [
62
]. However, this still
is limited, since the function of about 40–60% of the genes present in a given sample cannot
be functionally predicted [
63
]. Nevertheless, deep-sequenced metagenome samples are
certainly helpful in detecting bacteria, which might cause or have caused an aneurysm
(see below).
3. The Gut Microbiome and Intracranial Aneurysm Formation and Rupture
While several studies have investigated the microbiome’s influence on stroke
[17–19,64]
,
there are limited data on the role of the microbiome in IA formation and rupture. The
studies discussed here are depicted in Table 1.
Int. J. Mol. Sci. 2024,25, 48 5 of 14
Table 1. Overview of studies on the gut microbiome and IAs.
Study Type Medium Intervention Aim Method Result
Shikata et al.,
2019 [52]
interventional
study mice
gut depletion by
antibiotics in mice
with IA induction
vs. mice with
normal gut and
IA induction
- number and
rupture rate of
IAs;
- number of
macrophages in
IA tissue;
- mRNA levels of
cytokines in IA
tissue.
- immunohisto-
chemistry;
- RT-PCR.
- gut depletion reduced the
incidence of IA (83% vs. 6%,
p< 0.001) and rupture;
- macrophage infiltration
and mRNA levels of
inflammatory cytokines
were reduced with gut
depletion.
Li et al.,
2020 [65]
case–control
study
- humans
- mice
- analysis of fecal
samples of 140
UIA and 140
control patients;
- 20 mice treated
with UIA patient
feces and 20
treated
with control feces.
- comparison of
gut microbiome
of patients with
UIAs and
without;
- test, if changes
in the gut
microbiota
influence the
progression of
UIAs in vivo.
- metagenomic
shotgun
sequencing;
- serum
metabolomic
analysis.
-Bacteroides ssp., Odoribacter
splanchnicus,Clostridium ssp.
were significantly enriched
in the UIAs;
-Hungatella hathewayi was
enriched in the control
group;
- microbiome of UIAs was
significantly dominated by
unsaturated fatty acid
biosynthesis;
- microbiome of controls was
dominated by amino acid
synthesis;
- treatment with feces from
UIA patients increased the
overall incidence of IAs
(85% vs. 45%; p= 0.019) and
rupture rate (82% vs. 22%;
p= 0.009);
- serum concentrations of 2
of 8 fatty acids and 8 of
38 amino acids differed in
mice transplanted with feces
from UIA patients and
controls.
Kawabata et al.,
2022. [66]
multicenter,
prospective
case–control
humans
analysis of fecal
samples of 28
RAs vs. 33 UIAs
comparison of
gut microbiome
of patients with
UIAs and RAs
16S rRNA
sequencing
- gut microbiome profile of
UIAs and RAs were
significantly different;
-Campylobacter ssp. and
Campylobacter ureolyticus
were significantly higher in
the RA group.
He et al.,
2023. [67]
two-sample
Mendelian
randomization
study
humans
database analysis
of gut
microbiome of
patients with IA,
UIA, SAH
association
between the gut
microbiome and
the risk of
IA, UIA, and SAH
inverse variance
weighting
approach
- Candidatus Soleaferrea
decreased the risk of IA;
-Holdemania and Olsenella
increased risk of IA;
- Lentisphaeria,
Porphyromonadaceae,
Bilophila,Fusicatenibacter,
Ruminococcus sp. 1,
Victivallales decreased risk
of SAH;
- Streptococcaceae increased
risk of SAH;
- Porphyromonadaceae,
Bilophila decreased the risk
of UIA;
- Oxalobacteraceae,
Adlercreutzia,
Intestinimonas,Victivallis
increased the risk of UIA.
Int. J. Mol. Sci. 2024,25, 48 6 of 14
Table 1. Cont.
Study Type Medium Intervention Aim Method Result
Ma et al.,
2023. [68]
two-sample
Mendelian
randomization
study
humans
database
analysis of gut
microbiome of
UIA patients
association
between the gut
microbiome
and the risk of
UIA
inverse
variance
weighting
approach
-Clostridia,
Rhodospirillaceae,
Adlercreutzia,Sutterella,
Victivallis,Streptococcus,
Peptostreptococcaceae
increased risk of UIA;
-Oscillospira,
Paraprevotella decreased
the risk of UIA.
IA = intracranial aneurysm, UIA = unruptured intracranial aneurysm, RA = ruptured intracranial aneurysm,
SAH = subarachnoid hemorrhage, RT-PCR = real time polymerase chain reaction, sp. = species (sg.);
ssp. = species (pl.).
Kawabata et al. [
66
] observed significant differences in the gut microbiome between
patients with unruptured intracranial aneurysms (UIAs) and those with ruptured intracra-
nial aneurysms (RAs). The relative abundance of Campylobacter, especially Campylobacter
ureolyticus, was larger in the RA group compared to the UIA group [
66
]. Nevertheless, it is
well-established that brain injuries, such as ischemic stroke and ICH, can already have an
impact on the gut microbiome [
69
], which raises uncertainty about whether the distinctions
between the groups were influenced by the stress associated with SAH or had already
manifested prior to the onset of SAH. A cause–effect relationship between Campylobacter
and aneurysm rupture could not be shown.
In a database analysis of the gut microbiome of patients with UIAs and Ras, He
et al. [
67
] identified three bacterial traits causally related to IAs and six bacterial traits
related to UIAs (Table 1). Six bacterial traits were causally related to a decreased risk of
subarachnoid hemorrhage (SAH) (Table 1).
Another Mendelian randomization study by Ma et al. [
68
] indicated bacteria with
beneficial and detrimental effects on IAs as well (Table 1). However, both studies did not
reflect other individual risk factors.
Nevertheless, these three studies reveal significant differences in the composition
of the microbiome between patients with unruptured and ruptured IAs. They can even
identify specific protective or non-protective bacterial species, suggesting that certain
microbiome compositions could potentially serve as biomarkers for the risk of an aneurysm
rupture in the future. However, correlation does not automatically imply causation. To
identify biomarkers in the microbiome for the multifactorial event of a ruptured IA, it is
necessary to explore the causes of the observed changes in the microbiome, to understand
the mechanisms of signaling and interaction of the microbiome and the host, and to take
into account ethnic variations in the composition of the microbiome [
70
] and other patient-
specific risk factors.
Li et al. [
65
] observed that patients with UIAs had significant differences in their
microbiome composition compared to healthy patients. They also found differences in
metabolic pathways in the microbiome and differences in circulating amino acids between
UIA patients and healthy controls. Moreover, transplanting feces from UIA patients into
mice increased the incidence and rupture rate of IAs compared to mice treated with control
feces, while supplementation with taurine significantly reduced the aneurysm formation
and rupture rate. This study strongly indicates a role of the microbiome and of taurine on
the formation and rupture of IAs. However, the exact mechanism could not be identified.
Moreover, results from mice models should be transferred to the more complex human
context extremely carefully.
Int. J. Mol. Sci. 2024,25, 48 7 of 14
Shikata et al. [
52
] demonstrated that gut depletion by antibiotics significantly reduced
the incidence and rupture rate of IAs in mice, accompanied by a decrease in macrophages
within the aneurysm wall and reduced levels of IL-1
β
, IL-6, and inducible nitric oxide
synthase. Undoubtedly, this study indicates an influence of the microbiome on the devel-
opment of IAs and the associated inflammatory response. However, the following aspects
should be discussed: (I) The authors themselves acknowledge that they cannot explain the
exact mechanism through which the microbiome affects IA formation. They could only
rule out a direct migration of the bacteria into the cerebral arteries, as they did not find any
bacterial DNA in the vessels. (II) The gut microbiota was eliminated by a combination of
four antibiotics. However, these drugs themselves could have an influence on IA formation
and the observed inflammatory response. (III) Antibiotic application not only depletes
the gut microbiome but also microbiota in other sites, which also could contribute to the
observed effects. (IV) Results from mice models are generated in a highly controlled setting
that should be kept in mind when extrapolating the results to patients in a much more
complex setting.
4. The Oral Microbiome and Intracranial Aneurysm Formation and Rupture
The literature on the role of the oral microbiome in intracranial aneurysm formation is
limited. Table 2gives an overview.
Table 2. Overview of studies on the oral microbiome and IAs.
Study Type Medium Intervention Aim Method Result
Pyysalo et al.,
2013. [71]
prospective
cohort study humans
analysis of RA
tissue of
36 patients with
SAH
assess the
presence of oral
and pharyngeal
bacterial
genome in RAs
qRT-PCR
- bacterial DNA was
detected in 21/36 (58%);
- DNA from endodontic
bacteria was detected in
20/36 (56%) and from
periodontal bacteria in 17/36
(47%);
- DNA of the
Streptococcus-mitis group
was the most common.
Pyysalo et al.,
2016. [72]
prospective
cohort study humans
analysis of RA
tissue of
42 patients and
UIA tissue of
28 patients,
tissue from
healthy vessels
and cardiac
by-pass
operations as
controls
assess the
presence of oral
and pharyngeal
bacterial DNA
in RAs and
UIAs
qRT-PCR
- bacterial DNA was
detected in 49/70 (70%);
- 29/42 (69%) of the RA
tissue and 20/28 (71%) of the
UIA tissue contained
bacterial DNA of oral origin;
- RA and UIA samples
contained significantly more
bacterial DNA than control
samples.
Pyysalo et al.,
2018. [73]
prospective
cohort study humans
analysis of
tissue from
gingival
pockets of
30 patients with
RA and 60 with
UIA
assess the
presence of
dental
infectious foci
and
odontogenic
bacteria in
patients
before surgical
treatment of IA
qRT-PCR
- total of 43% had gingival
pockets of 6 mm or deeper;
- bacterial and Fusobacterium
nucleatum DNA were
significantly higher in the
patients with ≥6 mm
gingival pockets than
patients without them.
Int. J. Mol. Sci. 2024,25, 48 8 of 14
Table 2. Cont.
Study Type Medium Intervention Aim Method Result
Inenaga et al.,
2018. [74]
prospective
cohort study humans
analysis of
saliva from
48 patients with
CES, 151 with
non-CES infarct,
54 with ICH, 43
with RA, and 97
with UIA vs.
79 healthy
controls
assess the rate
of
Streptococcus
mutans
with collagen-
binding protein,
Cnm, in CES,
non-CES infarct,
ICH, RA, and
UIA
PCR
- significantly high
Cnm-positive rate was
observed in CES, non-CES
infarct, ICH and RA
compared to controls.
Aboukais
et al.,
2019. [75]
prospective
cohort study humans
analysis of IA
tissue from
10 patients with
RA and 20 with
UIA, samples
from STA, dura
mater, and
MCA as control
assess the
presence of
bacteria in the
walls of UIAs
and RAs
PCR
- no bacterial presence was
found in the wall of
aneurysms.
Hallikainen
et al.,
2019. [76]
case series,
case–control,
prospective
study
humans
oral
examination of
42 patients with
UIAs and 34
RAs compared
to 5170 from
prospective
database
association of
periodontitis
with IA
formation and
SAH
multivariate
logistic
regression
- periodontitis, severe
periodontitis, and gingival
bleeding increased the risk
of IAs significantly;
severe periodontitis in
≥3 teeth or gingival
bleeding increased the risk
of SAH significantly.
Hallikainen
et al.,
2021. [77]
prospective
cohort study humans
analysis of
serum of 227 IA
patients,
compared to
1096 from
prospective
database
association of
IgA and IgG
against
Porphyromonas
gingivalis and
Aggregatibacter
actinomycetem-
comitans with
IA and SAH
ELISA
- high IgA against
P. gingivalis and A.
actinomycetemcomitans
increased the risk of IA and
SAH significantly;
- high IgG levels against
P. gingivalis and A.
actinomycetemcomitans
decreased the risk of IA and
SAH significantly.
Hallikainen
et al.,
2023. [78]
case–control,
prospective
study
humans
oral
examination of
60 patients with
UIA and 30
with RA
compared to
5144 from
prospective
database
association of
caries with IA
formation and
SAH
multivariate
logistic
regression
- caries does not increase the
risk of IAs and SAH.
RA = ruptured intracranial aneurysm, UIA = unruptured intracranial aneurysm, SAH = subarachnoid hemor-
rhage, ICH = intracerebral hemorrhage, CES = cardioembolic stroke, qRT-PCR = real time quantitative poly-
merase chain reaction, STA = superficial temporal artery, MCA = middle meningeal artery, sp. = species (sg.),
ssp. = species (pl.).
Int. J. Mol. Sci. 2024,25, 48 9 of 14
Pyysalo et al. [
73
] performed a dental examination of 89 patients before elective surgery
for an IA. They detected gingiva pockets
≥
6 mm as dental infection foci in 43% of patients.
Moreover, total bacterial and Fusobacterium nucleatum DNA was significantly higher in
patients with
≥
6 mm gingival pockets than in patients without them. Nonetheless, it is
important to note that these data do not permit us to draw any conclusion regarding a
causal link between this observation and the formation of IAs.
In another study, Pyysalo et al. [
71
] detected DNA from endodontic and periodontal
bacteria in 56% (20/36) and 47% (17/36) of tissue samples from ruptured IAs, respectively.
The most frequently identified DNA belonged to the Streptococcus mitis group. Another
study by Pyysalo et al. [
72
] found oral bacterial DNA in 69% (29/42) of ruptured and in 71%
(20/28) of unruptured IA samples. Both tissue types contained significantly more bacterial
DNA than control samples from non-atherosclerotic vessels walls. While these findings sug-
gest a potential association between oral pathogens and IA formation, Aboukais et al. [
75
]
could not detect bacterial DNA in any sample from ten ruptured and 20 unruptured IAs.
A recently published review by Kennedy et al. [
79
] warns against interpreting studies
with bacterial detection from low-biomass tissue. Their analysis of studies on the presence
of bacteria in intrauterine prenatal tissue revealed that this is most likely contamination.
Similarly, in the case of brain tissue, which is regarded as low-to-zero-biomass tissue, there
is a high risk of results being distorted by contamination.
Hallikainen et al. [
76
] found that periodontitis was significantly associated with IAs
and significantly increased the risk of SAH, while caries did not [
78
]. The association of
periodontitis with the risk of IA formation and SAH was independent of gender, smoking
status, hypertension, or alcohol abuse. The authors suggest the following mechanism:
As periodontitis can accelerate the activation and mobilization of circulating neutrophils
or monocytes, resulting in a generalized inflammatory response, it has the potential to
influence the progression of cerebral artery remodeling and aneurysm pathology. This
influence may render the artery more susceptible to aneurysm development and rupture.
The lack of an association of caries with IAs and SAH could be explained by the fact
that caries, contrary to periodontitis, does not predispose to bacteremia. In the case of
periodontitis, there is a vulnerable surface of the gingiva that serves as an entry point for
bacteria into the systemic circulation.
Another study by Hallikainen et al. [
77
] detected that serum IgA antibody levels
against the two key periodontal pathogens, Porphyromonas gingivalis and Aggregatibacter
actinomycetemcomitans, were significantly higher in patients with IAs compared to control
patients. In a multivariate analysis, high IgA serum antibody levels against P. gingivalis and
A. actinomycetemcomitans were significantly associated with a higher risk of IA formation
and rupture, while IgG serum antibody levels against the same pathogens were signifi-
cantly associated with a lower risk. Regarding this discrepancy, the authors provide the
following rationale [
77
]: IgA levels predominantly signify recent or recurrent encounters
with P. gingivalis and A. actinomycetemcomitans, whereas IgG levels are indicative of the
development or triggering of an acquired immune response to these pathogens. Reduced
IgG levels observed in IA patients may arise from several potential factors. One explanation
is the capacity of P. gingivalis and A. actinomycetemcomitans to evade complement-mediated
immune activation. Alternatively, it is conceivable that individuals may have developed
immunity in response to prolonged pathogen exposure, without a concomitant increase in
IgG levels. Furthermore, it is plausible that the quantity of circulating bacteria or bacterial
metabolites/fragments may be insufficient to stimulate a significant elevation in IgG levels.
Nonetheless, a limitation of this investigation lies in the exclusive measurement of IgA
and IgG levels in serum, with no concurrent isolation of bacteria from the oral cavity.
Furthermore, the study did not find any correlation between the clinical oral condition and
the levels of serum antibodies.
Int. J. Mol. Sci. 2024,25, 48 10 of 14
Inenaga et al. [
74
] identified a significantly higher rate of Streptococcus mutans with
collagen-binding protein, a bacterium with hemorrhagic characteristics, such as the ac-
tivation of MMPs, in the saliva of patients with stroke, intracerebral hemorrhage, and
ruptured intracranial aneurysm compared to a healthy control group. However, this was
not a matched comparison, so the difference could be due to confounding factors.
5. Conclusions
The available evidence suggests that the gut and oral microbiome may play a role in the
formation and rupture of IAs. Several studies have identified associations between oral and
gut bacteria, periodontitis, gut microbiota depletion, unsaturated fatty acid biosynthesis,
and IA pathophysiology. However, most studies are limited by a small sample size, the
lack of matched controls, or are based on animal models, which hinder their ability to
establish causality. The process of aneurysm formation in humans is complex and involves
multiple factors, including genetics and exposure to risk factors. Animal aneurysm models
are artificially generated and cannot reflect all these factors sufficiently.
6. Future Directions
In the field of chronic inflammatory bowel diseases and cancer, promising strategies
have already emerged in the context of utilizing the microbiome [
80
,
81
]. Notably, fecal
microbiota transplantation, which involves transferring fecal material containing distal
gut microbiota from a healthy donor to a patient with an imbalanced gut microbiota,
has been established as an effective therapy for recurrent Clostridioides difficile (former
Clostridium difficile) colitis. Furthermore, the European Society of Clinical Microbiology and
Infectious Diseases (ESCMID) has granted approval for the utilization of fecal microbiota
transplantation in cases of recurrent diarrhea following antibiotic-associated diarrhea [
80
].
In the field of cancer research, there are numerous approaches to enhance the response to
immunotherapy through the transplantation of various bacterial strains [81].
For IAs, further research is necessary to elucidate the exact mechanisms by which
the gut and oral microbiome influence IA formation and rupture in humans. Prospective
cohort studies and randomized controlled trials would provide higher-quality evidence
for assessing these relationships. Moreover, the bias of contamination has to be addressed
through a thorough experimental design. Understanding the role of the microbiome could
potentially lead to new preventive strategies and therapeutic interventions for IAs.
Author Contributions: Conceptualization, M.W., V.R. and B.M.; methodology, A.-K.J.; data curation,
A.-K.J.; writing—original draft preparation, A.-K.J. and M.W.; writing—review and editing, A.-K.J.,
M.W., B.M., C.A., A.W., K.N. and V.R.; supervision, M.W. and B.M. All authors have read and agreed
to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data are provided in the text.
Conflicts of Interest: The authors declare no conflict of interest.
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